**3. Transgenics for agricultural traits**

**2. Developing transgenics: state-of-the-art strategies**

4 Transgenic Crops - Emerging Trends and Future Perspectives

ber of explant.

repeat regions of the plastome [17].

used for carrying out selection for transplastomic lines.

A plant cell is blessed with three major organelles with their own genomes, namely, nucleus, chloroplast, and mitochondria. Of these three, two genomes are routinely manipulated to incorporate new traits in cultivated plants. There are a number of approaches to transfer and introduce genes into the plant genome, depending upon the choice of explant to be used in transformation experiments, for example, *Agrobacterium*-mediated gene transfer, gene gun, agro-infiltration, sonication, and polyethylene glycol treatment. Of these, *Agrobacterium*mediated and gene gun methods are most commonly used approaches to develop transgenic plants. For nuclear transformation, *Agrobacterium* method is more successful than particle bombardment as a more number of transformed shoots can be recovered from the same num-

Genetic transformation process involves a number of steps, including selection of a gene that confers resistance to a particular antibiotic for selection and screening purposes, isolation of a trait-encoding gene, choice of promoters and terminating sequences to control the expression of the gene or genes, choice of explant, and an artificial medium to support explant to regenerate into a complete shoot. For selection and screening, usually two types of markers are used: (1) selectable marker and (2) visual marker [10, 12–14]. Selectable marker could be lethal or nonlethal in nature. Nuclear genome transformation is carried out using lethal markers. Regenerated shoots are normally hemi- or heterozygous and need either further purification of transgenome using selection medium or through selfing depending upon the crop used.

The second genome is the chloroplast genome, the plastome that has been modified in a number of plant species, including model, crop, and tree plants. Plastome is a doublestranded DNA molecule of 152 (*Cinnamomum campohra*) – 218 kb (*Pelargonium*) size [15, 16]. Approximately, 120 genes in various plant species are encoded by the plastome [17]. It looks like that most of the ancestral genes have either been lost during evolution or transferred to the nucleus. A mature mesophyll cell contains up to 100 chloroplasts, and each chloroplast contains 100 plastome copies; therefore, the ploidy number of plastome per cell reaches up to 10,000 copies. Furthermore, this number is doubled for genes that are located in inverted

When transforming a chloroplast, a universal antibiotic cannot be used given that different plants have variable sensitivity to selective agents; therefore, recovery of the transplastomic shoots is dependent on two things: (1) choice of the selective agent to be used and (2) the concentration of the selective agent that allows regeneration and development of shoots from the transformed cells while killing the non-transformed cells. For example, spectinomycin is used to select transformed cells on selection medium from tobacco, lettuce, tomato, potato, cabbage, oil rape seed, and carrot. However, several monocots, including rice and sugarcane, are naturally resistant to spectinomycin; therefore, streptomycin-containing medium was

Initially, only few copies of the plastid genome are transformed and maintained under continuous selection pressure. However, stable lines with uniformly transformed genome copies are recovered on selection medium through a repeated cycle of regeneration [6]. During the period, the wild-type and the transgenic plastids and their genomes gradually sort out;

#### **3.1. Transgenics to improve crop production**

Plants on earth synthesize their food by themselves, owing to harbor solar energy conversion and several other chemical reactions in their cells. One of the reactions carried out in plants is carbon fixation during a process, namely, photosynthesis. During photosynthesis Rubisco catalyzes the inefficient carbon fixation, reviewed extensively elsewhere [19, 20]. This raises a question why carbon fixation during photosynthesis is rate limiting. Major reasons are as follows: first, Rubisco's rate of catalysis is much low, and, second, it has to compete with a nonproductive reaction, oxygenation [21], depending upon the relative concentration of carbon dioxide and oxygen, as well as on temperature. Carboxylation results in CO<sup>2</sup> fixation. Therefore, plant growth and yield can be improved by two ways: (1) by increasing photosynthesis and (2) by reducing photorespiration.

