Transgenic Plants: Experiences and Challenges

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1 Transgenic Plants: Experiences and Challenges Dr Anil Kumar Associate Professor & Incharge Deptt. Of Molecular Biology & Genetic Engineering, CBSH GBPUAT Pantnagar Uttranchal Pin

2 Agriculture : a scenario Main stay of our economy Productivity enhancement by combined efforts of agricultural scientists Green revolution in mid 60 s and 70 s in 20 th century Alleviation of world hunger, poverty and malnutrition Food security Nutritional security

3 Enhanced Agricultural Productivity: Why? Burgeoning and upsurging population (160 persons per minute) Plateuing of productivity due to adopting traditional farming practices Sinking of cultivable land Decaying of natural agricultural resource base Catering need of over 11 billion people by doubling of grain yield by 2050

4 POPULATION GROWTH : URGENT NEED TO INCREASE AGRICULTURAL PRODUCTIVITY POPULATION (MILLION) INDIAN POPULATION IS EXPONENTIALLY GROWING AND EXPECTED TO REACH 1.38 BILLION BY 2020.

5 Biotechnology : An answer Crop improvement programme through new found wisdom of Biotechnology Remedy of stagnation of crop productivity Impact on food production system through balanced approach and knowledge of Plant tissue culture Molecular Biology Cell Biology Biochemical Engineering Gene technology Plant breeding

6 Plant Breeding Biotechnology Improved Genotypes/ Cultivars Integrated Farm Management Enhanced crop Productivity And Production

7 Gene technology An additional tool for crop improvement programme Two keys of plant improvement Creation of variation Selection for positive attribute The attributes : Improvement of important agricultural traits Food and nutritional security Producing specialty foods, biochemicals, pharamaceuticals and smart designer crops Protecting plants against the ever increasing threats of biotic stress Alleviating the hazards of abiotic stress

8 IS IT POSSIBLE? GENE TECHNOLOGY MAKES IT POSSIBLE PRO--PRO P P PRO--EUK P Bacteria EUK--EUK EUK--PRO GENE Plant Animal

9 Transgenic Technology : Art and science of creating designer crops Identification of useful gene (s) Creation of a suitable gene construct Transfer of this construct in to plant cells/tissues in vitro ( maintained as organized explants such as immature embryos, stem sections, cotyledons etc. Using tissue culture techniques, through a process called transformation. Selection of transformed cell lines or seedling using a suitable marker system and regeneration of fertile plants from the transformed cells. Analysis of transformed plants for several aspects including stable integration, expression and genetic behaviour of transgene(s)

10 Procedure to develop transgenic biotech crop.

11 How are Transgenic crops made? Locating Genes for Plant Traits Identifying & locating genes for agriculturally important traits : Most limiting step in transgenic process Little is Known about specific genes to enhance yield potential improve stress tolerance modify chemical properties of harvested products otherwise affect plant characters Identifying a single gene for a trait is not sufficient Must understand how the gene is regulated What other effects it might have on the plant How it interacts with other genes active in the same biochemical pathway

12 Designing Genes for Insertion Once a gene has been isolated & cloned : it must be suitably modified for effective insertion into a plant A promoter sequence must be added for the gene to be correctly expressed Sometimes, the cloned gene is modified to achieve greater expression in a plant The termination sequence signals to the cellular machinery that the end of the gene sequence has been reached A selectable marker gene is added to the gene "construct" in order to identify plant cells or tissues that have successfully integrated the transgene.

13 Simplified representation of a constructed transgene, containing necessary components for successful integration and expression

14 Promoters used for Transgene expression It is the ON/OFF switch : controls when & where in the plant the gene will be expressed Most promoters used are CONSTITUTIVE : cause gene expression throughout the life cycle of plant in most tissues. CaMV 35S Promoter : Most commonly used & gives high degree of expression in plants Other promoters : More Specific : respond to cues in the plant s internal & external environment Promoter from cab gene (encoding chlorophyll a/b binding proteins) : Light inducible Promoters from Arabidopsis : Specifically & rapidly induced by natural plant stress/wounding related semiochemical cis-jasmone

