Contents

## **Preface XI**


James H. Lee, David M. Chan and Rodney J. Dyer

Preface

Pollination is an important precedence to reproduction in flowering plants. The productivity of plants in terms of fruit and seed set depends on the efficiency of pollination. As a result, polli‐ nation is equally essential for the productivity of plants in crop production systems and ecosys‐ tems. In nature, this process has been perfected over the ages with plant evolving mechanisms to ensure pollen deposition on the stigma. Through coevolution, plants have developed mech‐ anisms of attracting pollinators and rewarding them for the performance of pollination.

Changing agro-ecological conditions—such as land degradation, deforestation, monoculture agricultural production systems, and global warming—result in low productivity of plants. This makes it necessary that pollination systems within these changing conditions be studied and appropriate measures taken to maintain productivity in the agro-ecological systems.

**Phatlane William Mokwala, PhD**

Department of Biodiversity University of Limpopo Polokwane, South Africa

Senior Lecturer

## Preface

Pollination is an important precedence to reproduction in flowering plants. The productivity of plants in terms of fruit and seed set depends on the efficiency of pollination. As a result, polli‐ nation is equally essential for the productivity of plants in crop production systems and ecosys‐ tems. In nature, this process has been perfected over the ages with plant evolving mechanisms to ensure pollen deposition on the stigma. Through coevolution, plants have developed mech‐ anisms of attracting pollinators and rewarding them for the performance of pollination.

Changing agro-ecological conditions—such as land degradation, deforestation, monoculture agricultural production systems, and global warming—result in low productivity of plants. This makes it necessary that pollination systems within these changing conditions be studied and appropriate measures taken to maintain productivity in the agro-ecological systems.

**Phatlane William Mokwala, PhD**

Senior Lecturer Department of Biodiversity University of Limpopo Polokwane, South Africa

**Chapter 1**

**Provisional chapter**

**Introductory Chapter: Pollination**

**Introductory Chapter: Pollination**

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77227

and animals coexist in same habitats [1].

**1. Introduction**

Phetole Mangena and Phatlane William Mokwala

Phetole Mangena and Phatlane William MokwalaAdditional information is available at the end of the chapter

DOI: 10.5772/intechopen.77227

Pollination is the transfer of pollen grains from the anther, which is the male part of the flower, to the stigma, which is on the female part. This process normally precedes fertilization. It is an important process in the reproduction of plants without which sexual reproduction will not take place. It is a process that has been coordinated and perfected over the ages as plants coevolved with animals, where the animals act as pollinators or pollination agents. The plants

The coevolution between plants and animals in respect of pollination led to the development of pollination syndromes. In pollination syndromes, specific pollination agents pollinate specific plants or flowers. This is, however, not a water tight arrangement as there are polyphilic flowers which attract and are pollinated by different types of pollination agents and polytrophic pollinators which are attracted and pollinated by different types of flowers. Pollination syndromes are a symbiotic relationship between the plants and the agents or animals in which both benefit. The agents get nutrients from the flowers and the plants benefit from improved

There are two types of pollination, namely, cross- and self-pollination. In cross-pollination, pollen grains are transferred from the anther to the sigma of a different plant. Crosspollination when followed by fertilization leads to the production offspring with heterosis or hybrid vigor. Self-pollination, on the other hand, is the transfer of pollen grains from an anther to a stigma of the same flower or plant. This often results in inbreeding depression where undesirable recessive traits are expressed in the offspring. Self-pollination occurs in species where pollination agents are scarce and in closed flowers where the pollination agents

reproduction (**Figure 1**). The latter is very important in crop production.

have limited access to the sexual structures of the flower.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

#### **Introductory Chapter: Pollination Introductory Chapter: Pollination**

Phetole Mangena and Phatlane William Mokwala

Additional information is available at the end of the chapter Phetole Mangena and Phatlane William MokwalaAdditional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.77227

## **1. Introduction**

Pollination is the transfer of pollen grains from the anther, which is the male part of the flower, to the stigma, which is on the female part. This process normally precedes fertilization. It is an important process in the reproduction of plants without which sexual reproduction will not take place. It is a process that has been coordinated and perfected over the ages as plants coevolved with animals, where the animals act as pollinators or pollination agents. The plants and animals coexist in same habitats [1].

DOI: 10.5772/intechopen.77227

The coevolution between plants and animals in respect of pollination led to the development of pollination syndromes. In pollination syndromes, specific pollination agents pollinate specific plants or flowers. This is, however, not a water tight arrangement as there are polyphilic flowers which attract and are pollinated by different types of pollination agents and polytrophic pollinators which are attracted and pollinated by different types of flowers. Pollination syndromes are a symbiotic relationship between the plants and the agents or animals in which both benefit. The agents get nutrients from the flowers and the plants benefit from improved reproduction (**Figure 1**). The latter is very important in crop production.

There are two types of pollination, namely, cross- and self-pollination. In cross-pollination, pollen grains are transferred from the anther to the sigma of a different plant. Crosspollination when followed by fertilization leads to the production offspring with heterosis or hybrid vigor. Self-pollination, on the other hand, is the transfer of pollen grains from an anther to a stigma of the same flower or plant. This often results in inbreeding depression where undesirable recessive traits are expressed in the offspring. Self-pollination occurs in species where pollination agents are scarce and in closed flowers where the pollination agents have limited access to the sexual structures of the flower.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lengths. Because animals can perceive color of specific wavelength, each type of animal will be attracted by a flower of different color [4]. A combination of floral structure, reward, color and scent from the plant and color perception and nutrient preference on the side of the animal will result in a specific pollination syndrome. The following pollination syndromes are recognized: cantharophily (beetles), melittophily (bees), myophily (by flies), psychophily (butterflies and moths), chiropterophily (bats), and ornithophily (birds). In these pollination syndromes, the pollen grains are attached on some parts of the animals and become brushed onto the stigma. On the other hand, some pollen grains are transferred by abiotic factors

Introductory Chapter: Pollination

3

http://dx.doi.org/10.5772/intechopen.77227

The flowers in beetle pollinated plants are unspecialized. They are dull in color, scented, and produce lots of pollen grains. The beetles feed on the pollen grains and other flower parts. The

Flowers pollinated by bats are large, bowl-shaped, dark to green in color, produce large amounts of nectar and pollen, and smell like rotten fruit. Bats pollinating the flowers are fru-

Bees are known to forage flowers for pollen and nectar for their hives. Such flowers are blue, yellow in color or reflect UV light and produce lots of nectar and pollen grains. Carneiro et al. [7] reported of oil gathering bees on flowers containing elaiophores or oil secreting bodies.

Flies are attracted by the mimicry of carrion and feces, a phenomenon called sapromyiophily [8].

Flowers pollinated by birds are red in color, tubular in shape, scented, and nectariferous (Medan). The birds are attracted by the red color and scent while they obtain nectar as reward. Bird pollinators are mainly hummingbirds and sunbirds [9]. These are hoverers which flip their winds while sucking. Others are perchers which sit on branches while suck-

In wind pollinated flowers (anemophily), the pollen grains are very fine released on dangling anthers. The stigmas are sticky and hang out of the flower like beard. This increases the chances of the pollen grains landing on the stigma. This type of pollen transfer occurs mainly

(wind and water) from anther to stigma.

scent is emitted from different parts of the flower [5].

**2.1. Cantharophily**

**2.2. Chiropterophily**

**2.3. Melittophily**

**2.4. Myophily**

**2.5. Ornithophily**

ing nectar.

**2.6. Other pollination syndromes**

givores and nectarivores [6].

**Figure 1.** Flowers of the watermelon (*Citrullus lanatus* Thunb.); A is the male flower and B is the bisexual flower; the species is andromonoecious producing male and bisexual flowers on the same plant; in C, a honeybee is foraging for pollen grains on a male flower and in D, it is foraging for nectar with pollen grains visible on its legs.

## **2. Pollination syndromes**

Within pollination syndromes, plants and animals exploit each other for their own advantage. Animals exploit the relationship to guarantee their reproduction and survival. They forage on the flowers for carbohydrates (in nectar) and proteins (in pollen grains), which are the rewards. On the other hand, the plant guarantees its pollination and reproduction by providing the rewards and offering other attractive features like color and scent [2].

Scent plays a major role in cases where the pollination agents are deceived. The deception involves mainly reproduction hues like pheromones and reproduction substrates or sites [3]. Different plants produce flowers of different colors. The colors reflect light of different wave lengths. Because animals can perceive color of specific wavelength, each type of animal will be attracted by a flower of different color [4]. A combination of floral structure, reward, color and scent from the plant and color perception and nutrient preference on the side of the animal will result in a specific pollination syndrome. The following pollination syndromes are recognized: cantharophily (beetles), melittophily (bees), myophily (by flies), psychophily (butterflies and moths), chiropterophily (bats), and ornithophily (birds). In these pollination syndromes, the pollen grains are attached on some parts of the animals and become brushed onto the stigma. On the other hand, some pollen grains are transferred by abiotic factors (wind and water) from anther to stigma.

## **2.1. Cantharophily**

The flowers in beetle pollinated plants are unspecialized. They are dull in color, scented, and produce lots of pollen grains. The beetles feed on the pollen grains and other flower parts. The scent is emitted from different parts of the flower [5].

### **2.2. Chiropterophily**

Flowers pollinated by bats are large, bowl-shaped, dark to green in color, produce large amounts of nectar and pollen, and smell like rotten fruit. Bats pollinating the flowers are frugivores and nectarivores [6].

#### **2.3. Melittophily**

Bees are known to forage flowers for pollen and nectar for their hives. Such flowers are blue, yellow in color or reflect UV light and produce lots of nectar and pollen grains. Carneiro et al. [7] reported of oil gathering bees on flowers containing elaiophores or oil secreting bodies.

## **2.4. Myophily**

Flies are attracted by the mimicry of carrion and feces, a phenomenon called sapromyiophily [8].

#### **2.5. Ornithophily**

**2. Pollination syndromes**

2 Pollination in Plants

Within pollination syndromes, plants and animals exploit each other for their own advantage. Animals exploit the relationship to guarantee their reproduction and survival. They forage on the flowers for carbohydrates (in nectar) and proteins (in pollen grains), which are the rewards. On the other hand, the plant guarantees its pollination and reproduction by provid-

**Figure 1.** Flowers of the watermelon (*Citrullus lanatus* Thunb.); A is the male flower and B is the bisexual flower; the species is andromonoecious producing male and bisexual flowers on the same plant; in C, a honeybee is foraging for

Scent plays a major role in cases where the pollination agents are deceived. The deception involves mainly reproduction hues like pheromones and reproduction substrates or sites [3]. Different plants produce flowers of different colors. The colors reflect light of different wave

ing the rewards and offering other attractive features like color and scent [2].

pollen grains on a male flower and in D, it is foraging for nectar with pollen grains visible on its legs.

Flowers pollinated by birds are red in color, tubular in shape, scented, and nectariferous (Medan). The birds are attracted by the red color and scent while they obtain nectar as reward. Bird pollinators are mainly hummingbirds and sunbirds [9]. These are hoverers which flip their winds while sucking. Others are perchers which sit on branches while sucking nectar.

#### **2.6. Other pollination syndromes**

In wind pollinated flowers (anemophily), the pollen grains are very fine released on dangling anthers. The stigmas are sticky and hang out of the flower like beard. This increases the chances of the pollen grains landing on the stigma. This type of pollen transfer occurs mainly in the grasses and some acacias. Water aided pollination occurs mainly in submerged water plants. Both water and wind pollinated flowers do not produce nectar and are not brightly colored. It is suggested that anemophily evolved from entomophily (pollination by insects) is a result of limitation of insect availability [10].

**6. Pollination in changing climatic conditions**

tainable and to feed an increasing world population.

Phetole Mangena\* and Phatlane William Mokwala

\*Address all correspondence to: phatlane.mokwala@ul.ac.za

Life Sciences, University of Limpopo, Sovenga, South Africa

2011;**72**:1655-1666. DOI: 10.1016/j.phytochem.2011.02.014

The author has no conflict of interest in the publication of this article.

**7. Conclusion**

**Conflict of interest**

**Author details**

**References**

10.1093/aob/mcn260

Management. 2014;**3**(12):625-631

Global warming will have an effect on both plants and pollinators. Bumble bees were found to be less sensitive to temperature change than managed honey bees [9]. According to [16], expected climate change will negatively affect the geographical distribution of five native

Introductory Chapter: Pollination

5

http://dx.doi.org/10.5772/intechopen.77227

Pollinators are necessary for ecosystem services and crop production productivity. Changes in ecosystems due to global warming as well as agricultural production systems will need to be studied and managed in order to keep ecosystem productivity and crop production sus-

Department of Biodiversity, Faculty of Science and Agriculture, School of Molecular and

[1] Muchhala N, Caiza A, Vizuete JC, Thomson JD. A generalized pollination system in the tropics: Bats, birds and *Aphelandra acanthus*. Annals of Botany. 2008;**103**:1481-1487. DOI:

[2] Awoke TG. Vertebrates' pollination in angiosperm: Floral syndrome, mode of pollen transfer and pollinators specialization. Research Journal of Agricultural and Environmental

[3] Urru I, Stensmyr MC, Hansson BS. Pollination by brood-site deception. Phytochemistry.

bees in Brazil which will potentially decrees tomato production by the year 2100.

## **3. Processes that ensure cross-pollination**

Nature devised a means of ensuring that cross-pollination succeeds and self-pollination is prevented. Some plants are dioecious. In dioecious plants, male plants produce female flowers and female ones produce female flowers. This is different from monoecious plants where plants produce both male (staminate) and female (pistillate) or bisexual flowers. Clearly, self-pollination cannot take place in dioecious plants since one plant produces either male or female flowers. Prevention of self-pollination in monoecious plants includes mechanisms like dichogamy, heterostyly, and self-incompatibility. Dichogamy is a condition in which either the anthers mature or release pollen grains before the stigmas are receptive (protandry) or the stigmas become receptive before the anthers mature (protogyny) [11]. In self-incompatible species, the pollen tube fails to reach the embryo sac. Either the pollen grain fails to germinate on the stigma or the pollen tube is hydrolyzed in the style after germination. In some cases, self-incompatibility is due to morphological characteristics. Some plant species produce longstyled flowers (pin morphs) and short-styled flowers (thrum morphs). Pin morphs can only fertilize thrum morphs and vice versa [12].

## **4. Systems in which self-pollination occurs**

Self-pollination occurs in species with cleistogamous flowers and in plants in which pollinators are scarce. In cleistogamous flowers, the sexual structures (androecium and gynoecium) are enclosed by the petals which form a keel. Pollination agents cannot access the stamens and pistils within the keel. Self-pollination occurs also in monoecious plants.

## **5. Pollination in agricultural production systems**

Pollinators play a major role in crop production. A balance between plants and pollination agents in ecosystems was maintained through the ages though disturbed now and then by natural disasters like wild fires and diseases. However, with the advent of monoculture and expanding agricultural land, the balance is constantly disturbed. Pollinators became supplemented with managed bee hives [13, 14]. On the other hand, an increase in managed honey bee hives has a negative impact on natural pollinators like bumble bees [14]. It is suggested that the introduction of honey bees needs to be managed in combination with pollinator habitat and pesticide use in a system called integrated crop pollination [15].

## **6. Pollination in changing climatic conditions**

Global warming will have an effect on both plants and pollinators. Bumble bees were found to be less sensitive to temperature change than managed honey bees [9]. According to [16], expected climate change will negatively affect the geographical distribution of five native bees in Brazil which will potentially decrees tomato production by the year 2100.

## **7. Conclusion**

in the grasses and some acacias. Water aided pollination occurs mainly in submerged water plants. Both water and wind pollinated flowers do not produce nectar and are not brightly colored. It is suggested that anemophily evolved from entomophily (pollination by insects) is

Nature devised a means of ensuring that cross-pollination succeeds and self-pollination is prevented. Some plants are dioecious. In dioecious plants, male plants produce female flowers and female ones produce female flowers. This is different from monoecious plants where plants produce both male (staminate) and female (pistillate) or bisexual flowers. Clearly, self-pollination cannot take place in dioecious plants since one plant produces either male or female flowers. Prevention of self-pollination in monoecious plants includes mechanisms like dichogamy, heterostyly, and self-incompatibility. Dichogamy is a condition in which either the anthers mature or release pollen grains before the stigmas are receptive (protandry) or the stigmas become receptive before the anthers mature (protogyny) [11]. In self-incompatible species, the pollen tube fails to reach the embryo sac. Either the pollen grain fails to germinate on the stigma or the pollen tube is hydrolyzed in the style after germination. In some cases, self-incompatibility is due to morphological characteristics. Some plant species produce longstyled flowers (pin morphs) and short-styled flowers (thrum morphs). Pin morphs can only

Self-pollination occurs in species with cleistogamous flowers and in plants in which pollinators are scarce. In cleistogamous flowers, the sexual structures (androecium and gynoecium) are enclosed by the petals which form a keel. Pollination agents cannot access the stamens and

Pollinators play a major role in crop production. A balance between plants and pollination agents in ecosystems was maintained through the ages though disturbed now and then by natural disasters like wild fires and diseases. However, with the advent of monoculture and expanding agricultural land, the balance is constantly disturbed. Pollinators became supplemented with managed bee hives [13, 14]. On the other hand, an increase in managed honey bee hives has a negative impact on natural pollinators like bumble bees [14]. It is suggested that the introduction of honey bees needs to be managed in combination with pollinator habi-

pistils within the keel. Self-pollination occurs also in monoecious plants.

tat and pesticide use in a system called integrated crop pollination [15].

**5. Pollination in agricultural production systems**

a result of limitation of insect availability [10].

4 Pollination in Plants

fertilize thrum morphs and vice versa [12].

**4. Systems in which self-pollination occurs**

**3. Processes that ensure cross-pollination**

Pollinators are necessary for ecosystem services and crop production productivity. Changes in ecosystems due to global warming as well as agricultural production systems will need to be studied and managed in order to keep ecosystem productivity and crop production sustainable and to feed an increasing world population.

## **Conflict of interest**

The author has no conflict of interest in the publication of this article.

## **Author details**

Phetole Mangena\* and Phatlane William Mokwala

\*Address all correspondence to: phatlane.mokwala@ul.ac.za

Department of Biodiversity, Faculty of Science and Agriculture, School of Molecular and Life Sciences, University of Limpopo, Sovenga, South Africa

## **References**


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**Chapter 2**

**Provisional chapter**

**The Regulation of Sperm Cells Delivery to the**

**The Regulation of Sperm Cells Delivery to the** 

DOI: 10.5772/intechopen.75359

Pollination, or the first contact between male and female gametophytes, is one of the most important steps in plant reproduction. After pollination, the pollen grains, male gametophytes, are hydrated and then germinate pollen tubes. The pollen tube initially penetrates and grows through the intercellular spaces of the stigma and then grows through the transmitting tract to the placenta connected to an ovule. The pollen tube grows along the surface of the ovule's funiculus, through the micropyle, and into the female gametophyte. After the pollen tube enters the female gametophyte, it ruptures and releases two sperm cells with its contents. The two sperm cells then move toward and fuse with the egg cell and central cell to produce embryo and endosperm, respectively. Multiple sperm cells typically strive to "win the race" and fertilize an egg cell during animal fertilization; however, in flowering plants, each ovule harboring an egg cell generally encounters only one of many pollen tubes conveying plant sperm cells. This chapter mainly addresses reproductive strategies of plants following pollination from the pollen tube extension and the guidance of two sperm cells to the female gametophyte for fertilization in the

**Keywords:** plant fertilization, pollen tube guidance, MYB98, LUREs, fertilization

Discussing the journey of the pollen tube first requires an introduction to the smallest fertilization units, namely, the male and female gametophytes (**Figure 1**). The male gametophyte (pollen) comprises two sperm cells and one vegetative cell and is found in the stamen of a flower. The two sperm cells fertilize the egg and central cells inside the female gametophyte

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Embryo Sac**

**Embryo Sac**

Ryushiro Dora Kasahara

Ryushiro Dora Kasahara

**Abstract**

ovule.

recovery system, POEM

**1. Male and female gametophytes**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75359


#### **The Regulation of Sperm Cells Delivery to the Embryo Sac The Regulation of Sperm Cells Delivery to the Embryo Sac**

DOI: 10.5772/intechopen.75359

#### Ryushiro Dora Kasahara Ryushiro Dora Kasahara

[4] Shrestha M, Dyer AG, Boyd-Gerny S, Wong BBM, Burd M. Shades of red: Bird-pollinated flowers target the specific colour discrimination abilities of avian vision. New Phytologist.

[5] Steenhuisen SL, Raguso RA, Jurgens A, Johnson SD. Variation in scent emission among floral parts and inflorescence developmental stages in beetle-pollinated Protea species (Proteaceae). South African Journal of Botany. 2010;**76**:779-787. DOI: 10.1016/j.sajb.2010.

[6] Matins ACM, Willig MR, Presley SJ, Marinho-Filho J. Effects of forest height and vertical complexity on abundance and biodiversity of bats in Amazonia. Forest Ecology and

[7] Carneiro LT, Aguiar AJC, Martins CF, Machado JC, Alves-dos-Santos I. *Rameria tomentosa* oil flowers and their pollinators: Bees specialised on trichome elaiophores exploit its

[8] Johnson SD, Jurgens A. Convergent evolution of carrion and faecal scent mimicry in flypollinated angiosperm flowers and a stinkhorn fungus. South African Journal of Botany.

[9] Medan D, Montaldo NH. Ornithophily in the Rhamnaceae: The pollination of the Chilean endemic *Colletia ulicina*. Flora. 2005;**200**:339-344. DOI: 10.1016/j.flora.2004.12.003

[10] Culley TM, Weller SG. The evolution of wind pollination in angiosperms. Trends in

[11] Lloyd DG, Webb CJ. The avoidance of interference between the presentation of pollen and stigmas in angiosperms I. Dichogamy. New Zealand Journal of Botany. 1986;**24**:

[12] Watanabe K, Kato H, Sugawara T. Distyly and incompatibility in *Psychotria homalosperma* (Rubiaceae), an endemic plant of the oceanic Bonin (Ogasawara) islands. Flora.

[13] Bailes EJ, Ollerton J, Pattrick JG, Glover BJ. How can an understanding of plant–pollinator interactions contribute to global food security? Current Opinion in Plant Biology. 2015;

[14] Nielsen A, Andreas TR, Rinvoll Brysting K. Effects of competition and climate on a crop pollinator community. Agriculture Ecosystems and Environment. 2017;**247**:253-260.

[15] Isaacs R, Williams N, Ellis J, Pitts-Singer TL, Bommarco R, Vaughan M. Integrated crop pollination: Combining strategies to ensure stable and sustainable yields of pollinationdependent crops. Basic and Applied Ecology. 2017;**22**:44-60. DOI: 10.1016/j.baae.2017.

[16] Elias MAS, Borges FJA, Bergamini LL, Franceschinelli EV, Sujii ER. Climate change threatens pollination services in tomato crops in Brazil. Agriculture, Ecosystems and

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epithelial oil glands. Flora. 2015;**215**:1-8. DOI: 10.1016/j.flora.2015.06.002

2013;**198**:301-310. DOI: 10.1111/nph.12135

2010;**76**:796-807. DOI: 10.1016/j.sajb.2010.07.012

Ecology & Evolution. 2002;**17**(8):361-368

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07.003

135-162. DOI: 10.1080/0028825X.1986.10409725

2014;**209**:641-648. DOI: 10.1016/j.flora.2014.09.006

08.008

6 Pollination in Plants

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75359

#### **Abstract**

Pollination, or the first contact between male and female gametophytes, is one of the most important steps in plant reproduction. After pollination, the pollen grains, male gametophytes, are hydrated and then germinate pollen tubes. The pollen tube initially penetrates and grows through the intercellular spaces of the stigma and then grows through the transmitting tract to the placenta connected to an ovule. The pollen tube grows along the surface of the ovule's funiculus, through the micropyle, and into the female gametophyte. After the pollen tube enters the female gametophyte, it ruptures and releases two sperm cells with its contents. The two sperm cells then move toward and fuse with the egg cell and central cell to produce embryo and endosperm, respectively. Multiple sperm cells typically strive to "win the race" and fertilize an egg cell during animal fertilization; however, in flowering plants, each ovule harboring an egg cell generally encounters only one of many pollen tubes conveying plant sperm cells. This chapter mainly addresses reproductive strategies of plants following pollination from the pollen tube extension and the guidance of two sperm cells to the female gametophyte for fertilization in the ovule.

**Keywords:** plant fertilization, pollen tube guidance, MYB98, LUREs, fertilization recovery system, POEM

## **1. Male and female gametophytes**

Discussing the journey of the pollen tube first requires an introduction to the smallest fertilization units, namely, the male and female gametophytes (**Figure 1**). The male gametophyte (pollen) comprises two sperm cells and one vegetative cell and is found in the stamen of a flower. The two sperm cells fertilize the egg and central cells inside the female gametophyte

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Male gametophyte and female gametophyte. The male gametophyte, also called the pollen grain or microgametophyte, develops within the anther and consists of two sperm cells encased within a vegetative cell (left). The mature female gametophyte (right) inside the pistil (center). The egg and central cells are polarized such that the nuclei of both cells lie very close to each other. This feature is important for double fertilization because these two nuclei are the targets of the two sperm nuclei. After double fertilization, the egg cell forms embryo and the central cell forms endosperm. The synergid cells have at least two functions associated with the fertilization process. First, the synergid cells secrete pollen tube attractants. In addition, the pollen tube enters the synergid cell, suggesting that the synergid cells are important for pollen tube reception. The black areas represent vacuoles of the cells in the female gametophyte.

gametophyte development. Particularly, these homozygous mutants mostly produce defective ovules. Although wild-type pollen tubes grow normally during initial phases from pollen hydration to tube emergence from the transmitting tract, they fail to grow toward the mutant ovules, which lack female gametophytes. In other words, although the female gametophyte does not influence early pollen tube growth, it appears to be required for subsequent pollen tube guidance to the ovule [11, 13–15]. These observations suggest that a molecular approach

**Figure 2.** Pollen tube guidance from the stigma to the funiculus. Soon after pollination, the male gametophyte becomes hydrated and then germinates a pollen tube. The pollen tube initially penetrates and grows through the intercellular spaces between the papillar cells of the stigma and then grows through the transmitting tract of the carpel's style and ovary. The pollen tube then emerges from the transmitting tract and grows along the surface of the placenta toward an

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The pollen tube is subsequently guided from the funiculus to the female gametophyte. Although the molecular mechanisms underlying this step have been relatively well elucidated, as shown in **Figure 3**, a complete understanding requires a discussion of synergid cell biology (**Figure 1**). Synergid cells within the female gametophyte are essential for reproduction. After the pollen tube grows from the stigma to the funiculus, it enters the female gametophyte by growing into one of the two synergid cells, which typically undergo cell death before or upon pollen tube arrival. Soon after arrival, the pollen tube ceases to grow and subsequently ruptures to release its sperm cells into the receptive synergid's cytoplasm, thus triggering the completion of degeneration. Finally, one sperm cell each migrates to the egg cell and central cell to complete double fertilization of the female

is essential for understanding pollen tube growth from the stigma to the funiculus.

**3. From the funiculus to the female gametophyte**

gametophyte [3, 16–19].

ovule.

via a guided pollen tube journey that is described later. The female gametophyte, which is embedded in an ovule within the pistil, contains seven cells of four different types: an egg cell, a central cell, two synergid cells, and three antipodal cells. The egg and central cells are polarized such that their nuclei lie in very close proximity, a feature facilitating double fertilization of these two sperm nuclei targets [1–3]. The synergid cells are extremely essential for the attraction of pollen tubes, as discussed below [4–8].

## **2. From the stigma to the funiculus**

Once a pollen grain reaches the stigma at the top of a carpel, the pollen tubes elongate toward the funiculus to form a bridge-like structure to an ovule, as shown in **Figure 2**. This pollen tube growth through the stigma to the funiculus is controlled via multiple signals from both sporophytic and gametophytic maternal tissues in the carpels. The roles of the female tissues in pollen tube guidance have been focused upon.

Light and transmission electron microscopy studies of *Arabidopsis* have led to several observations regarding pollen tube growth in the female tissues of carpels [9–12]. Although the morphologic features of the pollen tube journey are well understood, the underlying regulatory molecular mechanisms remain unclear. Accordingly, previous studies used sporophytic mutants to elucidate the relationship between pollen tube growth and ovule/female

**Figure 2.** Pollen tube guidance from the stigma to the funiculus. Soon after pollination, the male gametophyte becomes hydrated and then germinates a pollen tube. The pollen tube initially penetrates and grows through the intercellular spaces between the papillar cells of the stigma and then grows through the transmitting tract of the carpel's style and ovary. The pollen tube then emerges from the transmitting tract and grows along the surface of the placenta toward an ovule.

gametophyte development. Particularly, these homozygous mutants mostly produce defective ovules. Although wild-type pollen tubes grow normally during initial phases from pollen hydration to tube emergence from the transmitting tract, they fail to grow toward the mutant ovules, which lack female gametophytes. In other words, although the female gametophyte does not influence early pollen tube growth, it appears to be required for subsequent pollen tube guidance to the ovule [11, 13–15]. These observations suggest that a molecular approach is essential for understanding pollen tube growth from the stigma to the funiculus.

## **3. From the funiculus to the female gametophyte**

via a guided pollen tube journey that is described later. The female gametophyte, which is embedded in an ovule within the pistil, contains seven cells of four different types: an egg cell, a central cell, two synergid cells, and three antipodal cells. The egg and central cells are polarized such that their nuclei lie in very close proximity, a feature facilitating double fertilization of these two sperm nuclei targets [1–3]. The synergid cells are extremely essential for

**Figure 1.** Male gametophyte and female gametophyte. The male gametophyte, also called the pollen grain or microgametophyte, develops within the anther and consists of two sperm cells encased within a vegetative cell (left). The mature female gametophyte (right) inside the pistil (center). The egg and central cells are polarized such that the nuclei of both cells lie very close to each other. This feature is important for double fertilization because these two nuclei are the targets of the two sperm nuclei. After double fertilization, the egg cell forms embryo and the central cell forms endosperm. The synergid cells have at least two functions associated with the fertilization process. First, the synergid cells secrete pollen tube attractants. In addition, the pollen tube enters the synergid cell, suggesting that the synergid cells are important for pollen tube reception. The black areas represent vacuoles of the cells in the female gametophyte.

Once a pollen grain reaches the stigma at the top of a carpel, the pollen tubes elongate toward the funiculus to form a bridge-like structure to an ovule, as shown in **Figure 2**. This pollen tube growth through the stigma to the funiculus is controlled via multiple signals from both sporophytic and gametophytic maternal tissues in the carpels. The roles of the female tissues

Light and transmission electron microscopy studies of *Arabidopsis* have led to several observations regarding pollen tube growth in the female tissues of carpels [9–12]. Although the morphologic features of the pollen tube journey are well understood, the underlying regulatory molecular mechanisms remain unclear. Accordingly, previous studies used sporophytic mutants to elucidate the relationship between pollen tube growth and ovule/female

the attraction of pollen tubes, as discussed below [4–8].

**2. From the stigma to the funiculus**

8 Pollination in Plants

in pollen tube guidance have been focused upon.

The pollen tube is subsequently guided from the funiculus to the female gametophyte. Although the molecular mechanisms underlying this step have been relatively well elucidated, as shown in **Figure 3**, a complete understanding requires a discussion of synergid cell biology (**Figure 1**). Synergid cells within the female gametophyte are essential for reproduction. After the pollen tube grows from the stigma to the funiculus, it enters the female gametophyte by growing into one of the two synergid cells, which typically undergo cell death before or upon pollen tube arrival. Soon after arrival, the pollen tube ceases to grow and subsequently ruptures to release its sperm cells into the receptive synergid's cytoplasm, thus triggering the completion of degeneration. Finally, one sperm cell each migrates to the egg cell and central cell to complete double fertilization of the female gametophyte [3, 16–19].

data suggest that MYB98 functions as a transcription factor within the synergid cell gene regulatory network, where it particularly controls the expression of downstream genes required

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**Figure 4.** pMYB98::GFP expression and *myb98* phenotype. (A) MYB98 is expressed predominantly in the synergid cells (*pMYB98::GFP* photograph captured by Liyang Xie, HBMC, FAFU). *myb98* female gametophytes are defective in pollen tube guidance. (B) In the wild type, the pollen tube grew along the ovule's funiculus, through the ovule's micropyle, and into the female gametophyte. (C) By contrast, wild-type pollen tubes grew abnormally on *myb98* ovules. Pollen tubes grew from the placenta to the funiculus but then failed to grow into the micropyle. These data suggest that MYB98 functions as a transcription factor within the synergid cells to regulate the expression of genes required for pollen tube guidance. Bars = 30 μm.

for pollen tube guidance and filiform apparatus formation.

**Figure 3.** Pollen tube guidance from the funiculus to the micropyle. Synergid cells are required for pollen tube guidance. Several studies using *Arabidopsis thaliana* mutants have reported that pollen tubes fail to grow onto ovules containing abnormal female gametophytes, indicating that the embryo sac provides a guidance cue for the pollen tube. AtLURE1 peptides are attractants that guide pollen tubes to the ovular micropyle. These AtLURE1 peptides are particularly expressed in synergid cells and secreted toward the funicular surface through the micropyle. A transcription factor MYB98 is required for AtLURE1 since myb98 mutants do not express AtLURE1 peptides. PRK6 and MDIS1-MIK receptors are AtLURE1 pollen tube attractant counterparts.

Synergid cells are required for pollen tube guidance. Several studies using *Arabidopsis thaliana* mutants have reported that pollen tubes fail to grow onto ovules containing abnormal female gametophytes, indicating that the embryo sac provides a guidance cue for the pollen tube [11, 20, 21]. Higashiyama *et al*. used laser ablation in an *in vitro Torenia* pollen germination system to demonstrate that synergid cells, but not other female gametophyte cells, produce a pollen tube attractant [4]. Early in 2005, the requirement of a small protein, maize EA1, for pollen tube guidance was reported; however, maize EA1 has no homolog in *Arabidopsis* or other dicots and is unlikely to be a universal attractant [5].

MYB98, which is exclusively expressed in synergid cells (**Figure 4**), provides the first molecular evidence of pollen tube guidance in *Arabidopsis* [6]. Laser ablation studies have demonstrated that synergid cells secrete attractants that guide the pollen tube to the female gametophyte [4], suggesting that defects in pollen tube guidance should be observed in the *myb98* mutant. Accordingly, Kasahara *et al*. observed the pollen tubes of *myb98* mutant pistils pollinated with wild-type pollen. In the wild-type plant, the pollen tube grew along the funiculus of the ovule and through the micropyle to the female gametophyte; however, the wild-type pollen tubes grew abnormally on ovules containing *myb98* female gametophytes, specifically, the pollen tubes grew from the placenta to the funiculus but failed to grow into the micropyle (**Figure 4**).

MYB98 is expressed during the very early stage of synergid cellularization during female gametocyte development, consistent with the *myb98* mutant phenotype, in which pollen tube guidance and filiform apparatus structure are affected. However, several observations indicate that other synergid cell development aspects, including cell specification, remain normal in *myb98* mutants. Female gametophyte development and overall synergid cell morphology appear to be unaffected in *myb98* mutants. Additionally, the *myb98* mutation does not appear to affect the steps of fertilization process subsequent to pollen tube guidance, including the control of pollen tube growth cessation, pollen tube rupture, and sperm cell migration. These data suggest that MYB98 functions as a transcription factor within the synergid cell gene regulatory network, where it particularly controls the expression of downstream genes required for pollen tube guidance and filiform apparatus formation.

Synergid cells are required for pollen tube guidance. Several studies using *Arabidopsis thaliana* mutants have reported that pollen tubes fail to grow onto ovules containing abnormal female gametophytes, indicating that the embryo sac provides a guidance cue for the pollen tube [11, 20, 21]. Higashiyama *et al*. used laser ablation in an *in vitro Torenia* pollen germination system to demonstrate that synergid cells, but not other female gametophyte cells, produce a pollen tube attractant [4]. Early in 2005, the requirement of a small protein, maize EA1, for pollen tube guidance was reported; however, maize EA1 has no homolog in *Arabidopsis* or other

**Figure 3.** Pollen tube guidance from the funiculus to the micropyle. Synergid cells are required for pollen tube guidance. Several studies using *Arabidopsis thaliana* mutants have reported that pollen tubes fail to grow onto ovules containing abnormal female gametophytes, indicating that the embryo sac provides a guidance cue for the pollen tube. AtLURE1 peptides are attractants that guide pollen tubes to the ovular micropyle. These AtLURE1 peptides are particularly expressed in synergid cells and secreted toward the funicular surface through the micropyle. A transcription factor MYB98 is required for AtLURE1 since myb98 mutants do not express AtLURE1 peptides. PRK6 and MDIS1-MIK

MYB98, which is exclusively expressed in synergid cells (**Figure 4**), provides the first molecular evidence of pollen tube guidance in *Arabidopsis* [6]. Laser ablation studies have demonstrated that synergid cells secrete attractants that guide the pollen tube to the female gametophyte [4], suggesting that defects in pollen tube guidance should be observed in the *myb98* mutant. Accordingly, Kasahara *et al*. observed the pollen tubes of *myb98* mutant pistils pollinated with wild-type pollen. In the wild-type plant, the pollen tube grew along the funiculus of the ovule and through the micropyle to the female gametophyte; however, the wild-type pollen tubes grew abnormally on ovules containing *myb98* female gametophytes, specifically, the pollen tubes grew from the placenta to the funiculus but failed to grow into the micropyle (**Figure 4**). MYB98 is expressed during the very early stage of synergid cellularization during female gametocyte development, consistent with the *myb98* mutant phenotype, in which pollen tube guidance and filiform apparatus structure are affected. However, several observations indicate that other synergid cell development aspects, including cell specification, remain normal in *myb98* mutants. Female gametophyte development and overall synergid cell morphology appear to be unaffected in *myb98* mutants. Additionally, the *myb98* mutation does not appear to affect the steps of fertilization process subsequent to pollen tube guidance, including the control of pollen tube growth cessation, pollen tube rupture, and sperm cell migration. These

dicots and is unlikely to be a universal attractant [5].

receptors are AtLURE1 pollen tube attractant counterparts.

10 Pollination in Plants

**Figure 4.** pMYB98::GFP expression and *myb98* phenotype. (A) MYB98 is expressed predominantly in the synergid cells (*pMYB98::GFP* photograph captured by Liyang Xie, HBMC, FAFU). *myb98* female gametophytes are defective in pollen tube guidance. (B) In the wild type, the pollen tube grew along the ovule's funiculus, through the ovule's micropyle, and into the female gametophyte. (C) By contrast, wild-type pollen tubes grew abnormally on *myb98* ovules. Pollen tubes grew from the placenta to the funiculus but then failed to grow into the micropyle. These data suggest that MYB98 functions as a transcription factor within the synergid cells to regulate the expression of genes required for pollen tube guidance. Bars = 30 μm.

The female gametophyte pollen tube attractants LURE1 and LURE2 have also been identified in *Torenia* [7]. LUREs are cysteine-rich proteins (CRPs) within the defensin-like (DEFL) family. *LURE* genes are expressed in synergid cells, which secrete the encoded proteins into the filiform apparatus. Accordingly, LURE downregulation reduces pollen tube attraction, and recombinant mature proteins attract pollen tubes *in vitro* and in a species-specific manner [7]. As discussed above, *myb98* mutation affects the filiform apparatus within synergid cells. However, MYB98 is also required for the expression of at least 83 genes encoding CRPs similar to LURE1 and LURE2 [22, 23]. Many of these CRPs exhibit localization and diffusion patterns similar to those of ZmEA1 [5, 24]; particularly, the CRPs are secreted into the filiform apparatus and subsequently diffuse into the micropylar region [22]. In 2012, Takeuchi and Higashiyama [8] finally identified a recently evolved DEFL gene cluster in *Arabidopsis* and demonstrated that these DEFL [cysteine-rich peptide (CRP810\_1)] peptides, or AtLURE1 peptides, are attractants that guide pollen tubes to the ovular micropyle. These AtLURE1 peptides are particularly expressed in synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses have revealed that gametophytic mutants defective in micropylar guidance *myb98* [6], *magatama3* [21], and *central cell guidance* [25] do not express AtLURE1 peptides and that recombinant AtLURE1 peptides were found to preferentially attract *A. thaliana* pollen tubes vs. *A. lyrata* pollen tubes, indicating that these peptides act as species-preferential attractants in micropylar guidance [8]. Several female-secreted peptides have been identified as species-specific attractants directly controlling pollen tube growth direction. However, the method by which the pollen tubes precisely and promptly respond to guidance signals from their own species remains unknown. In 2016, two research groups reported AtLURE1 pollen tube attractant counterparts [26, 27]. Takeuchi and Higashiyama [26] reported that the tip-localized pollen-specific receptor-like kinase 6 (PRK6), featuring an extracellular leucine-rich repeat domain, serves as an essential sensor of LURE1 [8] in *Arabidopsis* under semi-*in vivo* conditions and is important for ovule targeting in the pistil. PRK6 interacts with pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which facilitates pollen tube growth by activating the Rho GTPase ROP1 [28, 29]. Particularly, PRK6 acts as a key membrane receptor for external AtLURE1 attractants and recruits core tip-growth machinery, including ROP signaling proteins. Furthermore, Wang *et al*. [27] identified that a cell-surface receptor heteromer, MDIS1-MIK, perceives the female attractant AtLURE1 on the pollen tube of *Arabidopsis*. MDIS1, MIK1, and MIK2 are plasma-membrane-localized receptorlike kinases containing extracellular leucine-rich repeats and an intracellular kinase domain. AtLURE1 particularly binds the extracellular domains of MDIS1, MIK1, and MIK2, whereas *mdis1* and *mik1 mik2* mutant pollen tubes respond less sensitively to AtLURE1.

a protein, GCS1 (generative cell specific 1), using generative cells isolated from *Lilium longiflorum* pollen. Homologs of GCS1, possessing a carboxy-terminal transmembrane domain, are present in various species, including non-angiosperms. Immunological assays have indicated that GCS1 accumulates during late gametogenesis and localizes on the plasma membranes of generative cells. Notably, *Arabidopsis gcs1* mutants exhibit male sterility because the male gametes fail to fuse with the egg or central cell (**Figure 5**). Mori *et al*. [31] identified another important male factor, GEX2 (gamete expressed 2), which encodes a sperm-expressed protein of unknown function that localizes to the sperm membrane and contains extracellular immunoglobulin-like domains, similar to the gamete interaction factors in algae and mammals. Using a novel *in vivo* assay, Mori *et al*. demonstrated the requirement of GEX2 for gamete

also fail to fuse with the egg or central cell but the frequency of failure is lower than the *gcs1* mutant.

**Figure 5.** Discharge of sperm cells from the pollen tube tip to double fertilization. (A) Upon reaching an ovule, the pollen tube grows along the surface of the ovule's funiculus, through the micropyle, and into the female gametophyte. The pollen tube enters the female gametophyte by growing through the synergid cells. The pollen tube then comes in contact with the synergid cells and ceases growth. One of the synergid cells then undergoes cell death. Finally, soon after synergid degeneration is initiated, the pollen tube ruptures and releases two sperm cells into the degenerating synergid cytoplasm. The two sperm cells then move toward and fuse with the egg cell and central cell to complete double fertilization. (B) The male gametes of *gcs1* mutant fail to fuse with the egg or central cell. GCS1 accumulates during late gametogenesis and localizes on the plasma membranes of generative cells. The male gametes of *gex2* mutant

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In angiosperms, double fertilization within the ovule occurs with the entry of two sperm cells, which are usually delivered by a single pollen tube. In 1904, Wylie [32] observed the insertion of two pollen tubes in an *Elodea canadensis* ovule and concluded, "It often happens that two pollen tubes pass into one ovule; in such cases both synergids disappear." Since this discovery, the reception of two pollen tubes in an embryo sac, although rare, has been reported in at least 12 species [33]. Similarly, the reception of two pollen tubes has been reported in several *Arabidopsis* mutants, including the *gcs1* mutant [30]. Although this phenomenon is interest-

attachment, as double fertilization is compromised in its absence.

**5. Fertilization recovery system**

## **4. Discharge of sperm cells from the pollen tube tip to fertilization**

Immediately after growth cessation, the pollen tube ruptures at or near its tip, leading to release of the pollen tube's contents, including the two sperm cells. In *Arabidopsis* and *Torenia*, rupture occurs within 1 min after entry of the pollen tube into the female gametophyte [16, 17]. Regarding the molecular mechanisms underlying this step, two proteins localized in the sperm cells have been reported (**Figure 5**): GCS1 [30] and GEX2 [31]. Mori *et al*. [30] identified

**Figure 5.** Discharge of sperm cells from the pollen tube tip to double fertilization. (A) Upon reaching an ovule, the pollen tube grows along the surface of the ovule's funiculus, through the micropyle, and into the female gametophyte. The pollen tube enters the female gametophyte by growing through the synergid cells. The pollen tube then comes in contact with the synergid cells and ceases growth. One of the synergid cells then undergoes cell death. Finally, soon after synergid degeneration is initiated, the pollen tube ruptures and releases two sperm cells into the degenerating synergid cytoplasm. The two sperm cells then move toward and fuse with the egg cell and central cell to complete double fertilization. (B) The male gametes of *gcs1* mutant fail to fuse with the egg or central cell. GCS1 accumulates during late gametogenesis and localizes on the plasma membranes of generative cells. The male gametes of *gex2* mutant also fail to fuse with the egg or central cell but the frequency of failure is lower than the *gcs1* mutant.

a protein, GCS1 (generative cell specific 1), using generative cells isolated from *Lilium longiflorum* pollen. Homologs of GCS1, possessing a carboxy-terminal transmembrane domain, are present in various species, including non-angiosperms. Immunological assays have indicated that GCS1 accumulates during late gametogenesis and localizes on the plasma membranes of generative cells. Notably, *Arabidopsis gcs1* mutants exhibit male sterility because the male gametes fail to fuse with the egg or central cell (**Figure 5**). Mori *et al*. [31] identified another important male factor, GEX2 (gamete expressed 2), which encodes a sperm-expressed protein of unknown function that localizes to the sperm membrane and contains extracellular immunoglobulin-like domains, similar to the gamete interaction factors in algae and mammals. Using a novel *in vivo* assay, Mori *et al*. demonstrated the requirement of GEX2 for gamete attachment, as double fertilization is compromised in its absence.

## **5. Fertilization recovery system**

The female gametophyte pollen tube attractants LURE1 and LURE2 have also been identified in *Torenia* [7]. LUREs are cysteine-rich proteins (CRPs) within the defensin-like (DEFL) family. *LURE* genes are expressed in synergid cells, which secrete the encoded proteins into the filiform apparatus. Accordingly, LURE downregulation reduces pollen tube attraction, and recombinant mature proteins attract pollen tubes *in vitro* and in a species-specific manner [7]. As discussed above, *myb98* mutation affects the filiform apparatus within synergid cells. However, MYB98 is also required for the expression of at least 83 genes encoding CRPs similar to LURE1 and LURE2 [22, 23]. Many of these CRPs exhibit localization and diffusion patterns similar to those of ZmEA1 [5, 24]; particularly, the CRPs are secreted into the filiform apparatus and subsequently diffuse into the micropylar region [22]. In 2012, Takeuchi and Higashiyama [8] finally identified a recently evolved DEFL gene cluster in *Arabidopsis* and demonstrated that these DEFL [cysteine-rich peptide (CRP810\_1)] peptides, or AtLURE1 peptides, are attractants that guide pollen tubes to the ovular micropyle. These AtLURE1 peptides are particularly expressed in synergid cells and secreted toward the funicular surface through the micropyle. Genetic analyses have revealed that gametophytic mutants defective in micropylar guidance *myb98* [6], *magatama3* [21], and *central cell guidance* [25] do not express AtLURE1 peptides and that recombinant AtLURE1 peptides were found to preferentially attract *A. thaliana* pollen tubes vs. *A. lyrata* pollen tubes, indicating that these peptides act as species-preferential attractants in micropylar guidance [8]. Several female-secreted peptides have been identified as species-specific attractants directly controlling pollen tube growth direction. However, the method by which the pollen tubes precisely and promptly respond to guidance signals from their own species remains unknown. In 2016, two research groups reported AtLURE1 pollen tube attractant counterparts [26, 27]. Takeuchi and Higashiyama [26] reported that the tip-localized pollen-specific receptor-like kinase 6 (PRK6), featuring an extracellular leucine-rich repeat domain, serves as an essential sensor of LURE1 [8] in *Arabidopsis* under semi-*in vivo* conditions and is important for ovule targeting in the pistil. PRK6 interacts with pollen-expressed ROPGEFs (Rho of plant guanine nucleotide-exchange factors), which facilitates pollen tube growth by activating the Rho GTPase ROP1 [28, 29]. Particularly, PRK6 acts as a key membrane receptor for external AtLURE1 attractants and recruits core tip-growth machinery, including ROP signaling proteins. Furthermore, Wang *et al*. [27] identified that a cell-surface receptor heteromer, MDIS1-MIK, perceives the female attractant AtLURE1 on the pollen tube of *Arabidopsis*. MDIS1, MIK1, and MIK2 are plasma-membrane-localized receptorlike kinases containing extracellular leucine-rich repeats and an intracellular kinase domain. AtLURE1 particularly binds the extracellular domains of MDIS1, MIK1, and MIK2, whereas

12 Pollination in Plants

*mdis1* and *mik1 mik2* mutant pollen tubes respond less sensitively to AtLURE1.

