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RESEARCH & INTERPRETATION

Modern Biotechnology as an Integral Supplement to Conventional Plant Breeding

The Prospects and Challenges

USDA–ARS, Northern Crop Science Laboratory, Fargo, ND 58105

^* Corresponding author (prem.jauhar{at}ndsu.edu)

It would be an unsound fancy to expect that things which have never yet been done can be done except by methods which have never been tried.

—Sir Francis Bacon

	ABSTRACT

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
The art of plant breeding was developed long before the laws of genetics became known. The advent of the principles of genetics at the turn of the last century catalyzed the growth of breeding, making it a science-based technology that has been instrumental in substantial improvements in crop plants. Largely through exploitation of hybrid vigor, grain yields of several cereal crops were substantially increased. Intervarietal and interspecific hybridizations, coupled with appropriate cytogenetic manipulations, proved useful in moving genes for resistance to diseases and insect pests from suitable alien donors into crop cultivars. Plant improvement has been further accelerated by biotechnological tools of gene transfer, to engineer new traits into plants that are very difficult to introduce by traditional breeding. The successful deployment of transgenic approaches to combat insect pests and diseases of important crops like rice (Oryza sativa L.), wheat (Triticum aestivum L.), maize (Zea mays L.), barley (Hordeum vulgare L.), and cotton (Gossypium hirsutum L.) is a remarkable accomplishment. Biofortification of crops constitutes another exciting development in tackling global hunger and malnutrition. Golden Rice, genetically enriched with vitamin A and iron, has, for example, the real potential of saving millions of lives. Yet another exciting application of transgenic technology is in the production of edible vaccines against deadly diseases. How these novel approaches to gene transfer can effectively supplement the conventional breeding programs is described. The current resistance to acceptance of this novel technology should be assessed and overcome so that its full potential in crop improvement can be realized.

Abbreviations: Bt, Bacillus thuringiensis • FHB, Fusarium head blight • fl-GISH, fluorescent genomic in situ hybridization • GM, genetically modified • PDR, pathogen-derived resistance • QPM, quality protein maize

	INTRODUCTION

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
A PARAMOUNT FACTOR in the evolution of human civilizations was a steady supply of food. Food production is therefore the oldest profession of humanity. The processes of crop cultivation and selection were an integral part of human activity. Although early "plant breeding" was developed essentially as an art, its scientific basis became well established with the rediscovery of laws of genetics at the turn of the last century. And with the application of the principles of genetics to crop improvement, the period from 1930 to 1970 witnessed a phenomenal increase in crop yields, particularly of cereal grains (Khush, 1999). Largely through exploitation of hybrid vigor, maize, pearl millet [Pennisetum glaucum (L.) R. Br.], and sorghum [Sorghum bicolor (L.) Moench] registered a considerable increase in grain yields during 1965 to 1990 (Khush, 2001; Jauhar and Hanna, 1998; Jauhar et al., 2006). Improved wheat and rice varieties with reduced height developed by incorporating dwarfing genes in the 1960s and 1970s launched the famous Green Revolution in Asia (Khush, 1999; see also Jauhar, 2006). Around the same period, the advent of the tools of cytogenetics greatly facilitated wide hybridization and chromosome-mediated gene transfers from wild species into crop plants (Jiang et al., 1994; Jauhar, 1993, 2003a; Friebe et al., 1996; Fedak, 1999). Chromosome engineering methodologies, based on the manipulation of pairing control mechanisms and induced translocations, were, for example, applied to transfer into wheat cultivars specific disease and pest resistance genes of alien origin (Ceoloni and Jauhar, 2006; Jauhar, 2006; Mujeeb-Kazi, 2006). Thus, cytogenetic tools were instrumental in the genetic improvement of several crop plants, particularly cereals.

The development, in the last decade and a half, of novel tools of direct gene transfer, collectively termed genetic engineering, has added new dimensions to breeding efforts. Genetic engineering is defined as any nonconventional tool aimed at mobilizing specific genetic information from one member of the plant kingdom (or, for that matter, any organism) into another. (Any nonconventional tool of today may of course become conventional in the future.) These asexual techniques of biotechnology help engineer into plants new characters that are otherwise very difficult to introduce by conventional breeding. The molecular techniques, including the recombinant DNA methods, involve the introduction of well-characterized alien DNA into the recipient plant cells of regenerable embryogenic calli to permanently transform the plant's genetic makeup. Genetic engineering has the potential to accelerate crop improvement and has already yielded encouraging results (e.g., Jauhar and Chibbar, 1999; Muthukrishnan et al., 2001; Repellin et al., 2001; Dahleen et al., 2001; Janakiraman et al., 2002; Patnaik and Khurana, 2003; Wesseler, 2003; Sharma et al., 2004). Value-added traits engineered into crop plants include resistance to fungal and viral diseases, and biofortification of their nutritional status (Jauhar and Khush, 2002; Schubert et al., 2004; Bajaj and Mohanty, 2005). However, as with any new technology, genetic engineering is encountering resistance from some sections of the public. There are concerns about the potential adverse impact of genetically modified (GM) foods or organisms on human health and the environment. Although some of the public concerns may not be well founded (Jauhar and Khush, 2002), they will need to be properly addressed. To alleviate some of these fears, perceived or real, we will need to do a better job of informing the public. Some of these issues are raised in this paper. The main objective of this article is to highlight the potential of the transgenic technology as a supplementary tool to plant breeding and to discuss its prospects and the challenges that lie ahead.

	Plant Breeding as an Art: The Man-Made Evolution

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
Plant breeding deals with the exploitation of existing genetic variability and the generation, manipulation, and combination of new variability into plant forms most useful to humans. The genetic and cytogenetic basis of plant breeding is now well understood. We must remember, however, that the art of plant breeding was developed long before the principles of genetics and cytogenetics became known. Even before Mendel (1822–1884), plant hybridizers, such as Kölreuter, Knight, Gärtner, and others, had produced improved strains of crop plants. Several thousand years ago, the early "plant breeders," although essentially unschooled, intuitively looked for, skillfully recovered, and successfully propagated genetic variants or recombinants that exhibited desirable traits.

Working under a myriad of cultural contexts, these early selectionists turned the relatively useless weedy species into useful crop plants that sustain us today. The exercise of a rigorous process of screening and selection brought about modification in the genetic makeup of the plant forms. Thus, plant breeding is essentially an exercise at manipulating plant genetic material to humanity's best advantage. An advance in yield, for example, must involve appropriate changes in the genetic constitution of the crop plant in question. In this regard, plant breeding is nothing but human-made evolution, which has brought about substantial increases in crop yields, for example, quantity and quality of cereal grains.

