Preface

Chapter 9 **Heavy Metal and Mineral Element-Induced Abiotic Stress in**

Chapter 10 **Abiotic Stress Tolerance in Rice (Oryza sativa L.): A Genomics**

Chapter 11 **Natural Resistance of Sri Lankan Rice (Oryza sativa L.) Varieties to Broad-Spectrum Herbicides (Glyphosate and**

Chapter 12 **Methanogens Harboring in Rice Rhizosphere Reduce Labile**

Chapter 13 **Farmers' Willingness to Cultivate Traditional Rice in Sri Lanka: A Case Study in Anuradhapura District 229**

Chapter 14 **Assessing the Impact of Collective Marketing of Paddy Rice in**

Ginigaddara Appuhamilage Sanjeewanie Ginigaddara and

**Innovation Platforms by Smallholder Producers in Benin 241**

Shyama R. Weerakoon, Seneviratnage Somaratne, E. M. Sachini I.

**Organic Carbon Compounds to Produce Methane Gas 211**

Anitha Mani and Kavitha Sankaranarayanan

**Perspective of Salinity Tolerance 181**

Ekanayaka and Sachithri Munasighe

Prabhat Pramanik and Pil Joo Kim

Sampath Priya Disanayake

Aminou Arouna

**Rice Plant 149**

**VI** Contents

Muhammad Saeed

**Glufosinate) 193**

Rice has been found in archaeological sites dating back to 8000 BC or even earlier. This crop is unique in a way that it is domesticated independently in several continents such as Asia, Africa, Australia and South America. Worldwide production of rice is the third highest after sugarcane and maize and can be thus regarded as central to the lives of millions of people on earth. The yield of rice can be greatly affected by both abiotic and biotic stresses, which under the changing environment are further threatening global food security. The major abiotic stresses include drought, excessive watering, extreme temperatures, salinity and mineral toxicity along with many others. A recent increase in global warming has exposed rice crops to elevated temperatures and drought, which in turn have already offset a sig‐ nificant portion of yield increase. Similarly, heavy metal contamination of agricultural land not only causes abiotic stress for the crop, but has also shown drastic effects on humans. Increased metal concentration in plants leads to the production of reactive oxygen species, which ultimately cause cell death and thus negatively affect overall crop productivity.

Also, the presence of pesticide residues and insect pest attack can reduce the quality and quantity of the rice grain. Some pathogens have the ability to cause devastating diseases in rice, especially in intensive production systems, such as double rice-cropping systems. For instance, the intensity of sheath blight in rice-growing regions has increased due to several agronomic practices, characterized by abundant nitrogenous fertilizer application, increased planting density, and use of popular high-yielding hybrid cultivars. Likewise, *Pomacea canal‐ iculata*, the golden apple snail, is well known as a major pest of rice as it can cause severe damage by completely eliminating the young leaves and stems from the plant base, which may result in the death of damaged plants. To combat such abiotic and biotic stresses, new rice cultivars must be developed, which are not only input and management responsive but are rich in macro- and micronutrients as well. Fortunately, molecular biology has made it possible to develop high-yielding resistant rice cultivars in a short span of time.

The present book is aimed at attracting a wider range of audience, ranging from rice grow‐ ers, students, and researchers to policymakers, who are somehow directly or indirectly in‐ volved with the rice industry. Although most of the chapters are well focused on the scientific aspects (biofortification, quality and quantity of grain, use of anther culture as a breeding tool, and response and management of crops under elevated temperature) of rice, some chapters may be of particular interest for marketing personnel also. Most of the con‐ tents of this book are very easy to read and understand. We are sure that this book will not only serve in the capacity building of fresh students, but will provide the basis for seasoned scientists to explore further in the relevant field. This concise book on rice will be an invalu‐ able teaching resource and reference text for all academic and practical workers engaged in rice production systems, which are extremely prone to climatic changes.

We are dedicating this book to one of the greatest living legends Professor Yuan Longping who is known as the "Father of Hybrid Rice". Indeed the whole world is greatly indebted to him for his valuable contribution in the field of rice breeding which has helped in ensuring global food security of not only the current but also future generations.

### **Dr. Farooq Shah, Dr. Zafar Hayat Khan, and Dr. Amjad Iqbal**

Department of Agriculture Abdul Wali Khan University Mardan, Pakistan **Chapter 1**

**Provisional chapter**

**Anther Culture as a Supplementary Tool for Rice**

**Anther Culture as a Supplementary Tool for Rice** 

DOI: 10.5772/intechopen.76157

There is a timely need to harness biotechnology and related tools to support conventional breeding strategies, overcoming the limitations in rice production and improving quantity and quality as well as climatic and disease stress tolerance of the crop. Anther culture allows immediate fixation of homozygosity through diploidization of regenerated haploid plants and therefore serves as an efficient path for inbred line development. Anther culture has been successfully used to hasten the breeding programs in several crop species including rice. However, associated constraints still prevent the realization of its full potential. Even though anther culture technique has been effective for Japonica rice breeding, applicability for Indica rice remains limited mainly due to inherent recalcitrant genetic background. Constraints associated with Indica rice can be identified as early anther necrosis, poor callus induction and proliferation, extremely low green plant regeneration and frequent albinism. Success of androgenesis is determined by factors such as genotype, physiological status of donor plant, pollen development stage at culture, composition and physical status of culture media, culture incubation conditions and anther pretreatments. This chapter has detailed out the scope for improving the applicability of anther culture technique on rice in order to develop it as a supplementary

**Keywords:** callus induction, plant regeneration, microspores, ploidy, homozygous lines

From ancient times the crop rice has served the human population as a staple food. Due to the steep increase in human population, rice growers need to increase the production as well. This has become more difficult due to the limitations in available resources. Therefore, other

> © 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.M. Ruwani G. Mayakaduwa and Tara D. Silva

D.M. Ruwani G. Mayakaduwa and Tara D. Silva

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.76157

**Breeding**

**Abstract**

breeding tool.

**1. Introduction**

**Breeding**

#### **Anther Culture as a Supplementary Tool for Rice Breeding Anther Culture as a Supplementary Tool for Rice Breeding**

DOI: 10.5772/intechopen.76157

D.M. Ruwani G. Mayakaduwa and Tara D. Silva D.M. Ruwani G. Mayakaduwa and Tara D. Silva

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.76157

#### **Abstract**

We are dedicating this book to one of the greatest living legends Professor Yuan Longping who is known as the "Father of Hybrid Rice". Indeed the whole world is greatly indebted to him for his valuable contribution in the field of rice breeding which has helped in ensuring

**Dr. Farooq Shah, Dr. Zafar Hayat Khan, and Dr. Amjad Iqbal**

Abdul Wali Khan University Mardan, Pakistan

Department of Agriculture

global food security of not only the current but also future generations.

VIII Preface

There is a timely need to harness biotechnology and related tools to support conventional breeding strategies, overcoming the limitations in rice production and improving quantity and quality as well as climatic and disease stress tolerance of the crop. Anther culture allows immediate fixation of homozygosity through diploidization of regenerated haploid plants and therefore serves as an efficient path for inbred line development. Anther culture has been successfully used to hasten the breeding programs in several crop species including rice. However, associated constraints still prevent the realization of its full potential. Even though anther culture technique has been effective for Japonica rice breeding, applicability for Indica rice remains limited mainly due to inherent recalcitrant genetic background. Constraints associated with Indica rice can be identified as early anther necrosis, poor callus induction and proliferation, extremely low green plant regeneration and frequent albinism. Success of androgenesis is determined by factors such as genotype, physiological status of donor plant, pollen development stage at culture, composition and physical status of culture media, culture incubation conditions and anther pretreatments. This chapter has detailed out the scope for improving the applicability of anther culture technique on rice in order to develop it as a supplementary breeding tool.

**Keywords:** callus induction, plant regeneration, microspores, ploidy, homozygous lines

### **1. Introduction**

From ancient times the crop rice has served the human population as a staple food. Due to the steep increase in human population, rice growers need to increase the production as well. This has become more difficult due to the limitations in available resources. Therefore, other

© 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.

than the conventional strategies, it is a timely need to harness biotechnology and related tools to overcome not only the productivity barrier but also the production efficiency, quality of the product, and abiotic and biotic stress tolerance of the crop. Among a number of modern biotechnological tools to improve rice crop, anther culture plays a useful role.

**3. Historical trajectory of anther culture**

Indica rice breeding is yet to be fully unraveled [3].

**4. Limitations associated with androgenesis**

The possibility of changing the normal gametophytic pathway of microspores to sporophytic pathway facilitating the haploid plant development through *in vitro* culture was first reported by reference [7] on culturing immature anthers of the Solanaceous species *Datura innoxia*. Reference [8] successfully obtained haploid plants from culturing isolated anthers of *Nicotiana*. Since then, haploid development using *in vitro* culture of anthers and isolated pollen has been successful with many other crop species such as rice, wheat, maize, *Brassica*, and pepper [2, 5]. Although microspore embryogenesis has been effective with model species such as barley, rapeseed, tobacco, and wheat, some other species that are scientifically or economically important, such as *Arabidopsis*, woody plants, and legume crops, continue to be less responsive for the technique. Extensive research has been performed in order to make this important technique of developing haploids and dihaploids more robust. For the technique to be practically applied in breeding programs, anther or microspore culture should be able to permit produc-

Anther Culture as a Supplementary Tool for Rice Breeding

http://dx.doi.org/10.5772/intechopen.76157

3

tion of haploids in very large quantities from almost any species or genotype [9].

Haploid plant production in rice through anther culture was first reported by reference [10]. Since then, many studies have been conducted improving various aspects of rice anther culture. Other than utilizing anther culture technique directly for dihaploid development, recently applications have been expanded to facilitate other biotechnological approaches such as gene transformation [3]. In Japan and China, where the Japonica rice varieties are mainly in use, anther culture technique has been extensively applied for improving the rice crop due to the amenability of Japonica rice varieties to *in vitro* anther culture [5]. However, the use of this technique as a tool for Indica rice breeding has been extremely limited due to the inherent recalcitrance associated with Indica varieties. Therefore, the potential of the technique for

Induction of haploids in rice is associated with a number of constraints. Fine tuning of anther culture process addressing the constraints is required in order to use this technique equally well for breeding of Japonica and Indica rice. Although anther culture technique has been used to produce haploids from an array of species, success of the technique cannot be proven in respect of all genotypes of a crop species [11]. Particularly when it comes to the anther culture of Indica rice, the response remains extremely variety or genotype specific [1]. The problem is further aggravated because anther culture response is affected even by the growing season [4]. Under *in vitro* conditions, many of the anthers fail to grow in culture and thus repress the pollen from forming calluses. Some reasons for failure are the early abortion of pollen and even in situations where pollen starts to divide and produce callus and necrosis or cell death occurs very early during callus proliferation. There is also a degree of uncertainty associated with the ploidy of the resulting callus tissue as it can comprise a chimera of diploid, tetraploid, and haploid cells. Another problem that seriously affects the anther culture of cereals is the

Anther culture can be considered as a technique for the rapid development of fully homozygous lines. Therefore, anther culture technique provides an efficient alternative to the conventional inbred line development which is usually achieved through several cycles of inbreeding. Even though anther culture has been efficiently used as a supplementary breeding tool with Japonica rice varieties, application of this technique for Indica rice varieties is limited due to their inherent recalcitrant genetic background. Limitations of androgenesis in Indica rice result from early anther necrosis, poor callus induction and proliferation, remarkably low green plant regenerability, and frequent albinism [1, 2]. Success of the technique is determined by numerous factors such as genotype and physiological status of donor plant, pollen development stage at culture, anther wall factors, composition of culture media including nutritional sources and growth regulators, physical status of culture media, and anther pretreatments [1, 3, 4].

This chapter serves as an insight to the practical aspects of anther culture technique for it to be fully exploited for improving rice breeding.

### **2. Technique of anther culture**

For androgenesis to be successful, normal gametophyte formation from microspores should be halted, and microspores are directed toward sporophyte development. Usually, pretreatments are required to alter the normal pollen development pathway and to trigger the androgenic response. The specific pretreatments for androgenesis that are required by different species and also varieties within species are quite variable. Therefore, a single standard method cannot be generalized for androgenesis for a given species or even a variety. However, some common protocols to be followed during anther culture are well known and documented [4].

Rice anther culture is carried out in two phases in which the initial step is to induce embryogenic calluses from microspores followed by green plant regeneration from the induced calluses [5]. Protocol for rice anther culture includes pretreatment given to panicles, surface sterilization and excision of anthers from panicles, and *in vitro* culture of anthers on a specific culture medium under aseptic conditions [6]. Response of anthers in culture is usually indicated by the gradual browning of the anther wall tissues and bursting or splitting of the anther to expose the pollen callus. Pollen callus can be expected to be formed in anthers after 3–8 weeks of culture [2]. The second phase is to regenerate green plants from the calluses using appropriate regeneration media [6]. The regenerated plants are then transplanted and acclimatized under controlled environmental conditions, and they can be subjected to chromosome doubling using antimitotic agents in order to obtain doubled haploids which can serve as homozygous lines.

### **3. Historical trajectory of anther culture**

than the conventional strategies, it is a timely need to harness biotechnology and related tools to overcome not only the productivity barrier but also the production efficiency, quality of the product, and abiotic and biotic stress tolerance of the crop. Among a number of modern

Anther culture can be considered as a technique for the rapid development of fully homozygous lines. Therefore, anther culture technique provides an efficient alternative to the conventional inbred line development which is usually achieved through several cycles of inbreeding. Even though anther culture has been efficiently used as a supplementary breeding tool with Japonica rice varieties, application of this technique for Indica rice varieties is limited due to their inherent recalcitrant genetic background. Limitations of androgenesis in Indica rice result from early anther necrosis, poor callus induction and proliferation, remarkably low green plant regenerability, and frequent albinism [1, 2]. Success of the technique is determined by numerous factors such as genotype and physiological status of donor plant, pollen development stage at culture, anther wall factors, composition of culture media including nutritional sources and growth regulators, physical status of culture media, and anther

This chapter serves as an insight to the practical aspects of anther culture technique for it to be

For androgenesis to be successful, normal gametophyte formation from microspores should be halted, and microspores are directed toward sporophyte development. Usually, pretreatments are required to alter the normal pollen development pathway and to trigger the androgenic response. The specific pretreatments for androgenesis that are required by different species and also varieties within species are quite variable. Therefore, a single standard method cannot be generalized for androgenesis for a given species or even a variety. However, some common protocols to be followed during anther culture are well known and

Rice anther culture is carried out in two phases in which the initial step is to induce embryogenic calluses from microspores followed by green plant regeneration from the induced calluses [5]. Protocol for rice anther culture includes pretreatment given to panicles, surface sterilization and excision of anthers from panicles, and *in vitro* culture of anthers on a specific culture medium under aseptic conditions [6]. Response of anthers in culture is usually indicated by the gradual browning of the anther wall tissues and bursting or splitting of the anther to expose the pollen callus. Pollen callus can be expected to be formed in anthers after 3–8 weeks of culture [2]. The second phase is to regenerate green plants from the calluses using appropriate regeneration media [6]. The regenerated plants are then transplanted and acclimatized under controlled environmental conditions, and they can be subjected to chromosome doubling using antimitotic agents in order to obtain doubled haploids which can

biotechnological tools to improve rice crop, anther culture plays a useful role.

pretreatments [1, 3, 4].

2 Rice Crop - Current Developments

documented [4].

serve as homozygous lines.

fully exploited for improving rice breeding.

**2. Technique of anther culture**

The possibility of changing the normal gametophytic pathway of microspores to sporophytic pathway facilitating the haploid plant development through *in vitro* culture was first reported by reference [7] on culturing immature anthers of the Solanaceous species *Datura innoxia*. Reference [8] successfully obtained haploid plants from culturing isolated anthers of *Nicotiana*. Since then, haploid development using *in vitro* culture of anthers and isolated pollen has been successful with many other crop species such as rice, wheat, maize, *Brassica*, and pepper [2, 5]. Although microspore embryogenesis has been effective with model species such as barley, rapeseed, tobacco, and wheat, some other species that are scientifically or economically important, such as *Arabidopsis*, woody plants, and legume crops, continue to be less responsive for the technique. Extensive research has been performed in order to make this important technique of developing haploids and dihaploids more robust. For the technique to be practically applied in breeding programs, anther or microspore culture should be able to permit production of haploids in very large quantities from almost any species or genotype [9].

Haploid plant production in rice through anther culture was first reported by reference [10]. Since then, many studies have been conducted improving various aspects of rice anther culture. Other than utilizing anther culture technique directly for dihaploid development, recently applications have been expanded to facilitate other biotechnological approaches such as gene transformation [3]. In Japan and China, where the Japonica rice varieties are mainly in use, anther culture technique has been extensively applied for improving the rice crop due to the amenability of Japonica rice varieties to *in vitro* anther culture [5]. However, the use of this technique as a tool for Indica rice breeding has been extremely limited due to the inherent recalcitrance associated with Indica varieties. Therefore, the potential of the technique for Indica rice breeding is yet to be fully unraveled [3].

### **4. Limitations associated with androgenesis**

Induction of haploids in rice is associated with a number of constraints. Fine tuning of anther culture process addressing the constraints is required in order to use this technique equally well for breeding of Japonica and Indica rice. Although anther culture technique has been used to produce haploids from an array of species, success of the technique cannot be proven in respect of all genotypes of a crop species [11]. Particularly when it comes to the anther culture of Indica rice, the response remains extremely variety or genotype specific [1]. The problem is further aggravated because anther culture response is affected even by the growing season [4].

Under *in vitro* conditions, many of the anthers fail to grow in culture and thus repress the pollen from forming calluses. Some reasons for failure are the early abortion of pollen and even in situations where pollen starts to divide and produce callus and necrosis or cell death occurs very early during callus proliferation. There is also a degree of uncertainty associated with the ploidy of the resulting callus tissue as it can comprise a chimera of diploid, tetraploid, and haploid cells. Another problem that seriously affects the anther culture of cereals is the formation of albino plants during regeneration, and this can be identified as the most limiting step in the anther culture process [12]. Detailed investigations of proplastids and the plastid genome of the regenerated albino plantlets revealed that albinism is mainly due to incomplete formation of the membrane structures and different blockages in the plastid development [13]. Molecular studies carried out on anther culture of cereals such as wheat, barley, and rice have attributed the associated albinism to large-scale deletions and rearrangements in the plastid genome [14].

four varieties produced regeneration response. Similarly, reference [23] verified the extremely low androgenic response of Indica varieties as they found only 1 out of 35 Indica varieties

for each of different genotypes, may help to improve the low response associated with some

Success of the anther culture is greatly influenced by the physiological condition of the anther donor plants. That is mainly because physiology affects the number of viable and healthy pollen grains produced within the anthers, the endogenous levels of hormones that regulate metabolic pathways, and the nutritional status of the anther tissues [4]. During maturation of the anther donor plants, environmental factors such as light intensity, photoperiod,

Also, pest infestations and control measures may have a detrimental effect on microspore

An improved androgenic response of an Indica rice variety induced by growing donor plants under specific conditions of light and day/night temperature regime was observed by reference [24]. The highest anther response was shown by anthers cultured from donor plants of variety IR43 grown until panicle emergence stage under long days (>12 h), high solar radiation (>18 Mj m−2), and sunshine (>8 h) and day/night temperature (34/24°C), and a declined response was observed when the plants were grown under an environment with low values of the above conditions. They also observed that the plants grown under the field conditions were significantly superior than those grown in controlled conditions such as glasshouse or in pots near the field. Similar observations have also been reported for other cereals, such as maize and wheat. Certain chemicals such as ethereal, when applied on the donor plants, have altered their physiology, thereby enhancing the androgenic response [1]. Further, the anthers collected from the primary tillers were more responsive for anther culture than the anthers from panicles on late tillers [3]. When the anther donor plants were starved of nitrogen, anthers were able to produce much better response in *in vitro* culture compared to those

The pollen development stage is a critical factor that strongly affects the success of anther culture. Induction of embryogenic calluses cannot be achieved by culturing pollen in any stage of development, and the potential is restricted to specific pollen maturity stages only [4, 25]. For rice, the best responsive stage for embryogenic induction has been reported to be the middle to late uninucleate stage of microspore division, and therefore anthers need to be cultured at these specific stages [26–28]. These are very early stages of microspore development. The highest response shown by these early stages is most likely due to the fact that they are cells which have not yet been committed to gametophytic development and therefore can be forced to become proliferative. The undifferentiated cells in the callus can then assume a new

medium. The use of optimal media, specifically formulated

Anther Culture as a Supplementary Tool for Rice Breeding

http://dx.doi.org/10.5772/intechopen.76157

5

concentration critically affect the growth and development.

exhibited pollen callusing on N<sup>6</sup>

temperature, nutrition, and CO2

**5.3. Pollen development stage**

development [13].

**5.2. Physiological status of donor plants**

that are given optimum levels of nitrogen fertilizers [2].

pathway of development leading to sporophyte formation [4].

high valued varieties [13].

### **5. Factors affecting rice anther culture**

Investigations on haploid induction through anther culture have been steadily increasing due to its importance as a supplementary breeding strategy. These studies are mainly driven with close monitoring of a number of factors that influence androgenesis in rice as described in detail below.

#### **5.1. Genotype of the donor plants**

Response to anther culture by Indica rice varieties is generally poor, and even among those that respond by producing callus, the *in vitro* morphogenic responses are highly genotypedependent. The recalcitrance associated with the Indica types can be characterized mainly by poor callus induction response, poor regenerability of green plants, and the occurrence of a large proportion of albinos [1]. By comparison, Japonica rice varieties respond much better. Anther culture ability of Japonica varieties, Indica varieties, and their hybrids can be indicated in the following order of Japonica/Japonica > Japonica > Indica/Japonica > Indica/Indica > Indica [15]. Thus, the Japonica varieties have benefitted more from this supplementary breeding approach, and extensive practical applications have been possible. For example, 67,000– 159,000 anthers from F1 hybrids of 25–36 rice crosses produced 1500–15,000 (2–10%) green plants per season for selection [16]. Anther culture technique has been used with a Japonica rice variety to purify it and in the process to develop stable new lines that are distinctly different to the parent variety [17]. On the other hand, utilizing anther culture technique for Indica rice breeding has been extremely limited due to its comparatively poor androgenic response [3] with a few exceptions [18]. However, anther culture performance in F1 hybrids and F2 plants could be improved when high yielding commercially grown Indica rice varieties were crossed with high anther culture-responsive Japonica varieties [19].

Reference [20] has very convincingly illustrated the extreme variability in anther culture response between Japonica and Indica varieties as 41% for a Japonica variety to 0% for an Indica variety. Not only between the subspecies but also among different varieties from the same subspecies, a considerable variation for callus induction and plant regeneration has been observed. Reference [21] stated that among seven Indica rice varieties on anther culture, callus induction frequencies varied extensively from 3.6 to 51.7%, while green plant regeneration efficiency ranged from 1.6 to 82.9%. Reference [22] reported that out of 18 Indica varieties subjected to anther culture, only five varieties were responsive for pollen callusing, and only four varieties produced regeneration response. Similarly, reference [23] verified the extremely low androgenic response of Indica varieties as they found only 1 out of 35 Indica varieties exhibited pollen callusing on N<sup>6</sup> medium. The use of optimal media, specifically formulated for each of different genotypes, may help to improve the low response associated with some high valued varieties [13].

#### **5.2. Physiological status of donor plants**

formation of albino plants during regeneration, and this can be identified as the most limiting step in the anther culture process [12]. Detailed investigations of proplastids and the plastid genome of the regenerated albino plantlets revealed that albinism is mainly due to incomplete formation of the membrane structures and different blockages in the plastid development [13]. Molecular studies carried out on anther culture of cereals such as wheat, barley, and rice have attributed the associated albinism to large-scale deletions and rearrangements in the plastid

Investigations on haploid induction through anther culture have been steadily increasing due to its importance as a supplementary breeding strategy. These studies are mainly driven with close monitoring of a number of factors that influence androgenesis in rice as described in

Response to anther culture by Indica rice varieties is generally poor, and even among those that respond by producing callus, the *in vitro* morphogenic responses are highly genotypedependent. The recalcitrance associated with the Indica types can be characterized mainly by poor callus induction response, poor regenerability of green plants, and the occurrence of a large proportion of albinos [1]. By comparison, Japonica rice varieties respond much better. Anther culture ability of Japonica varieties, Indica varieties, and their hybrids can be indicated in the following order of Japonica/Japonica > Japonica > Indica/Japonica > Indica/Indica > Indica [15]. Thus, the Japonica varieties have benefitted more from this supplementary breeding approach, and extensive practical applications have been possible. For example, 67,000– 159,000 anthers from F1 hybrids of 25–36 rice crosses produced 1500–15,000 (2–10%) green plants per season for selection [16]. Anther culture technique has been used with a Japonica rice variety to purify it and in the process to develop stable new lines that are distinctly different to the parent variety [17]. On the other hand, utilizing anther culture technique for Indica rice breeding has been extremely limited due to its comparatively poor androgenic response

[3] with a few exceptions [18]. However, anther culture performance in F1

crossed with high anther culture-responsive Japonica varieties [19].

plants could be improved when high yielding commercially grown Indica rice varieties were

Reference [20] has very convincingly illustrated the extreme variability in anther culture response between Japonica and Indica varieties as 41% for a Japonica variety to 0% for an Indica variety. Not only between the subspecies but also among different varieties from the same subspecies, a considerable variation for callus induction and plant regeneration has been observed. Reference [21] stated that among seven Indica rice varieties on anther culture, callus induction frequencies varied extensively from 3.6 to 51.7%, while green plant regeneration efficiency ranged from 1.6 to 82.9%. Reference [22] reported that out of 18 Indica varieties subjected to anther culture, only five varieties were responsive for pollen callusing, and only

hybrids and F2

genome [14].