**4. Discharge of sperm cells from the pollen tube tip to fertilization**

Immediately after growth cessation, the pollen tube ruptures at or near its tip, leading to release of the pollen tube's contents, including the two sperm cells. In *Arabidopsis* and *Torenia*, rupture occurs within 1 min after entry of the pollen tube into the female gametophyte [16, 17]. Regarding the molecular mechanisms underlying this step, two proteins localized in the sperm cells have been reported (**Figure 5**): GCS1 [30] and GEX2 [31]. Mori *et al*. [30] identified In angiosperms, double fertilization within the ovule occurs with the entry of two sperm cells, which are usually delivered by a single pollen tube. In 1904, Wylie [32] observed the insertion of two pollen tubes in an *Elodea canadensis* ovule and concluded, "It often happens that two pollen tubes pass into one ovule; in such cases both synergids disappear." Since this discovery, the reception of two pollen tubes in an embryo sac, although rare, has been reported in at least 12 species [33]. Similarly, the reception of two pollen tubes has been reported in several *Arabidopsis* mutants, including the *gcs1* mutant [30]. Although this phenomenon is interesting, it has long been considered anomalous. However, Kasahara *et al*. [34] investigated the mechanisms underlying this phenomenon in higher plants upon frequently observing ovules that accepted two pollen tubes in the fertilization-defective *hap2-1* (allelic to *gcs1*) mutant [35], as shown in **Figure 6**. As the *hap2-1* mutant pollen tubes were marked by the pollen tube-specific reporter gene *LAT52:GUS* [36], Kasahara *et al*. traced the tube behaviors *in vivo* by staining for GUS activity, followed by aniline blue staining, to trace the behaviors of the first and second pollen tubes. Accordingly, most ovules contained one pollen tube at 10 hours

after pollination (HAP), indicating that the reception of a second pollen tube is independent of sperm cell fertility until several hours after the arrival of the first pollen tube. This delay may represent a blocking system by which ovules avoid polysiphonogamy [34, 37]. However, after 10 HAP, ovules that failed to be fertilized by the first *hap2-1* pollen tube began to attract a second tube. In this case, the persistent synergid cell, which would degenerate upon successful fertilization, continued to attract pollen tubes, leading to a second pollen tube acceptance rate of ~80% among failed ovules by 28 HAP. Although no particular role has been proposed for synergid cell persistence after the arrival of the first pollen tube, Kasahara *et al*. demonstrated that the second synergid cell could retain its function and thus attract and accept a second tube to rescue fertilization. This might explain the presence of two synergid cells in

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Previously, several research groups [35, 38, 39] studied why several sperm cell–defective mutants exhibited an enhanced fertility phenotype (60–70% fertility); particularly, the frequency ratio of double pollen tube reception was almost completely consistent with the frequency of enhanced fertility (**Figure 6**). Additionally, the GUS staining experiment revealed that by 10 HAP, ~50% ovules had accepted a mutant allele, indicating that the mutant and wild-type pollen tubes were similarly competent to enter the embryo sac and release their contents. von Besser *et al*. [35] suggested that *hap2-1* sperm cells affect pollen tube guidance. However, our data led us to conclude that sperm cells are passive pollen tube cargo and do not influence pollen tube guidance in *hap2-1* mutants, consistent with the observation that sperm cell–defective mutants (i.e., no transmission via the male germline) exhibit only 30–35% sterility, instead of the expected 50%. Very recently, Zhang *et al*. [40] demonstrated that in the absence of two bHLH transcription factors, *Arabidopsis* produces an abnormal, sperm cell-free pollen exhibiting behavior similar to its wild-type counterpart, thus indicating that sperm cells are dispensable for normal pollen tube development. This result reinforced our concept of sperm cells as passive cargo, with no control over pollen tube growth and behavior.

According to previous report by Kasahara *et al*. [37], all hand pollination experiments were performed using large numbers of pollen grains. Particularly, *Arabidopsis* pistils, usually containing 50–60 ovules, were pollinated with approximately 20, 40, 80, 120, and 700 grains. Two days after pollination, the insertion of few second pollen tubes into ovules were observed among the pistils pollinated with 20 and 40 grains, indicating that under restricted conditions (ovules > pollen tubes), the pollen tubes selectively inserted into ovules that had not previously accepted any pollen tube. Conversely, when a wild-type pistil was pollinated with 80 (ovules ≤ pollen tubes) and 120 (ovules < pollen tubes) grains, approximately 12 and 25% of the ovules accepted second pollen tubes, respectively, suggesting that ovules accept second pollen tubes while under saturated conditions (ovules < pollen tubes) [37]. In other words, excess pollen is required to saturate the fertilization recovery system, and approximately 80% (not 100%) of the failed ovules can accept a second pollen tube to complete recovery, consistent with a previous report [34] that a substantial period of ~28 h is required for ovules to complete the fertilization recovery system. This delay in secondary guidance may be attributable to the functional synergid cell numbers; a previously penetrated ovule contains only one persistent synergid cell (the other would have been disrupted by a burst pollen tube) to provide guidance. Higashiyama *et al*. [4] reported that an ovule containing two synergid cells

many higher plants (**Figure 6**).

**Figure 6.** Fertilization recovery system. Upon insertion of a single pollen tube into an ovule, the pollen tube bursts and releases two sperm cells. When the sperm cells complete fertilization, the ovule blocks the entry of the other pollen tubes and develops into a seed by forming an embryo and endosperm. When fertilization fails, the ovule attracts a second pollen tube to rescue fertilization. The rescued ovule develops into a seed, resulting in increased fertility. In the case of failure of fertilization by the second pollen tube, the ovule does not attract a third pollen tube, possibly due to depletion of the pollen tube attractant from synergid cells, since both synergid cells collapse after entry of two pollen tubes.

after pollination (HAP), indicating that the reception of a second pollen tube is independent of sperm cell fertility until several hours after the arrival of the first pollen tube. This delay may represent a blocking system by which ovules avoid polysiphonogamy [34, 37]. However, after 10 HAP, ovules that failed to be fertilized by the first *hap2-1* pollen tube began to attract a second tube. In this case, the persistent synergid cell, which would degenerate upon successful fertilization, continued to attract pollen tubes, leading to a second pollen tube acceptance rate of ~80% among failed ovules by 28 HAP. Although no particular role has been proposed for synergid cell persistence after the arrival of the first pollen tube, Kasahara *et al*. demonstrated that the second synergid cell could retain its function and thus attract and accept a second tube to rescue fertilization. This might explain the presence of two synergid cells in many higher plants (**Figure 6**).

ing, it has long been considered anomalous. However, Kasahara *et al*. [34] investigated the mechanisms underlying this phenomenon in higher plants upon frequently observing ovules that accepted two pollen tubes in the fertilization-defective *hap2-1* (allelic to *gcs1*) mutant [35], as shown in **Figure 6**. As the *hap2-1* mutant pollen tubes were marked by the pollen tube-specific reporter gene *LAT52:GUS* [36], Kasahara *et al*. traced the tube behaviors *in vivo* by staining for GUS activity, followed by aniline blue staining, to trace the behaviors of the first and second pollen tubes. Accordingly, most ovules contained one pollen tube at 10 hours

14 Pollination in Plants

**Figure 6.** Fertilization recovery system. Upon insertion of a single pollen tube into an ovule, the pollen tube bursts and releases two sperm cells. When the sperm cells complete fertilization, the ovule blocks the entry of the other pollen tubes and develops into a seed by forming an embryo and endosperm. When fertilization fails, the ovule attracts a second pollen tube to rescue fertilization. The rescued ovule develops into a seed, resulting in increased fertility. In the case of failure of fertilization by the second pollen tube, the ovule does not attract a third pollen tube, possibly due to depletion of the pollen tube attractant from synergid cells, since both synergid cells collapse after entry of two pollen tubes.

Previously, several research groups [35, 38, 39] studied why several sperm cell–defective mutants exhibited an enhanced fertility phenotype (60–70% fertility); particularly, the frequency ratio of double pollen tube reception was almost completely consistent with the frequency of enhanced fertility (**Figure 6**). Additionally, the GUS staining experiment revealed that by 10 HAP, ~50% ovules had accepted a mutant allele, indicating that the mutant and wild-type pollen tubes were similarly competent to enter the embryo sac and release their contents. von Besser *et al*. [35] suggested that *hap2-1* sperm cells affect pollen tube guidance. However, our data led us to conclude that sperm cells are passive pollen tube cargo and do not influence pollen tube guidance in *hap2-1* mutants, consistent with the observation that sperm cell–defective mutants (i.e., no transmission via the male germline) exhibit only 30–35% sterility, instead of the expected 50%. Very recently, Zhang *et al*. [40] demonstrated that in the absence of two bHLH transcription factors, *Arabidopsis* produces an abnormal, sperm cell-free pollen exhibiting behavior similar to its wild-type counterpart, thus indicating that sperm cells are dispensable for normal pollen tube development. This result reinforced our concept of sperm cells as passive cargo, with no control over pollen tube growth and behavior.

According to previous report by Kasahara *et al*. [37], all hand pollination experiments were performed using large numbers of pollen grains. Particularly, *Arabidopsis* pistils, usually containing 50–60 ovules, were pollinated with approximately 20, 40, 80, 120, and 700 grains. Two days after pollination, the insertion of few second pollen tubes into ovules were observed among the pistils pollinated with 20 and 40 grains, indicating that under restricted conditions (ovules > pollen tubes), the pollen tubes selectively inserted into ovules that had not previously accepted any pollen tube. Conversely, when a wild-type pistil was pollinated with 80 (ovules ≤ pollen tubes) and 120 (ovules < pollen tubes) grains, approximately 12 and 25% of the ovules accepted second pollen tubes, respectively, suggesting that ovules accept second pollen tubes while under saturated conditions (ovules < pollen tubes) [37]. In other words, excess pollen is required to saturate the fertilization recovery system, and approximately 80% (not 100%) of the failed ovules can accept a second pollen tube to complete recovery, consistent with a previous report [34] that a substantial period of ~28 h is required for ovules to complete the fertilization recovery system. This delay in secondary guidance may be attributable to the functional synergid cell numbers; a previously penetrated ovule contains only one persistent synergid cell (the other would have been disrupted by a burst pollen tube) to provide guidance. Higashiyama *et al*. [4] reported that an ovule containing two synergid cells attracts more pollen tubes than does an ovule with one synergid cell, suggesting that the latter produces insufficient levels of attractant. This may explain why only approximately 80% ovules with one synergid cell will attract a second pollen tube.

fertilization and normal endosperm formation was required for the initiation of the seed coat formation. Contrarily, our observation of vanillin staining in *agl62* mutant ovules indicated that the central cell fertilization is not required for the initiation of the seed coat formation (**Figure 7**).

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**Figure 7.** Pollen tube–dependent ovule enlarged morphology (POEM). (A) After the pollen tube is inserted to the female gametophyte, the pollen tube bursts and releases its contents (yellow region) with two sperm cells. Double fertilization is accomplished by these sperm cells fertilizing egg cell and central cell, respectively. *gcs1* mutant sperm cells fail to fertilize and the ovule does not produce a seed. It had long been suggested that the ovule will just die if the ovule fails to fertilize. However, if the pollen tube contents are supplied to the ovule, the ovule will be enlarged and initiate seed coat formation without fertilization. (B) A wild-type seed stained by vanillin at 3DAP. Whole seed coat region is stained. (C) Ovules without pollen tubes. Seed coat is not stained by vanillin. (D) A wild-type ovule crossed by *gcs1*/*gcs1* pollen. The ovule is partially stained. (E) An *agl62* mutant ovule. The ovule is partially stained even though it has abnormal

endosperm. Arrows indicate the stained part by vanillin. Bars = 100 μm.

## **6. Pollen tube-dependent ovule enlargement morphology (POEM)**

In angiosperms, the pollen tube releases its contents (including sperm cells) into the embryo sac upon insertion into the ovule, thus completing double fertilization. Recently, Kasahara *et al*. [41, 42] reported that the expansion and initiation of seed coat formation occurred even in ovules wherein fertilization failed after pollen tube insertion. This phenomenon was designated as pollen tube–dependent ovule enlargement morphology (POEM), which occurs only when the ovule accepts the pollen tube content (PTC). POEM was the first report addressing the paternal functions of PTC in facilitating the ovule's maternal development without fertilization in plants.

In animals, once semen is discharged into the uterus, the seminal plasma carries the sperm to the egg [43, 44], whereas in plants, PTC, which transports sperm cells to the ovules, has an analogous function. In mice, fertilization requires seminal vesicle secretory protein 2, which localizes only in the seminal plasma [45]. As seminal plasma is essential for fertilization in animals, Kasahara *et al*. proposed that PTC should also be important for fertilization in plants. To understand the function of PTC, a *gcs1* mutant [30] was used, which fails to accomplish fertilization despite releasing PTC to evaluate transcriptional variations after PTC release into the embryo sac and compared the transcriptomes between two ovule RNA types: one after normal fertilization and the other after PTC release without fertilization. At 12 and 24 HAP, the observation of similar expression profiles for both RNAs was unexpected because early events after pollen tube insertion were thought to be fertilization-dependent. However, these events were instead found to depend on PTC. Notably, between 24 and 48 HAP, multiple genes associated with cell expansion, cell division, and seed coat formation were upregulated regardless of fertilization, suggesting that PTC can affect ovule shape. Hence, the ovule phenotype was investigated. Interestingly, ovules that accepted PTC expanded without fertilization because of cell expansion and division and the production of a partial seed coat, consistent with the results of the transcriptome analysis. Using data from the successful transcriptome analysis, Kasahara *et al*. identified that the novel plant phenomenon POEM occurs only when the ovule accepts PTC, irrespective of fertilization (**Figure 7**) [41].

In angiosperms, pollination is the first step toward fertilization. Once the pollen reaches the stigma, the grains elongate to form pollen tubes and move toward the synergid cells observed within the female gametophyte. Fertilization occurs when the pollen tubes pierce the female gametophyte; this action terminates pollen tube growth and induces bursting, resulting in the deposition of the two sperm cells inside the female gametophyte. The phenomenon represents a new reproductive phase between pollen tube guidance and fertilization because PTC release itself could induce POEM. Roszak and Köhler [46] demonstrated failure of seed coat synthesis in *agl62* mutant ovules (Kang *et al*. [47]), which exhibited early endosperm cellularization, resulting in abnormal endosperm formation. Roszak and Köhler suggested that central cell fertilization and normal endosperm formation was required for the initiation of the seed coat formation. Contrarily, our observation of vanillin staining in *agl62* mutant ovules indicated that the central cell fertilization is not required for the initiation of the seed coat formation (**Figure 7**).

attracts more pollen tubes than does an ovule with one synergid cell, suggesting that the latter produces insufficient levels of attractant. This may explain why only approximately 80%

In angiosperms, the pollen tube releases its contents (including sperm cells) into the embryo sac upon insertion into the ovule, thus completing double fertilization. Recently, Kasahara *et al*. [41, 42] reported that the expansion and initiation of seed coat formation occurred even in ovules wherein fertilization failed after pollen tube insertion. This phenomenon was designated as pollen tube–dependent ovule enlargement morphology (POEM), which occurs only when the ovule accepts the pollen tube content (PTC). POEM was the first report addressing the paternal functions of PTC in facilitating the ovule's maternal development without fertil-

In animals, once semen is discharged into the uterus, the seminal plasma carries the sperm to the egg [43, 44], whereas in plants, PTC, which transports sperm cells to the ovules, has an analogous function. In mice, fertilization requires seminal vesicle secretory protein 2, which localizes only in the seminal plasma [45]. As seminal plasma is essential for fertilization in animals, Kasahara *et al*. proposed that PTC should also be important for fertilization in plants. To understand the function of PTC, a *gcs1* mutant [30] was used, which fails to accomplish fertilization despite releasing PTC to evaluate transcriptional variations after PTC release into the embryo sac and compared the transcriptomes between two ovule RNA types: one after normal fertilization and the other after PTC release without fertilization. At 12 and 24 HAP, the observation of similar expression profiles for both RNAs was unexpected because early events after pollen tube insertion were thought to be fertilization-dependent. However, these events were instead found to depend on PTC. Notably, between 24 and 48 HAP, multiple genes associated with cell expansion, cell division, and seed coat formation were upregulated regardless of fertilization, suggesting that PTC can affect ovule shape. Hence, the ovule phenotype was investigated. Interestingly, ovules that accepted PTC expanded without fertilization because of cell expansion and division and the production of a partial seed coat, consistent with the results of the transcriptome analysis. Using data from the successful transcriptome analysis, Kasahara *et al*. identified that the novel plant phenomenon POEM occurs

only when the ovule accepts PTC, irrespective of fertilization (**Figure 7**) [41].

In angiosperms, pollination is the first step toward fertilization. Once the pollen reaches the stigma, the grains elongate to form pollen tubes and move toward the synergid cells observed within the female gametophyte. Fertilization occurs when the pollen tubes pierce the female gametophyte; this action terminates pollen tube growth and induces bursting, resulting in the deposition of the two sperm cells inside the female gametophyte. The phenomenon represents a new reproductive phase between pollen tube guidance and fertilization because PTC release itself could induce POEM. Roszak and Köhler [46] demonstrated failure of seed coat synthesis in *agl62* mutant ovules (Kang *et al*. [47]), which exhibited early endosperm cellularization, resulting in abnormal endosperm formation. Roszak and Köhler suggested that central cell

**6. Pollen tube-dependent ovule enlargement morphology (POEM)**

ovules with one synergid cell will attract a second pollen tube.

ization in plants.

16 Pollination in Plants

**Figure 7.** Pollen tube–dependent ovule enlarged morphology (POEM). (A) After the pollen tube is inserted to the female gametophyte, the pollen tube bursts and releases its contents (yellow region) with two sperm cells. Double fertilization is accomplished by these sperm cells fertilizing egg cell and central cell, respectively. *gcs1* mutant sperm cells fail to fertilize and the ovule does not produce a seed. It had long been suggested that the ovule will just die if the ovule fails to fertilize. However, if the pollen tube contents are supplied to the ovule, the ovule will be enlarged and initiate seed coat formation without fertilization. (B) A wild-type seed stained by vanillin at 3DAP. Whole seed coat region is stained. (C) Ovules without pollen tubes. Seed coat is not stained by vanillin. (D) A wild-type ovule crossed by *gcs1*/*gcs1* pollen. The ovule is partially stained. (E) An *agl62* mutant ovule. The ovule is partially stained even though it has abnormal endosperm. Arrows indicate the stained part by vanillin. Bars = 100 μm.

PTC was previously found to initiate central cell/endosperm nuclei division without fertilization when it was released to an autonomous endosperm mutant, *mea* [48, 49]. The finding that segmentation can be induced in a fertilization-independent manner by physical stimuli, leading to the development of some eggs into normal tadpoles, was first reported in 1910 [50]. In plants, Kasahara *et al*. [41] showed that PTC could increase central cell/ endosperm nuclei division in the absence of fertilization, suggesting a function parallel to the observed fertilization-independent division of animal germ cells in response to external stimuli. By inducing endosperm nuclear division, PTC facilitates apomixes [51] in important crops when the POEM phenomenon is combined with autonomous endosperm and embryo mutants. In plants, seed formation without fertilization, or apomixis, is agriculturally valuable because important genetic traits can be easily fixed in apomictic crops, which then propagate without interference from unfavorable environmental conditions. POEM could therefore be categorized as "pseudogamy," which is defined as any reproductive process requiring pollination but no inheritance from the male gametophyte [52]. Although Focke [53] first defined pseudogamy as a part of apomixis, the underlying cellular or molecular mechanisms have remained obscure. Given the conceptual similarities, POEM may therefore be a key in understanding pseudogamy, particularly concerning pollen and PTC stimuli.

**Author details**

**References**

Ryushiro Dora Kasahara

2011;**21**:497-502

Cell. 2005;**17**:2981-2992

cells. Nature. 2009;**458**:357-361

Address all correspondence to: kasahara@fafu.edu.cn

W.H. Freeman and Company; 1989. p. 49

Forestry University, Fuzhou, Fujian, China

FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and

The Regulation of Sperm Cells Delivery to the Embryo Sac

http://dx.doi.org/10.5772/intechopen.75359

19

[1] Gifford EM, Foster AS. Morphology and Evolution of Vascular Plants. New York:

[2] Drews GN, Koltunow AMG. The female gametophyte. The Arabidopsis Book. 2011;**9**:e0155

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[5] Márton ML, Cordts S, Broadhvest J, Dresselhaus T. Micropylar pollen tube guidance by

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interactions in *Arabidopsis*. Development. 1993:77-84

## **7. Summary**

This chapter discusses the journey of the pollen tube from the stigma to fertilization as well as the POEM phenomenon. Because very few factors related to pollen tube guidance from the stigma to the funiculus of the ovule have been elucidated, additional insights into this step are eagerly awaited. However, the molecular mechanisms underlying pollen tube guidance from the funiculus to the female gametophyte are well known in *Arabidopsis* because the pollen tube attractants AtLURE1 peptides had previously been identified downstream of the master synergid cell regulator MYB98. During the final step after pollen tube bursting, only two proteins, GCS1 and GEX2, have been identified as direct male-related key fertilization factors in the pollen tube. Accordingly, further molecular evidences are required to understand the final step for plant fertilization. Finally, very few factors related to new plant phenomena, fertilization recovery system, and POEM have been identified. New insights into the underlying molecular mechanisms are anticipated.

## **Acknowledgements**

I thank Liyang Xie and Xiaoyan Liu at HBMC, FAFU for technical assistance. These works were supported by the Precursory Research for Embryonic Science and Technology (13416724, Kasahara Sakigake Project), Japan Science and Technology Agency. These works were also supported by a grant-in-aid (25840106) from the Japanese Society for the promotion of Science (JSPS). These works were also supported by FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University.

## **Author details**

PTC was previously found to initiate central cell/endosperm nuclei division without fertilization when it was released to an autonomous endosperm mutant, *mea* [48, 49]. The finding that segmentation can be induced in a fertilization-independent manner by physical stimuli, leading to the development of some eggs into normal tadpoles, was first reported in 1910 [50]. In plants, Kasahara *et al*. [41] showed that PTC could increase central cell/ endosperm nuclei division in the absence of fertilization, suggesting a function parallel to the observed fertilization-independent division of animal germ cells in response to external stimuli. By inducing endosperm nuclear division, PTC facilitates apomixes [51] in important crops when the POEM phenomenon is combined with autonomous endosperm and embryo mutants. In plants, seed formation without fertilization, or apomixis, is agriculturally valuable because important genetic traits can be easily fixed in apomictic crops, which then propagate without interference from unfavorable environmental conditions. POEM could therefore be categorized as "pseudogamy," which is defined as any reproductive process requiring pollination but no inheritance from the male gametophyte [52]. Although Focke [53] first defined pseudogamy as a part of apomixis, the underlying cellular or molecular mechanisms have remained obscure. Given the conceptual similarities, POEM may therefore be a key in understanding pseudogamy, particularly concerning

This chapter discusses the journey of the pollen tube from the stigma to fertilization as well as the POEM phenomenon. Because very few factors related to pollen tube guidance from the stigma to the funiculus of the ovule have been elucidated, additional insights into this step are eagerly awaited. However, the molecular mechanisms underlying pollen tube guidance from the funiculus to the female gametophyte are well known in *Arabidopsis* because the pollen tube attractants AtLURE1 peptides had previously been identified downstream of the master synergid cell regulator MYB98. During the final step after pollen tube bursting, only two proteins, GCS1 and GEX2, have been identified as direct male-related key fertilization factors in the pollen tube. Accordingly, further molecular evidences are required to understand the final step for plant fertilization. Finally, very few factors related to new plant phenomena, fertilization recovery system, and POEM have been identified. New insights into the underlying

I thank Liyang Xie and Xiaoyan Liu at HBMC, FAFU for technical assistance. These works were supported by the Precursory Research for Embryonic Science and Technology (13416724, Kasahara Sakigake Project), Japan Science and Technology Agency. These works were also supported by a grant-in-aid (25840106) from the Japanese Society for the promotion of Science (JSPS). These works were also supported by FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and

pollen and PTC stimuli.

molecular mechanisms are anticipated.

Technology, Fujian Agriculture and Forestry University.

**Acknowledgements**

**7. Summary**

18 Pollination in Plants

Ryushiro Dora Kasahara

Address all correspondence to: kasahara@fafu.edu.cn

FAFU-UCR Joint Center and Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, Haixia Institute of Science and Technology, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China

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**Chapter 3**

**Provisional chapter**

**Pollination Ecology of the** *Manicaria saccifera*

**Pollination Ecology of the** *Manicaria saccifera*

Juan Carlos Copete, Danny Mosquera Flórez and

Juan Carlos Copete, Danny Mosquera Flórez and

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

Luis Alberto Núñez-Avellaneda

Luis Alberto Núñez-Avellaneda

http://dx.doi.org/10.5772/intechopen.76073

**Abstract**

**1. Introduction**

**(ARECACEAE): A Rare Case of Pollinator Exclusion**

We studied the reproductive biology and pollination ecology of the palm cabecinegro (*Manicaria saccifera*) in very wet tropical forest, in the Chocó, Pacific region of Colombia. We present data about the phenology, floral morphology, floral biology, reproductive system, and pollination. *M. saccifera* is monoecious, self-incompatible, lacks apomixis and has dichogamy in the form of protogyny. Flowering occurs all year round with a peak between April and May. A single individual may produce up to five inflorescences in its reproductive period. Each inflorescence has unisexual flowers grouped in dyads and triads; anthesis is diurnal and the flowers may be receptive for 72 h. Flowers are visited by 10 species of insects. The inflorescences in the female-phase do not offer reward and insects are attracted by olfactory mimicry; in the male-phase flowers reward visitors with pollen and a place to oviposit. The most efficient pollinator is *Mystrosp cercus* (Nitidulidae), the only visitor arriving in abundance during the female-phase. Other insects do not enter the flower because the peduncular bract and the petals act as barriers, blocking the entrance of insects greater than 2 mm. Having one exclusive pollinator which in turn depends on the palm for its survival is an example of extreme specialization and mutual dependence. **Keywords:** Arecaceae, beetle pollination, *Mystrops cercus*, neotropical palm, phenology

Palms are one of the most important plant families because they are a food source for wildlife [1]; they provide a variety of products that rural peoples use for construction, food, medicine and handicraft purposes [2, 3]; they are a source of raw materials for a great variety of products that communities exploit commercially at small or large scales, always in an extractive

**(ARECACEAE): A Rare Case of Pollinator Exclusion**

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

DOI: 10.5772/intechopen.76073


#### **Pollination Ecology of the** *Manicaria saccifera* **(ARECACEAE): A Rare Case of Pollinator Exclusion Pollination Ecology of the** *Manicaria saccifera* **(ARECACEAE): A Rare Case of Pollinator Exclusion**

DOI: 10.5772/intechopen.76073

Juan Carlos Copete, Danny Mosquera Flórez and Luis Alberto Núñez-Avellaneda Juan Carlos Copete, Danny Mosquera Flórez and Luis Alberto Núñez-Avellaneda

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.76073

#### **Abstract**

[40] Zhang J, Huang Q, Zhong S, Bleckmann A, Huang J, Guo X, Lin Q, Gu H, Dong J, Dresselhaus T, Qu LJ. Sperm cells are passive cargo of the pollen tube in plant fertiliza-

[41] Kasahara RD, Notaguchi M, Nagahara S, Suzuki T, Susaki D, Honma Y, Maruyama D, Higashiyama T. Pollen tube contents initiate ovule enlargement and enhance seed coat

[42] Kasahara RD, Notaguchi M, Honma Y.Discovery of pollen tube-dependent ovule enlargement morphology phenomenon, a new step in plant reproduction. Communicative and

[43] Sasanami T, Sugiura K, Tokumoto T, Yoshizaki N, Dohra H, Nishio S, Mizushima S, Hiyama G, Matsuda T. Sperm proteasome degrades egg envelope glycoprotein ZP1 during fertilization of Japanese quail (Coturnix japonica). Reproduction. 2012;**144**:423-431

[44] Sasanami T, Izumi S, Sakurai N, Hirata NT, Mizushima S, Matsuzaki M, Hiyama G, Yorinaga E, Yoshimura T, Ukena K, Tsutsui K. A unique mechanism of successful fertil-

[45] Kawano N, Araki N, Yoshida K, Hibino T, Ohnami N, Makino M, Kanai S, Hasuwa H, Yoshida M, Miyado K, Umezawa A. Seminal vesicle protein SVS2 is required for sperm survival in the uterus. Proceedings of the National Academy of Sciences of the United

[46] Roszak P, Köhler C. Polycomb group proteins are required to couple seed coat initiation to fertilization. Proceedings of the National Academy of Sciences of the United States of

[47] Kang IH, Steffen JG, Portereiko MF, Lloyd A, Drews GN. The AGL62 MADS domain protein regulates cellularization during endosperm development in *Arabidopsis*. Plant

[48] Grossniklaus U, Vielle-Calzada JP, Hoeppner MA, Gagliano WB. Maternal control of embryogenesis by MEDEA, a polycomb group gene in *Arabidopsis*. Science. 1998;**280**:

[49] Luo M, Bilodeau P, Koltunow A, Dennis ES, Peacock WJ, Chaudhury AM. Genes controlling fertilization-independent seed development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America. 1999;**96**:296-301 [50] Bataillon E. L'embryogénèse complète provoquèe chez les Amphibiens par figure de l′œuf vierge, larves parthénogénétiques de Rana fusca. Comptes Rendus. Académie des

[51] Koltunow AM, Grossniklaus U. Apomixis: A developmental perspective. Annual Review

[52] Nogler GA. Gametophytic apomixis. In: Embryology of Angiosperms. 1984. pp. 475-518 [53] Focke WO. Die Pflanzen–mischlinge, ein Beitrag zur Biologie der Gewächse. Berlin:

development without fertilization. Science Advances. 2016;**2**:e1600554

ization in a domestic bird. Scientific Reports. 2015;**9**:7700

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Integrative Biology. 2017;**10**:e1338989

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446-450

We studied the reproductive biology and pollination ecology of the palm cabecinegro (*Manicaria saccifera*) in very wet tropical forest, in the Chocó, Pacific region of Colombia. We present data about the phenology, floral morphology, floral biology, reproductive system, and pollination. *M. saccifera* is monoecious, self-incompatible, lacks apomixis and has dichogamy in the form of protogyny. Flowering occurs all year round with a peak between April and May. A single individual may produce up to five inflorescences in its reproductive period. Each inflorescence has unisexual flowers grouped in dyads and triads; anthesis is diurnal and the flowers may be receptive for 72 h. Flowers are visited by 10 species of insects. The inflorescences in the female-phase do not offer reward and insects are attracted by olfactory mimicry; in the male-phase flowers reward visitors with pollen and a place to oviposit. The most efficient pollinator is *Mystrosp cercus* (Nitidulidae), the only visitor arriving in abundance during the female-phase. Other insects do not enter the flower because the peduncular bract and the petals act as barriers, blocking the entrance of insects greater than 2 mm. Having one exclusive pollinator which in turn depends on the palm for its survival is an example of extreme specialization and mutual dependence.

**Keywords:** Arecaceae, beetle pollination, *Mystrops cercus*, neotropical palm, phenology

## **1. Introduction**

Palms are one of the most important plant families because they are a food source for wildlife [1]; they provide a variety of products that rural peoples use for construction, food, medicine and handicraft purposes [2, 3]; they are a source of raw materials for a great variety of products that communities exploit commercially at small or large scales, always in an extractive

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

way [3, 4], and finally, palms are culturally important because many species are essential to the cultural development of local peoples [5].

(**Figure 1**), and 90 m of elevation. With a mean annual temperature of 28° C, a relative humidity of 89% and a mean annual precipitation of 5000–7000 mm, this area is classified under very

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

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25

*Manicaria saccifera* Gaertn., known as "cabecinegro" in the Colombian Pacific (Galeano and Bernal [13]) is a monoecious palm that grows in swampy inundated areas, preferably near the edge of rivers and creeks. Its individuals are medium sized, solitary or cespitose, with large regularly pinnatisect or simple leaves and with a dentate margin. The inflorescences are interfoliar and solitary [13]. It is distributed in Central America, in the Pacific littoral of Colombia southwards to northern Ecuador, and in the Orinoco and Amazon basin of Colombia, Brazil

A morphological description of the inflorescence was performed based on 20 inflorescences of *M. saccifera*. We (i) recorded the height at which the inflorescences are found, (ii) measured the length of the inflorescence, rachillae and flowers; (iii) counted the number of rachillae, of female and male flowers per rachillae and of female and male flowers per inflorescence; and (iv) determined the position of the flowers in the rachillae, the number and shape of the

wet tropical forests in the Holdrige life zone system [22].

**2.2. Study species**

and Venezuela [12].

**Figure 1.** Study zone.

**2.3. Inflorescence morphology**

Due to this importance, studies on their reproductive biology have increased in the last decades [6]. Initially, most studies focused on cultivated palms or on widely used species [1, 7]; however, recent works have focused on a wider range of palms [8–10].

Research on the reproductive biology of this plant family has increased our knowledge on the great variety of pollination strategies and mechanisms found in plants and on the strong dependence and intimate association that most palm species share with the insects that pollinate them, mainly species of coleoptera belonging to the families Nitidulidae and Curculionidae [9–11]. Due to palms economic, ecologic and cultural importance, studies on palms reproductive biology are essential for their sustainable use, conservation and future domestication [10].

*Manicaria saccifera* is a widely distributed species [12], of ecological importance [1, 13] and great economic potential [14, 15]. It ranks among the most utilized and economically valuable palms for the Afro-American and indigenous communities of the Chocó biogeographic region: [16] reported 36 different uses for this species, evidencing its versatility as a non-timber forest product. The most significant uses of *M. saccifera* in the Pacific region of Colombia include: the use of the peduncular bract as a source of fiber for crafts and textiles [15–17]; weaving the leaf veins for basketry [15]; the use of unexpanded leaves for brooms; the leaves for thatch houses; the immature fruits filled with a liquid resembling coconut water are consumed while working in the forest. Additionally, fruits are sold in local markets for medicinal purposes [16]. In Venezuela the Warao Indians use *M. saccifera* for construction, sailing, food, medicine, and for crafting bags and hats [18, 19].

*M. saccifera* is a little studied palm in terms of its reproductive biology [20], who mentions the possibilities of auto-pollination and the great quantity of larvae inside the flowers. On the other hand, [21] reported several visiting insects, with *Mystrops cercus* and *Mystrops erviki* (Nitidulidae: Coleoptera) being frequent visitors. The most important aspect to highlight in this species is that, contrary to most species in the family, anthesis and all remaining reproductive mechanisms occur "hidden" within the interior of the peduncular bract, without opening or exposing the male and female flowers. This aspect makes an understanding of the species reproductive strategy even the more intriguing.

Our work constitutes the first significant contribution detailing the principal aspects of the reproductive biology and pollination ecology of one of the most important palms for the inhabitants of the Pacific region of Colombia and a key species of tropical pluvial rainforests. In this work we evaluate the periods of flower and fruit production, the reproductive system, floral biology, flower visitors and we quantify the role each visitor has in the pollination of the palm.

## **2. Materials and methods**

#### **2.1. Study area**

Our study was conducted in the Quibdó municipality, Pacific coast of Colombia, in an area adjacent to the road leading from Quibdó to the Atrato municipality, at 5° 39′ N, 76° 38′ W (**Figure 1**), and 90 m of elevation. With a mean annual temperature of 28° C, a relative humidity of 89% and a mean annual precipitation of 5000–7000 mm, this area is classified under very wet tropical forests in the Holdrige life zone system [22].

## **2.2. Study species**

way [3, 4], and finally, palms are culturally important because many species are essential to

Due to this importance, studies on their reproductive biology have increased in the last decades [6]. Initially, most studies focused on cultivated palms or on widely used species [1,

Research on the reproductive biology of this plant family has increased our knowledge on the great variety of pollination strategies and mechanisms found in plants and on the strong dependence and intimate association that most palm species share with the insects that pollinate them, mainly species of coleoptera belonging to the families Nitidulidae and Curculionidae [9–11]. Due to palms economic, ecologic and cultural importance, studies on palms reproductive biol-

*Manicaria saccifera* is a widely distributed species [12], of ecological importance [1, 13] and great economic potential [14, 15]. It ranks among the most utilized and economically valuable palms for the Afro-American and indigenous communities of the Chocó biogeographic region: [16] reported 36 different uses for this species, evidencing its versatility as a non-timber forest product. The most significant uses of *M. saccifera* in the Pacific region of Colombia include: the use of the peduncular bract as a source of fiber for crafts and textiles [15–17]; weaving the leaf veins for basketry [15]; the use of unexpanded leaves for brooms; the leaves for thatch houses; the immature fruits filled with a liquid resembling coconut water are consumed while working in the forest. Additionally, fruits are sold in local markets for medicinal purposes [16]. In Venezuela the Warao Indians use *M. saccifera* for construction, sailing, food,

*M. saccifera* is a little studied palm in terms of its reproductive biology [20], who mentions the possibilities of auto-pollination and the great quantity of larvae inside the flowers. On the other hand, [21] reported several visiting insects, with *Mystrops cercus* and *Mystrops erviki* (Nitidulidae: Coleoptera) being frequent visitors. The most important aspect to highlight in this species is that, contrary to most species in the family, anthesis and all remaining reproductive mechanisms occur "hidden" within the interior of the peduncular bract, without opening or exposing the male and female flowers. This aspect makes an understanding of the

Our work constitutes the first significant contribution detailing the principal aspects of the reproductive biology and pollination ecology of one of the most important palms for the inhabitants of the Pacific region of Colombia and a key species of tropical pluvial rainforests. In this work we evaluate the periods of flower and fruit production, the reproductive system, floral biology, flower visitors and we quantify the role each visitor has in the pollination of the palm.

Our study was conducted in the Quibdó municipality, Pacific coast of Colombia, in an area adjacent to the road leading from Quibdó to the Atrato municipality, at 5° 39′ N, 76° 38′ W

ogy are essential for their sustainable use, conservation and future domestication [10].

7]; however, recent works have focused on a wider range of palms [8–10].

the cultural development of local peoples [5].

24 Pollination in Plants

medicine, and for crafting bags and hats [18, 19].

species reproductive strategy even the more intriguing.

**2. Materials and methods**

**2.1. Study area**

*Manicaria saccifera* Gaertn., known as "cabecinegro" in the Colombian Pacific (Galeano and Bernal [13]) is a monoecious palm that grows in swampy inundated areas, preferably near the edge of rivers and creeks. Its individuals are medium sized, solitary or cespitose, with large regularly pinnatisect or simple leaves and with a dentate margin. The inflorescences are interfoliar and solitary [13]. It is distributed in Central America, in the Pacific littoral of Colombia southwards to northern Ecuador, and in the Orinoco and Amazon basin of Colombia, Brazil and Venezuela [12].

## **2.3. Inflorescence morphology**

A morphological description of the inflorescence was performed based on 20 inflorescences of *M. saccifera*. We (i) recorded the height at which the inflorescences are found, (ii) measured the length of the inflorescence, rachillae and flowers; (iii) counted the number of rachillae, of female and male flowers per rachillae and of female and male flowers per inflorescence; and (iv) determined the position of the flowers in the rachillae, the number and shape of the

**Figure 1.** Study zone.

stigmas and the number of anthers. To estimate the number of flowers per inflorescence, we multiplied the total number of flowers of each rachillae by the mean number of rachillae counted in 10 inflorescences.

inside the bags. This procedure was repeated three times each day during all the flowering phase. For each floral visitor we documented its (i) abundance, (ii) behavior and (iii) role in pollination. To document the behavior of floral visitors we performed observations on the female- and malephase of each palm, noting: the hour of arrival and departure to the flower, the activity within the flower, the utilized resources, and the permanence in the flower, and the contact with the stigmas in the female-phase. Following [10] we calculated the pollinator importance value (PIV) and the pollinator relative importance value (PRI). The variables used to calculate these indices were: the relative abundance of insects in the female flowers (AB), pollen-transport capacity (PTC), fidelity (F), constancy (C), and pollen-transport efficiency (PTE). For details on each variable refer to [10]. To determine the CTP and ETP, we collected five insects of each species, preserved them in 70% alcohol and took them to the laboratory to conduct the pollen load analyses following the methods in [9]. To evaluate fidelity we compared the pollinators of *M. saccifera* with pollinators of the palms *Attalea allenii, Attalea cuatrecasana, Oenocarpus bataua, Oenocarpus minor, Socratea exhorriza* and *Wettinia quinaria*, all of which are palms that grow in the study area together with *M. saccifera.*

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

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27

The inflorescences of *M. saccifera* are interfoliar and located at a mean of 1.89 ± 1.1 (SD, n = 20) meters above the ground (**Figure 2A**). An individual during the reproductive phase may produce 1–5 inflorescences (n = 45), which are found in different developing stages and which flower alternately. Each inflorescence can measure up to 1.50 m, including the peduncle, which measures 46 cm and the prophyll (35 cm). Each inflorescence is covered by a fibrous peduncular bract, which is closed without suture, is brown and has the shape of a long hood. Throughout all development phases of the inflorescence, the peduncular bract acts as a mesh or as a selective barrier to insect visitors (**Figure 2B** and **C**). The bract suffers changes in morphology and coloration at its exterior surface and thickening due to the development and growth of the fruits in the inner part. Inflorescence present a mean of 35 ± 19.5 (SD, n = 10) rachillae. The flowers in inflorescences are distributed in (i) triads, with a female flower in the center and two male flowers at the sides in the basal part; (ii) toward the apex as dyads, with two male flowers (**Figure 3**). This distribution of flowers distinguishes the subfamily Arecoideae from the other

palm subfamilies. Generally, the last rachillae to develop contain only male flowers.

female flowers and 15,085 ± 532 (SD, n = 10) male flowers.

stamens (mean of 35) (**Figure 2D**).

Each rachillae contains a mean of 2.88 ± 0.84 (SD, n = 504) female flowers and a mean of 218 ± 132 (SD, n = 504) male flowers so that each inflorescence can harbor 199 ± 32 (SD, n = 10)

The flowers are of rigid texture, yellowish in color, and the female flowers are greater, measuring 0.7 ± 0.3 cm (SD, n = 16) in length, and with a laminar stigma in the shape of a pyramid (**Figure 2E**); each male flower measures 0.8 ± 0.3 cm (SD, n = 16) in length and has numerous

The fruits are spherical, 5 cm in diameter and covered with woody pyramidal or pointed projections; occasionally the fruits have the shape of two or three united spheres forming a

**3. Results**

**3.1. Inflorescence morphology**

#### **2.4. Reproductive phenology**

We recorded the reproductive phenology of 48 individuals of *M. saccifera* during a 12-month period, marking individuals along a pre-defined trail in the forest. Each week we revisited the study area during 3 days to register in each individual if flowering and fruiting occurred. We calculated the monthly percentage of flowering and determined the flowering synchrony. Following [23] we defined flowering events as being: (i) asynchronous, when less than 20% of the individuals are in flower; (ii) low synchrony, between 21 and 60%; and (iii) high synchrony when over 60% of individuals flower at the same time.

## **2.5. Floral biology**

To view the reproductive structures and carry out our observations, a longitudinal slit was opened on the peduncular bract and was later covered with paper tape. Direct observation of the flowering buds and opened flowers were made at intervals of 6, 12, and 24 h in 10 inflorescences of 10 individuals. We registered the (i) hour of anthesis of the flowers, (ii) the daily rhythm of anthesis of the flowering buds, (iii) the presence and longevity of pollen, and (iv) the stigmatic receptivity. We used three methods to evaluate the stigmatic receptivity: (1) direct observation of the stigmas, noting changes in morphology, color and presence of exudates; (2) signs of peroxidase activity, using hydrogen peroxide tests [24]; (3) colorimetry tests, applying the Perex-Test solution by Merck [25]. Additionally, we registered the increase in temperature within the inflorescence using digital thermometers made by Cox Technologies Inc., with a range of temperature between −35 and 210°C. We performed measurements in five closed inflorescences of five individuals, introducing the thermometer's sensor to register the internal temperature of the inflorescence, which was compared with ambient temperature.

#### **2.6. Reproductive system**

We determined the reproductive system of *M. saccifera* by performing controlled pollinations on five inflorescences from different individuals using four different treatments: (i) Autopollination: we isolated inflorescences to avoid the entry of pollen; (ii) Open pollination: natural pollination without our intervention; (iii) Apomixis: without pollination; (iv) Allogamy: controlled pollinations using pollen from different individuals of *M. saccifera.* In all cases, the flowers were isolated with synthetic mesh at least 30 days. After each treatment we checked whether fruits were developing and recorded the percentage of fruits formed. The degree of genetic compatibility was evaluated using the self-incompatibility index proposed by [26].

#### **2.7. Floral visitors and pollinators**

The composition of floral visitors was studied in 10 inflorescences from 10 individuals. When the rate of visits was highest, inflorescences were covered with bags and shaken so insects would fall inside the bags. This procedure was repeated three times each day during all the flowering phase. For each floral visitor we documented its (i) abundance, (ii) behavior and (iii) role in pollination. To document the behavior of floral visitors we performed observations on the female- and malephase of each palm, noting: the hour of arrival and departure to the flower, the activity within the flower, the utilized resources, and the permanence in the flower, and the contact with the stigmas in the female-phase. Following [10] we calculated the pollinator importance value (PIV) and the pollinator relative importance value (PRI). The variables used to calculate these indices were: the relative abundance of insects in the female flowers (AB), pollen-transport capacity (PTC), fidelity (F), constancy (C), and pollen-transport efficiency (PTE). For details on each variable refer to [10]. To determine the CTP and ETP, we collected five insects of each species, preserved them in 70% alcohol and took them to the laboratory to conduct the pollen load analyses following the methods in [9]. To evaluate fidelity we compared the pollinators of *M. saccifera* with pollinators of the palms *Attalea allenii, Attalea cuatrecasana, Oenocarpus bataua, Oenocarpus minor, Socratea exhorriza* and *Wettinia quinaria*, all of which are palms that grow in the study area together with *M. saccifera.*

## **3. Results**

stigmas and the number of anthers. To estimate the number of flowers per inflorescence, we multiplied the total number of flowers of each rachillae by the mean number of rachillae

We recorded the reproductive phenology of 48 individuals of *M. saccifera* during a 12-month period, marking individuals along a pre-defined trail in the forest. Each week we revisited the study area during 3 days to register in each individual if flowering and fruiting occurred. We calculated the monthly percentage of flowering and determined the flowering synchrony. Following [23] we defined flowering events as being: (i) asynchronous, when less than 20% of the individuals are in flower; (ii) low synchrony, between 21 and 60%; and (iii) high syn-

To view the reproductive structures and carry out our observations, a longitudinal slit was opened on the peduncular bract and was later covered with paper tape. Direct observation of the flowering buds and opened flowers were made at intervals of 6, 12, and 24 h in 10 inflorescences of 10 individuals. We registered the (i) hour of anthesis of the flowers, (ii) the daily rhythm of anthesis of the flowering buds, (iii) the presence and longevity of pollen, and (iv) the stigmatic receptivity. We used three methods to evaluate the stigmatic receptivity: (1) direct observation of the stigmas, noting changes in morphology, color and presence of exudates; (2) signs of peroxidase activity, using hydrogen peroxide tests [24]; (3) colorimetry tests, applying the Perex-Test solution by Merck [25]. Additionally, we registered the increase in temperature within the inflorescence using digital thermometers made by Cox Technologies Inc., with a range of temperature between −35 and 210°C. We performed measurements in five closed inflorescences of five individuals, introducing the thermometer's sensor to register the internal temperature of the inflorescence, which was compared with ambient temperature.

We determined the reproductive system of *M. saccifera* by performing controlled pollinations on five inflorescences from different individuals using four different treatments: (i) Autopollination: we isolated inflorescences to avoid the entry of pollen; (ii) Open pollination: natural pollination without our intervention; (iii) Apomixis: without pollination; (iv) Allogamy: controlled pollinations using pollen from different individuals of *M. saccifera.* In all cases, the flowers were isolated with synthetic mesh at least 30 days. After each treatment we checked whether fruits were developing and recorded the percentage of fruits formed. The degree of genetic compatibility was evaluated using the self-incompatibility index proposed by [26].

The composition of floral visitors was studied in 10 inflorescences from 10 individuals. When the rate of visits was highest, inflorescences were covered with bags and shaken so insects would fall

chrony when over 60% of individuals flower at the same time.

counted in 10 inflorescences.

