Advanced Biotechnology: Transgenic Plants and Insect-Resistant Crops

# ADVANCED BIOTECHNOLOGY ## TRANSGENIC PLANTS - **Construct chimeric genes**: Construct chimeric genes for 3'- regulatory sequences of nopaline synthase (nos) - **Transformation of tobacco**: Introduce genes into the transformation of tobacco plants to increase their tolerance to high levels of hyg...

# ADVANCED BIOTECHNOLOGY ## TRANSGENIC PLANTS - **Construct chimeric genes**: Construct chimeric genes for 3'- regulatory sequences of nopaline synthase (nos) - **Transformation of tobacco**: Introduce genes into the transformation of tobacco plants to increase their tolerance to high levels of hygromycin sulphate - **Hygromycin B phosphotransferase**: Hygromycin B phosphotransferase, an antibiotic, inhibits protein synthesis in eukaryotes, animal cells and plant cells by catalysing the phosphorylation of a 7-hydroxyl group in a ribosome - **Aminoglycoside 3'-phosphotransferase**: Aminoglycoside 3'-phosphotransferase, also called aminoglycoside phosphotransferase, catalyses the phosphorylation of aminoglycoside antibiotics which inhibits the antibiotic's binding to ribosomes - **Phosphinothricin acetyltransferase**: Phosphinothricin acetyltransferase converts ammonia into protein and confers resistance to phosphinothricin ### **Selectable Marker Genes** - **Neomycin phosphotransferase**: a selectable marker in many dicot plants that provides resistance to high levels of kanamycin sulphate - **Hygromycin B phosphotransferase**: a selectable marker that provides resistance to high levels of hygromycin - **Phosphinothricin acetyltransferase**: a selectable marker that provides resistance to phosphinothricin, also known as the herbicide BastaⓇ, and is part of the herbicide RoundupⓇ formulations **Miki and McHugh (2004) have reviewed the selectable marker genes used in transgenic plants** and have described many mechanisms associated with glyphosate resistance, including: - **Over expression of a petunia EPSP synthase gene**: Over expression of a petunia EPSP synthase gene using the 35S promoter, resulting in glyphosate tolerance in transformed petunia - **Expression of mutant forms of the EPSP synthase gene aroA**: Expression of mutant forms of the EPSP synthase gene aroA in Salmonella typhimurium or E. coli targeted to chloroplasts, which confers glyphosate resistance to tobacco - **Naturally-glyphosate-resistant EPSP synthase gene from the A. tumefaciens strain CP4**: A naturally-glyphosate-resistant EPSP synthase gene from the A. tumefaciens strain CP4 fused to the transit peptide sequence of Arabidopsis EPSP synthase to target chloroplasts, which confers glyphosate resistance to several crop species - **Catabolism of glyphosate to glyoxylate and minomethylphosphonic acid (AMPA)**: Catabolism of glyphosate to glyoxylate and minomethylphosphonic acid (AMPA) by bacterial glyphosate oxidoreductase (GOX) targeted to the chloroplast, which confers glyphosate resistance to several different plants - **Acetolactate synthase (ALS) gene**: The als gene isolated from Arabidopsis thaliana encodes for the enzyme acetolactate synthase and provides resistance to the herbicide sulfonylurea. - **Mutant acetolactate synthase (ALS) gene**: A mutant acetolactate synthase (ALS) gene (csr1-1 gene) isolated from sulfonylurea herbicide-resistant Arabidopsis thaliana, placed under the control of a cauliflower mosaic virus 35S promoter, provided about 200-fold greater resistance (at extremely low chlorsulfuron concentrations of 10 nm) to transformed calli ### **Methods for Developing Genetically Modified (GM) Plants** **Steps for Developing GM Plants** 1. **Isolation of gene(s) of interest**: Isolate genes responsible for the desired trait in an organism. An example of this would be genes responsible for drought tolerance, or insect resistance. 2. **Insertion of gene(s) into a vector**: Insert genes into a vector which serves as a gene transfer tool into the cells of desired hosts. The most commonly used vector for plant transformations is the Ti plasmid derived from A. tumefaciens. 3. **Plant transformation**: Transform A. tumefaciens cells containing the modified plasmid with transgenes with plant cells, leaf discs or explants. Some of the cells take up a piece of the plasmid called T-DNA. 4. **Selection of transformants**: Select for transformed plant cells that have not incorporated the desired genes. Marker genes are used to favour the growth of transformed cells as compared to non-transformed cells. 