	Genetics and the Scientific Foundation of Plant Breeding: Some Accomplishments

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
With the discovery (or rediscovery) of the laws of genetics at the turn of the 20th century, the process of plant breeding was accelerated considerably and became a science-based technology. The principles of genetics found immediate application in crop improvement, as illustrated by a few examples given below.

Hybrid Vigor: A Boon to Plant Breeders
Grain yields of the major cereal crops, viz., wheat, maize, sorghum, and pearl millet, increased steadily since 1930, primarily because of genetic improvement of these crops (Duvick, 1984; Fehr, 1984). Largely because of exploitation of hybrid vigor, the maize yields in the United States registered a phenomenal increase from 1966 kg ha^–1 in 1930 to 4841 kg ha^–1 in 1982 (USDA's "Agricultural Statistics"). World maize yields increased at the rate of 0.7% per year during the period 1982 to 1990 (Duvick, 1992). Exploitation of hybrid vigor continues to be the most appropriate means to increase grain yields relatively rapidly (Vasal et al., 2006). Heterosis breeding has also significantly increased the grain yields of pearl millet (Jauhar, 1981; Jauhar and Hanna, 1998; Jauhar et al., 2006), sorghum (Reddy et al., 2006), and rice (Brar and Khush, 2006). It is remarkable that in 2001 more than 70 hybrids were under cultivation on 6 million hectares of the total 10 million hectares of pearl millet area in India (Jauhar et al., 2006).

The Dawn of Green Revolution
Improved, high-yielding varieties of wheat and rice developed in the 1960s and 1970s launched the Green Revolution in Asia. A product of an unprecedented international effort, the Green Revolution is certainly one of the most important accomplishments of the 20th century. Breakthroughs in wheat and rice yields were achieved with the development of semidwarf varieties characterized by lodging resistance and N responsiveness (Swaminathan, 1993; Borlaug, 1998; Khush, 1999, 2001). Thus, the introduction of dwarfing genes by conventional breeding revolutionized both wheat and rice production in Asia, averting mass-scale starvation.

Nutritional Enhancement of Food Crops
In addition to grain yields, improving nutritional quality of food crops such as cereals is an important goal because 842 million people worldwide are malnourished according to most recent reports (www.fao.org/english/newsroom/news/2003/24779-en.html; verified 10 May 2006). Nutritional upgrading of maize was achieved mainly by conventional breeding, and hybrid initiative proved to be exceedingly important for successful development of quality protein maize (QPM) (Vasal, 2002; Vasal et al., 2006). The development of QPM was an important scientific breakthrough, whose fruits are being reaped by several developing countries. Sorghum cultivars with high protein digestibility as well as high lysine content are being developed and offer prospects for combining high nutritional quality and grain yield (Oria et al., 2000; Reddy et al., 2006).

Harnessing Apomixis for Perpetuating Hybrid Vigor
As stated above, heterosis breeding has dramatically increased grain yields of maize and pearl millet. In the USA, hybrid maize was introduced in the mid-1930s and within 10 yr almost all maize fields were planted with hybrids. Similarly, pearl millet hybrids were widely accepted in India. However, the main problem with such hybrids is that their seed has to be produced year after year for distribution to growers and farmers. Introduction of apomixis in hybrids would enable them to maintain heterozygosity through seed production, thereby perpetuating hybrid vigor, and eliminating the need to produce commercial hybrids every year. Some researchers have attempted to transfer the apomictic mode of reproduction from eastern gama grass (Tripsacum dactyloides L.) into maize, although with limited success (Savidan, 2000; Kindinger, pers. comm., 2004; Bicknell and Koltunow, 2004). Attempts to make rice apomictic have also been underway. Both mutagenesis (see Brar and Khush, 2006) and molecular tools (Bennett et al., 2001) have been employed to introduce apomixis in rice, but with limited success so far.

Crosses between synthetic tetraploid pearl millet and its hexaploid, apomictic relative, Pennisetum squamulatum Fresen. have shown a high level of expression of apomixis in the progeny (Ozias-Akins et al., 1998), but producing a completely apomictic pearl millet remains problematic (Ozias-Akins et al., 2003; Akiyama et al., 2004; Jauhar et al., 2006). Fixation of apomixis would help clone superior hybrids and result in the decrease of seed production cost, benefiting resource-poor farmers. Only some progress in this direction has been made (Koltunow and Tucker, 2003) and ongoing research in this area may yield dividends.

	Cytogenetic Tools in Plant Breeding

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
The formulation of the laws of inheritance by Mendel in 1865 led to the foundation of genetics, the science of heredity, although it was born belatedly in 1900 with the rediscovery of Mendel's work. Soon afterward, the elucidation of parallelism between chromosome behavior during the course of meiosis and of genes during breeding experiments forged an alliance between cytology and genetics, resulting in the hybrid science cytogenetics. The establishment of the chromosome theory of heredity put chromosomes at the center of life sciences. And cytogenetics has already had a tremendous impact on agriculture, biology, and medicine.

General Applications in Plant Improvement
The tools of cytogenetics have played a pivotal role in accelerating crop improvement. Thus, the understanding of chromosome pairing and its regulation and manipulation, genome relationships between and within plant species, polyploidy, aneuploidy and haploidy—to name a few—have greatly aided plant breeding. The haploidy technique, for example, is very useful. The haploid-derived homozygous lines provide a rapid means of achieving homozygosity, thereby accelerating breeding programs. An understanding of genomic affinities facilitates the planning of effective hybridization programs designed to transfer desired genes or gene clusters from alien species into otherwise superior cultivars of crop plants. Chromosome engineering, involving chromosome-pairing manipulation in polyploid crop plants, leads to fruitful recombination of entire genomes, parts of genomes or chromosome segments resulting in superior cultivars. This form of plant breeding involves, in essence, genomic reconstructions to meet human needs.

Cytogenetic Architecture of a Crop Plant: Its Bearing on Breeding
A full understanding of the genomic constitution of a crop and its nature of polyploidy, if any, is very valuable in planning an effective breeding strategy. Thus, hexaploid bread wheat (T. aestivum L., 2n = 6x = 42; AABBDD) and tetraploid durum wheat (T. turgidum L., 2n = 4x = 28; AABB) are natural hybrids, having resulted from hybridization between related diploid wild species. Durum wheat is the forerunner of bread wheat. Although the constituent genomes of polyploid wheats are genetically similar or homoeologous, the Ph1 gene in the long arm of chromosome 5B ensures diploid-like pairing, that is, pairing only between homologous partners (Riley and Chapman, 1958; Sears and Okamoto, 1958).