4 Rice Crop - Current Developments

detail below.

**5. Factors affecting rice anther culture**

**5.1. Genotype of the donor plants**

Success of the anther culture is greatly influenced by the physiological condition of the anther donor plants. That is mainly because physiology affects the number of viable and healthy pollen grains produced within the anthers, the endogenous levels of hormones that regulate metabolic pathways, and the nutritional status of the anther tissues [4]. During maturation of the anther donor plants, environmental factors such as light intensity, photoperiod, temperature, nutrition, and CO2 concentration critically affect the growth and development. Also, pest infestations and control measures may have a detrimental effect on microspore development [13].

An improved androgenic response of an Indica rice variety induced by growing donor plants under specific conditions of light and day/night temperature regime was observed by reference [24]. The highest anther response was shown by anthers cultured from donor plants of variety IR43 grown until panicle emergence stage under long days (>12 h), high solar radiation (>18 Mj m−2), and sunshine (>8 h) and day/night temperature (34/24°C), and a declined response was observed when the plants were grown under an environment with low values of the above conditions. They also observed that the plants grown under the field conditions were significantly superior than those grown in controlled conditions such as glasshouse or in pots near the field. Similar observations have also been reported for other cereals, such as maize and wheat. Certain chemicals such as ethereal, when applied on the donor plants, have altered their physiology, thereby enhancing the androgenic response [1]. Further, the anthers collected from the primary tillers were more responsive for anther culture than the anthers from panicles on late tillers [3]. When the anther donor plants were starved of nitrogen, anthers were able to produce much better response in *in vitro* culture compared to those that are given optimum levels of nitrogen fertilizers [2].

#### **5.3. Pollen development stage**

The pollen development stage is a critical factor that strongly affects the success of anther culture. Induction of embryogenic calluses cannot be achieved by culturing pollen in any stage of development, and the potential is restricted to specific pollen maturity stages only [4, 25]. For rice, the best responsive stage for embryogenic induction has been reported to be the middle to late uninucleate stage of microspore division, and therefore anthers need to be cultured at these specific stages [26–28]. These are very early stages of microspore development. The highest response shown by these early stages is most likely due to the fact that they are cells which have not yet been committed to gametophytic development and therefore can be forced to become proliferative. The undifferentiated cells in the callus can then assume a new pathway of development leading to sporophyte formation [4].

Although it is stated that the best responsive stages are the middle to late uninucleate microspores, the precise stage of microspores that is best suited for producing a superior anther response can vary from one genotype to another. Therefore, application of the anther culture technique requires detailed examination of pollen before culture to determine its effective development stage [3]. When culturing rice anthers, determining the microspore development stage requires nuclear staining and cytological examination of microspores prior to culture. However, repeating nuclear staining of microspores with each rice panicle, before dissecting anthers for culture, greatly impedes the anther culture process. Therefore, a distinct morphological indicator trait that correlates well with the stage of microspore maturity is commonly used during *in vitro* culture. In rice anther culture, the morphological trait that has been used is the measured distance between the nodes of the last two leaves: the flag leaf, and the penultimate leaf [27, 29]. In some cases the panicle length at the time of harvest has also been used as a visually identifiable guide [30, 31]. The use of a direct cytological marker such as the degree of starch accumulation in microspores has been identified as more accurate than the internode distance and even more convenient than the laborious nuclear staining process to rapidly assess microspore maturity. The most appropriate stage is when pollen grains just begin to accumulate starch which can be simply tested with I2 /KI solution [32].

as the standard carbon source, different sources have also been tested and proven effective for cereals. Maltose has been identified as a superior source of carbohydrate compared to sucrose for androgenesis in cereals. Anther culture efficiency and green plant formation of highly recalcitrant Indica rice varieties could be improved significantly when sucrose was replaced by maltose [1]. Reference [23] reported an inferior anther culture response with sucrose, as only 1 out of 23 Indica rice varieties responded with pollen callusing and green plant production on N6 medium provided with 146 mM sucrose. When sucrose was replaced by equimolar amount of maltose, callus induction response improved from 6.3% to 10.1% and green plant regeneration from 0.6–1%. Reference [36] had observed that 20% maltose used for microspore isolation and 9% maltose used for culturing produced a genotype-independent plant regeneration response. In other cereals such as wheat, maize, and barley, maltose promoted direct embryogenesis from cultured pollen. Sucrose is rapidly broken down into glucose and fructose. The toxic effects of sucrose on androgenesis have been attributed to the sensitivity of microspores to fructose. This also causes depletion of sucrose in the medium with time [1]. Comparatively, long-term availability of maltose in the culture medium has been detected due to the slow rate of hydrolysis.

In culture media, inorganic nitrogen is usually supplied in the form of nitrate and/or ammo-

with both these sources of nitrogen at specific concentrations. However, Indica rice variet-

medium [1]. Reference [37], in which the response of eight Indica rice varieties were studied

+

to 1.5 mM has been recommended for Indica-Japonica hybrids [5]. Reference [38] reported that a significant improvement in anther culture could be made in Indica x Indica F1 hybrids

Reference [39] studied the effect of nitrogen source on androgenesis in another Indica variety IR24 using R-2 medium as the control. R-2 has been formulated with 40 mM KNO<sup>3</sup>

superior green plant regeneration could be achieved. In rice anther culture, amino acids such as proline and glutamine added to the culture media have been able to increase the rate of callus induction from cultured anthers while avoiding the degeneration of anther wall tissue [4].

Plant growth regulators have been widely investigated in anther culture. Supplementing *in vitro* culture media with effective growth regulators (auxins, cytokinins, or a combination of these) as appropriate is crucial for the success of androgenic response particularly from

except that the (NH<sup>4</sup>

+

and NH4

medium to be more effective than N<sup>6</sup>

) 2 SO4

− :NH4 +

+

basal medium which is most widely used for rice anther culture has been formulated

has been found to be critical for

ions than normal is used in the

medium is


and

medium. He2

concentration is reduced from 3.5

concentration to half strength. In Korea, N<sup>6</sup>

Anther Culture as a Supplementary Tool for Rice Breeding

http://dx.doi.org/10.5772/intechopen.76157

7

ions completely replaced by an organic source of

was combined with the amino acid 5 mM alanine,

*5.4.2. Nitrogen source*

The N6

nium ions. The ratio of the two nitrogen sources NO3

ies perform much better when lower concentration of NH<sup>4</sup>

. When 20 mM KNO<sup>3</sup>

medium by reducing NH4

the success of anther culture in rice [3].

on different media, found He<sup>2</sup>

medium which is similar to N6

using a medium with high KNO<sup>3</sup>

) 2 SO4

*5.4.3. Plant growth regulators*

recalcitrant genotypes [4].

nitrogen, casein hydrolysate, at 50 mgL−1.

derived from the N6

2.5 mM (NH4

#### **5.4. Culture media**

The two main phases of anther culture in rice, callus induction and shoot regeneration, require different nutrient regimes and growth regulators. The culture medium that best supports callus induction is often not suitable for regeneration. Therefore, the transfer of callus onto a suitable regeneration medium must be done at an appropriate time. Since the callus induction potential of a given rice variety is largely determined by the genetic makeup, significant levels of improvement in anther response cannot be expected by manipulation of nongenetic factors such as the culture medium. Nevertheless, the best responsive nutrient requirements must be chosen as an initial step in order to optimize anther culture, particularly if they are low responding Indica varieties [3].

The most commonly used basal media for anther culture are N6 medium [33], MS medium [34], and B5 medium [35]. Generally, basal N6 medium supplemented with plant growth regulators has been used extensively in cereal anther culture to initiate callus. Macronutrients of culture media comprises mainly of carbon and nitrogen sources. Embryogenic and morphogenic responses are elicited by supplementing the basal media with appropriate plant growth regulators at effective concentrations. Physical state of the culture medium and also culture maintenance conditions are equally important for the success of rice anther culture.

#### *5.4.1. Carbohydrate source*

A carbohydrate source is essential in tissue culture media because it serves as the main source of energy to the cultured explant tissue. Carbohydrates are also important as osmotic agents. In rice anther culture, osmotic pressure in the medium is generally regulated by applying the carbohydrate source to the medium at a particular concentration. Very high concentrations when used during the latter stages of culture seem to be deleterious for cereals [4]. The type of carbon source directly influences the anther response. Although many early studies have used sucrose as the standard carbon source, different sources have also been tested and proven effective for cereals. Maltose has been identified as a superior source of carbohydrate compared to sucrose for androgenesis in cereals. Anther culture efficiency and green plant formation of highly recalcitrant Indica rice varieties could be improved significantly when sucrose was replaced by maltose [1]. Reference [23] reported an inferior anther culture response with sucrose, as only 1 out of 23 Indica rice varieties responded with pollen callusing and green plant production on N6 medium provided with 146 mM sucrose. When sucrose was replaced by equimolar amount of maltose, callus induction response improved from 6.3% to 10.1% and green plant regeneration from 0.6–1%. Reference [36] had observed that 20% maltose used for microspore isolation and 9% maltose used for culturing produced a genotype-independent plant regeneration response. In other cereals such as wheat, maize, and barley, maltose promoted direct embryogenesis from cultured pollen. Sucrose is rapidly broken down into glucose and fructose. The toxic effects of sucrose on androgenesis have been attributed to the sensitivity of microspores to fructose. This also causes depletion of sucrose in the medium with time [1]. Comparatively, long-term availability of maltose in the culture medium has been detected due to the slow rate of hydrolysis.

#### *5.4.2. Nitrogen source*

Although it is stated that the best responsive stages are the middle to late uninucleate microspores, the precise stage of microspores that is best suited for producing a superior anther response can vary from one genotype to another. Therefore, application of the anther culture technique requires detailed examination of pollen before culture to determine its effective development stage [3]. When culturing rice anthers, determining the microspore development stage requires nuclear staining and cytological examination of microspores prior to culture. However, repeating nuclear staining of microspores with each rice panicle, before dissecting anthers for culture, greatly impedes the anther culture process. Therefore, a distinct morphological indicator trait that correlates well with the stage of microspore maturity is commonly used during *in vitro* culture. In rice anther culture, the morphological trait that has been used is the measured distance between the nodes of the last two leaves: the flag leaf, and the penultimate leaf [27, 29]. In some cases the panicle length at the time of harvest has also been used as a visually identifiable guide [30, 31]. The use of a direct cytological marker such as the degree of starch accumulation in microspores has been identified as more accurate than the internode distance and even more convenient than the laborious nuclear staining process to rapidly assess microspore maturity. The most appropriate stage is when pollen grains just

The two main phases of anther culture in rice, callus induction and shoot regeneration, require different nutrient regimes and growth regulators. The culture medium that best supports callus induction is often not suitable for regeneration. Therefore, the transfer of callus onto a suitable regeneration medium must be done at an appropriate time. Since the callus induction potential of a given rice variety is largely determined by the genetic makeup, significant levels of improvement in anther response cannot be expected by manipulation of nongenetic factors such as the culture medium. Nevertheless, the best responsive nutrient requirements must be chosen as an initial step in order to optimize anther culture, particularly if they are

ulators has been used extensively in cereal anther culture to initiate callus. Macronutrients of culture media comprises mainly of carbon and nitrogen sources. Embryogenic and morphogenic responses are elicited by supplementing the basal media with appropriate plant growth regulators at effective concentrations. Physical state of the culture medium and also culture

A carbohydrate source is essential in tissue culture media because it serves as the main source of energy to the cultured explant tissue. Carbohydrates are also important as osmotic agents. In rice anther culture, osmotic pressure in the medium is generally regulated by applying the carbohydrate source to the medium at a particular concentration. Very high concentrations when used during the latter stages of culture seem to be deleterious for cereals [4]. The type of carbon source directly influences the anther response. Although many early studies have used sucrose

maintenance conditions are equally important for the success of rice anther culture.

/KI solution [32].

medium supplemented with plant growth reg-

medium [33], MS medium

begin to accumulate starch which can be simply tested with I2

The most commonly used basal media for anther culture are N6

**5.4. Culture media**

6 Rice Crop - Current Developments

low responding Indica varieties [3].

*5.4.1. Carbohydrate source*

[34], and B5 medium [35]. Generally, basal N6

In culture media, inorganic nitrogen is usually supplied in the form of nitrate and/or ammonium ions. The ratio of the two nitrogen sources NO3 − :NH4 + has been found to be critical for the success of anther culture in rice [3].

The N6 basal medium which is most widely used for rice anther culture has been formulated with both these sources of nitrogen at specific concentrations. However, Indica rice varieties perform much better when lower concentration of NH<sup>4</sup> + ions than normal is used in the medium [1]. Reference [37], in which the response of eight Indica rice varieties were studied on different media, found He<sup>2</sup> medium to be more effective than N<sup>6</sup> medium. He2 medium is derived from the N6 medium by reducing NH4 + concentration to half strength. In Korea, N<sup>6</sup> -Y1 medium which is similar to N6 except that the (NH<sup>4</sup> )2 SO4 concentration is reduced from 3.5 to 1.5 mM has been recommended for Indica-Japonica hybrids [5]. Reference [38] reported that a significant improvement in anther culture could be made in Indica x Indica F1 hybrids using a medium with high KNO<sup>3</sup> and NH4 + ions completely replaced by an organic source of nitrogen, casein hydrolysate, at 50 mgL−1.

Reference [39] studied the effect of nitrogen source on androgenesis in another Indica variety IR24 using R-2 medium as the control. R-2 has been formulated with 40 mM KNO<sup>3</sup> and 2.5 mM (NH4 )2 SO4 . When 20 mM KNO<sup>3</sup> was combined with the amino acid 5 mM alanine, superior green plant regeneration could be achieved. In rice anther culture, amino acids such as proline and glutamine added to the culture media have been able to increase the rate of callus induction from cultured anthers while avoiding the degeneration of anther wall tissue [4].

#### *5.4.3. Plant growth regulators*

Plant growth regulators have been widely investigated in anther culture. Supplementing *in vitro* culture media with effective growth regulators (auxins, cytokinins, or a combination of these) as appropriate is crucial for the success of androgenic response particularly from recalcitrant genotypes [4].

The growth regulator 2,4-dichlorophenoxy acetic acid (2,4-D) is commonly used in the first phase of rice anther culture, and 2,4-D provided at fairly high concentrations (2 mgL−1) has produced improved rates of callus induction of up to 15% in some genotypes [24]. Also, applicability of some other auxins such as naphthalene acetic acid (NAA), phenyl acetic acid, picloram, and dicamba alone or in combination with 2,4-D has been tested for improving androgenic response. Not only the growth regulator combination but also the auxin/cytokinin balance has been found critically important for effective androgenesis. Reference [23] reported that the growth regulator regime of 2,4-D (2 mgL−1), picloram (0.07 mgL−1), and kinetin (0.5 mgL−1) was favorable for enhancing the anther response in a large number of genotypes. Further, the type of auxin and its concentration determine the microspore development pathway. For example, the use of 2,4-D favored callus formation, whereas indole-3-acetic acid and NAA promoted direct embryogenesis from cultured anthers without an intervening callus phase [4].

Bokra and Pokkali, callus induction frequencies and plant regeneration responses could be improved when cultures were incubated at alternating temperature regime of 30/20°C (14/10 h) instead of constant incubation at 25°C [5]. Light regulates morphogenesis of cultured pollen and specifically darkness (low intensity of light) or alternating light and dark conditions can be preferable for embryogenic induction. Reference [41] reported the effectiveness of culture conditions such as alternating periods of light with different temperatures (12–18 h;

even more specific incubation conditions to achieve success, particularly for green shoot formation. Shoot regeneration from scutellum-derived callus of Indica rice was stimulated by applying osmotic stress conditions. Osmotic stress was created in tissues by altering the water content of the medium with the use of agarose and mannitol or by partial desiccation of callus. It is possible to expect similar stimulatory effects in anther-derived callus also. With osmotic stress, water content in the calluses is reduced, thus converting the callus tissues into

The composition of the atmosphere in the culture vessel has not been thoroughly studied despite its importance shown with tobacco [4]. Explant density and explant orientation in the

In many crop varieties including cereals, usually a treatment applied to excised anthers, inflorescences, or anther donor plants prior to culture is important to trigger the sporophytic development deviating from normal pollen development pathway. The type of the effective pretreatment, duration, and the time of application vary with the species or even for different varieties [1, 4]. Reference [43] reviewed the different pretreatments which are in current applications for triggering the anther culture response, and they have been classified into three categories based on their utility as widely used, neglected, and novel. These pretreatments include high temperature and chilling, high humidity, water stress, anaerobic treatment, centrifugation, sucrose and nitrogen starvation, ethanol, γ-irradiation, use of microtubule dis-

Most frequently used effective method of pretreatment for rice anther culture is the low-temperature application. Harvested rice panicles are subjected to cold shock prior to the culture. However, the temperature and duration vary with the variety. Cold pretreatment given to rice anthers is known to enhance the androgenesis potential by delaying the degeneration of microspores and anther wall tissue in rice [1, 3]. Reference [44] reported that a pretreatment at 10°C for 10–30 days was necessary to induce sporophytic divisions in microspores of the Japonicas. Generally, temperatures from 8 to 10°C for 8 days have been recommended to be optimal for many varieties of rice [45]. Panicle pretreatments longer than 11 days tend to increase albino production [46]. Reference [37] reported a brief exposure to high temperature (35°C for 10 min) before the cold treatment to enhance callus induction although it adversely

more compact structures with better embryogenic and regeneration potential [42].

ruptive agents, electrostimulation, high medium pH, and heavy metal treatment.

culture medium also have been found to be critical in anther culture [2, 4].

**5.5. Pretreatments to trigger androgenesis in rice**

*5.5.1. Temperature pretreatment*

affected green plant production.

at 28°C and 12–6 h; in darkness at 22°C). Regeneration phase requires

Anther Culture as a Supplementary Tool for Rice Breeding

http://dx.doi.org/10.5772/intechopen.76157

9

5000–10,000 lux/m<sup>2</sup>

Although high levels of 2,4-D were useful for increasing callus production, it has proven to have a negative effect on the next phase of culture which is regeneration from callus, particularly from recalcitrant rice genotypes [22]. A lower level of 2,4-D (0.5 mgL−1) in combination with the milder auxin NAA (2.5 mgL−1) and kinetin (0.5 mgL−1) has been used effectively during both phases [9]. This suggests that the use of 2,4-D in the callus induction medium needs to be regulated with a compromise reached between callus induction and regeneration efficiency [24].

#### *5.4.4. Physical state of the medium*

Usually, rice anthers are cultured on solid media. However, reference [23] found increased necrosis of anther tissue when they were cultured on solid media and observed a better callusing response in liquid media. Liquid culture media are able to supply the anthers with an improved access to nutrients and plant growth regulators, and also toxic and degenerated material can be readily dispersed. During the culture of anthers from Indica×Basmati rice on liquid media, severalfold increment in green plant regeneration comparable with the rates reported for Japonica rice varieties/hybrids could be obtained [29].

However, since the rice anthers tend to settle at the bottom of the liquid cultures, this would affect respiration and result in loss of viability of the explants. These have been identified as barriers for the use of liquid media for androgenesis. When the liquid culture media was added with substances such as Ficoll, it was possible to avoid sinking of anthers due to the increased buoyancy, and therefore viability could be maintained [3]. In principle, the solidifying agent should not carry any nutritional effect. Agar is in extensive use as the gelling agent of solid culture media. However, more reproducible results have been obtained with the use of Gelrite. Starch also has been used for solidification despite the nutritional effects and its dissociation into sugar [13]. Some have found improved response by embedding anthers in agarose than culturing on semisolid or liquid media [40].

#### *5.4.5. Culture incubation conditions*

Culture temperature plays an important role in plant tissue culture. Anther cultures are usually incubated at the temperature range of 24–27°C. For two Indica rice varieties, Nona Bokra and Pokkali, callus induction frequencies and plant regeneration responses could be improved when cultures were incubated at alternating temperature regime of 30/20°C (14/10 h) instead of constant incubation at 25°C [5]. Light regulates morphogenesis of cultured pollen and specifically darkness (low intensity of light) or alternating light and dark conditions can be preferable for embryogenic induction. Reference [41] reported the effectiveness of culture conditions such as alternating periods of light with different temperatures (12–18 h; 5000–10,000 lux/m<sup>2</sup> at 28°C and 12–6 h; in darkness at 22°C). Regeneration phase requires even more specific incubation conditions to achieve success, particularly for green shoot formation. Shoot regeneration from scutellum-derived callus of Indica rice was stimulated by applying osmotic stress conditions. Osmotic stress was created in tissues by altering the water content of the medium with the use of agarose and mannitol or by partial desiccation of callus. It is possible to expect similar stimulatory effects in anther-derived callus also. With osmotic stress, water content in the calluses is reduced, thus converting the callus tissues into more compact structures with better embryogenic and regeneration potential [42].

The composition of the atmosphere in the culture vessel has not been thoroughly studied despite its importance shown with tobacco [4]. Explant density and explant orientation in the culture medium also have been found to be critical in anther culture [2, 4].

#### **5.5. Pretreatments to trigger androgenesis in rice**

In many crop varieties including cereals, usually a treatment applied to excised anthers, inflorescences, or anther donor plants prior to culture is important to trigger the sporophytic development deviating from normal pollen development pathway. The type of the effective pretreatment, duration, and the time of application vary with the species or even for different varieties [1, 4]. Reference [43] reviewed the different pretreatments which are in current applications for triggering the anther culture response, and they have been classified into three categories based on their utility as widely used, neglected, and novel. These pretreatments include high temperature and chilling, high humidity, water stress, anaerobic treatment, centrifugation, sucrose and nitrogen starvation, ethanol, γ-irradiation, use of microtubule disruptive agents, electrostimulation, high medium pH, and heavy metal treatment.

#### *5.5.1. Temperature pretreatment*

The growth regulator 2,4-dichlorophenoxy acetic acid (2,4-D) is commonly used in the first phase of rice anther culture, and 2,4-D provided at fairly high concentrations (2 mgL−1) has produced improved rates of callus induction of up to 15% in some genotypes [24]. Also, applicability of some other auxins such as naphthalene acetic acid (NAA), phenyl acetic acid, picloram, and dicamba alone or in combination with 2,4-D has been tested for improving androgenic response. Not only the growth regulator combination but also the auxin/cytokinin balance has been found critically important for effective androgenesis. Reference [23] reported that the growth regulator regime of 2,4-D (2 mgL−1), picloram (0.07 mgL−1), and kinetin (0.5 mgL−1) was favorable for enhancing the anther response in a large number of genotypes. Further, the type of auxin and its concentration determine the microspore development pathway. For example, the use of 2,4-D favored callus formation, whereas indole-3-acetic acid and NAA promoted

direct embryogenesis from cultured anthers without an intervening callus phase [4].

*5.4.4. Physical state of the medium*

8 Rice Crop - Current Developments

Although high levels of 2,4-D were useful for increasing callus production, it has proven to have a negative effect on the next phase of culture which is regeneration from callus, particularly from recalcitrant rice genotypes [22]. A lower level of 2,4-D (0.5 mgL−1) in combination with the milder auxin NAA (2.5 mgL−1) and kinetin (0.5 mgL−1) has been used effectively during both phases [9]. This suggests that the use of 2,4-D in the callus induction medium needs to be regulated with a compromise reached between callus induction and regeneration efficiency [24].

Usually, rice anthers are cultured on solid media. However, reference [23] found increased necrosis of anther tissue when they were cultured on solid media and observed a better callusing response in liquid media. Liquid culture media are able to supply the anthers with an improved access to nutrients and plant growth regulators, and also toxic and degenerated material can be readily dispersed. During the culture of anthers from Indica×Basmati rice on liquid media, severalfold increment in green plant regeneration comparable with the rates

However, since the rice anthers tend to settle at the bottom of the liquid cultures, this would affect respiration and result in loss of viability of the explants. These have been identified as barriers for the use of liquid media for androgenesis. When the liquid culture media was added with substances such as Ficoll, it was possible to avoid sinking of anthers due to the increased buoyancy, and therefore viability could be maintained [3]. In principle, the solidifying agent should not carry any nutritional effect. Agar is in extensive use as the gelling agent of solid culture media. However, more reproducible results have been obtained with the use of Gelrite. Starch also has been used for solidification despite the nutritional effects and its dissociation into sugar [13]. Some have found improved response by embedding anthers in

Culture temperature plays an important role in plant tissue culture. Anther cultures are usually incubated at the temperature range of 24–27°C. For two Indica rice varieties, Nona

reported for Japonica rice varieties/hybrids could be obtained [29].

agarose than culturing on semisolid or liquid media [40].