5. **Regeneration into whole plants**: Regenerate whole plants via tissue culture involving the explants placed onto nutrient media that induce development of the cells into various plant parts to regenerate into whole plants. 6. **Confirmation of transformation**: Confirm the inheritance and normal function of the gene inserted into transformed plants, such as determining the number of copies inserted, and interference (if any) of the insertion with other genes. 7. **Plant performance testing**: Test the plant's performance first in a greenhouse, then in a field, to determine that the desired new trait present in the modified plant exists, and to ensure that the plant does not have any unwanted new traits. 8. **Bio-safety Assessment**: Conduct food and environmental safety assessments to determine the safety of the GM crop, ensuring that the GM crop does not have any bad impact on human health or the environment. ## **Agroinfection and Transformation** - **Agroinfection**: Inject a host with viral DNA using Agrobacterium tumefaciens, an alternative method for viral infection of plants. - **Transgenic cereals**: Produce transgenic cereals by using Agrobacterium-based vector systems, which are not better than typical Agrobacterium applications. ## **Anti-viral Proteins of Other Plants** - **Pokeweed (Phytolacca americana)**: Pokeweed contains three antiviral proteins in its cell wall: - **PAPI (pokeweed antiviral protein I)**, found in spring leaves - **PAPII (pokeweed antiviral protein II)**, found in summer leaves - **PAP-S (pokeweed antiviral protein-seed)**, found in seeds. - **Ribosome-inactivating proteins (RIPs**: The proteins PAP and PAPII are ribosome-inactivating proteins that remove a specific adenine residue from the large ribosomal RNA of the 60S subunit of eukaryotic ribosomes. - **Transgenic plants**: Introducing the PAP gene into tobacco and potato plants through binary Ti plasmid vectors under the transcriptional control of the 35S promoter yielded a lower number of lesions in transformed plants compared to their non-transformed counterparts. ## **Transgenic Plants** ### **GM Papaya** - **International markets**: Papaya is consumed in many tropical and subtropical regions but has also entered international markets in recent years. - **Papaya ringspot virus (PRSV) disease**: The papaya industry is threatened by papaya ringspot virus (PRSV) disease, which spreads via aphids. - **Transgenic papaya**: Transgenic papaya cultivars were field-tested and the transgenic trees have exhibited varying levels of resistance against virus infections. - **Fruit production**: The papaya industry was revived in Hawaii after the commercialisation of PRSV resistant transgenic papaya, despite a subsequent decline in yield due to the transgenic papaya's deregulation in Canada. - **Deregulation in Japan**: Japan's Ministry of Agriculture, Forestry and Fisheries approved line 55-1 transgenic papaya in 2000, which allowed Hawaiian growers to expand their transgenic papaya markets and eliminate excessive costs associated with segregating transgenic and non-transgenic fruits. ### **GM Tomato** - **Potato virus X (PVX)**: Potato virus X (PVX) contains a single infectious genomic RNA of 2×10⁶ daltons; the 3'-region of the PVX RNA has been cloned and sequenced. ## **Insect-Resistant Transgenic Plants (BT Technology)** ### **Bacillus thuringiensis (Bt)** - **Biopesticide**: B. thuringiensis is a Gram-positive, soil-residing bacterium commonly used as a bio-pesticide. B. thuringiensis also occurs naturally in the gut of caterpillars of various types of moths and butterflies, as well as on the dark surface of plants. - **Endotoxin**: During sporulation, many strains of B. thuringiensis produce proteinaceous crystal inclusion bodies, called endotoxins. - **Cry (crystal) toxin**: The endotoxins are called Cry toxin because they are pore-forming toxins, therefore, Cry toxin crystals may be extracted and used as a pesticide. - **Cyt (cytotoxin) toxin**: Cry toxins are encoded by cry genes which are located on the plasmid in certain bacterial strains. Cyt toxins are encoded by cyt genes. - **Specific