26 Pollination in Plants

**2.4. Reproductive phenology**

**2.5. Floral biology**

**2.6. Reproductive system**

**2.7. Floral visitors and pollinators**

#### **3.1. Inflorescence morphology**

The inflorescences of *M. saccifera* are interfoliar and located at a mean of 1.89 ± 1.1 (SD, n = 20) meters above the ground (**Figure 2A**). An individual during the reproductive phase may produce 1–5 inflorescences (n = 45), which are found in different developing stages and which flower alternately. Each inflorescence can measure up to 1.50 m, including the peduncle, which measures 46 cm and the prophyll (35 cm). Each inflorescence is covered by a fibrous peduncular bract, which is closed without suture, is brown and has the shape of a long hood. Throughout all development phases of the inflorescence, the peduncular bract acts as a mesh or as a selective barrier to insect visitors (**Figure 2B** and **C**). The bract suffers changes in morphology and coloration at its exterior surface and thickening due to the development and growth of the fruits in the inner part. Inflorescence present a mean of 35 ± 19.5 (SD, n = 10) rachillae. The flowers in inflorescences are distributed in (i) triads, with a female flower in the center and two male flowers at the sides in the basal part; (ii) toward the apex as dyads, with two male flowers (**Figure 3**). This distribution of flowers distinguishes the subfamily Arecoideae from the other palm subfamilies. Generally, the last rachillae to develop contain only male flowers.

Each rachillae contains a mean of 2.88 ± 0.84 (SD, n = 504) female flowers and a mean of 218 ± 132 (SD, n = 504) male flowers so that each inflorescence can harbor 199 ± 32 (SD, n = 10) female flowers and 15,085 ± 532 (SD, n = 10) male flowers.

The flowers are of rigid texture, yellowish in color, and the female flowers are greater, measuring 0.7 ± 0.3 cm (SD, n = 16) in length, and with a laminar stigma in the shape of a pyramid (**Figure 2E**); each male flower measures 0.8 ± 0.3 cm (SD, n = 16) in length and has numerous stamens (mean of 35) (**Figure 2D**).

The fruits are spherical, 5 cm in diameter and covered with woody pyramidal or pointed projections; occasionally the fruits have the shape of two or three united spheres forming a

triangular structure. The seed is spherical, very hard, and is covered by a brownish or light

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

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29

*M. saccifera* flowered throughout the year (**Figure 4**), with a peak in inflorescence production in the month of May, when nearly 61% of individuals had one or two inflorescences; the remaining individuals flowered during the months of June until August. There was no relationship between flowering and precipitation, flowering being constant in months of low rainfall (February) and highest rainfall (November). The individuals of the studied population showed low synchrony in flowering, with 27% of the individuals flowering simultane-

The events that take place during floral biology can be summarized as: (1) flower buds emerge; (2) buds of pistillate flowers develop completely; (3) anthesis begins; in female flowers it occurs simultaneously whereas it is alternate in male flowers and lasts up to 2 days. During anthesis there is an increase in temperature above ambient temperature in female and male inflorescences of 4.0 and 4.7°C, respectively (both n = 3); (4) maximum stigmatic receptivity, evidenced by the white color of the stigmas covered by a hyalinebrilliant substance; (5) pollination, with subsequent loss of stigmatic receptivity evidenced

**Figure 4.** Monthly proportion of flowering and fruiting individuals of *Manicaria saccifera* compared with annual

ously. The fruiting period was relatively constant throughout the year (**Figure 4**).

purplish kernel of brittle texture (**Figure 2G**).

**3.2. Reproductive phenology**

**3.3. Floral biology**

precipitation.

**Figure 2.** Morphology of *Manicaria saccifera*. (A) Habit. (B) and (C) inflorescence covered by the peduncular bract. (D) Flowers of *M. saccifera* in triads. (E) Receptive female flower. (F) Open male flower.

**Figure 3.** Distribution of the female- and male-flowers at the rachillae of *Manicaria saccifera.*

triangular structure. The seed is spherical, very hard, and is covered by a brownish or light purplish kernel of brittle texture (**Figure 2G**).

#### **3.2. Reproductive phenology**

*M. saccifera* flowered throughout the year (**Figure 4**), with a peak in inflorescence production in the month of May, when nearly 61% of individuals had one or two inflorescences; the remaining individuals flowered during the months of June until August. There was no relationship between flowering and precipitation, flowering being constant in months of low rainfall (February) and highest rainfall (November). The individuals of the studied population showed low synchrony in flowering, with 27% of the individuals flowering simultaneously. The fruiting period was relatively constant throughout the year (**Figure 4**).

#### **3.3. Floral biology**

**Figure 3.** Distribution of the female- and male-flowers at the rachillae of *Manicaria saccifera.*

Flowers of *M. saccifera* in triads. (E) Receptive female flower. (F) Open male flower.

28 Pollination in Plants

**Figure 2.** Morphology of *Manicaria saccifera*. (A) Habit. (B) and (C) inflorescence covered by the peduncular bract. (D)

The events that take place during floral biology can be summarized as: (1) flower buds emerge; (2) buds of pistillate flowers develop completely; (3) anthesis begins; in female flowers it occurs simultaneously whereas it is alternate in male flowers and lasts up to 2 days. During anthesis there is an increase in temperature above ambient temperature in female and male inflorescences of 4.0 and 4.7°C, respectively (both n = 3); (4) maximum stigmatic receptivity, evidenced by the white color of the stigmas covered by a hyalinebrilliant substance; (5) pollination, with subsequent loss of stigmatic receptivity evidenced

**Figure 4.** Monthly proportion of flowering and fruiting individuals of *Manicaria saccifera* compared with annual precipitation.

by a change of color from white to brown to black; (6) fruit formation, with maturation lasting approximately 15 months.

**4. Discussion**

insects for pollination.

activity in male phase.

Results of the reproductive system indicate that self-pollination is unlikely because *M. saccifera* is a monoecious palm with unisexual flowers and anthesis time of the male and female phase do not match due to the type of protogynous dicogamia present. No fruit formation occurs via apomixis (**Table 1**), and the probability of geitonogamy is low due to the non-coincidence of two inflorescences in anthesis in the same individual; however, if the two inflorescences in anthesis manage to coincide, fertilization is prevented by the self-incompatibility found. And because the anthers and stigma remain covered avoiding pollen dispersion by wind anemophilia is unlikely to occur. Consequently, the non-presence of apomixis, the non-occurrence of self-pollination, the degree of self-incompatibility found and the fact that no wind pollination occurs, determine that *M. saccifera* should be considered a xenogamous palm, dependent on

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

http://dx.doi.org/10.5772/intechopen.76073

31

Cross pollination apparently works well in *M. saccifera* as open-pollination and allogamy testing showed 62 and 53% of fruit formation, respectively (**Table 1**), such efficiency indicates the importance of pollinators as carriers of pollen between individuals in the population, given the obligatory xenogamy of the palm. Cross-pollination is efficient despite that the pollination mechanism that occurs in *M. saccifera* is atypical to what usually happens in palms, in which visitors insects have full access to the flowers. In *M. saccifera* the peduncular bract keeps hidden and isolated male and female flowers, and only *Mystrops cercus* can cross the peduncular bract when the stigmas are receptive, the rest of the insects that are attracted at this time cannot enter; they do it in male phase when receptivity has passed and focus their

The fact that the peduncular bract does not open preventing access to a group of insects, mainly larger than 2 mm, becomes a selective filter that limits free access of insects into the inflorescence at a critical moment in the reproduction of the palm. Large insects like bees (Apidae) or with larger sizes cannot cross the peduncular bract, while small insects or smaller

The peduncular bract acts as a barrier or selective filter that restricts access of large insects to female flowers at the most important time for fertilization, but is not the only one: those insects that may cross the first barrier are immediately faced with a second barrier and therefore a second filter, this time generated by the petals of the female flowers which do not open completely either and only two small slots of 1–2 mm are the space between the petals that insects may use to enter the flower, access the stigma and deposit the needed pollen to fertilize each flower of the inflorescence. Consequently, the bract that covers the inflorescence and the petals of the female flowers become two barriers that act as filters for selecting by size the type of pollinator in *M. saccifera* and only *Mystrops cercus* is the insect that passes through the

Keep flowers hidden at the time of anthesis is a rare phenomenon in palms, presented only in other species of the genus *Pholydostachys* (personal observation), which have a fibrous small peduncular bract similar to *M. saccifera* and insects must pass through at the time of anthesis in a yet unknown mechanism. The closest thing to the selective filter imposed by the

than 2 mm, are the only ones who can access the flowers when they are receptive.

filters and access the stigma that are hidden for the rest of the visitors.

#### **3.4. Reproductive system**

Our controlled pollination treatments revealed that *M. saccifera* is strictly xenogamous. Thus, neither of the apomixis or auto-pollination treatments led to fruit formation. In contrast, with the open pollination and allogamy treatments the percentage of fruits (**Table 1**).

#### **3.5. Floral visitors**

The inflorescences of *M. saccifera* were visited by eight species of insects (**Table 2**), with a mean abundance of 716 ± 60 visitors per inflorescence (n = 10). *Mystrosp cercus* was the most abundant floral visitor and the only insect able to cross the two barriers imposed by the fibrous peduncular bract and the petals of the female flowers. It had the highest pollinator relative importance value, representing 99.9% of the pollination in *M. saccifera*. In contrast, the remaining species were occasional visitors with low abundances and were only present in the male-phase of the inflorescences. Among these frequent species were *Amazoncharis* sp.1 and *Xanthogypus* sp.1 (Staphylinidae).


**Table 1.** Percentage of fruits formed in *Manicaria saccifera* after four controlled pollination tests.


AB: abundance in female phase, PTC: pollen-transport capacity in female phase, PTE: pollen-transport efficiency, C: constancy in the phase female, F: fidelity, PIV: pollinator importance value and PRI: pollinator relative importance value

**Table 2.** Role of visitor in the pollination of *Manicaria saccifera*.

## **4. Discussion**

by a change of color from white to brown to black; (6) fruit formation, with maturation last-

Our controlled pollination treatments revealed that *M. saccifera* is strictly xenogamous. Thus, neither of the apomixis or auto-pollination treatments led to fruit formation. In contrast, with

The inflorescences of *M. saccifera* were visited by eight species of insects (**Table 2**), with a mean abundance of 716 ± 60 visitors per inflorescence (n = 10). *Mystrosp cercus* was the most abundant floral visitor and the only insect able to cross the two barriers imposed by the fibrous peduncular bract and the petals of the female flowers. It had the highest pollinator relative importance value, representing 99.9% of the pollination in *M. saccifera*. In contrast, the remaining species were occasional visitors with low abundances and were only present in the male-phase of the inflorescences. Among these frequent species were *Amazoncharis* sp.1 and *Xanthogypus* sp.1 (Staphylinidae).

**Floral visitors AB CTP ETP C F IVIP IRIP** *Mystrops cercus* 560 1564 569 1 1 498,352,960 99.9 *Mystrops erviki* 12 156 234 0.25 1 109,512 0.021 *Trigona fulviventris* 23 669 245 0.05 0.5 94245.3 0.018 *Trigona ferricauda* 12 456 123 0.05 0.5 16826.4 0.003 *Derelomini* sp.1 12 123 12 0.25 0.33 1461.24 0.0002 *Xanthopygus* 34 167 32 1 0.33 59959.68 0.012 *Atheta* sp.1 12 23 12 1 0.33 1092.96 0.0002 *Amazoncharis* sp.1 123 12 12 1 0.33 5844.96 0.001 Total 498,641,903 100

**Table 1.** Percentage of fruits formed in *Manicaria saccifera* after four controlled pollination tests.

AB: abundance in female phase, PTC: pollen-transport capacity in female phase, PTE: pollen-transport efficiency, C: constancy in the phase female, F: fidelity, PIV: pollinator importance value and PRI: pollinator relative importance value

**Table 2.** Role of visitor in the pollination of *Manicaria saccifera*.

the open pollination and allogamy treatments the percentage of fruits (**Table 1**).

**Treatments Nº palms/ N°flowers Nº fruits/% set fruits**

Apomixis(A) 5/410 0/0 Open pollination (OP) 5/410 225/62 Auto-pollination (AP) 5/410 0/0 Allogamy (AL) 5/410 220/53

ing approximately 15 months.

**3.4. Reproductive system**

**3.5. Floral visitors**

30 Pollination in Plants

Results of the reproductive system indicate that self-pollination is unlikely because *M. saccifera* is a monoecious palm with unisexual flowers and anthesis time of the male and female phase do not match due to the type of protogynous dicogamia present. No fruit formation occurs via apomixis (**Table 1**), and the probability of geitonogamy is low due to the non-coincidence of two inflorescences in anthesis in the same individual; however, if the two inflorescences in anthesis manage to coincide, fertilization is prevented by the self-incompatibility found. And because the anthers and stigma remain covered avoiding pollen dispersion by wind anemophilia is unlikely to occur. Consequently, the non-presence of apomixis, the non-occurrence of self-pollination, the degree of self-incompatibility found and the fact that no wind pollination occurs, determine that *M. saccifera* should be considered a xenogamous palm, dependent on insects for pollination.

Cross pollination apparently works well in *M. saccifera* as open-pollination and allogamy testing showed 62 and 53% of fruit formation, respectively (**Table 1**), such efficiency indicates the importance of pollinators as carriers of pollen between individuals in the population, given the obligatory xenogamy of the palm. Cross-pollination is efficient despite that the pollination mechanism that occurs in *M. saccifera* is atypical to what usually happens in palms, in which visitors insects have full access to the flowers. In *M. saccifera* the peduncular bract keeps hidden and isolated male and female flowers, and only *Mystrops cercus* can cross the peduncular bract when the stigmas are receptive, the rest of the insects that are attracted at this time cannot enter; they do it in male phase when receptivity has passed and focus their activity in male phase.

The fact that the peduncular bract does not open preventing access to a group of insects, mainly larger than 2 mm, becomes a selective filter that limits free access of insects into the inflorescence at a critical moment in the reproduction of the palm. Large insects like bees (Apidae) or with larger sizes cannot cross the peduncular bract, while small insects or smaller than 2 mm, are the only ones who can access the flowers when they are receptive.

The peduncular bract acts as a barrier or selective filter that restricts access of large insects to female flowers at the most important time for fertilization, but is not the only one: those insects that may cross the first barrier are immediately faced with a second barrier and therefore a second filter, this time generated by the petals of the female flowers which do not open completely either and only two small slots of 1–2 mm are the space between the petals that insects may use to enter the flower, access the stigma and deposit the needed pollen to fertilize each flower of the inflorescence. Consequently, the bract that covers the inflorescence and the petals of the female flowers become two barriers that act as filters for selecting by size the type of pollinator in *M. saccifera* and only *Mystrops cercus* is the insect that passes through the filters and access the stigma that are hidden for the rest of the visitors.

Keep flowers hidden at the time of anthesis is a rare phenomenon in palms, presented only in other species of the genus *Pholydostachys* (personal observation), which have a fibrous small peduncular bract similar to *M. saccifera* and insects must pass through at the time of anthesis in a yet unknown mechanism. The closest thing to the selective filter imposed by the peduncular bract occurs in some palms species of the genus *Attalea* in which as at the time of anthesis the peduncle bract leaves only a small slit that acts momentarily as a filter, but over time the bract exposes most of the flowers and insect access is complete [9].

In angiosperms the mechanism of pollination where flowers are not exposed and the androecium and gynoecium are hidden at the time of fertilization is rare but still occurs in several plant families and this type of pollination is called cleistogamy [27]. Cleistogamous plants are usually hermaphroditic, self-compatible and the release of pollen and stigmatic receptivity occurs at the same time therefore self-pollination and autogamy are predominant [28], freeing themselves from dependence on pollinators. Although *M. saccifera* reproductive structures remain hidden, cleistogamy is unlikely because the palm has unisexual flowers, with temporal phase separation and high values of self-incompatibility, therefore highly dependent on insects to fertilize the flowers.

The selective filter imposed by the peduncular bract of *M. saccifera* really influences the access of floral visitors as evidenced by the fact that the diversity of insects found within the inflorescence was low (only eight species). Comparing the rate of visitors to palms with characteristics similar to *M. saccifera* as size, rewards offered, type and location in the forest, these are visited by great diversity of visitors; for example, in *Oenocarpus batau*a [10] reported 81 species; *Phytelephas macrocarpa* is visited by 45 [29], as *P. seemanii*; and *Astrocaryum mexicanum* is visited on average by 35 species [30].

Therefore, the mechanism of isolating the reproductive structures and to have selective barriers can bring advantages and disadvantages for *M. saccifera*. Among the disadvantages, access by insects is limited and therefore the options of species that can act as pollinators is reduced, which may limit pollen flow with consequent pollination problem [31]. It has often been suggested that plants that display their flowers can attract more visitors and potential pollinators than those with few exposed flowers [32] and thus the pollination probabilities increase. Moreover, the filters presented in *M. saccifera* can bring advantage in the fact that there is a real selection of insects that prevents the entry of those who have little part in pollination, and thus an antagonist interaction with the palm, generating actions that directly or indirectly affect the reproductive success of the species. This phenomenon of insects that are not involved in pollination of palms is very common and widely reported for other species, where only a small number of visitors is actively involved in pollination and most visitors focus their activities exclusively on male stage or male flowers [9, 10, 33–35].

The low diversity of visitors generated by the selective filters in *M. saccifera* is balanced by an intimate and exclusive association of the palm with its main pollinator *Mystrops cercus*, which has easy access to the inflorescence through the selective filters and deposits pollen with efficiency values reaching 99% of the pollen transported and used for fertilization of the flowers of the palm (**Table 2**), ensuring fertilization of flowers and thus a constant fruit production. The pollination mechanism present in *M. saccifera* with *M. cercus* as main pollinator is summarized in a general model of pollination (**Figure 5**).

establishment of a relationship of mutual dependence or obligatory mutualism between the palm and its pollinator. In that mutualism, pollinators need the palm they host for food, find a mate, make their life cycle; while for the palm the benefit of having a close relationship with the pollinator ensuring their loyalty, perseverance and efficiency in pollen trans-

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

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33

fer necessary to achieve reproduction.

**Figure 5.** Pollination of *Manicaria saccifera* by *Mystrops cercus.*

Because *Mystrops cercus* depends *M. saccifera* flowers as an ideal environment for feeding with pollen, protection (isolated flowers) and an ideal microenvironment to develop part of their life cycle due to the thermogenesis of the flowers, such association entails the Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion http://dx.doi.org/10.5772/intechopen.76073 33

**Figure 5.** Pollination of *Manicaria saccifera* by *Mystrops cercus.*

peduncular bract occurs in some palms species of the genus *Attalea* in which as at the time of anthesis the peduncle bract leaves only a small slit that acts momentarily as a filter, but over

In angiosperms the mechanism of pollination where flowers are not exposed and the androecium and gynoecium are hidden at the time of fertilization is rare but still occurs in several plant families and this type of pollination is called cleistogamy [27]. Cleistogamous plants are usually hermaphroditic, self-compatible and the release of pollen and stigmatic receptivity occurs at the same time therefore self-pollination and autogamy are predominant [28], freeing themselves from dependence on pollinators. Although *M. saccifera* reproductive structures remain hidden, cleistogamy is unlikely because the palm has unisexual flowers, with temporal phase separation and high values of self-incompatibility, therefore highly dependent on

The selective filter imposed by the peduncular bract of *M. saccifera* really influences the access of floral visitors as evidenced by the fact that the diversity of insects found within the inflorescence was low (only eight species). Comparing the rate of visitors to palms with characteristics similar to *M. saccifera* as size, rewards offered, type and location in the forest, these are visited by great diversity of visitors; for example, in *Oenocarpus batau*a [10] reported 81 species; *Phytelephas macrocarpa* is visited by 45 [29], as *P. seemanii*; and *Astrocaryum mexicanum*

Therefore, the mechanism of isolating the reproductive structures and to have selective barriers can bring advantages and disadvantages for *M. saccifera*. Among the disadvantages, access by insects is limited and therefore the options of species that can act as pollinators is reduced, which may limit pollen flow with consequent pollination problem [31]. It has often been suggested that plants that display their flowers can attract more visitors and potential pollinators than those with few exposed flowers [32] and thus the pollination probabilities increase. Moreover, the filters presented in *M. saccifera* can bring advantage in the fact that there is a real selection of insects that prevents the entry of those who have little part in pollination, and thus an antagonist interaction with the palm, generating actions that directly or indirectly affect the reproductive success of the species. This phenomenon of insects that are not involved in pollination of palms is very common and widely reported for other species, where only a small number of visitors is actively involved in pollination and most visitors focus their

The low diversity of visitors generated by the selective filters in *M. saccifera* is balanced by an intimate and exclusive association of the palm with its main pollinator *Mystrops cercus*, which has easy access to the inflorescence through the selective filters and deposits pollen with efficiency values reaching 99% of the pollen transported and used for fertilization of the flowers of the palm (**Table 2**), ensuring fertilization of flowers and thus a constant fruit production. The pollination mechanism present in *M. saccifera* with *M. cercus* as main pollinator is sum-

Because *Mystrops cercus* depends *M. saccifera* flowers as an ideal environment for feeding with pollen, protection (isolated flowers) and an ideal microenvironment to develop part of their life cycle due to the thermogenesis of the flowers, such association entails the

activities exclusively on male stage or male flowers [9, 10, 33–35].

marized in a general model of pollination (**Figure 5**).

time the bract exposes most of the flowers and insect access is complete [9].

insects to fertilize the flowers.

32 Pollination in Plants

is visited on average by 35 species [30].

establishment of a relationship of mutual dependence or obligatory mutualism between the palm and its pollinator. In that mutualism, pollinators need the palm they host for food, find a mate, make their life cycle; while for the palm the benefit of having a close relationship with the pollinator ensuring their loyalty, perseverance and efficiency in pollen transfer necessary to achieve reproduction.

Obligate mutualisms *Mystrops* - *Palmae* are more and more recognized, sometimes in oneto-one relations as in *Attalea allenii* [9, 36, 37]; or a *Mystrops* species associated with several species of the same genus of palms as with *Mystrops rotundula* and *Mystrops pulcra*, which pollinate seven species of the genus *Ceroxylon* [36]. The mutual dependency between *Mystrops cercus* and *M. saccifera* ensures reproductive success of the palm and the permanence of pollinators through the coordination of several mechanisms of association including attraction, maintenance and fertilization of flowers by their primary pollinator *Mystrops cercus*, this leads us to suggest that a high degree of specialization exists between *Mystrops cercus* and *Manicaria saccifera*, which has also been reported in other palm species pollinated by *Mystrops* species [1, 7–9, 21, 36, 38]. Likewise, two additional evidences can support the degree of specialization found and suggested in this paper: specificity and distribution of interaction.

Niño (Staphilinidae) for identification of insect specimens; to Eva Ledezma for help in the

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

1 Maestría en Bosques y Conservación Ambiental, Universidad Nacional de Colombia, Sede

2 Departamento de Biología, Universidad Tecnológica del Chocó, Quibdó, Colombia

3 Grupo de Investigación en Bioprospección y Conservación Biológica, Departamento de Ciencias Básicas, Programa de Biología, Universidad de La Salle, Bogotá, Colombia

[1] Henderson A. Evolution and Ecology of Palms. The New York Botanical Garden Press;

[2] Araújo FR, Lopes MA. Diversity of use and local knowledge of palms (Arecaceae) in

[3] Macía MJ, Armesilla PJ, Cámara-Leret R, Paniagua-Zambrano N, Villalba S, Balslev H, Pardo-de Santayana M. Palm uses in Northwestern South America: A quantitative

[4] Bernal R, Torres C, García N, Isaza C, Navarro J, Vallejo MI, Galeano G, Balslev H. Palm

[5] Balick MJ, Beck HT. Useful Palms of the World: A Synoptic Bibliography. New York:

[6] Barfod AS, Hagen M, Borchsenius F. Twenty-five years of progress in understanding pollination mechanisms in palms (Arecaceae). Annals of Botany. 2011;**108**(8):1-14 [7] Henderson A. A review of pollination studies in the Palmae. The Botanical Review.

[8] Fava WS, Covre W, Sigrist MR. *Attalea phalerata* and *Bactris glaucescens* (Arecaceae, Arecoideae): Phenology and pollination ecology in the pantanal, Brazil. Flora. 2011;

[9] Núñez LA, Bernal R, Knudsen J. Diurnal palm pollination by Mytropine beetles: Is it

[10] Núñez LA, Rojas R. Biología reproductiva y ecología de la polinización de la palma milpesos *Oenocarpus bataua* en los *Andes colombianos*. Caldasia. 2008;**30**(1):99-122

weather-related? Plant Systematics and Evolution. 2005;**254**:149-171

eastern Amazonia. Biodiversity and Conservation. 2012;**21**:487-501

management in South America. The Botanical Review. 2011;**77**:607-646

review. The Botanical Review. 2011;**77**:462-570

Columbia University Press; 1990

1986;**52**:221-259

**206**:575-584

and Luis Alberto Núñez-Avellaneda3

http://dx.doi.org/10.5772/intechopen.76073

35

field, and to the communities of Kilometer 7 for their hospitality and collaboration.

\*, Danny Mosquera Flórez2

\*Address all correspondence to: jccopetem@unal.edu.co

**Author details**

Juan Carlos Copete1

Medellín, Colombia

**References**

2002

Regarding specificity [36] conducted a comparative study of *Mystrops* species in at least 80 species of palms including *Mystrops* species visiting and pollinating flowers of palms found in the Chocó (*Attalea allenii*, *Attalea cuatrecasana*, *Oenocarpus bataua*, *O. minor*, *Socratea exhorriza*, *Wettinia quinaria* and *M. saccifera*), and found that species of pollinators are not shared: each palm has its own association with a particular *Mystrops* species. In terms of distribution, we have found *Mystrops cercus* in five additional locations to our study area.

Participation of the genus *Mystrops* in the pollination of palms has been amply demonstrated, whether acting as principal pollinators, secondary or co-pollinators [9, 30, 34]. However, to the extent that detailed reproduction studies of tropical palms increase, species of the genus *Mystrop*s are showing greater relevance and importance as pollinators of palms: one or more species of *Mystrops* are the most important pollinators, and in some cases, are the solely responsible for the movement of pollen in a particular palm species, e.g., *Mystrops* sp. nov. 1 in *Attalea allenii* and *Mystrops sp*. nov. 2 in *Wettinia quinaria* [9], *Mystrop*s sp. in *Mauritia flexuosa* [37], *Mystrops* sp.15 in *Wettinia praemorsa*, and *Mystrops* sp. nov. 22 in *Cryosophila kalbreyeri* (Núñez [36]) *Mystrops* in *Wettinia kalbreyeri* [39].

In synthesis, *M. saccifera* presents a specialized pollination system with morphological barriers that blocks access of floral visitors when the stigmas are receptive, and is closely associated with *Mystrops cercus*, a kind of small beetle that, in an exclusive way, visits and pollinates the flowers of this important palm with morphological constraints. The most striking examples of highly specific mutualism relationships and close interdependence between plants and pollinating insects are given in cases where the flower morphology limits the visitor access to reproductive or floral rewards structures and consequently the possibilities of pollination are minimal, *M. saccifera* is a clear example.

We recommend further studies, mainly focused on evaluating features like the changes in time and if this mutual dependence is maintained throughout the disjunctive distribution of the palm.

## **Acknowledgements**

Our gratitude goes to Gloria Galeano, Rodrigo Bernal, Rodrigo Cámara and Henrik Balslev for revising the manuscript; to the entomologists Nazly Wilchéz (Himenóptera) and Alfredo Niño (Staphilinidae) for identification of insect specimens; to Eva Ledezma for help in the field, and to the communities of Kilometer 7 for their hospitality and collaboration.

## **Author details**

Obligate mutualisms *Mystrops* - *Palmae* are more and more recognized, sometimes in oneto-one relations as in *Attalea allenii* [9, 36, 37]; or a *Mystrops* species associated with several species of the same genus of palms as with *Mystrops rotundula* and *Mystrops pulcra*, which pollinate seven species of the genus *Ceroxylon* [36]. The mutual dependency between *Mystrops cercus* and *M. saccifera* ensures reproductive success of the palm and the permanence of pollinators through the coordination of several mechanisms of association including attraction, maintenance and fertilization of flowers by their primary pollinator *Mystrops cercus*, this leads us to suggest that a high degree of specialization exists between *Mystrops cercus* and *Manicaria saccifera*, which has also been reported in other palm species pollinated by *Mystrops* species [1, 7–9, 21, 36, 38]. Likewise, two additional evidences can support the degree of specialization

Regarding specificity [36] conducted a comparative study of *Mystrops* species in at least 80 species of palms including *Mystrops* species visiting and pollinating flowers of palms found in the Chocó (*Attalea allenii*, *Attalea cuatrecasana*, *Oenocarpus bataua*, *O. minor*, *Socratea exhorriza*, *Wettinia quinaria* and *M. saccifera*), and found that species of pollinators are not shared: each palm has its own association with a particular *Mystrops* species. In terms of distribution, we

Participation of the genus *Mystrops* in the pollination of palms has been amply demonstrated, whether acting as principal pollinators, secondary or co-pollinators [9, 30, 34]. However, to the extent that detailed reproduction studies of tropical palms increase, species of the genus *Mystrop*s are showing greater relevance and importance as pollinators of palms: one or more species of *Mystrops* are the most important pollinators, and in some cases, are the solely responsible for the movement of pollen in a particular palm species, e.g., *Mystrops* sp. nov. 1 in *Attalea allenii* and *Mystrops sp*. nov. 2 in *Wettinia quinaria* [9], *Mystrop*s sp. in *Mauritia flexuosa* [37], *Mystrops* sp.15 in *Wettinia praemorsa*, and *Mystrops* sp. nov. 22 in *Cryosophila kalbreyeri*

In synthesis, *M. saccifera* presents a specialized pollination system with morphological barriers that blocks access of floral visitors when the stigmas are receptive, and is closely associated with *Mystrops cercus*, a kind of small beetle that, in an exclusive way, visits and pollinates the flowers of this important palm with morphological constraints. The most striking examples of highly specific mutualism relationships and close interdependence between plants and pollinating insects are given in cases where the flower morphology limits the visitor access to reproductive or floral rewards structures and consequently the possibilities of pollination are

We recommend further studies, mainly focused on evaluating features like the changes in time and if this mutual dependence is maintained throughout the disjunctive distribution of

Our gratitude goes to Gloria Galeano, Rodrigo Bernal, Rodrigo Cámara and Henrik Balslev for revising the manuscript; to the entomologists Nazly Wilchéz (Himenóptera) and Alfredo

found and suggested in this paper: specificity and distribution of interaction.

have found *Mystrops cercus* in five additional locations to our study area.

(Núñez [36]) *Mystrops* in *Wettinia kalbreyeri* [39].

minimal, *M. saccifera* is a clear example.

the palm.

34 Pollination in Plants

**Acknowledgements**

Juan Carlos Copete1 \*, Danny Mosquera Flórez2 and Luis Alberto Núñez-Avellaneda3

\*Address all correspondence to: jccopetem@unal.edu.co

1 Maestría en Bosques y Conservación Ambiental, Universidad Nacional de Colombia, Sede Medellín, Colombia

2 Departamento de Biología, Universidad Tecnológica del Chocó, Quibdó, Colombia

3 Grupo de Investigación en Bioprospección y Conservación Biológica, Departamento de Ciencias Básicas, Programa de Biología, Universidad de La Salle, Bogotá, Colombia

## **References**


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[26] Ruiz-Zapata T, Arroyo MTK. Plant reproductive ecology of a secondary deciduous trop-

Pollination Ecology of the *Manicaria saccifera* (ARECACEAE): A Rare Case of Pollinator Exclusion

http://dx.doi.org/10.5772/intechopen.76073

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[27] Lord EM. Cleistogamy: A tool for the study of floral morphogenesis, function and evolu-

[28] Culley TM, Klooster M. The cleistogamous breeding system: A review of its frequency, evolution and ecology in angiosperms. The Botanical Review. 2007;**73**(1):1-30

[29] Bernal R, Ervik F. Floral biology and pollination of the dioecious palm *Phytelephas seemannii* in Colombia: An adaptation to staphylinid beetles. Biotropica. 1996;**28**:682-696

[30] Búrquez A, Sarukhán J, Pedraza A. Floral biology of a primary rain forest palm, *Astrocaryum mexicanum*. Botanical Journal of the Linnean Society. 1987;**94**:407-419 [31] Knight TM, Steets JA, Vamosi JC, Mazer SJ, Burd M, Campbell DR, Dudash RJ, Ashman TL. Pollen limitation of plant reproduction: Pattern and process. Annual Review of

[32] Feldman TS. Pollinator aggregative and functional responses to flower density: Does pollinator response to patches of plants accelerate at low-densities? Oikos. 2006;**115**:128-140

[33] Anderson AB, Overal W, Henderson A. Pollination ecology of a forest dominant palm

[34] Ervik F. Comparative studies of pollination biology in neotropical palms [PhD thesis].

[35] Küchmeister H, Silberbauer-Gottsberger I, Gottsberger G. Flowering, pollination, nectar standing crop, and nectaries of *Euterpe precatoria* (Arecaceae), an Amazonian rain forest

[36] Núñez LA. Patrones de asociación entre polinizadores y palmas en Colombia. Con énfasis en palmas de importancia económica [Tesis Doctoral]; Universidad Nacional de

[37] Núñez LA, Carreño J. Biología reproductiva de *Mauritia flexuosa* en Casanare, Orinoquia colombiana. In: Lasso CA, Rial A, González V, editors. VII: Morichales y Cananguchales de la Orinoquia y Amazonia (Colombia-Venezuela), Serie Editorial Recursos Hidrobiológicos y pesqueros Continentales de Colombia. Bogotá D.C. Colombia: Instituto de

Investigación de Recursos Biológicos Alexander von Humboldt; 2015. p. 450

[38] Silberbauer-Gottsbergeri I. Pollination and evolution in palms. Phyton. 1990;**30**:213-233 [39] Lara CE, Diez MC, Restrepo Z, Núñez LA, Moreno F. Flowering phenology and flower visitors of the Macana palm *Wettinia kalbreyeri* (Arecaceae) in an Andean montane forest.

(*Orbignya phalerata* Mart.) in Northern Brazil. Biotropica. 1998;**20**:192-205

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Colombia; 2014

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[26] Ruiz-Zapata T, Arroyo MTK. Plant reproductive ecology of a secondary deciduous tropical forest in Venezuela. Biotropica. 1978;**10**:221-230

[11] Núñez LA, Isaza C, Galeano G. Ecología de la polinización de tres especies de Oenocarpus (Arecaceae) simpátricas en la Amazonia Colombiana. International Journal of Tropical

[12] Henderson A, Galeano G, Bernal R. Field Guide to the Palms of the Americas. Vol. 144.

[13] Galeano G, Bernal R. Palmas de Colombia. Guía de Campo. Editorial Universidad Nacional de Colombia. Instituto de Ciencias Naturales. Facultad de Ciencias. Bogotá,

[14] Balick MJ. Jessenia y Oenocarpus: Palmas aceiteras neotropicales dignas de ser domesticadas. In: Estudio FAO Producción y Protección Vegetal. Vol. 88. Roma: FAO; 1982.

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[16] Ledezma E, Galeano G. Etnobotánica del Cabecinegro (*Manicaria saccifera*) en las tierras bajas del Pacífico Colombiano. Montpellier-Francia: Simposio Internacional de Palmas;

[17] Jiménez-Ortega AM, Ramos YA, García- Cossio F, Ríos-Hurtado A, Asprilla-Perea J. El Chocó: Una fuente de oportunidades comerciales, a partir del conocimiento, valoración y manejo de su biodiversidad. Revista Institucional. Universidad Tecnológica del Chocó

[18] Hoyos J, Braun A. Palmas en Venezuela: Autóctonas y Exóticas Caracas. La Salle: Socie-

[19] Wilbert J. *Manicaria saccifera* and its culture significance among the Warao Indians of Venezuela. In: Botanical Museum Leaflets. Vol. 24(10). Cambridge, Massachusetts:

[21] Ervik F. Notes on the phenology and pollination of the dioecious palms *Mauritia flexuosa* (Calamoideae) and *Aphandra natalia* in Ecuador (Phytelephantoideae). In: Barthlott W, Naumann C, Schmidt-Loske C, Schuchmann K, editors. Animal-Plant Interactions in

[23] Bencke C, Morellato P. Comparação de dois métodos de avaliação da fenología de plantas, sua interpretação representação. Revista Brasileira de Botânica. 2002;**25**:269-275 [24] Kearns CA, Inouye DW. Techniques for Pollination. Biology University Press of

[25] Dafni A. Pollination Ecology. A Practical Approach. Oxford: Oxford University Press;

[20] Wessels Boer JG. The Indigenous Palms of Suriname. Leiden: E. J. Brill; 1995

[22] Holdridge LR. Ecología Basada en Zonas de Vida. San José: IICA; 1978

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36 Pollination in Plants

2010

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**Chapter 4**

**Provisional chapter**

**Challenges in Cocoa Pollination: The Case of Côte**

**Challenges in Cocoa Pollination: The Case of Côte** 

DOI: 10.5772/intechopen.75361

Cocoa (*Theobroma cacao* L.) is mainly pollinated by ceratopogonid midges (*Forcipomyia* spp.). However, other insect species will also pollinate cocoa flowers when these midges are scarce. In Côte d'Ivoire, inadequate pest control practices (insecticide spraying, mostly against the mirids *Distantiella theobromae* and *Sahlbergella singularis*) and landscape degradation as a result of deforestation and cocoa monoculture, have decreased overall pollinator population levels and, as a result, pollination services to cocoa trees. The current low average Ivorian cocoa yield of 538 kg per ha (in 2016) is the result of global agricultural mismanagement (deteriorated soils, lack of fertilizers, inadequate or absent pest control, absence of shade trees and intercrops). However, there is also an evidence of a pollination gap that could cause low cocoa yield. More research is needed to understand: (i) which agro-ecological efforts to enhance cocoa pollination can improve yield, and (ii) which strategies are effective in enhancing cocoa pollination. In this chapter, we briefly describe the cocoa sector. Next, the cocoa flower and pollinator biology and phenology are presented, followed by an overview of current environmental and management constraints to cocoa pollination in the context of Côte d'Ivoire, the largest cocoa producer in the world. We conclude with explor-

ing possibilities to enhance pollination in the Ivorian small-scale cocoa sector.

**Keywords:** cocoa, pollination, *Theobroma cacao*, fructification, cherelle wilt, *Forcipomyia*,

Non-bee insect pollinators play a significant role in global crop production [1]. Cocoa (*Theobroma cacao* L.) is one of the 13 most important commercial crops in the world. It entirely

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**d'Ivoire**

**d'Ivoire**

Gregor Claus, Wouter Vanhove,

Gregor Claus, Wouter Vanhove,

http://dx.doi.org/10.5772/intechopen.75361

**Abstract**

**1. Introduction**

Patrick Van Damme and Guy Smagghe

Patrick Van Damme and Guy Smagghe

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

phenology, pesticides, IPM, Côte d'Ivoire

#### **Challenges in Cocoa Pollination: The Case of Côte d'Ivoire Challenges in Cocoa Pollination: The Case of Côte d'Ivoire**

DOI: 10.5772/intechopen.75361

Gregor Claus, Wouter Vanhove, Patrick Van Damme and Guy Smagghe Gregor Claus, Wouter Vanhove, Patrick Van Damme and Guy Smagghe

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75361

#### **Abstract**

Cocoa (*Theobroma cacao* L.) is mainly pollinated by ceratopogonid midges (*Forcipomyia* spp.). However, other insect species will also pollinate cocoa flowers when these midges are scarce. In Côte d'Ivoire, inadequate pest control practices (insecticide spraying, mostly against the mirids *Distantiella theobromae* and *Sahlbergella singularis*) and landscape degradation as a result of deforestation and cocoa monoculture, have decreased overall pollinator population levels and, as a result, pollination services to cocoa trees. The current low average Ivorian cocoa yield of 538 kg per ha (in 2016) is the result of global agricultural mismanagement (deteriorated soils, lack of fertilizers, inadequate or absent pest control, absence of shade trees and intercrops). However, there is also an evidence of a pollination gap that could cause low cocoa yield. More research is needed to understand: (i) which agro-ecological efforts to enhance cocoa pollination can improve yield, and (ii) which strategies are effective in enhancing cocoa pollination. In this chapter, we briefly describe the cocoa sector. Next, the cocoa flower and pollinator biology and phenology are presented, followed by an overview of current environmental and management constraints to cocoa pollination in the context of Côte d'Ivoire, the largest cocoa producer in the world. We conclude with exploring possibilities to enhance pollination in the Ivorian small-scale cocoa sector.

**Keywords:** cocoa, pollination, *Theobroma cacao*, fructification, cherelle wilt, *Forcipomyia*, phenology, pesticides, IPM, Côte d'Ivoire

#### **1. Introduction**

Non-bee insect pollinators play a significant role in global crop production [1]. Cocoa (*Theobroma cacao* L.) is one of the 13 most important commercial crops in the world. It entirely

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

depends on insects for pollination and successful production [2]. In cocoa, pollination is almost exclusively performed by ceratopogonid midges (Order Diptera) from genus *Forcipomyia* [3, 4]. In 2016, global cocoa production was 4.472 million tons of dry beans, of which 2.655 million tons (59%) were produced in West Africa and 1.472 million tons (33%) in Côte d'Ivoire [5]. In 2016, global average cocoa yield was 438 kg dry beans per ha and per year (480 kg per ha in West Africa), whereas it was shown in research stations that cocoa bean yield could attain up to 2000 kg per ha and per year [6].

cultivation area not larger than 3 ha) [5, 17]. As such, cocoa provides a livelihood to around 4.5 million farming families. Globally, 14 million people work in cocoa production [17]. In 2013, the total chocolate confectionary retail consumption had a value of 109,992 million USD [20]. Cocoa is produced on around 10 million ha, which is just 0.7% of the total global arable land, but 7% of the global permanent crop area. As a result, cocoa cultivation, and particularly cocoa agroforestry systems, play an important role in carbon sequestration and consequently

Challenges in Cocoa Pollination: The Case of Côte d'Ivoire

http://dx.doi.org/10.5772/intechopen.75361

41

Côte d'Ivoire comprises the main cocoa-producing region in the world. In 2016, the country provided one third (1.472 million tons) of global cocoa supplies on 2.851 million ha of land. Average cocoa yield in Côte d'Ivoire was thus 516 kg/ha of dry cocoa beans, which is slightly below the global average yield of 538 kg/ha for that year [5]. In Côte d'Ivoire, cocoa is exclusively produced by around 1,000,000 small-scale farmers, each cultivating on around 2–3 ha [22]. These smallholders operate in a difficult context. Between 2000 and 2011, Côte d'Ivoire was generally considered a failed state with frequent occurrences of violent conflicts, where cocoa tax revenues were often used to fuel the conflicts [23, 24]. Moreover, the cocoa production in Côte d'Ivoire has often been linked to child slavery on plantations [23]. As in most cocoa-producing regions, fluctuating prices (between 1500 and 3500 USD per ton in the period 2011–2018, see http://www.nasdaq.com/markets/cocoa.aspx) affect Ivorian cocoa smallholders because in a situation with volatile prices, it is difficult to make informed choices

Pollination intensity and fruit set largely determine cocoa yield [26]. If natural pollination is limiting cocoa yield, then enhancing pollinator population levels should result in increased

Cocoa flowers are hermaphrodite. They are produced on the trees' trunks and branches (cauliflory). After 2–3 years, so-called flower cushions, i.e., thickened flower-producing leaf axils, are formed. Every cushion bears up to 50 flowers per flowering season. There are two flowering seasons per year, which thus yields 100 flowers per year. The pentamerous flower is about 15 mm in diameter. A petal consists of a pouch—which conceals the anthers—and a wide tip. The function of the latter tip is unknown, but it does not specifically attract pollinators [27]. A particular aspect of cocoa flowers is the outer whorl of purple staminodes around the style. Right after anthesis, these staminodes align parallel to the style (**Figure 1**). Pollinators move around on the inner side of the staminodes, thereby rubbing their pollen grain-carrying bodies against the style. On older flowers, staminodes are somewhat withered and flexed away

have important climate mitigation potential [21].

**2.2. The cocoa sector in Côte d'Ivoire**

on the "right" crop investments [25].

**3. Cocoa pollination**

*3.1.1. Biology*

fruit set and consequently yield.

**3.1. Biology and phenology of cocoa flowers and fruit set**

Cocoa yield remains under the latter potential level due to: (i) an inadequate cropping system mainly consisting of full-sun monocultures without shade, leading to soil erosion, nutrient depletion, water shortages, weed growth, and increased pest and disease outbreaks [7–11]; and (ii) inadequate pest and disease management [12, 13]; both leading to (iii) below-optimum pollinator population levels. Earlier research showed that increasing pollination, either manually [14] or indirectly by improving breeding opportunities for pollinating midges [14–16], had a significant impact on cocoa yield compared with normal agricultural practices control plots.

Over the past 50 years, cocoa demand has consistently increased annually by some 2.5% [17]. Demand continues to rise, particularly as a result of newly emerging chocolate markets such as China and India [18]. However, cocoa production levels have decreased by 3–5% over the past five years (compared to 2012 levels), leading to unstable prices because of market shortages. Cocoa production could expand through increasing global cocoa acreage (as it has always been done in the past). However, this is not a sustainable solution as it is mostly achieved at the cost of deforestation in tropical areas [19]. The other, more sustainable, approach is increasing productivity per ha. The latter strategy not only increases overall cocoa production without further deforestation, but can also increase income of cocoa farmers who nowadays often leave the cocoa production sector because of its low profitability [9].

In this chapter, we explore how cocoa farmers—besides by improving soil conditions, adequate pruning and integrated pest and disease management—can increase cocoa yield by increasing pollination intensity of their cocoa trees. We will first present the global cocoa sector and then focus on Côte d'Ivoire, the leading cocoa-producing country in the world. Next, we give background information on the biology and phenology of both cocoa flowers and pollinating midges (*Forcipomyia* spp.), followed by a discussion of pollinator-reducing factors (environmental and managerial) in Côte d'Ivoire. We conclude by discussing some options for relieving the constraints on cocoa pollination.

## **2. The cocoa crop sector**

#### **2.1. Global production and economic value**

Although global yearly cocoa production quantities are below those of other tropical commodity crops such as sugar cane, rice, soybean, oil palm, cassava or banana, it is a unique crop because more than 90% of its production comes from small-scale farmers (each with a cultivation area not larger than 3 ha) [5, 17]. As such, cocoa provides a livelihood to around 4.5 million farming families. Globally, 14 million people work in cocoa production [17]. In 2013, the total chocolate confectionary retail consumption had a value of 109,992 million USD [20]. Cocoa is produced on around 10 million ha, which is just 0.7% of the total global arable land, but 7% of the global permanent crop area. As a result, cocoa cultivation, and particularly cocoa agroforestry systems, play an important role in carbon sequestration and consequently have important climate mitigation potential [21].

## **2.2. The cocoa sector in Côte d'Ivoire**

depends on insects for pollination and successful production [2]. In cocoa, pollination is almost exclusively performed by ceratopogonid midges (Order Diptera) from genus *Forcipomyia* [3, 4]. In 2016, global cocoa production was 4.472 million tons of dry beans, of which 2.655 million tons (59%) were produced in West Africa and 1.472 million tons (33%) in Côte d'Ivoire [5]. In 2016, global average cocoa yield was 438 kg dry beans per ha and per year (480 kg per ha in West Africa), whereas it was shown in research stations that cocoa bean yield could attain

Cocoa yield remains under the latter potential level due to: (i) an inadequate cropping system mainly consisting of full-sun monocultures without shade, leading to soil erosion, nutrient depletion, water shortages, weed growth, and increased pest and disease outbreaks [7–11]; and (ii) inadequate pest and disease management [12, 13]; both leading to (iii) below-optimum pollinator population levels. Earlier research showed that increasing pollination, either manually [14] or indirectly by improving breeding opportunities for pollinating midges [14–16], had a significant impact on cocoa yield compared with normal agricultural practices

Over the past 50 years, cocoa demand has consistently increased annually by some 2.5% [17]. Demand continues to rise, particularly as a result of newly emerging chocolate markets such as China and India [18]. However, cocoa production levels have decreased by 3–5% over the past five years (compared to 2012 levels), leading to unstable prices because of market shortages. Cocoa production could expand through increasing global cocoa acreage (as it has always been done in the past). However, this is not a sustainable solution as it is mostly achieved at the cost of deforestation in tropical areas [19]. The other, more sustainable, approach is increasing productivity per ha. The latter strategy not only increases overall cocoa production without further deforestation, but can also increase income of cocoa farmers who nowadays often leave the cocoa production sector because of its low profit-

In this chapter, we explore how cocoa farmers—besides by improving soil conditions, adequate pruning and integrated pest and disease management—can increase cocoa yield by increasing pollination intensity of their cocoa trees. We will first present the global cocoa sector and then focus on Côte d'Ivoire, the leading cocoa-producing country in the world. Next, we give background information on the biology and phenology of both cocoa flowers and pollinating midges (*Forcipomyia* spp.), followed by a discussion of pollinator-reducing factors (environmental and managerial) in Côte d'Ivoire. We conclude by discussing some options

Although global yearly cocoa production quantities are below those of other tropical commodity crops such as sugar cane, rice, soybean, oil palm, cassava or banana, it is a unique crop because more than 90% of its production comes from small-scale farmers (each with a

up to 2000 kg per ha and per year [6].

for relieving the constraints on cocoa pollination.

