**1.1 Pollination and plant reproduction**

The pollination process occurs in spermatophitic plants consists in pollen grain transfer to the stigma, which is the receptive part of feminine flowers of superior vegetal. The pollen grains are structures that contain the reproductive male cells, they are produced and storaged in anthers (part of male organ of flowers) until the deiscence, moment wherein are released. The indispensable factor in pollination process and that denotes the success is the need of ovule fertilization and subsequent fruit set and seeds formation.

The pollination of flowers beside the diaspore dispersion is a fundamental process in reproductive success of vegetal species. The involved animals in this process have an important role (Buckmann & Nabhan, 1997), then the efficience in the pollination process means rise in disponibility of food to the human being and animals.

The structure of plant (monoic or dioic) varies to the size and anatomic and physiological characterists of flower and their position in the plant, may occur the autopollination or cross pollination. This cross pollination provides a rise in gene flow between plants, spreading them and the results are favorable (Malerbo-Souza et al., 2004).

The animals that carried out the pollen transfer of anthers to the stigma flowers are known pollinators, and can be insects like bees, bettles, flies, butterflies, wasps and moth; birds hummingbirds and parakeets; and small mammals - bats, rodents, and marsupials (Malagodi-Braga, 2005). Among pollinators, animals of Insecta class are the most important, and in the order Hymenoptera you can find the major number of them. Honeybees are the most important pollinators available in the nature.

Unlike of other insects that visit flowers only to collect its own food, honeybees visit a bigger quantity of flowers, besides of food to own survival, they harvesting pollen and nectar to feed their larvae and storaging (Müller et al., 2006). Futhermore, the higher efficiency of honeybees as pollinators is as much as by their number in the nature, as by better adaptation to floral complex structures like mouthparts and adapted body to imbibe the nectar of flowers and harvesting pollen, respectively (Proctor et al., 1996). The bees of Apidae family have higher distinction because their morphological traits are representative, like special structure to load pollen - corbicula, located in tibia of hind leg, similar to basket

Other factors that must be considered are secreted sugars: sucrose, glucose and fructose. The quantities of them may vary depending on variety and type of vegetal. Honeybees have preference for nectar with more sucrose concentration. Sugars present in nectar are related with honey quality that will be produced by honeybees and, finally will be commercialized. The association of beekeeping and agriculture provide a rise in profits as for farmer as for beekeeper. However, the quantity and/or quality of the sugars in floral nectar like the pollination of cultivated crops by honeybees have an economic and social role well-

The proposal of this chapter is perform a review about spectrophotometry in sugar dosages of nectar of the main cultivated crops and show the importance of this tool (spectrophotometry) to improve crop production by honeybee pollination contributing as

The pollination process occurs in spermatophitic plants consists in pollen grain transfer to the stigma, which is the receptive part of feminine flowers of superior vegetal. The pollen grains are structures that contain the reproductive male cells, they are produced and storaged in anthers (part of male organ of flowers) until the deiscence, moment wherein are released. The indispensable factor in pollination process and that denotes the success is the

The pollination of flowers beside the diaspore dispersion is a fundamental process in reproductive success of vegetal species. The involved animals in this process have an important role (Buckmann & Nabhan, 1997), then the efficience in the pollination process

The structure of plant (monoic or dioic) varies to the size and anatomic and physiological characterists of flower and their position in the plant, may occur the autopollination or cross pollination. This cross pollination provides a rise in gene flow between plants, spreading

The animals that carried out the pollen transfer of anthers to the stigma flowers are known pollinators, and can be insects like bees, bettles, flies, butterflies, wasps and moth; birds hummingbirds and parakeets; and small mammals - bats, rodents, and marsupials (Malagodi-Braga, 2005). Among pollinators, animals of Insecta class are the most important, and in the order Hymenoptera you can find the major number of them. Honeybees are the

Unlike of other insects that visit flowers only to collect its own food, honeybees visit a bigger quantity of flowers, besides of food to own survival, they harvesting pollen and nectar to feed their larvae and storaging (Müller et al., 2006). Futhermore, the higher efficiency of honeybees as pollinators is as much as by their number in the nature, as by better adaptation to floral complex structures like mouthparts and adapted body to imbibe the nectar of flowers and harvesting pollen, respectively (Proctor et al., 1996). The bees of Apidae family have higher distinction because their morphological traits are representative, like special structure to load pollen - corbicula, located in tibia of hind leg, similar to basket

need of ovule fertilization and subsequent fruit set and seeds formation.

means rise in disponibility of food to the human being and animals.

them and the results are favorable (Malerbo-Souza et al., 2004).

most important pollinators available in the nature.

established and significant currently.

for agriculture as for beekeeping.

