**4. Transgenics for medicinal traits**

#### **4.1. Transgenics for nutraceuticals**

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 caused by deficiency such as iron-deficiency anemias, etc. Iron fortification of wheat has been proven transgenically by expressing phytase gene (*phy*A) from *Aspergillus japonicas* [40]. In situ degradation of phytates in the seed endosperm is considered desirable in order to increase bioavailability of micronutrients [40].

levels were very low because of a mutation in the critical region of the promoter during synthesis. Other examples of therapeutic expression are human serum albumin (HSA) that was expressed between 0.02 and 11.1%, depending upon the regulatory sequences used [45], oral and injectable insulin [46], HIV inhibitor cyanovirin [47], TGFb3 [48], and thioredoxins from plastids as modulators of recombinant therapeutic protein production [49]. Recently, different companies and foundations like the Bill and Melinda Gates Foundation or Juvenile Diabetes Research Foundation are undertaking well to advance such developments from labs

Introductory Chapter: Transgenics—Crops Tailored for Novel Traits

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9

Vaccination is an efficient strategy to control viral infections in both human and animal species. Different expression systems, having their own merits and demerits, are being used to produce recombinant vaccines. An ideal system would be that allows producing the desired functional product cost-effectively. Plant-based expression strategies encouraged biotechnologists to use this system to produce vaccines for both humans and animals. Moreover, plant system-derived subunit vaccines are heat stable, bio-encapsulated, and easy to scale up.

Oral vaccine term was introduced and extensively pursued after the successful expression of HBsAg in plants and recovery of the antigen as viruslike particles [50]. Interestingly, the antigen has the same properties as produced in yeast. Later on, the binding subunit of *E. coli* enterotoxin (LTB) and the capsid protein of norovirus genotype, which formed viruslike particles (NV-VLP), was expressed in plants that triggered mucosal immunization response in animals, hence, based on the data on approval clinical trials was obtained [7]. Diverse antigenic proteins were expressed in vegetable and fruit crops, and animal trials were successfully conducted. Plant species used are alfalfa, carrot, lettuce, tomato, potato, maize, soya bean, rice,

Development of an efficient plant-based system to express human antigenic proteins successfully has prompted its application to vaccinate livestock. Different attempts have been made by different research groups to address various diseases of livestock caused by viruses to increase the production in a cost-effective manner. Some of the examples are foot-andmouth disease virus (FMDV), bovine rotavirus, bovine viral diarrhea virus, bluetongue virus, and bovine papillomavirus. Of these viruses, foot-and-mouth disease virus (FMDV) has been addressed majorly as livestock is an inevitable part of the economy. Livestock productivity is compromised due to frequent occurrence of foot-and-mouth disease (FMD). Vaccination is one of the main strategies to control foot-and-mouth disease. Yet, lack of high-quality and effective vaccine in Pakistan warrants the development of genotype-matched vaccines. One of the approaches to develop such vaccines is reverse genetics that is very costly and laborious. This demands exploring other alternative approaches. Of the alternative approaches,

engineering edible plants with the pathogenicity-causing genes is more promising.

An oral vaccine against FMDV was developed by expressing structural protein VP1 in transgenic *Stylosanthes guianensis* [52]. In these experiments, the level of recombinant protein was

to the clinics.

and banana [51].

**4.2. Transgenics for antigenic proteins (vaccines)**

Golden rice and provitamin A-fortified maize are crops that have caught interest of nutritionists globally. Provitamin A deficiency that results in night blindness in masses may be addressed through genetic improvement of crops like maize. In a study where single-cross maize yellow hybrids were evaluated for carotenoid contents [3] since biofortification of maize, endosperm is found to be the most convenient solution addressing its deficiency. Hence, improved contents of provitamin A carotenoids in maize may help Pakistani populace to alleviate the subclinical symptoms of vitamin A deficiency [4].