**2.1. Global production and economic value**

**2. The cocoa crop sector**

control plots.

40 Pollination in Plants

ability [9].

Côte d'Ivoire comprises the main cocoa-producing region in the world. In 2016, the country provided one third (1.472 million tons) of global cocoa supplies on 2.851 million ha of land. Average cocoa yield in Côte d'Ivoire was thus 516 kg/ha of dry cocoa beans, which is slightly below the global average yield of 538 kg/ha for that year [5]. In Côte d'Ivoire, cocoa is exclusively produced by around 1,000,000 small-scale farmers, each cultivating on around 2–3 ha [22]. These smallholders operate in a difficult context. Between 2000 and 2011, Côte d'Ivoire was generally considered a failed state with frequent occurrences of violent conflicts, where cocoa tax revenues were often used to fuel the conflicts [23, 24]. Moreover, the cocoa production in Côte d'Ivoire has often been linked to child slavery on plantations [23]. As in most cocoa-producing regions, fluctuating prices (between 1500 and 3500 USD per ton in the period 2011–2018, see http://www.nasdaq.com/markets/cocoa.aspx) affect Ivorian cocoa smallholders because in a situation with volatile prices, it is difficult to make informed choices on the "right" crop investments [25].

## **3. Cocoa pollination**

Pollination intensity and fruit set largely determine cocoa yield [26]. If natural pollination is limiting cocoa yield, then enhancing pollinator population levels should result in increased fruit set and consequently yield.

#### **3.1. Biology and phenology of cocoa flowers and fruit set**

#### *3.1.1. Biology*

Cocoa flowers are hermaphrodite. They are produced on the trees' trunks and branches (cauliflory). After 2–3 years, so-called flower cushions, i.e., thickened flower-producing leaf axils, are formed. Every cushion bears up to 50 flowers per flowering season. There are two flowering seasons per year, which thus yields 100 flowers per year. The pentamerous flower is about 15 mm in diameter. A petal consists of a pouch—which conceals the anthers—and a wide tip. The function of the latter tip is unknown, but it does not specifically attract pollinators [27]. A particular aspect of cocoa flowers is the outer whorl of purple staminodes around the style. Right after anthesis, these staminodes align parallel to the style (**Figure 1**). Pollinators move around on the inner side of the staminodes, thereby rubbing their pollen grain-carrying bodies against the style. On older flowers, staminodes are somewhat withered and flexed away

anther dehiscence [32]. However, pollen release is maximum between 8 am and 2 pm [10]. Styles and stigmas mature later than anthers, and have maximum receptivity around 12 am–2 pm. Maximum stigma and style receptivity does not concur with maximum anther dehiscence, thus limiting the possibility of self-pollination. The period during which the stigma is receptive to pollen and consequently during which successful pollination is possible, only lasts one day. Non-fertilized flowers will abscise the next day. About 1–5% of all flowers

Challenges in Cocoa Pollination: The Case of Côte d'Ivoire

http://dx.doi.org/10.5772/intechopen.75361

43

Even after cocoa flowers are successfully pollinated and led to fruit set, not all young fruits (cherelles) will grow to mature cocoa fruits. Up to 80% of cherelles will shrivel, turn black, and become rapidly colonized by pathogens, while the pod remains on the tree. This so-called cherelle wilt is a physiological mechanism whereby the fruits are naturally thinned to balance nutrient allocation in the tree. Cherelles can wilt up to day 100 after fruit set [38]. Poor soils and impeded photosynthesis result in increased cherelle wilting [39, 40]. Leguminous shade trees, which supply nitrogen to the soil, can therefore lower cherelle wilt [7]. Wilting in an early stage saves energy that can be invested in the development of the remaining fruits [30, 31]. Apart from resource limitation, inadequate pollination (insufficient pollen grains deposited on the stigma surface) and incompatible pollen may also cause cherelle wilting [41].

There are 130–160 days between fertilization and pod harvest [32]. The cocoa fruit is an indehiscent drupe. During the first 40 days after fertilization, pod growth is slow. Afterward, growth accelerates. The first division of the zygote only takes place between day 40 and 50. Pod and ovule growth decrease from day 85 onwards, when embryos start to develop. On day

Most cocoa trees are self-incompatible. Self-pollination on a self-incompatible variety will not result in successful fertilization; as such, cross-pollination is then the only way for successful fertilization [42]. Self-incompatible trees are mostly cross-compatible; i.e., they are able to successfully fertilize flowers on other trees, including trees of the same variety. Incompatibility takes place at the stage of gamete fusion: incompatible gametes are unable to fuse. The underlying mechanism is of a genetic nature [32, 43]. Following unsuccessful fertilization due to incompatibility, the flower drops off after 2–3 days. Even within a single variety, not all trees are necessarily either self-incompatible or self-compatible. However, the proportion of selfincompatible trees of a certain variety is determined by the specific variety. Self-compatible varieties that are cross-incompatible can restrict bean yield. In commercial plantations, it is therefore recommended to always plant different varieties [31]. Self-compatible hybrids pro-

Viable pollen is able to germinate (producing a pollen tube) when it reaches the stigma. Pollen

viability lies between 80 and 90%, hence does not limit fertilization [32, 45].

140, the embryo has completed its development and pod ripening starts [38].

develop into a pod [31, 32].

*3.1.3. Cherelle wilt*

*3.1.4. Pod maturation*

*3.1.5. Self-incompatibility*

duce larger fruits with a higher dry bean yield [44].

**Figure 1.** Closed and open flowers as well as fruits (pods) on the trunk of *Theobroma cacao*. Flowers are produced in clusters directly on the trunk and older branches (this is known as cauliflory) and are small, 1–2 cm in diameter, with pink calyx. The floral formula is ✶ K5 C5 A(5° + 5) G(5) [31]. While many of the world's flowers are pollinated by bees (hymenoptera) or butterflies/moths (Lepidoptera), cocoa flowers are pollinated by tiny flies, *Forcipomyia* midges in the family Ceratopogonidae [16, 94]. The tree flowers profusely, but few flowers set particularly in the dry season. When the tree is under water stress, all flowers are dropped within about 5 days. Successful pollination requires the deposition of at least 35 suitable pollen grains on the receptive parts of the flower, and is dependent on the season [95]. (Photos by Guy Smagghe in cocoa plantation at Tiassalé, Côte d'Ivoire, 15/01/2018).

from the style, which obstructs pollen deposition on the style [28]. The ovary consists of 40–70 ovules with axile placentation [29]. At least 20 ovules need to be successfully fertilized for a pod to develop and mature. Maximum pollination is achieved when pollination intensity, i.e., the number of pollen grains deposited on the style, exceeds 115 [30]. Usually, a mature pod contains between 30 and 40 beans [31, 32]. Flower morphological characteristics (size, color, and shape) can differ greatly among varieties. However, even the most noticeable differences (e.g., white vs. red sepals) have no effect on pollination [33].

#### *3.1.2. Bud development and maturation*

Flower bud development from meristem to receptive flower takes at least 20 days and can take up to 30 days [31, 32, 34]. In India, it was shown [32] that flower bud development is faster in months with higher mean temperatures (e.g. June with a mean air temperature of 28°C) compared to colder months (e.g. November with mean air temperature of 25°C). Prolonged dry (<125 mm per month) or cold (mean monthly air temperature < 23°C) periods inhibit flowering [35]. Flowering is optimal during rainy days with high relative humidity and moderate temperatures (100 mm per month, 70% RH, and 27°C). High solar radiation incidence is linked with increased flower abscission [32]. Pollen grains are only able to germinate on a receptive stigma [36, 37]. The receptive period is at about 2–3 days after anthesis. Unsuccessful pollination leads to flower abscission. Reported flower abscission rates vary from 63% on the main trunk and 81% on the fan branches to over 90% for all flowers [27, 32, 35].

Anthesis starts at around 2–4 pm. The latter becomes evident through splitting of the five sepals [27]. The process of sepal splitting continues overnight and finishes at around 4–6 am. Complete anthesis (flower fully open) is quickly followed by pollen release from the anthers (also between 4 and 6 am). Higher air temperature, as well as low air humidity, facilitates anther dehiscence [32]. However, pollen release is maximum between 8 am and 2 pm [10]. Styles and stigmas mature later than anthers, and have maximum receptivity around 12 am–2 pm. Maximum stigma and style receptivity does not concur with maximum anther dehiscence, thus limiting the possibility of self-pollination. The period during which the stigma is receptive to pollen and consequently during which successful pollination is possible, only lasts one day. Non-fertilized flowers will abscise the next day. About 1–5% of all flowers develop into a pod [31, 32].

## *3.1.3. Cherelle wilt*

Even after cocoa flowers are successfully pollinated and led to fruit set, not all young fruits (cherelles) will grow to mature cocoa fruits. Up to 80% of cherelles will shrivel, turn black, and become rapidly colonized by pathogens, while the pod remains on the tree. This so-called cherelle wilt is a physiological mechanism whereby the fruits are naturally thinned to balance nutrient allocation in the tree. Cherelles can wilt up to day 100 after fruit set [38]. Poor soils and impeded photosynthesis result in increased cherelle wilting [39, 40]. Leguminous shade trees, which supply nitrogen to the soil, can therefore lower cherelle wilt [7]. Wilting in an early stage saves energy that can be invested in the development of the remaining fruits [30, 31]. Apart from resource limitation, inadequate pollination (insufficient pollen grains deposited on the stigma surface) and incompatible pollen may also cause cherelle wilting [41].

#### *3.1.4. Pod maturation*

from the style, which obstructs pollen deposition on the style [28]. The ovary consists of 40–70 ovules with axile placentation [29]. At least 20 ovules need to be successfully fertilized for a pod to develop and mature. Maximum pollination is achieved when pollination intensity, i.e., the number of pollen grains deposited on the style, exceeds 115 [30]. Usually, a mature pod contains between 30 and 40 beans [31, 32]. Flower morphological characteristics (size, color, and shape) can differ greatly among varieties. However, even the most noticeable differences

**Figure 1.** Closed and open flowers as well as fruits (pods) on the trunk of *Theobroma cacao*. Flowers are produced in clusters directly on the trunk and older branches (this is known as cauliflory) and are small, 1–2 cm in diameter, with pink calyx. The floral formula is ✶ K5 C5 A(5° + 5) G(5) [31]. While many of the world's flowers are pollinated by bees (hymenoptera) or butterflies/moths (Lepidoptera), cocoa flowers are pollinated by tiny flies, *Forcipomyia* midges in the family Ceratopogonidae [16, 94]. The tree flowers profusely, but few flowers set particularly in the dry season. When the tree is under water stress, all flowers are dropped within about 5 days. Successful pollination requires the deposition of at least 35 suitable pollen grains on the receptive parts of the flower, and is dependent on the season [95]. (Photos by Guy

Flower bud development from meristem to receptive flower takes at least 20 days and can take up to 30 days [31, 32, 34]. In India, it was shown [32] that flower bud development is faster in months with higher mean temperatures (e.g. June with a mean air temperature of 28°C) compared to colder months (e.g. November with mean air temperature of 25°C). Prolonged dry (<125 mm per month) or cold (mean monthly air temperature < 23°C) periods inhibit flowering [35]. Flowering is optimal during rainy days with high relative humidity and moderate temperatures (100 mm per month, 70% RH, and 27°C). High solar radiation incidence is linked with increased flower abscission [32]. Pollen grains are only able to germinate on a receptive stigma [36, 37]. The receptive period is at about 2–3 days after anthesis. Unsuccessful pollination leads to flower abscission. Reported flower abscission rates vary from 63% on the

Anthesis starts at around 2–4 pm. The latter becomes evident through splitting of the five sepals [27]. The process of sepal splitting continues overnight and finishes at around 4–6 am. Complete anthesis (flower fully open) is quickly followed by pollen release from the anthers (also between 4 and 6 am). Higher air temperature, as well as low air humidity, facilitates

main trunk and 81% on the fan branches to over 90% for all flowers [27, 32, 35].

(e.g., white vs. red sepals) have no effect on pollination [33].

Smagghe in cocoa plantation at Tiassalé, Côte d'Ivoire, 15/01/2018).

*3.1.2. Bud development and maturation*

42 Pollination in Plants

There are 130–160 days between fertilization and pod harvest [32]. The cocoa fruit is an indehiscent drupe. During the first 40 days after fertilization, pod growth is slow. Afterward, growth accelerates. The first division of the zygote only takes place between day 40 and 50. Pod and ovule growth decrease from day 85 onwards, when embryos start to develop. On day 140, the embryo has completed its development and pod ripening starts [38].

#### *3.1.5. Self-incompatibility*

Most cocoa trees are self-incompatible. Self-pollination on a self-incompatible variety will not result in successful fertilization; as such, cross-pollination is then the only way for successful fertilization [42]. Self-incompatible trees are mostly cross-compatible; i.e., they are able to successfully fertilize flowers on other trees, including trees of the same variety. Incompatibility takes place at the stage of gamete fusion: incompatible gametes are unable to fuse. The underlying mechanism is of a genetic nature [32, 43]. Following unsuccessful fertilization due to incompatibility, the flower drops off after 2–3 days. Even within a single variety, not all trees are necessarily either self-incompatible or self-compatible. However, the proportion of selfincompatible trees of a certain variety is determined by the specific variety. Self-compatible varieties that are cross-incompatible can restrict bean yield. In commercial plantations, it is therefore recommended to always plant different varieties [31]. Self-compatible hybrids produce larger fruits with a higher dry bean yield [44].

Viable pollen is able to germinate (producing a pollen tube) when it reaches the stigma. Pollen viability lies between 80 and 90%, hence does not limit fertilization [32, 45].

#### *3.1.6. Flower phenology*

The number of flowers per tree varies throughout the season and is a function of climatic factors, such as photoperiod and temperature regime [46], whereas it is also cultivar dependent [31]. Furthermore, it seems that fruit production in the previous year determines flower production in the following year. Years of high pod production alternate with years with a low level of flowering [47, 48]. In most tropical countries, flowering occurs year-round. Flowering peaks are often preceded by increased temperature and rainfall, and occur at the onset of the rainy season, after which flower numbers gradually decline [45]. In West Africa, the major rainy season commences in April and climaxes in June, a period that is characterized by intense flowering (flowers on branches and trunks) [6]. In the minor rainy season (September–November), flowering intensity is lower (flowers on branches only). Few flowers are observed during the dry season (December–March) [47]. When pods are developing and this sink for assimilates is increasing, new flower production diminishes [40].

that genus, the most frequently reported pollinators belong to the subgenera *Euprojoannisia*

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It is well-documented that ceratopogonids breed in humid, decaying organic material such as cocoa leaf litter, decomposing cocoa pod husks, banana pseudostems, and bromeliads [4, 54]. Besides being moist, these breeding substrates are cooler than the ambient environment and

In the 1970s, cage experiments [28, 52, 53] were performed to characterize the pollination capacity of different ceratopogonid species. However, results of these experiments have little value as they were performed under unrealistic conditions (exposure of a high number of flowers to a single midge and use of small cages, both causing pollination levels that the same midges would not achieve in nature). The only valid method to determine whether a species is a pollinator is through field observation [26]. It has been shown that artificial circumstances bias lab experiment results considerably; for example, successful pollination by *Tyora tessmanni* was shown under lab conditions, but could not be confirmed under field conditions,

There is weak evidence for the indirect influence of the ant *Azteca chartifex spiriti* Forel (in Brazil) on cocoa pollination, as it has been shown to attract ceratopogonid midges [56]. There is some evidence of the pollination potential of stingless bees *Tetragona jaty* (Smith), *T. testaceicornis* Lep., *T. coryina* Ckll., *T. pallida* Latr.; *Nannotrigona testaceicornis punctata* (Smith); *Paratrigona lineata subnuda* Moure, and *Plebeia mosquita* (Smith). However, cocoa pollination by the latter species is merely coincidental. Sweat bees (*Lasioglossum* spp.) have also been sug-

*Forcipomyia* eggs hatch 3 days after deposition. Twelve days later, larvae transform into pupae. Pupation lasts 3 days. Adults live 1–12 days (under laboratory conditions) [59, 60]. A complete

Female ceratopogonids, in search for sugary nectar, start pollinating cocoa flowers early in the morning (5–8 am) and also actively visit flowers in the afternoon (4–6 pm) [3, 52]. Ceratopogonids carry cocoa pollen grains on their thoracic hairs. Weather conditions affect their flower visiting activities: rain and clouds decrease their activity whereas sunny weather increases it [3]. Some trees receive more attention from pollinators than others, resulting in a greater fruit set in some trees as compared to others. The interest for particular trees shifts with time. Why this happens, is not clear. Female ceratopogonids commonly visit cocoa and

Ceratopogonid midge flights might cover long distances, but it is not known how far exactly [26]. Distance traveled during one foraging event, and consequently during which pollination is performed, can reach up to 50 m. However, midges mostly deposit pollen from a certain cocoa tree on flower stigmas of neighboring cocoa trees [31, 62]. It has been shown that there are 5–7 times more *Forcipomyia* specimens above the cocoa canopy than below the

(before: *Proforcipomyia* and *Euforcipomyia*), *Thyridomyia,* and *Forcipomyia* [26].

provide dark conditions which all benefit ceratopogonid breeding [31].

where the putative pollinator was abundantly present [55].

gested as possible cocoa pollinators [57, 58].

*3.2.2. Biology and phenology of Forcipomyia spp.*

other flowers everywhere in the world [4, 61].

life cycle thus covers about 28 days [31].

## **3.2. Biology and phenology of cocoa pollinators**

#### *3.2.1. Overview of cocoa pollinating species*

Early studies have ruled out wind as a pollinating agent—pollen grains form chunks, due to their viscosity and become too heavy to travel on their own [49]. However, in South America, experiments have been conducted to increase pollination by artificially increasing air currents in the field with motorized knapsack sprayers, thus stimulating wind pollination. This technique, however, only proved to be effective (doubling of cocoa bean yield) on self-compatible varieties [50].

Cocoa is almost exclusively pollinated by insects. The most important pollinators are midges from the family Ceratopogonidae. In reference [26], the author claims based on a review of five papers that female specimens are the main pollinators, although in reference [28], four times more males than females were collected in cocoa flowers. Ceratopogonids are biting midges of 1–4 mm length [51]. Males also pollinate, but to a lesser extent. It is not clear why females visit cocoa flowers more frequently than males [28, 52, 53]. Females presumably visit cocoa flowers to feed on the protein-rich pollen grains, necessary for egg maturation.

Besides ceratopogonids, other small dipteran insects such as Cecidomyiidae (gall midges), Chironomidae (non-biting midges), Drosophilidae (fruit flies), Psychodidae (moth flies), and Sphaeroceridae (small dung flies) have been documented to visit cocoa flowers. Other insects, such as aphids, coccids and cicadellids (Hemiptera), thrips (Thysanoptera), and ants (Hymenoptera), also occasionally visit cocoa flowers. However, their contribution to pollination is most probably very low. Up to date, pollen grains have not been detected by microscopic observation on insects other than *Forcipomyia* spp. In some cases, observations suggest that cecidomyiids (in Cameroon) and drosophilids (in Ghana) may contribute to some extent to pollination [26].

Only Diptera, and particularly genus *Forcipomyia* (Fam. Ceratopogonidae), are morphologically able to pollinate cocoa. *Forcipomyia* holds the largest number of cocoa pollinators. Within that genus, the most frequently reported pollinators belong to the subgenera *Euprojoannisia* (before: *Proforcipomyia* and *Euforcipomyia*), *Thyridomyia,* and *Forcipomyia* [26].

It is well-documented that ceratopogonids breed in humid, decaying organic material such as cocoa leaf litter, decomposing cocoa pod husks, banana pseudostems, and bromeliads [4, 54]. Besides being moist, these breeding substrates are cooler than the ambient environment and provide dark conditions which all benefit ceratopogonid breeding [31].

In the 1970s, cage experiments [28, 52, 53] were performed to characterize the pollination capacity of different ceratopogonid species. However, results of these experiments have little value as they were performed under unrealistic conditions (exposure of a high number of flowers to a single midge and use of small cages, both causing pollination levels that the same midges would not achieve in nature). The only valid method to determine whether a species is a pollinator is through field observation [26]. It has been shown that artificial circumstances bias lab experiment results considerably; for example, successful pollination by *Tyora tessmanni* was shown under lab conditions, but could not be confirmed under field conditions, where the putative pollinator was abundantly present [55].

There is weak evidence for the indirect influence of the ant *Azteca chartifex spiriti* Forel (in Brazil) on cocoa pollination, as it has been shown to attract ceratopogonid midges [56]. There is some evidence of the pollination potential of stingless bees *Tetragona jaty* (Smith), *T. testaceicornis* Lep., *T. coryina* Ckll., *T. pallida* Latr.; *Nannotrigona testaceicornis punctata* (Smith); *Paratrigona lineata subnuda* Moure, and *Plebeia mosquita* (Smith). However, cocoa pollination by the latter species is merely coincidental. Sweat bees (*Lasioglossum* spp.) have also been suggested as possible cocoa pollinators [57, 58].

#### *3.2.2. Biology and phenology of Forcipomyia spp.*

*3.1.6. Flower phenology*

44 Pollination in Plants

The number of flowers per tree varies throughout the season and is a function of climatic factors, such as photoperiod and temperature regime [46], whereas it is also cultivar dependent [31]. Furthermore, it seems that fruit production in the previous year determines flower production in the following year. Years of high pod production alternate with years with a low level of flowering [47, 48]. In most tropical countries, flowering occurs year-round. Flowering peaks are often preceded by increased temperature and rainfall, and occur at the onset of the rainy season, after which flower numbers gradually decline [45]. In West Africa, the major rainy season commences in April and climaxes in June, a period that is characterized by intense flowering (flowers on branches and trunks) [6]. In the minor rainy season (September–November), flowering intensity is lower (flowers on branches only). Few flowers are observed during the dry season (December–March) [47]. When pods are developing and

Early studies have ruled out wind as a pollinating agent—pollen grains form chunks, due to their viscosity and become too heavy to travel on their own [49]. However, in South America, experiments have been conducted to increase pollination by artificially increasing air currents in the field with motorized knapsack sprayers, thus stimulating wind pollination. This technique, however, only proved to be effective (doubling of cocoa bean yield) on self-compatible

Cocoa is almost exclusively pollinated by insects. The most important pollinators are midges from the family Ceratopogonidae. In reference [26], the author claims based on a review of five papers that female specimens are the main pollinators, although in reference [28], four times more males than females were collected in cocoa flowers. Ceratopogonids are biting midges of 1–4 mm length [51]. Males also pollinate, but to a lesser extent. It is not clear why females visit cocoa flowers more frequently than males [28, 52, 53]. Females presumably visit

Besides ceratopogonids, other small dipteran insects such as Cecidomyiidae (gall midges), Chironomidae (non-biting midges), Drosophilidae (fruit flies), Psychodidae (moth flies), and Sphaeroceridae (small dung flies) have been documented to visit cocoa flowers. Other insects, such as aphids, coccids and cicadellids (Hemiptera), thrips (Thysanoptera), and ants (Hymenoptera), also occasionally visit cocoa flowers. However, their contribution to pollination is most probably very low. Up to date, pollen grains have not been detected by microscopic observation on insects other than *Forcipomyia* spp. In some cases, observations suggest that cecidomyiids (in Cameroon) and drosophilids (in Ghana) may contribute to some extent

Only Diptera, and particularly genus *Forcipomyia* (Fam. Ceratopogonidae), are morphologically able to pollinate cocoa. *Forcipomyia* holds the largest number of cocoa pollinators. Within

cocoa flowers to feed on the protein-rich pollen grains, necessary for egg maturation.

this sink for assimilates is increasing, new flower production diminishes [40].

**3.2. Biology and phenology of cocoa pollinators**

*3.2.1. Overview of cocoa pollinating species*

varieties [50].

to pollination [26].

*Forcipomyia* eggs hatch 3 days after deposition. Twelve days later, larvae transform into pupae. Pupation lasts 3 days. Adults live 1–12 days (under laboratory conditions) [59, 60]. A complete life cycle thus covers about 28 days [31].

Female ceratopogonids, in search for sugary nectar, start pollinating cocoa flowers early in the morning (5–8 am) and also actively visit flowers in the afternoon (4–6 pm) [3, 52]. Ceratopogonids carry cocoa pollen grains on their thoracic hairs. Weather conditions affect their flower visiting activities: rain and clouds decrease their activity whereas sunny weather increases it [3]. Some trees receive more attention from pollinators than others, resulting in a greater fruit set in some trees as compared to others. The interest for particular trees shifts with time. Why this happens, is not clear. Female ceratopogonids commonly visit cocoa and other flowers everywhere in the world [4, 61].

Ceratopogonid midge flights might cover long distances, but it is not known how far exactly [26]. Distance traveled during one foraging event, and consequently during which pollination is performed, can reach up to 50 m. However, midges mostly deposit pollen from a certain cocoa tree on flower stigmas of neighboring cocoa trees [31, 62]. It has been shown that there are 5–7 times more *Forcipomyia* specimens above the cocoa canopy than below the canopy [26]. Since wind speed above the canopy is higher than below, it can be expected that wind could play an important role in horizontal cocoa pollinator distribution over the cocoa field.

**4. Constraints to cocoa pollination in Côte d'Ivoire**

Cocoa is a shade-tolerant tree. Traditionally, cocoa is grown in shaded, agroforestry systems where it is intercropped with forest trees that were spared when the forest was cleared for cocoa cultivation. However, it was shown that—provided soil nutrition levels are adequate—cocoa production with shade trees is lower when compared to full-sun production [31, 67–69]. As a consequence, agroforestry systems have globally been replaced by monoculture systems with low shade provision [70]. Over the past few decades, cocoa cultivation has intensified not only by removing shade trees but also by extensive application of fertilizers and pesticides. As a result, the insect assemblage of cocoa cultivation systems has changed considerably. When compared to agroforestry systems or natural forests, insect biodiversity has decreased in present-day cocoa plantations, often at

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In Côte d'Ivoire, the cocoa sector is largely responsible for landscape degradation [19]. Over the past few decades, cocoa was typically cultivated on freshly cleared land where its production rapidly expanded, after which the land was abandoned 10–15 years later due to declining yields. Since the 1970s, such continuous so-called boom-and-bust cycles, as well as cocoa expansion from the southeast to the southwest of Côte d'Ivoire, have led to massive deforestation in the country [9, 22, 76]. In the 1960s, total tropical primary forest cover amounted to around 8.14 million ha. In the 1980s, that area had dropped to 2.6 million ha, whereas in the 2000s, primary forest cover was just over 1.35 million ha, meaning that since its independence,

Almost all cocoa plantations in Côte d'Ivoire have less than 50% of shade, meaning that the majority of trees are fully exposed to sunlight, leading to biodiversity loss and soil deterioration, often resulting in reduced addition of organic matter to the cocoa plantation soils [78]. It has been extensively shown that *Forcipomyia* spp., which are the predominant pollinating midges, require moist and decaying organic material to breed [15, 28, 52, 79]. Also, the vicinity of natural forest and moist refuges promote diversity of *Forcipomyia* spp. and cocoa pollinators in general [52, 80]. It is therefore fair to assume that in Côte d'Ivoire, massive landscape degradation has led to decreased breeding opportunities and consequently to lower population levels of cocoa pollinating midges.

The major cocoa pest problem in West Africa is caused by mirids (Order: Hemiptera, Fam. Miridae). *Sahlbergella singularis* and *Distantiella theobromae* suck the sap from cocoa pods

the expense of predators, leading to increased pest outbreaks and pollinators [71–75].

**4.1. Deteriorating pollinator environment**

*4.1.1. Cocoa monoculture*

*4.1.2. Landscape degradation*

Côte d'Ivoire has lost 80% of its forest cover [77].

**4.2. Pesticide use in the Ivorian cocoa sector**

*4.2.1. Target pests and insecticide products used*

Besides feeding on flower nectar, adult ceratopogonids also suck the blood of other insects and mammals. In general, pollinating activity is very limited in time during the lifetime of these pollinators.

Ceratopogonid pollinator populations can be abundant and exceed one million individuals per ha [3]. Moist environments favor ceratopogonid midge abundance. In fact, there is a positive correlation between soil moisture and ceratopogonid population levels [26]. Stable moist conditions are indispensable for successful development of eggs and larvae [63]. It is suggested that the West African harmattan (dry, hot wind from the north) results in withered breeding places, rendering them unsuitable for insect breeding [26]. Pollinator populations thus increase with each rainy period, to decrease again with the onset of a drier period [31].

#### **3.3. Pollination gap in cocoa**

The yield gap in cocoa (i.e., the difference between yield at optimal, experimentally determined growing conditions and the current cocoa farm yield) is caused by multiple factors including disease, pest and weed pressure as well as inadequate phytosanitary practices, lack of improved varieties, low soil fertility, etc. [64]. However, there is increasing evidence that the present yield gap is also linked with inadequate pollination. This so-called pollination gap was already observed in the late 1970s when it was found that during the dry season, the number of ceratopogonid pollinators, as well the relative number of pollinated flowers were lower than in the wet season [3, 4, 26]. Because rotten, moist organic material is an ideal breeding substrate for ceratopogonid midges, attempts have been made to increase reproduction opportunities for these midges by adding such organic material in cocoa plantations. In an experiment in Ghana, banana pseudostems, cocoa pod husks and leaf litter were added as pollinator breeding substrates next to cocoa trees. It was found that midge population increased to 500% of the control tree levels whereas fruit set in treated trees was four times higher than in control trees. Cherelle wilt also increased in treated trees but was lower than increased fruit set rates so that the final number of mature fruits was twice as high for all substrate-treated trees compared to the control trees [65]. A more direct proof of the pollination gap was found when cocoa trees in Sulewesi (Indonesia) were artificially pollinated. Optimum dry bean yield was achieved when 40% of flowers were hand-pollinated [14]. The latter treatment increased dry bean yield by 350 kg per ha as compared to a pollination intensity of 10%, which concurs with natural pollination intensities observed over the past 20 years [30, 66]. In North Queensland (Australia), it was recently shown that adding cocoa pod husks as a pollinator breeding substrate considerably increased fruit set (110 times more cherelles) and yield (60 times more fresh fruit production). However, hand pollination in fields where breeding substrate had been added did not result in extra yield, indicating that breeding substrates had already increased pollination intensities to optimum levels [16].

## **4. Constraints to cocoa pollination in Côte d'Ivoire**

## **4.1. Deteriorating pollinator environment**

## *4.1.1. Cocoa monoculture*

canopy [26]. Since wind speed above the canopy is higher than below, it can be expected that wind could play an important role in horizontal cocoa pollinator distribution over the

Besides feeding on flower nectar, adult ceratopogonids also suck the blood of other insects and mammals. In general, pollinating activity is very limited in time during the lifetime of

Ceratopogonid pollinator populations can be abundant and exceed one million individuals per ha [3]. Moist environments favor ceratopogonid midge abundance. In fact, there is a positive correlation between soil moisture and ceratopogonid population levels [26]. Stable moist conditions are indispensable for successful development of eggs and larvae [63]. It is suggested that the West African harmattan (dry, hot wind from the north) results in withered breeding places, rendering them unsuitable for insect breeding [26]. Pollinator populations thus increase with each rainy period, to decrease again with the onset of a

The yield gap in cocoa (i.e., the difference between yield at optimal, experimentally determined growing conditions and the current cocoa farm yield) is caused by multiple factors including disease, pest and weed pressure as well as inadequate phytosanitary practices, lack of improved varieties, low soil fertility, etc. [64]. However, there is increasing evidence that the present yield gap is also linked with inadequate pollination. This so-called pollination gap was already observed in the late 1970s when it was found that during the dry season, the number of ceratopogonid pollinators, as well the relative number of pollinated flowers were lower than in the wet season [3, 4, 26]. Because rotten, moist organic material is an ideal breeding substrate for ceratopogonid midges, attempts have been made to increase reproduction opportunities for these midges by adding such organic material in cocoa plantations. In an experiment in Ghana, banana pseudostems, cocoa pod husks and leaf litter were added as pollinator breeding substrates next to cocoa trees. It was found that midge population increased to 500% of the control tree levels whereas fruit set in treated trees was four times higher than in control trees. Cherelle wilt also increased in treated trees but was lower than increased fruit set rates so that the final number of mature fruits was twice as high for all substrate-treated trees compared to the control trees [65]. A more direct proof of the pollination gap was found when cocoa trees in Sulewesi (Indonesia) were artificially pollinated. Optimum dry bean yield was achieved when 40% of flowers were hand-pollinated [14]. The latter treatment increased dry bean yield by 350 kg per ha as compared to a pollination intensity of 10%, which concurs with natural pollination intensities observed over the past 20 years [30, 66]. In North Queensland (Australia), it was recently shown that adding cocoa pod husks as a pollinator breeding substrate considerably increased fruit set (110 times more cherelles) and yield (60 times more fresh fruit production). However, hand pollination in fields where breeding substrate had been added did not result in extra yield, indicating that breeding substrates had already increased pollination intensities to opti-

cocoa field.

46 Pollination in Plants

these pollinators.

drier period [31].

mum levels [16].

**3.3. Pollination gap in cocoa**

Cocoa is a shade-tolerant tree. Traditionally, cocoa is grown in shaded, agroforestry systems where it is intercropped with forest trees that were spared when the forest was cleared for cocoa cultivation. However, it was shown that—provided soil nutrition levels are adequate—cocoa production with shade trees is lower when compared to full-sun production [31, 67–69]. As a consequence, agroforestry systems have globally been replaced by monoculture systems with low shade provision [70]. Over the past few decades, cocoa cultivation has intensified not only by removing shade trees but also by extensive application of fertilizers and pesticides. As a result, the insect assemblage of cocoa cultivation systems has changed considerably. When compared to agroforestry systems or natural forests, insect biodiversity has decreased in present-day cocoa plantations, often at the expense of predators, leading to increased pest outbreaks and pollinators [71–75].

### *4.1.2. Landscape degradation*

In Côte d'Ivoire, the cocoa sector is largely responsible for landscape degradation [19]. Over the past few decades, cocoa was typically cultivated on freshly cleared land where its production rapidly expanded, after which the land was abandoned 10–15 years later due to declining yields. Since the 1970s, such continuous so-called boom-and-bust cycles, as well as cocoa expansion from the southeast to the southwest of Côte d'Ivoire, have led to massive deforestation in the country [9, 22, 76]. In the 1960s, total tropical primary forest cover amounted to around 8.14 million ha. In the 1980s, that area had dropped to 2.6 million ha, whereas in the 2000s, primary forest cover was just over 1.35 million ha, meaning that since its independence, Côte d'Ivoire has lost 80% of its forest cover [77].

Almost all cocoa plantations in Côte d'Ivoire have less than 50% of shade, meaning that the majority of trees are fully exposed to sunlight, leading to biodiversity loss and soil deterioration, often resulting in reduced addition of organic matter to the cocoa plantation soils [78]. It has been extensively shown that *Forcipomyia* spp., which are the predominant pollinating midges, require moist and decaying organic material to breed [15, 28, 52, 79]. Also, the vicinity of natural forest and moist refuges promote diversity of *Forcipomyia* spp. and cocoa pollinators in general [52, 80]. It is therefore fair to assume that in Côte d'Ivoire, massive landscape degradation has led to decreased breeding opportunities and consequently to lower population levels of cocoa pollinating midges.

#### **4.2. Pesticide use in the Ivorian cocoa sector**

#### *4.2.1. Target pests and insecticide products used*

The major cocoa pest problem in West Africa is caused by mirids (Order: Hemiptera, Fam. Miridae). *Sahlbergella singularis* and *Distantiella theobromae* suck the sap from cocoa pods and young shoots, causing commercial cocoa losses of up to 30% [81, 82]. In West Africa in general, more than 75% (in some areas 100%) of cocoa farmers use chemicals to control mirid infestation [83]. Nowadays, most frequently used insecticides in cocoa cultivation are the pyrethroids bifenthrin, cypermethrin, deltamethrin and lambda-cyhalothrin, and the neonicotinoids acetamiprid, imidacloprid and thiacloprid [84]. In Côte d'Ivoire, almost all farmers who use insecticides, apply commercial products containing a systemic neonicotinoid insecticide, usually in combination with a contact pyrethroid insecticide two times per year (July–August and January–February). The pyrethroid would thereby kill the mirid adults as well as the nymphal instars, whereas the systemic neonicotinoid would ensure that mirids that hatch after insecticide applications are also killed (personal communication with local pesticide dealers) (**Table 1**). However, the precise impact of these specific insecticides on cocoa pollinators in Côte d'Ivoire is unclear and should be further investigated.

insecticide sprayings by leaves and other organic material. However, residual effects of insecticides might affect cocoa pollinators [3, 54]. A study in West Africa on the effect of large-scale insecticide treatments (against mirids; Fam. Miridae) on both pollinator population levels and cocoa pod production showed that there is only a short-term negative impact of insecticide treatments on pollinator population levels [31]. Also in West Africa, it was shown that fogging instead of spraying insecticides is less harmful for pollinators, as fogging only negatively influences the population level for 2 days compared to 8 days with spraying [85]. Alternative approaches are to (i) reduce insecticide dosages during the period that pollinator population

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49

Despite the currently widely applied spraying programs, mirid infestation remains the most severe cocoa production limitation factor [81]. Although the precise impact of pyrethroids and neonicotinoids on cocoa pollinators in Côte d'Ivoire is unknown, it cannot be excluded that apart from these regular pests, pollinators are also affected by these products. Therefore, novel and more integrated pest management (IPM) approaches should be tested against mirids. The latter approaches might include: (i) further development and testing of mirid pheromones [86]; (ii) increasing shade levels by planting shade trees to avoid so-called "mirid pockets" (i.e., mirids particularly occurring in non-shade areas of the plantations) [81]; and (iii) enhancing ant populations as they are most probably natural mirid predators [87].

In cocoa plantations, pollinator population levels can be increased by augmenting the amount of natural pollinator breeding sites or by adding artificial breeding substrates. Since it is known that *Forcipomyia* spp. breed in moist and rotting organic material, introducing such material in the cocoa field will most likely enhance pollinator breeding and subsequently their population levels. Banana pseudostems are preferred as a pollinator breeding substrate over cocoa husks, because the latter are a possible source of black pod disease [88]. Intercropping with fruit trees will not only provide shade, but (provided that not all fruit is harvested), will also introduce rotting fruit in the plantations as potential pollinator breeding sites. As shown in **Figure 2**, in Côte d'Ivoire, we currently investigate, together with the cocoa farmers of the local cooperatives, the effect on pollination levels of squared pits (0.5 × 0.5 m and 0.3 m deep) that are spaced in 10 × 10 m squares and in which organic material such as fresh empty pod husks, cut banana pseudostems, and fruits from intercropped trees such as *Citrus* spp. will be

Mass breeding and subsequent mass release of *Forcipomyia* spp. at times when cocoa flowering peaks, might also have a significant effect on effective cocoa flower pollination. The idea

levels are low, and (ii) use narrow-spectrum insecticides.

**5.2. Enhancing cocoa pollinator environment**

deposited to enhance pollinator breeding.

**5.3. Pollinator mass breeding and mass release**

**5.1. Integrated pest management (IPM) options in the Ivorian cocoa sector**

**5. Recommendations**

#### *4.2.2. Impact of pesticides on cocoa pollinators*

Broad-spectrum insecticides (such as β-hexachlorocyclohexane and dichlorodiphenyltrichloroethane), which were historically widely applied in cocoa crop production, did not affect pollinator population levels [26]. It is suggested that breeding sites are protected from


**Table 1.** Insecticides, designed for application in cocoa cultivation, randomly collected by the authors from pesticide shops in Abidjan, Côte d'Ivoire in October 2016.

insecticide sprayings by leaves and other organic material. However, residual effects of insecticides might affect cocoa pollinators [3, 54]. A study in West Africa on the effect of large-scale insecticide treatments (against mirids; Fam. Miridae) on both pollinator population levels and cocoa pod production showed that there is only a short-term negative impact of insecticide treatments on pollinator population levels [31]. Also in West Africa, it was shown that fogging instead of spraying insecticides is less harmful for pollinators, as fogging only negatively influences the population level for 2 days compared to 8 days with spraying [85]. Alternative approaches are to (i) reduce insecticide dosages during the period that pollinator population levels are low, and (ii) use narrow-spectrum insecticides.

## **5. Recommendations**

and young shoots, causing commercial cocoa losses of up to 30% [81, 82]. In West Africa in general, more than 75% (in some areas 100%) of cocoa farmers use chemicals to control mirid infestation [83]. Nowadays, most frequently used insecticides in cocoa cultivation are the pyrethroids bifenthrin, cypermethrin, deltamethrin and lambda-cyhalothrin, and the neonicotinoids acetamiprid, imidacloprid and thiacloprid [84]. In Côte d'Ivoire, almost all farmers who use insecticides, apply commercial products containing a systemic neonicotinoid insecticide, usually in combination with a contact pyrethroid insecticide two times per year (July–August and January–February). The pyrethroid would thereby kill the mirid adults as well as the nymphal instars, whereas the systemic neonicotinoid would ensure that mirids that hatch after insecticide applications are also killed (personal communication with local pesticide dealers) (**Table 1**). However, the precise impact of these specific insecticides on cocoa pollinators in Côte d'Ivoire is unclear and should be further

Broad-spectrum insecticides (such as β-hexachlorocyclohexane and dichlorodiphenyltrichloroethane), which were historically widely applied in cocoa crop production, did not affect pollinator population levels [26]. It is suggested that breeding sites are protected from

**Pesticide brand Neonicotinoid Conc. (g/L) Pyrethroid Conc. (g/L)**

**Table 1.** Insecticides, designed for application in cocoa cultivation, randomly collected by the authors from pesticide

Thiodalm Super Acetamiprid 20 Bifenthrin 20 Callifan Super BD Acetamiprid 20 Bifenthrin 20 Gourou Super 45 EC Acetamiprid 25 Cypermethrin 25 Onex Super 40 EC Acetamiprid 20 Cypermethrin 20 Caomine 40 EC Acetamiprid 20 Cypermethrin 20 Blinde 20 EC Acetamiprid 10 Lambda-Cyhalothrin 10 Gawa 30 SC BTE Imidacloprid 30 — — Thiosulfan 60 EC Imidacloprid 60 — — Caostar 60 EC Imidacloprid 60 — — Gawa Pro 80 SC Imidacloprid 60 Bifenthrin 20 Koumabana Imidacloprid 30 Bifenthrin 20 Grosudine Super 50 Imidacloprid 30 Bifenthrin 20 Tropinex Ultra Imidacloprid 30 Lambda-Cyhalothrin 20 Actara 240 SC BTE Thiamethoxam 240 — — Azudine 50 SC Thiamethoxam 30 Deltamethrin 20 Boradyne 45 ZC Thiamethoxam 30 Lambda-Cyhalothrin 15

investigated.

48 Pollination in Plants

*4.2.2. Impact of pesticides on cocoa pollinators*

shops in Abidjan, Côte d'Ivoire in October 2016.

## **5.1. Integrated pest management (IPM) options in the Ivorian cocoa sector**

Despite the currently widely applied spraying programs, mirid infestation remains the most severe cocoa production limitation factor [81]. Although the precise impact of pyrethroids and neonicotinoids on cocoa pollinators in Côte d'Ivoire is unknown, it cannot be excluded that apart from these regular pests, pollinators are also affected by these products. Therefore, novel and more integrated pest management (IPM) approaches should be tested against mirids. The latter approaches might include: (i) further development and testing of mirid pheromones [86]; (ii) increasing shade levels by planting shade trees to avoid so-called "mirid pockets" (i.e., mirids particularly occurring in non-shade areas of the plantations) [81]; and (iii) enhancing ant populations as they are most probably natural mirid predators [87].

#### **5.2. Enhancing cocoa pollinator environment**

In cocoa plantations, pollinator population levels can be increased by augmenting the amount of natural pollinator breeding sites or by adding artificial breeding substrates. Since it is known that *Forcipomyia* spp. breed in moist and rotting organic material, introducing such material in the cocoa field will most likely enhance pollinator breeding and subsequently their population levels. Banana pseudostems are preferred as a pollinator breeding substrate over cocoa husks, because the latter are a possible source of black pod disease [88]. Intercropping with fruit trees will not only provide shade, but (provided that not all fruit is harvested), will also introduce rotting fruit in the plantations as potential pollinator breeding sites. As shown in **Figure 2**, in Côte d'Ivoire, we currently investigate, together with the cocoa farmers of the local cooperatives, the effect on pollination levels of squared pits (0.5 × 0.5 m and 0.3 m deep) that are spaced in 10 × 10 m squares and in which organic material such as fresh empty pod husks, cut banana pseudostems, and fruits from intercropped trees such as *Citrus* spp. will be deposited to enhance pollinator breeding.

#### **5.3. Pollinator mass breeding and mass release**

Mass breeding and subsequent mass release of *Forcipomyia* spp. at times when cocoa flowering peaks, might also have a significant effect on effective cocoa flower pollination. The idea

identified as cocoa flower visitors and the fact that some are restricted to either Africa, Central America, or South America (only one cocoa flower pollinator, *F. fuliginosa* was observed in all regions), it can be assumed that specific pollinating midges are restricted to certain areas [4]. It is therefore recommended that *Forcipomyia* spp. mass breeding for use in a certain cocoa area would start with locally sampled *Forcipomyia* midges, as exotic midges might disturb local biotic equilibria. Obviously, as a precondition to adoption of commercial mass breeding of pollinating midges by resource-poor smallholders in Côte d'Ivoire, the technology should be cost-effective.

Challenges in Cocoa Pollination: The Case of Côte d'Ivoire

http://dx.doi.org/10.5772/intechopen.75361

51

Since cocoa production essentially depends on insect pollination, any threat to pollinators will have a negative impact on cocoa production. There is evidence that currently, cocoa pollination is below the optimum level and that enhancing pollinator populations in cocoa fields could increase cocoa production [14–16]. It is clear that cocoa pollinators are threatened by the currently predominant cocoa production system, which consists of full-sun cultivation on often deforested land with degraded soils and chemical pest control. Pest control, shade tree planting, and landscape management all influence cocoa pollinator presence, making pollina-

Many research questions on cocoa pollination remain. They include: (i) quantification of the pollination gap (only in [14] attempts have been made, but just by comparing hand-pollination treatments with unpollinated controls); (ii) evolution of the pollination gap throughout the year (e.g., in West Africa, the gap might be narrower during the dry season when flowering is less abundant); (iii) the relation between pollination and cherelle wilt (can cherelle wilt be decreased by improving pollination efficiency?); (iv) success rates of artificial pollination (a difficult task requiring a lot of agility and experience); (v) influence of insecticide applications on pollinator and other insect population levels; (vi) role of landscape and cocoa cropping systems (agroforestry, intercropping, soil mulching) on pollinator species composition and abundance; (vii) pollen load and pollination efficiency of cocoa flower visitors other than *Forcipomyia* spp.; (viii) evaluation of pollinator roles in self-compatible as compared to selfincompatible cocoa trees; (ix) promotion of self-pollinating self-compatible trees; and (x) effec-

As final conclusion, we believe that the answers to these research questions will undoubtedly lead to decreasing the current cocoa yield gap, which is the only sustainable solution to

The authors acknowledge support from the local cooperatives of Ivorian cocoa producers in Côte d'Ivoire as well as Barry-Callebaut SA for financing cocoa pollination research in Côte d'Ivoire. Guy Smagghe also thanks the Special Research Fund of the Ghent University and the Research Foundation-Flanders (FWO-Vlaanderen) in supporting research with pollinators.

tiveness of enhancing ant populations to improve cocoa pollination.

**6. Conclusion**

tion management very complex.

increasing global cocoa supplies.