*5.4.5. Culture incubation conditions*

Most frequently used effective method of pretreatment for rice anther culture is the low-temperature application. Harvested rice panicles are subjected to cold shock prior to the culture. However, the temperature and duration vary with the variety. Cold pretreatment given to rice anthers is known to enhance the androgenesis potential by delaying the degeneration of microspores and anther wall tissue in rice [1, 3]. Reference [44] reported that a pretreatment at 10°C for 10–30 days was necessary to induce sporophytic divisions in microspores of the Japonicas. Generally, temperatures from 8 to 10°C for 8 days have been recommended to be optimal for many varieties of rice [45]. Panicle pretreatments longer than 11 days tend to increase albino production [46]. Reference [37] reported a brief exposure to high temperature (35°C for 10 min) before the cold treatment to enhance callus induction although it adversely affected green plant production.

#### *5.5.2. Osmotic stress*

Osmotic shock has been identified as another pretreatment, which can substitute or be used in combination with cold treatment for the induction of androgenesis. Reference [47] have reported the treatment of anthers in 0.4 M mannitol solution to be effective for inducing androgenesis in microspore cultures of Indica and Japonica varieties. Sole mannitol treatment without the cold pretreatment given to anthers promoted androgenesis in anther cultures of variety IR43 from 3 to 33.4% [48]. It is described that when the anthers or isolated microspores are subjected to high osmolarity by incubating in metabolizable carbohydrates for short time, they start divisions during stress treatment and tolerate the following stress conditions [43]. Further, regenerability of callus could also be improved markedly by osmotic treatment. It is supposed to regulate the endogenous levels of auxin interacting with abscisic acid affecting the carbohydrate metabolism and thereby trigger both callus initiation and shoot regeneration responses in rice [49].

Some species are associated with a great tendency for spontaneous chromosome doubling. In such cases haploid cells are directly converted to homozygous DH plants. If spontaneous doubling is completely absent or occurs at a low frequency, the haploid plants resulted in cultures need to be converted to dihaploids by some other means such as application of chromosome doubling agents [53]. Among many such chemicals, colchicine is the most widely used anti-microtubule agent *in vivo* and *in vitro*, and oryzalin and trifluralin have also been

Anther Culture as a Supplementary Tool for Rice Breeding

http://dx.doi.org/10.5772/intechopen.76157

11

The ploidy status of regenerated plants can be determined through direct and indirect measures. Direct determinations include mitotic chromosome counts or use of the flow cytometry technique [54]. Initial attempts to ploidy determination relied on karyotypical assessments which was highly time-consuming, laborious, and difficult due to the requirement of skilled operators and was most of the time a failure due to the unavailability of dividing cells. Recently, flow cytometry has hastened the ploidy status analysis. As an alternative to chromosome counting, some other correlated measurements which do not require actively dividing cells have been reported for estimating the ploidy level. These include leaf stomatal density and size. However, these measurements alone cannot be granted sufficiently reliable [54]. When the diploids arisen from anther cultures were analyzed using SSR markers to determine their source of origin, homozygosity has been detected for all in 150 DHs except for one which turned out as a heterozygote [31]. Therefore, microsatellites and other molecular tools such as isozyme analyses and RAPD markers can be utilized to ascertain the homozygosis confirming that the calluses and plantlets have been arisen from gamete itself and not from

Anther culture technique has been recognized as an efficient alternative to the conventional inbred line development which is usually achieved through a number of inbreeding cycles. A number of critical factors have been addressed for directing the anther culture process for its optimum condition since it can be potentially developed as a supplementary breeding tool

This research on rice anther culture was graciously funded by the National Science Foundation

(NSF) of Sri Lanka under the research grant RG/2011/BT/10.

No other condition or relationship has a potential conflict of interest.

used.

other somatic tissues.

**Acknowledgements**

**Conflict of interest**

**6. Conclusion**

for rice.

#### *5.5.3. Sugar starvation*

Not only in rice but also in many other crop species such as tobacco, wheat, and barley, sugar starvation has been found effective in induction of embryogenesis [43]. Reference [39] reported that cold pretreatment could be partially substituted by subjecting microspores for sugar starvation for 3 days during androgenesis of Indica rice. Reference [47] also confirmed that sugar starvation could be applied for Indica and Japonica rice in obtaining high-frequency embryogenesis and plantlet regeneration. Many changes induced in starved microspores at cytoplasmic and nuclear levels have been described in detail by reference [43].

#### *5.5.4. Irradiation*

Penetration of irradiation varies with the species and dependent pollen morphology and the thickness of the pollen wall [50]. Reference [51] demonstrated the stimulation of green plant regeneration from rice anther culture with the application of gamma rays at the dose of 20 Gy. Enhancement of the green plant regeneration from two- to threefold could be possible by the use of irradiation of the 137Cs gamma rays, and the maximum response was elicited with the dose of 15 Gy [52].

#### **5.6. Ploidy-level determination and doubled haploid production**

Ideally, the plants developed from anther culture can be considered as haploids as they are arisen from haploid microspores. However, the actual plants resulted during the regeneration could be a mixture of haploid, diploid, or mixoploid [13]. Occurrence of non-haploids can be due to different malformations. Tissues formed from somatic tissue of anther walls, the fusion of nuclei, endomitosis within the pollen grain, or irregular microspores formed during irregular meiosis lead to the development of plants other than the haploids. Also, when the vegetative and generative nuclei are not separated by cell wall formation, non-haploids could be originated [4].

Some species are associated with a great tendency for spontaneous chromosome doubling. In such cases haploid cells are directly converted to homozygous DH plants. If spontaneous doubling is completely absent or occurs at a low frequency, the haploid plants resulted in cultures need to be converted to dihaploids by some other means such as application of chromosome doubling agents [53]. Among many such chemicals, colchicine is the most widely used anti-microtubule agent *in vivo* and *in vitro*, and oryzalin and trifluralin have also been used.

The ploidy status of regenerated plants can be determined through direct and indirect measures. Direct determinations include mitotic chromosome counts or use of the flow cytometry technique [54]. Initial attempts to ploidy determination relied on karyotypical assessments which was highly time-consuming, laborious, and difficult due to the requirement of skilled operators and was most of the time a failure due to the unavailability of dividing cells. Recently, flow cytometry has hastened the ploidy status analysis. As an alternative to chromosome counting, some other correlated measurements which do not require actively dividing cells have been reported for estimating the ploidy level. These include leaf stomatal density and size. However, these measurements alone cannot be granted sufficiently reliable [54]. When the diploids arisen from anther cultures were analyzed using SSR markers to determine their source of origin, homozygosity has been detected for all in 150 DHs except for one which turned out as a heterozygote [31]. Therefore, microsatellites and other molecular tools such as isozyme analyses and RAPD markers can be utilized to ascertain the homozygosis confirming that the calluses and plantlets have been arisen from gamete itself and not from other somatic tissues.

### **6. Conclusion**

*5.5.2. Osmotic stress*

10 Rice Crop - Current Developments

tion responses in rice [49].

*5.5.3. Sugar starvation*

*5.5.4. Irradiation*

dose of 15 Gy [52].

be originated [4].

Osmotic shock has been identified as another pretreatment, which can substitute or be used in combination with cold treatment for the induction of androgenesis. Reference [47] have reported the treatment of anthers in 0.4 M mannitol solution to be effective for inducing androgenesis in microspore cultures of Indica and Japonica varieties. Sole mannitol treatment without the cold pretreatment given to anthers promoted androgenesis in anther cultures of variety IR43 from 3 to 33.4% [48]. It is described that when the anthers or isolated microspores are subjected to high osmolarity by incubating in metabolizable carbohydrates for short time, they start divisions during stress treatment and tolerate the following stress conditions [43]. Further, regenerability of callus could also be improved markedly by osmotic treatment. It is supposed to regulate the endogenous levels of auxin interacting with abscisic acid affecting the carbohydrate metabolism and thereby trigger both callus initiation and shoot regenera-

Not only in rice but also in many other crop species such as tobacco, wheat, and barley, sugar starvation has been found effective in induction of embryogenesis [43]. Reference [39] reported that cold pretreatment could be partially substituted by subjecting microspores for sugar starvation for 3 days during androgenesis of Indica rice. Reference [47] also confirmed that sugar starvation could be applied for Indica and Japonica rice in obtaining high-frequency embryogenesis and plantlet regeneration. Many changes induced in starved microspores at

Penetration of irradiation varies with the species and dependent pollen morphology and the thickness of the pollen wall [50]. Reference [51] demonstrated the stimulation of green plant regeneration from rice anther culture with the application of gamma rays at the dose of 20 Gy. Enhancement of the green plant regeneration from two- to threefold could be possible by the use of irradiation of the 137Cs gamma rays, and the maximum response was elicited with the

Ideally, the plants developed from anther culture can be considered as haploids as they are arisen from haploid microspores. However, the actual plants resulted during the regeneration could be a mixture of haploid, diploid, or mixoploid [13]. Occurrence of non-haploids can be due to different malformations. Tissues formed from somatic tissue of anther walls, the fusion of nuclei, endomitosis within the pollen grain, or irregular microspores formed during irregular meiosis lead to the development of plants other than the haploids. Also, when the vegetative and generative nuclei are not separated by cell wall formation, non-haploids could

cytoplasmic and nuclear levels have been described in detail by reference [43].

**5.6. Ploidy-level determination and doubled haploid production**

Anther culture technique has been recognized as an efficient alternative to the conventional inbred line development which is usually achieved through a number of inbreeding cycles. A number of critical factors have been addressed for directing the anther culture process for its optimum condition since it can be potentially developed as a supplementary breeding tool for rice.

### **Acknowledgements**

This research on rice anther culture was graciously funded by the National Science Foundation (NSF) of Sri Lanka under the research grant RG/2011/BT/10.

### **Conflict of interest**

No other condition or relationship has a potential conflict of interest.

### **Author details**

D.M. Ruwani G. Mayakaduwa\* and Tara D. Silva

\*Address all correspondence to: ruwani.mayakaduwa@pts.cmb.ac.lk

University of Colombo, Sri Lanka

### **References**

[1] Bhojwani SS, Pande H, Raina A. Factors Affecting Androgenesis in Indica Rice. Delhi, India: Department of Botany, University of Delhi; 2001

[13] Foroughi-Wehr B, Wenzel G. Andro- and parthenogenesis. In: Hayward MD, Bosemark NO, Romagosa I, Cerezo M, editors. Plant Breeding-Principles and Prospects. Dordrecht:

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14 Rice Crop - Current Developments

pp. 43-50


**Chapter 2**

**Provisional chapter**

**Genetic Analysis of Biofortification of Micronutrient**

Rice is a staple food for millions of people and has great importance in food and nutritional security. Rice is the second most widely consumed in the world next to wheat. The poorest to the richest person in the world consumes rice in one or other form. New research on the importance of micronutrients, vitamins and proteins aims at biological and genetic enrichment. Vital nutrients that the farmer can grow indefinitely without any additional input to produce nutrient-packed rice grains in a sustainable way is the only feasible way of reaching the malnourished population in India. In the present study, an

Rice is the very life and the main staple food for more than 50% of the world's population. It provides more carbohydrate and protein in the average daily diet to supplement with essential micronutrients. The per capita consumption of rice is very high, ranging from 62 to 190 kg/ year. Therefore, it is one of the most important crop plants on Earth. It provides 35–75% of the calories consumed by more than three billion Asians. Major advances have occurred in rice production during the last four decades due to the adoption of green revolution technologies. Rice production increased 136%, from 257 million tonnes in 1966 to 600 million tonnes in 2000. Rice production grew at the rate of 2% during 1970–1980 and 1.1% during the 1990s. There has been no substantial increase in rice production during the last four years. Worldwide, rice is cultivated in an area of about 152.99 million hectares with a production and productivity of 418 million metric tonnes and 4.07 tonnes per hectare, respectively. In India, rice is cultivated in 43.7 million hectares with production of 93.35 million metric tonnes and productivity of 3.18

attempt has been made to improve the nutritional quality of rice.

**Keywords:** rice, biofortification, iron, zinc, malnutrition

**Genetic Analysis of Biofortification of Micronutrient** 

DOI: 10.5772/intechopen.72810

© 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,

© 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.

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

**Breeding in Rice (***Oryza sativa* **L.)**

**Breeding in Rice (***Oryza sativa* **L.)**

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.72810

Savitha Palanisamy

**Abstract**

**1. Introduction**

Savitha Palanisamy

**Provisional chapter**

### **Genetic Analysis of Biofortification of Micronutrient Breeding in Rice (***Oryza sativa* **L.) Breeding in Rice (***Oryza sativa* **L.)**

**Genetic Analysis of Biofortification of Micronutrient** 

DOI: 10.5772/intechopen.72810

Savitha Palanisamy Savitha Palanisamy 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.72810

#### **Abstract**

Rice is a staple food for millions of people and has great importance in food and nutritional security. Rice is the second most widely consumed in the world next to wheat. The poorest to the richest person in the world consumes rice in one or other form. New research on the importance of micronutrients, vitamins and proteins aims at biological and genetic enrichment. Vital nutrients that the farmer can grow indefinitely without any additional input to produce nutrient-packed rice grains in a sustainable way is the only feasible way of reaching the malnourished population in India. In the present study, an attempt has been made to improve the nutritional quality of rice.

**Keywords:** rice, biofortification, iron, zinc, malnutrition

### **1. Introduction**

Rice is the very life and the main staple food for more than 50% of the world's population. It provides more carbohydrate and protein in the average daily diet to supplement with essential micronutrients. The per capita consumption of rice is very high, ranging from 62 to 190 kg/ year. Therefore, it is one of the most important crop plants on Earth. It provides 35–75% of the calories consumed by more than three billion Asians. Major advances have occurred in rice production during the last four decades due to the adoption of green revolution technologies. Rice production increased 136%, from 257 million tonnes in 1966 to 600 million tonnes in 2000. Rice production grew at the rate of 2% during 1970–1980 and 1.1% during the 1990s. There has been no substantial increase in rice production during the last four years. Worldwide, rice is cultivated in an area of about 152.99 million hectares with a production and productivity of 418 million metric tonnes and 4.07 tonnes per hectare, respectively. In India, rice is cultivated in 43.7 million hectares with production of 93.35 million metric tonnes and productivity of 3.18

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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

metric tonnes per hectare (USDA, World Agriculture production, 2008). In terms of area under cultivation, India ranks first in the world, and with respect to production, China ranks first. To be healthy, human beings require more than 20 mineral elements and more than 40 nutrients, particularly vitamins and essential amino acids, all of which can be supplied by an appropriate diet. Malnutrition deficiencies common in rice consuming countries are iron (Fe) and zinc (Zn) deficiencies that occur mostly in developing countries.

world health consequences, to concentrate poor people to balance the daily diet with micronutrient enriched diet. The major micronutrient deficiencies common in rice-consuming

Genetic Analysis of Biofortification of Micronutrient Breeding in Rice (*Oryza sativa* L.)

http://dx.doi.org/10.5772/intechopen.72810

19

According to the FAO (2010), lack of micronutrients in food, leading to hidden hunger, is seriously damaging the health of billions of people all over the world. Over 9 billion people are affected by undernourishment and the numbers are on the rise. Nearly 5 billion people from Asia and Pacific region and billions of people in developing and underdeveloped countries suffer due to lack of micronutrients, of which 40% of people are affected by lack of iron and zinc.

The nutritional disorder anemia due to iron deficiency is widespread in rice-consuming countries. South East Asia shows the highest prevalence of anemia in women with over 50% of pregnant women being affected. Anemia lowers work performance. It has been linked to reduced resistance to infection. Severe anemia is a significant cause of maternal deaths, while mild anemia may also affect cognitive functions. The magnitude of micronutrient (Fe and Zn) deficiency is alarming, particularly among children, women of reproductive age and pregnant and lactating women. Zinc is a critical micronutrient needed for structural and functional integrity of biological membrane and for diversification of highly

Decrease in zinc concentration in the human body results in a number of cellular disturbances and impairments such as immune dysfunctions and high susceptibility to infection of dis-

The main strategies addressing micronutrient disorders are dietary diversification, food fortification, supplementation and biofortification. A sustainable solution for micronutrient deficiency is to enhance the level of micronutrients in major staple food crop through plant breeding strategies, that is, biofortification. According to high yielding cultivars combine

Successful biofortification strategies must be initiated with screening of diverse germplasm for desired micronutrient content, followed by suitable breeding methods. Genetic engineering through modification of functional pathways to improve the functional novel genes is another major approach. Genetic studies generally reveal the information on the mode of inheritance of the targeted trait. Variability and heritability are the true key phenomena determining the efficiency of the breeding program. Estimates of heritability would be helped in predicting the parents to advance generation for further selection improvement. It facilitates the breeder in finding out the heritable portion of phenotypic variance for effective selection. Genetic advance is yet another parameter for knowing the quantum of desired genes transferred to the progenies. Information on inter-relationships existing among these traits and their association with yield is also important for suggesting suitable guide for selection. Crop

Major micronutrient deficiency affected people to lose the valuable life. Since rice is the principal cereal crop in most rice-growing countries and is the staple food of the world's population, it bears significant impact on human health. Biofortification has emerged as a new way to eradicate

and F<sup>3</sup>

segregat-

effort of enriched micro nutrient rice possible through rice improvement strategies.

improvement for specific trait has been achieved through effective use of F<sup>2</sup>

ing population and fixing desirable combinations [1].

eases, retardation of mental development and stunted growth in children.

countries are iron and zinc deficiencies.

aggressive radicals.

### **2. Micronutrient deficiencies**

Iron deficiency anemia is by far the most common micronutrient deficiency in the world, affecting more than two billion people. It is estimated that about 79% of the children aged between 6 and 35 months and 56% of women between the age of 15 and 49 years are anemic in India. Iron deficiency during childhood and adolescence impairs physical growth, mental development and learning capacity. In adults, iron deficiency anemia reduces the capacity to do physical labor and increases the risk of women dying during childbirth or in the postpartum period.

Zinc is required as a co-factor in over 300 enzymes and plays critical structural roles in many protein and transcriptional factors. Zinc deficiency is more extensive in developing countries, where more than 60% of the population is at risk. Zn deficiency in older children and adolescent males causes retarded growth and dwarfism, retarded sexual development, impaired sense of taste, poor appetite and mental lethargy.

To address micronutrient deficiencies, nutritionists focus on supplementation, fortification and dietary diversification. Fortified food and food supplements do not reach all those affected in the developing countries, because of weak market infrastructure and high recurring cost. Sustainable solutions to the micronutrient problem in these countries can be developed through agricultural approaches. One such approach is crop diversification and the other is to enhance the level of micronutrients in major staple food crops through plant breeding strategies, that is, biofortification of staple in rice production for both rural and urban people.

"Harvest Plus" is one of the programs to motivate the breed crop varieties to enriching in iron and zinc. The ultimate goal of the biofortification strategy is to reduce mortality and morbidity rates related to micronutrient malnutrition and to increase food security, productivity and the quality of life for poor populations of developing countries.

Exploiting genetic variation in crop plants for micronutrient content is one of the most powerful tools to change the nutrient level of a given diet on a large scale. Genetic studies are required to obtain information on the mode of inheritance of the targeted trait, variability and heritability, which are the true key phenomena determining the efficiency of the breeding programs. Based on the information generated through genetic analysis studies, desirable plants with improved levels of Fe and Zn could be fixed from advanced generation breeding materials. A human being needs 49 essential nutrients for normal metabolic activities. Inadequate consumption of even one of these nutrients will result in adverse metabolic disturbances leading to malnutrition. To consider the micronutrient malnutrition to arising world health consequences, to concentrate poor people to balance the daily diet with micronutrient enriched diet. The major micronutrient deficiencies common in rice-consuming countries are iron and zinc deficiencies.

metric tonnes per hectare (USDA, World Agriculture production, 2008). In terms of area under cultivation, India ranks first in the world, and with respect to production, China ranks first. To be healthy, human beings require more than 20 mineral elements and more than 40 nutrients, particularly vitamins and essential amino acids, all of which can be supplied by an appropriate diet. Malnutrition deficiencies common in rice consuming countries are iron (Fe) and zinc (Zn)

Iron deficiency anemia is by far the most common micronutrient deficiency in the world, affecting more than two billion people. It is estimated that about 79% of the children aged between 6 and 35 months and 56% of women between the age of 15 and 49 years are anemic in India. Iron deficiency during childhood and adolescence impairs physical growth, mental development and learning capacity. In adults, iron deficiency anemia reduces the capacity to do physical labor and increases the risk of women dying during childbirth or in the postpartum period.

Zinc is required as a co-factor in over 300 enzymes and plays critical structural roles in many protein and transcriptional factors. Zinc deficiency is more extensive in developing countries, where more than 60% of the population is at risk. Zn deficiency in older children and adolescent males causes retarded growth and dwarfism, retarded sexual development, impaired

To address micronutrient deficiencies, nutritionists focus on supplementation, fortification and dietary diversification. Fortified food and food supplements do not reach all those affected in the developing countries, because of weak market infrastructure and high recurring cost. Sustainable solutions to the micronutrient problem in these countries can be developed through agricultural approaches. One such approach is crop diversification and the other is to enhance the level of micronutrients in major staple food crops through plant breeding strategies, that is, biofortification of staple in rice production for both rural

"Harvest Plus" is one of the programs to motivate the breed crop varieties to enriching in iron and zinc. The ultimate goal of the biofortification strategy is to reduce mortality and morbidity rates related to micronutrient malnutrition and to increase food security, productivity and

Exploiting genetic variation in crop plants for micronutrient content is one of the most powerful tools to change the nutrient level of a given diet on a large scale. Genetic studies are required to obtain information on the mode of inheritance of the targeted trait, variability and heritability, which are the true key phenomena determining the efficiency of the breeding programs. Based on the information generated through genetic analysis studies, desirable plants with improved levels of Fe and Zn could be fixed from advanced generation breeding materials. A human being needs 49 essential nutrients for normal metabolic activities. Inadequate consumption of even one of these nutrients will result in adverse metabolic disturbances leading to malnutrition. To consider the micronutrient malnutrition to arising

deficiencies that occur mostly in developing countries.

sense of taste, poor appetite and mental lethargy.

the quality of life for poor populations of developing countries.

and urban people.

**2. Micronutrient deficiencies**

18 Rice Crop - Current Developments

According to the FAO (2010), lack of micronutrients in food, leading to hidden hunger, is seriously damaging the health of billions of people all over the world. Over 9 billion people are affected by undernourishment and the numbers are on the rise. Nearly 5 billion people from Asia and Pacific region and billions of people in developing and underdeveloped countries suffer due to lack of micronutrients, of which 40% of people are affected by lack of iron and zinc.

The nutritional disorder anemia due to iron deficiency is widespread in rice-consuming countries. South East Asia shows the highest prevalence of anemia in women with over 50% of pregnant women being affected. Anemia lowers work performance. It has been linked to reduced resistance to infection. Severe anemia is a significant cause of maternal deaths, while mild anemia may also affect cognitive functions. The magnitude of micronutrient (Fe and Zn) deficiency is alarming, particularly among children, women of reproductive age and pregnant and lactating women. Zinc is a critical micronutrient needed for structural and functional integrity of biological membrane and for diversification of highly aggressive radicals.

Decrease in zinc concentration in the human body results in a number of cellular disturbances and impairments such as immune dysfunctions and high susceptibility to infection of diseases, retardation of mental development and stunted growth in children.

The main strategies addressing micronutrient disorders are dietary diversification, food fortification, supplementation and biofortification. A sustainable solution for micronutrient deficiency is to enhance the level of micronutrients in major staple food crop through plant breeding strategies, that is, biofortification. According to high yielding cultivars combine effort of enriched micro nutrient rice possible through rice improvement strategies.

Successful biofortification strategies must be initiated with screening of diverse germplasm for desired micronutrient content, followed by suitable breeding methods. Genetic engineering through modification of functional pathways to improve the functional novel genes is another major approach. Genetic studies generally reveal the information on the mode of inheritance of the targeted trait. Variability and heritability are the true key phenomena determining the efficiency of the breeding program. Estimates of heritability would be helped in predicting the parents to advance generation for further selection improvement. It facilitates the breeder in finding out the heritable portion of phenotypic variance for effective selection. Genetic advance is yet another parameter for knowing the quantum of desired genes transferred to the progenies. Information on inter-relationships existing among these traits and their association with yield is also important for suggesting suitable guide for selection. Crop improvement for specific trait has been achieved through effective use of F<sup>2</sup> and F<sup>3</sup> segregating population and fixing desirable combinations [1].

Major micronutrient deficiency affected people to lose the valuable life. Since rice is the principal cereal crop in most rice-growing countries and is the staple food of the world's population, it bears significant impact on human health. Biofortification has emerged as a new way to eradicate micronutrient deficiencies. Biofortification is likely to reach rural households, especially subsistence farmers who grow and consume the harvested cereal grains, which in turn is expected to have impact in an affordable and sustainable manner.