The role of Ph1 in enforcing homologous chromosome pairing is best illustrated by studying pairing in wheat haploids with and without Ph1. These haploids with half the chromosome complement do not have their homologous partners to pair with and hence show no or very little pairing (Fig. 1A ). Thus, Ph1 haploids show mostly univalents, although some superficial pairing, termed chromosome "dating" (Jauhar, 1990, p. 528), may be observed (Fig. 1A). However, in the absence of Ph1, the wheat haploids show extensive homoeologous pairing (Fig. 1B) because control on pairing is relaxed. It is remarkable that one gene can make such a difference in the pattern of chromosome pairing. Such a disciplined pairing ensures disomic inheritance and confers meiotic regularity and reproductive stability to polyploid wheats. Oat (Avena sativa L.) is another important allohexaploid cereal with a genomic makeup similar to that of bread wheat and also the genetic regulation of chromosome pairing (Rajhathy and Thomas, 1972; Jauhar, 1977) similar to the Ph1 system of wheat. Appropriate cytogenetic manipulation of the chromosome pairing control mechanism helps transfer alien genes into wheat, as stated below.

View larger version (74K): [in this window] [in a new window]   Fig. 1. Effects of Ph1 on chromosome pairing at meiosis in bread wheat haploids (2n = 3x = 21; ABD genomes). Pollen mother cells (PMCs) with and without Ph1 are shown. (A) PMC with Ph1 showing 1 rod II and 19 I. Such a superficial pairing in the presence of Ph1 has been called "chromosome dating." (B) PMC of a ph1b-haploid showing 6 II (3 ring and 3 rod bivalents) + 9 I. Note extensive homoeologous pairing because of the absence of Ph1. Interestingly, one gene can make such a difference. (Fig. 1B from Jauhar et al., 1991).

Wild relatives of crop plants are also reservoirs of genes for superior traits, which can be incorporated into crop species via wide hybridization. Cytogenetic techniques are useful in bringing about such alien gene transfers. As early as 1956, Ernest Sears translocated onto wheat chromosome 6B a small segment from Aegilops umbellulata Zhuk. that carried a gene for resistance to leaf rust (caused by Puccinia triticina Eriks.) (Sears, 1956). This pioneering work heralded an era of utilization, by chromosome manipulation, of wild gene resources for improvement of crop plants. During the last few decades, interspecific and intergeneric hybridization have been widely used to develop wheat cultivars with improved agronomic performance, pest tolerance, and high yields (Friebe et al., 1996; Jauhar and Chibbar, 1999). Such alien transfers following wide hybridization with donor species coupled with manipulation of chromosome pairing, by suppressing or removing the pairing regulator Ph1, are called chromosome-mediated gene transfers that have resulted in several commercial cultivars with genes of alien origin. And such cultivars have been widely accepted for human consumption.

Chromosome Pairing: The Key to Genetic Enhancement
Crop diseases and pests pose a serious threat to global food security. Close relatives of crop species are rich reservoirs of genes for resistance to pathogens and insect pests and those genes can be transferred to crop cultivars through hybridization. Pairing among chromosomes of parental species in their hybrids is the key to gene transfer across species. Cytogenetic manipulations for suppressing the Ph1-pairing regulation of polyploid wheats and oat, for example, would be necessary to bring about the desired chromosome pairing and hence alien gene transfers into these crop species. The methods of circumventing the Ph1 system include: (i) using substitution lines lacking chromosome 5B and hence Ph1; (ii) suppressing the activity of the Ph1 or Ph1-like genes by crossing polyploid cereal crops with appropriate genotypes of wild donors that partially or fully inactivate the regulatory genes; and (iii) using the ph1bph1b mutant of wheat as the female parent in crosses with the wild donor species (see Jauhar, 2006). Such cytogenetic manipulations, including the suppression of the Ph1 system, for recombining desirable alien chromatin into wheat were termed chromosome engineering (Sears, 1972, 1981). Essentially similar cytogenetic manipulations to effect gene transfer can be done in hexaploid oat (Jellen and Leggett, 2006). Some examples are given below.

Fusarium head blight (FHB) or scab, caused by the fungus Fusarium graminearum Schwabe, is a devastating fungal disease of durum wheat, an important cereal used for human consumption worldwide. Current durum cultivars have very little FHB resistance; therefore, we transferred resistance from wild relatives into durum germplasm. By crossing a durum wheat 5D(5B) disomic substitution with a diploid wheatgrasses, Lophopyrum elongatum (Host) Á. Löve (2n = 2x = 14; EE genome), we realized substantial chromosome pairing among the parental chromosomes in the intergeneric hybrids and transferred alien chromatin into the durum genome (Jauhar and Peterson, 2000). We adopted the same approach for transferring chromosome segments from another diploid wheatgrass, Thinopyrum bessarabicum (Savul. & Rayss) Á. Löve (2n = 2x = 14; JJ genome), into the durum genome (Fig. 2 ). In the presence of Ph1, the intergeneric hybrids (Fig. 2A) showed very little pairing, if at all (Fig. 2B). However, in the absence of Ph1, extensive homoeologous pairing occurred (Fig. 2C). Multicolor fluorescent genomic in situ hybridization revealed some pairing between durum and grass chromosomes (Fig. 2D).

View larger version (56K): [in this window] [in a new window]   Fig. 2. Durum wheat x Th. bessarabicum hybrid, and its chromosome pairing in the presence and absence of Ph1. (A) Spikes of parental species—durum wheat (left), Th. bessarabicum (right)—and their intergeneric hybrid (center). (B) PMC of the triploid intergeneric hybrid (2n = 3x = 21; ABJ genomes) with Ph1 showing 21 I. Note complete absence of pairing because of the presence of Ph1. (C) PMC of the intergeneric hybrid durum Langdon (LDN) disomic substitution 5D (5B) x Th. bessarabicum, showing 2 III [one V-shaped (arrow) and one frying pan–shaped (arrowhead)] + 4 II + 7 I. Note extensive homoeologous pairing, a welcome feature from the breeding standpoint. Some pairing takes place between the wheat and grass chromosomes (see Fig. 2D). (D) Same hybrid as in (C) with meiotic chromosomes after fluorescent genomic in situ hybridization when the durum wheat A-genome (colored green) was probed with Triticum urartu DNA labeled with FITC, the J-genome was probed with Th. bessarabicum DNA labeled with Rhodamine (colored red), and the remaining chromosomes counterstained with DAPI (colored blue) belong to the B-genome with one D-genome chromosome from 5D. Note wheat–grass pairing (A-J pairing).

Recent improvements in methods of characterizing alien chromatin introgressed into crop genomes are facilitating crop improvement by chromosome engineering. Thus, fl-GISH is very helpful in characterizing such introgression products (Jauhar et al., 2004; Ceoloni and Jauhar, 2006; Jauhar, 2006). These techniques also help retain the desired alien chromosome segments while eliminating the undesirable ones.