**Acknowledgements**

**Figure 2.** Midges of *Forcipomyia squamipennis* in the family Ceratopogonidae are believed to be the most important pollinators of cocoa globally, based on field observations and laboratory rearings [53, 96]. Indeed, early on, scientists figured out that most *Theobroma cacao* trees are not able to self-pollinate, but for years, they could not figure out what moved cocoa pollen between trees. It turned out that cocoa flowers are pollinated by midges not much bigger than tiny specks of airborne dust. Midge populations are greatest in the rainy season. Adult midges spend the day in shady spots such as between the buttress roots of large trees, in crevices in logs, in hollow stumps or in piles of husk debris. They emerge at variable times of the day to swarm near their hiding locations, and disperse in the early morning and late afternoon. Most midges do not move further than about 6 m. The females lay batches of eggs on damp piles of plant debris, on moist decomposing wood, cocoa husks and other plant debris, in batches of 40–90 eggs. Eggs hatch after 2–3 days and the larvae pass through four instar stages before pupating at about 12 days; the pupal stage lasts 2–3 days. The adults survive for about a week and there are thought to be about 12 midge generations per year. Adult females require liquid plant food for survival and oviposition, although ovary maturation is independent of adult food intake or mating. In a joint project between Ghent University and Barry-Callebaut, and in collaboration with local cocoa smallholders and their cooperatives, we introduced squared pits of 50 × 50 cm and 30 cm deep, filled with organic material such as cut banana pseudostems, fresh empty pod husks and fruits from intercropped trees such as *Citrus* spp., at a density of 1 pit per 100 m2 (spaced at 10 × 10 m as based on presumed midge flight radius of the midges) to enhance the establishment of the cocoa pollinating midge populations in the field (main picture is a photo by Guy Smagghe in cocoa plantation at Tiassalé, Côte d'Ivoire, 15/01/2018; inset photo is of a mating pair of *Forcipomyia* midges by Christophe Quintin, https://www.flickr.com/photos/34878947@N04/).

is based on similar practices commonly applied in the horticultural sector where bumblebees (*Bombus terrestris*) are commercially bred and subsequently released in tomato (*Solanum lycopersicon*) greenhouses for tomato flower pollination [89]. As compared to the earlier used vibrating sticks to induce pollen release from tomato flowers, bumblebees increase tomato fruit set by 45%. Another example is the black soldier fly (*Hermetia illucens*) (Order: Diptera, Fam: Stratiomyidae) that is used to enhance composting of food waste and reduction of organic manure volumes, and which can be mass bred prior to release on organic material [90]. To our knowledge, no mass breeding attempts for *Forcipomyia* spp. have been undertaken up to date. The hematophagous nature of *Forcipomyia* midges can be a constraint to their mass breeding success [91]. Laboratory experiments showed that *F. townsvillensis* eggs will not develop without complete blood meals [92]. Research is needed to test the most appropriate midge rearing conditions (temperature, humidity and feeding).

*Forcipomyia* spp. mostly pollinate flowers neighboring the ones where they have collected pollen [93]. We assume therefore that they do not swarm further than 10 m from their breeding sites. Under that assumption, mass release should be performed at least each 20 × 20 m in cocoa plantations (25 releases per ha). Given the wide diversity of *Forcipomyia* spp. that have been identified as cocoa flower visitors and the fact that some are restricted to either Africa, Central America, or South America (only one cocoa flower pollinator, *F. fuliginosa* was observed in all regions), it can be assumed that specific pollinating midges are restricted to certain areas [4]. It is therefore recommended that *Forcipomyia* spp. mass breeding for use in a certain cocoa area would start with locally sampled *Forcipomyia* midges, as exotic midges might disturb local biotic equilibria. Obviously, as a precondition to adoption of commercial mass breeding of pollinating midges by resource-poor smallholders in Côte d'Ivoire, the technology should be cost-effective.

## **6. Conclusion**

Since cocoa production essentially depends on insect pollination, any threat to pollinators will have a negative impact on cocoa production. There is evidence that currently, cocoa pollination is below the optimum level and that enhancing pollinator populations in cocoa fields could increase cocoa production [14–16]. It is clear that cocoa pollinators are threatened by the currently predominant cocoa production system, which consists of full-sun cultivation on often deforested land with degraded soils and chemical pest control. Pest control, shade tree planting, and landscape management all influence cocoa pollinator presence, making pollination management very complex.

Many research questions on cocoa pollination remain. They include: (i) quantification of the pollination gap (only in [14] attempts have been made, but just by comparing hand-pollination treatments with unpollinated controls); (ii) evolution of the pollination gap throughout the year (e.g., in West Africa, the gap might be narrower during the dry season when flowering is less abundant); (iii) the relation between pollination and cherelle wilt (can cherelle wilt be decreased by improving pollination efficiency?); (iv) success rates of artificial pollination (a difficult task requiring a lot of agility and experience); (v) influence of insecticide applications on pollinator and other insect population levels; (vi) role of landscape and cocoa cropping systems (agroforestry, intercropping, soil mulching) on pollinator species composition and abundance; (vii) pollen load and pollination efficiency of cocoa flower visitors other than *Forcipomyia* spp.; (viii) evaluation of pollinator roles in self-compatible as compared to selfincompatible cocoa trees; (ix) promotion of self-pollinating self-compatible trees; and (x) effectiveness of enhancing ant populations to improve cocoa pollination.

As final conclusion, we believe that the answers to these research questions will undoubtedly lead to decreasing the current cocoa yield gap, which is the only sustainable solution to increasing global cocoa supplies.

## **Acknowledgements**

is based on similar practices commonly applied in the horticultural sector where bumblebees (*Bombus terrestris*) are commercially bred and subsequently released in tomato (*Solanum lycopersicon*) greenhouses for tomato flower pollination [89]. As compared to the earlier used vibrating sticks to induce pollen release from tomato flowers, bumblebees increase tomato fruit set by 45%. Another example is the black soldier fly (*Hermetia illucens*) (Order: Diptera, Fam: Stratiomyidae) that is used to enhance composting of food waste and reduction of organic manure volumes, and which can be mass bred prior to release on organic material [90]. To our knowledge, no mass breeding attempts for *Forcipomyia* spp. have been undertaken up to date. The hematophagous nature of *Forcipomyia* midges can be a constraint to their mass breeding success [91]. Laboratory experiments showed that *F. townsvillensis* eggs will not develop without complete blood meals [92]. Research is needed to test the most appropriate midge rearing

to enhance the establishment of the cocoa pollinating midge populations in the field (main picture is a photo by Guy Smagghe in cocoa plantation at Tiassalé, Côte d'Ivoire, 15/01/2018; inset photo is of a mating pair of *Forcipomyia* midges

(spaced at 10 × 10 m as based on presumed midge flight radius of the midges)

**Figure 2.** Midges of *Forcipomyia squamipennis* in the family Ceratopogonidae are believed to be the most important pollinators of cocoa globally, based on field observations and laboratory rearings [53, 96]. Indeed, early on, scientists figured out that most *Theobroma cacao* trees are not able to self-pollinate, but for years, they could not figure out what moved cocoa pollen between trees. It turned out that cocoa flowers are pollinated by midges not much bigger than tiny specks of airborne dust. Midge populations are greatest in the rainy season. Adult midges spend the day in shady spots such as between the buttress roots of large trees, in crevices in logs, in hollow stumps or in piles of husk debris. They emerge at variable times of the day to swarm near their hiding locations, and disperse in the early morning and late afternoon. Most midges do not move further than about 6 m. The females lay batches of eggs on damp piles of plant debris, on moist decomposing wood, cocoa husks and other plant debris, in batches of 40–90 eggs. Eggs hatch after 2–3 days and the larvae pass through four instar stages before pupating at about 12 days; the pupal stage lasts 2–3 days. The adults survive for about a week and there are thought to be about 12 midge generations per year. Adult females require liquid plant food for survival and oviposition, although ovary maturation is independent of adult food intake or mating. In a joint project between Ghent University and Barry-Callebaut, and in collaboration with local cocoa smallholders and their cooperatives, we introduced squared pits of 50 × 50 cm and 30 cm deep, filled with organic material such as cut banana pseudostems, fresh empty pod husks and fruits from intercropped trees such as *Citrus*

*Forcipomyia* spp. mostly pollinate flowers neighboring the ones where they have collected pollen [93]. We assume therefore that they do not swarm further than 10 m from their breeding sites. Under that assumption, mass release should be performed at least each 20 × 20 m in cocoa plantations (25 releases per ha). Given the wide diversity of *Forcipomyia* spp. that have been

conditions (temperature, humidity and feeding).

by Christophe Quintin, https://www.flickr.com/photos/34878947@N04/).

spp., at a density of 1 pit per 100 m2

50 Pollination in Plants

The authors acknowledge support from the local cooperatives of Ivorian cocoa producers in Côte d'Ivoire as well as Barry-Callebaut SA for financing cocoa pollination research in Côte d'Ivoire. Guy Smagghe also thanks the Special Research Fund of the Ghent University and the Research Foundation-Flanders (FWO-Vlaanderen) in supporting research with pollinators.

## **Conflict of interest**

The authors have no conflict of interest.

## **Author details**

Gregor Claus1 , Wouter Vanhove1 \*, Patrick Van Damme1,2 and Guy Smagghe1

\*Address all correspondence to: wouter.vanhove@ugent.be

1 Department of Plant and Crops, Ghent University, Ghent, Belgium

2 Faculty of Tropical AgriSciences, Czech University of Life Sciences Prague, Czech Republic

[8] Beer J, Muschler R, Kass D, Somarriba E. Shade management in coffee and cacao planta-

Challenges in Cocoa Pollination: The Case of Côte d'Ivoire

http://dx.doi.org/10.5772/intechopen.75361

53

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**Conflict of interest**

**Author details**

Gregor Claus1

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**Chapter 5**

**Provisional chapter**

**Artificial Pollination in Kiwifruit and Olive Trees**

**Artificial Pollination in Kiwifruit and Olive Trees**

DOI: 10.5772/intechopen.74831

In the last 10 years, kiwifruit vine artificial pollination became a widespread practice useful to increase fruit quality. Kiwifruit size is directly proportional to the number of seeds, i.e., to the number of fertilized ovaries. However, artificial pollination efficiency depends on many parameters such as pollen quality (germinability, humidity, and conservation), pollination system (dry or liquid), coadjuvants, and flowering stage. Those parameters were well defined in *Actinidia* in recent studies, however, they remain quite undefined for other anemophilous pollinated trees such as olive tree, hazelnut, pistachio, and palm. In these plants, the flowers are very small and extremely numerous, so the pollination was difficult to study. In addition, there are incompatibility factors (genetic and physic), long lap time from pollination to fertilization, and alternate bearing, lower economic gain for these fruits, low agronomic input, and low innovation level in the field. All these aspects had reduced the application of pollination technique for these cultivations. The experiences developed in kiwifruit lead to define a new model crop fruit set that could be applied to anemophilous pollinated plants such as olive tree, where the fruit set are lower than 2%. The first experiences have shown a great potential and have encouraged

**Keywords:** kiwifruit, olive, pollination, equipment, quality, flowering stage,

Pollination of crop plants is often the major requirement in achieving sufficient crop set [1, 2]. Insufficient pollination has been found to be one of the important causative factors of low yield and low quality in many fruit tree species [3]. Supplementary pollination is a valid support to increase productivity in crop species such as strawberry [4, 5], olive [6], kiwifruit [7, 8], almond [9, 10], pistachio [11, 12], hazelnut [13], macadamia [14, 15] and date palm [16, 17]. Artificial pollination

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Tacconi Gianni and Michelotti Vania

Tacconi Gianni and Michelotti Vania

http://dx.doi.org/10.5772/intechopen.74831

the development of this technique.

germinability, humidity

**1. Introduction**

**Abstract**

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter


#### **Artificial Pollination in Kiwifruit and Olive Trees Artificial Pollination in Kiwifruit and Olive Trees**

DOI: 10.5772/intechopen.74831

#### Tacconi Gianni and Michelotti Vania Tacconi Gianni and Michelotti Vania

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74831

#### **Abstract**

[85] Lucas P, Decazy B. Influence Des Traitements Insecticides Contre Les Mirides Du Cacaoyer Sur Les Conditions De Pollinisation. Actes, Douala, Cameroun. Nov.

[86] Mahob RJ, Babin R, ten Hoopen GM, Dibog L, Yede DRH, Bilong Bilong CF. Field evaluation of synthetic sex pheromone traps for the cocoa Mirid Sahlbergella Singularis

[87] Wheeler AG. Biology of the Plant Bugs (Hemiptera: Miridae): Pests, Predators,

[88] Ndoumbe-Nkeng M, Cilas C, Nyemb E, Nyasse S, Bieysse D, Flori A, Sache I. Impact of removing diseased pods on cocoa black pod caused by *Phytophthora megakarya* and on

[89] Velthuis HHW, van Doorn A. A century of advances in bumblebee domestication and the economic and environmental aspects of its commercialization for pollination.

[90] Nakamura S, Ichiki RT, Shimoda M, Morioka S. Small-scale rearing of the black soldier fly, *Hermetia illucens* (Diptera: Stratiomyidae), in the laboratory: Low-cost and yearround rearing. Applied Entomology and Zoology. February 01 2016;**51**(1):161-166 [91] Gibson G, Torr SJ. Visual and olfactory responses of haematophagous diptera to host

[92] Cribb BW. Oviposition and maintenance of Forcipomyia (Lasiohelea) Townsvillensis (Diptera: Ceratopogonidae) in the laboratory. Journal of Medical Entomology.

[93] Young AM. Seasonal differences in abundance and distribution of cocoa-pollinating midges in relation to flowering and fruit set between shaded and sunny habitats of the

[95] Frimpong-Anin K, Adjaloo MK, Kwapong PK, Oduro W. Structure and stability of cocoa

[96] Deheuvels O, Cordoba Obando C, Cerda R, Somarriba Chavez E. The Efficiency of Cocoa Pollination in Agroforestry Systems under Bio-Fertilizer Application. Paper Presented at

the World Congress on Agroforestry, Delhi, Inde, 2014-02-10/2014-02-14; 2014

la Lola cocoa farm in Costa Rica. Journal of Applied Ecology. 1983;**20**(3):801-831

[94] Hernández B. Insect Pollination of Cacao (*Theobroma cacao* L.) in Costa Rica; 1965

flowers and their response to pollination. Journal of Botany. 2014;**2014**:6

cocoa production in Cameroon. Crop Protection. 2004/05/01;**23**(5):415-424

(Hemiptera: Miridae). Pest Management Science. 2011;**67**(6):672-676

Opportunists. London: Cornell University Press; 2001

stimuli. Medical and Veterinary Entomology. 1999;**13**(1):2-23

1979;**4-12**(1981):447

58 Pollination in Plants

Apidologie. 2006;**37**(4):421-451

2000;**37**(3):316-318

In the last 10 years, kiwifruit vine artificial pollination became a widespread practice useful to increase fruit quality. Kiwifruit size is directly proportional to the number of seeds, i.e., to the number of fertilized ovaries. However, artificial pollination efficiency depends on many parameters such as pollen quality (germinability, humidity, and conservation), pollination system (dry or liquid), coadjuvants, and flowering stage. Those parameters were well defined in *Actinidia* in recent studies, however, they remain quite undefined for other anemophilous pollinated trees such as olive tree, hazelnut, pistachio, and palm. In these plants, the flowers are very small and extremely numerous, so the pollination was difficult to study. In addition, there are incompatibility factors (genetic and physic), long lap time from pollination to fertilization, and alternate bearing, lower economic gain for these fruits, low agronomic input, and low innovation level in the field. All these aspects had reduced the application of pollination technique for these cultivations. The experiences developed in kiwifruit lead to define a new model crop fruit set that could be applied to anemophilous pollinated plants such as olive tree, where the fruit set are lower than 2%. The first experiences have shown a great potential and have encouraged the development of this technique.

**Keywords:** kiwifruit, olive, pollination, equipment, quality, flowering stage, germinability, humidity

#### **1. Introduction**

Pollination of crop plants is often the major requirement in achieving sufficient crop set [1, 2]. Insufficient pollination has been found to be one of the important causative factors of low yield and low quality in many fruit tree species [3]. Supplementary pollination is a valid support to increase productivity in crop species such as strawberry [4, 5], olive [6], kiwifruit [7, 8], almond [9, 10], pistachio [11, 12], hazelnut [13], macadamia [14, 15] and date palm [16, 17]. Artificial pollination

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

leads also to increase final set, weight, kernel recovery, and, in many cases, fruit quality in terms of nutritional characteristics and shelf life [4]. Moreover, in olive tree, a greater pollination and fruiting cause a slower ripening of the drupes, and consequently harvest times are more suitable to the improvement of olive and oil quality. In many cases, natural pollination (both wind and bee) is often unsatisfactory or not constant in the years (**Figure 1**), because it can be affected by climatic factors, wrong synchronization of male and female flowering, and low attraction for bee since the absence of nectar in the flowers of wind-pollinated (anemophily) plants.

Kiwifruit artificial pollination was first studied by Dr. Hopping in 70 years [7, 18, 19] in New Zealand and in Italy, in collaboration with Dr. Cacioppo and Dr. Galimberti in Latina, in 1987 (**Figure 2**).

Kiwifruit (*Actinidia chinensis* var. *deliciosa* and *A. chinensis* var. *chinensis*) is a dioecious plant, and, in order to have good pollination, in orchard, there are female and male plants in 6:1 ratio. The pollination is mainly anemophilous (wind-pollinated), and the fruits size depends on the number of seeds: a 100 g fruit has more than 1000 seeds, and it is estimated that about 10fold pollen grains are necessary to reach this seed number [21, 22]. Also, an increase in male:female ratio to 1:1 (**Figure 3**) was not enough, in many cases, to optimize the pollination.

Moreover, in many specialized orchards, there are installed anti-hail net or plastic tunnel to protect the plants from climate injuries or from the bacterial disease *Pseudomonas syringae* pv*. actinidiae* [23]. These installations reduce the ventilation and indeed pollen movement. Furthermore, in yellow flesh kiwifruit (but also in green ones), often male plants were not planted in the orchard in order to have a higher yield (male occupy 16% of the surface) and an easier management of the plants (treatments for plant protection due the higher disease susceptibility of male, pruning, fertilization, and irrigation). In the cases where male plants are absent in the orchard, pollen are kept from specialized male orchards or buy on market (following plant protection rules to avoid diseases contaminations).

**Figure 1.** Kiwifruit with opposite size due to the pollination efficiency (left), perfect pollination with many pollinated ovules (center), and abundant pollination in olive (right).

Kiwifruit artificial pollination is nowadays a consolidate technique to increase kiwifruit quality and size [8, 24]. However, pollination not always reacts with maximum efficiency: the results could change in different years and depend on the pollen harvesting system, pollen storage technique, pollination system (dry or wet), added substance to dilute pollen (dry or liquid) or

**Figure 3.** *A. deliciosa* cv. Hayward in T-bar orchard in Verona with the male permanent leader in the middle. Despite this configuration, ideated by Tacconi Lorenzo in 1987, the pollination efficiency is low and is necessary in collecting and

**Figure 2.** M. E. Hopping who developed, in New Zealand, artificial pollination in kiwifruit (left). Hopping's group of

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 61

researchers during the experiment of spray pollination in Latina in 1987 (right) [20].

distributing pollen to obtain fruits with good size.

leads also to increase final set, weight, kernel recovery, and, in many cases, fruit quality in terms of nutritional characteristics and shelf life [4]. Moreover, in olive tree, a greater pollination and fruiting cause a slower ripening of the drupes, and consequently harvest times are more suitable to the improvement of olive and oil quality. In many cases, natural pollination (both wind and bee) is often unsatisfactory or not constant in the years (**Figure 1**), because it can be affected by climatic factors, wrong synchronization of male and female flowering, and low attraction for bee since the

Kiwifruit artificial pollination was first studied by Dr. Hopping in 70 years [7, 18, 19] in New Zealand and in Italy, in collaboration with Dr. Cacioppo and Dr. Galimberti in Latina, in 1987

Kiwifruit (*Actinidia chinensis* var. *deliciosa* and *A. chinensis* var. *chinensis*) is a dioecious plant, and, in order to have good pollination, in orchard, there are female and male plants in 6:1 ratio. The pollination is mainly anemophilous (wind-pollinated), and the fruits size depends on the number of seeds: a 100 g fruit has more than 1000 seeds, and it is estimated that about 10fold pollen grains are necessary to reach this seed number [21, 22]. Also, an increase in male:female ratio to 1:1 (**Figure 3**) was not enough, in many cases, to optimize the pollination. Moreover, in many specialized orchards, there are installed anti-hail net or plastic tunnel to protect the plants from climate injuries or from the bacterial disease *Pseudomonas syringae* pv*. actinidiae* [23]. These installations reduce the ventilation and indeed pollen movement. Furthermore, in yellow flesh kiwifruit (but also in green ones), often male plants were not planted in the orchard in order to have a higher yield (male occupy 16% of the surface) and an easier management of the plants (treatments for plant protection due the higher disease susceptibility of male, pruning, fertilization, and irrigation). In the cases where male plants are absent in the orchard, pollen are kept from specialized male orchards or buy on market

**Figure 1.** Kiwifruit with opposite size due to the pollination efficiency (left), perfect pollination with many pollinated

absence of nectar in the flowers of wind-pollinated (anemophily) plants.

(following plant protection rules to avoid diseases contaminations).

ovules (center), and abundant pollination in olive (right).

(**Figure 2**).

60 Pollination in Plants

**Figure 2.** M. E. Hopping who developed, in New Zealand, artificial pollination in kiwifruit (left). Hopping's group of researchers during the experiment of spray pollination in Latina in 1987 (right) [20].

**Figure 3.** *A. deliciosa* cv. Hayward in T-bar orchard in Verona with the male permanent leader in the middle. Despite this configuration, ideated by Tacconi Lorenzo in 1987, the pollination efficiency is low and is necessary in collecting and distributing pollen to obtain fruits with good size.

Kiwifruit artificial pollination is nowadays a consolidate technique to increase kiwifruit quality and size [8, 24]. However, pollination not always reacts with maximum efficiency: the results could change in different years and depend on the pollen harvesting system, pollen storage technique, pollination system (dry or wet), added substance to dilute pollen (dry or liquid) or to help the germination, pollination equipment, moment of pollination, floral stage of application, and economic impact of the operation (cost and gain). The analysis of these aspects could be applied to other crops and could be summarized in a flowchart where physiological aspects and human practices/decisions are integrated (**Figure 4**). Given the optimal pollen quality and optimal agronomical management (irrigation, fertilization, pruning), the results could vary in relation to the choice of the floral stage of intervention in relation to the type of pollination. In the reported studies, many parameters were analyzed alone and in interaction in different environments in Italy and for many years: pollen quality, pollination system, and flowering stage.

**2. Pollen quality for kiwifruit pollination**

**2.1. Materials and methods**

The first parameter that is considered to define the pollen quality is the germinability. Pollen could germinate but stop early in the tube growth: must it be considered right for pollination or not? Many grains germinate, but in different ways due to their different germination energies (germinability related to the time or germination tube length), and it is evident recording pollen germination under microscope (**Figure 5** and related movie). Other parameters must be considered in order to evaluate pollen quality, as humidity and germination energy [26]. These parameters were evaluated under different conditions of pollen harvesting, conservation at different temperatures and different times, exposition at different temperatures, and manipulation before and during pollination. For example, stresses against pollen during harvest and manipulation result in decrease of germination energy more than decrease of germinability.

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 63

The experiment was performed on *Actinidia deliciosa* cv. Hayward (female) and cv. Tumuri (male) in the Tacconi Lorenzo's farm in Verona (North Italy), on a plantation built in 1982, (T-bar system, 4.5 × 3 m) having a permanent corded male suspended in the middle of the inter-row (**Figure 3**). The pollen samples were collected in two seasons (2008 and 2009) having opposite conditions of high relative humidity (RH) and low temperatures and low RH and high temperatures, respectively. Pollen samples were collected with two different systems (**Figure 6**): filter separator (Aspir@ Polline TR Biotac, Verona, Italy, www.biotac.it) and cyclone separator (AspiraPollineMini2 Biotac, Verona, Italy). The pollens were extracted from the machine and placed at 4°C every 45 min.

The germination temperature test was made with fresh Tumuri pollen (collected with Aspir@ Polline TR Biotac) on standard substrate (sucrose 85 g/l, boric acid 0.5 g/l) by taking a photo of the same field of view under microscope every minute for 14 h. The temperatures considered were 18, 24, and 30°C. To test the in vitro germination conditions, the germination of two

**Figure 5.** Time lapse during pollen germination at 20°C: the number indicates hours.

High-quality pollen is basic for good results: germinability, germination energy, and humidity were evaluated under different conditions of pollen harvesting, conservation at different temperatures and time of exposition at different temperatures, and manipulation before and during pollination in different pollination systems (dry and liquid).

The interaction of the pollination systems and the flowering stage were also evaluated.

Many aspects are in common with olive (*Olea europaea* L.) and can be applied to its pollination. Olive fruit set are very low, less than 2% of flowers, which in Northern Italy means about 10 kg of fruit per plant [25]. The main problems are self-incompatibility, scarce pollen from wild, wrong pollinator cultivars in the orchard, pollen quality and quantity, lack of coincidence of blooming period, and adverse climate conditions.

This observation leads to define kiwifruit pollination as a new model for crop fruit pollination that could be applied in other wind pollination (anemophily) trees such as olive tree, hazelnut, and pistachio that were studied but without a practical application (**Figure 4**) [6, 12, 13].

This chapter does not want be a review on biology of the pollination in kiwifruit and olive but an update of the research applied in the field supported by scientific data. Here, publicized works but also original researches data are reported.

**Figure 4.** Flowchart of the main phases ranging from pollen collection to pollination and critical points analyzed in this chapter. Physiological aspects, climatic conditions, and human practices are integrated.

## **2. Pollen quality for kiwifruit pollination**

The first parameter that is considered to define the pollen quality is the germinability. Pollen could germinate but stop early in the tube growth: must it be considered right for pollination or not? Many grains germinate, but in different ways due to their different germination energies (germinability related to the time or germination tube length), and it is evident recording pollen germination under microscope (**Figure 5** and related movie). Other parameters must be considered in order to evaluate pollen quality, as humidity and germination energy [26].

These parameters were evaluated under different conditions of pollen harvesting, conservation at different temperatures and different times, exposition at different temperatures, and manipulation before and during pollination. For example, stresses against pollen during harvest and manipulation result in decrease of germination energy more than decrease of germinability.

## **2.1. Materials and methods**

to help the germination, pollination equipment, moment of pollination, floral stage of application, and economic impact of the operation (cost and gain). The analysis of these aspects could be applied to other crops and could be summarized in a flowchart where physiological aspects and human practices/decisions are integrated (**Figure 4**). Given the optimal pollen quality and optimal agronomical management (irrigation, fertilization, pruning), the results could vary in relation to the choice of the floral stage of intervention in relation to the type of pollination. In the reported studies, many parameters were analyzed alone and in interaction in different environments in Italy and for many years: pollen quality, pollination system, and flowering

High-quality pollen is basic for good results: germinability, germination energy, and humidity were evaluated under different conditions of pollen harvesting, conservation at different temperatures and time of exposition at different temperatures, and manipulation before and

Many aspects are in common with olive (*Olea europaea* L.) and can be applied to its pollination. Olive fruit set are very low, less than 2% of flowers, which in Northern Italy means about 10 kg of fruit per plant [25]. The main problems are self-incompatibility, scarce pollen from wild, wrong pollinator cultivars in the orchard, pollen quality and quantity, lack of coinci-

This observation leads to define kiwifruit pollination as a new model for crop fruit pollination that could be applied in other wind pollination (anemophily) trees such as olive tree, hazelnut, and pistachio that were studied but without a practical application (**Figure 4**)

This chapter does not want be a review on biology of the pollination in kiwifruit and olive but an update of the research applied in the field supported by scientific data. Here, publicized

**Figure 4.** Flowchart of the main phases ranging from pollen collection to pollination and critical points analyzed in this

chapter. Physiological aspects, climatic conditions, and human practices are integrated.

The interaction of the pollination systems and the flowering stage were also evaluated.

during pollination in different pollination systems (dry and liquid).

dence of blooming period, and adverse climate conditions.

works but also original researches data are reported.

stage.

62 Pollination in Plants

[6, 12, 13].

The experiment was performed on *Actinidia deliciosa* cv. Hayward (female) and cv. Tumuri (male) in the Tacconi Lorenzo's farm in Verona (North Italy), on a plantation built in 1982, (T-bar system, 4.5 × 3 m) having a permanent corded male suspended in the middle of the inter-row (**Figure 3**). The pollen samples were collected in two seasons (2008 and 2009) having opposite conditions of high relative humidity (RH) and low temperatures and low RH and high temperatures, respectively. Pollen samples were collected with two different systems (**Figure 6**): filter separator (Aspir@ Polline TR Biotac, Verona, Italy, www.biotac.it) and cyclone separator (AspiraPollineMini2 Biotac, Verona, Italy). The pollens were extracted from the machine and placed at 4°C every 45 min.

The germination temperature test was made with fresh Tumuri pollen (collected with Aspir@ Polline TR Biotac) on standard substrate (sucrose 85 g/l, boric acid 0.5 g/l) by taking a photo of the same field of view under microscope every minute for 14 h. The temperatures considered were 18, 24, and 30°C. To test the in vitro germination conditions, the germination of two

**Figure 5.** Time lapse during pollen germination at 20°C: the number indicates hours.

**Figure 6.** Schematic representation of the operating principle of the two pollen separation systems and related critical points.

observation that the pollen tube length is higher if the germination appends at about 18–24°C, whereas at higher temperature (30°C), the germination stops early and tube length is lower. Moreover, the suspension of pollen in water must be sprayed before the germination starts, in

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 65

The different media used in vitro can give useful indications for pollen suspensions in the case of liquid pollination and for analysis. The different media showed a different percentage of germination and germination energy (given by start time of germination and final lengths of

The analyses carried out show how the result evaluation of germination could vary according to the growth media used and depending on the moment in which the observation is made. Furthermore, a media that is too nutritious (i.e., n. 6 and 7) could overestimate the real germination that would occur in vivo in field condition, whereas a less stimulating substrate (i.e., n. 2) would be more useful as it highlights any weakness (less germination energy). PollenAid and Biotac solution could be useful in liquid pollination because they encourage germination [26].

Different pollination machines are available in the market, and these fall in two categories basing on the separation system: filter and cyclone (centrifugation). The comparison of these systems in two different climatic conditions during pollen collection, in particular relative air humidity (RH), reveals some differences in the pollen quality. Regarding cyclone system, pollen RH increases with air RH increasing (RH), whereas in the case of filter system, pollen RH is about 10% independently to the air RH (**Figure 8**). This difference is due to the lower pressure inside the filter system and, therefore, lower temperature in comparison to external

The humidity of the pollen is important for pollen long-term storage. One advantage of artificial pollination is the possibility to store pollen at −18°C for years maintaining its viability.

practical within 40 min, to avoid pollen damage.

**Figure 7.** Dynamic of pollen germination at different temperatures and tube elongation.

*2.2.2. Pollen harvest systems, pollen humidity, and pollen viability*

one, such that water vapor in the air is condensed and extracted.

pollen tubes; **Table 1**).

pollen samples (collected with Aspir@Polline TR and AspiraPollineMini2, Biotac) on different agar growth media was compared (**Table 1**). The germination was observed at intervals of about 1 h for 15 h under the microscope (Olympus BX51 microscope at 200 magnifications with Olympus DP50 camera). The germination was made at a constant temperature of 20°C in a growth chamber (Sanyo Gallenkamp PLC, Loughborough, UK) with RH 100% and with cold light. The germination was calculated as percentage of germinated pollen counting about 100 pollen grains in three different optical field; tube length was evaluated using UTHSCSA ImageTool software and reported as fold grain diameter (D, about 30 micron).

#### **2.2. Results and discussion**

#### *2.2.1. Germination: effect of temperature and growth media*

Germination latency period and tube length are inversely proportional to the temperature of germination (**Figure 7**). During pollen application in field lower temperature is useful due the


**Table 1.** Percentage of germination and germination energy of pollen collected with different systems and germinated on various media.

**Figure 7.** Dynamic of pollen germination at different temperatures and tube elongation.

pollen samples (collected with Aspir@Polline TR and AspiraPollineMini2, Biotac) on different agar growth media was compared (**Table 1**). The germination was observed at intervals of about 1 h for 15 h under the microscope (Olympus BX51 microscope at 200 magnifications with Olympus DP50 camera). The germination was made at a constant temperature of 20°C in a growth chamber (Sanyo Gallenkamp PLC, Loughborough, UK) with RH 100% and with cold light. The germination was calculated as percentage of germinated pollen counting about 100 pollen grains in three different optical field; tube length was evaluated using UTHSCSA

**Figure 6.** Schematic representation of the operating principle of the two pollen separation systems and related critical

Germination latency period and tube length are inversely proportional to the temperature of germination (**Figure 7**). During pollen application in field lower temperature is useful due the

**Table 1.** Percentage of germination and germination energy of pollen collected with different systems and germinated

ImageTool software and reported as fold grain diameter (D, about 30 micron).

*2.2.1. Germination: effect of temperature and growth media*

**2.2. Results and discussion**

on various media.

points.

64 Pollination in Plants

observation that the pollen tube length is higher if the germination appends at about 18–24°C, whereas at higher temperature (30°C), the germination stops early and tube length is lower. Moreover, the suspension of pollen in water must be sprayed before the germination starts, in practical within 40 min, to avoid pollen damage.

The different media used in vitro can give useful indications for pollen suspensions in the case of liquid pollination and for analysis. The different media showed a different percentage of germination and germination energy (given by start time of germination and final lengths of pollen tubes; **Table 1**).

The analyses carried out show how the result evaluation of germination could vary according to the growth media used and depending on the moment in which the observation is made. Furthermore, a media that is too nutritious (i.e., n. 6 and 7) could overestimate the real germination that would occur in vivo in field condition, whereas a less stimulating substrate (i.e., n. 2) would be more useful as it highlights any weakness (less germination energy). PollenAid and Biotac solution could be useful in liquid pollination because they encourage germination [26].

#### *2.2.2. Pollen harvest systems, pollen humidity, and pollen viability*

Different pollination machines are available in the market, and these fall in two categories basing on the separation system: filter and cyclone (centrifugation). The comparison of these systems in two different climatic conditions during pollen collection, in particular relative air humidity (RH), reveals some differences in the pollen quality. Regarding cyclone system, pollen RH increases with air RH increasing (RH), whereas in the case of filter system, pollen RH is about 10% independently to the air RH (**Figure 8**). This difference is due to the lower pressure inside the filter system and, therefore, lower temperature in comparison to external one, such that water vapor in the air is condensed and extracted.

The humidity of the pollen is important for pollen long-term storage. One advantage of artificial pollination is the possibility to store pollen at −18°C for years maintaining its viability.

**3.1. Materials and methods**

All the experiments were performed on *Actinidia deliciosa* cv. Hayward in field condition with three repetitions per treatment, among 5 years (2009–2013) in three different environments:

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 67

The comparing of pollination-systems (**Figures 9** and **10**) was conducted in collaboration with Agrion (Cuneo, www.agrion.it); the pollination was carried out with 90% of flowers at the stage of petal fall (with white pistils) with 600 g of pollen per hectare with a single-step distribution. The experimental design was a randomized block in standard orchards (female:male rate 1:6) with T-bar (Verona and Cuneo) and pergola (in Latina) trellis systems. The liquid distribution was 12 g/l of pollen in deionized water and 5 ml/l of activator PollenAid (Kiwi Pollen, New Zealand) for a total of 50 l/ha of water suspension. The machines used in the pol-

The role of *Lycopodium* was evaluated in 2013 in Verona by comparing two systems of dry pollination with and without *Lycopodium* added. *Lycopodium* was added to pollen in dry pollination as inert in some machines like Speedy. Experimental design consisted of three theses (two rows each): pollination with the Soffi@Polline system with pure pollen, pollination with SoffiaPolline with pollen:*Lycopodium* mixture (55%:45%), and pollination with Speedy with

To understand the relation between flowering stage and the type of pollination, dry or liquid, just before pollination the flowers were labeled according to their flowering stage (**Figure 11**). The signed stages were, according to BBCH scale [28] are the following: closed flower (55– 59), white petals (60–64), ocher petals (65–66), early petal fall (67), and petal fall (68) with most of the pistils white and stigmas viscous, just before pistils dry and ovary increasing (69). To understand the success of the pollination, about 100 fruits for three biological replicates were weighed at harvesting time (end of October). This experiment was repeated for 4 years (2010–2013) in Verona, Cuneo, and Latina using Soffi@PollineZ for dry pollination and

Usually, the best pollination method considered is the manual method of pon-pon but because of its considerable employment of labor is rarely used in commercial orchards. As shown in **Figure 10**, it was overcome by Soffi@PollineZ pollinator, probably because with pon-pon some flowers were not touched, whereas the pollen powder blown reaches all the canopy. Analogously, with Speedy some flowers were not pollinated and, in addition the role of *Lycopodium*, will be analyzed in another experiment. Good results were also obtained with liquid pollination applied 2 days before the other when the 90% of flowers at the stage of petal fall. In other terms, it seems that liquid pollination could be better before petal fall. The

Cuneo (NO Italy), Verona (NE Italy), and Latina (Central Italy).

lination system's comparative test were reported in **Figure 9**.

"ElettroEASY" (or similar diaphragm pump) for liquid pollination.

pollen:*Lycopodium* mixture (55%:45%).

**3.2. Result and discussion**

*3.2.1. Comparison of equipments for pollination*

following experiments will elucidate this aspect [8].

**Figure 8.** Pollen humidity related to collecting systems and environmental conditions (left) and effect of pollen humidity on germination during years of storage at −18°C (right).

For this purpose, pollen RH must be about 10–12%; in other ways its germinability decreases in direct proportionality with RH and years (**Figure 8**). For practical usage, it could be considered that pollen can be stored about 3 years if its humidity is low or after drying with silica gel.

#### **2.3. Conclusion**

The highest pollen quality was obtained when the pollen was picked up from the collecting machine frequently during the day (about every hour), to avoid any stresses, and stored at 4°C for no more than 7 days. Pollen can be stored at −18°C up to 3 years, better with low humidity or pre-dried to 10–12% with silica gel at 4°C. A recent method to estimate pollen viability was developed and is based on physical analysis of the single cells by impedance flow cytometry [27], and it could be interesting to compare the two methods especially during pollen storage.

## **3. The interaction between pollination systems and flowering stage in** *Actinidia*

High-quality pollen is essential for good pollination, but pollination efficiency depends also on the equipments used and the time of application: dry pollination with pure pollen or diluted with lycopodium, liquid pollination in water suspension with adjuvants, handing application, or with mechanized tools [8]. In this paragraph, the interaction between pollination systems and the flowering stage will be elucidated, in order to understand which is the best flowering stage in relation to the pollination system adopted.

#### **3.1. Materials and methods**

All the experiments were performed on *Actinidia deliciosa* cv. Hayward in field condition with three repetitions per treatment, among 5 years (2009–2013) in three different environments: Cuneo (NO Italy), Verona (NE Italy), and Latina (Central Italy).

The comparing of pollination-systems (**Figures 9** and **10**) was conducted in collaboration with Agrion (Cuneo, www.agrion.it); the pollination was carried out with 90% of flowers at the stage of petal fall (with white pistils) with 600 g of pollen per hectare with a single-step distribution. The experimental design was a randomized block in standard orchards (female:male rate 1:6) with T-bar (Verona and Cuneo) and pergola (in Latina) trellis systems. The liquid distribution was 12 g/l of pollen in deionized water and 5 ml/l of activator PollenAid (Kiwi Pollen, New Zealand) for a total of 50 l/ha of water suspension. The machines used in the pollination system's comparative test were reported in **Figure 9**.

The role of *Lycopodium* was evaluated in 2013 in Verona by comparing two systems of dry pollination with and without *Lycopodium* added. *Lycopodium* was added to pollen in dry pollination as inert in some machines like Speedy. Experimental design consisted of three theses (two rows each): pollination with the Soffi@Polline system with pure pollen, pollination with SoffiaPolline with pollen:*Lycopodium* mixture (55%:45%), and pollination with Speedy with pollen:*Lycopodium* mixture (55%:45%).

To understand the relation between flowering stage and the type of pollination, dry or liquid, just before pollination the flowers were labeled according to their flowering stage (**Figure 11**). The signed stages were, according to BBCH scale [28] are the following: closed flower (55– 59), white petals (60–64), ocher petals (65–66), early petal fall (67), and petal fall (68) with most of the pistils white and stigmas viscous, just before pistils dry and ovary increasing (69). To understand the success of the pollination, about 100 fruits for three biological replicates were weighed at harvesting time (end of October). This experiment was repeated for 4 years (2010–2013) in Verona, Cuneo, and Latina using Soffi@PollineZ for dry pollination and "ElettroEASY" (or similar diaphragm pump) for liquid pollination.

#### **3.2. Result and discussion**

For this purpose, pollen RH must be about 10–12%; in other ways its germinability decreases in direct proportionality with RH and years (**Figure 8**). For practical usage, it could be considered that pollen can be stored about 3 years if its humidity is low or after drying with

**Figure 8.** Pollen humidity related to collecting systems and environmental conditions (left) and effect of pollen humidity

The highest pollen quality was obtained when the pollen was picked up from the collecting machine frequently during the day (about every hour), to avoid any stresses, and stored at 4°C for no more than 7 days. Pollen can be stored at −18°C up to 3 years, better with low humidity or pre-dried to 10–12% with silica gel at 4°C. A recent method to estimate pollen viability was developed and is based on physical analysis of the single cells by impedance flow cytometry [27], and it could be interesting to compare the two methods especially during

**3. The interaction between pollination systems and flowering stage** 

best flowering stage in relation to the pollination system adopted.

High-quality pollen is essential for good pollination, but pollination efficiency depends also on the equipments used and the time of application: dry pollination with pure pollen or diluted with lycopodium, liquid pollination in water suspension with adjuvants, handing application, or with mechanized tools [8]. In this paragraph, the interaction between pollination systems and the flowering stage will be elucidated, in order to understand which is the

silica gel.

66 Pollination in Plants

**2.3. Conclusion**

on germination during years of storage at −18°C (right).

pollen storage.

**in** *Actinidia*

#### *3.2.1. Comparison of equipments for pollination*

Usually, the best pollination method considered is the manual method of pon-pon but because of its considerable employment of labor is rarely used in commercial orchards. As shown in **Figure 10**, it was overcome by Soffi@PollineZ pollinator, probably because with pon-pon some flowers were not touched, whereas the pollen powder blown reaches all the canopy. Analogously, with Speedy some flowers were not pollinated and, in addition the role of *Lycopodium*, will be analyzed in another experiment. Good results were also obtained with liquid pollination applied 2 days before the other when the 90% of flowers at the stage of petal fall. In other terms, it seems that liquid pollination could be better before petal fall. The following experiments will elucidate this aspect [8].

**Figure 9.** The commercial available equipments used in the pollination system's comparative test and their working capacity. (A) "speedy" (Dall'Agata, Forlì, Italy) is a battery dry distributor for pollen: *Lycopodium* (45–55%) mix (5–7 h/ ha); (B) "ElettroEASY" (Volpi, Mantova, Italy) is a battery diaphragm pump for liquid pollination (4 h/ha); (C) "Soffi@ PollineZ" (Biotac, Verona, Italy) is an engine blower with dry distributor for pure pollen for dry pollination (1 h/ha); (D) "Spruzz@Polline TR" (Gerbaudo, Cuneo, Italy) is a sprayer with fogger-type nozzles attached to the tractor, for liquid pollination (2 h/ha); (E) "pon-pon" (homemade ball covered with velluto) for flower-to-flower manual dry pollination) (25 h/ha); and (F) "Ventole" (Romani, Verona, Italy) consists of two fans attached to the tractor for air and pollen shuffling (0.5 h/ha).

106 g with pure pollen, and 75 g free pollinated fruit (data not shown). That result indicates that the presence of this inert may adversely affect fertilization, regardless of the distribution

**Figure 11.** Fruits marked at the time of harvesting with the ribbon attached during pollination in order to go back the

**Figure 10.** Average weight of the fruit pollinated with different equipments. Blue color is for liquid pollination systems: Spruzz@Polline TR 2x means double pollen dose, and Spruzz@Polline TR-2 means that the application was made 2 days before the other pollination. Yellow color is for dry pollination system: Green color is for natural pollination (control).

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 69

Different letters indicate statistically significant differences (ANOVA Tukey P 0.05).

After the first evidence where liquid pollination appears more efficient before petal fall, the interaction between flowering stage and pollination system was investigated. *Actinidia* flowering is scalar, and the same flower is viable for about 4 days, in normal climatic condition, after that the pistil degenerates and starts the fruit set (**Figure 11** and related movie). Regarding liquid pollination, the best results were at full bloom and at early petal fall (**Figures 11** and **12A**), whereas for dry pollination, the best results were reached at petal fall (**Figures 1** and **12B**) before pistil

system.

*3.2.3. Flowering stage*

original flowering stage during pollination.

#### *3.2.2. Role of Lycopodium in pollination*

The low pollination rate observed using the pollen-*Lycopodium* mix Speedy machine (**Figure 9A**) is due to the drying effect of the *Lycopodium* on pistils and does not depend on the machine: the addition of *Lycopodium* to the Soffi@Polline gave the same results.

The fruit size obtained in the thesis pollinated with pollen-*Lycopodium* mixture was lower than the thesis pollinated with pure pollen: average weight 96 g with the addition of lycopodium,

**Figure 10.** Average weight of the fruit pollinated with different equipments. Blue color is for liquid pollination systems: Spruzz@Polline TR 2x means double pollen dose, and Spruzz@Polline TR-2 means that the application was made 2 days before the other pollination. Yellow color is for dry pollination system: Green color is for natural pollination (control). Different letters indicate statistically significant differences (ANOVA Tukey P 0.05).

**Figure 11.** Fruits marked at the time of harvesting with the ribbon attached during pollination in order to go back the original flowering stage during pollination.

106 g with pure pollen, and 75 g free pollinated fruit (data not shown). That result indicates that the presence of this inert may adversely affect fertilization, regardless of the distribution system.

#### *3.2.3. Flowering stage*

*3.2.2. Role of Lycopodium in pollination*

(0.5 h/ha).

68 Pollination in Plants

addition of *Lycopodium* to the Soffi@Polline gave the same results.

The low pollination rate observed using the pollen-*Lycopodium* mix Speedy machine (**Figure 9A**) is due to the drying effect of the *Lycopodium* on pistils and does not depend on the machine: the

**Figure 9.** The commercial available equipments used in the pollination system's comparative test and their working capacity. (A) "speedy" (Dall'Agata, Forlì, Italy) is a battery dry distributor for pollen: *Lycopodium* (45–55%) mix (5–7 h/ ha); (B) "ElettroEASY" (Volpi, Mantova, Italy) is a battery diaphragm pump for liquid pollination (4 h/ha); (C) "Soffi@ PollineZ" (Biotac, Verona, Italy) is an engine blower with dry distributor for pure pollen for dry pollination (1 h/ha); (D) "Spruzz@Polline TR" (Gerbaudo, Cuneo, Italy) is a sprayer with fogger-type nozzles attached to the tractor, for liquid pollination (2 h/ha); (E) "pon-pon" (homemade ball covered with velluto) for flower-to-flower manual dry pollination) (25 h/ha); and (F) "Ventole" (Romani, Verona, Italy) consists of two fans attached to the tractor for air and pollen shuffling

The fruit size obtained in the thesis pollinated with pollen-*Lycopodium* mixture was lower than the thesis pollinated with pure pollen: average weight 96 g with the addition of lycopodium, After the first evidence where liquid pollination appears more efficient before petal fall, the interaction between flowering stage and pollination system was investigated. *Actinidia* flowering is scalar, and the same flower is viable for about 4 days, in normal climatic condition, after that the pistil degenerates and starts the fruit set (**Figure 11** and related movie). Regarding liquid pollination, the best results were at full bloom and at early petal fall (**Figures 11** and **12A**), whereas for dry pollination, the best results were reached at petal fall (**Figures 1** and **12B**) before pistil

and the artificial pollination must be done every 1–2 days, depending on the climate conditions. It is notable also that, due to physical properties, the pistil's exudate increases the pollen attached if it is powder, whereas decreases pollen adhesion if it is conveyed with water. Moreover, in dry pollination the fruit size is higher with respect to liquid pollination (**Figure 12**). This observation indicates indeed the receptivity of the ovules in the flower that is maximum just before pistil senescence (change from white to brown color) after petal fall. Often, early pollination leads to ovary-growing and pistil senescence even if not all ovules were fertilized, thus precluding the possibility of a complete pollination of the fruit. This phenomenon is visible observing the longitudinal section of the fruit (**Figure 13**) because, excluding phenomena of water stress, the ovule's maturation is not simultaneous and starts from the petiole side to the tip side of the flower.

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 71

New histological analysis is under way in order to study the relationship between flower stage and ovary maturation. The process from pollen adhesion to fertilization could be observed

In *Actinidia*, fertilization appends within only 6 h after pollination (**Figure 15**), and this aspect facilitates the study of the relation between the moment of pollination and the flower stage. The *Actinidia* floral biology could be useful as model of wind-pollinated trees in field condition.

Kiwifruit artificial pollination, in conventional orchard, increases the production up to 30% (**Figures 10** and **12**) due to bigger fruit size. Pollen is collected from male plants, and to maintain its viability is necessary to avoid high temperature and high humidity. In practice it is picked up from the collecting machine every 45 min and stored at 4°C for ready usage (up to 7 days) or for long storage at −18°C (3 years if its humidity is lower than 12%) (**Figure 8**). Both liquid and dry pollinations are effective if done at the right flowering stage: liquid pollination not later than early petal fall stage, dry pollination, with pure pollen, at petal fall stage (in cv. Hayward) when the pistils exudate is maximum (**Figures 11**–**13**), at early morning with high air humidity. In order to pollinate early and late flowering flowers, the pollination must be done in two steps or more in particular in yellow flash kiwifruit. In any cases, dry pollination seems to be most suitable because it is applied when the number of mature ovaries in the flower is maximum.