activity** The toxins have specific activities against insect species of the orders Lepidoptera (moths and butterflies), Diptera (flies and mosquitoes), Coleoptera (beetles), Hymenoptera (wasps, bees, ants and sawflies) and nematodes. - **Cry toxin crystal**: When insects ingest toxin crystals, the alkaline pH of their digestive tract activates the Cry toxin. The Cry toxin crystals enter into the cell membrane of insect gut and form a pore that results in cell lysis and death of the insect. ### **Bt Cotton** - **White Gold**: Cotton is the most important commercial crop of India which is often referred to as 'white gold', and it provides employment to about 60 million people. - **Bollworms**: The important insect pests attacking cotton crops are classified into two broad groups: (i) sap sucking insect pests and (ii) leaf chewing insect pests. Bollworms alone reduce the yield up to 40 – 70%. - **Monsanto Holdings P Ltd**: Monsanto Holdings P Ltd (a US-based multinational agricultural biotechnology corporation) first released the commercial variety of Bt cotton (named Bollgard), which contains cry1A(c) gene of B. thuringiensis. - **Second Generation Bt Cotton**: Monsanto developed the 'second generation' of improved Bt-cotton by introducing the two Bt-genes, which is designated as Bollgard II'. Bollgard II cotton contains cryIIA(b) and crylA(c) genes. - **Bt cotton in India**: Mahyco (Maharastra Hybrid Seed Corporation) took the initiative to introduce Bt cotton technology into India in 1995. - **Bollgard Bt gene**: Mahyco received 100 grams of Bt cotton seeds (variety Cocker 312) containing the Bollgard Bt gene (cryIAc). - **Field trials**: In 1998, over 500 field trials were conducted in different agro-climatic regions to assess the efficacy of MON 531 against bollworms and the concomitant agronomic benefits. - **All India Coordinated Cotton Improvement Project (AICCIP)**: The Indian Council of Agricultural Research (ICAR) also conducted 55 multi-location field trials through its AICCIP program. - **Safety**: Several studies have shown that Bt cotton is safe to goats, cows, buffaloes, fish and poultry. Feed-safety studies with Bt cotton seed meal were carried out to show that the animals fed with Bt cotton seed meal were comparable to the control animals in various tests and showed no adverse effects. The Cry1A(c) protein of Bt-cotton did not have any direct effect on any of the non-target beneficial insects. - **Economic Impact**: The most extensive studies of transgenic crop adoption in developing countries have been conducted for insect-resistant (IR) cotton in Argentina, China, India, Mexico, and South Africa. The average percent difference between IR and conventional cotton for all farmers over all seasons is shown in the table below. | | **India** | **Argentina** | **China** | **Mexico** | **South Africa** | |---|---|---|---|---|---| | **Yield** | 34 | 33 | 19 | 11 | 65 | | **Revenue** | 33 | 34 | 23 | 9 | 65 | | **Pesticide costs** | - 41 | -47 | - 67 | - 77 | - 58 | | **Seed costs** | 17 | 530 | 95 | 165 | 89 | | **Profit** | 69 | 31 | 31 | 340 | 12 | 299 | ### **Bt Brinjal Controversy** - **Controversy**: Monsanto's promotion of GM brinjal crops in India resulted in controversy after reports of the failure of genetically modified cotton seeds. - **Safety concerns**: Environmentalists are of the opinion that the use of Bt brinjal on rats could be fatal. - **Convention on Biodiversity**: India, as a party to the Convention on Biodiversity has ratified the Cartagena Protocol, which provides a broad framework on biosafety, especially focusing on trans-boundary movements of GMOs and also covers seeds that are meant for intentional release into the environment. - **Bt cotton**: The first transgenic crop to be released in India was in 2002, which was the Bt cotton. After its introduction, there has been a lot of controversy. - **Large scale field trials**: The GEAC approved large-scale field trials for Bt brinjal in India in 2007. - **Moratorium**: In 2010, the Union Minister of Environment and Forest placed a moratorium on the release of genetically modified brinjal. ## **Modification in Nutrient Quality** - **Transgenic Arabidopsis thaliana**: A transgenic Arabidopsis thaliana has been developed that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant. - **Glycinin