**1.1 Pollination and plant reproduction** 

– pollen is loaded in this structure in association with nectar or oil, absence of ventral scopa and long tongue (Teixeira & Zampieron, 2008).

Each group among animals that visit flowers is associated to some particular type of floral reward, that is the morphological traits of flowers reflect adaptation to diversified pollinators. The contemporaneous interactions between plants with flowers and insects can be because of long and closer coevolutionary relation (Backer & Hurd, 1968; Prince, 1997). This process of coevolution or the interaction between plants and pollinators is based on a system of mutual dependence. This system was detailed by the first time by Christian Konrad Spengel (1750-1816), in which plants show their rewards like nectar, pollen, oil and resins by floral arrangement, colour, size and odour of flowers, while the pollinators in change of provided resources by plants transfer pollen between flowers increasing the gene flow and promoting the diversification of the species, named by this as key mutualism (Morgado et al., 2002).

Plants, year by year, specialized in attract more efficient pollinators and make transportation of their reproductive cells, and therewith could be benefited with cross pollination. The disposition of flowers is an important factor that can be isolated in the branches or grouped in the same floral axis forming inflorescence, colour, odour, size, nectar, oil, pollen and resins.

The floral rewards provided by Angiosperms are required to attract pollinators, nectar seems the most searched in crop cultivated commercially, however, in searching by this reward many animals, mainly worker honeybees *Apis mellifera* have pollen adhered to their body, and so, later deposit accidentally their loads on the stigma of other flower of the same specie, performing indirectly the cross pollination.

Assays carried out in Marechal Cândido Rondon, Paraná, Brazil, with sunflower make clear worker honeybees that hoarding nectar are more frequent (mean 2.28 honeybees/capitulum) than pollen foragers (0.40 honeybees by capitulum) in anthesis period and schedule of higher visitation in the crop (Chambó et al., 2011). Other experimental results make clear more frequency of nectar foragers than pollen foragers in sunflower crop (Paiva et al., 2002; Teixeira & Zampieron, 2008).

Moreover, it must be considered that quantity of honeybee visitors to different species of superior vegetal can be related to concentration and volume of nectar in the flowers during all day (Pham-Delegue et al., 1990). Experiments with attractiveness sunflower genotypes show significative difference in relation to the number of honeybee visitation, mean of 3.40 (genotypes Helio 360 and Aguará) and 1.60 (genotype Multissol) visits of *A. mellifera* by capitulum in third day of anthesis (Chambó et al., 2011, in press). The researchers did not tested the concentration and volume secreted during all day, but assign to these causes the difference of sunflower genotypes assayed. In hybrid of ornamental coloured sunflower BRS-OASIS had an increase (*p* < 0.05) in four times in number of honeybee visitors using sucrose solution in two concentrations 5% and 7.5% as attractive. These solutions were pulverized on sunflower capitulum in relation to concentration of 2.5% (Martin et al., 2005).

Despite of efficiency of pollination process, it depends on numerous received visits by pollinators (Schirmer, 1985). Vidal et al. (2010) studying the pollination and set fruit in *Cucurbita pepo* by honyebees reported that percentage of set fruit was maximum (100%)

Spectrophotometry as a Tool for Dosage Sugars in Nectar of Crops Pollinated by Honeybees 273

determination, also can be used the technique of DNS and the reaction with p-

For specific determination of some sugars also are available some other methods. Fructose can be determinated by cysteine/tryptophan/sulphuric acid method (Messineo & Musarra, 1972) and resorcinol method (Roe et al., 1949). Sucrose can be determined by anthrone method after destruction of monosaccharides with KOH (Sala Jr. et al., 2007). Besides these methods, the glucose, fructose and sucrose can be selectively determined by methods using

