Transgenic Plants in IPM: Transgenic Plants in IPM . INTRODUCTION: INTRODUCTION Transgenic plant is simply a normal plant with one or more genes integrated from diverse sources. This is developed by cloning additional genes into plant genome by GE techniques. Added genes impart resistance to various factors affecting plants growth and yield. HISTORY: HISTORY Development of first TP i.e Tobacco with Bt gene for control of Manduca sexta in 1987. But 1996 is considered as landmark in agricultural biotechnology as 4 transgenic varieties of which 3 are IR cotton and a HR soybean varieties developed by Monsanto Company, received regulatory approvals. These were commercially grown & harvested for the first time in USA. PowerPoint Presentation: India made its entry into commercial agricultural biotechnology when Genetic Engineering Approval Committee (GEAC), Ministry of Environment & Forests and Govt. of India on 26th March 2002 approved three Bt -cotton hybrids for commercial cultivation. This is a historic decision as Bt -cotton became first transgenic crop to receive such an approval in India. STEPS INVOLVED IN DEVELOPMENT: STEPS INVOLVED IN DEVELOPMENT Locating Genes for Plant Traits Designing Genes for Insertion Transforming Plants Selection of plants Regeneration of whole plants Locating Genes for Plant Traits: Locating Genes for Plant Traits Identifying and locating genes for agriculturally important traits is most limiting step in transgenic process . First crop variety/accession having resistance is to be identified, then gene responsible for that resistance is identified in laboratory through DNA sequencing. Designing Genes for Insertion: Designing Genes for Insertion Once a gene has been isolated & cloned ( amplified in a bacterial vector), it must undergo several modifications before it can be effectively inserted into a plant. Simplified representation of a constructed transgene, containing necessary components for successful integration & expression. Transforming Plants: Transforming Plants It is the heritable change in a cell or organism brought about by the uptake & establishment of introduced DNA. There are two main methods of transforming plant cells & tissues : Gene gun method Agrobacterium method PowerPoint Presentation: Gene Gun method (micro projectile bombardment or biolistics) has been useful in transforming monocot species Agrobacterium method has been successfully practiced in dicots, but recently it has been found effective in monocots. This method is preferable than gene gun, because of the greater efficiency of single-site insertions of the foreign DNA, making it easier to monitor. Selection of Plants: Selection of Plants After gene insertion process, plant tissues are transferred to a selective medium containing an antibiotic or herbicide (depending on which selectable marker was used). Only plants expressing selectable marker gene will survive and possess the transgene of interest. Thus, subsequent steps in the process will only use these surviving plants. Regeneration of whole plants: Regeneration of whole plants To obtain whole plants from transgenic tissues such as immature embryos, they are grown under controlled environmental conditions in a series of media containing nutrients & hormones (Tissue Culture). Once whole plants are generated and produce seed, evaluation of progeny begins. Gene Components of TP’s: Gene Components of TP’s TP’s contain mainly following components Bt endotoxins Protease Inhibitors α-Amylase Inhibitors Lectins Enzymes Bt endotoxins: Bt endotoxins Bacillus thurigiensis is a gram positive entomocidal spore forming bacteria. Sporulating cells of Bt produce an inclusion body which are proteinaceous parasporal crystals (Cry). These crystal proteins when breakdown produce δ -endotoxin which is having toxic effects on some insects. PowerPoint Presentation: δ -endotoxin is the principal component of Bt’s insecticidal property. It is not a single toxin but a group of proteins produced from crystal protein in gut of insects of several orders. By introducing gene/s responsible for expression of Bt into plants controlling of some pests is achieved effectively. History of Bt: History of Bt Bt was first isolated from diseased larva of silkworm in Japan (1902) by Ishiwata . Name Bt was given by Berliner (1915) who isolated it from Mediterranean Flour Moth ( Ephestia kuehniella ) in Thuringinia of Germany. First commercial Bt product “ Sporeine ” was released from France in 1938. Parasporal inclusions of Bt were first described by Hannay (1953) later he demonstrated that these are proteinaceous in nature. PowerPoint Presentation: In 1959 Heimpel & Augus demonstrated that parasporal inclusions are responsible for insecticidal activity. Active and sustainable strain Bt kurstaki was isolated in France in 1972. In July, 1987 Plant Genetic systems, a Belgian Biotechnology Company developed first Transgenic Tobacco plant containing Bt endotoxin against Manduca sexta & Heliothis virescens . Later Monsanto (Tomato against Fruit worm & Pinworm) and Agrautus Company (Tobacco) developed transgenic plants with Bt gene. Strains of Bt used in TP’s: Strains of Bt used in TP’s About 68 strains have been identified & isolated from Bt, but only few are effective & they are exploited for commercial purpose. Bacillus thurigiensis var kurstaki (Lepidoptera) Bacillus thurigiensis var galleriae (Lepidoptera) Bacillus thurigiensis var israelensis (Diptera) Bacillus thurigiensis var tenebrionis (Coleoptera) Bt Proteins: Bt Proteins 5 main types of protein spores (Cry) are been isolated from Bt which have insecticidal effect on specific pest. Cry 1 – Lepidoptera Cry 2 – Lepidoptera & Diptera Cry 3 – Coleoptera Cry 4 – Diptera Cry 5 – Lepidoptera & Coleoptera PowerPoint Presentation: Several sub-strains of each protein particle has been identified and isolated. There are about 22 protein strains of Bt but most commonly used are as follows Cry 1Ab Cry 1Ac Cry 2Ab Spodoptera in Cotton Cry 3Ba Leptinotarsa decemlineata in Brinjal Helicoverpa, Pectinophora, Spodoptera (Cotton) Scirphophaga, Cnaphalocrosis (Rice) CryIAb for Phthorimaea (Potato), Heliothis (Tobacco) Mode of Action of Bt: Mode of Action of Bt Bt kurstaki : Protein crystals dissolve in midgut of lepidopteron insect’s which is alkaline (p H >9) by breaking of inter-protein disulphide bonds liberating toxins of 130 KDa. Proteolytic enzymes breakdown toxins size to 65-67 KDa. Then toxins diffuses easily across peritrophic membrane which acts as barrier for toxins of >100 KDa size. Toxins bind irreversibly with specific receptors/binding sites (glycoprotein of 120-180 KDa size) of midgut epithelial cells. Then cell lysis, midgut lesions, destruction of gut wall and finally death of insect occurs. PowerPoint Presentation: Bt tenebrionis : Proteins dissolve directly producing toxins of 73 KDa in coleopteron insect’s midgut which is almost neutral without requiring any activation, because it is soluble at both high (>10) & low (<4) p H as it lacks disulphide bonds between each of them. Effect of toxin is similar to Bt kurstaki however membrane lesions are not observed and first cellular response like swelling, elongation is slow. Bt israelensis : Require high p H >11 (dipterans) for dissolution and produces toxins of 27, 70, 135, 140 KDa and a 27 KDa cytolytic toxin (Cyt A) appears to synergize other toxins. Effect of toxin is same Advantages of Bt Crops: Advantages of Bt Crops The level of toxin expression can be very high thus efficient control of pest is possible. Toxin expression is contained within whole plant system and hence all plant parts are protected from insect pest attack. Act rapidly in the insect system. Safe to humans and non-target organisms. Completely bio-degradable thus eco-friendly. Labour cost is reduced as no insecticidal spray. No problem of drift or ground contamination. Disadvantages of Bt Crops: Disadvantages of Bt Crops Development of resistance to Bt toxin cannot be ruled out. Only some major pests are controlled but other & minor pests may outbreak. Control of pest population in one region using TP’s may affect population dynamics of pest in another region. Pollen produced is toxic & affect social insects especially pollinators of crop. Gene introduced may not be stable. Whether Bt toxins are safe to humans & animals?: Whether Bt toxins are safe to humans & animals? Bt proteins require specific p H for dissolution and release of toxins and toxins require specific receptors for binding and expressing their toxicity. But in the human or animal intestine proteins of Bt cannot dissolve because intestine is highly acidic (p H 1.5), if also dissolved and released toxins will not express because there are no receptors to bind toxins released. Protease Inhibitors: Protease Inhibitors Insects have proteases in their gut which help in digestion of protein. Protease inhibitors are enzymes synthesized in plants in response to insect attack which inhibit foreign proteases to start proteolysis and affect digestion of insects. PI’s are found in all classes of organisms and found in storage organs (seeds, tubers). Two types Serine Protease Inhibitors Thiol/Cysteine Protease Inhibitors PowerPoint Presentation: SPI: Many insects especially lepidopterans depend on serine proteases (trypsianse, chymotrypsinase) as their primary protein digestive enzymes. SPI is mostly found in legumes. Gene identified and transferred successfully was Cowpea Trypsin inhibitor (CpTi) found in Cowpea. This was identified by Gatehouse et al. (1979) at IITA while searching for resistant genotypes of Cowpea to Bruchid beetle and accession TVu 2027 which contained 2-4 times more CpTi than susceptible lines. This is found effective against some pest of Lepidoptera, Coleoptera and Orthoptera. PowerPoint Presentation: Mode of Action: Inhibits essential digestive proteases resulting in abnormal development & death of insect due to deficiency of essential amino acids. Crop Target Insect Pest Tobacco Heliothis virescens Rice Chilo suppressalis Tomato Lacanobia oleracea Potato Lacanobia oleracea Apple Cydia pomonella PowerPoint Presentation: CPI: These are found in cereals. CPI of Rice called Oryzacystatin found to inhibit nearly all proteolytic activity of Rice Weevil. Recently Cystatis from Corn also found to inhibit protease activity of weevil when introduced into Rice. OC-I: Rice Cysteine Inhibitor Plant Gene Target Insect Pest Rapeseed OC-I Coleoptera Poplar OC-I Chrysomela tremulae PowerPoint Presentation: Crop Gene Target Insect Pest Rapeseed & Poplar CII Lepidoptera and Diptera Lettuce Pot PI I&II Teleogryllus commodus Rice Pot PI I&II Sesamia inferens, Chilo suppressalis Tobacco Pot PI I&II Heliothis virescens Tomato Pot PI I&II Helicoverpa armigera, Teleogryllus commodus Tobacco Na PI Helicoverpa punctigera Additional PI’s: Double headed SPI from Soybean (CII). Potato Protease Inhibitor I & II (Pot PI I&II). Nicotiana alata Protease Inhibitor (Na PI). Mungbean Trypsin Inhibitor. PowerPoint Presentation: Advantages: They are anti-metabolite to wide range of pests. More effective and non-toxic to mammals. Resistance is stably inherited. Disadvantages: Varying degree of resistance is found in different crops. Effective control requires higher level of gene products (1% protein accumulation in leaves). α-amylase Inhibitors: α-amylase Inhibitors α-amylase is a digestive enzyme present in insects for digestion of CHO’s. α-AI’s inhibit activity of those enzymes and disrupt insect digestion. More than 10 α-AI’s were identified from Wheat and Rice plants that exhibit unique selectivities towards insect. Several were specific inhibitors of Rice Weevil, Red Flour Beetle & Yellow Mealworm enzymes. Recently transgenic Tobacco having α-AI encoding gene of wheat has shown resistance to Spodoptera litura & Agrotis ipsilon . PowerPoint Presentation: Crop Target Insect Pest Tobacco Spodoptera litura, Agrostis ipsilon Pea Zabrotes subfaciatus Azuki Bean Callosobruchus sinensis Lectins: Lectins These are group of plant proteins that bind to CHO’s including chitin. When insects feed on lectin containing food they bind with chitin in peritrophic membrane of midgut & prevents uptake of nutrients. Seeds of Common Bean (P. vulgaris ) contain CHO binding lectin called Phytohemagglutinin (PHA) found to be effective against some pests but ineffective against Bean Weevil and Bruchid pest. Whereas Acerlin-1 protein found in Wild Bean has shown effective against the above pests. Pea lectin gene (P-lec) in Tobacco plants is effective against Heliothis virescens . PowerPoint Presentation: Recently mannose specific lectin gene ( GNA ) from Snowdrops ( Galanthus nivalis ) introduced into Tobacco and transgenics showed resistance against Aphid ( Myzus persicae ). Similarly transgenic Potato expressing GNA shown resistance to Aphid, Tuber moth & Tomato moth ( Lacanobia oleracea ). Crop Gene Target Insect Pest Potato GNA Lacanobia oleracea, Myzus persicae Tobacco GNA Heliothis virescens , Myzus persicae Tomato GNA Lacanobia oleracea Tobacco p-lec Heliothis virescens Enzymes: Enzymes Genes expressing various enzymes has been proposed as a crop protection strategy. Most important is Chitinase enzyme. Transgenic Tobacco expressing Bean chitinase (BCH) enhanced resistance against some lepidopterans and similarly Transgenic Potato to Aphid ( A. solani ). Recently a bacterial endochitinase from Serratia marcesens shown synergism with Bt toxin against Serratia littoralis but not still introduced in commercial plants. Cholestrol oxidase secreted from Streptomyces has shown acute toxicity to larva of Cotton Boll Weevil ( Anthonomus grandis ). Advantages & Disadvantages of TP’s: Advantages & Disadvantages of TP’s Advantages: Effective against specific pest so safe to others. Biodegradable thus safe to ecosystem. All plant parts are having genes thus no insecticidal usage and less labour cost. Disadvantages: Development of