gene**: The glycinin gene from soybean has been introduced into rice and successfully expressed because glycinin is a lysine-rich protein. ### **Golden Rice (Oryza sativa)** - **Beta-carotene**: Golden rice is genetically engineered rice that biosynthesises beta-carotene (a precursor of pro-vitamin A) in its edible parts, i.e. the endosperm. - **Vitamin A deficiency**: Golden rice was developed for those countries where people suffer from vitamin A-deficiency. - **Environmental activists**: Concerns were raised by environmental activists and anti-globalisation activists that the genetically engineered rice was not safe. The development team introduced three genes involved in the biosynthetic pathway for carotenoid, synthesized in the endosperm as: - **Phytoene synthase (psy)**: The first transgene encodes phytoene synthase (psy), which utilises the endogenously synthesised geranylgeranyl-diphosphate to form phytoene. - **Carotene desaturase (crtl)**: The second encodes a bacterial carotene desaturase (cril) that introduces conjugation by adding four double bonds. - **B-Lycopene (B-lcy)**: The cDNA coding for phytoene synthase (psy) and lycopene B-cyclase (B-lcy) was from wild daffodil and the genes were introduced in a single combined transformation effort to obtain a functioning provitamin A (B-carotene) biosynthetic pathway. - **Genetically modified golden rice**: The combined activity of psy and crtl leads to the formation of lycopene, which is a red compound due to its undecaene chromophore. Lycopene has never been observed in any rice transformant. ## **Pests and Insect Resistance** ### **Bt Brinjal** - **Bt Brinjal**: Bt brinjal is genetically engineered brinjal that contains the cry1A(c) gene from **Bacillus thuringiensis**, which produces a protein that is toxic to the insect pest called **lepidoptera** (moths and butterflies), specifically the **bollworm**. - **Cry1A(c) gene**: The Bt gene is inserted into the plant's DNA. The protein produced by this gene kills the bollworm, therefore, the plant is protected from the pest. - **Resistance**: Brinjal has a high level of resistance to insect pests, particularly the **fruit borer** (a pest that attacks the fruit of the brinjal plant). - **Resistance mechanism**: When the insect eats the brinjal plant, the Cry1A(c) protein is ingested. The protein is toxic to the insect, but it is not toxic to humans or other animals or plants. - **Environmental impact**: The Bt brinjal is considered to be environmentally friendly because it reduces the need for chemical pesticides. - **Controversy**: The use of Bt brinjal is a contentious issue. Critics argue that it could have negative impacts on biodiversity and that it could lead to the development of resistant pests. - **Field trials**: Bt brinjal has been field-tested in India, but it is yet to be commercially available. - **Benefits**: Bt brinjal has the potential to reduce the use of pesticides, increase the yield of brinjal, and improve the livelihoods of farmers. ### **Bt Cotton** - **Bollgard**: Bollgard is the first commercial variety of Bt cotton released by the Monsanto Company. It contains the cry1A(c) gene of B. thuringiensis, and it is highly effective in controlling bollworms. - **Bollgard II**: Bollgard II cotton is the second generation of Bt cotton released by Monsanto. It contains two Bt genes, cryIIA(b) and crylA(c), which provide broader control over various insect pests. - **Bt-cotton in India**: Mahyco (Maharastra Hybrid Seed Corporation) introduced the Bt cotton technology into India in collaboration with Monsanto. ### **Biosafety Testing** - **Biosafety Tests**: Biosafety tests of Bt cotton were carried out extensively in India by several institutions, including the National Dairy Research Institute (Karnal), Central Avian Research Nutrition (Hyderabad), Central Institute of Fisheries Education (Mumbai), and G.B. Pant University for Agriculture and Technology (Pantnagar, Nainital). - **Safety to animals**: These studies have resulted in absolute safety to goats, cows, buffaloes, fish and poultry. - **Safety for human consumption**: Feed-safety studies with Bt cotton seed meal were carried out to show that animals fed with Bt cotton seed meal were comparable to the control animals in various tests and showed no adverse effects. - **Cry1A(c) protein**: The Cry1A(c) protein of Bt-cotton did not have any direct effect on any of the non-target beneficial insects. - **Non-target organisms**: Studies have shown that the Cry1A(c) protein of Bt-cotton is non-toxic to all other non-target organisms, such as beneficial insects, birds, fish, animals and human beings. ### **Economic Impact of Bt Cotton** - **Developing countries**: The most extensive studies of transgenic crop adoption in developing countries have been conducted for insect-resistant (IR) cotton in Argentina, China, India, Mexico, and South Africa. - **Farm-level impacts**: Each of the studies was based on data from two or three seasons of commercial farm production. - **Yields**: The studies estimated potential yield benefits of 80%. - **Economic benefits**: The economic studies were based on farm-level field trial data, and as such did not reflect the actual farm experience with commercial cultivation. - **National level benefits**: The studies found large net gains from IR cotton adoption at the national level in India, although significant variation was observed across states. - **Andhra Pradesh**: One state, Andhra Pradesh, experienced negative results, which may be due to the lack of locally adapted cultivars. - **Biosafety authorities**: The Indian Biosafety authorities had approved only four IR cotton varieties for use throughout the country. By 2005, that number had increased to 20, and the area of the country planted with IR cotton almost tripled from the previous year. ### **Genetically Modified (GM) Rice** - **Golden rice**: Golden rice is a genetically engineered rice that biosynthesises β-carotene (a precursor of pro-vitamin A) in its edible parts, i.e. the endosperm. - **Vitamin A deficiency**: Golden rice was developed for those countries where people suffer from vitamin A deficiency. - **Environmental concerns**: Concerns were raised by environmental activists and anti-globalisation activists that the genetically engineered rice was not safe. However, the development team addressed these concerns by introducing three genes involved in the biosynthetic pathway for carotenoid: - **Phytoene synthase (psy)**: The first transgene encodes phytoene synthase (psy), which utilises the endogenously synthesised geranylgeranyl-diphosphate to form phytoene. - **Carotene desaturase (crtl)**: The second encodes a bacterial carotene desaturase (cril) that introduces conjugation by adding four double bonds. - **B-Lycopene (B-lcy)**: The cDNA coding for phytoene synthase (psy) and lycopene B-cyclase (B-lcy) was from wild daffodil and the genes were introduced in a single combined transformation effort to obtain a functioning provitamin A (B-carotene) biosynthetic pathway. - **Biosynthetic pathway**: The combined activity of psy and crtl leads to the formation of lycopene, which is a red compound due to its undecaene chromophore. Lycopene has never been observed in any rice transformant. - **Pro-vitamin A:** Golden rice got enriched in pro-vitamin A. ### **Other GMO Crops** - **Transgenic crops with improved nutritional qualities**: Transgenic crops have been developed that can produce ten-fold higher vitamin E (alpha-tocopherol) than the native plant, by engineering the vitamin's metabolic pathway in the plant. - **Glycine**: Glycine is a lysine-rich protein found in soybean. The glycinin gene from soybean has been introduced into rice and successfully expressed. - **GM crops affecting insect resistance**: GMOs like Bt cotton and Bt brinjal aim at protecting the host plants from agricultural insects by expressing specific genes from Bacillus thuringiensis that are toxic to insects. The development of genetically modified organisms has a great potential to address challenges related to food security, environmental sustainability, and human health. However, it is essential to conduct thorough research, risk assessments, and public engagement to ensure the safe and responsible use of these technologies. The scientific and technological advancements in the field of genetic engineering continue to evolve, offering new possibilities for addressing global challenges. It is imperative to constantly evaluate the ethical, social, and environmental implications of these technologies and ensure that they are used for the benefit of humanity and the planet.