**2.2. Heritability and genetic advance**

**i.** The amount of genetic variability

**ii.** The intensity of selection

**2.3. Association of characters**

**2.4. Path coefficient analysis**

of materials.

parameter to evaluate the effectiveness of selection [2].

effect through selection of the best individuals.

Knowledge of heritability serves as an effective tool to the plant breeder to estimate the relative importance of the inheritance and environment on the variation observed for a character. The concept of heritability helps to discern whether phenotypic differences among individuals are due to genetic differences or due to environmental causes. Heritability in a narrow sense is defined as that fraction of the observed variance which is caused by additive genetic effect.

Genetic Analysis of Biofortification of Micronutrient Breeding in Rice (*Oryza sativa* L.)

http://dx.doi.org/10.5772/intechopen.72810

21

Similarly, the estimate of genetic advance or genetic gain for a particular character is an important

Knowledge of variability, heritability and genetic advance is of great value in both stages. Estimation of heritability along with genetic gain is more useful in predicting the resultant

Estimates of heritability for different traits of economic importance are available on a variety

Inter-relationship of yield with other traits is considered as the most valuable while formulating selection program for yield improvement in any crop. Correlation studies pave the way to know the association between highly heritable characters with the most economic, namely the grain yield. Several authors have worked in this aspect to bring about the relationship of

A number of independent components will influence yield, since it is a complex quantitative trait. Simultaneous selection for more characters can easily be done with the knowledge of association between yield and yield components. This association between highly heritable characters with the most economic character, the yield, can be obtained by correlation studies. Various authors have brought out the relationship between yield and yield attributing

Path coefficient analysis provides an efficient means of partitioning of correlation coefficients into direct and indirect effects of the component character. Selection on the basis of direct and indirect effect is much more useful than selection for yield *per se* alone. Several authors have

Parent-offspring regression analysis in advancing generations makes it possible to study how far the genetic potentials from one generation is transferred to the next; the higher the values of

different characters with yield and also within the yield contributing character.

economic traits by computing genotypic and phenotypic correlation coefficients.

reported the extent of direct and indirect influence of characters on yield in rice.

**2.5. Studies on parent-progeny regression analysis**

Rice (*Oryza sativa* L.) is basically a starchy crop and it has low nutritional elements compared to millets. Though rice provides 50–80% of the energy intake of the poor, it does not provide enough essential micronutrients to eliminate hidden hunger, iron deficiency anemia (IDA) and zinc deficiency. Sufficient micronutrient in the daily diet is one of the prerequisites for human health. The main strategies addressing micronutrient disorders are dietary diversification, food fortification, supplementation and biofortification. Most of the micronutrient deficiencies can be addressed, to some extent, through biofortification. One of the ways to enhance the micronutrient level in the staple crops is biofortification breeding.

To develop new varieties with high amount of micronutrients in the rice kernel, a population with high variability serves as prime source for effective selection. Particularly, the role played by F<sup>2</sup> segregants, contributing much variability, is highly recognized. The F<sup>2</sup> and F<sup>3</sup> generation is the correct stage for selection in any hybridization program and fixing desirable traits in the early segregants of rice by selecting and evaluating them for desirable characters.

A scrutiny of available literature is invaluable in gaining an insight into the research problem under study. This review helps to acquire broad and general background in the given field or discipline. Comparative views of past approaches and findings can also be had through this compiled information. This could orient researchers in the desired lines of thinking, which is supposed to be a prerequisite for a scientific study.

A sincere attempt has been made to review the available literature relevant to the study and is presented under the following sub-heads:


### **2.1. Variability**

The development of an effective plant breeding program is dependent upon the existence of genetic variability and it is a prerequisite for a plant breeder to work with any crop species. Genetic improvement for quantitative traits can be achieved through a clear understanding of the nature and extent of variability present in the material. The efficiency of selection in any crop largely depends on the magnitude of genetic variability available in the population.

The simple measures of variability partitions the variation into phenotypic, genotypic and environmental components. Phenotypic coefficient of variation (PCV) is the measure of total variability resulting from the genotype, environment and interaction of both. Phenotypic and genotypic coefficients of variation give the real picture of variability concealed in a population.

### **2.2. Heritability and genetic advance**

micronutrient deficiencies. Biofortification is likely to reach rural households, especially subsistence farmers who grow and consume the harvested cereal grains, which in turn is expected to

Rice (*Oryza sativa* L.) is basically a starchy crop and it has low nutritional elements compared to millets. Though rice provides 50–80% of the energy intake of the poor, it does not provide enough essential micronutrients to eliminate hidden hunger, iron deficiency anemia (IDA) and zinc deficiency. Sufficient micronutrient in the daily diet is one of the prerequisites for human health. The main strategies addressing micronutrient disorders are dietary diversification, food fortification, supplementation and biofortification. Most of the micronutrient deficiencies can be addressed, to some extent, through biofortification. One of the ways to

To develop new varieties with high amount of micronutrients in the rice kernel, a population with high variability serves as prime source for effective selection. Particularly, the role

generation is the correct stage for selection in any hybridization program and fixing desirable traits in the early segregants of rice by selecting and evaluating them for desirable characters. A scrutiny of available literature is invaluable in gaining an insight into the research problem under study. This review helps to acquire broad and general background in the given field or discipline. Comparative views of past approaches and findings can also be had through this compiled information. This could orient researchers in the desired lines of thinking, which is

A sincere attempt has been made to review the available literature relevant to the study and

The development of an effective plant breeding program is dependent upon the existence of genetic variability and it is a prerequisite for a plant breeder to work with any crop species. Genetic improvement for quantitative traits can be achieved through a clear understanding of the nature and extent of variability present in the material. The efficiency of selection in any crop largely depends on the magnitude of genetic variability available in the population. The simple measures of variability partitions the variation into phenotypic, genotypic and environmental components. Phenotypic coefficient of variation (PCV) is the measure of total variability resulting from the genotype, environment and interaction of both. Phenotypic and genotypic coefficients of variation give the real picture of variability concealed in a population.

segregants, contributing much variability, is highly recognized. The F<sup>2</sup>

and F<sup>3</sup>

enhance the micronutrient level in the staple crops is biofortification breeding.

have impact in an affordable and sustainable manner.

supposed to be a prerequisite for a scientific study.

is presented under the following sub-heads:

**2.** Studies on heritability and genetic advance

**3.** Studies on association of characters **4.** Studies on path coefficient analysis

**5.** Studies on parent-progeny regression

**1.** Studies on variability

**2.1. Variability**

played by F<sup>2</sup>

20 Rice Crop - Current Developments

Knowledge of heritability serves as an effective tool to the plant breeder to estimate the relative importance of the inheritance and environment on the variation observed for a character. The concept of heritability helps to discern whether phenotypic differences among individuals are due to genetic differences or due to environmental causes. Heritability in a narrow sense is defined as that fraction of the observed variance which is caused by additive genetic effect.

Similarly, the estimate of genetic advance or genetic gain for a particular character is an important parameter to evaluate the effectiveness of selection [2].


Knowledge of variability, heritability and genetic advance is of great value in both stages. Estimation of heritability along with genetic gain is more useful in predicting the resultant effect through selection of the best individuals.

Estimates of heritability for different traits of economic importance are available on a variety of materials.

### **2.3. Association of characters**

Inter-relationship of yield with other traits is considered as the most valuable while formulating selection program for yield improvement in any crop. Correlation studies pave the way to know the association between highly heritable characters with the most economic, namely the grain yield. Several authors have worked in this aspect to bring about the relationship of different characters with yield and also within the yield contributing character.

A number of independent components will influence yield, since it is a complex quantitative trait. Simultaneous selection for more characters can easily be done with the knowledge of association between yield and yield components. This association between highly heritable characters with the most economic character, the yield, can be obtained by correlation studies. Various authors have brought out the relationship between yield and yield attributing economic traits by computing genotypic and phenotypic correlation coefficients.

### **2.4. Path coefficient analysis**

Path coefficient analysis provides an efficient means of partitioning of correlation coefficients into direct and indirect effects of the component character. Selection on the basis of direct and indirect effect is much more useful than selection for yield *per se* alone. Several authors have reported the extent of direct and indirect influence of characters on yield in rice.

### **2.5. Studies on parent-progeny regression analysis**

Parent-offspring regression analysis in advancing generations makes it possible to study how far the genetic potentials from one generation is transferred to the next; the higher the values of regression, the higher will be the genetic effect with less environmental influence. The analysis will be helpful to select the early fixing characters in a segregating population. Parent-progeny regression analysis method helps us to ascertain the influence of environment on different characters in progenies obtained from individual selection from early generation and to study the real genetic potentiality of the progenies, which was inherited from their parents [3]. Parentprogeny regression analysis assumes no environment association between generations, so genotype × environment interaction and co-variances between parents and offspring will be zero [4].

Among the methods of increasing the frequency of desirable recombinants, the biparental mating or disruptive mating gets importance in the improvement of self-pollinated crops, because it increases the possibility of obtaining desirable and valuable recombinants [7]. The system of biparental mating is reported to alter the phase of linkage through forced recombination. As a result, greater amount of concealed genetic variation is released, particularly of the additive type. Since the additive genetic variance is the only variance which responds to

Genetic Analysis of Biofortification of Micronutrient Breeding in Rice (*Oryza sativa* L.)

http://dx.doi.org/10.5772/intechopen.72810

23

Quality breeding in rice has assumed greater significance due to varied consumer preference in recent years. Developing a better variety with respect to higher yield with good grain quality and multiple resistances to biotic and abiotic tolerance than the already existing ones is the prime goal of plant breeders. Thus, high yield is the foremost goal of any crop improvement program but consumer preference varies from region to region. Next to grain yield, grain quality is the important criterion considered by the plant breeders. If the newly developed variety is not accepted by the consumers due to its poor taste, texture, aroma or appearance, its usefulness is greatly impaired. In developed countries, as well as in the rice-exporting countries, physical appearance of the grain or kernel is often more important than the yield [8]. In developing countries, like India, grain quality has become greatly important as the

Quality of rice is determined by a combination of many physio-chemical properties. Rice kernel size and shape are important quality components that directly influence the market value. Gelatinization temperature and amylose content are the principal determinants for cooking and eating quality of milled rice. In addition to this trait, kernel length after cooking and linear elongation ratio is desirable quality traits. Properties of cooked rice are controlled by amylose content and gelatinization temperature. Varieties with intermediate values for both parameters remain nonsticky and tender after cooking and low values for both make the rice

Plant Breeding and Genetics, PGP College of Agricultural Sciences, Namakkal, Tamil Nadu,

[1] Sangeetha LNE. Genetic variability studies for grain yield and grain quality traits in F<sup>2</sup>

population of rice (*Oryza sativa* L.) [M.Sc. (Ag.) Thesis (Unpubl.)]. TNAU, Madurai;

selection, a perceptible genetic gain can be expected after biparental mating.

country has become more prosperous and self-sufficient in food production.

sticky on cooking.

**Author details**

Savitha Palanisamy

India

**References**

and F<sup>3</sup>

2013

Address all correspondence to: saviagri@gmail.com

Multiple regression analysis to fix yield attributing characters found that percentage of filled grains per panicle, biological yield and harvest index were major selection criteria for yield improvement [5]. A comparative analysis to estimate heritability for grain yield and plant height using parent-offspring regression and variance components in maize revealed broad sense heritability estimated from variance components of the progeny greater than those based on the parent–offspring regression. It was found that selection is not effective for other traits like plant height.

High narrow sense and realized heritability for the characters, 1000 grain weight, and number of grains per panicle in early generation showed the prospects of selecting for these traits in early generation itself. Multiple regression analysis in pearl millet to fix yield-attributing characters found that number of grains per spike, 1000 grain weight, totally contributed a 60% variation in grain yield, suggesting that selection should be based on the above characters for improvement in grain yield.

With regard to heritability and environmental effects of yield and yield-related traits using parent-offspring regression in F<sup>1</sup> progenies, environmental effects and heritability estimates were high for culm length, tillers per plant, panicles per plant and 1000 grain weight. Based on the variance estimates of the parent-offspring regression model, it was suggested that these traits with high heritability, considerable phenotypic correlation and low seasonal variability could be used for further improvement of the F<sup>1</sup> progenies [6].

The most widely used breeding method in rice is the hybridization of homozygous diverse genotypes followed by pedigree method of handling the segregating population in order to isolate genotypes possessing desirable characteristics of parents. The usefulness of these methods is limited because of limited parental participation, low genetic diversity, reduced recombination and rapid fixation of genes. The selection and choice of breeding method for the improvement of quantitative or qualitative character largely depends on the nature and magnitude of additive and dominance variance. The success in the improvement of cultivated variety for yield, grain quality and resistance to biotic and abiotic resistance largely depends upon the natural variability present in the population. Through hybridization among the selected genotypes, it is possible to reshuffle desired characteristics, provided the segregating generations contain large variability. Selection for quantitative characters is generally taken up in the early segregating generations. For characters like grain yield, the selection is continued till the material becomes homozygous, because such characters are controlled by large number of genes and huge number of population has to be raised for making the selection effective. This is not always true, because the effective selection was known to be restricted by close linkages between desirable and undesirable component characters and these undesirable linkages delay the utilization of full recombination potential.

Among the methods of increasing the frequency of desirable recombinants, the biparental mating or disruptive mating gets importance in the improvement of self-pollinated crops, because it increases the possibility of obtaining desirable and valuable recombinants [7]. The system of biparental mating is reported to alter the phase of linkage through forced recombination. As a result, greater amount of concealed genetic variation is released, particularly of the additive type. Since the additive genetic variance is the only variance which responds to selection, a perceptible genetic gain can be expected after biparental mating.

Quality breeding in rice has assumed greater significance due to varied consumer preference in recent years. Developing a better variety with respect to higher yield with good grain quality and multiple resistances to biotic and abiotic tolerance than the already existing ones is the prime goal of plant breeders. Thus, high yield is the foremost goal of any crop improvement program but consumer preference varies from region to region. Next to grain yield, grain quality is the important criterion considered by the plant breeders. If the newly developed variety is not accepted by the consumers due to its poor taste, texture, aroma or appearance, its usefulness is greatly impaired. In developed countries, as well as in the rice-exporting countries, physical appearance of the grain or kernel is often more important than the yield [8]. In developing countries, like India, grain quality has become greatly important as the country has become more prosperous and self-sufficient in food production.

Quality of rice is determined by a combination of many physio-chemical properties. Rice kernel size and shape are important quality components that directly influence the market value. Gelatinization temperature and amylose content are the principal determinants for cooking and eating quality of milled rice. In addition to this trait, kernel length after cooking and linear elongation ratio is desirable quality traits. Properties of cooked rice are controlled by amylose content and gelatinization temperature. Varieties with intermediate values for both parameters remain nonsticky and tender after cooking and low values for both make the rice sticky on cooking.

### **Author details**

regression, the higher will be the genetic effect with less environmental influence. The analysis will be helpful to select the early fixing characters in a segregating population. Parent-progeny regression analysis method helps us to ascertain the influence of environment on different characters in progenies obtained from individual selection from early generation and to study the real genetic potentiality of the progenies, which was inherited from their parents [3]. Parentprogeny regression analysis assumes no environment association between generations, so genotype × environment interaction and co-variances between parents and offspring will be zero [4]. Multiple regression analysis to fix yield attributing characters found that percentage of filled grains per panicle, biological yield and harvest index were major selection criteria for yield improvement [5]. A comparative analysis to estimate heritability for grain yield and plant height using parent-offspring regression and variance components in maize revealed broad sense heritability estimated from variance components of the progeny greater than those based on the parent–offspring regression. It was found that selection is not effective for other

High narrow sense and realized heritability for the characters, 1000 grain weight, and number of grains per panicle in early generation showed the prospects of selecting for these traits in early generation itself. Multiple regression analysis in pearl millet to fix yield-attributing characters found that number of grains per spike, 1000 grain weight, totally contributed a 60% variation in grain yield, suggesting that selection should be based on the above characters for

With regard to heritability and environmental effects of yield and yield-related traits using

were high for culm length, tillers per plant, panicles per plant and 1000 grain weight. Based on the variance estimates of the parent-offspring regression model, it was suggested that these traits with high heritability, considerable phenotypic correlation and low seasonal variability

The most widely used breeding method in rice is the hybridization of homozygous diverse genotypes followed by pedigree method of handling the segregating population in order to isolate genotypes possessing desirable characteristics of parents. The usefulness of these methods is limited because of limited parental participation, low genetic diversity, reduced recombination and rapid fixation of genes. The selection and choice of breeding method for the improvement of quantitative or qualitative character largely depends on the nature and magnitude of additive and dominance variance. The success in the improvement of cultivated variety for yield, grain quality and resistance to biotic and abiotic resistance largely depends upon the natural variability present in the population. Through hybridization among the selected genotypes, it is possible to reshuffle desired characteristics, provided the segregating generations contain large variability. Selection for quantitative characters is generally taken up in the early segregating generations. For characters like grain yield, the selection is continued till the material becomes homozygous, because such characters are controlled by large number of genes and huge number of population has to be raised for making the selection effective. This is not always true, because the effective selection was known to be restricted by close linkages between desirable and undesirable component characters and these undesir-

progenies [6].

progenies, environmental effects and heritability estimates

traits like plant height.

22 Rice Crop - Current Developments

improvement in grain yield.

parent-offspring regression in F<sup>1</sup>

could be used for further improvement of the F<sup>1</sup>

able linkages delay the utilization of full recombination potential.

Savitha Palanisamy

Address all correspondence to: saviagri@gmail.com

Plant Breeding and Genetics, PGP College of Agricultural Sciences, Namakkal, Tamil Nadu, India

### **References**

[1] Sangeetha LNE. Genetic variability studies for grain yield and grain quality traits in F<sup>2</sup> and F<sup>3</sup> population of rice (*Oryza sativa* L.) [M.Sc. (Ag.) Thesis (Unpubl.)]. TNAU, Madurai; 2013

[2] Comstock RE, Robinson HF. Estimation of the Average Dominance of Genes. Heterosis, Ames, Iowa: Iowa State College Press; 1952. pp. 494-516

**Chapter 3**

**Provisional chapter**

**Iron Biofortification of Rice: Progress and Prospects**

Biofortification is the process of improving the bioavailability of essential nutrients in food crops either through conventional breeding or modern biotechnology techniques. Rice is one of the most demanding staple foods worldwide. Most global population live on a diet based on rice as the main carbohydrate source that serve as suitable target for biofortification. In general, polished grain or white rice contains nutritionally insufficient concentration of iron (Fe) to meet the daily requirements in diets. Therefore, iron biofortification in rice offers an inexpensive and sustainable solution to mitigate iron deficiency. However, understanding on the mechanism and genes involved in iron uptake in rice is a prerequisite for successful iron biofortification. In this chapter, the overview of iron uptake strategies in plants and as well as different iron-biofortified approaches used in rice will be outlined. Then, the challenges and future prospects of rice iron biofortifica-

**Keywords:** agronomic practices, conventional plant breeding, genetic engineering,

Rice is one of the most consumed staple foods worldwide. In developing countries, people often rely on rice as their sole source of nutrition [1]. However, polished grain, known as white rice, contains limited amount of essential nutrients to sustain a good health and development [2]. Hence, those who are incapable to afford other micronutrients-rich nonstaple food for their balance diet are often at the highest risk for micronutrients deficiencies [3].

**Iron Biofortification of Rice: Progress and Prospects**

© 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.73572

Andrew De-Xian Kok, Low Lee Yoon, Rogayah Sekeli, Wee Chien Yeong,

Andrew De-Xian Kok, Low Lee Yoon, Rogayah Sekeli, Wee Chien Yeong,

http://dx.doi.org/10.5772/intechopen.73572

**Abstract**

**1. Introduction**

Zetty Norhana Balia Yusof and Lai Kok Song

Zetty Norhana Balia Yusof and Lai Kok Song

Additional information is available at the end of the chapter

Additional information is available at the end of the chapter

tion to improve global human health will also be discussed.

iron biofortification, *Oryza sativa*, transgenic rice


#### **Iron Biofortification of Rice: Progress and Prospects Iron Biofortification of Rice: Progress and Prospects**

DOI: 10.5772/intechopen.73572

Andrew De-Xian Kok, Low Lee Yoon, Rogayah Sekeli, Wee Chien Yeong, Zetty Norhana Balia Yusof and Lai Kok Song Andrew De-Xian Kok, Low Lee Yoon, Rogayah Sekeli, Wee Chien Yeong, Zetty Norhana Balia Yusof and Lai Kok Song

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.73572

#### **Abstract**

[2] Comstock RE, Robinson HF. Estimation of the Average Dominance of Genes. Heterosis,

[3] Lush JL. Correlation and regression of offspring on dams as a method of estimating heritability of characters. Proceedings of American Society of Animal Production. 1940;

[4] Sala M. Rice breeding for biofortification with high iron and zinc content in segregating

[5] Rao NK, Reddy KB, Naik K. Studies on genetic variability, correlation and path coefficient analysis in rice under saline soil conditions. The Andhra Agricultural Journal. 2010;

eration of rice (*Oryza sativa* L.) **[**M.Sc. (Ag.) Thesis (Unpubl.)]. TNAU, Coimbatore; 2011

[7] Johnson HW, Robinson HF, Comstock RE. Estimates of genetic and environmental vari-

[8] Savitha P. Genetic analysis of different generation involving traditional landraces of rice

(*Oryza sativa* L.) [P.hD. (Ag.) Thesis (Unpubl.)]. TNAU, Coimbatore; 2014

gen-

[6] Kalaimaghal R. Studies on genetic variability of grain iron and zinc content in F2, F<sup>3</sup>

Ames, Iowa: Iowa State College Press; 1952. pp. 494-516

ability in soybean. Agronomy Journal. 1955;**47**:314-318

population [M.Sc. (Ag.) Thesis (Unpubl.)]. TNAU, Madurai; 2012

**33**:293-301

24 Rice Crop - Current Developments

**57**(4):335-338

Biofortification is the process of improving the bioavailability of essential nutrients in food crops either through conventional breeding or modern biotechnology techniques. Rice is one of the most demanding staple foods worldwide. Most global population live on a diet based on rice as the main carbohydrate source that serve as suitable target for biofortification. In general, polished grain or white rice contains nutritionally insufficient concentration of iron (Fe) to meet the daily requirements in diets. Therefore, iron biofortification in rice offers an inexpensive and sustainable solution to mitigate iron deficiency. However, understanding on the mechanism and genes involved in iron uptake in rice is a prerequisite for successful iron biofortification. In this chapter, the overview of iron uptake strategies in plants and as well as different iron-biofortified approaches used in rice will be outlined. Then, the challenges and future prospects of rice iron biofortification to improve global human health will also be discussed.

**Keywords:** agronomic practices, conventional plant breeding, genetic engineering, iron biofortification, *Oryza sativa*, transgenic rice

### **1. Introduction**

Rice is one of the most consumed staple foods worldwide. In developing countries, people often rely on rice as their sole source of nutrition [1]. However, polished grain, known as white rice, contains limited amount of essential nutrients to sustain a good health and development [2]. Hence, those who are incapable to afford other micronutrients-rich nonstaple food for their balance diet are often at the highest risk for micronutrients deficiencies [3].

© 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.

Iron deficiency is a common health disorder affecting nearly 2 billion people worldwide with other mineral and vitamin deficiency [4, 5]. Common effects of iron deficiency include anemia and impaired growth development in pregnant women and preschool children [6]. It can be easily addressed through dietary diversification, micronutrient supplements, medicines, and surgery depending of the severity of the condition [5, 7]. However, such treatments may not be available to everyone due to limitations such as geographical and financial capabilities [4]. In addition, iron is the most difficult mineral to be used in food fortification because the most soluble and absorbable compounds (e.g., FeSO<sup>4</sup> ) alters the taste or color of fortified food making it unappetizing while the least soluble compounds (e.g., Fe<sup>4</sup> (P2 O7 ) 3 ) are poorly absorbed by human body [8, 9]. Hence, food fortification is not a sustainable solution to mitigate iron deficiency.

**2. Iron uptake strategies in plants**

**2.1. Strategy I: reduction-based strategy**

**2.2. Strategy II: chelation-based strategy**

controlled by iron regulated transporter 1 (IRT1) [22].

transported into the root via yellow stripe 1 (YS1) transporter [22, 24].

**Figure 1** [22, 23].

pH and they can be readily absorbed by the plant roots [20].