A number of examples are available in the literature, reporting different versions of Rubisco that would improve photosynthesis [22], but considerable success has not been achieved yet. Introducing C<sup>4</sup> pathway in C<sup>3</sup> plants appears to be more promising, but due to the leakage of gases, the advantages of concentrating carbon dioxide in the chloroplasts of C<sup>3</sup> plants are objectionable [23]. Glycolate catabolic pathway was introduced in chloroplasts for alleviation of photorespiratory losses in *Arabidopsis thaliana*. Photosynthesis is markedly increased in this engineered pathway, thereby widening the applicability of the technique to cereals, for example, wheat and rice, described in detail elsewhere [24].

Developing chlorophyll in the dark, and chloroplasts that are competent for photosynthesis upon exposure to light, is another promising technique that can be implied to improve the photosynthesis in plants [25, 26]. In a study, *chlB* gene from *Pinus thunbergii* was introduced into the plastome of *Nicotiana tabacum* [27]. Transgenic plants when shifted to light from dark in early development of chlorophyll pigments were observed in leaves of transgenic compared to wild-type plants. This helps us to understand the molecular biology of transgenic angiosperms. Another effort is underway to introduce C<sup>4</sup> pathway in rice, a C<sup>3</sup> plant [28], using various techniques.

#### **3.2. Transgenics for weed management**

Weeds compete with crops for food, thereby lowering the crop yield and affecting farmers. There are two types of herbicides, (1) selective in nature and (2) used before and after emergence of plants from seeds, and are specific to leaf morphology.

A pioneering concept to engineer a crop for herbicide tolerance was developed in the 1980s [29] when it was observed that few herbicides kill plants by blocking photosynthetic electron transport. For example, triazine herbicides bind to a photosystem-II protein (D1) in the chloroplasts that appeared to be the first molecular target to develop a commercial herbicide [30]. However, tolerance to herbicides in transgenic plants is considered the best approach in weed control in crops. Glyphosate is a nonselective broad-spectrum herbicide that kills narrow-leaf grasses and broad-leaf weeds. Glyphosate competitively inhibits 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSP) in the amino acid biosynthetic pathway. This proves to be a standard strategy to overcome the problem of herbicide selectivity. Yet, this strategy raises the concern of gene transfer to other plants or weeds.

*Bt* toxin only in green tops with no residues in the juice [36]. Lately, a different version of the *cry1Ab* gene was again used to develop transgenic sugarcane, and similar results were

Introductory Chapter: Transgenics—Crops Tailored for Novel Traits

http://dx.doi.org/10.5772/intechopen.81372

7

Plant pathogens are damaging plants and causing yield losses exceptionally; it is therefore highly desirable to develop transgenics that would be resistant to pathogenic bacteria and fungi. There are a number of examples available in literature where pathogens have been targeted to control diseases in plants. Arrieta and colleagues in 1996 reported co-expression of genes encoding glucanase- and thaumitin-like proteins, and a low level of fungal infection was observed [37]. In other studies when snakin-1 gene was overexpressed transgenically, an enhanced resistance to *Rhizoctonia solani* and *Erwinia carotovora* was observed. Similarly, chitinase gene from *Streptomyces griseus* showed resistance against *Alternaria solani*, while expression of mycoparasitic chitinase and glucanase enzymes developed improved resistance to *Rhizoctonia solani*. Five novel thionin genes were isolated from plants belonging to the *Brassicaceae* family, and when expressed transgenically in potato, a high-degree resistance to gray mold (*Botrytis cinerea*) was observed [38]. Literature review suggests that broad-spectrum resistance could be attained in valuable plant species through transgenic technology. Mycoparasites can be controlled using glucanases, chitinases, proteases, cellu-

Amphipathic peptides such as magainin are known to control microbe infections; Daniell and his colleagues expressed MSI-99 in chloroplasts of tobacco and reported a varied degree resistance to microbes [39] with no changes in growth and development of the transgenic plants compared to wild-type plants. But using such genes in crops warrants extensive biosafety