Genotype-Induced Homoeologous Chromosome Pairing: Breeding for Cold Tolerance
Certain genotypes of wild grasses are known to suppress the activity of Ph1 in their hybrids with wheat, thereby accelerating homoeologous chromosome pairing and hence alien gene transfers. Aegilops speltoides Tausch, for example, has been known to induce high homoeologous pairing (Dvoák, 1972). Genotype-induced interference with the regulatory mechanism is an efficient means of promoting homoeologous chromosome pairing and we have used this attribute in our wheat improvement program. Tetraploid crested wheatgrass, Agropyron cristatum (L.) Gaertner (2n = 4x = 28; PPPP), is a valuable source of genes for drought and cold tolerance that could be transferred into bread wheat via hybridization. In pentaploid hybrids (2n = 5x = 35; ABDPP) (Fig. 3A ) between bread wheat and crested wheatgrass, we observed substantial chromosome pairing (Fig. 3B) induced by the grass genotype, offering the possibility of alien gene transfer into the wheat chromosome complement (Jauhar, 1992).

View larger version (37K): [in this window] [in a new window]   Fig. 3. Intergeneric hybrid (2n = 5x = 35; ABDPP genomes) between bread wheat (2n = 6x = 42; AABBDD) wheatgrass, Agropyron cristatum (2n = 4x = 28; PPPP), and genotype-induced homoeologous chromosome pairing. (A) Bread wheat var. Fukuhokomugi (left), crested wheatgrass (right), and their hybrid (center). Note the intermediate phenotype of the hybrid. (B) Meiotic metaphase in the hybrid showing 1 III + 10 II + 12 I. The genotype of the grass parent induces higher pairing than is expected based on chromosome homology.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Intergeneric F1 hybrids between bread wheat cultivar Fukuhokomugi (Fuko) and decaploid tall wheatgrass, Thinopyrum ponticum var., ‘Alkar’ (2n = 10x = 70). This hybridization offers prospects for breeding "perennial" wheat. (A) Seeds of the female parent Fuko (top row), intergeneric F1 hybrid (middle row), and the male parent Alkar (bottom row). Note large size of the seeds (with husk) of the hybrid. (B) Seeds of Fuko (top row), dehusked seeds of intergeneric F1 hybrid (middle row), and dehusked seeds of the grass parent Alkar (bottom row). (From Jauhar, 1995).

Cytogenetic tools help transfer chromatin from one crop species into another. Using oat x maize crosses, for example, maize chromatin has been added to the oat genome (Riera-Lizarazu et al., 2000; Kynast et al., 2002). Through hybridization with other cereals, followed by chromosome elimination, maize offers an excellent mechanism for producing haploids of cereal crops (e.g., Jauhar, 2003b), thereby facilitating their cytogenetic manipulation and enhancement.

Gametocidal Chromosomes and Induction of Translocations in Hybrids
The use of chromosome breaking action of some alien chromosomes offers a novel technique of inducing crop–alien chromosome translocations. Certain Aegilops chromosomes become gametocidal and induce chromosome breakage when introduced into wheat (Endo, 2003). Thus, by introducing these gametocidal chromosomes into a wheat–alien addition or substitution line, random wheat–alien translocations were recovered in the selfed progenies. Shi and Endo (1999) were able to induce structural changes in barley chromosomes added to wheat utilizing gametocidal chromosomes derived from Ae. cylindrica. Using such a gametocidal system, Masoudi-Nejad et al. (2002) transferred rye chromosome segments in wheat.

	Modern Biotechnology: A Means of Genetic Enrichment of Crop Plants

TOP NOTES ABSTRACT INTRODUCTION Plant Breeding as an... Genetics and the Scientific... Cytogenetic Tools in Plant... Modern Biotechnology: A Means... Biotechnology and Human Health Novel Applications of Transgenic... Resistance to Acceptance of... Modern Biotechnology as a... Conclusion and Perspectives REFERENCES

 
Conventional plant breeding (Duvick, 1984; Jauhar, 1988; Khush, 1999, 2001), sometimes assisted by marker-assisted selection (Dubcovsky, 2004; Lapitan and Jauhar, 2006), and wide hybridization coupled with manipulation of chromosome pairing (Friebe et al., 1996; Fedak, 1999; Jauhar and Chibbar, 1999; Jauhar, 2003a) has clearly been instrumental in producing superior crop cultivars. However, these procedures are time consuming. Conventional breeding may take 10 or more years to transfer a trait from a donor species into a crop cultivar. Wide hybridization is undoubtedly an effective means of incorporating desirable alien genes into crop cultivars, but it has several limitations. It results in transmission of unwanted alien chromosomes and adverse genetic interactions can lead to sterility. Other efficient means of gene transfer have therefore been explored.

Dawn of Genetic Engineering: Direct Gene Transfers
Recent biotechnological tools of direct gene transfer help engineer into plants new characters that are otherwise very difficult to transfer by breeding programs. The world's major crops are being transformed by direct DNA delivery by microprojectile bombardment and other methods of direct gene transfer (e.g., Jauhar and Chibbar, 1999; Dahleen et al., 2001; Muthukrishnan et al., 2001; Patnaik and Khurana, 2001; Repellin et al., 2001; Gelvin, 2003; Sharma et al., 2003; Sahrawat et al., 2003; Altpeter et al., 2005).

Prerequisites for Successful Genetic Transformation
The main prerequisites for efficient genetic transformations are: (i) in vitro regeneration; (ii) a DNA delivery system; and (iii) functional introduced DNA.

In Vitro Regeneration
An efficient in vitro regeneration system must be standardized before introducing exogenous DNA into single cells. A major limitation to cereal transformation has been the lack of an efficient in vitro regeneration by somatic embryogenesis. Protoplasts prepared from immature embryo-derived suspension cultures of pearl millet, for example, were among the first cereal cells that were shown to possess totipotency (Vasil and Vasil, 1980). Soon afterward, regeneration procedures for immature embryos, young inflorescences, anthers, and shoot apices were developed (Vasil, 1987) and most of these explant tissues or cells are used for transformation (Jauhar and Chibbar, 1999; Repellin et al., 2001; Cho et al., 2004). Thus, standardization of a suitable regeneration protocol of durum wheat from scutellar cells (Bommineni and Jauhar, 1996) facilitated the production of transgenic durum (Bommineni et al., 1997). Since then there have been several reports of durum transformation (He et al., 1999; Pellegrineschi et al., 2002) that allowed incorporation of value-added traits into this important cereal. Because regeneration efficiency varies among species and often among cultivars of the same species, genotype-specific regeneration and transformation protocols will need to be established for best results (Machii et al., 1998; Repellin et al., 2001).