**Figure 14.** (A) Male flower, (B) pollen during germination under optical microscope (40×), and (C) magnification of

germinated pollen stained with aniline blue under UV light (100×).

in vivo by staining the pollen with aniline blue under UV light (**Figures 14** and **15**).

**3.3. Conclusion**

**Figure 12.** Average weights of the fruits pollinated by spray pollination system (A) and by dry pollination system (B). This experiment was repeated for 4 years in Verona, Cuneo, and Latina using Soffi@PollineZ for dry pollination and a sprayer diaphragm pump for liquid pollination. Different letters indicate statistically significant differences (ANOVA Tukey P 0.05).

senescence [8]. The pollination efficiency is evident at the harvest but could be useful approximately within 30 days after pollination (see related movie). In this period there are endosperm cellularizations that define the final fruit size and are important to proceed with the thinning of the bad pollinated fruit to avoid loose of energy and favorite the growing of the best pollinated fruit.

The flowering stage is easily described observing the petals, but it reflects more important aspect of the flower and in particular the pistil exudate, essential for pollen adhesion, germination, and the ovary receptivity. The pistil's exudate production increases during flower life and, in cv. Hayward, is maximum at the petal fall stage (**Figure 13**). For yellow flash kiwifruit, it is less evident, and the flower has a lower self-life compared with Hayward and evolves within 1–2 days to late flowering stages. In this case, it is not possible to wait that all flowers reach the petal fall stage

**Figure 13.** Easy test for the evaluation of pistil exudate production: the maximum dry pollen receptivity is at early morning with flowers at the petal fall stage when almost all ovules are receptive (left). Longitudinal section of the fruit showing lack of seeds on the tip due to not fertilized ovules because of an early pollination (right).

and the artificial pollination must be done every 1–2 days, depending on the climate conditions. It is notable also that, due to physical properties, the pistil's exudate increases the pollen attached if it is powder, whereas decreases pollen adhesion if it is conveyed with water. Moreover, in dry pollination the fruit size is higher with respect to liquid pollination (**Figure 12**). This observation indicates indeed the receptivity of the ovules in the flower that is maximum just before pistil senescence (change from white to brown color) after petal fall. Often, early pollination leads to ovary-growing and pistil senescence even if not all ovules were fertilized, thus precluding the possibility of a complete pollination of the fruit. This phenomenon is visible observing the longitudinal section of the fruit (**Figure 13**) because, excluding phenomena of water stress, the ovule's maturation is not simultaneous and starts from the petiole side to the tip side of the flower.

New histological analysis is under way in order to study the relationship between flower stage and ovary maturation. The process from pollen adhesion to fertilization could be observed in vivo by staining the pollen with aniline blue under UV light (**Figures 14** and **15**).

In *Actinidia*, fertilization appends within only 6 h after pollination (**Figure 15**), and this aspect facilitates the study of the relation between the moment of pollination and the flower stage. The *Actinidia* floral biology could be useful as model of wind-pollinated trees in field condition.

#### **3.3. Conclusion**

senescence [8]. The pollination efficiency is evident at the harvest but could be useful approximately within 30 days after pollination (see related movie). In this period there are endosperm cellularizations that define the final fruit size and are important to proceed with the thinning of the bad pollinated fruit to avoid loose of energy and favorite the growing of the best pollinated fruit. The flowering stage is easily described observing the petals, but it reflects more important aspect of the flower and in particular the pistil exudate, essential for pollen adhesion, germination, and the ovary receptivity. The pistil's exudate production increases during flower life and, in cv. Hayward, is maximum at the petal fall stage (**Figure 13**). For yellow flash kiwifruit, it is less evident, and the flower has a lower self-life compared with Hayward and evolves within 1–2 days to late flowering stages. In this case, it is not possible to wait that all flowers reach the petal fall stage

Tukey P 0.05).

70 Pollination in Plants

**Figure 12.** Average weights of the fruits pollinated by spray pollination system (A) and by dry pollination system (B). This experiment was repeated for 4 years in Verona, Cuneo, and Latina using Soffi@PollineZ for dry pollination and a sprayer diaphragm pump for liquid pollination. Different letters indicate statistically significant differences (ANOVA

**Figure 13.** Easy test for the evaluation of pistil exudate production: the maximum dry pollen receptivity is at early morning with flowers at the petal fall stage when almost all ovules are receptive (left). Longitudinal section of the fruit

showing lack of seeds on the tip due to not fertilized ovules because of an early pollination (right).

Kiwifruit artificial pollination, in conventional orchard, increases the production up to 30% (**Figures 10** and **12**) due to bigger fruit size. Pollen is collected from male plants, and to maintain its viability is necessary to avoid high temperature and high humidity. In practice it is picked up from the collecting machine every 45 min and stored at 4°C for ready usage (up to 7 days) or for long storage at −18°C (3 years if its humidity is lower than 12%) (**Figure 8**). Both liquid and dry pollinations are effective if done at the right flowering stage: liquid pollination not later than early petal fall stage, dry pollination, with pure pollen, at petal fall stage (in cv. Hayward) when the pistils exudate is maximum (**Figures 11**–**13**), at early morning with high air humidity. In order to pollinate early and late flowering flowers, the pollination must be done in two steps or more in particular in yellow flash kiwifruit. In any cases, dry pollination seems to be most suitable because it is applied when the number of mature ovaries in the flower is maximum.

**Figure 14.** (A) Male flower, (B) pollen during germination under optical microscope (40×), and (C) magnification of germinated pollen stained with aniline blue under UV light (100×).

competition among growing fruits and other sinks [39]. After petal fall, about 25% of the ovaries are retained, but only a small percentage of fruits reach maturity. It was estimated that a good commercial yield could be reacted if at least 1% of the total number of flowers set fruits

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 73

Many steps of pollination were optimized, and many parameters were evaluated during the experimentations. Artificial pollination was tested by taking advantage of previous expertise developed in artificial pollination of *Actinidia*, using Aspir@PollineMini2 (**Figure 16**) to suck pollen and Soffi@PollineZ (**Figures 9C** and **16**) (Biotac, Verona, Italy). Pollen was stored at 4°C for short-term usage and at low temperature (−20 and −80°C) for long-term usage. The influence of the time of distribution was evaluated using a completely randomized block design, with four replicates on cv. Leccino. Pollen germinability was evaluated as described for *Actinidia*. The pollination experiment design will be aimed at understanding: the influence of the pollinizer on productivity of fruit, the influence of artificial pollination on alternate bearing, the optimization of distribution in relation to the flowering stage, and the influence of the amount

**Figure 16.** Mr. D'Isola during pollen sucking from olive tree varieties compatible with Leccino (left) and during

and remaining until harvest [31].

**4.1. Materials and methods**

pollination (right).

**Figure 15.** (A) Female flower, (B) schematic section and flower organs, (C) section of flower under optical microscope (40×), (D) magnification of ovules (100×), and (E) the same section stained with aniline blue under UV light 6 h after pollination.

## **4. Artificial pollination in olive tree**

Olive trees (*Olea europaea* L.) bear both hermaphrodite and staminate flowers [29, 30] in the form of panicles [31]. Hermaphrodite flowers generally have two stamens and a bilocular ovary with a short style and stigma. Artificial pollination seems particularly suited also to olive tree because of a wind-pollinated crop, hermaphrodite but with many flowers specialized for pollen production, and in many cases self-incompatible [32]. In staminate flowers, the pistil is either rudimentary or absent. The flowers are not entomophilous pollinated, in fact they produce large quantity of pollen and don't have nectaries [33, 34]. The problems linked to pollination/fertilization olive cultivation are numerous: the blooming period of male and female trees does not overlap, and pistillate flowers are usually unable to receive pollen grain [35]; adverse climatic conditions during fruit set; compatibility relationships among cultivars; pollinizers could be bad oriented and/or in a non-satisfactory ratio with the cultivar of interest; and even if pollen is abundant, it could have low viability [35] and can be scarcely retained by the stigma surface. Moreover, depending on the cultivar, the environmental conditions, the specific tree and shoot, and ovary abortion could occur many weeks after pollination [36]. Shedding of staminate flowers begins just after full bloom [37] and partially overlaps the abscission of unfertilized flowers, triggered by pollination and fertilization of adjacent flowers. It takes place in the days after petal drop [38]. Most fertilized ovary abscission, occurring after 2 weeks and until about 6 weeks after full bloom, is affected by substrate competition among growing fruits and other sinks [39]. After petal fall, about 25% of the ovaries are retained, but only a small percentage of fruits reach maturity. It was estimated that a good commercial yield could be reacted if at least 1% of the total number of flowers set fruits and remaining until harvest [31].

#### **4.1. Materials and methods**

**4. Artificial pollination in olive tree**

pollination.

72 Pollination in Plants

Olive trees (*Olea europaea* L.) bear both hermaphrodite and staminate flowers [29, 30] in the form of panicles [31]. Hermaphrodite flowers generally have two stamens and a bilocular ovary with a short style and stigma. Artificial pollination seems particularly suited also to olive tree because of a wind-pollinated crop, hermaphrodite but with many flowers specialized for pollen production, and in many cases self-incompatible [32]. In staminate flowers, the pistil is either rudimentary or absent. The flowers are not entomophilous pollinated, in fact they produce large quantity of pollen and don't have nectaries [33, 34]. The problems linked to pollination/fertilization olive cultivation are numerous: the blooming period of male and female trees does not overlap, and pistillate flowers are usually unable to receive pollen grain [35]; adverse climatic conditions during fruit set; compatibility relationships among cultivars; pollinizers could be bad oriented and/or in a non-satisfactory ratio with the cultivar of interest; and even if pollen is abundant, it could have low viability [35] and can be scarcely retained by the stigma surface. Moreover, depending on the cultivar, the environmental conditions, the specific tree and shoot, and ovary abortion could occur many weeks after pollination [36]. Shedding of staminate flowers begins just after full bloom [37] and partially overlaps the abscission of unfertilized flowers, triggered by pollination and fertilization of adjacent flowers. It takes place in the days after petal drop [38]. Most fertilized ovary abscission, occurring after 2 weeks and until about 6 weeks after full bloom, is affected by substrate

**Figure 15.** (A) Female flower, (B) schematic section and flower organs, (C) section of flower under optical microscope (40×), (D) magnification of ovules (100×), and (E) the same section stained with aniline blue under UV light 6 h after Many steps of pollination were optimized, and many parameters were evaluated during the experimentations. Artificial pollination was tested by taking advantage of previous expertise developed in artificial pollination of *Actinidia*, using Aspir@PollineMini2 (**Figure 16**) to suck pollen and Soffi@PollineZ (**Figures 9C** and **16**) (Biotac, Verona, Italy). Pollen was stored at 4°C for short-term usage and at low temperature (−20 and −80°C) for long-term usage. The influence of the time of distribution was evaluated using a completely randomized block design, with four replicates on cv. Leccino. Pollen germinability was evaluated as described for *Actinidia*. The pollination experiment design will be aimed at understanding: the influence of the pollinizer on productivity of fruit, the influence of artificial pollination on alternate bearing, the optimization of distribution in relation to the flowering stage, and the influence of the amount

**Figure 16.** Mr. D'Isola during pollen sucking from olive tree varieties compatible with Leccino (left) and during pollination (right).

of pollen spread (standard application was 2 g per plant). To get a detailed experimentation, many parameters must be taken into account. During pollen collection the data recorded were air temperature and RH, amount of pollen per hour, cultivars collected, germinability, and pollen RH. Regarding pollination, the data were number of flowers per panicle, number of panicle per twig, position of the twig on the branch, position of the branch in the canopy, and cardinal orientation. In particular, two levels in the canopy and one shoot in each cardinal point per tree were considered. These parameters were recorded just before flowering and during all flowering time every day and once a week after the end of flowering for 6 weeks to take in account abortion and fruitlet abscission. The pollination was made one time in the early morning with moderate air temperature and higher RH, at the middle of flowering when 95% of flowers were open and many corollas were fallen down but with white pistils. After pollination other parameters were recorded: shoot length in order to evaluate the vegetative and reproductive competition and number of growing ovaries and fruits twice the time in the season. In order to evaluate the influence of the fruit, the number on the fruit quality, at harvesting was measured: harvesting time, total fruit collected per tree, fruit diameter, average weight, and oil yield. All these parameters were recorded also for some pollinizer plants in order to understand the influence of pollen collection in the hypothesis of a yield reduction due to pollen subtraction or not. Open-pollinated plants were used as control in all the experiments.

#### **4.2. Results and discussion**

Many data are available in literature about olive flower and fruit set biology; anyway, most of the knowledge are not adopted in the field. The experimentations started on 2014 thanks to the resourcefulness of an olive grower, Gianfranco D'Isola, on Lake of Garda (Brescia, North Italy). His 23 plants had never yielded anything being all Leccino, a self-incompatible cultivar without other pollinators nearby: the compatible cv. Frantoio was dead because of frost winter. The idea of artificial pollination came from kiwifruit pollination technique. The principle is simple: taking pollen from compatible varieties and, at the time of flowering, "blow" it on the target plants (**Figure 16**). Also, a modest improvement in the percentage of fruit setting leads to significant production increase [25]. Using the equipment employed in kiwifruit, the results were surprising: an average yield of 48 kg of olives per plant (**Figure 17**), an exceptional value compared to 10 kg, and the average production of North Italy [25, 40].

Despite the pollen collected during the day when air RH is low, olive pollen has a higher RH than kiwifruit pollen, and the incubation at 4°C for 24 h with silica gel before storing or spreading is suitable. Pollen germinability ranges from 35 to 68% and RH from 15 to 26% just after collection. After dehydration for 12 h at 4°C with silica gel, the RH decreases to 12–15%. During storing, the germinability decreases about 1.8% per day at 4°C and 3.4% per year during 3 years of observation when stored at −18°C. The collected pollen quantity seems not to be a problem thanks to the abundant production of olive tree in the "charging" season [35, 41] which are not economically sustainable during "off" season [40]. Anyway, pollen can be easily stored for several years in domestic fridge with a very low viability decrement. During harvesting period it was noted that the pollen collected at the beginning of the flowering is very low (few grams per hour), while after full blooming, when 20–30% of the corollas drop down, the amount reaches 100 g/h in many cultivars (also in "olivastro") and also 200 g/h in Ascolana. In these experiments, the fruit set improvement ranges from 10 to 30% more than the control free pollinated. Moreover, the pollinizer plant where pollen was collected, often self-compatible, showed a fruit set about 10% higher than the control (**Table 2**). It has been hypothesized that the movement of the braches and the panicles made during pollen suction increases pol-

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 75

**Table 2.** Example of data collected by AiPol on 2016 in sale Marasino (Brescia, IT) in a field trial of 20 plants of cv. Leccino with five cv. Casaliva as pollinizer. Different letters indicate statistically significant differences (ANOVA Tukey P 0.05).

**Figure 17.** Result of olive tree pollination on self-sterile cv. Leccino in 2014 (left) and 2016 (right).

len dispersion inside the tree.

Pollen was collected from olive cv. Pendolino, Moraiolo, and Casaliva and stored at 4°C for a few days until the time of pollination. Pollination was carried out with pure pollen in the early morning by delivering a total of about 2 g of pollen per plant in two steps within 2 days.

The experiment was successfully repeated in 2016 (**Figure 17**) [25], whereas in 2017 the extremely high temperature during olive flowering (the subsequently in weeks) and the absence of rain until the end of the season have provoked a copious fruit abscission.

These experiments have led to a larger trials on Lake of Garda cultivation conducted by AiPol (www.aipol.bs.it) in collaboration with CREA and new trials in other regions [40] and in other countries like in Japan by Associazione Italia Giappone and Biotac (www.biotac.it). Because these experiments aim to a practical application for farmers, the field trials were done in conventional orchards with pollinizers present in order to evaluate the effective gains given by the pollination technique in the real situations.

**Figure 17.** Result of olive tree pollination on self-sterile cv. Leccino in 2014 (left) and 2016 (right).

of pollen spread (standard application was 2 g per plant). To get a detailed experimentation, many parameters must be taken into account. During pollen collection the data recorded were air temperature and RH, amount of pollen per hour, cultivars collected, germinability, and pollen RH. Regarding pollination, the data were number of flowers per panicle, number of panicle per twig, position of the twig on the branch, position of the branch in the canopy, and cardinal orientation. In particular, two levels in the canopy and one shoot in each cardinal point per tree were considered. These parameters were recorded just before flowering and during all flowering time every day and once a week after the end of flowering for 6 weeks to take in account abortion and fruitlet abscission. The pollination was made one time in the early morning with moderate air temperature and higher RH, at the middle of flowering when 95% of flowers were open and many corollas were fallen down but with white pistils. After pollination other parameters were recorded: shoot length in order to evaluate the vegetative and reproductive competition and number of growing ovaries and fruits twice the time in the season. In order to evaluate the influence of the fruit, the number on the fruit quality, at harvesting was measured: harvesting time, total fruit collected per tree, fruit diameter, average weight, and oil yield. All these parameters were recorded also for some pollinizer plants in order to understand the influence of pollen collection in the hypothesis of a yield reduction due to pollen subtraction or

Many data are available in literature about olive flower and fruit set biology; anyway, most of the knowledge are not adopted in the field. The experimentations started on 2014 thanks to the resourcefulness of an olive grower, Gianfranco D'Isola, on Lake of Garda (Brescia, North Italy). His 23 plants had never yielded anything being all Leccino, a self-incompatible cultivar without other pollinators nearby: the compatible cv. Frantoio was dead because of frost winter. The idea of artificial pollination came from kiwifruit pollination technique. The principle is simple: taking pollen from compatible varieties and, at the time of flowering, "blow" it on the target plants (**Figure 16**). Also, a modest improvement in the percentage of fruit setting leads to significant production increase [25]. Using the equipment employed in kiwifruit, the results were surprising: an average yield of 48 kg of olives per plant (**Figure 17**), an excep-

tional value compared to 10 kg, and the average production of North Italy [25, 40].

until the end of the season have provoked a copious fruit abscission.

the pollination technique in the real situations.

Pollen was collected from olive cv. Pendolino, Moraiolo, and Casaliva and stored at 4°C for a few days until the time of pollination. Pollination was carried out with pure pollen in the early morning by delivering a total of about 2 g of pollen per plant in two steps within 2 days. The experiment was successfully repeated in 2016 (**Figure 17**) [25], whereas in 2017 the extremely high temperature during olive flowering (the subsequently in weeks) and the absence of rain

These experiments have led to a larger trials on Lake of Garda cultivation conducted by AiPol (www.aipol.bs.it) in collaboration with CREA and new trials in other regions [40] and in other countries like in Japan by Associazione Italia Giappone and Biotac (www.biotac.it). Because these experiments aim to a practical application for farmers, the field trials were done in conventional orchards with pollinizers present in order to evaluate the effective gains given by

not. Open-pollinated plants were used as control in all the experiments.

**4.2. Results and discussion**

74 Pollination in Plants

Despite the pollen collected during the day when air RH is low, olive pollen has a higher RH than kiwifruit pollen, and the incubation at 4°C for 24 h with silica gel before storing or spreading is suitable. Pollen germinability ranges from 35 to 68% and RH from 15 to 26% just after collection. After dehydration for 12 h at 4°C with silica gel, the RH decreases to 12–15%. During storing, the germinability decreases about 1.8% per day at 4°C and 3.4% per year during 3 years of observation when stored at −18°C. The collected pollen quantity seems not to be a problem thanks to the abundant production of olive tree in the "charging" season [35, 41] which are not economically sustainable during "off" season [40]. Anyway, pollen can be easily stored for several years in domestic fridge with a very low viability decrement. During harvesting period it was noted that the pollen collected at the beginning of the flowering is very low (few grams per hour), while after full blooming, when 20–30% of the corollas drop down, the amount reaches 100 g/h in many cultivars (also in "olivastro") and also 200 g/h in Ascolana.

In these experiments, the fruit set improvement ranges from 10 to 30% more than the control free pollinated. Moreover, the pollinizer plant where pollen was collected, often self-compatible, showed a fruit set about 10% higher than the control (**Table 2**). It has been hypothesized that the movement of the braches and the panicles made during pollen suction increases pollen dispersion inside the tree.


**Table 2.** Example of data collected by AiPol on 2016 in sale Marasino (Brescia, IT) in a field trial of 20 plants of cv. Leccino with five cv. Casaliva as pollinizer. Different letters indicate statistically significant differences (ANOVA Tukey P 0.05).

#### **4.3. Conclusions**

Regarding the practical application, these preliminary results confirm the possibility to improve olive yield by pollination and aim to understand the better moment for pollination in relation to the flowering stage during flowering. The pollination technique can also be used to balance the vegetative-reproductive balance of the plant and mitigate the alternation of production. Of course, the pollination must be inserted in a context of adequate agronomic management in order to support the greater production. In this sense, plants with a great fruit set must be "prepared" to support a larger production: fertilization and irrigation must adequately satisfy the major removal of the plant, pruning must balance vegetative and reproductive aspects and must be done every year, and, during the summer, plant protection must be applied in order not to vain all the improvements. Using the methods described (**Figure 4**), it is possible to set up experiments that allow to investigate the pollination process in more detail in order to understand, also at the molecular level (**Figures 14** and **15**), the fertilization, the fruitlet drop, and the alternate bearing. These experiments will help also to elucidate the real cultivar cross compatibility: conflicting reports about the classification of pollen compatibility exist. Often, these classifications came from field observation with contradictory results obtained in different locations and years [33, 34, 36, 42], and one of the main constraints is the overlapping of flowering period. The possibility to collect and store pollen for many years allows to test the compatibility of varieties with very different flowering times: it is possible also pollinate an early flowering variety with the pollen taken from a late flowering ones. Moreover, it is possible to establish a pollen bank for the farmers and for research purposes and identify some varieties with the higher compatibility producing the higher amount of pollen and perhaps a universal pollinizer. It could be interesting to develop also a super-intensive orchard where mechanized pollen collecting will be possible, analogously to the mechanical fruit harvesting, whereas the pollen spreading could be easily mechanized with blower for the tractor (already used in kiwifruit as reported in www.biotac.it) or with drones. Unlike happen in kiwifruit, in olive tree the pollination technique is at the begin but with very interesting perspectives.

**Competing interests**

**Author details**

**References**

1994. pp. 141-163

Institut. 2016;**183**:49

pp. 41-50

2016;**6**:417-426

Tacconi Gianni\* and Michelotti Vania

\*Address all correspondence to: gianni.tacconi@crea.gov.it

The authors declare that the research was conducted in the absence of any commercial or

Artificial Pollination in Kiwifruit and Olive Trees http://dx.doi.org/10.5772/intechopen.74831 77

financial relationships that could be construed as a potential conflict of interest.

CREA Research Centre for Genomics and Bioinformatics, Consiglio per la Ricerca in Agricoltura e l'analisi dell'economia agraria (CREA), Fiorenzuola d'Arda, Piacenza, Italy

[1] MacDaniels LH. The possibilities of hand pollination in the orchard on a commercial scale. Proceedings of the American Society for Horticultural Science. 1930;**27**:370-373 [2] Calderone NW. Insect pollinated crops, insect pollinators and US agriculture: Trend

[3] Sedgley M. Self-incompatibility in woody horticultural species. Genetic Co. E. G. Williams AEC RBK, editor. London: Kluwer Academic Publishers, Dordrecht; Boston;

[4] Klatt BK, Holzschuh A, Westphal C, Clough Y, Smit I, Pawelzik E, et al. Bee pollination improves crop quality, shelf life and commercial value. Philosophical Transactions of the Royal Society B: Biological Sciences. 2013;**281**(1775):20132440-20132440. Available

[5] Wietzke A, Westphal C, Kraft M, Gras P, Tscharntke T, Pawelzik E, et al. Pollination as a key factor for strawberry fruit physiology and quality. Berichte aus dem Jul Kühn-

[6] Ayerza R, Coates W. Supplemental pollination - increasing olive (Olea europaea) yields

[7] Hopping ME, Hacking NJ. A comparison of pollen application methods for the artificial pollination of kiwifruit. Vol. 139, Fruit Set and Development XXI IHC 139. 1982.

[8] Tacconi G, Michelotti V, Cacioppo O, Vittone G. Kiwifruit pollination: The interaction between pollen quality, pollination systems and flowering stage. Journal of Berry Research.

analysis of aggregate data for the period 1992-2009. PLoS One. 2012;**7**(5):1-27

from: http://rspb.royalsocietypublishing.org/cgi/doi/10.1098/rspb.2013.2440

in hot, arid environments. Experimental Agriculture. 2004;**40**(4):481-491

## **Acknowledgements**

We thank the cooperation of the following: for kiwifruit trials Graziano Vittone and Luca Nari (www.agrion.it), Laura Astegiano, Andrea Bonetti, Lorenzo Tacconi, and Ottavio Cacioppo (www.kiwiinforma.it); for olive trials in Italy Simone Frusca, Nicola Berini, Elena Savoldi, Emanuele Ghirardelli, Matteo Ghilardi, Elia Belotti, Paolo Zani (www.aipol.bs.it), and Gianfranco D'Isola; and for olive trials in Japan Yoko Nakajima and Vincenzo Andreacchio (http://www.aisitalia.it/olio-extra-vergine-di-oliva-made-in-japan.aspx#.WlueCjdJnFg). All the experiments and field trials were done without specific founding, except for the trials conducted by AIPOL. Parts of the chapter was taken from our previously published papers, with the permission of the coauthors.

## **Competing interests**

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

## **Author details**

**4.3. Conclusions**

76 Pollination in Plants

perspectives.

**Acknowledgements**

with the permission of the coauthors.

Regarding the practical application, these preliminary results confirm the possibility to improve olive yield by pollination and aim to understand the better moment for pollination in relation to the flowering stage during flowering. The pollination technique can also be used to balance the vegetative-reproductive balance of the plant and mitigate the alternation of production. Of course, the pollination must be inserted in a context of adequate agronomic management in order to support the greater production. In this sense, plants with a great fruit set must be "prepared" to support a larger production: fertilization and irrigation must adequately satisfy the major removal of the plant, pruning must balance vegetative and reproductive aspects and must be done every year, and, during the summer, plant protection must be applied in order not to vain all the improvements. Using the methods described (**Figure 4**), it is possible to set up experiments that allow to investigate the pollination process in more detail in order to understand, also at the molecular level (**Figures 14** and **15**), the fertilization, the fruitlet drop, and the alternate bearing. These experiments will help also to elucidate the real cultivar cross compatibility: conflicting reports about the classification of pollen compatibility exist. Often, these classifications came from field observation with contradictory results obtained in different locations and years [33, 34, 36, 42], and one of the main constraints is the overlapping of flowering period. The possibility to collect and store pollen for many years allows to test the compatibility of varieties with very different flowering times: it is possible also pollinate an early flowering variety with the pollen taken from a late flowering ones. Moreover, it is possible to establish a pollen bank for the farmers and for research purposes and identify some varieties with the higher compatibility producing the higher amount of pollen and perhaps a universal pollinizer. It could be interesting to develop also a super-intensive orchard where mechanized pollen collecting will be possible, analogously to the mechanical fruit harvesting, whereas the pollen spreading could be easily mechanized with blower for the tractor (already used in kiwifruit as reported in www.biotac.it) or with drones. Unlike happen in kiwifruit, in olive tree the pollination technique is at the begin but with very interesting

We thank the cooperation of the following: for kiwifruit trials Graziano Vittone and Luca Nari (www.agrion.it), Laura Astegiano, Andrea Bonetti, Lorenzo Tacconi, and Ottavio Cacioppo (www.kiwiinforma.it); for olive trials in Italy Simone Frusca, Nicola Berini, Elena Savoldi, Emanuele Ghirardelli, Matteo Ghilardi, Elia Belotti, Paolo Zani (www.aipol.bs.it), and Gianfranco D'Isola; and for olive trials in Japan Yoko Nakajima and Vincenzo Andreacchio (http://www.aisitalia.it/olio-extra-vergine-di-oliva-made-in-japan.aspx#.WlueCjdJnFg). All the experiments and field trials were done without specific founding, except for the trials conducted by AIPOL. Parts of the chapter was taken from our previously published papers, Tacconi Gianni\* and Michelotti Vania

\*Address all correspondence to: gianni.tacconi@crea.gov.it

CREA Research Centre for Genomics and Bioinformatics, Consiglio per la Ricerca in Agricoltura e l'analisi dell'economia agraria (CREA), Fiorenzuola d'Arda, Piacenza, Italy

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[10] Dicenta F, Ortega E, Cánovas JA, Egea J. Self-pollination vs. cross-pollination in almond: Pollen tube growth, fruit set and fruit characteristics. Plant Breeding. 2002;**121**(2):163-167

[11] Karimi HR, Mohammadi N, Estaji A, Esmaeilizadeh M. Effect of supplementary pollination using enriched pollen suspension with Zn on fruit set and fruit quality of pistachio.

[12] Vaknin Y, Gan-Mor S, Bechar A, Ronen B, Eisikowitch D. Effects of supplementary pollination on cropping success and fruit quality in pistachio. Plant Breeding. 2002;**121**(5):451-455

[13] Ellena M, Sandoval P, Gonzalez A, Galdames R, Jequier J, Contreras M, et al. Preliminary results of supplementary pollination on hazelnut in south Chile. Acta Horticulturae.

[14] Wallace HM, Vithanage V, Exley EM. The effect of supplementary pollination on nut set

[15] Howlett BG, Nelson WR, Pattemore DE, Gee M. Pollination of macadamia: Review and opportunities for improving yields. Scientia Horticulturae. 2015;**197**:411-419

[16] Awad MA. Pollination of date palm {*Phoenix dactylifera* L. cv. Khenazy by pollen grainwater suspension spray. Journal of Food, Agriculture and Environment. 2010;**8**(3-4

[17] Chao CCT, Krueger RR. The date palm (*Phoenix dactylifera* L.): Overview of biology, uses,

[18] Hopping ME, Jerram EM. Pollination of kiwifruit (*Actinidia chinensis* planch.): Stigmastyle structure and pollen tube growth. New Zealand Journal of Botany. 1979;**17**:233-240

[19] King MJ, Ferguson AM. Collection and use of dry pollen for pollination of kiwifruit.

[20] Cacioppo O. Impollinazione artificiale dell'actinidia con il metodo neozelandese a spru-

[21] Costa G, Testolin R, Vizzotto G. Kiwifruit pollination: An unbiased estimate of wind and bee contribution. New Zealand Journal of Crop and Horticultural Science. 1993;**21**(2):189-195

[23] Donati I, Buriani G, Cellini A, Mauri S, Costa G, Spinelli F. New insights on the bacterial canker of kiwifruit (*Pseudomonas syringae* pv. Actinidiae). Journal of Berry Research.

[24] Antunes MDC, Oliveira M, Teixeira M, Veloso A, Veloso F, Panagopoulos T. Evaluation of the effect of complementary pollination on Actinidia deliciosa "Hayward" in north-

[22] Spada G. Note sulla biologia fiorale dell'Actinidia. Kiwi Informa. 2005;**10-12**:35-43

New Zealand Journal of Crop and Horticultural Science. 1991;**19**(4):385-389

of macadamia (Proteaceae). Annals of Botany. 1996;**78**(6):765-773

of Horticultural Science and Biotechnology. 2001;**76**(2):208-212

Scientia Horticulturae (Amsterdam). 2017;**216**:272-7

and cultivation. HortScience. 2007;**42**(5):1077-1082

zzo vaporizzato. L'Informatore Agrar. 1992;XL VIII(4)

west Portugal. Acta Horticulturae. 2007:347-352

2014;**1052**:121-128

78 Pollination in Plants

PART 1):313-317

2014;**4**(2):53-67


[41] Ferrara G, Camposeo S, Palasciano M, Godini A. Comparison between two pollen quality evaluation methods and germination in Olive (*Olea europaea* L.). Acta Horticulturae. 2012;**949**:227-230

**Chapter 6**

**Provisional chapter**

**Pollen Germination** *in vitro*

**Pollen Germination** *in vitro*

http://dx.doi.org/10.5772/intechopen.75360

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

pigeonpea and its wild relatives are discussed.

DOI: 10.5772/intechopen.75360

Pollen germination *in vitro* is a reliable method to test the pollen viability. It also addresses many basic questions in sexual reproduction and particularly useful in wide hybridization. Many pollen germination medium ranging from simple sugars to complex one having vitamins, growth regulators, etc. in addition to various minerals have been standardized to germinate pollen artificially. The different media, successful pollen germination methods, procedures from pollen germination studies with wheat, rye, brinjal,

**Keywords:** *in vitro* pollen germination, grasses, PGM, pollen germination method

Pollen germination in the stigmatic tissue of *Portulaca* was first observed as early as 1824 by Amici and later he observed the germinating pollen tube entering ovule. Pollen acts as a vehicle for the transfer of male gametes to embryo of female plant. Pollen viability is an important factor in successful hybridization which may last from few minutes mostly in selfpollinated crops to many hours or days in cross pollinated crops. Assessing pollen viability is very crucial in artificial pollination especially involving different species or genera. Among diverse techniques used to assess pollen viability, *in vitro* pollen germination is the most reliable method. Pollen has been germinated in variety of media which differs from species to species and even for different varieties of a crop [1, 2]. Linskens [3] used simple sucrose/boric acid media and later many complex medium have been reported with addition of polyethylene glycol, various amino acids, etc. [4–6]. Among the many PGMs the one developed by

Brewbaker and Kwack [7] has been widely used with some alterations.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Jayaprakash P

Jayaprakash P

**Abstract**

**1. Introduction**

[42] Bartolini S, Guerriero R. Self-compatibility in several clones of oil olive cv. Leccino. Advances in Horticultural Science. 1995;**9**(2):71-74

#### **Chapter 6 Provisional chapter**

#### **Pollen Germination** *in vitro* **Pollen Germination** *in vitro*

#### Jayaprakash P Jayaprakash P

[41] Ferrara G, Camposeo S, Palasciano M, Godini A. Comparison between two pollen quality evaluation methods and germination in Olive (*Olea europaea* L.). Acta Horticulturae.

[42] Bartolini S, Guerriero R. Self-compatibility in several clones of oil olive cv. Leccino.

Advances in Horticultural Science. 1995;**9**(2):71-74

2012;**949**:227-230

80 Pollination in Plants

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.75360

**Abstract**

Pollen germination *in vitro* is a reliable method to test the pollen viability. It also addresses many basic questions in sexual reproduction and particularly useful in wide hybridization. Many pollen germination medium ranging from simple sugars to complex one having vitamins, growth regulators, etc. in addition to various minerals have been standardized to germinate pollen artificially. The different media, successful pollen germination methods, procedures from pollen germination studies with wheat, rye, brinjal, pigeonpea and its wild relatives are discussed.

DOI: 10.5772/intechopen.75360

**Keywords:** *in vitro* pollen germination, grasses, PGM, pollen germination method

## **1. Introduction**

Pollen germination in the stigmatic tissue of *Portulaca* was first observed as early as 1824 by Amici and later he observed the germinating pollen tube entering ovule. Pollen acts as a vehicle for the transfer of male gametes to embryo of female plant. Pollen viability is an important factor in successful hybridization which may last from few minutes mostly in selfpollinated crops to many hours or days in cross pollinated crops. Assessing pollen viability is very crucial in artificial pollination especially involving different species or genera. Among diverse techniques used to assess pollen viability, *in vitro* pollen germination is the most reliable method. Pollen has been germinated in variety of media which differs from species to species and even for different varieties of a crop [1, 2]. Linskens [3] used simple sucrose/boric acid media and later many complex medium have been reported with addition of polyethylene glycol, various amino acids, etc. [4–6]. Among the many PGMs the one developed by Brewbaker and Kwack [7] has been widely used with some alterations.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A brief discussion was made in this chapter to develop a pollen germination protocol for a crop/species.

• 100 mg l

• 300 mg l

• 19% Maltose • 13% PEG 6000

• 50 mg l

• 30 mg l

• 19% Maltose • 13% PEG 6000

• 50 mg l

−1 boric acid,

−1 calcium chloride

Peptone PGM of Wheat [2] (**Figure 1**)

Tryptone PGM of rye [10]

−1 boric acid

−1 boric acid

−1 calcium nitrate

Agarose medium [11]:

• 0.5% agarose • 18% sucrose

• 0.01% boric acid

• 1 mM CaCl2

• 1 mM Ca(NO<sup>3</sup>

**2.2. Pollen germination methods**

)2

and culture by any one of the following methods:

A basic medium contains a sugar, calcium nitrate and boric acid to which poly ethylene glycol, vitamins, amino acids, growth regulators etc. are added to make a complete pollen germi-

• 0.01% ferric ammonium citrate

• 0.03% casein enzymatic hydrolysate

• 30 mg l

• BK salts • 1% agar

• 80–100 mg l

−1 calcium nitrate

• BK salts • 1% agar −1 peptone

Pollen Germination *in vitro*

83

http://dx.doi.org/10.5772/intechopen.75360

Pollen is collected from freshly opened flowers and enough pollen is dusted over medium

• **Cavity slide technique (Rangaswamy and Shivanna** [12, 13]**)** : It is used for liquid pollen germination medium(PGM). A drop of medium is placed in the cavity, pollen is dusted over and it is covered with a dust free cover slip with its periphery sealed with Vaseline. It creates a required relative humidity inside. The slide is placed inversely over a pair of glass

nation medium. The pH and temperatures are also important factors.

• 1 mM KCl

• 0.01% myoinositol

• 0.25 mM spermidine

## **2. Different pollen germination media and methods**

#### **2.1. Pollen germination medium**

The composition of some of the successful media used pollen germination are given below

Brewbaker and Kwack medium [7]


Roberts medium [8]


PEG medium [4]


EACA medium [1]


• 250 mg l−1 boric acid

	- 1% agar

## Raffinose medium [9].

• 0.75 M raffinose,


### Peptone PGM of Wheat [2] (**Figure 1**)

• 19% Maltose

A brief discussion was made in this chapter to develop a pollen germination protocol for a

The composition of some of the successful media used pollen germination are given below

• 100 mg l−1 potassium nitrate • 200 mg l−1 magnesium sulfate

1000 mg l−1

• 1% agar

• E amino caproic acid (EACA)-0, 100, 250, 500, 750 or

**2. Different pollen germination media and methods**

crop/species.

82 Pollination in Plants

• 10% sucrose

• 100 mg l−1 boric acid

Roberts medium [8]

• 10 mg l−1 boric acid

• Tris 60–130 mg l−1

• 0.1 to 1.1 M PEG 400, • 100 mg l−1 Boric acid

PEG medium [4]

EACA medium [1]

• 37.5% sucrose • 15% PEG 4000

nitrate

Raffinose medium [9].

• 250 mg l−1 boric acid • 300 mg l−1 calcium

• 0.75 M raffinose,

• 362 mg l−1 calcium chloride • 100 mg l−1 potassium nitrate

• 20% sucrose

• 300 mg l−1 calcium nitrate

• 200 mg l−1 magnesium sulfate • 100 mg l−1 potassium nitrate

**2.1. Pollen germination medium**

Brewbaker and Kwack medium [7]


Tryptone PGM of rye [10]


Agarose medium [11]:


A basic medium contains a sugar, calcium nitrate and boric acid to which poly ethylene glycol, vitamins, amino acids, growth regulators etc. are added to make a complete pollen germination medium. The pH and temperatures are also important factors.

#### **2.2. Pollen germination methods**

Pollen is collected from freshly opened flowers and enough pollen is dusted over medium and culture by any one of the following methods:

• **Cavity slide technique (Rangaswamy and Shivanna** [12, 13]**)** : It is used for liquid pollen germination medium(PGM). A drop of medium is placed in the cavity, pollen is dusted over and it is covered with a dust free cover slip with its periphery sealed with Vaseline. It creates a required relative humidity inside. The slide is placed inversely over a pair of glass


rods in a humid chamber. A Petri dish is used as humid chamber where a moist filter paper is placed inside the lid.



Further in order to have a better visualization, a drop of stain was placed carefully over the medium droplet before observation. The extra stain was removed with a piece of dried filter paper. (Acetocarmine, and components of Alexander stain *viz*., Malachite green, Orange G, Aniline Blue and acid fuchsine may be used).

In our experiments initially liquid medium was used but profuse pollen bursting was seen due to quick hydration. To overcome this problem later agarified media were used by adding 0.5 to 1% agar to pollen germination medium. PGM droplet technique is a further refined technique which may be used effectively to fix the initial levels of inorganic salts level for factorial experiments. Each season, at least 200–300 fresh media were prepared and tested. This allowed large number of media to be tested in a single day, thus avoiding preparation of fresh medium each time and furthermore, more than two genotypes or species could be tested simultaneously.

**Figure 2.** PGM droplet technique. (a) Incubation chamber showing 3 × 3 combination of media; (b) unstained pollen

Pollen Germination *in vitro*

85

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**Sugars**: The sugar in the medium acts as an osmotic regulant which regulates the diffusion rate of water from the medium into pollen grains [16] and sucrose is generally used in PGM. The failure of pollen to germinate and its bursting may indicate lack of sugars within pollen grains and critical dependence on external supply. Sucrose has given satisfactory pollen germination for pigeonpea, wild pigeonpeas and wild brinjals whereas it was a poor osmatic regulant for

**Boron and calcium nitrate**: The mineral requirements are also different for any two species. Pollen grains are believed to be deficient in boron, which is normally compensated by high levels of boron present in stigma and style. Boron combines with sugar to form a sugar-borate complex which facilitates translocation of sugar molecules. Boron is reported to be toxic to plants even at as low as 5.1 ppm [17]. However, pollen seems to tolerate very high concentration of boron. Visser [18] showed that certain species of crop plants require as much as 1200 ppm boric acid for optimal germination and tube growth. Boron deficiency leads to pollen tube bursting as its required in the pollen wall structure [19]. Calcium is involved in cationic balance and is essential for tube elongation [7]. Pollen germination involves many ions with Ca2+ as the key player and extracellular calcium proved essential for pollen tip growth [20]. It was observed that the grass pollen (wheat and rye) requires very low levels of these minerals (30–50 mg l−1) as compared to the dicots (pigeonpea and brinjal) (300–400 mg l−1).

**3. Effect of different components of pollen germination medium** 

pollen of wheat, rye and brinjal. Maltose was used as carbon source for these species.

**(PGM) on pollen germination**

tubes; (c) pollen tube with acid fuchsine.

• **The cellulose membrane/agarose culture apparatus** [11]: Rectangular agarose pad was prepared over a microscopic slide with PGM containing 0.5% agarose. Appropriate size of cellulose membrane was cut and layered over agarose pad. The pollen is placed over the membrane and cultured in a moist chamber.

**Figure 1.** Wheat *in vitro* pollen germination (a) initiation of pollen germination (b, c, d) pollen germination after 5, 10, and 15 min of incubation.

rods in a humid chamber. A Petri dish is used as humid chamber where a moist filter paper

• **Agarified medium in Petri dishes** [14]**:** Pollen was extracted from fresh buds (with bud break) and was spread evenly on a drop of medium in a Petri dish and covered with Petri plate lined with moist filter paper. The plats were incubated in a BOD incubator with a

• **PGM droplet technique** [15]**:** In a Petri dish a droplet of pollen germination medium was placed using a glass rod. Thus drops of different media could be placed within few centimeter distance between them For example nine media can be placed in 3 × 3 fashion (**Figure 2**).

> 3-Medium C 6-Medium F 9-Medium I

Further in order to have a better visualization, a drop of stain was placed carefully over the medium droplet before observation. The extra stain was removed with a piece of dried filter paper. (Acetocarmine, and components of Alexander stain *viz*., Malachite green, Orange G,

• **The cellulose membrane/agarose culture apparatus** [11]: Rectangular agarose pad was prepared over a microscopic slide with PGM containing 0.5% agarose. Appropriate size of cellulose membrane was cut and layered over agarose pad. The pollen is placed over the

**Figure 1.** Wheat *in vitro* pollen germination (a) initiation of pollen germination (b, c, d) pollen germination after 5, 10,

temperature of 18°C/20.5°C depending on the pollen sample

2-Medium B 5-Medium E 8-Medium H

Aniline Blue and acid fuchsine may be used).

membrane and cultured in a moist chamber.

is placed inside the lid.

1-Medium A 4-Medium D 7-Medium G

84 Pollination in Plants

and 15 min of incubation.

**Figure 2.** PGM droplet technique. (a) Incubation chamber showing 3 × 3 combination of media; (b) unstained pollen tubes; (c) pollen tube with acid fuchsine.

In our experiments initially liquid medium was used but profuse pollen bursting was seen due to quick hydration. To overcome this problem later agarified media were used by adding 0.5 to 1% agar to pollen germination medium. PGM droplet technique is a further refined technique which may be used effectively to fix the initial levels of inorganic salts level for factorial experiments. Each season, at least 200–300 fresh media were prepared and tested. This allowed large number of media to be tested in a single day, thus avoiding preparation of fresh medium each time and furthermore, more than two genotypes or species could be tested simultaneously.

## **3. Effect of different components of pollen germination medium (PGM) on pollen germination**

**Sugars**: The sugar in the medium acts as an osmotic regulant which regulates the diffusion rate of water from the medium into pollen grains [16] and sucrose is generally used in PGM. The failure of pollen to germinate and its bursting may indicate lack of sugars within pollen grains and critical dependence on external supply. Sucrose has given satisfactory pollen germination for pigeonpea, wild pigeonpeas and wild brinjals whereas it was a poor osmatic regulant for pollen of wheat, rye and brinjal. Maltose was used as carbon source for these species.

**Boron and calcium nitrate**: The mineral requirements are also different for any two species. Pollen grains are believed to be deficient in boron, which is normally compensated by high levels of boron present in stigma and style. Boron combines with sugar to form a sugar-borate complex which facilitates translocation of sugar molecules. Boron is reported to be toxic to plants even at as low as 5.1 ppm [17]. However, pollen seems to tolerate very high concentration of boron. Visser [18] showed that certain species of crop plants require as much as 1200 ppm boric acid for optimal germination and tube growth. Boron deficiency leads to pollen tube bursting as its required in the pollen wall structure [19]. Calcium is involved in cationic balance and is essential for tube elongation [7]. Pollen germination involves many ions with Ca2+ as the key player and extracellular calcium proved essential for pollen tip growth [20]. It was observed that the grass pollen (wheat and rye) requires very low levels of these minerals (30–50 mg l−1) as compared to the dicots (pigeonpea and brinjal) (300–400 mg l−1).

**Polyethylene glycol (PEG)**: It is known to be a non-penetrating osmotic agent that decreases water potential of culture medium [21]. In pollen grains, PEG is considered to regulate the permeability of plasma membrane and to give stability to the pollen tube membrane [6] and to give stability to the pollen tube membrane. PEG of different molecular weight has been in pollen cultures of different species and in all cases it has a promontory effect [4, 6, 22]. Generally 10–15% of PEG was added to PGM. PEG 4000 was preferred for pigeonpea and brinjal whereas wheat and rye pollen germination satisfactorily with addition of PEG 6000.

**Peptone**: Inclusion of peptone arrested pollen tip bursting in wheat at concentration 80–120 mg l −1which was used in PGM for the first time [2]. Peptone has been used in plant tissue culture for various effects such as induction of shoot regeneration in avocado [23], promotion of hairy root formation in ginseng [24] etc. Addition of peptone, contained pollen tube burst to a great extent, it was less than 20% as compared to >85% in the medium which is devoid of peptone however, the level of peptone varied with genotypes.

Key media

Sucrose 10%

**Set I Set II**

Boric acid 100 mg l−1 Calcium nitrate 300 mg l−1 Potassium nitrate 100 mg l−1 Magnesium sulfate 200 mg l−1

boric acid and calcium nitrate.

tube growth of *C. platycarpus* was

Based on the germination/initiation of germination and bursting of pollen, one of the key media was first selected (**Figure 3**). The complete medium for each species was then standardized by altering the concentration of sucrose, boric acid and/or calcium nitrate one by one to obtain maximum pollen germination and good pollen tube growth. First sucrose concentration and temperature were varied to prevent bursting of pollen before germination. Secondly, varying concentrations of boric acid was tried *viz*., 50, 100, … 300 ppm keeping 300 ppm of calcium nitrate (as in BK medium) and other components unchanged. Lastly, the optimum concentration of calcium nitrate was determined (100, 200, … 600 ppm). The observations were recoded for three best concentrations of each

Pollen Germination *in vitro*

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http://dx.doi.org/10.5772/intechopen.75360

A. 10% sucrose + BK salts + 1% agar E. Medium A + 15% PEG 4000 B. 20% sucrose + BK salts + 1% agar F. Medium B + 15% PEG 4000 C. 30% sucrose + BK salts + 1% agar G. Medium B + 15% PEG 4000 D. 40% sucrose + BK salts + 1% agar H. Medium B + 15% PEG 4000

**4.1. Standardization of** *in vitro* **pollen germination medium for** *C. platycarpus*

the tube length was reduced considerably (33 μm), also pollen tube burst at the tips.