One of the items on the wish list of biotechnologists is to engineer genomes of plants to tailor high-value traits other than agronomic, pathological, and entomological in nature. Among high-value traits are the introduction of nutrition and related characters. "Nutraceuticals" is a portmanteau of "nutrition" and "pharmaceuticals"; hence, the word implies that nutraceuticals are products regulated as medicine, food ingredients, and dietary supplements. These products not only provide protection against various diseases caused due to the deficiency of the nutrients but also have physiological benefits. Traditionally, nutraceuticals have been employed in the form of medicinal plants, etc., but in this modern era, nutraceuticals are being used in a variety of perspectives, such as nutrition and medicine. Iron-fortified products are the prime examples of it. Addition of iron-containing compounds during the grinding of wheat, otherwise deficient in iron, protects the wheat-dependant populace from diseases

obtained.

studies.

**3.4. Transgenics for pathogen resistance**

lases, kinases, and certain antibiotics.

**4. Transgenics for medicinal traits**

**4.1. Transgenics for nutraceuticals**

An antibiotic bialaphos inhibits glutamine synthetase (GS) upon removal of alanine residues in the nitrogen assimilation pathway; resultantly, accumulation of toxic levels of ammonia in both bacteria and plant cells occurs. This antibiotic was used as an herbicide that appeared to be nonselective in nature. In two different studies, transgenic tobacco plants exhibited fieldlevel tolerance to phosphinothricin (PPT) when bar was expressing from chloroplast genome [31, 32]. Further, development of glufosinate-resistant traits has been reported worldwide in corn, soybean, and cotton until now. The trait has also been developed by Khan and his team in sugarcane, and the transgenic plants were tolerant to BASTA [33]. The extensive and continuous use of a single herbicide should be avoided to exclude the possibilities of resistance development in plants, and precautionary measures should be taken to safeguard human health.

#### **3.3. Transgenics for insect resistance**

Engineering plant genomes for useful traits leads toward sustainable agriculture. Among useful traits, resistance against insects is developed by using Cry proteins from *Bacillus thuringiensis* (*Bt*). *Bacillus thuringiensis* is a soilborne bacterium, having crystal (Cry) proteins in the cytoplasm of the cells at sporulating stage. These proteins are toxic to some chewing and sucking insects.

Genes encoding Cry proteins have been expressed in a number of crops worldwide to control major pests. This has reduced the pesticide usage and has lowered the production costs of crops. First, transgenic crops developed were corn and cotton that expressed *cry1Ab* and *cry1Ac* genes, respectively [34]. Afterward, other crops including soybean, maize, cotton, canola, squash, papaya, tomato, sugar beet, and sugarcane were transformed using *Bt* genes to control insects. Almost all global biotech crop area is because of soybean, corn, cotton, and canola crops [35]. Since the first commercial cultivation of GM crops in 1996, farmers (16.7 million) from 29 countries cultivated 160 million hectares of biotech crops in 2011. Out of this number, about 90% were small and resource poor farmers belonging to developing countries. The United States and Brazil were major producers who adopted GM crops.

In Pakistan, first indigenously developed transgenic crop was sugarcane, carrying *cry1Ab* gene that was approved by the Technical Advisory and National Biosafety Committees after the approval of biosafety rules and guidelines in 2005. Developed sugarcane plants carry *Bt* toxin only in green tops with no residues in the juice [36]. Lately, a different version of the *cry1Ab* gene was again used to develop transgenic sugarcane, and similar results were obtained.

### **3.4. Transgenics for pathogen resistance**

A pioneering concept to engineer a crop for herbicide tolerance was developed in the 1980s [29] when it was observed that few herbicides kill plants by blocking photosynthetic electron transport. For example, triazine herbicides bind to a photosystem-II protein (D1) in the chloroplasts that appeared to be the first molecular target to develop a commercial herbicide [30]. However, tolerance to herbicides in transgenic plants is considered the best approach in weed control in crops. Glyphosate is a nonselective broad-spectrum herbicide that kills narrow-leaf grasses and broad-leaf weeds. Glyphosate competitively inhibits 5-enol-pyruvyl shikimate-3-phosphate synthase (EPSP) in the amino acid biosynthetic pathway. This proves to be a standard strategy to overcome the problem of herbicide selectivity. Yet, this strategy