DNA Delivery System
Gene transfers in plants have been achieved through electroporation (Shillito, 1999), polyethylene glycol treatment (Funatsuki et al., 1995), particle bombardment (Altpeter et al., 2005), and Agrobacterium-mediated methods (Komari and Kubo, 1999; Gelvin, 2003), among others. The first transgenic cereals were produced from rice and maize protoplast cultures into which exogenous DNA was introduced by electroporation (Shimamoto et al., 1989) or by polyethylene glycol treatment (Zhang and Wu, 1988). The most commonly used method of DNA delivery into plant tissues is particle bombardment or biolistics, which facilitates simultaneous introduction of several genes (Vasil et al., 1992). Chen et al. (1998), for example, achieved the co-introduction of 13 genes into rice. Microprojection has become an important method for cereal transformation (Jauhar and Chibbar, 1999) and for study of gene expression and regulation (Klein and Jones, 1999).

Introduced DNA to Be Functional
For successful commercialization of transgenic crops, a stable and consistent expression of the gene(s) of interest is necessary (Kathuria et al., 2003). Several cases of transgene silencing have been reported in plants (Yu and Kumar, 2003), both monocots (Iyer et al., 2000) and dicots (Fu et al., 2005). Mechanisms of gene silencing are under study to learn how to avoid this phenomenon.

Genetic Transformation: A Rapid Tool for Crop Improvement
Traditional breeding is generally notoriously slow in transferring a desired trait into an otherwise superior crop cultivar. The time needed to transfer a desired gene into a crop plant depends on the source of the gene and the evolutionary distance of that source to the recipient crop plant. If the gene source is a landrace or a related species, forming a primary gene pool with the crop species in question, the gene transfer may take 5 to 8 yr if not longer. Less related wild species belonging to the secondary or even tertiary gene pool may be rich reservoirs of genes for agronomic traits like disease or pest resistance, but to transfer such genes into crop cultivars may take 10 to 15 yr or even longer, if they are at all possible. Pre- and post-fertilization barriers may impede sexual hybridization between the donor and the crop species and compound the problem of alien gene transfers. In some cases, it may not even be possible to incorporate a certain trait by conventional means because a suitable donor may not be available or, if available, it may not be possible to hybridize the donor species with the crop plant.

Genetic engineering offers an excellent tool for asexually inserting a well-characterized gene(s) of unrelated organisms into plant cells, which on regeneration produce full plants with the inserted gene(s) integrated into their genome. This process may take less than a year to about 18 mo in some cases, thus accelerating the process of genetic improvement of crop plants. Moreover, this exciting technology allows access to an unlimited gene pool without the constraint of sexual compatibility. Genetic transformation by microprojectile bombardment has, for example, been demonstrated in wheat and other cereals (Jauhar and Chibbar, 1999; Dahleen et al., 2001; Jauhar and Khush, 2002; Altpeter et al., 2005). Some instances of this rapid crop improvement are described below.

Resistance to Insect Pests
Numerous insect pests attack crop plants and cause enormous losses, threatening global food security. European corn borer [ECB, Ostrinia nubilalis (Hübner)], for example, causes a loss of up to 2000 million dollars annually in the USA alone (Hyde et al., 1999). Resistance breeding by conventional means is cumbersome and fraught with uncertainty. To breed a corn cultivar with resistance or even partial resistance to ECB may well take 10 to 15 yr by traditional breeding, provided a suitable resistance donor is available. Thus, through 12 yr of breeding, Syngenta, a Swiss agrochemical company, was able to produce a corn cultivar with only 10% resistance to ECB (pers. comm., 2002). However, a gene from a soil-borne bacterium, Bacillus thuringiensis (Bt), when bioengineered into the corn genome, confers almost complete resistance to ECB. This is an efficient means of eliminating the pest damage and pesticide application without affecting grain yields. Thus, Bt-corn acquired the capacity of an efficient pesticide—a biopesticide. It took Syngenta only 5 yr to engineer the Bt gene into corn.

Scientists at the University of Minnesota estimated that farmers averaged several times greater returns on their investment by using Bt corn for insect control, compared to the use of a chemical insecticide (Ostlie et al., 1997). The Bt corn hybrids had 4 to 8% higher grain yields than standard hybrids when infested with ECB (Lauer and Wedberg, 1999). Moreover, Bt corn is beneficial to the environment and the Bt-induced insect resistance in corn is much safer to farmers and other field workers, compared with the use of a chemical insecticide. Based on safety data, the U.S. Environmental Protection Agency (EPA) authorized commercial planting of Bt corn varieties (Palevitz, 2001). Several transgenic crops with insecticidal genes have been introduced in temperate regions of the world (Sharma et al., 2004). Transgenic rice varieties resistant to yellow stem borer [Scirpophaga incertulas (Walker)] have been produced in India (Ramesh et al., 2004).

Because of its higher productivity and positive health effects through reduced pesticide use, Bt cotton has been commercialized aggressively especially in Asian countries like China (Huang et al., 2002a) and India (Whitfield, 2003). Carrière et al. (2003) found long-term regional suppression of pink bollworm [Pectinophora gossypiella (Saunders)] by Bt cotton. Bt rice has the potential to eliminate yield losses caused by lepidopteran insects, estimated at 2 to 10% of Asia's annual rice yield of 523 million tons (High et al., 2004). Field trials of transgenic rice suggested high tolerance of transgenic rice against yellow stem borer (Bashir et al., 2004). Most recently, an insect-resistant variety GM Xianyou 63 that was produced by inserting a Chinese-created B. thuringiensis gene, showed resistance to rice stem borer (S. incertulas) and leaf roller [Cnaphalocrocis exigua(Butler)] and is on the threshold of being released for commercial cultivation in China. This insect-resistant variety is reported to benefit small farmers because of higher crop yields and reduced use of pesticides, which is important for health reasons (Huang et al., 2005).

Resistance to Diseases
Various fungal, bacterial, and viral diseases pose a major threat to global food security. Conventional plant breeding offers a useful means of breeding disease-resistant cultivars, provided a reliable donor of resistance is available. Chromosome engineering through wide hybridization has been successfully employed to transfer specific disease resistance genes from alien donors into wheat cultivars (Friebe et al., 1996; Jauhar and Chibbar, 1999; Jauhar, 2006). However, resistance breeding through these techniques can be painfully slow. Tools of biotechnology offer great promise for accelerating this process.