In the medium (B) containing 20% sucrose, 1% agar and standard Brewbaker and Kwack medium's salts, pollen of *C. platycarpus* showed 99.15% germination and tube length over 172 μm in 1 h duration at 20.5°C. Lowering the concentration of sucrose to 10% (medium A) and leaving other constituents unchanged gave 61.60% germination with a mean tube length of over 83.88 μm. Though the medium (C) containing 30% sucrose gave 94.25% germination,

The best medium which gave 99.15% germination for *in vitro* pollen germination and pollen

**ε‐Amino caproic acid (EACA)**: It is an amino acid derivative, a saturated six carbon fatty acid (C6 H12O2 , CH<sup>3</sup> (CH2 )4 -COOH) which occurs in milk fats. This immunosuppressor has been used to overcome self-incompatibility and incongruity in *Phaseolus* [25] and *Vigna* [26, 27]. It has been used as a component of PGM of pigeonpea for the first time [1] and subsequently in the medium of wheat and rye [2, 10].

**Tryptone**: It was good growth-stimulating nitrogen sources used in the cultivation of *Trichoderma hamatum* and *T. harzianum* [28]. It was found to be the best organic nitrogen source for kefiran production by *Lactobacillus kefiranofaciens* (Dainiel et al., 2015), asparaginase production from *Enterobacter cloacae* [29], production of biosurfactant by *Bacillus subtilis* [30]. In rye pollen germination, tryptone at 50–75 mg concentration gave satisfactory and reproducible level of pollen germination [2].

Besides many organic supplements were added to the PGM to enhance smooth pollen tube growth such as casein hydrolysate, vitamins etc.

## **4. A protocol to standardize** *in vitro* **pollen germination medium**

To standardize pollen germination medium for any crop initially a set of media are used. Based on the response, one of the media is picked up and modified. Here a procedure to develop a PGM for pigeonpea and its wild species is discussed. Initially two sets of key media(PGM) were used Set I consisted of Brewbaker and Kwack (BK) medium [7] with 1% agar at different levels of sucrose *viz*., 10, 20, 30 and 40% (A–D). With the addition of polyethylene glycol (PEG) 4000 at 15% concentration to each of A, B, C and D; the media E, F, G and H, respectively, were obtained. These were designated as Set II.

Composition of Brewbaker and Kwack's (BK) medium


Key media

**Polyethylene glycol (PEG)**: It is known to be a non-penetrating osmotic agent that decreases water potential of culture medium [21]. In pollen grains, PEG is considered to regulate the permeability of plasma membrane and to give stability to the pollen tube membrane [6] and to give stability to the pollen tube membrane. PEG of different molecular weight has been in pollen cultures of different species and in all cases it has a promontory effect [4, 6, 22]. Generally 10–15% of PEG was added to PGM. PEG 4000 was preferred for pigeonpea and brinjal whereas wheat and rye pollen germination satisfactorily with

**Peptone**: Inclusion of peptone arrested pollen tip bursting in wheat at concentration

tissue culture for various effects such as induction of shoot regeneration in avocado [23], promotion of hairy root formation in ginseng [24] etc. Addition of peptone, contained pollen tube burst to a great extent, it was less than 20% as compared to >85% in the medium which is

**ε‐Amino caproic acid (EACA)**: It is an amino acid derivative, a saturated six carbon fatty acid

used to overcome self-incompatibility and incongruity in *Phaseolus* [25] and *Vigna* [26, 27]. It has been used as a component of PGM of pigeonpea for the first time [1] and subsequently in

**Tryptone**: It was good growth-stimulating nitrogen sources used in the cultivation of *Trichoderma hamatum* and *T. harzianum* [28]. It was found to be the best organic nitrogen source for kefiran production by *Lactobacillus kefiranofaciens* (Dainiel et al., 2015), asparaginase production from *Enterobacter cloacae* [29], production of biosurfactant by *Bacillus subtilis* [30]. In rye pollen germination, tryptone at 50–75 mg concentration gave satisfactory

Besides many organic supplements were added to the PGM to enhance smooth pollen tube

To standardize pollen germination medium for any crop initially a set of media are used. Based on the response, one of the media is picked up and modified. Here a procedure to develop a PGM for pigeonpea and its wild species is discussed. Initially two sets of key media(PGM) were used Set I consisted of Brewbaker and Kwack (BK) medium [7] with 1% agar at different levels of sucrose *viz*., 10, 20, 30 and 40% (A–D). With the addition of polyethylene glycol (PEG) 4000 at 15% concentration to each of A, B, C and D; the media E, F, G and

**4. A protocol to standardize** *in vitro* **pollen germination medium**

H, respectively, were obtained. These were designated as Set II.

Composition of Brewbaker and Kwack's (BK) medium

devoid of peptone however, the level of peptone varied with genotypes.

−1which was used in PGM for the first time [2]. Peptone has been used in plant


addition of PEG 6000.

, CH<sup>3</sup>

(CH2 ) 4

the medium of wheat and rye [2, 10].

and reproducible level of pollen germination [2].

growth such as casein hydrolysate, vitamins etc.

80–120 mg l

86 Pollination in Plants

(C6 H12O2


Based on the germination/initiation of germination and bursting of pollen, one of the key media was first selected (**Figure 3**). The complete medium for each species was then standardized by altering the concentration of sucrose, boric acid and/or calcium nitrate one by one to obtain maximum pollen germination and good pollen tube growth. First sucrose concentration and temperature were varied to prevent bursting of pollen before germination. Secondly, varying concentrations of boric acid was tried *viz*., 50, 100, … 300 ppm keeping 300 ppm of calcium nitrate (as in BK medium) and other components unchanged. Lastly, the optimum concentration of calcium nitrate was determined (100, 200, … 600 ppm). The observations were recoded for three best concentrations of each boric acid and calcium nitrate.

#### **4.1. Standardization of** *in vitro* **pollen germination medium for** *C. platycarpus*

In the medium (B) containing 20% sucrose, 1% agar and standard Brewbaker and Kwack medium's salts, pollen of *C. platycarpus* showed 99.15% germination and tube length over 172 μm in 1 h duration at 20.5°C. Lowering the concentration of sucrose to 10% (medium A) and leaving other constituents unchanged gave 61.60% germination with a mean tube length of over 83.88 μm. Though the medium (C) containing 30% sucrose gave 94.25% germination, the tube length was reduced considerably (33 μm), also pollen tube burst at the tips.

The best medium which gave 99.15% germination for *in vitro* pollen germination and pollen tube growth of *C. platycarpus* was


**4.2. Standardization of** *in vitro* **pollen germination medium for** *C. volubilis*

On key media G (from Set II) the pollen of *C. volubilis* showed initiation of germination (budding) but almost all pollen grain showed bursting before or after budding. To contain bursting of pollen, sucrose level was increased to 35% and also the temperature was reduced to 18°C.

In order to improve germination, first boric acid concentration was altered keeping Ca(NO<sup>3</sup>

250 ppm to further improve the germination of *C. volubilis* pollen. At 100 ppm Ca(NO<sup>3</sup>

Sucrose 35%

Polyethylene glycol 15% Agar 1%

Temperature 18 ± 1°C

Calcium nitrate 100 mg l−1 Potassium nitrate 100 mg l−1 Magnesium sulfate 200 mg l−1

**4.3. Standardization of** *in vitro* **pollen germination media for** *C. cajan* **vars. Pusa 33** 

nitrate, it was observed that the medium with 40% sucrose, 250 ppm H<sup>3</sup>

In the medium D (Set I) only initiation of germination was observed whereas no germination was observed in media A, B and C. On varying the concentrations of boric acid and calcium

, 1% agar and BK salts gave 43.80% germination with abnormal pollen tubes.

From Set II, medium G showed over 45% germination (**Figure 2**) but there was a lot of bursting of pollen grains; medium F also showed little germination. To control the bursting of pollen grains sucrose level was raised to 37.5% and further, different combinations of boric acid and calcium nitrate were tried. Even though over 87% germination was seen, almost all the pollen grains showed bursting after germination. Among the permutations

has the following constituents:

This gives 94.26% pollen germination.

**and IMS‐1**

(NO<sup>3</sup> )2

at 300 ppm. At 250 ppm, maximum pollen germination was obtained (approximately 63%) and increasing or decreasing boric acid concentration over 250 ppm did not improve germination. Then, the concentration of calcium nitrate was modified keeping boric acid concentration at

len of *C. volubilis* showed 94.6% germination and pollen tube growth of 27.47 μm. *C. volubilis* showed 94.26% germination and pollen tube growth of 27.47 μm. Increasing the Ca(NO<sup>3</sup>

concentration to 200 ppm showed. The complete pollen germination medium for *C. volubilis*

)2

89

)2

) 2 , pol-

Pollen Germination *in vitro*

http://dx.doi.org/10.5772/intechopen.75360

BO<sup>3</sup>

, 300 ppm Ca

**Figure 3.** Procedure for standardization of *in vitro* pollen germination medium of *Cajanus* spp.

## **4.2. Standardization of** *in vitro* **pollen germination medium for** *C. volubilis*

On key media G (from Set II) the pollen of *C. volubilis* showed initiation of germination (budding) but almost all pollen grain showed bursting before or after budding. To contain bursting of pollen, sucrose level was increased to 35% and also the temperature was reduced to 18°C.

In order to improve germination, first boric acid concentration was altered keeping Ca(NO<sup>3</sup> )2 at 300 ppm. At 250 ppm, maximum pollen germination was obtained (approximately 63%) and increasing or decreasing boric acid concentration over 250 ppm did not improve germination.

Then, the concentration of calcium nitrate was modified keeping boric acid concentration at 250 ppm to further improve the germination of *C. volubilis* pollen. At 100 ppm Ca(NO<sup>3</sup> )2 , pollen of *C. volubilis* showed 94.6% germination and pollen tube growth of 27.47 μm. *C. volubilis* showed 94.26% germination and pollen tube growth of 27.47 μm. Increasing the Ca(NO<sup>3</sup> )2 concentration to 200 ppm showed. The complete pollen germination medium for *C. volubilis* has the following constituents:


This gives 94.26% pollen germination.

Sucrose 20%

88 Pollination in Plants

Agar 1%

Temperature 20.5 ± 2°C

**Figure 3.** Procedure for standardization of *in vitro* pollen germination medium of *Cajanus* spp.

Boric acid 100 mg l−1 Calcium nitrate 300 mg l−1 Magnesium sulfate 200 mg l−1 Potassium nitrate 100 mg l−1

## **4.3. Standardization of** *in vitro* **pollen germination media for** *C. cajan* **vars. Pusa 33 and IMS‐1**

In the medium D (Set I) only initiation of germination was observed whereas no germination was observed in media A, B and C. On varying the concentrations of boric acid and calcium nitrate, it was observed that the medium with 40% sucrose, 250 ppm H<sup>3</sup> BO<sup>3</sup> , 300 ppm Ca (NO<sup>3</sup> )2 , 1% agar and BK salts gave 43.80% germination with abnormal pollen tubes.

From Set II, medium G showed over 45% germination (**Figure 2**) but there was a lot of bursting of pollen grains; medium F also showed little germination. To control the bursting of pollen grains sucrose level was raised to 37.5% and further, different combinations of boric acid and calcium nitrate were tried. Even though over 87% germination was seen, almost all the pollen grains showed bursting after germination. Among the permutations and combinations of media tried by changing the concentration of boric acid and calcium nitrate, six combinations showed better germination (70–80%) but accompanied with bursting of pollen tubes.

#### *4.3.1. Pretreatments*

In order to standardize optimal media for pollen germination different methods of preconditioning the pollen were attempted. Heat treatment of pollen at 40°C for 30 min or at 60°C for 20 min did not improve germination of *C. cajan* pollen. Pre-hydration for 30 min in Petri dish lined with moist filter paper also did not improve germination. The stamen of pigeonpea is diadelphous (five short and five long stamens). Pollen extracted separately from short and long stamens showed approximately 70% germination. Pollen extracted from single anthers also showed about 70% germination. Thus, germination percentage did not vary between different anthers. Out of six combinations of media tried, the PGM with 250 ppm boric acid and 300 ppm calcium nitrate showed maximum normal pollen tubes.

Another pre-treatment of pollen was tried by incubating young buds (12 h or 36 h before anthesis) at 20.5°C in

To the above medium EACA at a concentration of 250–750 mg l−1 was added to arrest pollen

Pollen Germination *in vitro*

91

http://dx.doi.org/10.5772/intechopen.75360

The results of studies on *in vitro* pollen germination of *Cajanus cajan* and its wild species are

Based on the initial tests on *C. lineatus* it was found that modification of medium "G" could give optimum germination but due to non-availability of pollen, PGM could not be optimized.

**Optimal media composition** *C. platycarpus C. volubilis C. cajan* **vars. Pusa‐33 and IMS‐1**

Sucrose (%) 20 35 27.5 Boric acid (ppm) 100 250 250 Calcium nitrate (ppm) 300 100 300 Potassium nitrate 100 100 100 Magnesium sulfate 200 200 200 PEG 4000 (%) — 15 15 Agar (%) 1 1 1 Incubation period (h) 1 3 3

Sucrose 37.5%

PEG 4000 15% Agar 1%

Temperature 20.5 ± 20°C

Boric acid 250 mg l−1 Calcium nitrate 300 mg l−1 Potassium nitrate 100 mg l−1 Magnesium sulfate 300 mg l−1

Incubation temperature (°C) 20.5 18 20.5 % Pollen germination 99.15 94.26 92.52

**Table 1.** Results of *in vitro* pollen germination of *C. cajan* and its two wild relatives.

Pollen tube length (μm) 172.56 ± 0.315 27.47 ± 0.933 16.34 ± 0.264

tip bursting.

summarized in **Table 1**.


Then upon anther dehiscence, pollen was collected and germination was tested on six different media. The pollen from treatments (a), (b) and (c) showed good germination on medium G5 but burst soon after.

For treatments (d) and (e) media G<sup>1</sup> –G6 were used both for pre-treatments and germination tests. The pollen from the treatment (d) showed much variation for germination and bursting. Pollen extracted from the treatment (e) showed improved germination for *C. cajan* var. Pusa 33 and IMS-1. Among the media tested (G1 to G6 ) the medium (G<sup>5</sup> ) with 250 ppm H<sup>3</sup> BO<sup>3</sup> and 300 ppm Ca(NO<sup>3</sup> )2 showed over 92% germination for both varieties. Besides media G<sup>3</sup> , G<sup>4</sup> and G6 also showed over 90% germination but there was pollen tube bursting. The result indicated pre-treatment of young buds (36 h before anthesis) in PGM (G<sup>5</sup> ) for 36 h and germinating pollen in the same PGM gave over 92% germination Ca(NO<sup>3</sup> )2 some other media (G3 , G<sup>4</sup> and G6 ) also showed over 90% pollen germination but there was bursting of pollen tubes at tips and variation in pollen tube length.

Thus, the complete media for both Pusa-33 and IMS-1 should have the following composition:


and combinations of media tried by changing the concentration of boric acid and calcium nitrate, six combinations showed better germination (70–80%) but accompanied with

In order to standardize optimal media for pollen germination different methods of preconditioning the pollen were attempted. Heat treatment of pollen at 40°C for 30 min or at 60°C for 20 min did not improve germination of *C. cajan* pollen. Pre-hydration for 30 min in Petri dish lined with moist filter paper also did not improve germination. The stamen of pigeonpea is diadelphous (five short and five long stamens). Pollen extracted separately from short and long stamens showed approximately 70% germination. Pollen extracted from single anthers also showed about 70% germination. Thus, germination percentage did not vary between different anthers. Out of six combinations of media tried, the PGM with 250 ppm boric acid and 300 ppm calcium nitrate showed maximum

Another pre-treatment of pollen was tried by incubating young buds (12 h or 36 h before

Then upon anther dehiscence, pollen was collected and germination was tested on six different media. The pollen from treatments (a), (b) and (c) showed good germination on medium

tests. The pollen from the treatment (d) showed much variation for germination and bursting. Pollen extracted from the treatment (e) showed improved germination for *C. cajan* var. Pusa

to G6

also showed over 90% germination but there was pollen tube bursting. The result indicated

also showed over 90% pollen germination but there was bursting of pollen tubes at tips and

Thus, the complete media for both Pusa-33 and IMS-1 should have the following composition:

were used both for pre-treatments and germination

some other media (G3

) with 250 ppm H<sup>3</sup>

) for 36 h and germinating pol-

BO<sup>3</sup> and

, G<sup>4</sup> and

 and G6 )

, G<sup>4</sup>

) the medium (G<sup>5</sup>

)2

showed over 92% germination for both varieties. Besides media G<sup>3</sup>

bursting of pollen tubes.

*4.3.1. Pretreatments*

90 Pollination in Plants

normal pollen tubes.

anthesis) at 20.5°C in

G5

G6

(a) Petri dish lined with moist filter paper for 36 h.

(d) Pollen germination media for 12 h

(e) Pollen germination media for 36 h.

For treatments (d) and (e) media G<sup>1</sup>

)2

variation in pollen tube length.

33 and IMS-1. Among the media tested (G1

but burst soon after.

300 ppm Ca(NO<sup>3</sup>

(b) Agarified medium containing 37.5% sucrose in (a) for 36 h.

pre-treatment of young buds (36 h before anthesis) in PGM (G<sup>5</sup>

len in the same PGM gave over 92% germination Ca(NO<sup>3</sup>

(c) Agarified medium containing 37.5% sucrose +15% PEG in (a) for 36 h

–G6

To the above medium EACA at a concentration of 250–750 mg l−1 was added to arrest pollen tip bursting.

The results of studies on *in vitro* pollen germination of *Cajanus cajan* and its wild species are summarized in **Table 1**.

Based on the initial tests on *C. lineatus* it was found that modification of medium "G" could give optimum germination but due to non-availability of pollen, PGM could not be optimized.


**Table 1.** Results of *in vitro* pollen germination of *C. cajan* and its two wild relatives.

#### **4.4. Varietal response to PGMs**

It was noticed that there are genotypic differences for pollen germination. In pigeonpea, among the genotypes tested, IDTSP51 alone did not require EACA, but for others the requirement varied(250–750 mg l −1). Similar response was seen with wheat pollen germination. The medium supporting more than 90% pollen germination were selected for each genotype though some of them showed pollen tube bursting which indicated the need for fine tuning of the medium. The genotypes HW 971 and HW 741 responded well in M19 giving >95% pollen germination and pollen tube length of >400 μm. This is adjudged as the best medium since it did not require supplementing either EACA or peptone. It is noted that some genotypes (HD 2833, HW 971, HW 1095 etc.) require EACA alone in PGM whose response were at par with medium having EACA + peptone. Some varieties such as MACS 6195, HW 1095 etc. showed better responses (>92% germination) in PGM with a combination of EACA (500 or 750 mg) and peptone water (100 mg). These varieties responded poorly when peptone water was increased beyond 100 mg which reduced the pollen tube length (**Table 2**).

Among the varieties tested, two of them *viz*., HD 2833 and HW 1095 showed more that 94% in all the media tested. The genotype HD 2833 showed the maximum pollen tube length of

**Pollen germination** 

**(%)**

M19 + 750 E + 100 P 92.91 ± 4.53 371.87 ± 16.36 M19 + 500E + 120 P 90.82 ± 4.76 278.125 ± 18.13 M19 + 750 E + 120 P 96.37 ± 3.75 190 ± 3.65

achieved a mean pollen tube length of 190 μm in the same medium The variety HD 2833 showed a mean pollen tube length in the range of 334.37 to 713 μm followed by MACS6145 with 360.58 to 603.13 μm and HW671 with 462.50 to 551.25 μm for mean pollen length. Similar kind of genotypic differences for pollen germination requirement have also been reported [1].

A simple or a complex medium may be developed to suit the germination requirement of pollen (binucleate or trinucleate pollen). The protocol suggested would give a better guidance in development of PGM. The author has developed pollen germination medium (PGM) for many crops *viz.* pigeonpea and its wild species, wheat, rye and brinjal by using the base constituents of PGM of Brewbaker and Kwack [7]. These media were supplemented with polyethylene glycol, e-amino caproic acid, peptone etc. besides the addition of BK salts. Grasses pollen so far considered as recalcitrant can also be germinated in the artificial medium e.g. wheat and rye. The grass pollen is released at high moisture content (30–40%) as compared to 1–5% in the case of orthodox species. This trait makes it unsuitable for *in vitro* germination. With combination of maltose, PEG and tryptone/peptone contained the initial pollen bursting and resulted in successful pollen germination of wheat and rye. The grasses pollen was so far classified as recalcitrant [31]. It is observed that monocots require low salt content than the dicots in the pollen germination medium. Also wild species require minimal medium than the domesticate ones.

−1 peptone and the pollen of HW1095

**Pollen tube length (μm)** 

http://dx.doi.org/10.5772/intechopen.75360

**Range for pollen tube length (μm)** 93

Pollen Germination *in vitro*

−1 calcium nitrate + BK

**(mean ± S.E)**

−1 boric acid + 30 mg l

−1 EACA and 120 mg l

713 μm in PGM with 750 mg l

**Variety Pollen germination** 

**medium**

P = peptone; E = EACA.\*M19 = 19% Maltose + 13% PEG 6000 + 50 mg l

**Table 2.** Response of wheat genotypes in pollen germination medium.

**5. Conclusion**

salts + 1% agar.

**Author details**

Address all correspondence to: jpsarit@iari.res.in

ICAR-Indian Agricultural Research Institute, The Nilgiris, Tamil Nadu, India

Jayaprakash P



**Table 2.** Response of wheat genotypes in pollen germination medium.

Among the varieties tested, two of them *viz*., HD 2833 and HW 1095 showed more that 94% in all the media tested. The genotype HD 2833 showed the maximum pollen tube length of 713 μm in PGM with 750 mg l −1 EACA and 120 mg l −1 peptone and the pollen of HW1095 achieved a mean pollen tube length of 190 μm in the same medium The variety HD 2833 showed a mean pollen tube length in the range of 334.37 to 713 μm followed by MACS6145 with 360.58 to 603.13 μm and HW671 with 462.50 to 551.25 μm for mean pollen length. Similar kind of genotypic differences for pollen germination requirement have also been reported [1].

## **5. Conclusion**

**4.4. Varietal response to PGMs**

92 Pollination in Plants

requirement varied(250–750 mg l

**Variety Pollen germination** 

**medium**

length (**Table 2**).

It was noticed that there are genotypic differences for pollen germination. In pigeonpea, among the genotypes tested, IDTSP51 alone did not require EACA, but for others the

tion. The medium supporting more than 90% pollen germination were selected for each genotype though some of them showed pollen tube bursting which indicated the need for fine tuning of the medium. The genotypes HW 971 and HW 741 responded well in M19 giving >95% pollen germination and pollen tube length of >400 μm. This is adjudged as the best medium since it did not require supplementing either EACA or peptone. It is noted that some genotypes (HD 2833, HW 971, HW 1095 etc.) require EACA alone in PGM whose response were at par with medium having EACA + peptone. Some varieties such as MACS 6195, HW 1095 etc. showed better responses (>92% germination) in PGM with a combination of EACA (500 or 750 mg) and peptone water (100 mg). These varieties responded poorly when peptone water was increased beyond 100 mg which reduced the pollen tube

**Pollen germination** 

M19 + 500 E + 100 P 97.51 ± 2.98 553.75 ± 23.14 334.37–713.00

**(%)**

M19 + 750E 94.32 ± 3.67 667.7142 ± 32.18

M19 + 750 E + 100 P 95.38 ± 3.09 334.37 ± 18.39 M19 + 500 E + 120 P 97.12 ± 1.87 680.00 ± 24.255 M19 + 750 E + 120 P 97.22 ± 1.74 713.0 ± 21.55 HW2044 M19 + 500 E 98.02 ± 1.03 265.60 ± 9.88 265.60–467.50

M19 + 750E 98.01 ± 0.076 467.5 ± 9.55 HW5207 M19 + 750E 98.0 ± 0.043 313.529 ± 12.45 246–350.62 M19 + 500 E + 120 P 98 ± 0.054 350.625 ± 14.91 MACS 6145 M19 98.11 ± 0.87 360.58 ± 12.05 360.58–603.13 M19 + 750 E + 100 P 97.21 ± 1.54 603.13 ± 22.48 M19 + 750 E + 120 P 97 2.63 ± 2.11 546.25 ± 26.89 HW 971 M19 + 500 E 98 .13 ± 1.42 462.50 ± 18.76 462.50–551.25 M19 + 500 E + 100 P 98.0 ± 0.6 551.25 ± 25.85 HW 741 M19 98.23 ± 0.042 403.75 ± 21.13 241.87–403.75 M19 + 500 E 97.23 ± 1.99 241.87 ± 8.55

HD2833 \*M19 + 500 E 97.9 418.8 ± 15.39

HW1085 M19 + 500 E 98.18 ± 1.11 244.375 ± 11.44

HW1095 M19 + 500 E 98.01 ± 0.43 268.75 ± 11.83 190–371.87

−1). Similar response was seen with wheat pollen germina-

**Pollen tube length (μm)** 

**Range for pollen tube length (μm)**

**(mean ± S.E)**

A simple or a complex medium may be developed to suit the germination requirement of pollen (binucleate or trinucleate pollen). The protocol suggested would give a better guidance in development of PGM. The author has developed pollen germination medium (PGM) for many crops *viz.* pigeonpea and its wild species, wheat, rye and brinjal by using the base constituents of PGM of Brewbaker and Kwack [7]. These media were supplemented with polyethylene glycol, e-amino caproic acid, peptone etc. besides the addition of BK salts. Grasses pollen so far considered as recalcitrant can also be germinated in the artificial medium e.g. wheat and rye. The grass pollen is released at high moisture content (30–40%) as compared to 1–5% in the case of orthodox species. This trait makes it unsuitable for *in vitro* germination. With combination of maltose, PEG and tryptone/peptone contained the initial pollen bursting and resulted in successful pollen germination of wheat and rye. The grasses pollen was so far classified as recalcitrant [31]. It is observed that monocots require low salt content than the dicots in the pollen germination medium. Also wild species require minimal medium than the domesticate ones.

## **Author details**

Jayaprakash P

Address all correspondence to: jpsarit@iari.res.in

ICAR-Indian Agricultural Research Institute, The Nilgiris, Tamil Nadu, India

## **References**

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[2] Jayaprakash P, Annapoorani S, Vikas VK, Sivasamy M, Kumar1 J, Saravannan K, Punniakotti, Sheeba D. An improved *in vitro* germination medium for recalcitrant bread wheat pollen (*Triticum aestivum* L.). Indian Journal of Genetics and Plant Breeding.

[3] Linskens HF. Pollen. Handbuch der Pflanzenphysiologie. Vol. XVIII1967. pp. 368-406

[4] Zhang HQ, Croes AF. A new medium for pollen germination *in vitro*. Acta Botanica

[5] Shivanna KR, Saxena NP, Seetharama N. An improvised medium for *in vitro* pollen germination and pollen tube growth of chickpea. International Chickpea and Pigeonpea

[6] Read SM, Clarke A, Bacic A. Stimulation of growth of cultured *Nicotiana tabacum* W-38 pollen tubes by polyethylene glycol and Cu(II) salts. Protoplasma. 1993;**177**:1-14

[7] Brewbaker JL, Kwack BH. The calcium ion and substances influencing pollen growth. In: Linskens HF, editor. Pollen Physiology and Fertilization. Amsterdam: Elsevier North

[8] Roberts IN, Gaunde TC, Harrod G, Dickinson HG. Pollen stigma interaction in *Bracssica oleracea*: A new pollen germination medium and its use in elucidating the mechanism of

[9] Cheng C, McComb JA. *In vitro* germination of wheat pollen on raffinose medium. New

[10] Jayaprakash P, Sheeba D, Vikas VK, Sivasamy M, Kumar J, Annapoorani S, Krishan N. A reproducible *in vitro* pollen germination medium for recalcitrant cereal rye pollen (*Secale cereale* L.): Effect of tryptone. Indian Journal of Genetics and Plant Breeding.

[11] Rodriguez-Enriquez MJ, Mehdi S, Dickinson HG, Grant-Downton RT. A novel method for efficient *in vitro* germination and tube growth of *Arabidopsis thaliana* pollen. New

[12] Shivanna KR, Rangaswamy NS. Test for pollen viability. In: Pollen Biology: A Laboratory

[14] Singh IS, Bharti IS, Nandwal AS, Goswami C, Verma SK. Effect of temperature on *in vitro*

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[30] Gudina JE, Elisabete CF, Rodrigues AI, José AT, Lígia RR. Biosurfactant production by *Bacillus subtilis* using corn steep liquor as culture medium. Frontiers Microbiology. 2015:6-59

**Chapter 7**

Provisional chapter

. The greatest effect of

**Comparison of Pollination Graphs**

Comparison of Pollination Graphs

James H. Lee, David M. Chan and Rodney J. Dyer

James H. Lee, David M. Chan and Rodney J. Dyer

From the agent-based, correlated random walk model presented, we observe the effects of varying the maximum turning angle, δmax, tree density, ω, and pollen carryover, κmax, on the distribution of pollen within a tree population by examining pollination graphs. Varying maximum turning angle and pollen carryover alters the dispersal of pollen, which affects many measures of connectivity of the pollination graph. Among these measures the cluster-

DOI: 10.5772/intechopen.74553

varying ω is not on the clustering coefficient of fathers, but on the other measures of genetic diversity. In particular when comparing simulations with randomly placed trees with that of actual tree placement of C. florida at the VCU Rice Center, it is clear that having specific tree

Keywords: pollination network, correlated random walk, agent-based model, pollen

While the movement of genes from one generation to the next ensures the cohesiveness of plant species through time and space [1–3], the extent to which individual sites and populations are functionally connected is mitigated by both biotic and abiotic factors [4]. For wind dispersed pollen, features such as the direction and speed of the wind and physical properties of individual pollen grains [5] play prominent roles in how genes are carried across the landscape. In addition to intrinsic factors, site-specific features, such as the structural

> © 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

ing coefficient of fathers is largest when δmax is between 60 and 90<sup>∘</sup>

locations is crucial in determining the properties of a pollination graph.

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74553

Abstract

carryover, tree density

1. Introduction

[31] Franchi GG, Piotto B, Nepi M, Baskin CC, Baskin J, Pacini E. Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. Journal of Experimental Botany. 2011:1-15

## **Chapter 7**

Provisional chapter

## **Comparison of Pollination Graphs**

Comparison of Pollination Graphs

James H. Lee, David M. Chan and Rodney J. Dyer

James H. Lee, David M. Chan and Rodney J. Dyer

Additional information is available at the end of the chapter Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.74553

#### Abstract

[30] Gudina JE, Elisabete CF, Rodrigues AI, José AT, Lígia RR. Biosurfactant production by *Bacillus subtilis* using corn steep liquor as culture medium. Frontiers Microbiology.

[31] Franchi GG, Piotto B, Nepi M, Baskin CC, Baskin J, Pacini E. Pollen and seed desiccation tolerance in relation to degree of developmental arrest, dispersal, and survival. Journal

2015:6-59

96 Pollination in Plants

of Experimental Botany. 2011:1-15

From the agent-based, correlated random walk model presented, we observe the effects of varying the maximum turning angle, δmax, tree density, ω, and pollen carryover, κmax, on the distribution of pollen within a tree population by examining pollination graphs. Varying maximum turning angle and pollen carryover alters the dispersal of pollen, which affects many measures of connectivity of the pollination graph. Among these measures the clustering coefficient of fathers is largest when δmax is between 60 and 90<sup>∘</sup> . The greatest effect of varying ω is not on the clustering coefficient of fathers, but on the other measures of genetic diversity. In particular when comparing simulations with randomly placed trees with that of actual tree placement of C. florida at the VCU Rice Center, it is clear that having specific tree locations is crucial in determining the properties of a pollination graph.

DOI: 10.5772/intechopen.74553

Keywords: pollination network, correlated random walk, agent-based model, pollen carryover, tree density

## 1. Introduction

While the movement of genes from one generation to the next ensures the cohesiveness of plant species through time and space [1–3], the extent to which individual sites and populations are functionally connected is mitigated by both biotic and abiotic factors [4]. For wind dispersed pollen, features such as the direction and speed of the wind and physical properties of individual pollen grains [5] play prominent roles in how genes are carried across the landscape. In addition to intrinsic factors, site-specific features, such as the structural

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

complexity of the landscape and co-occurring species [6], also influence connectivity to an extent that it is easy to discern.

2.1. Field characteristics

2.2. Tree characteristics

simulated on this landscape, ω, is determined as

Simulations were also run for a field size of <sup>3100</sup> ffiffiffiffiffi

<sup>ω</sup> <sup>¼</sup> <sup>τ</sup>

trees per square unit, of ω ∈f g 0:0250; 0:0500; 0:0750; 0:1000; 0:1500 .

it 'bounces' off of the barrier at the opposite angle from which it approached.

For tracking purposes, each tree, <sup>T</sup>, is numbered such that 1 <sup>≤</sup> <sup>T</sup> <sup>≤</sup> <sup>τ</sup>. Let <sup>Y</sup>ð Þ <sup>T</sup> <sup>¼</sup> <sup>y</sup>

sired those seeds, and the frequency each tree fathered seeds on other trees.

heterogeneity influences broad trends in pollen connectivity.

The field size for model runs is set as a square 100 � 100 units grid. The density of the trees

where τ is the number of trees. In our simulation runs, we used tree densities, measured in

Previous simulation and empirical work has shown that density of pollen donors can have significant impacts on the genetic structure and diversity of offspring [6, 12], and as such should be a parameter across which we evaluate the other features of this model. The simulation field has rigid boundaries, and is considered impermeable. As such pollinators cannot leave the field nor are new pollinators allowed to enter the field during a model run. When an pollinator comes into contact with the edge of the field, its subsequent heading is set such that

<sup>541</sup> <sup>p</sup> � <sup>1100</sup> ffiffiffiffiffi

used based on data for trees sampled from the experimental natural population at the Virginia Commonwealth University, Rice Rivers Center (http://ricerivers.vcu.edu). This population was used as it has been the focus of previous work on pollen-mediated gene flow [11]. The tree density that results from this data set was ω ¼ 0:071552 trees per square unit, which was also simulated as a uniformly distributed scenario along with others previously mentioned.

the location of tree T, which is static. For the initial model runs, trees are randomly placed using a uniform random distribution within the allotted field size. For both simplicity and comparison to temperate tree species, we assume that all trees are self-incompatible and that all successful pollination and fertilization produces non-inbred individuals. This means that all pollination distances will be strictly greater than zero since at most one tree can occupy any location on the landscape. We also applied the model to a spatial arrangement of trees mimicking a natural population for which we have already conducted extensive empirical studies of insect-mediated pollination and gene flow. To create the pollination graph, data was collected to determine the number of seeds fertilized on each tree, which pollen donor trees are

For the second part of the study, coordinates of the trees at the VCU Rice Center were provided by the Dyer Laboratory [13]. These coordinates were used to create pollination graphs to compare with the random location pollination graphs to gauge the extent to which spatial

<sup>10</sup>; <sup>000</sup> <sup>¼</sup> <sup>0</sup>:0001τ, (1)

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<sup>541</sup> <sup>p</sup> <sup>≈</sup> <sup>133</sup>:<sup>2794</sup> � <sup>47</sup>:2927 units. This was

ð Þ T <sup>1</sup> ; y ð Þ T 2 � � be

<sup>100</sup> � <sup>100</sup> <sup>¼</sup> <sup>τ</sup>

Genes that are dispersed via active agents—animal mediated pollination—add increasing layers of involvement for at least two reasons. First, the way in which an animal disperser identifies, perceives, and interacts with features in the environment directly impact realized genetic connectivity. Over the last decade, enough work has been focused on this topic to denote a new subdiscipline of population genetic research, dubbed landscape genetics [7, 8], has been is devoted to developing methods for this task. From the perspective of the plant population, the characteristics of the intervening landscape determine the overall porosity of the landscape. Second, while the determination of which subset of features is important as it specifies the matrix through which pollinators move, the consequences to plant population genetic structure from alternative modes of pollinator movement is largely unexplored—even for the same plant species, alternative pollinating species leave discernibly distinct genetic structure [4, 9].

In this manuscript, we examine how the way in which pollinating individuals move across the landscape may influences population genetic structure. Here we develop an agent-based model (ABM) to simulate pollinator movement across a spatially explicit landscape. Individual pollinators are tracked as they pick up and disperse pollen among a set of individual plants. We adopt an underlying model of a correlated random walk (CRW) [10], where the direction and rate of movement is both temporally autocorrelated, though constrained. Within this framework, we explore the extent to which the spatial arrangement of trees interacts with variation in model parameters in producing variation in pollination statistics. We then apply this model to a data set from an natural population of the understory tree, Cornus florida L. (Cornaceae) [11]. This data set consists of both spatial and genetic information upon which previous landscape genetic studies have been conducted [11].

## 2. Background and methods

The agent based model developed herein uses two different categories of actors; trees act as the source and destination of pollen, and pollinating agents move individual pollen grains across the landscape. While the trees are spatially fixed on the landscape, the movement characteristics of the pollinating agents determine the ability of the plant population to maintain population genetic structure and determine relative reproductive output for individual trees. The movement of pollinating agents across the landscape is defined as a correlated random walk parameterized by inertia and speed. Across model runs, the aggregate movements of pollinating agents define a de facto pollination network whose characteristics are used to infer the robustness of the overall mating network and provide insights into population genetic stability. Across replicate runs, we extract parameters describing pollinator-movement dynamics parameters (average and maximum dispersal distances), pollen network robustness (pollen donor connectance and spatial clustering), and future population genetic structure (pollen donor density and diversity).

#### 2.1. Field characteristics

complexity of the landscape and co-occurring species [6], also influence connectivity to an

Genes that are dispersed via active agents—animal mediated pollination—add increasing layers of involvement for at least two reasons. First, the way in which an animal disperser identifies, perceives, and interacts with features in the environment directly impact realized genetic connectivity. Over the last decade, enough work has been focused on this topic to denote a new subdiscipline of population genetic research, dubbed landscape genetics [7, 8], has been is devoted to developing methods for this task. From the perspective of the plant population, the characteristics of the intervening landscape determine the overall porosity of the landscape. Second, while the determination of which subset of features is important as it specifies the matrix through which pollinators move, the consequences to plant population genetic structure from alternative modes of pollinator movement is largely unexplored—even for the same plant species, alterna-

In this manuscript, we examine how the way in which pollinating individuals move across the landscape may influences population genetic structure. Here we develop an agent-based model (ABM) to simulate pollinator movement across a spatially explicit landscape. Individual pollinators are tracked as they pick up and disperse pollen among a set of individual plants. We adopt an underlying model of a correlated random walk (CRW) [10], where the direction and rate of movement is both temporally autocorrelated, though constrained. Within this framework, we explore the extent to which the spatial arrangement of trees interacts with variation in model parameters in producing variation in pollination statistics. We then apply this model to a data set from an natural population of the understory tree, Cornus florida L. (Cornaceae) [11]. This data set consists of both spatial and genetic information upon which

The agent based model developed herein uses two different categories of actors; trees act as the source and destination of pollen, and pollinating agents move individual pollen grains across the landscape. While the trees are spatially fixed on the landscape, the movement characteristics of the pollinating agents determine the ability of the plant population to maintain population genetic structure and determine relative reproductive output for individual trees. The movement of pollinating agents across the landscape is defined as a correlated random walk parameterized by inertia and speed. Across model runs, the aggregate movements of pollinating agents define a de facto pollination network whose characteristics are used to infer the robustness of the overall mating network and provide insights into population genetic stability. Across replicate runs, we extract parameters describing pollinator-movement dynamics parameters (average and maximum dispersal distances), pollen network robustness (pollen donor connectance and spatial clustering), and future population genetic structure (pollen

tive pollinating species leave discernibly distinct genetic structure [4, 9].

previous landscape genetic studies have been conducted [11].

2. Background and methods

donor density and diversity).

extent that it is easy to discern.

98 Pollination in Plants

The field size for model runs is set as a square 100 � 100 units grid. The density of the trees simulated on this landscape, ω, is determined as

$$
\omega = \frac{\tau}{100 \times 100} = \frac{\tau}{10,000} = 0.0001\tau,\tag{1}
$$

where τ is the number of trees. In our simulation runs, we used tree densities, measured in trees per square unit, of ω ∈f g 0:0250; 0:0500; 0:0750; 0:1000; 0:1500 .

Previous simulation and empirical work has shown that density of pollen donors can have significant impacts on the genetic structure and diversity of offspring [6, 12], and as such should be a parameter across which we evaluate the other features of this model. The simulation field has rigid boundaries, and is considered impermeable. As such pollinators cannot leave the field nor are new pollinators allowed to enter the field during a model run. When an pollinator comes into contact with the edge of the field, its subsequent heading is set such that it 'bounces' off of the barrier at the opposite angle from which it approached.

Simulations were also run for a field size of <sup>3100</sup> ffiffiffiffiffi <sup>541</sup> <sup>p</sup> � <sup>1100</sup> ffiffiffiffiffi <sup>541</sup> <sup>p</sup> <sup>≈</sup> <sup>133</sup>:<sup>2794</sup> � <sup>47</sup>:2927 units. This was used based on data for trees sampled from the experimental natural population at the Virginia Commonwealth University, Rice Rivers Center (http://ricerivers.vcu.edu). This population was used as it has been the focus of previous work on pollen-mediated gene flow [11]. The tree density that results from this data set was ω ¼ 0:071552 trees per square unit, which was also simulated as a uniformly distributed scenario along with others previously mentioned.

#### 2.2. Tree characteristics

For tracking purposes, each tree, <sup>T</sup>, is numbered such that 1 <sup>≤</sup> <sup>T</sup> <sup>≤</sup> <sup>τ</sup>. Let <sup>Y</sup>ð Þ <sup>T</sup> <sup>¼</sup> <sup>y</sup> ð Þ T <sup>1</sup> ; y ð Þ T 2 � � be the location of tree T, which is static. For the initial model runs, trees are randomly placed using a uniform random distribution within the allotted field size. For both simplicity and comparison to temperate tree species, we assume that all trees are self-incompatible and that all successful pollination and fertilization produces non-inbred individuals. This means that all pollination distances will be strictly greater than zero since at most one tree can occupy any location on the landscape. We also applied the model to a spatial arrangement of trees mimicking a natural population for which we have already conducted extensive empirical studies of insect-mediated pollination and gene flow. To create the pollination graph, data was collected to determine the number of seeds fertilized on each tree, which pollen donor trees are sired those seeds, and the frequency each tree fathered seeds on other trees.

For the second part of the study, coordinates of the trees at the VCU Rice Center were provided by the Dyer Laboratory [13]. These coordinates were used to create pollination graphs to compare with the random location pollination graphs to gauge the extent to which spatial heterogeneity influences broad trends in pollen connectivity.

#### 2.3. Pollinator movement

Both natural and managed landscapes contain a broad range of species that are commonly distributed with a high degree of spatial heterogeneity. For tree species, reproductive structures may be nestled among several other taxa both below and above the target species in a mixed forest canopy. Under these conditions, a movement model based upon correlated random walk is preferred over alternatives such as Levy walks due to the complexity of the intervening landscape and the lack of long thoroughfares in the forest. Correlated Random Walk (CRW) models have been widely used to describe foraging behavior across a range of animal taxa [14–18].

In our simulations, we begin with an allotment of 1000 pollinators starting at random location with a random direction of travel on the simulated landscape. At each discrete time step, each pollinating agents will obtain a new heading based upon its previous heading with a specified random deviance. The individual will then move in this new direction 1 distance unit. This process continued for nmax time steps.

If a pollinator is within one unit distance of a tree, it will visit flowers on the tree. Each flower on a tree can be pollinated with equal probability. Pollinators visit one tree at a time. If multiple trees are within 1 unit the closest one is chosen. When visiting a tree, the pollinator may both gather pollen and deposit pollen from other trees. Due to the short length of the simulation we assume there are a sufficient number of flowers to gather pollen from and deposit pollen to on each tree.

Let β be the total number of pollinators in a simulation, and let Xð Þ<sup>i</sup> <sup>n</sup> ¼ x ð Þi <sup>1</sup>,n; x ð Þi 2,n be the location of the i th pollinator, 1 ≤ i ≤ β, at time step n, 0 ≤ n ≤ nmax. For all simulations, we assume β ¼ 1000 and nmax ¼ 600.

The initial position of each pollinator, Xð Þ<sup>i</sup> <sup>0</sup> is uniformly distributed throughout the field. Each pollinator's initial heading, �180<sup>∘</sup> <sup>≤</sup> <sup>θ</sup>ð Þ<sup>i</sup> <sup>1</sup> ≤ 180<sup>∘</sup> is chosen from a uniform random distribution. At each subsequent time step, the pollinator's new heading θð Þ<sup>i</sup> <sup>n</sup>þ<sup>1</sup> is dependent upon its current heading θð Þ<sup>i</sup> <sup>n</sup> and a random number δ ð Þi <sup>n</sup>þ<sup>1</sup>. That is

$$
\Theta\_{n+1}^{(i)} = \Theta\_n^{(i)} + \delta\_{n+1}^{(i)} \tag{2}
$$

Sample paths based on different values of δmax are shown in Figure 1. As δmax increases the paths do not generally travel away as far from the starting point and loop around more often.

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If a pollinator visits a flower, it will collect pollen from that individual. Pollen will be deposited with a probability of Pκ, where κ is the number of previously visited flowers, to account for pollen carryover. Carryover probability P<sup>κ</sup> ¼ 0 if κ > κmax where κmax is the maximum pollen

carryover. In the simulations the pollination carryovers used were κmax ∈ f g 1; 3; 5; 7; ∞ .

A path with δmax ¼ 0 would be along a straight line, which is not shown here.

2.4. Pollination

Figure 1. Sample paths based on δmax.

where δ ð Þi <sup>n</sup>þ<sup>1</sup> <sup>∈</sup> ð Þ �δmax; <sup>δ</sup>max for each <sup>n</sup> <sup>¼</sup> <sup>1</sup>, …, nmax. Similarly, the initial step size of each pollinator, r ð Þi <sup>1</sup> ¼ 1 at time n ¼ 1. Each subsequent step size, r ð Þi <sup>n</sup>þ<sup>1</sup> <sup>∈</sup> ð Þ <sup>0</sup>; <sup>r</sup>max uniformly distributed for each n ¼ 1, … , nmax. In Cartesian coordinates, the position of the i th pollinator at each subsequent time step will be

$$\mathbf{X}\_{n+1}^{(i)}\left(\mathbf{X}\_{n}^{(i)};\ r\_{n+1}^{(i)},\ \ \boldsymbol{\theta}\_{n+1}^{(i)}\right) = \left(\mathbf{x}\_{1,n}^{(i)} + r\_{n+1}^{(i)}\cos\left(\boldsymbol{\theta}\_{n+1}^{(i)}\right),\ \ \mathbf{x}\_{2,n}^{(i)} + r\_{n+1}^{(i)}\sin\left(\boldsymbol{\theta}\_{n+1}^{(i)}\right)\right) \tag{3}$$

for each <sup>n</sup> <sup>¼</sup> <sup>1</sup>, … , nmax. In our simulations we used values of <sup>δ</sup>max <sup>∈</sup> <sup>0</sup><sup>∘</sup> ; <sup>15</sup><sup>∘</sup> ; <sup>30</sup><sup>∘</sup> ; <sup>45</sup><sup>∘</sup> <sup>f</sup> ; 60<sup>∘</sup> ; 75<sup>∘</sup> ; 90<sup>∘</sup> ; 120<sup>∘</sup> ; <sup>150</sup><sup>∘</sup> ; <sup>180</sup><sup>∘</sup> <sup>g</sup>.

Sample paths based on different values of δmax are shown in Figure 1. As δmax increases the paths do not generally travel away as far from the starting point and loop around more often. A path with δmax ¼ 0 would be along a straight line, which is not shown here.

#### 2.4. Pollination

2.3. Pollinator movement

100 Pollination in Plants

animal taxa [14–18].

location of the i

heading θð Þ<sup>i</sup>

where δ

; 60<sup>∘</sup>

pollinator, r

ð Þi

; 75<sup>∘</sup> ; 90<sup>∘</sup>

ð Þi

Xð Þ<sup>i</sup> <sup>n</sup>þ<sup>1</sup> <sup>X</sup>ð Þ<sup>i</sup> <sup>n</sup> ; r ð Þi <sup>n</sup>þ<sup>1</sup>; <sup>θ</sup>ð Þ<sup>i</sup> nþ1

each subsequent time step will be

; 120<sup>∘</sup>

β ¼ 1000 and nmax ¼ 600.

process continued for nmax time steps.