This method is based on the fact that simple or complex sugar and their derivatives, including methyl esters with free reducing groups or potentially free, when treated with phenol and concentrated sulphuric acid will generate a yellow-orange colour, the reaction is sensible and this colour is stable. The method is simple, quick, sensible, and the results are

The changing in colour of solution is measured in visible range and proportional to the

The carbohydrates are hydrolyzed under heating in strongly acid pH, this reaction produces furan derivative compounds, that when condensed with phenolic compounds produce

The total sugar concentration is determined by spectrophotometry in 490 nm wavelength. Sensitivity of this method range from 10 to 100µg of total sugar and the quantification is made from calibration curve using glucose or pentose as standard and calculation are

Get an aliquot of 100µg.mL-1 from the sample, add 0.5mL of phenol solution 5%, shake in vortex, add 2.5mL, shake again and keep in water-bath 25°C for 15 minutes. After this

The anthrone is the reduction product of anthraquinone and was recognized first as specific reagent for several carbohydrates by Dreywood (1946) because sugar solution in concentrate sulphuric acid form the blue-greenish colour characteristic and since anthrone has been

The anthrone reaction is based on hydrolitic and dehydrating action of concentrate sulphuric acid on carbohydrates, in which glicosidic linkages are broken releasing free reducing sugar that are dehydrated and converted to furfural by pentoses and

performed by equation of the linear regression obtained from calibration curve.

Reagents: phenol solution 5% (w/v) and concentrate sulphuric acid (95%, p/v).

used widely as suitable and specific reagent for sugar colorimetric dosage.

period, read the absorbance in spectrophotometer at 490nm.

**3.2 Anthrone method (Dreywood, 1946)** 

hydroxymethylfurfural by hexoses (Figure 1).

hydroxybenzoic acid hydrazide - *PAHBAH* (Blakeney & Mutton, 1980).

enzymatic reactions (Moernan et al., 2004; Amaral et al., 2007).

**3.1 Phenol-sulphuric method (Dubois et al., 1956)** 

**3. Total sugar determination** 

quantity of sugar inside the sample.

coloured substances (Figure 1).

**3.1.1 Methodology** 

reproductible.

when the flowers received 12 visits of *A. mellifera*, corresponding to load of 1.253 pollen grains deposited on the stigma. In comparision, with two visits of honeybees, 174 pollen grains were deposited on the stigma, and the set fruit only of 50%. Other important factor is the attraction by pollen and nectar of male flower was arised by opening grade of nectary pore of flower (Vidal et al., 2010).

Models of pollen transfer depends on the specific pollinator rate about pollen remotion of anthers and deposition on the stigmas. The pollinators have a high remotion and low deposition (HRLD) of pollen on the stigma of flowers will benefite a plant wherever it is not better available pollinator. In case of pollinators have high remotion of pollen of anthers and high deposition (HRHD) of pollen on the stigmas also visit a plant population, the visits of pollinators HRLD can reduce the total pollen transfer. The HRLDs parasite the plants, displace the pollen grains that would delivery by HRHDs. When two visitors remove equal quantities of pollen, the pollinator more efficient will be that with higher delivery rate. In case of different quantities of remotion, the better do not depends on the deposition rates only, but another variables including schedule of visitation to pollen deposition (Thomson & Goodell, 2002).

The volume and sugar concentration of nectar, important factor in attraction of pollinators alos are known by varing between plant species and affect the answer of pollinator (Lanza et al*.*, 1995). Besides, different varieties of the same species can range to the sugar concentration in the nectar (Free, 1993). In some vegetal species, for example, *Curcubita pepo,*  the periodical remotion of nectar of the flower by pollinators do not arise the total volume of produced nectar by plant, then the nectar secretion is not stimulated or inhibited by successed harvesting of this reward. However, the nectar remotion of flowers can reduce or stimulate the process of secretion in several plant species.

The pollinator that drag out the spent time in flowers to nectar hoarding, specially if it is available, increase the probability of pollen deposition and, consequently, the pollination can be well successed. Nevertheless, cultivated commercially crops that do not stimulate the nectar production after several visits of pollinators can have an advantage in pollination process, so the number of honeybee visits to flowers is positively correlated to the nectar secretion in all flower duration (Vidal et al., 2006). The evaluation of nectar secretion rate is an important component in ecological studies related to the pollination process, mainly in that about flower-insect interaction.