Transgenic Approaches to Control Fungal and Bacterial Pathogens
Engineering with antifungal genes could help produce crop plants resistant to fungal pathogens (Datta and Muthukrishnan, 1999; Dahleen et al., 2001; Jauhar and Khush, 2002; Sahrawat et al., 2003). The role of chitinases in fungal protection has been documented in rice (Datta et al., 2001; Itoh et al., 2003). Genetic engineering has been employed to contain FHB, a ravaging disease of wheat (Anand et al., 2003, 2004). Some proteins, called defensins, are small cysteine-rich peptides with antimicrobial activity. Gao et al. (2000) demonstrated that an antifungal defensin isolated from alfalfa (Medicago sativa L.) displayed a strong activity against the fungus Verticillium dahliae Kleb. and that expression of this peptide in transgenic potato (Solanum tuberosum L.) conferred resistance to the fungus. Another alfalfa defensin was shown to inhibit the growth of the FHB pathogen Fusarium graminearum in vitro (Spelbrink et al., 2004), and we are attempting to express this peptide in transgenic wheat.

Dutch elm disease (DED) has destroyed more than 20 million elm trees (Ulmus procera Salisb.) in the UK over the last three decades, and more than 70% of the elms (Ulmus americana L.) in the USA have perished because of the DED fungal pathogen in the past 70 yr (Gartland, 2002). According to Professor K. Gartland of Scotland, genetically modified elms with resistance to the fungal pathogen "could help tackle damaged landscapes and ecosystems blighted by tree fungal diseases, such as Dutch elm disease and Chestnut blight, throughout the world" (The Independent, Scotland, 28 Aug 2001). This is an example of environmentally friendly biotechnology that could save the threatened landscapes and ecosystems worldwide (Gartland et al., 2002, 2003).

Transgenic approaches have also helped combat bacterial diseases. Thus, bacterial blight of rice [caused by Xanthomonas oryzae pv. oryzae (Ishiyama) Swings et al.] occurs throughout the rice-growing areas and causes serious yield losses. Xa21, a gene with broad-spectrum resistance to bacterial blight, was cloned through map-based cloning (Song et al., 1995). Tu et al. (2000) introduced the cloned gene into a widely grown rice variety IR72 with promising results. An attractive strategy to produce multiple pest tolerance is to stack up genes (Datta et al., 2002).

Pathogen-Derived Resistance to Viral Diseases
Transgenic technology also offers an excellent option to protect crop plants against devastating viral pathogens. Transformation of plants with nucleotide sequences derived from viral genomes has been shown to provide protection against the virus from which the sequences were derived. The evidence for such a pathogen-derived resistance (PDR) was provided by Powell-Abel et al. (1986), who demonstrated that transgenic tobacco plants expressing Tobacco mosaic virus (TMV) coat protein were resistant to the virus. Beachy et al. (1990) suggested that expression of a virus coat protein as a transgene in a plant confers resistance to the virus in direct proportion to the quantity of coat protein produced by the transformed plant. This novel technique opened up new avenues of controlling viral diseases (Lomonossoff, 1995; Bendahmane and Beachy, 1999) in crop plants and fruit trees. Rice yellow mottle virus (RYMV) is a serious viral disease causing enormous losses in rice yields. Because of lack of a conventional solution to this problem, a transgenic approach based on PDR was successfully employed to produce an RYMV-resistant rice variety (Pinto et al., 1999).

Transgenic wheat plants, engineered with the coat-protein gene of Wheat streak mosaic virus (WSMV) conferred protection against some WSMV strains (Sivamani et al., 2002). The PDR technology offers a promising means for inducing virus resistance in a variety of plants (Wesseler, 2003) including potato (Schubert et al., 2004). Coat-protein–mediated resistance has helped to control Papaya ring spot virus (PRSV) in papaya (Carica papaya L.) in Hawaii (Gonsalves, 1998; Ferreira et al., 2002) and the papaya industry was spared from disaster posed by PRSV (Gonsalves, 2003).

Tolerance to Abiotic Stresses
Abiotic stresses, including drought and salinity, are estimated to cause yield losses worldwide of more than 50% (Bray et al., 2000). Transgenic approaches offer an option to enhance drought (Abebe et al., 2003) and salt tolerance (Apse and Blumwald, 2002; Flowers, 2004). Although several abiotic stresses pose a limitation on yields of crop plants like wheat, drought is a major cause of yield loss and it is very difficult to breed drought tolerance through conventional breeding (Trethowan et al., 2001). Abebe et al. (2003) demonstrated that wheat engineered with the mtlD gene from Escherichia coli had improved tolerance to water stress and salinity. Garg et al. (2002) showed that overexpression of E. coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene provided increased tolerance to abiotic stress in rice, resulting in elevated capacity for photosynthesis under drought and low-temperature stress conditions. Thus, transgenic technology holds the possibility of engineering abiotic stress tolerance into cereal crops. Genetic engineering in conjunction with marker-assisted traditional breeding could help engineer plant tolerance to abiotic stresses (Vinocur and Altman, 2005).

Biofortification of Crops to Combat Nutritional Deficiency
Some 842 million people worldwide are malnourished (http://www.fao.org/english/newsroom/news/2003/26659-en.html; verified 10 May 2006). Most of these people live in the impoverished countries of Asia and Africa. Therefore, improvement of the nutritive value of food crops should be a high priority to alleviate deficiencies for protein, minerals, and vitamins, in addition to increasing crop yields. Iron deficiency, a most common dietary deficiency among the poor nations, affects especially children and women of reproductive age. In pregnant women, severe anemia may cause fetal growth retardation and large-scale maternal deaths (Gillespie, 1998). Nearly 400 million people in the world are reported to be at risk of vitamin A deficiency, which leads to blindness and premature death. Some 100 to 250 million children under 5 suffer from vitamin A deficiency, and half a million children become partially or totally blind each year (Conway and Toenniessen, 1999; Toenniessen, 2000, 2002).

It may be very difficult to improve nutritional deficiency, particularly for iron and vitamin A, using traditional crop breeding. Efforts are being made toward biofortification of crop plants using tools of biotechnology, and levels of essential nutrients have been increased. Genetic engineering was employed to raise the micronutrient content of rice, the stable food of more than one-third of the world population. Rice grains do not normally contain ß-carotene, which is the precursor of vitamin A. However, they contain geranylgeranyl pyrophosphate that can be sequentially converted to ß-carotene by four enzymes. By engineering rice with the four genes for these enzymes, two genes from daffodil and two from the bacterium Erwinia uredovora, Potrykus and his collaborators "instructed" rice to produce vitamin A. Later, by incorporating the iron-synthesizing ability in it, they were able to produce rice grains rich in vitamin A as well as iron (Ye et al., 2000; Beyer et al., 2002). The resulting rice, called Golden Rice, has the potential of saving millions of lives and averting blindness among millions of children, and is therefore referred to as the "grains of hope." Other transgenic strains with improved nutritional quality have been produced in both japonica and indica rices (Datta et al., 2003), and this strategy is being applied to other cereal crops (Poletti et al., 2004). Paine et al. (2005) developed Golden Rice 2 by incorporating a phytoene synthase gene (psy) from maize in combination with the Erwinia uredovora gene used to generate the original Golden Rice. They observed up to 23-fold increase in total carotenoids compared to Golden Rice.