Plants acquire iron mainly from the rhizosphere. There are abundant of iron in the soil, but only minute quantities of iron are absorbed by the plants. The availability of iron is dependent on the soil pH and soil redox potential [7]. Iron becomes less soluble in higher pH and it can be found in the form of insoluble ferric oxides. In contrast, iron becomes more soluble in low

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572 27

Micronutrient uptake and distribution in plants are heavily controlled and regulated by different uptake strategies. This allows the required amount of micronutrients to be absorbed into the plant but not high enough to exhibit toxicity effect [21]. Similarly for iron uptake in plants, there are two strategies used for iron uptake from the soil, namely reduction-based strategy and chelation-based strategy [22, 23]. Graminaceous plants are able to utilize chelation-based strategy while nongraminaceous plants utilize reduction-based strategy. However, rice is able to utilize combination of both reduction-based and chelation-based strategies as shown in

Reduction-based strategy is utilized by nongraminaceous plants. This strategy involves reducing available Fe3+ through the reduction activity into Fe2+ before being absorbed into the plant system. In reduction-based strategy, nongraminaceous plant will release protons toward the rhizosphere to decrease the pH in the surrounding soil under Fe-deficient condition. Kim [20] suggested that ATPase are responsible for releasing protons into the rhizosphere and reducing the pH of surrounding rhizosphere. The decrease in pH will increase the solubility of Fe3+ in the rhizosphere. In addition, NADPH-dependent Fe3+ chelate reductase reduces Fe3+ into a more soluble form of Fe2+ with the help of ferric reductase oxidase 2 (FRO2). Then, Fe2+ will be transported into the roots via ferric ion transporter

Grasses families such as maize, wheat, and rice are known as graminaceous plants. In response to iron deficiency, these plants are able to increase iron uptake through chelationbased strategy. Chelation-based strategy transports Fe3+ from rhizosphere into the roots with the help of soluble siderophores. Mugineic acid (MA) family phytosiderophores are natural iron chelators and they have a higher affinity toward Fe3+ [7]. Depending on different species, different sets of MAs will be released by the plant to surrounding rhizosphere via transporter of MAs (TOM1). For instance, rice will secrete only 2′-deoxymugineic acid (DMA), while barley secretes different types of MA such as MA, 3-epihydroxymugineic acid (epi-HMA), and 3-epihydroxy-2′-deoxymugineic acid (epi-HDMA) [22]. During iron deficiency, graminaceous plants will secrete MAs into the rhizosphere to solubilize sparingly soluble iron in rhizosphere. MAs will bind Fe3+ efficiently forming Fe3+-MA complexes. The complexes will be

Government bodies and nonprofit organization could play an important role to combat micronutrient deficiency by providing adequate food, supplements, and medicine supplies to rural areas. Nevertheless, it may not be an effective long-term solution because it is highly dependent on continuous investment, appropriate infrastructures, and transportation [10]. Hence, an alternative solution through biofortification is seen to be more efficient and costfriendly in mitigating to micronutrient deficiency.

Iron biofortification, the process of improving the bioavailability of iron in food crops can be achieved via agronomic practices, conventional breeding, and genetic engineering. Biofortification through agronomic practices can be performed through fertilizer or foliar feeding [11]. Agronomic practices need to take bioavailability of iron at different stages into account as not all of the nutrients are transferred [12]. Several crucial factors may contribute to the nutrient loss at different stages such as bioavailability of nutrient uptake from the soil, nutrient distribution in different parts of the plants, milling or dehusking during food processing, and the ability of human to absorb and utilize the nutrients [13]. These factors need to be considered carefully to ensure successful iron biofortification through agronomic practices.

Meanwhile, conventional plant breeding involves identifying and selection of parent line, which contains desirable traits found in both parent plants. Parent lines are then crossed over for a few generations until daughter plants with both desirable nutrient and agronomic traits are observed and selected [14]. For instance, iron bean is one of the successful products through conventional plant breeding with high iron content, high bioavailability, and high yield [15, 16]. In addition, the advancement of modern biotechnology techniques, such as marker-assisted selection, improves the efficiency and precision in identification of potential lines in daughter plants [17].

To date, with the recent advancement of genetic engineering technologies served as a platform, which inspires many researchers in exploring alternative solution through genetic modification. Genetic engineering involves in removing, altering, or inserting specific sequence into the plant genome, which provides a better flexibility by silencing or overexpressing desirable gene sequences for desirable traits [18, 19]. Genetic engineering is an excellent method to obtain desirable micronutrient levels in a more effective manner by targeting specific gene of interest. However, successful biofortification via genetic engineering requires extensive knowledge and understanding of iron uptake, trafficking, and homeostasis mechanisms in plants to prevent undesirable side effects.

### **2. Iron uptake strategies in plants**

Iron deficiency is a common health disorder affecting nearly 2 billion people worldwide with other mineral and vitamin deficiency [4, 5]. Common effects of iron deficiency include anemia and impaired growth development in pregnant women and preschool children [6]. It can be easily addressed through dietary diversification, micronutrient supplements, medicines, and surgery depending of the severity of the condition [5, 7]. However, such treatments may not be available to everyone due to limitations such as geographical and financial capabilities [4]. In addition, iron is the most difficult mineral to be used in food fortification because the most sol-

body [8, 9]. Hence, food fortification is not a sustainable solution to mitigate iron deficiency.

Government bodies and nonprofit organization could play an important role to combat micronutrient deficiency by providing adequate food, supplements, and medicine supplies to rural areas. Nevertheless, it may not be an effective long-term solution because it is highly dependent on continuous investment, appropriate infrastructures, and transportation [10]. Hence, an alternative solution through biofortification is seen to be more efficient and cost-

Iron biofortification, the process of improving the bioavailability of iron in food crops can be achieved via agronomic practices, conventional breeding, and genetic engineering. Biofortification through agronomic practices can be performed through fertilizer or foliar feeding [11]. Agronomic practices need to take bioavailability of iron at different stages into account as not all of the nutrients are transferred [12]. Several crucial factors may contribute to the nutrient loss at different stages such as bioavailability of nutrient uptake from the soil, nutrient distribution in different parts of the plants, milling or dehusking during food processing, and the ability of human to absorb and utilize the nutrients [13]. These factors need to be considered carefully to ensure successful iron biofortification through agronomic practices. Meanwhile, conventional plant breeding involves identifying and selection of parent line, which contains desirable traits found in both parent plants. Parent lines are then crossed over for a few generations until daughter plants with both desirable nutrient and agronomic traits are observed and selected [14]. For instance, iron bean is one of the successful products through conventional plant breeding with high iron content, high bioavailability, and high yield [15, 16]. In addition, the advancement of modern biotechnology techniques, such as marker-assisted selection, improves the efficiency and precision in identification of potential

To date, with the recent advancement of genetic engineering technologies served as a platform, which inspires many researchers in exploring alternative solution through genetic modification. Genetic engineering involves in removing, altering, or inserting specific sequence into the plant genome, which provides a better flexibility by silencing or overexpressing desirable gene sequences for desirable traits [18, 19]. Genetic engineering is an excellent method to obtain desirable micronutrient levels in a more effective manner by targeting specific gene of interest. However, successful biofortification via genetic engineering requires extensive knowledge and understanding of iron uptake, trafficking, and homeostasis mechanisms in

) alters the taste or color of fortified food making it

) are poorly absorbed by human

(P2 O7 ) 3

uble and absorbable compounds (e.g., FeSO<sup>4</sup>

26 Rice Crop - Current Developments

unappetizing while the least soluble compounds (e.g., Fe<sup>4</sup>

friendly in mitigating to micronutrient deficiency.

lines in daughter plants [17].

plants to prevent undesirable side effects.

Plants acquire iron mainly from the rhizosphere. There are abundant of iron in the soil, but only minute quantities of iron are absorbed by the plants. The availability of iron is dependent on the soil pH and soil redox potential [7]. Iron becomes less soluble in higher pH and it can be found in the form of insoluble ferric oxides. In contrast, iron becomes more soluble in low pH and they can be readily absorbed by the plant roots [20].

Micronutrient uptake and distribution in plants are heavily controlled and regulated by different uptake strategies. This allows the required amount of micronutrients to be absorbed into the plant but not high enough to exhibit toxicity effect [21]. Similarly for iron uptake in plants, there are two strategies used for iron uptake from the soil, namely reduction-based strategy and chelation-based strategy [22, 23]. Graminaceous plants are able to utilize chelation-based strategy while nongraminaceous plants utilize reduction-based strategy. However, rice is able to utilize combination of both reduction-based and chelation-based strategies as shown in **Figure 1** [22, 23].

### **2.1. Strategy I: reduction-based strategy**

Reduction-based strategy is utilized by nongraminaceous plants. This strategy involves reducing available Fe3+ through the reduction activity into Fe2+ before being absorbed into the plant system. In reduction-based strategy, nongraminaceous plant will release protons toward the rhizosphere to decrease the pH in the surrounding soil under Fe-deficient condition. Kim [20] suggested that ATPase are responsible for releasing protons into the rhizosphere and reducing the pH of surrounding rhizosphere. The decrease in pH will increase the solubility of Fe3+ in the rhizosphere. In addition, NADPH-dependent Fe3+ chelate reductase reduces Fe3+ into a more soluble form of Fe2+ with the help of ferric reductase oxidase 2 (FRO2). Then, Fe2+ will be transported into the roots via ferric ion transporter controlled by iron regulated transporter 1 (IRT1) [22].

### **2.2. Strategy II: chelation-based strategy**

Grasses families such as maize, wheat, and rice are known as graminaceous plants. In response to iron deficiency, these plants are able to increase iron uptake through chelationbased strategy. Chelation-based strategy transports Fe3+ from rhizosphere into the roots with the help of soluble siderophores. Mugineic acid (MA) family phytosiderophores are natural iron chelators and they have a higher affinity toward Fe3+ [7]. Depending on different species, different sets of MAs will be released by the plant to surrounding rhizosphere via transporter of MAs (TOM1). For instance, rice will secrete only 2′-deoxymugineic acid (DMA), while barley secretes different types of MA such as MA, 3-epihydroxymugineic acid (epi-HMA), and 3-epihydroxy-2′-deoxymugineic acid (epi-HDMA) [22]. During iron deficiency, graminaceous plants will secrete MAs into the rhizosphere to solubilize sparingly soluble iron in rhizosphere. MAs will bind Fe3+ efficiently forming Fe3+-MA complexes. The complexes will be transported into the root via yellow stripe 1 (YS1) transporter [22, 24].

**3. Iron biofortification via agronomic practices**

Agronomic biofortification is a traditional biofortification approach, which involves micronutrient uptake from the surrounding soil and translocation into the edible parts of the plants. Effective agronomic biofortification are determined by various factors due to the potential nutrient loss during the transition at different stages such as from the soil to the plants, plants to food, and finally to humans [13, 25, 26]. Soil conditions such as pH, soil composition, aeration, and moisture are important for iron availability and uptake in plants [13, 27]. As mentioned in Section 2.1, higher plants are able to release protons to the surrounding soil to increase iron solubility and pH of surrounding rhizosphere in order to enhance iron availability and uptake. Similarly through soil management, properties of the soil could be altered to increase iron availability and uptake in plants by utilizing organic wastes such as plant residues and animal manure [27, 28]. Besides, organic wastes is able to enhance the soil properties, nutrient bioavailability, cation exchange capacity, and water holding capacity while providing a constant and slower nutrient release [13, 29]. However, application of organic wastes alone is insufficient to mitigate iron deficiency and it requires combination application with iron fertilizer [13].

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572 29

Iron availability in the soil can be enhanced through fertilizer application onto the soil or foliar feeding application directly onto the leaves of the crop. Iron fertilizer via foliar feeding enhance iron uptake and efficient translocation into rice as compared to soil fertilizer [30–32]. However, the fertilizers are often removed by the rain and they require reapplication each time after raining, which are costly and dangerous to the environment [13, 33]. Conversely, the application of iron fertilizer through the soil is inefficient due to strong bind-

In addition, macronutrient also plays a crucial role in iron biofortification in plants. Previously, several studies on positive interactions between iron and zinc concentration in grains with nitrogen, phosphorus, and potassium (NPK) fertilizer have been reported [10, 27, 32, 34]. The presence of nitrogen alone was reported to increase iron content in brown rice by 15% and addition of potassium is able to further increase the iron content in rice grain [35]. This is because nitrogen and phosphorus are involved in root development, shoot transport and re-localization, which improves the translocation of iron into rice grain [13, 15, 27, 33]. On the other hand, the presence of phosphorus is able to reduce toxicity in plants at the cost of reduce uptake of both iron and zinc uptake in plant due to dilution effect [13]. Hence, combined application of both NPK fertilizer and iron fertilizer could be a potential approach to increase iron bioavailability in rice [13].

Conventional plant breeding has been practiced for centuries to improve the properties of food crops by identifying and developing parent plants with desired characteristics, crossing the parent plants, and selecting offspring with desired agronomic traits inherited from both parent plants [14]. An example of a product developed via plant breeding is high iron rice variety (IR68144) with high yield, disease tolerant, good tolerant to mineral deficient, and excellent seed vigor. The IR68144 rice variety was developed through crossing between

ing between iron and the soil, which reduces iron uptake efficiency in plants [13, 15].

**4. Iron biofortification via conventional plant breeding**

**Figure 1.** Iron uptake strategy by graminaceous plants and nongraminaceous plants.

### **2.3. Iron uptake mechanism in rice**

Some graminaceous plants, in particular rice, can undergo combined strategies of reductionbased strategy and chelation-based strategy for iron uptake. Rice acquires Fe3+ via strategy I-like system and Fe2+ directly from the surroundings via IRT1 or IRT2. However, there is no increase in Fe3+-chelate reductase levels detected in the roots as compared to nongraminaceous plants [20]. Possible explanation is that adaptation of rice when grown in submerged and anaerobic environment rich in Fe2+ compared to Fe3+ [10]. Similarly to strategy II, MAs will be secreted into the rhizosphere to bind with Fe3+ and the complexes will be transported into the root via YS-like 15 (YSL15). Between both strategies, rice is able to uptake iron from the surrounding more efficiently through Fe3+-MA complexes as compared to direct Fe2+ uptake [22].

### **3. Iron biofortification via agronomic practices**

Agronomic biofortification is a traditional biofortification approach, which involves micronutrient uptake from the surrounding soil and translocation into the edible parts of the plants. Effective agronomic biofortification are determined by various factors due to the potential nutrient loss during the transition at different stages such as from the soil to the plants, plants to food, and finally to humans [13, 25, 26]. Soil conditions such as pH, soil composition, aeration, and moisture are important for iron availability and uptake in plants [13, 27]. As mentioned in Section 2.1, higher plants are able to release protons to the surrounding soil to increase iron solubility and pH of surrounding rhizosphere in order to enhance iron availability and uptake. Similarly through soil management, properties of the soil could be altered to increase iron availability and uptake in plants by utilizing organic wastes such as plant residues and animal manure [27, 28]. Besides, organic wastes is able to enhance the soil properties, nutrient bioavailability, cation exchange capacity, and water holding capacity while providing a constant and slower nutrient release [13, 29]. However, application of organic wastes alone is insufficient to mitigate iron deficiency and it requires combination application with iron fertilizer [13].

Iron availability in the soil can be enhanced through fertilizer application onto the soil or foliar feeding application directly onto the leaves of the crop. Iron fertilizer via foliar feeding enhance iron uptake and efficient translocation into rice as compared to soil fertilizer [30–32]. However, the fertilizers are often removed by the rain and they require reapplication each time after raining, which are costly and dangerous to the environment [13, 33]. Conversely, the application of iron fertilizer through the soil is inefficient due to strong binding between iron and the soil, which reduces iron uptake efficiency in plants [13, 15].

In addition, macronutrient also plays a crucial role in iron biofortification in plants. Previously, several studies on positive interactions between iron and zinc concentration in grains with nitrogen, phosphorus, and potassium (NPK) fertilizer have been reported [10, 27, 32, 34]. The presence of nitrogen alone was reported to increase iron content in brown rice by 15% and addition of potassium is able to further increase the iron content in rice grain [35]. This is because nitrogen and phosphorus are involved in root development, shoot transport and re-localization, which improves the translocation of iron into rice grain [13, 15, 27, 33]. On the other hand, the presence of phosphorus is able to reduce toxicity in plants at the cost of reduce uptake of both iron and zinc uptake in plant due to dilution effect [13]. Hence, combined application of both NPK fertilizer and iron fertilizer could be a potential approach to increase iron bioavailability in rice [13].

### **4. Iron biofortification via conventional plant breeding**

**2.3. Iron uptake mechanism in rice**

28 Rice Crop - Current Developments

Some graminaceous plants, in particular rice, can undergo combined strategies of reductionbased strategy and chelation-based strategy for iron uptake. Rice acquires Fe3+ via strategy I-like system and Fe2+ directly from the surroundings via IRT1 or IRT2. However, there is no increase in Fe3+-chelate reductase levels detected in the roots as compared to nongraminaceous plants [20]. Possible explanation is that adaptation of rice when grown in submerged and anaerobic environment rich in Fe2+ compared to Fe3+ [10]. Similarly to strategy II, MAs will be secreted into the rhizosphere to bind with Fe3+ and the complexes will be transported into the root via YS-like 15 (YSL15). Between both strategies, rice is able to uptake iron from the surrounding more effi-

ciently through Fe3+-MA complexes as compared to direct Fe2+ uptake [22].

**Figure 1.** Iron uptake strategy by graminaceous plants and nongraminaceous plants.

Conventional plant breeding has been practiced for centuries to improve the properties of food crops by identifying and developing parent plants with desired characteristics, crossing the parent plants, and selecting offspring with desired agronomic traits inherited from both parent plants [14]. An example of a product developed via plant breeding is high iron rice variety (IR68144) with high yield, disease tolerant, good tolerant to mineral deficient, and excellent seed vigor. The IR68144 rice variety was developed through crossing between semi dwarf rice cultivar, IR8 and Taichung (Native)-1. Meanwhile, IR8 is a product developed through crossing between Chinese dwarf rice variety "Dee-Geo-woo-gen" (DGWG) and Indonesia high yield rice variety "Peta" [36]. The Taichung (Native)-1 is a product of crossing between DGWG and a traditional tall variety 'Tsai-Yuan-Chung', which produces high yield and dwarf variety. Crossing between IR8 and Taichung (Native)-1 allows the development of new rice cultivar, which is semi-dwarf and contains high yield properties [37]. This rice variety is able to produce 21 μg/g (2-fold) of iron concentration in brown rice [35]. In addition, IR68144 is able to retain most of the iron content (approximate 80%) after polishing for 15 minutes compared to other varieties [10]. Furthermore, consumption of IR68144 was reported to have improvement in iron status of women [38]. This rice cultivar can serve as a stepping stone for further transgenic enhancement [10].

Even though conventional breeding is able to develop high yield and semi dwarf IR68144, this approach alone in iron biofortification is insufficient in developing a sustainable agronomic plant in terms of yield and quality [39]. This is due to the possibility of inheriting undesirable traits from the parent line as the selection process is done based on the phenotypes and new traits can only be developed after performing extensive back crossing or wide crossing [40]. For instance, low phytic acid (PA) maize mutant (*lpa241*) has demonstrated its ability to reduce PA concentration by 90% in exchange of reduced germination rate by 30% [41]. Hence, conventional breeding is best coupled with other approach such as genetic engineering and agronomic practices to enhance iron content in grains [32, 37, 42, 43].

### **5. Iron biofortification via genetic engineering**

The advancement of genetic engineering technologies allows the advancement in molecular field including the development of transgenic plants. Characterization and analysis of gene function are performed via genetic engineering by the manipulation of gene expression. These include introducing gene of interest from other closely-related organism, RNA interference (RNAi) gene silencing, and overexpression of gene of interest [18]. Genetic engineering technologies is able to provide a more efficient and reliable method to study the relationship between genotype and the phenotype as compared to agronomic and conventional plant breeding [44, 45]. As a result, genetic engineering is preferred as an alternative for biofortification to increase the iron content in rice grains. There are five different transgenic approaches (**Table 1**) and as well as combination of different transgenic approaches (**Table 2**), which have been attempted and successfully used to enhance the iron content in rice grain.

### **5.1. Enhancement of iron storage in rice via** *ferritin* **genes**

Ferritin is an iron storage protein ubiquitously present in most organisms, which is capable to store up to 4500 iron atoms in a complex and nontoxic form [65, 66]. Iron complex in soybean ferritin is readily available for human body absorption via iron uptake mechanism in the intestine [46, 62]. Thus, the first approach in iron biofortification is to enhance the expression of ferritin by introducing soybean *ferritin* (*SoyferH1* and *SoyferH2*) genes into rice.

In soybean, there are two types of ferritin proteins, known as *SoyferH1* and *SoyferH2*, and both *ferritin* genes are controlled by endosperm specific promoters [47]. However, expression of multiple endosperm specific promoters (*Oryza sativa Globulin* (*OsGlb*) and *Oryza sativa Glutelin* (*OsGluB1*) promoters) did not produce a significant increase of iron concentration in

**Table 1.** Iron biofortification approach in rice targeting genes responsible for iron storage, iron transport, iron influx,

**Approach Genes-promoter** 

Improving iron storage via

Enhancing iron transport

Enhancing iron influx via

Enhancing iron uptake and translocation via *IDS3* gene

translocation via silencing

iron uptake and translocation.

via *NAS* gene

*OsYSL2* gene

Enhancing iron

*OsVITs* genes

*ferritin* genes

**used**

*OsGluB1* pro-*SoyferH1*

*OsGluB1* pro-*SoyferH1*

*OsGluB1* pro-*SoyferH1*

*OsGluB1* pro-*SoyferH1*

*OsGluB4* pro-*SoyferH1*

Maize *Ubiquitin* pro-*OsNAS2*

Maize *Ubiquitin* pro-*OsNAS3*

*35S* pro-barley 20-kb *IDS3* genome

*35S* pro-barley 20-kb *IDS3* genome

*OsVIT1* or *OsVIT2* T-DNA insertion line

*OsVIT2* T-DNA insertion line

fragment

fragment

**Rice cultivar Fold of Fe** 

*Japonica* cv. Kitaake 1.5 fold (brown

*Japonica* cv. Kitaake 2 fold (polished

*Japonica* cv. Taipei 309 2.2 fold (brown

*Indica* cv. IR68144 3.7 fold (polished

*Indica* cv. IR64 3.4 fold (polished

*Japonica* cv. Kitaake 2.9 fold (polished

*Japonica* cv. Dongjin 2.9 fold (polished

*Japonica* cv. Tsukinohikari 1.4 fold (polished

*Japonica* cv. Tsukinohikari 1.3 fold (brown

*Japonica* cv. Dongjin 1.8 fold (polished

*OsGluA2* pro-*Osfer2 Indica* cv. Pusa-Sugandh II 2.1 fold (polished

*35S* pro-*OsNAS1, 2, 3 Japonica* cv. Nipponbare 4 fold (polished

*35S* pro-*HvNAS1 Japonica* cv. Tsukinohikari 2.3 fold (polished

*OsSUT1* pro-*OsYSL2 Japonica* cv. Tsukinohikari 4.4 fold (polished

*Japonica* cv. Zhonghua11 and *Japonica* cv. Dongjin

**increase**

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

grain)

1.4 fold (brown grain)

grain)

**References**

31

[48]

[49]

[51]

[50]

[64]

[67]

[2]

[54]

[55]

[74]

[75]

[58]

[78]

[59]

[80]


semi dwarf rice cultivar, IR8 and Taichung (Native)-1. Meanwhile, IR8 is a product developed through crossing between Chinese dwarf rice variety "Dee-Geo-woo-gen" (DGWG) and Indonesia high yield rice variety "Peta" [36]. The Taichung (Native)-1 is a product of crossing between DGWG and a traditional tall variety 'Tsai-Yuan-Chung', which produces high yield and dwarf variety. Crossing between IR8 and Taichung (Native)-1 allows the development of new rice cultivar, which is semi-dwarf and contains high yield properties [37]. This rice variety is able to produce 21 μg/g (2-fold) of iron concentration in brown rice [35]. In addition, IR68144 is able to retain most of the iron content (approximate 80%) after polishing for 15 minutes compared to other varieties [10]. Furthermore, consumption of IR68144 was reported to have improvement in iron status of women [38]. This rice cultivar can serve as a

Even though conventional breeding is able to develop high yield and semi dwarf IR68144, this approach alone in iron biofortification is insufficient in developing a sustainable agronomic plant in terms of yield and quality [39]. This is due to the possibility of inheriting undesirable traits from the parent line as the selection process is done based on the phenotypes and new traits can only be developed after performing extensive back crossing or wide crossing [40]. For instance, low phytic acid (PA) maize mutant (*lpa241*) has demonstrated its ability to reduce PA concentration by 90% in exchange of reduced germination rate by 30% [41]. Hence, conventional breeding is best coupled with other approach such as genetic engineering and

The advancement of genetic engineering technologies allows the advancement in molecular field including the development of transgenic plants. Characterization and analysis of gene function are performed via genetic engineering by the manipulation of gene expression. These include introducing gene of interest from other closely-related organism, RNA interference (RNAi) gene silencing, and overexpression of gene of interest [18]. Genetic engineering technologies is able to provide a more efficient and reliable method to study the relationship between genotype and the phenotype as compared to agronomic and conventional plant breeding [44, 45]. As a result, genetic engineering is preferred as an alternative for biofortification to increase the iron content in rice grains. There are five different transgenic approaches (**Table 1**) and as well as combination of different transgenic approaches (**Table 2**), which have

Ferritin is an iron storage protein ubiquitously present in most organisms, which is capable to store up to 4500 iron atoms in a complex and nontoxic form [65, 66]. Iron complex in soybean ferritin is readily available for human body absorption via iron uptake mechanism in the intestine [46, 62]. Thus, the first approach in iron biofortification is to enhance the expression

been attempted and successfully used to enhance the iron content in rice grain.

of ferritin by introducing soybean *ferritin* (*SoyferH1* and *SoyferH2*) genes into rice.

stepping stone for further transgenic enhancement [10].