resistance to TP’s also observed. Gene expression in subsequent generations is reduced due to natural mutations & others. Out-crossing of gene with weeds may create problem. Requirements and Priorities: Requirements and Priorities Factors for resistance should be controlled by single gene. Standardization of methods for transfer of such genes can be easily accomplished. Expression of transferred gene should occur in desired tissues at appropriate time. TP product should be safe for consumption. Inheritance of gene in successive generations should be very stable. There should be no penalty for yield in terms of other quarantine characters. Global Cultivation of TP’s: As for the data from International Service for Acquisition of Agri-Boitech Application (ISAAA) global cultivated area of transgenic crops during 2008-09 is as follows Global Cultivation of TP’s Transgenic Plants Research in India: Transgenic Plants Research in India Traits Crops Insect resistance Blackgram , Brinjal , Cabbage , Cauliflower , Chickpea , Cotton , Maize , Pigeon pea, Potato , Rice , Tobacco , Tomato , Wheat Disease resistance Blackgram , Brinjal , Chickpea , Coffee , Rice , Tomato Herbicide tolerance Blackgram , Cotton , Mustard , Rice Stress tolerance Mustard, Rice Edible vaccines Muskmelon, Tomato Fruit ripening Banana, Potato , Tomato Nutrition enhancement Mustard, Potato Regulation of Transgenic crops: Regulation of Transgenic crops Potential for food toxicity, food allergenicity , cross pollination and effect on non-target beneficial organisms including biological control agents. Govt of India, Ministry of Science & Technology ( MoST ), Ministry of Environment & Forests ( MoEF ). Department of Biotechnology (DBT), Institutional Bio-Safety Committee (IBSC) and Review Committee on Genetic Manipulation (RCGM), Genetic Engineering Approval Committee (GEAC) Ministry of Agriculture, Indian Council of Agricultural Research (ICAR), Indian Agricultural Research Institute (IARI), Agricultural Universities. Pyramiding of Genes: Pyramiding of Genes It is “Development of TP’s with more than one gene to get multi-mechanistic resistance”. Boulter (1990) first made such an approach by introduction of both CpTi and P-lec genes into Tobacco by cross breeding 2 primary transformed lines. He observed insecticidal effect of those two genes although not synergistic but they were additive. Gatehouse (1998) developed transgenic Potato expressing both GNA and BCH which shown synergistic effect in control of A. solani . PowerPoint Presentation: Points to be considered: Each component should act on different target within insect system. Mechanisms of resistance of 2 genes should be compatible. Transgenic Tobacco having SPI & GNA has not shown additive effect because GNA acts as antifeedant that reduces intake of SPI by target pest. Future Prospects of Transgenic Plants: Future Prospects of Transgenic Plants Identification of traits for control of other major and minor pests is required. Better marker genes to replace use of antibiotic resistant genes. Better control of gene expression through more specific promoters, so that inserted gene will be active only when and where needed. More efficient transformation, that is higher percentage of plant cells will be successfully incorporated with transgene. PowerPoint Presentation: Control of illegal transgenic seeds in country through enforcement of strict policies. Certain agencies are exploiting TP’s through sales of unapproved or spurious seeds. It was first discovered in Gujarat in 2000 when Navbharat Seeds Pvt. Limited was identified as offender. Later such illegal seeds were found in several other states also & continued to occupy several thousand hectares. Understanding public about uses & safety of TP’s in better way through different medias is required. PowerPoint Presentation: Karnataka Rajya Raitha Sangha (KRRS), uprooted & burnt few approved experimental crops in 1998 & 1999, wrongly accusing that Bt -cotton gene would escape & cause ‘Gene pollution’ and sterility in other plants. They also alleged that Bt protein is harmful to humans, farm animals, other beneficial organisms and soil. Development of TP’s with multi-genes using pyramiding of genes which are effective against wide range of pest is required. Monsanto Co. released Bollgard II® which contains two genes Cry1Ac & Cry2Ab which is better than Bollgard I® which is resistant to one pest. Because Bollgard II® apart from bollworm, gives protection against cutworm caterpillar. It was approved for US and Australia in 2002 and 2003 respectively. GEAC recommended Bollgard II® for Gujarat, Maharashtra and Madhya Pradesh.