15 Selectable Marker Genes Gene that facilitates the detection of genetically modified plant tissue during development Two major types of genes Conferring resistance to antibiotics Conferring tolerance to herbicides Other genes coming up

16 Drug (Antibiotic) resistance Marker npt II (Neomycin Phosphotransferase) hpt (Hygromycin Phosphotransferase) dhfr (Dihydrofolate reductase) ble gat (Gentamycin Acetyltransferase) Substrate for selection G418, Kanamycin, Neomycin Hygromycin Methotrexate, Trimethoprim Bleomycin Gentamycin

17 Herbicide resistance marker bar & pat (Phosphinothricin acetyltransferase) epsps (5-enolpyruvylshikhimate-3- phosphate synthase) Substrate for Selection Phosphenothricin (Bialaphos, Basta) Glyphosate

18 Risks & Concerns of using Antibiotic Resistance Genes Horizontal Gene Transfer Transfer of these genes from the transgenic plants to the microbes Make the bacteria in the guts of animals and humans resistant to antibiotics Make antibiotic medicines less effective Transfer to soil microbes : A field study shows that transgenic DNA persists for 2 years after the GM crop has been harvested

19 Other genes used as Selectable Marker Genes man A gene from E coli encodes mannose phosphate transferase and confer upon transformed cells ability to use mannose as a sole carbon source Ipt (isopentenyl transferase) gene from Agrobacterium located on T-DNA and induces cytokinin synthesis. Plants selected on the basis of their ability to produce shoots from callus on medium lacking cytokinins. Betaine aldehyde dehydrogenase gene (BADH) from Spinach Used with chloroplast genome.converts toxic betaine aldehyde to non-toxic glycine betaine, which also serves as an osmoprotectant and confer drought/salt tolerance

20 Selection of successfully transformed tissues Following the 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 the selectable marker gene will survive, and it is assumed that these plants will also possess the transgene of interest. Thus, subsequent steps in the process will only use these surviving plants.

21 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 and hormones, a process known as tissue culture. Once whole plants are generated and produce seed, evaluation of the progeny begins. This regeneration step has been a stumbling block in producing transgenic plants in many species, but specific varieties of most crops can now be transformed and regenerated.

22 PRODUCTS WHEAT TRANSGENIC CROPS DEVELOPED SO FAR AT PANTNAGAR GENE INTROGR -ESSED PAT GENETICALLY ALTERED TRAITS HERBICIDE RESISTANCE (BASTA) BRASSICA OSMOTIN TOLERANCE TO ALTERNARIA BLIGHT AND SALINITY BRASSICA TOMATO CARROT ANNEXIN Pr M E Pr M E GUS MODULATION OF HYPERSENSITIVE RESPONSE AGAINST ALTERNARIA BLIGHT AND POWDERY MILDEW PRODUCTION OF EDIBLE VACCINE AGAINST JAPANESE ENCEPHALITIS VIRUS

23 TRANSGENIC TECHNOLOGY AN APPROACH FOR ENGINEERING RESISTANCE CELL PROLIFERATION CELL DEATH PLANT DEFENSE?? SYSTEMIC RESISTANT GENE OSMOTIN & ANNEXIN?? DIFFERENTIATION & DEVELOPMENT AT LEAST FOUR DIFFERENT SIGNALLING PATHWAYS ARE KNOWN TO EXIST IN PLANT CELL. ONE OR MORE OF SIGNALLING COMPONENTS CAN BE MODULATED BY TRANSFERRING STRESS RELATED USEFUL GENE. THIS MAY ENGINEER RESISTANCE.

24 Different stages of hardening of transformed plants. (a) Rooted PCR positive shoots transferred in plastic pots covered with polybags kept for hardening at transgenic glass house. (b) A PCR positive plant growing in the pot. (c) A hardened PCR positive plant growing in plantation pot after 2 months. (d) Hardened PCR positive plants growing at transgenic glass house.