30 Rice Crop - Current Developments

agronomic practices to enhance iron content in grains [32, 37, 42, 43].

**5. Iron biofortification via genetic engineering**

**5.1. Enhancement of iron storage in rice via** *ferritin* **genes**

**Table 1.** Iron biofortification approach in rice targeting genes responsible for iron storage, iron transport, iron influx, iron uptake and translocation.

In soybean, there are two types of ferritin proteins, known as *SoyferH1* and *SoyferH2*, and both *ferritin* genes are controlled by endosperm specific promoters [47]. However, expression of multiple endosperm specific promoters (*Oryza sativa Globulin* (*OsGlb*) and *Oryza sativa Glutelin* (*OsGluB1*) promoters) did not produce a significant increase of iron concentration in


*ferritin* genes as a single transgene approach may be insufficient in combating iron deficiency

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572 33

The second approach involves enhancing iron transport in the plant via overexpression of genes involves in biosynthesis of MA such as *nicotianamine synthase* (*NAS*). *NAS* is able to catalyze the synthesis of nicotianamine (NA) from *S*-adenosyl methionine [23]. NA, a natural metal chelators for Fe(II) and Fe(III), are found in all higher plants and involved with metal

Rice comprises of three *NAS* genes, *OsNAS1*, *OsNAS2*, and *OsNAS3*. These genes are involved in long-distance transportation in plants and each *NAS* gene is regulated at different parts of the plants in response to iron deficiency [9, 52]. Overexpression of *NAS* gene enhances MA secretion into the rhizosphere, and thus, increasing iron uptake into the plant via chelationbased strategy [23, 53, 72]. It has been demonstrated that overexpression of rice *OsNAS1, OsNAS2* and *OsNAS3* [2], *OsNAS2* [54], *OsNAS3* [55], and barley *HvNAS1* [74] genes are able

A total of 18 different *YSL* (*yellow stripe-like*) genes were identified by Koike [73] in rice. The rice *YSL2* (*OsYSL2*) is the main focus in this approach as this gene plays an important role as a metal-chelator transporter involved in translocation and accumulation of iron in endosperm [73, 75]. *OsYSL2* was found to be highly expressed in leaves of iron-deficient rice plants in contrast to other parts of the plant where no expression was detected. Therefore, Koike [73] hypothesized that this transporter is involved in long-distance transport of iron-NA com-

Consistently, it was discovered that the iron influx into the rice endosperm could be controlled through iron nicotianamine transporter *OsYSL2* [60]. Ishimaru [75] successfully demonstrated that disruption of *OsYSL2* gene in rice decreased the iron content in both brown rice and polished grain by 18 and 39%, respectively with increased iron accumulation in roots as compared to wild-type rice. Moreover, Ishimaru [75] also able to increase the iron content in rice grain up to 4-fold in polished grain through enhanced expression of *OsYSL2* using the rice sucrose transporter (*OsSUT1*) promoter. However, overexpression of *OsYSL2* may cause opposite effect similar to *OsYSL2* gene silencing in transgenic rice such that the iron concentration in roots was found higher than in both shoot and rice grain. Undoubtedly, the expression of *OsYSL2* with *OsSUT1* promoter is a promising approach in iron biofortification of rice grains.

As mentioned in Section 2.2, MAs are natural iron chelators, which are involved in translocation of iron from the rhizosphere into the plant by forming complexes with iron. Different sets of *MAs* genes were found in barley, which confers the ability to synthesize different types of MAs via biosynthetic pathway of MAs [76, 77]. In addition, the presence of iron deficiency

**5.2. Enhancement of iron transport in rice via** *NAS* **genes**

translocation and homeostasis in plants [47, 71–73].

to increase the iron content by more than twofold in polished grain.

**5.3. Enhancement of iron influx into seeds via** *OsYSL2* **gene**

plexes via phloem in response to iron deficiency in rice plant.

**5.4. Enhancement of iron uptake and translocation via** *IDS3* **gene**

[48, 70].

**Table 2.** Combinational of multiple transgene used for iron biofortification in rice.

rice grains when compared to transgenic rice with *ferritin* genes expression driven by single endosperm specific promoter [48]. On the other hand, the overexpression of soybean *ferritin* in rice has been demonstrated with at least twofold increase in iron concentration in endosperm compared to the wild-type rice [49–51, 64, 67].

Nevertheless, introducing *SoyferH2* into rice plants is preferred as *SoyferH1* is more susceptible to protease digestion causing alteration in structure in comparison to *SoyferH2*, which is more resistant to protease digestion [68, 69]. Interestingly, rice plants introduced with single soybean *ferritin* gene did not increase iron concentration in rice grain [48, 68]. This suggests that expressions of *ferritin* genes are dependent on soil composition and overexpression of *ferritin* genes as a single transgene approach may be insufficient in combating iron deficiency [48, 70].

### **5.2. Enhancement of iron transport in rice via** *NAS* **genes**

The second approach involves enhancing iron transport in the plant via overexpression of genes involves in biosynthesis of MA such as *nicotianamine synthase* (*NAS*). *NAS* is able to catalyze the synthesis of nicotianamine (NA) from *S*-adenosyl methionine [23]. NA, a natural metal chelators for Fe(II) and Fe(III), are found in all higher plants and involved with metal translocation and homeostasis in plants [47, 71–73].

Rice comprises of three *NAS* genes, *OsNAS1*, *OsNAS2*, and *OsNAS3*. These genes are involved in long-distance transportation in plants and each *NAS* gene is regulated at different parts of the plants in response to iron deficiency [9, 52]. Overexpression of *NAS* gene enhances MA secretion into the rhizosphere, and thus, increasing iron uptake into the plant via chelationbased strategy [23, 53, 72]. It has been demonstrated that overexpression of rice *OsNAS1, OsNAS2* and *OsNAS3* [2], *OsNAS2* [54], *OsNAS3* [55], and barley *HvNAS1* [74] genes are able to increase the iron content by more than twofold in polished grain.

### **5.3. Enhancement of iron influx into seeds via** *OsYSL2* **gene**

A total of 18 different *YSL* (*yellow stripe-like*) genes were identified by Koike [73] in rice. The rice *YSL2* (*OsYSL2*) is the main focus in this approach as this gene plays an important role as a metal-chelator transporter involved in translocation and accumulation of iron in endosperm [73, 75]. *OsYSL2* was found to be highly expressed in leaves of iron-deficient rice plants in contrast to other parts of the plant where no expression was detected. Therefore, Koike [73] hypothesized that this transporter is involved in long-distance transport of iron-NA complexes via phloem in response to iron deficiency in rice plant.

Consistently, it was discovered that the iron influx into the rice endosperm could be controlled through iron nicotianamine transporter *OsYSL2* [60]. Ishimaru [75] successfully demonstrated that disruption of *OsYSL2* gene in rice decreased the iron content in both brown rice and polished grain by 18 and 39%, respectively with increased iron accumulation in roots as compared to wild-type rice. Moreover, Ishimaru [75] also able to increase the iron content in rice grain up to 4-fold in polished grain through enhanced expression of *OsYSL2* using the rice sucrose transporter (*OsSUT1*) promoter. However, overexpression of *OsYSL2* may cause opposite effect similar to *OsYSL2* gene silencing in transgenic rice such that the iron concentration in roots was found higher than in both shoot and rice grain. Undoubtedly, the expression of *OsYSL2* with *OsSUT1* promoter is a promising approach in iron biofortification of rice grains.

#### **5.4. Enhancement of iron uptake and translocation via** *IDS3* **gene**

rice grains when compared to transgenic rice with *ferritin* genes expression driven by single endosperm specific promoter [48]. On the other hand, the overexpression of soybean *ferritin* in rice has been demonstrated with at least twofold increase in iron concentration in endo-

**Genes-promoter used Rice cultivar Fold of Fe increase References**

*Japonica* cv. Taipei 309 6.3 fold

*Japonica* cv. Tsukinohikari 4 fold

*Japonica* cv. Taipei 309 4.3 fold

*Japonica* cv. Nipponbare 4.7 fold

*Japonica* cv. Tsukinohikari 6 fold

Tropical *Japonica* cv. Paw San Yin 3.4 fold

IR64 6 fold

(polished grain)

(polished grain)

(polished grain)

(polished grain)

(brown grain)

(polished grain)

(polished grain)

[23]

[77]

[86]

[66]

[47]

[88]

[69]

*OsGlb1* pro-*Pvferritin 35S* pro-*AtNAS1 OsGlb* pro-*Afphytase*

32 Rice Crop - Current Developments

*OsGluB1* pro-*SoyferH2 OsGlb1* pro-*SoyferH2*

genome fragments

*MsENOD12B* pro-*AtIRT1 OsGlb1* pro-*Pvferritin 35S* pro-*AtNAS1 OsGlb* pro-*Afphytase*

Native *AtIRT1* pro-*AtIRT1 OsGlb1* pro-*Pvferritin 35S* pro-*AtNAS1*

*OsGluB1* pro-*SoyferH2 OsGlb1* pro-*SoyferH2 OsAct* pro-*HvNAS1 OsSUT1* pro-*OsYSL2 OsGlb1* pro-*OsYSL2*

*OsGluB1* pro-*SoyferH2 OsGlb1* pro-*SoyferH2 OsAct* pro-*HvNAS1 OsSUT1* pro-*OsYSL2 OsGlb1* pro-*OsYSL2*

*GluA2* pro-*SoyferH1 35S* pro-*OsNAS2*

*HvNAS1, HvNAAT-A,-B* and *IDS3*

Nevertheless, introducing *SoyferH2* into rice plants is preferred as *SoyferH1* is more susceptible to protease digestion causing alteration in structure in comparison to *SoyferH2*, which is more resistant to protease digestion [68, 69]. Interestingly, rice plants introduced with single soybean *ferritin* gene did not increase iron concentration in rice grain [48, 68]. This suggests that expressions of *ferritin* genes are dependent on soil composition and overexpression of

sperm compared to the wild-type rice [49–51, 64, 67].

**Table 2.** Combinational of multiple transgene used for iron biofortification in rice.

As mentioned in Section 2.2, MAs are natural iron chelators, which are involved in translocation of iron from the rhizosphere into the plant by forming complexes with iron. Different sets of *MAs* genes were found in barley, which confers the ability to synthesize different types of MAs via biosynthetic pathway of MAs [76, 77]. In addition, the presence of iron deficiency specific clone no. 2 (*IDS2*) and no. 3 (*IDS3*) in barley play an important role in combating iron deficiency [77]. The *IDS* genes enable the synthesis of different types of MAs via DMA and these genes are highly expressed in roots in response to iron deficiency [56]. On the contrary, rice lacks the ability to synthesize other types of MAs apart of DMA as rice does not contain both *IDS2* and *IDS3* genes. Having different sets of MAs enable barley become more tolerant to iron-deficient conditions as compared to rice.

antinutrients playing important roles in both plant metabolism and human diet [21, 32]. In plants, antinutrients involve in resistance toward pests, pathogens and abiotic stress and at the same time function as anticarcinogens in human diets [61, 63, 82–85]. For instance, PA is able to reduce the risk for both colon cancer and mammary cancer through its strong metal cations binding capabilities [63, 83]. Moreover, PA display antioxidant capability by acting as inhibitor of iron-mediated hydroxyl radical (-OH) formation in food and gastrointestinal tract, which would result in lipid peroxidation and tissue damage [83, 84]. On the other hand, antinutrient lectin was found to be responsible for plant defense system by exhibiting cyto-

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572 35

Masuda [77] has demonstrated that introducing a combination of different genes responsible for MA synthesis into rice (Fer-NAS-NAAT-IDS3 lines) and result in 4-fold increase of iron accumulation in endosperm. Likewise, transgenic line expressing *AtIRT1*, *Pvferritin*, *AtNAS1*, and *Afphytase* was shown to cause a 4-fold increase of iron accumulation in polished grain [66, 86]. The *OsYSL15* or *OsIRT1* genes are predominantly expressed in roots with enhanced expression in response to iron deficiency [86]. *OsIRT1* gene encode for Fe2+ transporter involved in both strategy I and II. Although overexpression of *OsIRT1* alone could increase the iron content in rice grain by 1.3-fold, *OsIRT1* has the potential to further enhance the iron

On the other hand, combination approaches were also demonstrated to increase the iron content in rice grain by 3.4- and 6-fold when introduced into Myanmar and Japanese rice cultivar respectively [47, 88]. Both *SoyFerH2* and *OsYSL2* were strongly expressed in transgenic rice due to the vector inserted contains two gene cassettes for each gene expression driven by different promoters for each gene cassettes (*OsSUT1* promoter-*OsYSL2*, *OsGlb* promoter-*OsYSL2*, *OsGluB1* promoter-*SoyferH2*, *OsGlb* promoter-*SoyferH2*). Interestingly, Trijatmiko [69] was able to develop transgenic rice expressing *OsNAS2* and *SoyferH1* genes result in 15 μg Fe/g increased (6-fold) in polished grain. In the transgenic rice line, the transgene construct was found to be inserted with inverted repeats in a single locus. This concludes that multiple transgene insertion was able to increase the iron concentration in rice [47, 69, 88]. However, transgene cassette with duplicated or inverted repeats of transgene may not be stable and inherited after several generations due to possibility of epigenetic silencing in transgenic plants [66, 89–91]. Hence, further investigation should be conducted to elucidate the stability of transgene or different approach to maintain multiple transgene over multiple generations.

Biofortification is a promising strategy for sustainable long-term approach in combating micronutrient deficiency but successful biofortification at the cost of the environmental damage is not acceptable. In agronomic practice, leaching is one of the main concerns in application of fertilizer as it will damage the environment, but most micronutrients are not susceptible to leaching as they are able to form a strong bond with the soil [13]. However, continuous application of micronutrient fertilizer may cause accumulation of these minerals which result in

toxic activities when ingested by pests and animals [85].

content when it is expressed with other genes [87].

**6. Challenges and future prospect**

Introducing *IDS3* gene from barley enables the synthesis and secretion of different types of MAs from transgenic rice into the rhizosphere [56]. In addition, formation of Fe(III)-MA complex has a better stability as compared to Fe(III)-DMA complex when grown in a slightly acidic soil [57]. This may enhance iron translocation in rice in combating iron deficiency while increasing tolerance toward iron deficiency in rice plants. Furthermore, Masuda [58] and Suzuki [78] demonstrated that *IDS3* rice lines are able to increase Fe concentrations to 1.4 and 1.3-fold for both polished and brown grains respectively compared to wild-type rice when grown in either Fe-sufficient soil or Fe-deficient soil. Thus, presence of *IDS3* gene is able to enhance iron accumulation in rice grain even when it is grown in iron-sufficient soil and as well as enhancing tolerance toward iron deficiency.

### **5.5. Enhancement of iron translocation via** *OsVIT* **gene**

Zhang [59] reported on the functional characterization of rice vacuolar iron transporter genes (*OsVIT1* and *OsVIT2*). These genes were found to be expressed ubiquitously in different parts of the plants at low levels but high level expression of *OsVIT* genes were detected in the flag leaves. These genes play an important role in transportation of Zn2+ and Fe2+ into vacuoles via tonoplast [79]. In addition, knockdown of *OsVIT* genes increases Fe and Zn accumulation in the rice grains significantly while decreases Fe and Zn accumulation in the flag leaves correspondingly [80]. Knockout of *OsVIT1* and *OsVIT2* genes were able to increase the iron content in rice grain by at least 1.4-fold [59, 80]. However, this approach is only applicable when the transgenic rice is grown in unpolluted soil. This is because studies had shown that accumulation of Cd2+ concentration was detected in rice when it is grown in polluted soil [59]. Hence, further understanding of regulatory mechanism is required to prevent toxic metal accumulation and to ensure the crops are safe for consumption.

#### **5.6. Combinational of multiple transgenes**

Multiple gene manipulation has been successfully carried out in rice. Wirth [23] has proven the synergism of three different genes expression with the increased of iron content in rice by 6-fold through introducing *Arabidopsis thaliana NAS1* (*AtNAS1)*, *Phaseolus vulgaris ferritin* (*Pvferritin*), and *Aspergillus fumigatus phytase* (*Afphytase*) genes into rice. The main purpose of introducing *phytase* genes is to reduce iron antinutrient phytate in rice. Some food may contain antinutrients like PA, which binds strongly to metal cations, such as iron and zinc, which render them insoluble [81]. Phytase is able to catalyze the hydrolysis of PA releasing the phosphate and chelated minerals [21]. Human digestive system lacks enzyme responsible for breakdown of such components [23]. Reducing antinutrients is a feasible approach to increase nutrient content in crops but it should be exercised with cautions due to many antinutrients playing important roles in both plant metabolism and human diet [21, 32]. In plants, antinutrients involve in resistance toward pests, pathogens and abiotic stress and at the same time function as anticarcinogens in human diets [61, 63, 82–85]. For instance, PA is able to reduce the risk for both colon cancer and mammary cancer through its strong metal cations binding capabilities [63, 83]. Moreover, PA display antioxidant capability by acting as inhibitor of iron-mediated hydroxyl radical (-OH) formation in food and gastrointestinal tract, which would result in lipid peroxidation and tissue damage [83, 84]. On the other hand, antinutrient lectin was found to be responsible for plant defense system by exhibiting cytotoxic activities when ingested by pests and animals [85].

Masuda [77] has demonstrated that introducing a combination of different genes responsible for MA synthesis into rice (Fer-NAS-NAAT-IDS3 lines) and result in 4-fold increase of iron accumulation in endosperm. Likewise, transgenic line expressing *AtIRT1*, *Pvferritin*, *AtNAS1*, and *Afphytase* was shown to cause a 4-fold increase of iron accumulation in polished grain [66, 86]. The *OsYSL15* or *OsIRT1* genes are predominantly expressed in roots with enhanced expression in response to iron deficiency [86]. *OsIRT1* gene encode for Fe2+ transporter involved in both strategy I and II. Although overexpression of *OsIRT1* alone could increase the iron content in rice grain by 1.3-fold, *OsIRT1* has the potential to further enhance the iron content when it is expressed with other genes [87].

On the other hand, combination approaches were also demonstrated to increase the iron content in rice grain by 3.4- and 6-fold when introduced into Myanmar and Japanese rice cultivar respectively [47, 88]. Both *SoyFerH2* and *OsYSL2* were strongly expressed in transgenic rice due to the vector inserted contains two gene cassettes for each gene expression driven by different promoters for each gene cassettes (*OsSUT1* promoter-*OsYSL2*, *OsGlb* promoter-*OsYSL2*, *OsGluB1* promoter-*SoyferH2*, *OsGlb* promoter-*SoyferH2*). Interestingly, Trijatmiko [69] was able to develop transgenic rice expressing *OsNAS2* and *SoyferH1* genes result in 15 μg Fe/g increased (6-fold) in polished grain. In the transgenic rice line, the transgene construct was found to be inserted with inverted repeats in a single locus. This concludes that multiple transgene insertion was able to increase the iron concentration in rice [47, 69, 88]. However, transgene cassette with duplicated or inverted repeats of transgene may not be stable and inherited after several generations due to possibility of epigenetic silencing in transgenic plants [66, 89–91]. Hence, further investigation should be conducted to elucidate the stability of transgene or different approach to maintain multiple transgene over multiple generations.

### **6. Challenges and future prospect**

specific clone no. 2 (*IDS2*) and no. 3 (*IDS3*) in barley play an important role in combating iron deficiency [77]. The *IDS* genes enable the synthesis of different types of MAs via DMA and these genes are highly expressed in roots in response to iron deficiency [56]. On the contrary, rice lacks the ability to synthesize other types of MAs apart of DMA as rice does not contain both *IDS2* and *IDS3* genes. Having different sets of MAs enable barley become more tolerant

Introducing *IDS3* gene from barley enables the synthesis and secretion of different types of MAs from transgenic rice into the rhizosphere [56]. In addition, formation of Fe(III)-MA complex has a better stability as compared to Fe(III)-DMA complex when grown in a slightly acidic soil [57]. This may enhance iron translocation in rice in combating iron deficiency while increasing tolerance toward iron deficiency in rice plants. Furthermore, Masuda [58] and Suzuki [78] demonstrated that *IDS3* rice lines are able to increase Fe concentrations to 1.4 and 1.3-fold for both polished and brown grains respectively compared to wild-type rice when grown in either Fe-sufficient soil or Fe-deficient soil. Thus, presence of *IDS3* gene is able to enhance iron accumulation in rice grain even when it is grown in iron-sufficient soil and as

Zhang [59] reported on the functional characterization of rice vacuolar iron transporter genes (*OsVIT1* and *OsVIT2*). These genes were found to be expressed ubiquitously in different parts of the plants at low levels but high level expression of *OsVIT* genes were detected in the flag leaves. These genes play an important role in transportation of Zn2+ and Fe2+ into vacuoles via tonoplast [79]. In addition, knockdown of *OsVIT* genes increases Fe and Zn accumulation in the rice grains significantly while decreases Fe and Zn accumulation in the flag leaves correspondingly [80]. Knockout of *OsVIT1* and *OsVIT2* genes were able to increase the iron content in rice grain by at least 1.4-fold [59, 80]. However, this approach is only applicable when the transgenic rice is grown in unpolluted soil. This is because studies had shown that accumulation of Cd2+ concentration was detected in rice when it is grown in polluted soil [59]. Hence, further understanding of regulatory mechanism is required to prevent toxic metal accumula-

Multiple gene manipulation has been successfully carried out in rice. Wirth [23] has proven the synergism of three different genes expression with the increased of iron content in rice by 6-fold through introducing *Arabidopsis thaliana NAS1* (*AtNAS1)*, *Phaseolus vulgaris ferritin* (*Pvferritin*), and *Aspergillus fumigatus phytase* (*Afphytase*) genes into rice. The main purpose of introducing *phytase* genes is to reduce iron antinutrient phytate in rice. Some food may contain antinutrients like PA, which binds strongly to metal cations, such as iron and zinc, which render them insoluble [81]. Phytase is able to catalyze the hydrolysis of PA releasing the phosphate and chelated minerals [21]. Human digestive system lacks enzyme responsible for breakdown of such components [23]. Reducing antinutrients is a feasible approach to increase nutrient content in crops but it should be exercised with cautions due to many

to iron-deficient conditions as compared to rice.

34 Rice Crop - Current Developments

well as enhancing tolerance toward iron deficiency.

**5.5. Enhancement of iron translocation via** *OsVIT* **gene**

tion and to ensure the crops are safe for consumption.

**5.6. Combinational of multiple transgenes**

Biofortification is a promising strategy for sustainable long-term approach in combating micronutrient deficiency but successful biofortification at the cost of the environmental damage is not acceptable. In agronomic practice, leaching is one of the main concerns in application of fertilizer as it will damage the environment, but most micronutrients are not susceptible to leaching as they are able to form a strong bond with the soil [13]. However, continuous application of micronutrient fertilizer may cause accumulation of these minerals which result in toxicity. Excessive intake of iron may cause Fe2+ and Fe3+ to act as a catalyst to form noxious reactive oxygen species (ROS). ROS are strong oxidizing agents, which are able to cause detrimental effect on DNA, proteins, and lipids in plants [33]. Therefore, fertilization strategies should be devised and optimized to ensure adequate supply of iron for proper growth of agronomic plants while minimizing accumulation of iron [92]. For instance, the 4R Nutrient Stewardship principle (application of fertilizer at the right place, right rate, right time and right source) could be implemented with fertilizer application [34, 93]. Based on HarvestPlus breeding programs, the iron-biofortified rice are to meet a recommended target iron level which is approximate 30% of the estimated average requirement (EAR) or 15 μg/g (dry weight) in polished grain [69, 94]. The recommended 30% EAR could be achieved via genetic engineering approaches listed in both **Tables 1** and **2**, however, the iron concentration in rice grain decreases when evaluated under field conditions as compared to iron concentration achieved in rice grown in greenhouse [47, 69]. This demonstrates that interactions between genetic and environment play an important role in iron concentration in rice grain [69, 95]. Field experiments should be included in evaluating iron levels in iron-biofortified rice for several growing seasons as evaluating iron levels in rice grown under strictly controlled environment conditions does not simulate the conditions when grown in the field [69, 94, 96, 97].

breeders, biofortified crops are unable to be produced despite the crop has the potential to alleviate micronutrient deficiency. Hence, to gain plant breeder acceptance, biofortified crops should contain visible and favorable traits such as increased in yield, higher stress tolerant, disease resistance, and other important agronomic traits [10]. Plant breeders may be reluctant to produce the biofortified crops with the potential income risk if the consumer does not adopt with the new crop variety especially with biofortified crops having their sensory characteristics altered such as the color and taste [12]. Some biofortified crops have been introduced for production and accepted by the public in some countries despite the change in sensory characteristics [14]. These biofortified crops are orange flesh sweet potato, orange maize, yellow cassava, iron pearl millet, and iron beans. Consumer acceptance on biofortified crops is not easy and achievable in a short duration of time but it can be accomplished through thoroughly planned strategies such as spreading knowledge among the people, raising awareness of micronutrient deficiency, creating new market opportunities, and creating a demand on biofortified variety [103]. On the contrary, the success of iron biofortification would results in improved nutritional value of micronutrient-deficient affected areas in developing countries and as a first step toward improving nutritional status worldwide.