The initial position of each pollinator, Xð Þ<sup>i</sup>

<sup>n</sup> and a random number δ

; <sup>150</sup><sup>∘</sup> ; <sup>180</sup><sup>∘</sup> <sup>g</sup>.

pollinator's initial heading, �180<sup>∘</sup> <sup>≤</sup> <sup>θ</sup>ð Þ<sup>i</sup>

Both natural and managed landscapes contain a broad range of species that are commonly distributed with a high degree of spatial heterogeneity. For tree species, reproductive structures may be nestled among several other taxa both below and above the target species in a mixed forest canopy. Under these conditions, a movement model based upon correlated random walk is preferred over alternatives such as Levy walks due to the complexity of the intervening landscape and the lack of long thoroughfares in the forest. Correlated Random Walk (CRW) models have been widely used to describe foraging behavior across a range of

In our simulations, we begin with an allotment of 1000 pollinators starting at random location with a random direction of travel on the simulated landscape. At each discrete time step, each pollinating agents will obtain a new heading based upon its previous heading with a specified random deviance. The individual will then move in this new direction 1 distance unit. This

If a pollinator is within one unit distance of a tree, it will visit flowers on the tree. Each flower on a tree can be pollinated with equal probability. Pollinators visit one tree at a time. If multiple trees are within 1 unit the closest one is chosen. When visiting a tree, the pollinator may both gather pollen and deposit pollen from other trees. Due to the short length of the simulation we assume there are a sufficient number of flowers to gather pollen from and deposit pollen to on each tree.

th pollinator, 1 ≤ i ≤ β, at time step n, 0 ≤ n ≤ nmax. For all simulations, we assume

<sup>n</sup> þ δ ð Þi

<sup>n</sup>þ<sup>1</sup> <sup>∈</sup> ð Þ �δmax; <sup>δ</sup>max for each <sup>n</sup> <sup>¼</sup> <sup>1</sup>, …, nmax. Similarly, the initial step size of each

<sup>n</sup> ¼ x ð Þi <sup>1</sup>,n; x ð Þi 2,n 

<sup>n</sup>þ<sup>1</sup> is dependent upon its current

<sup>n</sup>þ<sup>1</sup> <sup>∈</sup> ð Þ <sup>0</sup>; <sup>r</sup>max uniformly distrib-

nþ1

th pollinator at

; 15<sup>∘</sup> ; 30<sup>∘</sup> ; <sup>45</sup><sup>∘</sup> <sup>f</sup>

(3)

<sup>n</sup>þ<sup>1</sup> (2)

<sup>0</sup> is uniformly distributed throughout the field. Each

<sup>1</sup> ≤ 180<sup>∘</sup> is chosen from a uniform random distribution.

ð Þi

; x ð Þi <sup>2</sup>,n þ r ð Þi <sup>n</sup>þ<sup>1</sup>sin <sup>θ</sup>ð Þ<sup>i</sup>

nþ1  be the

Let β be the total number of pollinators in a simulation, and let Xð Þ<sup>i</sup>

ð Þi <sup>n</sup>þ<sup>1</sup>. That is

<sup>1</sup> ¼ 1 at time n ¼ 1. Each subsequent step size, r

¼ x ð Þi <sup>1</sup>,n þ r ð Þi <sup>n</sup>þ<sup>1</sup>cos <sup>θ</sup>ð Þ<sup>i</sup>

θð Þ<sup>i</sup> <sup>n</sup>þ<sup>1</sup> <sup>¼</sup> <sup>θ</sup>ð Þ<sup>i</sup>

uted for each n ¼ 1, … , nmax. In Cartesian coordinates, the position of the i

for each <sup>n</sup> <sup>¼</sup> <sup>1</sup>, … , nmax. In our simulations we used values of <sup>δ</sup>max <sup>∈</sup> <sup>0</sup><sup>∘</sup>

At each subsequent time step, the pollinator's new heading θð Þ<sup>i</sup>

If a pollinator visits a flower, it will collect pollen from that individual. Pollen will be deposited with a probability of Pκ, where κ is the number of previously visited flowers, to account for pollen carryover. Carryover probability P<sup>κ</sup> ¼ 0 if κ > κmax where κmax is the maximum pollen carryover. In the simulations the pollination carryovers used were κmax ∈ f g 1; 3; 5; 7; ∞ .

Figure 1. Sample paths based on δmax.

As a pollinator visits multiple flowers, the chances that it deposits pollen from a previous flower diminishes with each successive flower visited [19]. It was shown by [20] that from a given flower, a pollinator will deposit roughly <sup>γ</sup>ð Þ <sup>1</sup> � <sup>γ</sup> <sup>k</sup>�<sup>1</sup> pollen grains onto the <sup>k</sup> th flower visited, where γ depends upon the type of pollen as well as the type of pollinator. In this study, the probability that an pollinator distributes pollen from one tree to another tree is given by

$$P\_{\kappa} = \begin{cases} \rho (1 - \rho)^{\kappa - 1} & \text{if } \kappa \le \kappa\_{\text{max}} \\ 0 & \text{otherwise} \end{cases} \tag{4}$$

The connectance, L, of a graph is defined as the proportion of realized pollination events to the

As with the previous parameters, connectance has been used in a number of settings, see [23, 25–28], where the graphs is between species. In this study the focus is on individuals, and so the connectance is the proportion of realized pollination events between individual plants.

Furthermore, if there is an edge between tree m and tree f where tree f ∈ ϕm, then denote bf ,m to be the weight of that edge, which is equal to the number of times tree f fathers seeds on tree m.

seeds that tree f fathers on tree m. The weighted diversity of fathers for a mother tree m is a weighted measurement of the number of fathers that contribute pollen to seeds on m, accounting for the various number of seeds fathered by each father tree. The weighted diversity of

> Pj j <sup>ϕ</sup><sup>m</sup> <sup>f</sup> <sup>¼</sup><sup>1</sup> bf ,m � �<sup>2</sup>

Pj j <sup>ϕ</sup><sup>m</sup> <sup>f</sup> <sup>¼</sup><sup>1</sup> bf ,m

The average weighted diversity of fathers is the mean average of the weighted diversity of fathers

X μ

m¼1

The average pollination distance for an pollinator i is the average of the distances between any

� � be the location of mother tree <sup>m</sup>ð Þ<sup>i</sup> where pollinator <sup>i</sup> delivers pollen from tree

y <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>1</sup> � y

� � be the location of father tree <sup>f</sup>

f ð Þ<sup>i</sup> ð Þ 1 � �<sup>2</sup>

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

<sup>þ</sup> <sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>2</sup> � y

f ð Þ<sup>i</sup> ð Þ 2

τ τð Þ � <sup>1</sup> : (6)

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� �, which is a <sup>τ</sup> � <sup>τ</sup> matrix such that bf ,m is the number of

� �<sup>2</sup> : (7)

bFm, (8)

, achieved by pollinator i for all such

f ð Þ<sup>i</sup> ð Þ 2 � �<sup>2</sup>

ð Þ<sup>i</sup> and <sup>Y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>¼</sup>

, (9)

, for each

<sup>m</sup> are the number of

number of possible pollination events [24]. The connectance of the graph is given by

P<sup>τ</sup> f ¼1 P<sup>τ</sup> <sup>m</sup>¼<sup>1</sup> af ,m

L ¼

fathers, bFm, is computed for each m in the graph by the formula

over all mother trees, and is given by the formula

two trees mated by i. Let Y <sup>f</sup>

<sup>D</sup>ð Þ<sup>i</sup> <sup>¼</sup> <sup>1</sup> μð Þ<sup>i</sup>

<sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>1</sup> ; <sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ 2

pairings is

f

where 0 ≤ μ ≤ τ is the total number individuals in the graph.

ð Þ<sup>i</sup> ð Þ <sup>¼</sup> <sup>y</sup>

ð Þ<sup>i</sup> to tree mð Þ<sup>i</sup> . Then the average pollination distance, Dð Þ<sup>i</sup>

X μð Þ<sup>i</sup>

X ϕð Þ<sup>i</sup> m

f ¼1

m¼1

f ð Þ<sup>i</sup> ð Þ <sup>1</sup> ; y

1 ϕð Þ<sup>i</sup> m

where μð Þ<sup>i</sup> is the number of mother trees pollinated by pollinator i, and ϕ<sup>i</sup>

s

father trees pollinating tree m by pollinator i. The average pollination distance, Dð Þ<sup>i</sup>

bFm ¼

<sup>E</sup> <sup>¼</sup> <sup>1</sup> μ

With this we create the matrix B ¼ bi,j

where κ is the number of previously visited flowers, and r is the chance of pollination when κ ¼ 1. For the simulation work we used r ¼ 0:30 based on work by [21].

#### 2.5. Statistics

To characterize pollen movement and how it responds to the parameters of the models, pollination graphs were constructed. The connectivity network is based upon the physical location of individual trees and the pattern of spatial pollen movement created by the pollinators. In this network, each tree is represented as a node and the edges designate the movement of pollen from donor (paternal individual) to recipient (maternal individual), creating a directed pollination graph.

The parameters we vary in constructing these networks include tree density, ω, pollination carryover, κ, and pollinator maximum turning angle, δmax. To determine the effect of these parameters on the landscape pattern of connectivity, we estimated the number of fathers per mother, Φm, connectance, L, average weighted diversity of fathers, E, average pollination distance, D, average maximum pollination distance, D~ , and the weak and strong clustering coefficients of fathers, Cweak and Cstrong.

Each tree has the ability to contribute pollen to other trees and to accept pollen from other trees. When applicable, we will refer to a tree as a father tree, f , if that tree contributes pollen to another tree. We will refer to a tree as a mother tree, m, if that tree is accepts pollen from another tree. Let ϕ<sup>m</sup> be the set of trees which father seeds on tree m, then the number of fathers for each tree m in the graph is ϕ<sup>m</sup> � � � �, m ¼ 1, 2, … , τ, where ∣ � ∣ denotes cardinality. The set containing the number of fathers per mother for all trees in the graph is

$$\Phi\_m = \{ |\phi\_1|, \ |\phi\_2|, \ \dots, \ |\phi\_\tau| \}. \tag{5}$$

This value is similar to the degree distribution in the studies by Ramos-Jilberto et al. [22] and Valdovions et al. [23].

From this construct, we then create the τ � τ adjacency matrix, A ¼ ai,j � �, where af ,m <sup>¼</sup> 1 if tree f fathers at least one seed on tree m, and 0 if not. Thus A is a binary representation of the connectance of the graph. Since the trees do not self-pollinate, the number of possible interactions on this matrix is τ τð Þ � 1 .

The connectance, L, of a graph is defined as the proportion of realized pollination events to the number of possible pollination events [24]. The connectance of the graph is given by

$$L = \frac{\sum\_{f=1}^{\tau} \sum\_{m=1}^{\tau} a\_{f,m}}{\tau(\tau - 1)}. \tag{6}$$

As with the previous parameters, connectance has been used in a number of settings, see [23, 25–28], where the graphs is between species. In this study the focus is on individuals, and so the connectance is the proportion of realized pollination events between individual plants.

Furthermore, if there is an edge between tree m and tree f where tree f ∈ ϕm, then denote bf ,m to be the weight of that edge, which is equal to the number of times tree f fathers seeds on tree m. With this we create the matrix B ¼ bi,j � �, which is a <sup>τ</sup> � <sup>τ</sup> matrix such that bf ,m is the number of seeds that tree f fathers on tree m. The weighted diversity of fathers for a mother tree m is a weighted measurement of the number of fathers that contribute pollen to seeds on m, accounting for the various number of seeds fathered by each father tree. The weighted diversity of fathers, bFm, is computed for each m in the graph by the formula

$$\widehat{F}\_m = \frac{\left(\sum\_{f=1}^{\left|\phi\_m\right|} b\_{f,m}\right)^2}{\sum\_{f=1}^{\left|\phi\_m\right|} \left(b\_{f,m}\right)^2}.\tag{7}$$

The average weighted diversity of fathers is the mean average of the weighted diversity of fathers over all mother trees, and is given by the formula

$$E = \frac{1}{\mu} \sum\_{m=1}^{\mu} \quad \widehat{F}\_{m\prime} \tag{8}$$

where 0 ≤ μ ≤ τ is the total number individuals in the graph.

As a pollinator visits multiple flowers, the chances that it deposits pollen from a previous flower diminishes with each successive flower visited [19]. It was shown by [20] that from a

visited, where γ depends upon the type of pollen as well as the type of pollinator. In this study, the probability that an pollinator distributes pollen from one tree to another tree is given by

> <sup>P</sup><sup>κ</sup> <sup>¼</sup> <sup>r</sup>ð Þ <sup>1</sup> � <sup>r</sup> <sup>κ</sup>�<sup>1</sup> if <sup>κ</sup> <sup>≤</sup> <sup>κ</sup>max 0 otherwise

where κ is the number of previously visited flowers, and r is the chance of pollination when

To characterize pollen movement and how it responds to the parameters of the models, pollination graphs were constructed. The connectivity network is based upon the physical location of individual trees and the pattern of spatial pollen movement created by the pollinators. In this network, each tree is represented as a node and the edges designate the movement of pollen from donor (paternal individual) to recipient (maternal individual), creating a

The parameters we vary in constructing these networks include tree density, ω, pollination carryover, κ, and pollinator maximum turning angle, δmax. To determine the effect of these parameters on the landscape pattern of connectivity, we estimated the number of fathers per mother, Φm, connectance, L, average weighted diversity of fathers, E, average pollination distance, D, average maximum pollination distance, D~ , and the weak and strong clustering coefficients of fathers,

Each tree has the ability to contribute pollen to other trees and to accept pollen from other trees. When applicable, we will refer to a tree as a father tree, f , if that tree contributes pollen to another tree. We will refer to a tree as a mother tree, m, if that tree is accepts pollen from another tree. Let ϕ<sup>m</sup> be the set of trees which father seeds on tree m, then the number of fathers

This value is similar to the degree distribution in the studies by Ramos-Jilberto et al. [22] and

f fathers at least one seed on tree m, and 0 if not. Thus A is a binary representation of the connectance of the graph. Since the trees do not self-pollinate, the number of possible interac-

�; … ; ϕτ � � �

�, m ¼ 1, 2, … , τ, where ∣ � ∣ denotes cardinality. The set

� � �: (5)

� �, where af ,m <sup>¼</sup> 1 if tree

th flower

(4)

given flower, a pollinator will deposit roughly <sup>γ</sup>ð Þ <sup>1</sup> � <sup>γ</sup> <sup>k</sup>�<sup>1</sup> pollen grains onto the <sup>k</sup>

(

κ ¼ 1. For the simulation work we used r ¼ 0:30 based on work by [21].

2.5. Statistics

102 Pollination in Plants

directed pollination graph.

for each tree m in the graph is ϕ<sup>m</sup>

� � �

containing the number of fathers per mother for all trees in the graph is

Φ<sup>m</sup> ¼ ϕ<sup>1</sup> � � � �; ϕ<sup>2</sup> � � �

From this construct, we then create the τ � τ adjacency matrix, A ¼ ai,j

Cweak and Cstrong.

Valdovions et al. [23].

tions on this matrix is τ τð Þ � 1 .

The average pollination distance for an pollinator i is the average of the distances between any two trees mated by i. Let Y <sup>f</sup> ð Þ<sup>i</sup> ð Þ <sup>¼</sup> <sup>y</sup> f ð Þ<sup>i</sup> ð Þ <sup>1</sup> ; y f ð Þ<sup>i</sup> ð Þ 2 � � be the location of father tree <sup>f</sup> ð Þ<sup>i</sup> and <sup>Y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>¼</sup> <sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>1</sup> ; <sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ 2 � � be the location of mother tree <sup>m</sup>ð Þ<sup>i</sup> where pollinator <sup>i</sup> delivers pollen from tree f ð Þ<sup>i</sup> to tree mð Þ<sup>i</sup> . Then the average pollination distance, Dð Þ<sup>i</sup> , achieved by pollinator i for all such pairings is

$$\overline{D}^{(i)} = \frac{1}{\mu^{(i)}} \sum\_{m=1}^{\mu^{(i)}} \sum\_{f=1}^{\phi\_m^{(i)}} \frac{1}{\phi\_m^{(i)}} \sqrt{\left(y\_1^{(m^{(i)})} - y\_1^{(f^{(i)})}\right)^2 + \left(y\_2^{(m^{(i)})} - y\_2^{(f^{(i)})}\right)^2},\tag{9}$$

where μð Þ<sup>i</sup> is the number of mother trees pollinated by pollinator i, and ϕ<sup>i</sup> <sup>m</sup> are the number of father trees pollinating tree m by pollinator i. The average pollination distance, Dð Þ<sup>i</sup> , for each pollinator is averaged over the total number of pollinators, β, to obtain the average pollination distance, D, for the graph

$$
\overline{D} = \frac{1}{\beta} \sum\_{i=1}^{\beta} \ \overline{D}^{(i)}.\tag{10}
$$

<sup>D</sup><sup>~</sup> <sup>¼</sup> <sup>1</sup> β

other (Figure 2(l)).

densely connected groups.

3. Results and discussion

of parameters. The results follow.

3.1. Number of fathers

pollination events.

X β

D~ ð Þ<sup>i</sup> : (12)

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Comparison of Pollination Graphs

105

number of fathering triplets : (13)

number of fathering triplets : (14)

i¼1

A fathering triplet is the relationship between three trees such that tree f is a father to seeds on both tree m<sup>1</sup> and tree m2. These seeds are half-siblings on the paternal side. In particular a weak fathering triangle, as a subset of fathering triplets, is one such that m<sup>1</sup> also fathers seeds on m<sup>2</sup> (Figure 2(a)–(d)), m<sup>2</sup> fathers seeds on m<sup>1</sup> (Figure 2(e)–(h)), or both (Figure 2(i)–(l)). A strong fathering triangle is defined as fathering triangle where f , m1, and m<sup>2</sup> all father seeds on each

The weak clustering coefficient of fathers, Cweak, is the number of weak fathering triangles in the

<sup>C</sup>weak <sup>¼</sup> number of weak fathering triangles

The strong clustering coefficient of fathers, Cstrong is the number of strong fathering triangles in the

<sup>C</sup>strong <sup>¼</sup> number of strong fathering triangles

Cweak and Cstrong are measurements of the tendency of parent trees to be clustered together in

We examine the effect of varying parameters on the graph statistics: the number of fathers per mother, Φm, connectance, L, the average weighted diversity of fathers, E, the clustering coefficient of fathers, C, the average pollination distance, D, and the average maximum pollination distance, D~ . The model was simulated a total of 10 replicate runs for each unique combination

One way to analyze the genetic structure and connectivity within a local plant population is to examine the number of different fathers per mother tree, Φm. In Figure 3, the number of different fathers per mother in a randomized placement of trees is distributed in a Gaussianlike distribution. This is an expected outcome since gene flow should be directly proportional to the distance traveled by a pollinator. It has been shown, see [29], that the distribution range resulting from a CRW would necessarily result in Gaussian-like behavior for a large number of

pollination graph over the total number of fathering triplets

pollination graph over the total number of fathering triplets

The maximum pollination distance for an pollinator i is

$$\tilde{D}^{(i)} = \max\_{\substack{\boldsymbol{m}^{(i)} \le \boldsymbol{\mu}^{(i)}, \ \boldsymbol{f}^{(i)} \le \boldsymbol{\phi}\_{\text{w}}^{(i)}}} \left( \sqrt{\left( \boldsymbol{y}\_{1}^{(\boldsymbol{m}^{(i)})} - \boldsymbol{y}\_{1}^{(\boldsymbol{f}^{(i)})} \right)^{2} + \left( \boldsymbol{y}\_{2}^{(\boldsymbol{m}^{(i)})} - \boldsymbol{y}\_{2}^{(\boldsymbol{f}^{(i)})} \right)^{2}} \right). \tag{11}$$

The maximum pollination distance for each pollinator is averaged over all of the pollinators to obtain the average maximum pollination distance for the graph

Figure 2. Fathering triangles. Arrows indicate direction of gene flow.

$$
\tilde{D} = \frac{1}{\beta} \sum\_{i=1}^{\beta} \quad \tilde{D}^{(i)}.\tag{12}
$$

A fathering triplet is the relationship between three trees such that tree f is a father to seeds on both tree m<sup>1</sup> and tree m2. These seeds are half-siblings on the paternal side. In particular a weak fathering triangle, as a subset of fathering triplets, is one such that m<sup>1</sup> also fathers seeds on m<sup>2</sup> (Figure 2(a)–(d)), m<sup>2</sup> fathers seeds on m<sup>1</sup> (Figure 2(e)–(h)), or both (Figure 2(i)–(l)). A strong fathering triangle is defined as fathering triangle where f , m1, and m<sup>2</sup> all father seeds on each other (Figure 2(l)).

The weak clustering coefficient of fathers, Cweak, is the number of weak fathering triangles in the pollination graph over the total number of fathering triplets

$$\mathcal{C}\_{\text{weak}} = \frac{\text{number of weak father triangles}}{\text{number of father triangles}}.\tag{13}$$

The strong clustering coefficient of fathers, Cstrong is the number of strong fathering triangles in the pollination graph over the total number of fathering triplets

$$\mathcal{C}\_{\text{strong}} = \frac{\text{number of strong father triangles}}{\text{number of father triangles}}.\tag{14}$$

Cweak and Cstrong are measurements of the tendency of parent trees to be clustered together in densely connected groups.

## 3. Results and discussion

pollinator is averaged over the total number of pollinators, β, to obtain the average pollination

X β

Dð Þ<sup>i</sup>

f ð Þ<sup>i</sup> ð Þ 1 � �<sup>2</sup>

ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi

<sup>þ</sup> <sup>y</sup> <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>2</sup> � y

: (10)

1

A: (11)

f ð Þ<sup>i</sup> ð Þ 2 � �<sup>2</sup>

i¼1

<sup>D</sup> <sup>¼</sup> <sup>1</sup> β

> y <sup>m</sup>ð Þ<sup>i</sup> ð Þ <sup>1</sup> � y

The maximum pollination distance for each pollinator is averaged over all of the pollinators to

The maximum pollination distance for an pollinator i is

ð Þ<sup>i</sup> ≤ ϕð Þ<sup>i</sup> m

obtain the average maximum pollination distance for the graph

Figure 2. Fathering triangles. Arrows indicate direction of gene flow.

0s @

<sup>D</sup><sup>~</sup> ð Þ<sup>i</sup> <sup>¼</sup> max <sup>m</sup>ð Þ<sup>i</sup> <sup>≤</sup> <sup>μ</sup>ð Þ<sup>i</sup> , <sup>f</sup>

distance, D, for the graph

104 Pollination in Plants

We examine the effect of varying parameters on the graph statistics: the number of fathers per mother, Φm, connectance, L, the average weighted diversity of fathers, E, the clustering coefficient of fathers, C, the average pollination distance, D, and the average maximum pollination distance, D~ . The model was simulated a total of 10 replicate runs for each unique combination of parameters. The results follow.

#### 3.1. Number of fathers

One way to analyze the genetic structure and connectivity within a local plant population is to examine the number of different fathers per mother tree, Φm. In Figure 3, the number of different fathers per mother in a randomized placement of trees is distributed in a Gaussianlike distribution. This is an expected outcome since gene flow should be directly proportional to the distance traveled by a pollinator. It has been shown, see [29], that the distribution range resulting from a CRW would necessarily result in Gaussian-like behavior for a large number of pollination events.

Figure 3. Number of fathers per mother. Tree density ω ¼ 0:071552 trees per square unit. Maximum pollinator turning radius <sup>δ</sup>max <sup>¼</sup> <sup>45</sup><sup>∘</sup> . Pollination chance diminishing with larger carryover. Maximum pollen carryover <sup>κ</sup>max <sup>¼</sup> f g <sup>1</sup>; <sup>3</sup>; <sup>5</sup>; <sup>7</sup>; <sup>∞</sup> .

is attributed to the spatial heterogeneity of the research site. This influences the genetic structure

Figure 5. Tree coordinates (scaled) at the VCU Rice Center. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552

The spatial heterogeneity is evident in Figure 5. The bimodal distribution of the number of fathers per mother is caused by the variation of tree density across the landscape. There is a high density group of trees in the center of the region, and the density decreases toward the boundary of the region. This region is bounded by a river and a lake on two sides, and a major

These two different density regions create the two peaks shown in Figure 4. In particular when κmax ¼ ∞, the first peak is approximately 3 fathers per mother on 29 mother trees, and the second peak is 32 fathers per mother on 12 mother trees. This bimodality is present whether an

<sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup> . Clearly, knowing the locations of tree is critical in understanding the gene flow in

The connectance is a measure of how complete a pollination graph is in terms of individuals mating with others. In Figures 6 and 7, we see the effect of density and maximum turning angle. With higher density, and thus more individuals, the connectance is reduced due to a much larger number of potential pairs of individuals. As the maximum turning angle increases the connectance also decreases. If an pollinator travels in a straight line, it will cover a greater spatial distance as it visits more different trees than it would if it just spun around in circles locally. Smaller δmax increases the potential for mating to occur between trees with greater distance between them. The graph connectance is three to five times greater with small δmax

If an pollinator does not venture very far from its starting location, as would be the case when

decreases. When δmax ¼ 0, the connectance of the graph is nearly four times greater if the

, the effect of maximum pollen carryover on the connectance of the graph

, or with pure dispersal, i.e.,

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and connectivity in C. florida populations [11] at the Rice Center.

road and a farm on the other two sides, which are not pictured here.

pollinator is restricted to moving in a straight line, i.e., <sup>δ</sup>max <sup>¼</sup> <sup>0</sup><sup>∘</sup>

a particular area (Figure 5).

than it is with large δmax.

δmax is close to 180<sup>∘</sup>

3.2. Connectance

trees per square units.

Of interest here is that as the maximum pollen carryover increases, the mean number of fathers increases. This is due to the increase in the variability in pollen that each pollinator can distribute, which would increase diversity. The variance in the distribution also increases as pollen carryover increases, which is also due to this increase in diversity.

However, when this distribution is compared with the tree placement at the Rice Center, see Figure 4, we observe a bi-modal distribution of the number of fathers per mother. This distribution

Figure 4. Number of fathers per mother. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Maximum pollinator turning radius <sup>δ</sup>max <sup>¼</sup> <sup>45</sup><sup>∘</sup> . Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

Figure 5. Tree coordinates (scaled) at the VCU Rice Center. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square units.

is attributed to the spatial heterogeneity of the research site. This influences the genetic structure and connectivity in C. florida populations [11] at the Rice Center.

The spatial heterogeneity is evident in Figure 5. The bimodal distribution of the number of fathers per mother is caused by the variation of tree density across the landscape. There is a high density group of trees in the center of the region, and the density decreases toward the boundary of the region. This region is bounded by a river and a lake on two sides, and a major road and a farm on the other two sides, which are not pictured here.

These two different density regions create the two peaks shown in Figure 4. In particular when κmax ¼ ∞, the first peak is approximately 3 fathers per mother on 29 mother trees, and the second peak is 32 fathers per mother on 12 mother trees. This bimodality is present whether an pollinator is restricted to moving in a straight line, i.e., <sup>δ</sup>max <sup>¼</sup> <sup>0</sup><sup>∘</sup> , or with pure dispersal, i.e., <sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup> . Clearly, knowing the locations of tree is critical in understanding the gene flow in a particular area (Figure 5).

#### 3.2. Connectance

Of interest here is that as the maximum pollen carryover increases, the mean number of fathers increases. This is due to the increase in the variability in pollen that each pollinator can distribute, which would increase diversity. The variance in the distribution also increases as

Figure 3. Number of fathers per mother. Tree density ω ¼ 0:071552 trees per square unit. Maximum pollinator turning radius <sup>δ</sup>max <sup>¼</sup> <sup>45</sup><sup>∘</sup> . Pollination chance diminishing with larger carryover. Maximum pollen carryover <sup>κ</sup>max <sup>¼</sup> f g <sup>1</sup>; <sup>3</sup>; <sup>5</sup>; <sup>7</sup>; <sup>∞</sup> .

However, when this distribution is compared with the tree placement at the Rice Center, see Figure 4, we observe a bi-modal distribution of the number of fathers per mother. This distribution

Figure 4. Number of fathers per mother. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Maximum pollinator turning radius <sup>δ</sup>max <sup>¼</sup> <sup>45</sup><sup>∘</sup> . Pollination chance diminishing with larger carryover. Maximum

pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

106 Pollination in Plants

pollen carryover increases, which is also due to this increase in diversity.

The connectance is a measure of how complete a pollination graph is in terms of individuals mating with others. In Figures 6 and 7, we see the effect of density and maximum turning angle. With higher density, and thus more individuals, the connectance is reduced due to a much larger number of potential pairs of individuals. As the maximum turning angle increases the connectance also decreases. If an pollinator travels in a straight line, it will cover a greater spatial distance as it visits more different trees than it would if it just spun around in circles locally. Smaller δmax increases the potential for mating to occur between trees with greater distance between them. The graph connectance is three to five times greater with small δmax than it is with large δmax.

If an pollinator does not venture very far from its starting location, as would be the case when δmax is close to 180<sup>∘</sup> , the effect of maximum pollen carryover on the connectance of the graph decreases. When δmax ¼ 0, the connectance of the graph is nearly four times greater if the

Pollen carryover is important in connectance as well. In Figure 7, it is clear that if the maximum pollen carryover is limited, the connectance of the pollination graph is also limited due to the diversity of pollen an individual would have access to via the pollinator. With smaller κmax the diversity of pollen distributed is greatly decreased and thus the diversity of pollination

When considering the actual tree locations at the Rice Center, the connectance of the graph is close to half of the connectance value with randomly-placed trees. The differences between the

The average weighted diversity of fathers is clearly affected by density and the maximum turning angle, though the effects of maximum turning angle are more pronounced. In Figure 9, as the maximum turning angle increases, the average weighted diversity of fathers decreases. The greatest change occurs between 15 and 90<sup>∘</sup> and tends to even out at the extremes. As described earlier, with greater potential for a variety of fathers, the average number of fathers contributing pollen to mother trees increases as the maximum turning angle of pollinators decreases. This adds a greater genetic diversity to the tree population. Pollinators that travel in straighter paths not only distribute pollen greater distances, but also with greater diversity. The effects of density are more pronounced at smaller maximum turning angles, where we see

Figure 8. Connectance. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Pollination

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

a reduction of the average weighted diversity of fathers as the density increases.

. Connectance values for simulations run with the

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events is reduced as well.

graphs is greatest when δmax is close to 0<sup>∘</sup>

3.3. Average weighted diversity of fathers

Rice Center data are shown in Figure 8.

Figure 6. Connectance. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

Figure 7. Connectance. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f0:025; 0:050; 0:075; 0:100; ; 0:125; 0:150g (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly-placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

maximum pollen carryover is unlimited versus the case when pollen carryover is limited to only one flower. However, when <sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup> , the connectance of the graph is only about twice if the maximum pollen carryover is unlimited.

Pollen carryover is important in connectance as well. In Figure 7, it is clear that if the maximum pollen carryover is limited, the connectance of the pollination graph is also limited due to the diversity of pollen an individual would have access to via the pollinator. With smaller κmax the diversity of pollen distributed is greatly decreased and thus the diversity of pollination events is reduced as well.

When considering the actual tree locations at the Rice Center, the connectance of the graph is close to half of the connectance value with randomly-placed trees. The differences between the graphs is greatest when δmax is close to 0<sup>∘</sup> . Connectance values for simulations run with the Rice Center data are shown in Figure 8.

#### 3.3. Average weighted diversity of fathers

maximum pollen carryover is unlimited versus the case when pollen carryover is limited to only one flower. However, when <sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup> , the connectance of the graph is only about twice

Figure 7. Connectance. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f0:025; 0:050; 0:075; 0:100; ; 0:125; 0:150g (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly-placed trees). Pollination chance diminishing with larger car-

Figure 6. Connectance. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Pollination

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

if the maximum pollen carryover is unlimited.

ryover. Maximum pollen carryover κmax ¼ ∞.

108 Pollination in Plants

The average weighted diversity of fathers is clearly affected by density and the maximum turning angle, though the effects of maximum turning angle are more pronounced. In Figure 9, as the maximum turning angle increases, the average weighted diversity of fathers decreases. The greatest change occurs between 15 and 90<sup>∘</sup> and tends to even out at the extremes. As described earlier, with greater potential for a variety of fathers, the average number of fathers contributing pollen to mother trees increases as the maximum turning angle of pollinators decreases. This adds a greater genetic diversity to the tree population. Pollinators that travel in straighter paths not only distribute pollen greater distances, but also with greater diversity. The effects of density are more pronounced at smaller maximum turning angles, where we see a reduction of the average weighted diversity of fathers as the density increases.

Figure 8. Connectance. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit. Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

The differences in average weighted diversity of fathers as pollen carryover increases are exactly as one would expect, see Figure 10. With higher κmax, the larger the diversity in pollen increases the average weighted diversity in fathers. This increase is small for large δmax due to the increase in the number of multiple visits by pollinators to the

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When comparing the random tree distribution to that of the Rice Center, the random distribution has a larger average weighted diversity of fathers (not shown here) the average weighted diversity of fathers at the Rice Center is about 60% of the randomly placed trees. Trees that are in densely packed groups are going to be greatly influenced by surrounding trees, but trees at greater distances will have less of a comparative impact on the fatherhood

The average and maximum pollination distances behave similar to the connectance. As with the connectance, as the density increases both distances decrease, which is due to a greater number of shorter pollination events lowering the averages, see Figures 11 and 12. The change in density has a smaller effect on the maximum pollination distance. Also as the maximum turning angle increase both distances decreases as well since the pollinators do not travel as

Figure 11. Average pollination distance. Field size 100 � 100 units. Tree density in trees per square area, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

3.4. Average and maximum average pollination distances

far. This decrease is more dramatic at lower densities.

same trees.

of seeds.

Figure 9. Average weighted diversity of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

Figure 10. Average weighted diversity of fathers. Field size 133:2794 � 47:2927 units. Tree density in trees per square unit, ω ¼ 0:071552 (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

The differences in average weighted diversity of fathers as pollen carryover increases are exactly as one would expect, see Figure 10. With higher κmax, the larger the diversity in pollen increases the average weighted diversity in fathers. This increase is small for large δmax due to the increase in the number of multiple visits by pollinators to the same trees.

When comparing the random tree distribution to that of the Rice Center, the random distribution has a larger average weighted diversity of fathers (not shown here) the average weighted diversity of fathers at the Rice Center is about 60% of the randomly placed trees. Trees that are in densely packed groups are going to be greatly influenced by surrounding trees, but trees at greater distances will have less of a comparative impact on the fatherhood of seeds.

#### 3.4. Average and maximum average pollination distances

Figure 9. Average weighted diversity of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination

Figure 10. Average weighted diversity of fathers. Field size 133:2794 � 47:2927 units. Tree density in trees per square unit, ω ¼ 0:071552 (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

110 Pollination in Plants

The average and maximum pollination distances behave similar to the connectance. As with the connectance, as the density increases both distances decrease, which is due to a greater number of shorter pollination events lowering the averages, see Figures 11 and 12. The change in density has a smaller effect on the maximum pollination distance. Also as the maximum turning angle increase both distances decreases as well since the pollinators do not travel as far. This decrease is more dramatic at lower densities.

Figure 11. Average pollination distance. Field size 100 � 100 units. Tree density in trees per square area, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

Figure 12. Average maximum pollination distance. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

and do not stay in a small neighborhood. Since the weak clustering coefficient of fathers is an average measure of clustering at a local level, it is natural for Cweak to be low if pollinators do

Figure 13. Weak clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen

At the other extreme, for large δmax, pollinators do not move around enough to increase the value of Cweak. When δmax is not at an extreme value, the displacement of the pollinators is high enough to visit many trees, but low enough so more of the trees that it visits are within a closer

Modeled data on the weak clustering coefficient of fathers, Cweak, from the Rice Center is shown in Figure 14. The Cweak values slowly increase as κmax increases over the entire interval from

the same general area. Thus, in locally dense patches of trees, clustering will naturally be higher. Unexpectedly, varying the tree density, ω, did not have a major effect on Cweak. It would seem that varying ω would have the same quantitative effect on Cweak as varying the maximum pollinator turning angle, δmax. We suspect that the reason for the relative consistency of values for Cweak is due to the having both low and high density regions. The values for Cweak, varying

Figures 16–18 are corresponding plots for strong clustering coefficient of fathers, Cstrong. In these plots we see similar results as with the weak clustering coefficient. Numerically the Cstrong results are a magnitude smaller than the Cweak results since they are a measure of a subset of possible combinations of Cweak. This is however a greater increase in these values

with increasing density, which allows for these fathering triangles to occur.

. As δmax approaches 180<sup>∘</sup>

, pollinators remain in

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not remain in a small neighborhood.

ω and δmax, are shown in Figure 15.

, mostly flattening out for κmax > 75<sup>∘</sup>

proximity to one another.

carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

0 to 180<sup>∘</sup>

Using Rice Center locations (not shown here) if δmax is close to 0<sup>∘</sup> , the average pollination distance is greater than that of randomly placed trees. However, if δmax is close to 180<sup>∘</sup> , the average pollination distance using Rice Center data is less than that of randomly placed trees. This is again due to the combination of the low and high density distribution of trees at the Rice Center. At low δmax pollinators interact with both densities of trees, whereas at high δmax the interaction between these groups is greatly diminished.

We see the effects of pollen carryover in the average pollination distance is shown in Figure 11 and in the maximum pollination distance in Figure 12. As expected, increasing κmax increases the average pollination distance. If the pollen carryover increases that allows pollen grains to travel further since pollen can be deposited after visiting several intermediate trees.

The results are similar with the Rice Center model. The results at the Rice Center are slightly higher in distances due to the potential longer distances at the edges of the region.

#### 3.5. Clustering coefficients of fathers

The clustering coefficient is a measure of the interconnectedness of the graph, as well as how the genes are shared within the graph. For a field of randomly placed trees, there is a maximum value for the weak clustering coefficient of fathers, Cweak, see Figure 13, that occurs between 60 and 90<sup>∘</sup> for all maximum pollen carryover values, κmax. This is explained by examining the extreme values of δmax. For small δmax, pollinators travel across the landscape

Figure 13. Weak clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

and do not stay in a small neighborhood. Since the weak clustering coefficient of fathers is an average measure of clustering at a local level, it is natural for Cweak to be low if pollinators do not remain in a small neighborhood.

Using Rice Center locations (not shown here) if δmax is close to 0<sup>∘</sup> , the average pollination distance is greater than that of randomly placed trees. However, if δmax is close to 180<sup>∘</sup>

Figure 12. Average maximum pollination distance. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination

average pollination distance using Rice Center data is less than that of randomly placed trees. This is again due to the combination of the low and high density distribution of trees at the Rice Center. At low δmax pollinators interact with both densities of trees, whereas at high δmax

We see the effects of pollen carryover in the average pollination distance is shown in Figure 11 and in the maximum pollination distance in Figure 12. As expected, increasing κmax increases the average pollination distance. If the pollen carryover increases that allows pollen grains to

The results are similar with the Rice Center model. The results at the Rice Center are slightly

The clustering coefficient is a measure of the interconnectedness of the graph, as well as how the genes are shared within the graph. For a field of randomly placed trees, there is a maximum value for the weak clustering coefficient of fathers, Cweak, see Figure 13, that occurs between 60 and 90<sup>∘</sup> for all maximum pollen carryover values, κmax. This is explained by examining the extreme values of δmax. For small δmax, pollinators travel across the landscape

travel further since pollen can be deposited after visiting several intermediate trees.

higher in distances due to the potential longer distances at the edges of the region.

the interaction between these groups is greatly diminished.

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

3.5. Clustering coefficients of fathers

112 Pollination in Plants

, the

At the other extreme, for large δmax, pollinators do not move around enough to increase the value of Cweak. When δmax is not at an extreme value, the displacement of the pollinators is high enough to visit many trees, but low enough so more of the trees that it visits are within a closer proximity to one another.

Modeled data on the weak clustering coefficient of fathers, Cweak, from the Rice Center is shown in Figure 14. The Cweak values slowly increase as κmax increases over the entire interval from 0 to 180<sup>∘</sup> , mostly flattening out for κmax > 75<sup>∘</sup> . As δmax approaches 180<sup>∘</sup> , pollinators remain in the same general area. Thus, in locally dense patches of trees, clustering will naturally be higher.

Unexpectedly, varying the tree density, ω, did not have a major effect on Cweak. It would seem that varying ω would have the same quantitative effect on Cweak as varying the maximum pollinator turning angle, δmax. We suspect that the reason for the relative consistency of values for Cweak is due to the having both low and high density regions. The values for Cweak, varying ω and δmax, are shown in Figure 15.

Figures 16–18 are corresponding plots for strong clustering coefficient of fathers, Cstrong. In these plots we see similar results as with the weak clustering coefficient. Numerically the Cstrong results are a magnitude smaller than the Cweak results since they are a measure of a subset of possible combinations of Cweak. This is however a greater increase in these values with increasing density, which allows for these fathering triangles to occur.

Figure 14. Weak clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

Figure 16. Strong clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen

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Figure 17. Strong clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover

carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

κmax ¼ f g 1; 3; 5; 7; ∞ .

Figure 15. Weak clustering coefficient of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

Figure 16. Strong clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

Figure 14. Weak clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover

Figure 15. Weak clustering coefficient of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

κmax ¼ f g 1; 3; 5; 7; ∞ .

114 Pollination in Plants

Figure 17. Strong clustering coefficient of fathers. Field size 133:2794 � 47:2927 units. Tree density ω ¼ 0:071552 trees per square unit (τ ¼ 451 Rice Center trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ f g 1; 3; 5; 7; ∞ .

diversity in the C. florida population. While neither of these extremes may be biologically relevant in C. florida populations, we note that the clustering coefficient of fathers, C, is

Major changes are observed when comparing simulations using randomly-placed trees with simulations using the tree-placement at the Rice Center. When using the Rice Center data, we see a bimodal distribution in the number of fathers per mother, the connectance values are halved, the average weighted diversity of fathers is lower, the average pollination distance is lower when δmax is close to 0<sup>∘</sup> and higher when δmax is close to 180<sup>∘</sup> , and the clustering coefficient of fathers exhibits both quantitative and qualitative differences. All of these differences highlight the need for specificity in describing the tree locations within a specific ecosystem in order to truly understand how pollen is distributed within that ecosystem. The differences in these graph

The collection of data and construction of an initial model were supported by a National Science Foundation grant (DEB-0640803) to Rodney J. Dyer and David M. Chan. James H. Lee was supported by a grant from the VCU Rice Center to complete an advance model. This

\* and Rodney J. Dyer2

1 Department of Mathematics and Applied Mathematics, Virginia Commonwealth University,

2 Center for Environmental Studies, Virginia Commonwealth University, Richmond, Virginia,

[1] Slatkin M. Gene flow in natural populations. Annual Review of Ecology and Systematics.

[2] Morris WF. Predicting the consequence of plant spacing and biased movement for pollen

indicators is due to the non-uniform distribution of trees at the Rice Center.

, which could help illuminate some of the

Comparison of Pollination Graphs

117

http://dx.doi.org/10.5772/intechopen.74553

maximized when δmax is between 60 and 90<sup>∘</sup>

manuscript is VCU Rice Center Manuscript #69.

, David M. Chan<sup>1</sup>

\*Address all correspondence to: dmchan@vcu.edu

dispersal by honey bees. Ecology. 1993;74:493-500

biological processes at work in the system.

Funding

Author details

Richmond, Virginia, USA

1985;16:393-430

James H. Lee1

United States

References

Figure 18. Strong clustering coefficient of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

## 4. Conclusions

From the agent-based, correlated random walk model presented, we observe the effects of varying the maximum pollen carryover, κmax, the maximum pollinator turning area, δmax, and the density of trees, ω, on the distribution of pollen within a population of Cornus florida.

When κmax increases we see that the mean number of fathers per mother, the connectance, the average weighted diversity of fathers, the average and maximum average pollination distances, and the clustering coefficients all increase. The percentage increase varied between the measured, though the largest effect was seen in the connectance of the pollination graph. These increases are due to the increased capability of the pollinators to carry pollen farther from their source.

Changing the pollinator movement by increasing the maximum turning angle, δmax, affected each of the measures as well. With most of the measures decreasing with increasing δmax. These include the connectance, the average weighted diversity of fathers, and the average and maximum average pollination distances. On the other hand, the weak and strong clustering coefficients had maximal values between angles of 45 and 90<sup>∘</sup> .

When <sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup> , pollen is distributed in a purely random walk, and is more representative of pollen dispersal by wind. When <sup>δ</sup>max <sup>¼</sup> <sup>0</sup><sup>∘</sup> , pollinators travel in a straight line, only changing direction when bouncing off of the boundary. This leads to a greater spatial displacement for each pollinator and thus a greater distance that pollen travels, resulting in greater genetic diversity in the C. florida population. While neither of these extremes may be biologically relevant in C. florida populations, we note that the clustering coefficient of fathers, C, is maximized when δmax is between 60 and 90<sup>∘</sup> , which could help illuminate some of the biological processes at work in the system.

Major changes are observed when comparing simulations using randomly-placed trees with simulations using the tree-placement at the Rice Center. When using the Rice Center data, we see a bimodal distribution in the number of fathers per mother, the connectance values are halved, the average weighted diversity of fathers is lower, the average pollination distance is lower when δmax is close to 0<sup>∘</sup> and higher when δmax is close to 180<sup>∘</sup> , and the clustering coefficient of fathers exhibits both quantitative and qualitative differences. All of these differences highlight the need for specificity in describing the tree locations within a specific ecosystem in order to truly understand how pollen is distributed within that ecosystem. The differences in these graph indicators is due to the non-uniform distribution of trees at the Rice Center.

## Funding

4. Conclusions

116 Pollination in Plants

source.

When <sup>δ</sup>max <sup>¼</sup> <sup>180</sup><sup>∘</sup>

From the agent-based, correlated random walk model presented, we observe the effects of varying the maximum pollen carryover, κmax, the maximum pollinator turning area, δmax, and the density of trees, ω, on the distribution of pollen within a population of Cornus florida.

Figure 18. Strong clustering coefficient of fathers. Field size 100 � 100 units. Tree density in trees per square unit, ω ¼ f g 0:025; 0:050; 0:075; 0:100; 0:125; 0:150 (τ ¼ f g 250; 500; 750; 1000; 1250; 1500 randomly placed trees). Pollination

When κmax increases we see that the mean number of fathers per mother, the connectance, the average weighted diversity of fathers, the average and maximum average pollination distances, and the clustering coefficients all increase. The percentage increase varied between the measured, though the largest effect was seen in the connectance of the pollination graph. These increases are due to the increased capability of the pollinators to carry pollen farther from their

Changing the pollinator movement by increasing the maximum turning angle, δmax, affected each of the measures as well. With most of the measures decreasing with increasing δmax. These include the connectance, the average weighted diversity of fathers, and the average and maximum average pollination distances. On the other hand, the weak and strong clustering

ing direction when bouncing off of the boundary. This leads to a greater spatial displacement for each pollinator and thus a greater distance that pollen travels, resulting in greater genetic

.

, pollinators travel in a straight line, only chang-

, pollen is distributed in a purely random walk, and is more representative

coefficients had maximal values between angles of 45 and 90<sup>∘</sup>

chance diminishing with larger carryover. Maximum pollen carryover κmax ¼ ∞.

of pollen dispersal by wind. When <sup>δ</sup>max <sup>¼</sup> <sup>0</sup><sup>∘</sup>

The collection of data and construction of an initial model were supported by a National Science Foundation grant (DEB-0640803) to Rodney J. Dyer and David M. Chan. James H. Lee was supported by a grant from the VCU Rice Center to complete an advance model. This manuscript is VCU Rice Center Manuscript #69.

## Author details

James H. Lee1 , David M. Chan<sup>1</sup> \* and Rodney J. Dyer2

\*Address all correspondence to: dmchan@vcu.edu

1 Department of Mathematics and Applied Mathematics, Virginia Commonwealth University, Richmond, Virginia, USA

2 Center for Environmental Studies, Virginia Commonwealth University, Richmond, Virginia, United States

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## *Edited by Phatlane William Mokwala*

Plants are the basic source of food for both humans and animals. Most of the food is made of fruits and seeds. For these to be formed, pollination must first take place. This process is the transfer of pollen grains from the anther, which is the male structure of the flower, to the sigma on the female structure of the flower. The transfer process requires agents to be carried out. The agents can be either biotic or abiotic. Nature perfected this arrangement between the pollination agents and the plants. As ecosystems and agricultural systems are changing, this balanced arrangement becomes disturbed. This makes it necessary that pollination systems be studied so that necessary measures can be undertaken to ensure productivity.

The chapters of this book present results in research undertaken to improve productivity in crops such as *Actinidia chinensis* (the kiwifruit), *Theobroma cacao*  (cocoa), and *Manicaria saccifera* (a tropical forest palm). Some results are presented on tests to check the viability of pollen grains and the delivery of sperm cells through pollen tubes to the embryo sac. These results can serve as guidelines to any person seeking to improve pollination and productivity or to check the efficiency on pollination in ecosystems or agricultural production systems.

Published in London, UK © 2018 IntechOpen © paulbein / iStock

Pollination in Plants

Pollination in Plants

*Edited by Phatlane William Mokwala*