Perhaps, the most researched aspects of nutraceuticals are their use in medicine, to cure a variety of diseases such as cancer, osteoarthritis, cardiovascular disorders, etc. Over the years, several plants have been shown to contain compounds which, if incorporated into lifestyle early on, reduce the risk of cancer by as much as 33%. For example, blue maize has been found to be an effective nutraceutical in prevention of several types of cancers, such as colon cancer, etc. [41].

This era of rapid urbanization has seen an emerging trend of expressing many medicinal and nutritional traits into other food crops transgenically. Although several people have shown their concerns as to its biosafety, such drawbacks have not been reported to this date. Chloroplast transformation addresses biosafety issues as chloroplasts are not transmitted through pollens in most cultivated plants. Hence, the transgenics are a promising way forward to develop cost-effective nutraceuticals.

#### *4.1.1. Transgenics for therapeutics*

A number of pharmaceutical proteins have been synthesized exploiting plant genetic systems with overriding impact on conventional approaches used to manufacture pharmaceuticals. Some advantages of using plant system are low cost of production of pharmaceuticals and their processing. Commercial-scale production in bringing therapeutics to the clinic has been observed in the last 6–7 years. Manufacturing facilities of different capacities have been constructed in addition to the development of plant-made pharmaceuticals to meet current manufacturing standards [42]. Large Scale Biology Corporation (LSBC) in Owensboro, KY, USA, has designed the first manufacturing facility that plant virus transient expression system was developed to meet the current good manufacturing practice [43, 44].

However, synthesizing pharmaceuticals in plants by engineering the chloroplast genome is more advantageous as explained elsewhere in this chapter; therefore, attempts have been made to express different pharmaceuticals proteins. For example, interferons α2 and 5 were expressed from tobacco chloroplasts [5, 6]. In these studies the interferon α2 and 5 genes were synthesized and expressed. It was observed that fully expanded mature leaves contained high levels of interferon hen compared to young and senescence leaves; however, expression levels were very low because of a mutation in the critical region of the promoter during synthesis. Other examples of therapeutic expression are human serum albumin (HSA) that was expressed between 0.02 and 11.1%, depending upon the regulatory sequences used [45], oral and injectable insulin [46], HIV inhibitor cyanovirin [47], TGFb3 [48], and thioredoxins from plastids as modulators of recombinant therapeutic protein production [49]. Recently, different companies and foundations like the Bill and Melinda Gates Foundation or Juvenile Diabetes Research Foundation are undertaking well to advance such developments from labs to the clinics.

#### **4.2. Transgenics for antigenic proteins (vaccines)**

caused by deficiency such as iron-deficiency anemias, etc. Iron fortification of wheat has been proven transgenically by expressing phytase gene (*phy*A) from *Aspergillus japonicas* [40]. In situ degradation of phytates in the seed endosperm is considered desirable in order to

Golden rice and provitamin A-fortified maize are crops that have caught interest of nutritionists globally. Provitamin A deficiency that results in night blindness in masses may be addressed through genetic improvement of crops like maize. In a study where single-cross maize yellow hybrids were evaluated for carotenoid contents [3] since biofortification of maize, endosperm is found to be the most convenient solution addressing its deficiency. Hence, improved contents of provitamin A carotenoids in maize may help Pakistani populace

Perhaps, the most researched aspects of nutraceuticals are their use in medicine, to cure a variety of diseases such as cancer, osteoarthritis, cardiovascular disorders, etc. Over the years, several plants have been shown to contain compounds which, if incorporated into lifestyle early on, reduce the risk of cancer by as much as 33%. For example, blue maize has been found to be an effective nutraceutical in prevention of several types of cancers, such as colon cancer,

This era of rapid urbanization has seen an emerging trend of expressing many medicinal and nutritional traits into other food crops transgenically. Although several people have shown their concerns as to its biosafety, such drawbacks have not been reported to this date. Chloroplast transformation addresses biosafety issues as chloroplasts are not transmitted through pollens in most cultivated plants. Hence, the transgenics are a promising way for-