The authors thank Putra Grant (GP/2017/9572000) from Universiti Putra Malaysia and Fundamental Research Grant Scheme (FRGS/1/2014/SG05/MOSTI/1) from Ministry of Higher

, Rogayah Sekeli<sup>2</sup>

\*

1 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular

2 Malaysian Agricultural Research and Development Institute (MARDI), Kuala Lumpur,

[1] Yang Q, Zhang C, Chan M, Zhao D, Chen J, Wang Q, et al. Biofortification of rice with the essential amino acid lysine: Molecular characterization, nutritional evaluation, and field

3 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences,

performance. Journal of Experimental Botany. 2016 Jul;**67**(14):4285-4296

, Wee Chien Yeong<sup>2</sup>

,

Iron Biofortification of Rice: Progress and Prospects http://dx.doi.org/10.5772/intechopen.73572 37

**Acknowledgements**

**Author details**

Malaysia

**References**

Andrew De-Xian Kok<sup>1</sup>

Zetty Norhana Balia Yusof<sup>3</sup>

Education, Malaysia for the support.

, Low Lee Yoon<sup>1</sup>

\*Address all correspondence to: laikoksong@upm.edu.my

Sciences, Universiti Putra Malaysia, Selangor, Malaysia

Universiti Putra Malaysia, Selangor, Malaysia

and Lai Kok Song<sup>1</sup>

Biofortified crops still face strict regulatory hurdles and a lack of consumer acceptance, especially in Europe, even though there have been reports on improvement in nutritional status after consuming biofortified crops [16, 38, 98, 99]. For instance, golden rice, a product developed via genetic engineering in combating vitamin A deficiency, has been announced since early 2000, but it has yet to be seen in the market [100]. Although these transgenic plants has demonstrated its high nutritional content in combating micronutrient deficiency, but as far as public safety concern, additional regulations and more stringent monitoring are implemented onto transgenic crops before being available to the public compared to conventional breeding which is more widely accepted [3]. In addition, there are possibilities of irreversibility effect on health and environment due to the effects of GM crops on health and environment are not fully understood and not sustainable in the long run [100, 101]. Furthermore, there are possibilities of the transgene in biofortified crops survived through human digestion system which allows transgenic plant DNA such as antibiotic resistance genes to be transferred into small intestine microflora [102]. Therefore, additional researches from different disciplines are required in order to elucidate the effect of biofortified crops consumption on human health. This may appease public anxiety and to gain consumer acceptance [3]. On the other hand, the recent advancement of genetic engineering technologies, such as zinc-finger nucleases, TALENs and CRISPR-Cas9, could be a potential approach in iron biofortification, which allows efficient and effective gene editing without affecting the plant as a whole [18, 44, 45]. Moreover, gene-edited crops are subjected to different regulations and monitoring from government bodies and nongovernment organizations, which are not as stringent as genetically modified crops. As a result, gene-edited crops will have a higher consumer acceptance compared to conventional genetic engineering.

While iron biofortification in rice is a promising approach in combating iron deficiency, the success of biofortification is dependent on various factors and it requires the collaboration between different parties ranging from consumer, plant breeder, multilateral organizations, national governments, and researchers from various disciplines. Without the help and adoption from plant breeders, biofortified crops are unable to be produced despite the crop has the potential to alleviate micronutrient deficiency. Hence, to gain plant breeder acceptance, biofortified crops should contain visible and favorable traits such as increased in yield, higher stress tolerant, disease resistance, and other important agronomic traits [10]. Plant breeders may be reluctant to produce the biofortified crops with the potential income risk if the consumer does not adopt with the new crop variety especially with biofortified crops having their sensory characteristics altered such as the color and taste [12]. Some biofortified crops have been introduced for production and accepted by the public in some countries despite the change in sensory characteristics [14]. These biofortified crops are orange flesh sweet potato, orange maize, yellow cassava, iron pearl millet, and iron beans. Consumer acceptance on biofortified crops is not easy and achievable in a short duration of time but it can be accomplished through thoroughly planned strategies such as spreading knowledge among the people, raising awareness of micronutrient deficiency, creating new market opportunities, and creating a demand on biofortified variety [103]. On the contrary, the success of iron biofortification would results in improved nutritional value of micronutrient-deficient affected areas in developing countries and as a first step toward improving nutritional status worldwide.

### **Acknowledgements**

toxicity. Excessive intake of iron may cause Fe2+ and Fe3+ to act as a catalyst to form noxious reactive oxygen species (ROS). ROS are strong oxidizing agents, which are able to cause detrimental effect on DNA, proteins, and lipids in plants [33]. Therefore, fertilization strategies should be devised and optimized to ensure adequate supply of iron for proper growth of agronomic plants while minimizing accumulation of iron [92]. For instance, the 4R Nutrient Stewardship principle (application of fertilizer at the right place, right rate, right time and right source) could be implemented with fertilizer application [34, 93]. Based on HarvestPlus breeding programs, the iron-biofortified rice are to meet a recommended target iron level which is approximate 30% of the estimated average requirement (EAR) or 15 μg/g (dry weight) in polished grain [69, 94]. The recommended 30% EAR could be achieved via genetic engineering approaches listed in both **Tables 1** and **2**, however, the iron concentration in rice grain decreases when evaluated under field conditions as compared to iron concentration achieved in rice grown in greenhouse [47, 69]. This demonstrates that interactions between genetic and environment play an important role in iron concentration in rice grain [69, 95]. Field experiments should be included in evaluating iron levels in iron-biofortified rice for several growing seasons as evaluating iron levels in rice grown under strictly controlled environment condi-

36 Rice Crop - Current Developments

tions does not simulate the conditions when grown in the field [69, 94, 96, 97].

compared to conventional genetic engineering.

Biofortified crops still face strict regulatory hurdles and a lack of consumer acceptance, especially in Europe, even though there have been reports on improvement in nutritional status after consuming biofortified crops [16, 38, 98, 99]. For instance, golden rice, a product developed via genetic engineering in combating vitamin A deficiency, has been announced since early 2000, but it has yet to be seen in the market [100]. Although these transgenic plants has demonstrated its high nutritional content in combating micronutrient deficiency, but as far as public safety concern, additional regulations and more stringent monitoring are implemented onto transgenic crops before being available to the public compared to conventional breeding which is more widely accepted [3]. In addition, there are possibilities of irreversibility effect on health and environment due to the effects of GM crops on health and environment are not fully understood and not sustainable in the long run [100, 101]. Furthermore, there are possibilities of the transgene in biofortified crops survived through human digestion system which allows transgenic plant DNA such as antibiotic resistance genes to be transferred into small intestine microflora [102]. Therefore, additional researches from different disciplines are required in order to elucidate the effect of biofortified crops consumption on human health. This may appease public anxiety and to gain consumer acceptance [3]. On the other hand, the recent advancement of genetic engineering technologies, such as zinc-finger nucleases, TALENs and CRISPR-Cas9, could be a potential approach in iron biofortification, which allows efficient and effective gene editing without affecting the plant as a whole [18, 44, 45]. Moreover, gene-edited crops are subjected to different regulations and monitoring from government bodies and nongovernment organizations, which are not as stringent as genetically modified crops. As a result, gene-edited crops will have a higher consumer acceptance

While iron biofortification in rice is a promising approach in combating iron deficiency, the success of biofortification is dependent on various factors and it requires the collaboration between different parties ranging from consumer, plant breeder, multilateral organizations, national governments, and researchers from various disciplines. Without the help and adoption from plant The authors thank Putra Grant (GP/2017/9572000) from Universiti Putra Malaysia and Fundamental Research Grant Scheme (FRGS/1/2014/SG05/MOSTI/1) from Ministry of Higher Education, Malaysia for the support.

## **Author details**

Andrew De-Xian Kok<sup>1</sup> , Low Lee Yoon<sup>1</sup> , Rogayah Sekeli<sup>2</sup> , Wee Chien Yeong<sup>2</sup> , Zetty Norhana Balia Yusof<sup>3</sup> and Lai Kok Song<sup>1</sup> \*

\*Address all correspondence to: laikoksong@upm.edu.my

1 Department of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor, Malaysia

2 Malaysian Agricultural Research and Development Institute (MARDI), Kuala Lumpur, Malaysia

3 Department of Biochemistry, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, Selangor, Malaysia

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[2] Johnson AAT, Kyriacou B, Callahan DL, Carruthers L, Stangoulis J, Lombi E, et al. Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of Rice endosperm. Baxter I, editor. PLoS One 2011 Sep;**6**(9):e24476

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[22] Ishimaru Y, Suzuki M, Tsukamoto T, Suzuki K, Nakazono M, Kobayashi T, et al. Rice

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**Chapter 4**

**Provisional chapter**

**Improving Rice Grain Quality by Enhancing**

**Improving Rice Grain Quality by Enhancing** 

**Cadmium and Lead**

**Cadmium and Lead**

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.72826

Lei Gao and Jie Xiong

**Abstract**

**1. Introduction**

Lei Gao and Jie Xiong

**Accumulation of Iron and Zinc While Minimizing**

Iron (Fe) and zinc (Zn) are important trace elements for people's health around the globe. A lot of people, especially children and woman, are suffering from malnutrition caused by Fe and/or Zn deficiency. The deficiency is more pronounced in some parts of Africa and Asia due to low income, which makes it difficult to afford meat or sea foods that are rich in Fe and Zn. Biofortification of Fe and Zn in rice is the most economical and convenient way to supplement these important minerals in the diet of poor people. However, besides Fe and Zn, rice also can accumulate heavy metals, such as cadmium (Cd) and lead (Pb), which are harmful to people, especially for kids' health. Previous researches have shown that there are connections and discrepancies for metal absorption, translocation, and accumulation in rice. So it is imperative to review these issues. This chapter compares the physiological and molecular mechanisms of Fe, Zn, Cd, and Pb uptake, mobilization, and accumulation in rice and discusses the progress and strategies for not

only increasing Fe/Zn but also decreasing Cd/Zn accumulation in rice.

**Keywords:** biofortification, heavy metal, iron, zinc, cadmium, lead, stress

**Accumulation of Iron and Zinc While Minimizing** 

DOI: 10.5772/intechopen.72826

© 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,

© 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.

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

Metal elements, such as Fe, Zn, Mn and Cu, are essential for living organisms and humans' growth. Various metal nutrients supplied by food contributed to maintain metabolism normally. Unfortunately, iron (Fe) and zinc (Zn), as the most important metal elements, are present in low quantities of staple food, such as rice and wheat [1]. What is worse is that, in some parts of Africa and Asia, people even cannot afford enough food for their kids and families. Fe deficiency is one of the leading risk factors of disability and death worldwide. It is estimated to affect two billion


**Provisional chapter**

### **Improving Rice Grain Quality by Enhancing Accumulation of Iron and Zinc While Minimizing Cadmium and Lead Accumulation of Iron and Zinc While Minimizing Cadmium and Lead**

**Improving Rice Grain Quality by Enhancing** 

DOI: 10.5772/intechopen.72826

Lei Gao and Jie Xiong Additional information is available at the end of the chapter

Lei Gao and Jie Xiong

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yield potential. 2017;**11**(4):1-7

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Opinion in Biotechnology. 2017 Apr;**44**(**1)**:8-15

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.72826

#### **Abstract**

Iron (Fe) and zinc (Zn) are important trace elements for people's health around the globe. A lot of people, especially children and woman, are suffering from malnutrition caused by Fe and/or Zn deficiency. The deficiency is more pronounced in some parts of Africa and Asia due to low income, which makes it difficult to afford meat or sea foods that are rich in Fe and Zn. Biofortification of Fe and Zn in rice is the most economical and convenient way to supplement these important minerals in the diet of poor people. However, besides Fe and Zn, rice also can accumulate heavy metals, such as cadmium (Cd) and lead (Pb), which are harmful to people, especially for kids' health. Previous researches have shown that there are connections and discrepancies for metal absorption, translocation, and accumulation in rice. So it is imperative to review these issues. This chapter compares the physiological and molecular mechanisms of Fe, Zn, Cd, and Pb uptake, mobilization, and accumulation in rice and discusses the progress and strategies for not only increasing Fe/Zn but also decreasing Cd/Zn accumulation in rice.

**Keywords:** biofortification, heavy metal, iron, zinc, cadmium, lead, stress

### **1. Introduction**

Metal elements, such as Fe, Zn, Mn and Cu, are essential for living organisms and humans' growth. Various metal nutrients supplied by food contributed to maintain metabolism normally. Unfortunately, iron (Fe) and zinc (Zn), as the most important metal elements, are present in low quantities of staple food, such as rice and wheat [1]. What is worse is that, in some parts of Africa and Asia, people even cannot afford enough food for their kids and families. Fe deficiency is one of the leading risk factors of disability and death worldwide. It is estimated to affect two billion

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.

© 2016 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons

people in the world [2]. Also, it has been estimated that about 30% population of the world suffers from Zn deficiency [3]. Fe-deficiency anemia can impair cognitive and physical development in children and the reduction of daily productivity in adults. Adequate Zn nutrition is also important for the growth of children, immune function, and neurobehavioral development [4]. Thus, Fe and Zn deficiency has emerged as a major and common problem for the health of humans [5, 6].

strategy [15]. In rice, phytochelatins (PC) acting as Cd chelator plays a key role in Cd detoxification [22]. PC chelates Cd in the cytosol and forms complexes with Cd. Then the Cd-PCs complexes are sequestered in the vacuoles via specific transporters located at tonoplast [23, 24].

Improving Rice Grain Quality by Enhancing Accumulation of Iron and Zinc While Minimizing…

http://dx.doi.org/10.5772/intechopen.72826

47

Pb after arsenic (As) is ranked as the second most harmful element due to its occurrence, toxicity, and exposure potential [25]. Although Pb occurs naturally only in small amounts within the Earth's crust [26], a large amount of industrial activities are the primary sources of Pb in soil [27]. Pb is released into soil in general forms of Pb(II), lead oxide and lead-metal oxyanion, among which Pb(II) is the most common form [27]. Pb stress can produce reactive oxygen species (ROS) and trigger some antioxidative enzymes, accompanied by the increased level of lipid peroxidation [25, 27]. In rice, Pb toxicity reduces leaf chlorophyll and nitrogen content and increases antioxidative enzymes. Its subsequent translocation to grain causes a great threat to humans' health [28, 29]. High levels of Pb can cause brain and kidney damage, accompanied with central nervous prostration [27]. Therefore, higher plants possess preventive mechanisms against Pb. Similar to Cd detoxification, cell walls, and compartmentaliza-

tion in vacuoles have been suggested as an important detoxification mechanism [27].

ing grain Cd/Pb accumulation and enhancing Fe/Zn content in rice.

plex formation); and (3) uptake transporters [11].

line soil is immobilized as Fe(OH)3

**2. Uptake of Fe/Zn and Cd/Pb from rhizosphere to root in rice**

that *NAAT1* mutant was not able to produce DMA and take up Fe(III) efficiently.

Many researches show that the mechanisms of Fe/Zn/Cd/Pb uptake and accumulation in rice share commons in some aspects as a result of similar entry routes (transporters) within rice cells. However, an increasing number of studies discovered distinct pathways and mechanisms of Fe/Zn/Cd/Pb uptake and accumulation in rice recently. In this chapter, we mainly systematically elaborate and compare physiological and cellular mechanisms of Fe/Zn/Cd/ Pb uptake and accumulation in rice. In addition, we also review the mechanisms of maintaining Fe/Zn homeostasis and Cd/Pb detoxification in rice. Effects of fertilizers on Fe/Zn/Cd /Pb accumulation in rice are also discussed. Finally, we enumerate various approaches for reduc-

Mobility and availability of metals from soil are controlled by three following factors: (1) soil conditions (upland or flooded soil, soil solution pH); (2) mineralization (ionization and com-

Although Fe in acidic soil is ionized as Fe2+/Fe3+ and easily utilized by plants, Fe in aerobic alka-

methionine (SAM) is catalyzed by nicotianamine synthase (NAS) and produces nicotianamine (NA), which is an intermediate for the biosynthesis of MA family and a vital substance of nicotianamine aminotransferase (NAAT) [6]. Three rice NAS genes, *OsNAS1*, *OsNAS2*, and *OsNAS3*, have been identified to play different roles in Fe uptake and translocation in rice several years ago [30]. NAAT is a critical enzyme converting NA to 2′-deoxymugineic acid (DMA). Six rice *NAAT* genes (*OsNAAT1*–*6*) have been identified, but *OsNAAT1* was the only one highly upregulated under Fe deficiency, suggesting that *OsNAAT1* rather than *OsNAAT2–6* encodes the unique functional enzyme possessing NAAT activity [31]. Cheng et al. [10] demonstrated

. Rice absorbs Fe3+ via strategy II.In strategy II, *S*-adenosyl-l-*L*-

Rice is the staple food and provides energy to almost half of the world's population, especially in Asia and Africa. Thus, increasing Fe/Zn content of rice has a great potential to mitigate widespread Fe/Zn deficiency problem in humans [7]. Therefore, it is essential to understand the mechanisms through which rice uptakes, mobilizes, and accumulates Fe/Zn. In response to Fe deficiency, higher plants have developed two strategies for acquiring Fe from the rhizosphere [8, 9]. Strategy I is employed in nongraminaceous plants, and Fe(III) is reduced to soluble Fe(II) through activating membrane-bound Fe(III)-chelate reductases, then the reduced Fe(II) was transported into cytoplasm via Fe(II) transporters [10]. In contrast, Strategy II is only applied by graminaceous plants, such as rice and wheat. The root of strategy II plants secretes phytosiderophores (PSs) to rhizosphere and chelates Fe(III) to form Fe(III)-PS complexes. Subsequently, the Fe(III)-PS complexes are transported via specific plasma membrane transporters [7]. Rice not only employs strategy II to acquire Fe from rhizosphere but also utilizes strategy I-like system to uptake Fe(II) directly [10]. Besides Fe uptake, mugineic acid (MA) family also plays crucial roles in chelating Zn from rhizosphere, followed by uptake of Zn-PS complexes via specific plasma membrane transporters [7]. Moreover, Zn can be ionized as Zn(II) and directly enters into root [11]. In spite of rice can apply specific strategies to acquire Fe and Zn, these mechanisms have limited accessibility to resource poor people faced with Fe and Zn deficiency from certain areas of the world. To deal with limited Fe/Zn and improve human Fe/Zn nutritional status, rice with enhanced Fe/Zn absorption will be an effective method for populations consuming rice as their staple foods.

Besides necessary metal elements, such as Fe and Zn, rice also absorbs and accumulates toxic metals such as cadmium (Cd) and lead (Pb), which are harmful for both rice and humans. Cd enters into environment, such as soil and river, mainly through industrial activities or fertilizers [12]. As a highly mobile and soluble metal [13, 14], Cd exposure causes crops yield reduction and does harm to humans' health even at low concentrations [15]. Due to daily consumption, Cd in rice grains poses a latent health problem to humans through food chains and leads to chronic toxicity. The outbreak of "Itai-Itai disease" in the mid-twentieth century in Japan is due to consumption of Cd-contaminated rice [16]. A person with "Itai-Itai" has symptoms of weakness and softening of the bones. Even in recent years, Cd exposure in general Japanese population can be as high as 3–4 mg kg−1 body weight every week [17]. The directly observable toxic symptoms of Cd on plants are as follows: reduced rate of transpiration and photosynthesis, growth retardation and declining metabolic activities [15]. In response to Cd toxicity, plants also have evolved several protective mechanisms against Cd toxicity, including avoidance and tolerance strategies [18]. Plants can prevent Cd from entering into plant cells, which is refered to as avoidance strategy. Cell walls serve as the first barrier against Cd entrance [15, 19]. Root exudates, which are majorly consisted of sugars, proteins and organic acids are secreted from root to soil to combine with Cd, keeping Cd apart from root [20, 21]. After entered into the cells, the abilities of resistance to Cd stress are referred to as tolerance strategy [15]. In rice, phytochelatins (PC) acting as Cd chelator plays a key role in Cd detoxification [22]. PC chelates Cd in the cytosol and forms complexes with Cd. Then the Cd-PCs complexes are sequestered in the vacuoles via specific transporters located at tonoplast [23, 24].

people in the world [2]. Also, it has been estimated that about 30% population of the world suffers from Zn deficiency [3]. Fe-deficiency anemia can impair cognitive and physical development in children and the reduction of daily productivity in adults. Adequate Zn nutrition is also important for the growth of children, immune function, and neurobehavioral development [4]. Thus, Fe and Zn deficiency has emerged as a major and common problem for the health of humans [5, 6]. Rice is the staple food and provides energy to almost half of the world's population, especially in Asia and Africa. Thus, increasing Fe/Zn content of rice has a great potential to mitigate widespread Fe/Zn deficiency problem in humans [7]. Therefore, it is essential to understand the mechanisms through which rice uptakes, mobilizes, and accumulates Fe/Zn. In response to Fe deficiency, higher plants have developed two strategies for acquiring Fe from the rhizosphere [8, 9]. Strategy I is employed in nongraminaceous plants, and Fe(III) is reduced to soluble Fe(II) through activating membrane-bound Fe(III)-chelate reductases, then the reduced Fe(II) was transported into cytoplasm via Fe(II) transporters [10]. In contrast, Strategy II is only applied by graminaceous plants, such as rice and wheat. The root of strategy II plants secretes phytosiderophores (PSs) to rhizosphere and chelates Fe(III) to form Fe(III)-PS complexes. Subsequently, the Fe(III)-PS complexes are transported via specific plasma membrane transporters [7]. Rice not only employs strategy II to acquire Fe from rhizosphere but also utilizes strategy I-like system to uptake Fe(II) directly [10]. Besides Fe uptake, mugineic acid (MA) family also plays crucial roles in chelating Zn from rhizosphere, followed by uptake of Zn-PS complexes via specific plasma membrane transporters [7]. Moreover, Zn can be ionized as Zn(II) and directly enters into root [11]. In spite of rice can apply specific strategies to acquire Fe and Zn, these mechanisms have limited accessibility to resource poor people faced with Fe and Zn deficiency from certain areas of the world. To deal with limited Fe/Zn and improve human Fe/Zn nutritional status, rice with enhanced Fe/Zn absorption will be an

46 Rice Crop - Current Developments

effective method for populations consuming rice as their staple foods.

Besides necessary metal elements, such as Fe and Zn, rice also absorbs and accumulates toxic metals such as cadmium (Cd) and lead (Pb), which are harmful for both rice and humans. Cd enters into environment, such as soil and river, mainly through industrial activities or fertilizers [12]. As a highly mobile and soluble metal [13, 14], Cd exposure causes crops yield reduction and does harm to humans' health even at low concentrations [15]. Due to daily consumption, Cd in rice grains poses a latent health problem to humans through food chains and leads to chronic toxicity. The outbreak of "Itai-Itai disease" in the mid-twentieth century in Japan is due to consumption of Cd-contaminated rice [16]. A person with "Itai-Itai" has symptoms of weakness and softening of the bones. Even in recent years, Cd exposure in general Japanese population can be as high as 3–4 mg kg−1 body weight every week [17]. The directly observable toxic symptoms of Cd on plants are as follows: reduced rate of transpiration and photosynthesis, growth retardation and declining metabolic activities [15]. In response to Cd toxicity, plants also have evolved several protective mechanisms against Cd toxicity, including avoidance and tolerance strategies [18]. Plants can prevent Cd from entering into plant cells, which is refered to as avoidance strategy. Cell walls serve as the first barrier against Cd entrance [15, 19]. Root exudates, which are majorly consisted of sugars, proteins and organic acids are secreted from root to soil to combine with Cd, keeping Cd apart from root [20, 21]. After entered into the cells, the abilities of resistance to Cd stress are referred to as tolerance Pb after arsenic (As) is ranked as the second most harmful element due to its occurrence, toxicity, and exposure potential [25]. Although Pb occurs naturally only in small amounts within the Earth's crust [26], a large amount of industrial activities are the primary sources of Pb in soil [27]. Pb is released into soil in general forms of Pb(II), lead oxide and lead-metal oxyanion, among which Pb(II) is the most common form [27]. Pb stress can produce reactive oxygen species (ROS) and trigger some antioxidative enzymes, accompanied by the increased level of lipid peroxidation [25, 27]. In rice, Pb toxicity reduces leaf chlorophyll and nitrogen content and increases antioxidative enzymes. Its subsequent translocation to grain causes a great threat to humans' health [28, 29]. High levels of Pb can cause brain and kidney damage, accompanied with central nervous prostration [27]. Therefore, higher plants possess preventive mechanisms against Pb. Similar to Cd detoxification, cell walls, and compartmentalization in vacuoles have been suggested as an important detoxification mechanism [27].

