Genetic engineering & transgenic breeding

Genetic engineering & transgenic breeding

1. PRESEN TED BY: Pawan Nagar Reg. no.: 04-2690-2015 M.Sc. (Fruit Science) Genetic engineering & Transgenic breeding

2. Introduction Cell culture and transformation Cell Culture and Somatic Embryogenesis, a Means to Obtain Transgenic Plants 1 . Somatic Embryogenesis 2 . Organogenesis Methods of Plant Transformation 1 . Direct Transformation 2. Biological Delivery Methods 3. Selecting Transgenic Events Molecular Biology for Plant Transformation 1. Overexpression Vectors 2. Promoters for the Expression of Transgenes 3. RNA Interference 4. Zinc-Finger Nucleases Breeding with Transgenics / Transgenic breeding CONTENT

3. What is Transgenic Breeding ? Genetic improvement of crop plants, domestic animals, microbes through Biotechnology. What is Transgenics ? A genotype developed by the process of genetic engineering. Or A genotype containing foreign gene or modified gene of different species transferred by process of genetic engineering. What is Transgene ? Foreign gene or modified gene of any species which is used for development of transgenics . These may be from related wild species, microbes (Bacteria, viruses and fungi) or unrelated species.

4. Fig 1: A diagram re-drawn from Harlan and de Wet (1971) to include a description of gene pools as they relate to the use of transgenes . Circles are representations of the primary- (GP-1), secondary- (GP-2), tertiary- (GP-3), and quaternary-gene pools (GP-4)

5. Why Create Transgenic Plants? 1. Improve agricultural value of plant increase yield (herbicide-resistance, pest-resistance) enhance nutrition enhance taste 2. Plants can produce proteins for human needs (pharmaceutics) 3. Modified plants can be used to study effects of genes An entire plant can be regenerated from a single cell ***TOTIPOTENCY*** No separation of germ and somatic cells

6. Overview of The Process There are five major steps involved in genetically engineering plants. These are DNA isolation, single gene cloning, gene designing, cell transformation, and backcross breeding. DNA is extracted from an organism that has the desired trait. The desired gene is located and copied. The gene is inserted into a single plant cell using a transformation method. If the transgene successfully lands in the cells nucleus and is incorporated into one of the chromosomes, then the trait that it codes for will be expressed in the cell's offspring. The cell multiplies and grows a new plant that contains the transgene in all of its cells. Through backcross breeding the transgenic plant is crossed with a plant from a high yielding line. The resulting hybrids are the genetically modified plants that can enter the marketplace.

7. General procedure of recombinant DNA technology

8. Cell culture and transformation Cell Culture and Somatic Embryogenesis, a Means to Obtain Transgenic Plants Scheiden and Schwann made a critical observation, that plants possess a remarkable ability to generate free-living cells from plant tissues. Plant cell and tissue culture using sterile technique and in vitro (within glass) conditions are key elements to obtaining transgenic crop plants. Somatic Embryogenesis Totipotentcy : plant cell or cells are able to live independently, and also possess the ability to regenerate into a whole plant under the right environmental conditions. The first methodology of generating plants from cell culture is through the process of somatic embryo formation, whereby somatic cells (those not involved in sexual reproduction), produce an embryo similar to one produced by zygotic embryogenesis it is called somatic embryogenesis. Somatic embryos formed with root and shoot apical meristems are termed a bipolar embryo, and germinate into whole plants (Parrott, 2000). Organogenesis The second methodology of regenerating single cells into whole plants is termed organogenesis, where a meristematic cell from a root or shoot primordiums used to form organs (e.g. shoots, leaves or roots); these recovered organs can then be cultured into whole plants.

9. Methods of Plant Transformation The process of introducing DNA into living cells is called transformation. Introduced DNA is a viral DNA ,the process is called transfection. MICROINJECTION DNA solution is injected directly inside the cell using capillary glass micropipettes with the help of micromanipulators of a microinjection assembly. Involves the use of a micro-capillary to inject either the target DNA directly into the nucleus or the target RNA directly into the cytoplasm Partical Gun / Biolistics / Macroprojectile A device for injecting cells with genetic information The payload is an elemental particle of a heavy metal ( tungsten,gold,silver ) coated with plasmid DNA. This technique is often simply referred to as bioballistics or biolistics . Helium propellant and a multi-disk-collision delivery mechanism is used. Target of a gene gun is often a callus on gel medium in a petri dish

10. ELECTROPORATION Exposing cells for very brief period of electric pulses-induce transient pores in plasma lemma. Fiber mediated transformation Using silicon carbide fibres of 0.6micro m in diameter,10-80 micro m length. Chemical methods involves………. 1) Lipofection 2) PEG mediated transfer 3) Capo 4 precipitation method 4) DEAE Dextran method

11. Vector mediated transfer……… Agrobacterium mediated transformation Transformation of plant cells by Agrobacterium tumefaciens harboring a wild-type Ti plasmid

12. Molecular Biology for Plant Transformation Overexpression Vectors The importance of promoters in vector construction; as the choice of a promoter sequence and its optimization determines the constitutive, spatial, and/or temporal expression of transgenes Promoters for the Expression of Transgenes The regulation of gene expression is one of the goals in the development of transgenic cultivars. Promoters, enhancers and cis -acting regulatory sequences are all DNA elements, which control gene expression. Biotechnology has developed the ability to engineer chimeric expression cassettes, by combining promoters and coding regions from different genes. RNA Interference Gene silencing has resulted in the production of transgene cassettes capable of silencing specific genes in a dominant fashion. This process is termed RNA interference, or RNAi , and results from the production of double-stranded RNA ( dsRNA ) in the plant cell. The dsRNA produced specifically prevents the production of protein from messenger RNA of the target gene Zinc-Finger Nucleases Zinc-finger nuclease technology allows for constructs to be designed to target transgene insertion into a specific locus within the plant genome , used to produce knockout mutations by disrupting the sequence of the target gene; i.e. causing a frame-shift mutation, elimination of the start codon or important motif, or a complete removal of the target gene.