A number of pharmaceutical proteins have been synthesized exploiting plant genetic systems with overriding impact on conventional approaches used to manufacture pharmaceuticals. Some advantages of using plant system are low cost of production of pharmaceuticals and their processing. Commercial-scale production in bringing therapeutics to the clinic has been observed in the last 6–7 years. Manufacturing facilities of different capacities have been constructed in addition to the development of plant-made pharmaceuticals to meet current manufacturing standards [42]. Large Scale Biology Corporation (LSBC) in Owensboro, KY, USA, has designed the first manufacturing facility that plant virus transient expression sys-

However, synthesizing pharmaceuticals in plants by engineering the chloroplast genome is more advantageous as explained elsewhere in this chapter; therefore, attempts have been made to express different pharmaceuticals proteins. For example, interferons α2 and 5 were expressed from tobacco chloroplasts [5, 6]. In these studies the interferon α2 and 5 genes were synthesized and expressed. It was observed that fully expanded mature leaves contained high levels of interferon hen compared to young and senescence leaves; however, expression

tem was developed to meet the current good manufacturing practice [43, 44].

increase bioavailability of micronutrients [40].

8 Transgenic Crops - Emerging Trends and Future Perspectives

ward to develop cost-effective nutraceuticals.

*4.1.1. Transgenics for therapeutics*

etc. [41].

to alleviate the subclinical symptoms of vitamin A deficiency [4].

Vaccination is an efficient strategy to control viral infections in both human and animal species. Different expression systems, having their own merits and demerits, are being used to produce recombinant vaccines. An ideal system would be that allows producing the desired functional product cost-effectively. Plant-based expression strategies encouraged biotechnologists to use this system to produce vaccines for both humans and animals. Moreover, plant system-derived subunit vaccines are heat stable, bio-encapsulated, and easy to scale up.

Oral vaccine term was introduced and extensively pursued after the successful expression of HBsAg in plants and recovery of the antigen as viruslike particles [50]. Interestingly, the antigen has the same properties as produced in yeast. Later on, the binding subunit of *E. coli* enterotoxin (LTB) and the capsid protein of norovirus genotype, which formed viruslike particles (NV-VLP), was expressed in plants that triggered mucosal immunization response in animals, hence, based on the data on approval clinical trials was obtained [7]. Diverse antigenic proteins were expressed in vegetable and fruit crops, and animal trials were successfully conducted. Plant species used are alfalfa, carrot, lettuce, tomato, potato, maize, soya bean, rice, and banana [51].

Development of an efficient plant-based system to express human antigenic proteins successfully has prompted its application to vaccinate livestock. Different attempts have been made by different research groups to address various diseases of livestock caused by viruses to increase the production in a cost-effective manner. Some of the examples are foot-andmouth disease virus (FMDV), bovine rotavirus, bovine viral diarrhea virus, bluetongue virus, and bovine papillomavirus. Of these viruses, foot-and-mouth disease virus (FMDV) has been addressed majorly as livestock is an inevitable part of the economy. Livestock productivity is compromised due to frequent occurrence of foot-and-mouth disease (FMD). Vaccination is one of the main strategies to control foot-and-mouth disease. Yet, lack of high-quality and effective vaccine in Pakistan warrants the development of genotype-matched vaccines. One of the approaches to develop such vaccines is reverse genetics that is very costly and laborious. This demands exploring other alternative approaches. Of the alternative approaches, engineering edible plants with the pathogenicity-causing genes is more promising.