Many researches show that the mechanisms of Fe/Zn/Cd/Pb uptake and accumulation in rice share commons in some aspects as a result of similar entry routes (transporters) within rice cells. However, an increasing number of studies discovered distinct pathways and mechanisms of Fe/Zn/Cd/Pb uptake and accumulation in rice recently. In this chapter, we mainly systematically elaborate and compare physiological and cellular mechanisms of Fe/Zn/Cd/ Pb uptake and accumulation in rice. In addition, we also review the mechanisms of maintaining Fe/Zn homeostasis and Cd/Pb detoxification in rice. Effects of fertilizers on Fe/Zn/Cd /Pb accumulation in rice are also discussed. Finally, we enumerate various approaches for reducing grain Cd/Pb accumulation and enhancing Fe/Zn content in rice.

### **2. Uptake of Fe/Zn and Cd/Pb from rhizosphere to root in rice**

Mobility and availability of metals from soil are controlled by three following factors: (1) soil conditions (upland or flooded soil, soil solution pH); (2) mineralization (ionization and complex formation); and (3) uptake transporters [11].

Although Fe in acidic soil is ionized as Fe2+/Fe3+ and easily utilized by plants, Fe in aerobic alkaline soil is immobilized as Fe(OH)3 . Rice absorbs Fe3+ via strategy II.In strategy II, *S*-adenosyl-l-*L*methionine (SAM) is catalyzed by nicotianamine synthase (NAS) and produces nicotianamine (NA), which is an intermediate for the biosynthesis of MA family and a vital substance of nicotianamine aminotransferase (NAAT) [6]. Three rice NAS genes, *OsNAS1*, *OsNAS2*, and *OsNAS3*, have been identified to play different roles in Fe uptake and translocation in rice several years ago [30]. NAAT is a critical enzyme converting NA to 2′-deoxymugineic acid (DMA). Six rice *NAAT* genes (*OsNAAT1*–*6*) have been identified, but *OsNAAT1* was the only one highly upregulated under Fe deficiency, suggesting that *OsNAAT1* rather than *OsNAAT2–6* encodes the unique functional enzyme possessing NAAT activity [31]. Cheng et al. [10] demonstrated that *NAAT1* mutant was not able to produce DMA and take up Fe(III) efficiently.

In rice, gene that encodes DMA efflux transporters (OsTOM1) is highly expressed under Fe deficiency stress [32]. TOM1 transporter localizes at plasma membrane and mediates DMA secretion to rhizosphere, followed by Fe(III)-DMA complexes formation [32]. *Yellow stripe 1* (*YS1*) gene that encodes Fe(III)-MAs transporters was first acquired in maize [33], and *YS1*-like (*OsYSL*) genes in rice have been subsequently identified over decades. *OsYSL15* has been demonstrated to be upregulated in rice root and shoot under Fe deficiency to transport Fe(III)-DMA complexes [34]. In addition, *OsYSL* genes that encode transporters are also involved in Fe translocation within rice [35, 36]. Once transported into cytosol, Fe(III)-DMA is reduced by ascorbate to form Fe(II)-NA [37]. NA is not only an important intermediate for the biosynthesis of MAs but also a significant metal chelator that can take part in translocation of Fe within plants [38]. Fe may be excreted to the xylem in the form of Fe(II)–NA and shift to make complexes predominantly with citrate (Fe2 Cit<sup>2</sup> , Fe3 Cit<sup>3</sup> ) and some with DMA [39]. The excretion of citrate from the root cells to the xylem is partly operated by OsFRDL1 (rice ferric reductase defective1-like) to enhance Fe-transport in the xylem as Fe(III)–citrate complexes [40].

characteristic of Cd uptake could be mediated by transporters. In fact, entrance of Cd into root cells via transporter OsNRAMP5 or OsIRT1 has been proved, and OsNRAMP5 is predominantly applied [52, 53]. *OsNRAMP5* expression is identified in root epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem [54]. Knockout of *OsNRAMP5* reduces Cd accumulation both in straw and grains slightly [4]. Slamet-Loedin et al. [4] also proposed that downregulation of *OsNRAMP5* is a preferential strategy to decrease Cd uptake by root. OsNRAMP5 not only mediates Cd uptake but also regulates manganese (Mn) uptake and has relatively a minor effect on Fe uptake under Fe starvation [54]. In addition, higher expression of *OsNRAMP1* in root could enhance Cd accumulation in shoot of rice, indicating that OsNRAMP1 was also related with Cd uptake and transport [55]. When exposed to Cd contamination, rice supplied with Fe3+ generally represents weaker toxic symptoms than rice supplied with Fe2+. The phenomenon is largely attributed to divalent metal transporters that are nonselective for Fe2+ uptake. Rice can uptake either Fe2+ or Cd2+ consequently (**Figure 1**). In contrast, Fe3+ transporters are selective for Fe3+ with no affinity for other divalent cations, which decreases Cd entrance into rice root to a great extent and

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**Figure 1.** Schematic diagram of Fe and Cd uptake mechanisms in rice root. OsNRAMP1 and OsIRT1 can uptake either divalent ion Fe2+ or Cd2+. NA is catalyzed by NAAT1 and finally forms DMA, which is secreted into rhizosphere via transporter OsTOM1 and chelates with Fe3+ to form complex. The complex is transported into cell via transporter

reduces Cd toxicity accordingly (**Figure 1**).

OsYSL15.

In addition to Fe(III)-DMA uptake, rice also absorbs Fe(II) via iron-regulated transporter 1 (OsIRT1) and natural resistance-associated macrophage protein 1 (OsNRAMP1) under flooded conditions [16]. Seven rice *NRAMP* genes have been identified so far [41]. Recent research indicated that plasma membrane-localized protocatechuic acid (PCA) transporter and phenolic efflux zero1/2 (PEZ1/2) also participated in Fe uptake [42]. Such transporters played a role in absorbing apoplasmic precipitated Fe by secreting phenolics like PCA or caffeic acid. Suppression of *PEZ1/2* expression resulted in reduced Fe concentrations [42, 43].

Zn in both drained and flooded soil is largely ionized as (Zn2+) though some Zn may be bound to organic substances and immobilized as Zn-sulfide (ZnS) in the anaerobic layer [7]. Zn-regulated transporters and iron-regulated transporters like protein (ZIP) family participate in Zn uptake [44]. OsZIP1-8 transporters have been characterized for Zn uptake and translocation in rice [7]. OsZIP1, OsZIP3, and OsZIP4 are required for Zn acquisition from rhizosphere [45]. OsZIP1 and OsZIP3 are mainly located in vascular bundles and epidermal cells, while OsZIP4 is located in apical meristem and phloem cells [45]. Under Zn deficiency stress, expression of *OsZIP1, OsZIP3*, and *OsZIP4* are upregulated [44, 45]. In addition, ironregulated transporters (OsIRT) are also involved in Zn uptake [11]. OsIRT1 is characterized for Zn uptake in rice besides Fe acquisition [46, 47]. After transported into cytosol, Zn can be sequestered into the vacuoles via transporter OsZIP1 [11]. OsZIP1 is located to tonoplast and mediates influx of Zn into vacuoles [11]. MAs are also characterized for their role in Zn uptake in plants [7].

In comparison with Fe and Zn, Cd in paddy alkaline soil is present in the immobilized forms of CdCO<sup>3</sup> and humic acid-bound Cd [48]. Cd is immobilized as Cd-sulfide (CdS) and colloids-bound Cd in flooded soil [49]. Cd in drained acidic soil is ionized as Cd2+, and drainage converts CdS to Cd2+ dramatically, increasing its availability to plants [11]. Cd uptake from rhizosphere is a dose-dependent process and exhibits saturable kinetic characteristics in rice [16, 50]. After analyzing the kinetics of Cd uptake by root in rice, Fujimaki et al*.* [50] suggested that uptake rate of Cd was proportional to Cd concentration in the culture solution within range from 0.05 to 100 nM, demonstrating a linear relationship between uptake rate and Cd concentration in a certain range. Ishikawa et al. [51] suggested that this kinetic characteristic of Cd uptake could be mediated by transporters. In fact, entrance of Cd into root cells via transporter OsNRAMP5 or OsIRT1 has been proved, and OsNRAMP5 is predominantly applied [52, 53]. *OsNRAMP5* expression is identified in root epidermis, exodermis, and outer layers of the cortex as well as in tissues around the xylem [54]. Knockout of *OsNRAMP5* reduces Cd accumulation both in straw and grains slightly [4]. Slamet-Loedin et al. [4] also proposed that downregulation of *OsNRAMP5* is a preferential strategy to decrease Cd uptake by root. OsNRAMP5 not only mediates Cd uptake but also regulates manganese (Mn) uptake and has relatively a minor effect on Fe uptake under Fe starvation [54]. In addition, higher expression of *OsNRAMP1* in root could enhance Cd accumulation in shoot of rice, indicating that OsNRAMP1 was also related with Cd uptake and transport [55].

In rice, gene that encodes DMA efflux transporters (OsTOM1) is highly expressed under Fe deficiency stress [32]. TOM1 transporter localizes at plasma membrane and mediates DMA secretion to rhizosphere, followed by Fe(III)-DMA complexes formation [32]. *Yellow stripe 1* (*YS1*) gene that encodes Fe(III)-MAs transporters was first acquired in maize [33], and *YS1*-like (*OsYSL*) genes in rice have been subsequently identified over decades. *OsYSL15* has been demonstrated to be upregulated in rice root and shoot under Fe deficiency to transport Fe(III)-DMA complexes [34]. In addition, *OsYSL* genes that encode transporters are also involved in Fe translocation within rice [35, 36]. Once transported into cytosol, Fe(III)-DMA is reduced by ascorbate to form Fe(II)-NA [37]. NA is not only an important intermediate for the biosynthesis of MAs but also a significant metal chelator that can take part in translocation of Fe within plants [38]. Fe may be excreted to the xylem in the form of Fe(II)–NA and

[39]. The excretion of citrate from the root cells to the xylem is partly operated by OsFRDL1 (rice ferric reductase defective1-like) to enhance Fe-transport in the xylem as Fe(III)–citrate

In addition to Fe(III)-DMA uptake, rice also absorbs Fe(II) via iron-regulated transporter 1 (OsIRT1) and natural resistance-associated macrophage protein 1 (OsNRAMP1) under flooded conditions [16]. Seven rice *NRAMP* genes have been identified so far [41]. Recent research indicated that plasma membrane-localized protocatechuic acid (PCA) transporter and phenolic efflux zero1/2 (PEZ1/2) also participated in Fe uptake [42]. Such transporters played a role in absorbing apoplasmic precipitated Fe by secreting phenolics like PCA or caffeic acid. Suppression of *PEZ1/2* expression resulted in reduced Fe concentrations [42, 43]. Zn in both drained and flooded soil is largely ionized as (Zn2+) though some Zn may be bound to organic substances and immobilized as Zn-sulfide (ZnS) in the anaerobic layer [7]. Zn-regulated transporters and iron-regulated transporters like protein (ZIP) family participate in Zn uptake [44]. OsZIP1-8 transporters have been characterized for Zn uptake and translocation in rice [7]. OsZIP1, OsZIP3, and OsZIP4 are required for Zn acquisition from rhizosphere [45]. OsZIP1 and OsZIP3 are mainly located in vascular bundles and epidermal cells, while OsZIP4 is located in apical meristem and phloem cells [45]. Under Zn deficiency stress, expression of *OsZIP1, OsZIP3*, and *OsZIP4* are upregulated [44, 45]. In addition, ironregulated transporters (OsIRT) are also involved in Zn uptake [11]. OsIRT1 is characterized for Zn uptake in rice besides Fe acquisition [46, 47]. After transported into cytosol, Zn can be sequestered into the vacuoles via transporter OsZIP1 [11]. OsZIP1 is located to tonoplast and mediates influx of Zn into vacuoles [11]. MAs are also characterized for their role in Zn uptake

In comparison with Fe and Zn, Cd in paddy alkaline soil is present in the immobilized forms

loids-bound Cd in flooded soil [49]. Cd in drained acidic soil is ionized as Cd2+, and drainage converts CdS to Cd2+ dramatically, increasing its availability to plants [11]. Cd uptake from rhizosphere is a dose-dependent process and exhibits saturable kinetic characteristics in rice [16, 50]. After analyzing the kinetics of Cd uptake by root in rice, Fujimaki et al*.* [50] suggested that uptake rate of Cd was proportional to Cd concentration in the culture solution within range from 0.05 to 100 nM, demonstrating a linear relationship between uptake rate and Cd concentration in a certain range. Ishikawa et al. [51] suggested that this kinetic

and humic acid-bound Cd [48]. Cd is immobilized as Cd-sulfide (CdS) and col-

Cit<sup>2</sup> , Fe3 Cit<sup>3</sup>

) and some with DMA

shift to make complexes predominantly with citrate (Fe2

complexes [40].

48 Rice Crop - Current Developments

in plants [7].

of CdCO<sup>3</sup>

When exposed to Cd contamination, rice supplied with Fe3+ generally represents weaker toxic symptoms than rice supplied with Fe2+. The phenomenon is largely attributed to divalent metal transporters that are nonselective for Fe2+ uptake. Rice can uptake either Fe2+ or Cd2+ consequently (**Figure 1**). In contrast, Fe3+ transporters are selective for Fe3+ with no affinity for other divalent cations, which decreases Cd entrance into rice root to a great extent and reduces Cd toxicity accordingly (**Figure 1**).

**Figure 1.** Schematic diagram of Fe and Cd uptake mechanisms in rice root. OsNRAMP1 and OsIRT1 can uptake either divalent ion Fe2+ or Cd2+. NA is catalyzed by NAAT1 and finally forms DMA, which is secreted into rhizosphere via transporter OsTOM1 and chelates with Fe3+ to form complex. The complex is transported into cell via transporter OsYSL15.

After influx into cytosol, Cd is sequestered into the vacuoles via transporter OsHMA3 [56] and transiently stored in the form of complexes [15]. This pathway decreases Cd mobility in the cytosol and translocation from root to shoot [15, 57]. Enhancement of OsHMA3 activity has been found to increase storage of Cd in root and decrease the transport of Cd to the shoot and the final accumulation of Cd in rice grains [23]. *OsHMA3* is mainly expressed in root [24], and OsHMA3 localized at tonoplast belongs to P1B-ATPases [58]. A high rate of root-to-shoot transport and subsequent accumulation in the grains of 107Cd, which was administered from a culture solution, was observed in OsHMA3-depleted rice lines [51].

[6, 35, 43]. Thereafter, Fe is delivered to grains via phloem in forms of Fe(III)-DMA or binds

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The Zn chemical forms in xylem sap are free ions and Zn partially bound to unidentified chelators [40], while the Zn in phloem sap was dominantly bound to NA [70]. Obata and Kitagishi [71] indicated that some Zn in the xylem (transpiration stream) is transferred to the phloem at the vegetative nodes in addition to the mobilization of Zn from mature leaves in rice. Xylem transfer cells have been found in rice vegetative nodes, and localized metal transporters may support the active xylem-to-phloem transfer of xylem sap Zn [72] and Cd [73].

After entered into root cells, part of Cd present as Cd-phytochelatin (Cd-PC) complexes are sequestered in the vacuoles, and the others are transported to xylem mediated by OsHMA2 transporter in root pericycle cells [11, 74, 75]. In the phloem, Cd primarily bounds to specific proteins and slightly to thiol-compounds [76]. In contrast to Fe translocation that is mainly derived from leaves by remobilization, xylem-to-phloem transfer system of Cd mainly occurs at the nodes [50]. In rice nodes, the diffuse vascular bundles (DVBs) that encircle the enlarged elliptical vascular bundles (EVBs) are connected to the panicle [77]. A study demonstrated that Cd was predominantly transported toward the panicle instead of other tissues at the panicle-initiation stage through the nodes and ultimately reached grains by positron-emitting <sup>107</sup>Cd tracer imaging system (PETIS) [50]. Node I, the uppermost node, is connected to both flag leaf and panicles. Yamaguchi et al. [77] found that Cd concentration was higher in node I than in blade, culm and panicle due to the accumulation of Cd. In addition, a low-affinity cation transporter (OsLCT1), which is highly expressed in the node I, also takes part in Cd transport to grains [16].

**4. Effects of culture managements on Fe/Zn and Cd/Pb accumulation** 

In rice seeds, Fe localizes to dorsal vascular bundle, aleurone layer and endosperm, and it localizes to the scutellum and vascular bundle of the scutellum of embryo [78]. Zn is distributed to all parts of the seed with a significantly high value for the aleurone layer and embryo [79]. Low Fe and Zn contents in rice are often restricted due to low available pools of Fe or Zn in soil. Enriching Fe or Zn concentration in grains through either fertilization or water management is referred to as agronomic biofortification, which is a short-term strategy for

Fe is abundant in mineral soil, but Fe deficiency still can occur in aerobic condition [80]. The major problem with Fe uptake is solubility. Fe in the soil (usually in the form of Fe2+, either chelated or as a sulfate salt) is easily converted to unavailable Fe3+ under aerobic condition. Thus, application of Fe as fertilizer is not an effective strategy for increasing rice seed Fe [81]. Otherwise, foliar application is a better option to overcome Fe deficiency, increasing grains Fe and its bioavailability in rice [82]. In contrast, as soil changes from aerobic to anaerobic conditions after flooding, Fe-oxides are dissolved when the Fe3+ is reduced to Fe2+, which weakens the oxide stability and increases its water solubility [83]. In fact, irrigation management in rice strongly influences soil redox potential, which affects the availability of Fe, so flooded soil

to some citrates and proteins [11].

**in rice grains**

complementing the breeding programs.

nearly always has sufficient Fe for rice uptake [4].

Lead forms various complexes with soil components, and only small parts of the lead present as these complexes in the soil solution are phytoavailable [59]. In soil, Pb may occur as a free metal ion or make complexes with inorganic constituents, such as HCO<sup>3</sup> − , CO<sup>3</sup> 2−, SO4 2−, and Cl− . Pb also may exist as organic ligands with amino acids, fulvic acids, and humic acids [59]. Lead behavior in soil is mainly controlled by factors, such as pH [60], redox conditions [61], cation-exchange capacity, and organic and inorganic ligand levels [62]. Once adsorbed onto the rhizoderm roots surface, Pb may enter into the root passively, followed by translocating water streams while the mechanism by which Pb enters into root at the molecular level is still unknown. It is suggested that Pb enters into the root through several pathways, and a particular pathway is through ionic channels [59]. Several authors have demonstrated that Ca2+-permeable channels are the main pathway by which Pb enters into root [63, 64]. Ca2+ from rhizosphere will compete with Pb2+ for common uptake position [25].

### **3. Translocation of Fe/Zn and Cd/Pb in rice**

Following uptake by root, Fe, Zn and Cd are transported to shoot via xylem and phloem, where a large amount of vascular bundles exist [11]. This radial transport system includes symplasmic and apoplasmic pathways, but the former pathway is predominantly utilized as a result of impediment by Casparian strips occurring in apoplasmic pathway [65]. After Fe(II)-NA formation in the cytosol, Fe(II)-NA is transported to xylem and exchanges NA with citrate, transforming into Fe(III)-citrate preferentially [39, 40]. Fe in the xylem is largely in the form of Fe-citrate and then allocated to all leaves, whereas Fe in the phloem is mainly bound to DMA, citrate, and proteins [11]. The translocation of citrate from root pericycle cells to xylem is mediated by ferric reductase defective 1-like transporter OsFRDL1 [40].

Transportation of metals from plant root to shoot requires movement through the xylem [66] and is probably driven by transpiration [67]. Fe, Zn, Pb, and their chemical forms are in rice xylem and phloem saps, and phloem loading is the first step. OsYSL2 plays a role in Fe distribution in the phloem, localizing at the plasma membrane and is responsible for Fe(II)-NA or Mn(II)-NA transport, but not for Fe(III)-DMA transport [68]. Nozoye et al. [32] proposed that the NA efflux transporters (ENA1/2) are responsible for the efflux of NA into xylem or intracellular compartments in order to redistribute Fe. Under Fe deficiency, both *OsYSL2* and *ENA1* are strongly induced [68, 69]. In addition to transporter OsYSL2, OsYSL15 is considered to transport Fe(III)-DMA for phloem trafficking and expressed in the phloem companion cells [6, 35, 43]. Thereafter, Fe is delivered to grains via phloem in forms of Fe(III)-DMA or binds to some citrates and proteins [11].

After influx into cytosol, Cd is sequestered into the vacuoles via transporter OsHMA3 [56] and transiently stored in the form of complexes [15]. This pathway decreases Cd mobility in the cytosol and translocation from root to shoot [15, 57]. Enhancement of OsHMA3 activity has been found to increase storage of Cd in root and decrease the transport of Cd to the shoot and the final accumulation of Cd in rice grains [23]. *OsHMA3* is mainly expressed in root [24], and OsHMA3 localized at tonoplast belongs to P1B-ATPases [58]. A high rate of root-to-shoot transport and subsequent accumulation in the grains of 107Cd, which was administered from

Lead forms various complexes with soil components, and only small parts of the lead present as these complexes in the soil solution are phytoavailable [59]. In soil, Pb may occur as a free

. Pb also may exist as organic ligands with amino acids, fulvic acids, and humic acids [59]. Lead behavior in soil is mainly controlled by factors, such as pH [60], redox conditions [61], cation-exchange capacity, and organic and inorganic ligand levels [62]. Once adsorbed onto the rhizoderm roots surface, Pb may enter into the root passively, followed by translocating water streams while the mechanism by which Pb enters into root at the molecular level is still unknown. It is suggested that Pb enters into the root through several pathways, and a particular pathway is through ionic channels [59]. Several authors have demonstrated that Ca2+-permeable channels are the main pathway by which Pb enters into root [63, 64]. Ca2+ from

Following uptake by root, Fe, Zn and Cd are transported to shoot via xylem and phloem, where a large amount of vascular bundles exist [11]. This radial transport system includes symplasmic and apoplasmic pathways, but the former pathway is predominantly utilized as a result of impediment by Casparian strips occurring in apoplasmic pathway [65]. After Fe(II)-NA formation in the cytosol, Fe(II)-NA is transported to xylem and exchanges NA with citrate, transforming into Fe(III)-citrate preferentially [39, 40]. Fe in the xylem is largely in the form of Fe-citrate and then allocated to all leaves, whereas Fe in the phloem is mainly bound to DMA, citrate, and proteins [11]. The translocation of citrate from root pericycle cells to xylem is mediated by ferric reductase defective 1-like transporter

Transportation of metals from plant root to shoot requires movement through the xylem [66] and is probably driven by transpiration [67]. Fe, Zn, Pb, and their chemical forms are in rice xylem and phloem saps, and phloem loading is the first step. OsYSL2 plays a role in Fe distribution in the phloem, localizing at the plasma membrane and is responsible for Fe(II)-NA or Mn(II)-NA transport, but not for Fe(III)-DMA transport [68]. Nozoye et al. [32] proposed that the NA efflux transporters (ENA1/2) are responsible for the efflux of NA into xylem or intracellular compartments in order to redistribute Fe. Under Fe deficiency, both *OsYSL2* and *ENA1* are strongly induced [68, 69]. In addition to transporter OsYSL2, OsYSL15 is considered to transport Fe(III)-DMA for phloem trafficking and expressed in the phloem companion cells

− , CO<sup>3</sup>

2−, SO4

2−, and

a culture solution, was observed in OsHMA3-depleted rice lines [51].

rhizosphere will compete with Pb2+ for common uptake position [25].

**3. Translocation of Fe/Zn and Cd/Pb in rice**

Cl−

50 Rice Crop - Current Developments

OsFRDL1 [40].

metal ion or make complexes with inorganic constituents, such as HCO<sup>3</sup>

The Zn chemical forms in xylem sap are free ions and Zn partially bound to unidentified chelators [40], while the Zn in phloem sap was dominantly bound to NA [70]. Obata and Kitagishi [71] indicated that some Zn in the xylem (transpiration stream) is transferred to the phloem at the vegetative nodes in addition to the mobilization of Zn from mature leaves in rice. Xylem transfer cells have been found in rice vegetative nodes, and localized metal transporters may support the active xylem-to-phloem transfer of xylem sap Zn [72] and Cd [73].

After entered into root cells, part of Cd present as Cd-phytochelatin (Cd-PC) complexes are sequestered in the vacuoles, and the others are transported to xylem mediated by OsHMA2 transporter in root pericycle cells [11, 74, 75]. In the phloem, Cd primarily bounds to specific proteins and slightly to thiol-compounds [76]. In contrast to Fe translocation that is mainly derived from leaves by remobilization, xylem-to-phloem transfer system of Cd mainly occurs at the nodes [50]. In rice nodes, the diffuse vascular bundles (DVBs) that encircle the enlarged elliptical vascular bundles (EVBs) are connected to the panicle [77]. A study demonstrated that Cd was predominantly transported toward the panicle instead of other tissues at the panicle-initiation stage through the nodes and ultimately reached grains by positron-emitting <sup>107</sup>Cd tracer imaging system (PETIS) [50]. Node I, the uppermost node, is connected to both flag leaf and panicles. Yamaguchi et al. [77] found that Cd concentration was higher in node I than in blade, culm and panicle due to the accumulation of Cd. In addition, a low-affinity cation transporter (OsLCT1), which is highly expressed in the node I, also takes part in Cd transport to grains [16].