The potato is the most important noncereal food crop for human consumption and, therefore, the need to improve its nutritional quality cannot be overemphasized. Chakraborty et al. (2000) demonstrated that expression of the AmAl gene (from amaranth, Amaranthus hypochondriacus L.) in transgenic tubers resulted in a significant increase in most essential amino acids as well as in higher protein content in tubers compared with nontransgenic potato plants. Through metabolic engineering, Ducreux et al. (2004) produced high carotenoid potato tubers containing enhanced levels of ß-carotene and lutein. By incorporating three genes from algae and mushroom species, a "super-healthy" cress was created (Pilcher, 2004). Using a novel transgenic approach involving organ-specific gene silencing on tomato, Davuluri et al. (2005) have significantly increased the content of both carotenoids and flavonoids, which are highly beneficial for human health. It is encouraging to note that numerous GM food crops are making a valuable contribution to human nutrition (Bouis et al., 2003).

	Biotechnology and Human Health

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As stated above, biotechnology is playing a significant role in nutritional enhancement of certain human foods, which has a direct bearing on human health. There are some other areas, also directly related to human health, in which modern technology has potential applications. Tools of biotechnology can help accomplish genetic modifications and improvements in plants, hitherto impossible to achieve by cytogenetics or conventional plant breeding. Some of these remarkable applications of biotechnology are outlined below.

Edible Vaccines
Vaccines have saved millions of lives and thus played a tremendous role in human health for almost 200 yr. Such vaccines could help save animal lives as well by providing protection against contagious viral diseases such as rinderpest. Khandelwal et al. (2004) developed transgenic peanut (Arachis hypogaea L.) expressing hemagglutinin (H) protein of rinderpest virus. Oral immunization of mice with transgenic peanut induced H-specific antibodies, indicating potential for producing an edible vaccine for rinderpest. Modern biotechnology may contribute toward the production of inexpensive edible vaccines. Lack of proper refrigeration poses a major problem for vaccinating the poor in less developed countries because the heat makes drugs lose their efficacy. Researchers worldwide have been focusing on producing plant-based vaccines that can be eaten uncooked in such fruits and vegetables as melons, tomatoes, and banana.

Edible vaccines administered orally through GM foods could become available at a fraction of the current costs, estimated at two cents instead of the usual $15 for an injectable dose (DaSilva, 2001). Fully immunizing one person against hepatitis B can cost as much as $450 (American Medical Assoc., 2001). Edible vaccines are also safe because they can be administered without refrigeration, syringes, or needles. They may save millions of people who die because of lack of access to traditional inoculants (Langridge, 2000). With appropriate genetic engineering, certain food crops could provide immunization against deadly diseases like hepatitis or tuberculosis. Edible vaccines against measles, cholera, and hepatitis B are being developed in India (Tripurani et al., 2003; Kumar et al, 2005). Charles Arntzen, of the Biodesign Institute at Arizona State University, has genetically engineered potatoes to produce a vaccine against hepatitis B virus, which kills one million people every year. He reported that in a trial of an edible vaccine, up to 60% of volunteers who ate chunks of the raw potato developed antibodies against the virus (Ariza, 2005; pers comm., Feb 2006). Vaccines against pneumonia and bubonic plague orally immunogenic to mice have also been developed (Alvarez et al., 2006). Horticultural crops may well serve as vaccine factories and we may see a day when, instead of taking an injection, one may only need to eat a banana or perhaps a tomato.

Genetic Decaffeination of Coffee
Tea [Camelia sinensis (L.) Kuntze] and coffee (Coffea arabica L.) provide some of the most widely used beverages in the world. As much as people like to have tea or coffee, some of them would like to have little or no intake of caffeine, an important stimulant in both tea leaves and coffee beans. Caffeine can cause occasional side effects, including elevated blood pressure and heart palpitations (see Kato et al., 2000). Therefore, the demand for decaffeinated coffee and tea has been increasing in recent years. The commercial process of decaffeination currently available is not only expensive, it leaves certain chemical residues, and may also lead to loss of flavor for discerning consumers.

Methods of genetically decaffeinating coffee have been tried with remarkable success. Caffeine synthase is an enzyme that catalyses the final two steps in the caffeine biosynthesis pathway. Kato et al. (2000) cloned the gene encoding caffeine synthase from young leaves of tea, paving the way for creating tea and coffee plants that are naturally deficient in caffeine. Decaffeinated coffee is growing on genetically modified bushes that could yield low-caffeine beans in 3 or 4 yr (Ogita et al., 2003; Pilcher, 2003a) and these transgenic beans could rival industrial decaffeination if they gain public approval (Silvarolla et al., 2004).

	Novel Applications of Transgenic Technology

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Phytoremediation
Widespread contamination of the environment caused by manufacture, testing, and disposal of explosives is becoming a matter of great concern. Certain soil bacteria are known to have biodegradative capability. Scientists in England successfully introduced pentaerythritol tetranitrate reductase, the bacterial enzyme initiating degradation of explosive residues, into plants, and the transgenic plants so created were used for bioremediation of contaminated soils (Rosser et al., 2001; Wong, 2001). Such an application of biotechnology has great promise for cleaning the environment.

Reducing Allergenicity of Crop Plants
The public has some concerns about creation of new allergens in GM foods, although natural foods like peanuts are known to produce allergic reaction in some people. It has been shown that genetic engineering can in fact make a food less allergenic. Soybean, for example, is known to cause allergies in humans. Herman et al. (2003) used the transgene-induced gene silencing to shut down the gene that codes for the protein believed to cause most soybean allergies. This novel approach to reducing allergies should add nutritional value to crops.

Genetic Engineering of Christmas Trees
Another novel application of modern biotechnology has been explored and has resulted from the isolation of genes for light emission from insects and jellyfish. Scientists at the Institute of Biotechnology at Zurich's Swiss Federal Institute of Technology and researchers at Michigan State University in the USA have engineered a Christmas tree that could light up on its own, putting an end to the frustrating ritual of manually putting on lights every year (Lean, 2001; Read, 2001).