An oral vaccine against FMDV was developed by expressing structural protein VP1 in transgenic *Stylosanthes guianensis* [52]. In these experiments, the level of recombinant protein was varied from 0.1 to 0.5% of total soluble protein. These levels were enough to induce a protective systemic antibody response in mice. In another attempt, capsid precursor polypeptide (P1) was expressed in rice, and 0.6–1.3 mg/g of TSP was observed that induced a protective immune response in mice [53]. Further, in mice when vaccinated orally, FMDV-specific mucosal immune responses were detected. However, partial virus clearance after challenge was observed. To address these low-expression problems, chloroplast transformation approach can be used. In a study, VP1 was expressed in tobacco chloroplasts and 2–3% values were recorded [54]. In another study, epitopes (B cell) of structural proteins VP1 and VP4 and of nonstructural proteins 2C and 3D (T cell) were produced in *N. benthamiana* plants using a plant virus expression system [55]. More recently, tandem-linked VP1 proteins of two serotypes, A and O, are expressed in forage crop *Crotalaria juncea* and fed to guinea pigs that produced humoral as well as cell-mediated immune responses [56]. From all these studies and experiments being carried out in the laboratory of the author of this chapter demonstrate that plant-based overexpression of antigenic proteins to control FMDV is an effective way but needs further experimentation to improve efficacy of edible vaccines by engineering epitopic proteins with different adjuvants (Khan MS, unpublished).

**Author details**

Faisalabad, Pakistan

**References**

Muhammad Sarwar Khan<sup>1</sup>

2000;**287**:303-305

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\* and Kauser Abdulla Malik<sup>2</sup>

1 Center of Agricultural Biochemistry and Biotechnology (CABB), University of Agriculture,

Introductory Chapter: Transgenics—Crops Tailored for Novel Traits

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11

[1] Ye X, Al-Babili S, Kloti A, Zhang J, Lucca P, Beyer P, et al. Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm. Science.

[2] Diretto G, Welsch R, Tavazza R, Mourgues F, Pizzichin D, Beyer P, et al. Silencing of beta-carotene hydroxylase increases total carotenoid and beta-carotene levels in potato

[3] Maqbool MA, Aslam M, Khan MS, Beshir B, Ahsan M. Evaluation of single cross yellow maize hybrids for agronomic and carotenoid traits. International Journal of Agriculture

[4] Maqbool MA, Aslam M, Beshir A, Khan MS. Breeding for provitamin A biofortification

[5] Nurjis F, Khan MS. Expression of recombinant interferon alpha-2a in tobacco chloroplasts using microprojectile bombardment. The African Journal of Biotechnology.

[6] Khan MS, Nurjis F. Synthesis and expression of recombinant interferon alpha-5 gene in tobacco chloroplasts, a non-edible plant. Molecular Biology Reports. 2012;**39**:4391-4400

[7] Giorgi C, Franconi R, Rybicki EP. Human papillomavirus vaccines in plants. Expert

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\*Address all correspondence to: sarwarkhan\_40@hotmail.com

2 Forman Christian College (A chartered University), Lahore, Pakistan

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## **5. Conclusions**

Nuclear transformation, achieved using microprojectile bombardment or *Agrobacterium* strains, is predominantly carried out to tailor agronomic traits in crops. However, this technology is not successful to transform upland cotton where cells are recalcitrant to regeneration, despite genome mixing through crosses between different genotypes. Biotechnologists rely on this technology though genes escape and pollinate other related crops or weeds, developing weeds or super weeds, respectively. An alternate strategy to develop transgenics is the chloroplast transformation technology since this technology offers natural gene containment, highlevel transgene expression with bona fide structures of proteins, and allows all transformation events to be uniform as far as gene integration into the plastome is concerned. High-level gene expression is due to the polyploid nature of chloroplasts in a cell and of plastomes in each chloroplast, biologically active proteins are due to the presence of chaperon proteins, and uniform integration of transgenes into the plastome is due to the homologous recombination. Hence, chloroplast transformation is more suitable for expression of health-related traits in plants rather agronomic in crops. In either case, transgenics should be grown in the field following approved biosafety guidelines and strict stewardship.

### **Acknowledgements**

The corresponding author would like to acknowledge Punjab Agricultural Research Board (PARB) and Higher Education Commission (HEC) for providing funds to MSK for research.