An antibiotic bialaphos inhibits glutamine synthetase (GS) upon removal of alanine residues in the nitrogen assimilation pathway; resultantly, accumulation of toxic levels of ammonia in both bacteria and plant cells occurs. This antibiotic was used as an herbicide that appeared to be nonselective in nature. In two different studies, transgenic tobacco plants exhibited fieldlevel tolerance to phosphinothricin (PPT) when bar was expressing from chloroplast genome [31, 32]. Further, development of glufosinate-resistant traits has been reported worldwide in corn, soybean, and cotton until now. The trait has also been developed by Khan and his team in sugarcane, and the transgenic plants were tolerant to BASTA [33]. The extensive and continuous use of a single herbicide should be avoided to exclude the possibilities of resistance development in plants, and precautionary measures should be taken to safeguard human

Engineering plant genomes for useful traits leads toward sustainable agriculture. Among useful traits, resistance against insects is developed by using Cry proteins from *Bacillus thuringiensis* (*Bt*). *Bacillus thuringiensis* is a soilborne bacterium, having crystal (Cry) proteins in the cytoplasm of the cells at sporulating stage. These proteins are toxic to some chewing and

Genes encoding Cry proteins have been expressed in a number of crops worldwide to control major pests. This has reduced the pesticide usage and has lowered the production costs of crops. First, transgenic crops developed were corn and cotton that expressed *cry1Ab* and *cry1Ac* genes, respectively [34]. Afterward, other crops including soybean, maize, cotton, canola, squash, papaya, tomato, sugar beet, and sugarcane were transformed using *Bt* genes to control insects. Almost all global biotech crop area is because of soybean, corn, cotton, and canola crops [35]. Since the first commercial cultivation of GM crops in 1996, farmers (16.7 million) from 29 countries cultivated 160 million hectares of biotech crops in 2011. Out of this number, about 90% were small and resource poor farmers belonging to developing countries.

In Pakistan, first indigenously developed transgenic crop was sugarcane, carrying *cry1Ab* gene that was approved by the Technical Advisory and National Biosafety Committees after the approval of biosafety rules and guidelines in 2005. Developed sugarcane plants carry

The United States and Brazil were major producers who adopted GM crops.

raises the concern of gene transfer to other plants or weeds.

6 Transgenic Crops - Emerging Trends and Future Perspectives

health.

sucking insects.

**3.3. Transgenics for insect resistance**

Plant pathogens are damaging plants and causing yield losses exceptionally; it is therefore highly desirable to develop transgenics that would be resistant to pathogenic bacteria and fungi. There are a number of examples available in literature where pathogens have been targeted to control diseases in plants. Arrieta and colleagues in 1996 reported co-expression of genes encoding glucanase- and thaumitin-like proteins, and a low level of fungal infection was observed [37]. In other studies when snakin-1 gene was overexpressed transgenically, an enhanced resistance to *Rhizoctonia solani* and *Erwinia carotovora* was observed. Similarly, chitinase gene from *Streptomyces griseus* showed resistance against *Alternaria solani*, while expression of mycoparasitic chitinase and glucanase enzymes developed improved resistance to *Rhizoctonia solani*. Five novel thionin genes were isolated from plants belonging to the *Brassicaceae* family, and when expressed transgenically in potato, a high-degree resistance to gray mold (*Botrytis cinerea*) was observed [38]. Literature review suggests that broad-spectrum resistance could be attained in valuable plant species through transgenic technology. Mycoparasites can be controlled using glucanases, chitinases, proteases, cellulases, kinases, and certain antibiotics.

Amphipathic peptides such as magainin are known to control microbe infections; Daniell and his colleagues expressed MSI-99 in chloroplasts of tobacco and reported a varied degree resistance to microbes [39] with no changes in growth and development of the transgenic plants compared to wild-type plants. But using such genes in crops warrants extensive biosafety studies.