	Resistance to Acceptance of Modern Biotechnology

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As documented above, tools of modern biotechnology have already produced encouraging results in accelerating crop improvement in terms of resistance to insect pests and diseases, tolerance to abiotic stresses, and nutritional enhancement of food crops (Cook, 2000; Jauhar and Khush, 2002). Moreover, this technology has potential applications in producing food vaccines, in genetic decaffeination of coffee, and could have several other novel applications. Such genetic modifications or improvements in crop plants would be impossible to achieve by conventional tools of cytogenetics or plant breeding. Unfortunately, however, this relatively new technology is facing resistance from certain sectors. Attempts have been made to create fear about the potential adverse impact of GM foods or plants on human health and the environment (Borlaug, 2000; Marris, 2001; Falk et al., 2002; Jauhar and Khush, 2002) to the extent that GM experimental materials are being destroyed (Pilcher, 2003b). Although the concerns or perhaps misconceptions of certain groups may not be valid, these issues must be adequately addressed to satisfy the general public. Some of the perceived dangers of transgenic technology are discussed below.

Issues of Human Health
A major concern is the possibility or perception of health risks posed by GM foods. However, the safety record of transgenic crops and their products testifies to their wholesomeness. There is no report so far of anyone falling ill by consuming GM food, which millions of people consume everyday. In the USA, more than 60% of all processed foods contain transgenic ingredients, but not a single transgenic food product has been shown to have any harmful effects (Vasil, 2003). Thus, regardless of consumer concerns, it remains true that genetically engineered foods have not made anybody sick (Radin, 2003). Additionally, the British Medical Association reaffirmed that there is no evidence that GM foods pose any threat to human safety (The Observer, 25 May 2003). There is overwhelming evidence that the bacterium B. thuringiensis and the transgenic crops expressing cry genes do not pose a threat to mammalian health (de Maagd et al., 2005). In contrast, in an extensive study by the American Medical Association, 20% of the 548 drugs approved for human use during the past 25 yr were later found to have serious or life-threatening effects, some possibly contributing to 1002 deaths (Vasil, 2003).

Genetic Pollution of Related Plants
Another major concern is the potential for unwanted movement of a transgene from a genetically engineered crop plant to its relatives—whether cultivated or wild. Thus, a transgene for herbicide resistance could get incorporated into a wild relative, thereby creating a "super weed" that might be hard to control. The possibility of such genetic pollution through a misplaced transgene exists in some cases (Messeguer, 2003; Stewart et al., 2003; Armstrong et al., 2005), but in most cases it is unlikely to happen because of the difficulty of hybridization between a transgenic crop plant and its wild relatives and the need for embryo rescue to obtain a hybrid under laboratory conditions (Jauhar and Khush, 2002). Nevertheless, some gene flow does occur from transgenic rice to its wild relatives or to other rice cultivars (Song et al., 2003; Zhang et al., 2003; Chen et al., 2004; Messeguer et al., 2004), but this should not be a serious cause for concern. Several genes for insect pest and disease resistance have, for example, been transferred to cultivated rice using traditional breeding. However, it must be noted, that so far there is no known case where wild or weedy rice biotypes of rice, such as red rice, have become more resistant by out-crossing with cultivated rice (High et al., 2004).

Even then, insertion of transgenes into a crop plant's chloroplast DNA (plastid engineering) would alleviate the risk of transgene escape via pollen, which is the most common mode of genetic pollution. This and related issues have been discussed by Jauhar and Khush (2002).

"Tinkering" with Nature
An underlying argument of opponents of modern technology is that it is unnatural and therefore unsafe. Thus, transgenic technology as a means of introducing new genes into plants is considered by some as "tinkering" with nature. This technology is considered to be "inherently and morally wrong" (Hackett, 2002). Even Prince Charles of England contends that creating genetic modifications by transgenic techniques takes "mankind into realms that belong to God, and God alone" and that "life on earth could be wiped out by scientists playing God" (Daily Telegraph, London, 28 April 2003). Such expressions of concern can in fact adversely influence the growth of biotechnology. Arntzen et al. (2003) discuss the politics involved in adopting GM crops.

We must remember that humans, in their efforts to ensure and improve food production, have been tinkering with nature for centuries. Traditional plant breeding, involving selection pressure for the most suitable and desirable crop cultivars, constitutes tinkering with nature. It is, in essence, human-made evolution. Any breeding activity is accompanied by genetic modifications, which in the last analysis involve changes at the DNA level. The newer biotechnological tools of gene transfer into crop cultivars are, in fact, a refinement of earlier ones, and genetic enhancement by those techniques poses no greater risk to the consumer. Many of the current crop cultivars we consume do, after all, contain genes of alien origin (Jauhar and Khush, 2002).

	Modern Biotechnology as a Supplement to Conventional Plant Breeding

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In the quest for food, early humans practiced crude methods of improving food production. These methods included selection of superior strains for cultivation and further selection as and when needed. And those early cultivators and selectionists could in a way be called the first "plant breeders." In that sense, plant breeding is as old as agriculture itself, the beginnings of which are believed to have occurred about 10 000 yr ago. Thus, the art of plant breeding was developed long before the principles of genetics and cytogenetics became known. Several thousand years ago, the early "plant breeders" intuitively looked for, successfully recovered, and skillfully propagated genetic variants or recombinants that showed desirable traits. Working under a myriad of cultural contexts, these breeders or perhaps selectionists turned the relatively useless weedy species into crop plants that sustain us today.

Tackling Complex Problems
As civilizations progressed and the demand for food and other necessities increased, better methods of plant improvement were devised or became available. With the discovery of the laws of genetics and the advent of the techniques of cytogenetics at the turn of the 20th century, the art of plant breeding became a science-based technology and the process of genetic improvement was accelerated considerably. Thus, the discovery of hybrid vigor and sophisticated methods of selection helped raise crop yields.

The Evolution of Plant Breeding
The process of plant breeding has continued to evolve over the years (Fig. 5 ). As the complexities of plant improvement increased, and several insect pests and diseases became a threat to steady and adequate food supply, even better methods were adopted to tackle these problems. Wild genetic resources as sources of insect pest and disease resistance were tapped. With the means of genomic reconstruction that became available, the process of plant improvement was further accelerated. Wide hybridization coupled with cytogenetic manipulation of chromosome pairing helped to introduce desirable traits from wild relatives to crop plants such as polyploid cereals like wheat and oat. Specific examples of breeding for disease resistance using tools of cytogenetics are described in earlier sections. Sophisticated cytological techniques such as fluorescent-GISH for characterizing alien chromatin into the crop genomes helped the process of alien gene transfer (Ceoloni and Jauhar, 2006). Thus, the application of cytogenetic tools strengthened the scientific basis of plant breeding.