25 Alternaria blight tolerance in Brassica

26 Five Brassica transformants harboring Annexin(T0 plants) were scored for various diseases, no. of pods, no. of seeds etc. PLANT NO. Natural infection in plants growing in glass house under high humidity and high temp No. of pods obtained No. of seeds obtained Control Alternaria blight (++) and powdery mildew(++) D38 Only powdery mildew (+++) D14 Only powdery mildew (+++) D11 (1) Alternaria blight (++) and powdery mildew (++) D62 Died early (only powdery mildew) 6 35 D17 Alternaria blight (+) and powdery mildew (++)

27 INHERITANCE OF ANNEXIN TRANSGENE IN T 1 PLANTS M M kb 1.0 kb Annexin PCR of T 1 progeny of T 0 plant Lane M = 100 bp ladder Lanes 1-10 = T 1 progeny plants of T 0 plant THIS EXPERIMENT HAS FURTHER REVEALED THAT A TRANSFORMANT PLANT HAS MOSAICS OF GENE WHEN GROWN IN VITRO FROM CALLUS. THE GENE MOSAICS COULD OCCUR AT BOTH ORGAN AND TISSUE OR CELLULAR LEVEL.

28 Edible vaccine against Japanese Encephalitis virus Callusing Shoot initiation Different stages of regeneration in tomato Rooting in solid medium Rooting in liquid medium

29 Transformed plant kept for hardening Presence of Transgene in tomato by PCR amplification

30 Problems Associated with production of Transgenic plants Low regeneration frequency associated with albinism and anthocyanin pigmentation which varied from explants to explants and crop to crop and resolved by the use of additives Low transformation frequency after cocultivation, varied from to 5% in our experiments due to induction of hypersensitive response by Agrobacterium Large number of escapes when Kanamycin is used as a selectable marker

31 Production of gene mosaics especially in the transgenic plants developed through organogenesis during callus culture Pleiotropic effects of transgene insertions like dwarfism, seed germination, developmental and differentiation changes and alteration of other important processes and traits Segregation problems in subsequent generations if homozygosity is not obtained Problems of stability in subsequent generations due to gene silencing

32 Transgene silencing Reduced/abolished expression of foreign gene Loss of expression : Not due to loss of transgene but due to their inactivation Concept of gene Space : Genomes are made of isochores (long stretches of DNA with high compositional homogeneity) If a GC rich transgene is integrated into a GC isochore or an AT rich transgene is integrated into an AT isochore : It is Transcribed If a GC rich transgene is integrated into the AT rich gene space or vice versa : It is Inactivated, as there is no compositional homogeneity with the neighboring sequences

33 Position Dependent & Sequence Dependent Gene silencing Transgene integrates into a genomic region containing heterochromatin. The repressive chromatin structure & DNA methylation can spread into the transgenic locus from the flanking genomic DNA Homology Dependent Gene Silencing Caused by Multiple copies of transgene (Repeat induced gene silencing) Affect not only the stability of transgene but also alter the activity of endogenous gene (Cosuppression)

34 Transcriptional Gene Silencing No mrna is produced from silenced gene Affected loci : nucleation points for heterochromatin formation & DNA methylation Post Transcriptional Gene Silencing Transcription is required for silencing to take place Induce degradation of mrna : very little accumulate in cytoplasm

35 Genetically Modified Organisms Pitfalls Possible cause(s) Multiple transgene copies. Loss of proper feedback control. Bad expression: Level, tissue, time. Possible outcome(s) Organism out of harmony Low viability or death. Should be detectable during development. Risk minimization Use progressive methods Disturbance at insertion site Danger is with occasional need for normal function, such as resistance to a rare pathogen. Insertion in appropriately benign region.

36 Genetically Modified Organisms Pitfalls Possible cause(s) Resulting organism conflicts with environment and/or interacting organisms. Possible outcome(s) Threatened insect populations. Resistant pests. GMOs could spread out of control, either directly or via their gametes. Risk minimization Understand the species, its modes of propagation, and its interactions with other species and the environment. Resulting organism generates inappropriate food product. Hormones, pesticides, residues, allergens etc. in product. Understand risks and test widely for safety.

37 Genetically Modified Organisms Pitfalls Possible cause(s) Possible outcome(s) Risk minimization Public perception on safety, ethics, welfare. Market failure Generate arguably safe GMOs and educate public maybe difficult. Other unknown causes Other unknown outcomes Keep an open and critical mind.

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