13. Breeding with Transgenics After obtaining a transgenic event, the genetic engineer hands the seeds over to the plant breeder. Lines that survive transformation and tissue culture well are typically lower yielding than current elite lines. To make these transgenic lines marketable, plant breeders must use breeding techniques to transfer the transgene into a high yielding elite line

14. First, the breeder obtains an inbred line by self-pollinating the transgenic line . Each plant cell in the transgenic inbred line now contains two copies of the gene. The transgenic seeds produced are planted along with seeds from an elite inbred. Due to a lack of hybrid vigor, both of the inbred plants that grow from these seeds will be smaller than current hybrids. When the plants reach the proper stage, they are cross-pollinated.

15. The seed from this crop, the F1 seed , is harvested. All of the offspring have one copy of the transgene , as well as 50% elite and 50% non-elite genes. The F1 seed is planted near another elite inbred seed. The plants grow. Due to hybrid vigor, the F1 plant is larger than the elite inbred, but it still contains many undesirable genes. The F1 plant is mated back to the elite inbred. This process is known as backcrossing .

16. This seed, the backcross 1 (BC1) generation is harvested. The plants that grow from these seeds will have 75% elite genes , and half will contain the transgene . Again, BC1 plants are grown along with elite inbreds . Those that express the transgene are cross-pollinated with the elite inbreds .

17. This process is continued until the plants contain at least 98% of the elite genes and the transgene . This takes approximately 5 to 6 generations .

18. Comparison Classical breeding Transgenic breeding Desired gene is incorporated along with many other genes ,which then have to be bred out Desired gene only is transformed from one organism to another Crosses limited by species barriers Species barriers may be overcome

19. Transgenic Breeding for Increasing Yield Crop Non transgenic (yield per ha.) Transgenic (yield per ha.) Improvement in yield by % 1. Cotton 8-10 qtl . 14 qtl . 14 2. Corn (Forage) 50-60 qtl . 70 qtl . 7 3.Soybean 20-22 qtl . 28-30 qtl . 20 ( Source- CICR Technical bulletin – 22).

20. Transgenic Breeding for Insect Resistance Crop Gene inserted Resistance to Vector Achievement Year 1) Cotton Cry gene complex Bollworm complex Agrobacterium tumefacience 1 st Bt cotton in world by Monsanto Ltd. USA. 2) 1 st Bt cotton in india -1996 by MAHYCO -Monsanto Ltd. 1987 1996 2) Brinjal Cry 1 Ab Fruit and shoot borer Agrobacterium tumefacience 1) 1 st transgenic (Bt) Brinjal in world ( USA) 2004 3) Cabbage Cry gene complex Cabbage worm and cabbage looper Agrobacterium tumefacience 1) 1 st transgenic (Bt) Cabbage in world (USA) 2002 4)Maize Cry gene complex European Corn Borer Agrobacterium tumefacience 1) 1 st Transgenic maize in world is Maximizer (Spain) by Company Ciba Geigy 1995 5)Okra Cry 1 Ab Fruit and shoot borer Agrobacterium tumefacience 1) 1 st Transgenic okra in world by U.S.A. 2002

21. Crop Gene inserted Resistance to Vector Achievement Year 6) Potato Cry- gene complex Colorado potato beetle Agrobacterium tumefacience First transgenic potato in world – Monsanto Ltd. U.S.A. 1995 7) Apple Cry-1-Ac Codling moth Agrobacterium tumefacience First transgenic Apple in world by U.S.A. 2000 8) Soybean Cry-1-Ac Leaf eating catterpillar Agrobacterium tumefacience First transgenic Soybean in world – Monsanto Ltd. U.S.A. 1996 9) Sugar cane Cry-1-Ab Stem borer Agrobacterium tumefacience First transgenic Sugar cane in world – U.S.A. DNA plant technology company 1997 10) Tomato Cry-1-Ac Tobacco horn worm Agrobacterium tumefacience First transgenic Tomato in world – U.S.A by Calgene company 1987 11)Tobacco Trypsin inhibitor gene from cowpea Leaf eating catterpillar Agrobacterium tumefacience First transgenic tobacco resistant to leaf eating catterpillar was developed in USA. 2001. Source – Esseentials of Plant Breeding

22. Transgenic breeding for keeping quality Crop Gene inserted Traits Vector Achievement 1) Tomato - Delay ripening b) Delay fruit softening c) Thicker skin and Alter pectin content a) Agrobacterium tumefacience b) Antisense RNA technology c) Agrobacterium tumefacience Variety : Endless summer by DNA plant technology company 1995 b) Flavr Savr by Calgene company in 1994 c) By Zeneca company 1995

23. Crop Gene inserted Traits Vector Achievement 2) Apple ACC oxidase gene Delay ripening Agrobacterium tumefacience In scion cultivar ROYAL GALA is developed 3) Banana ACC gene Delay ripening Agrobacterium tumefacience Cavendish 1 4) Mango Rol C gene Storage and delay ripening Agrobacterium tumefacience Golden yellow ( Source - AGROBIAS Newsletter. 2006).

24. Transgenic breeding for disease resistance Crop Gene inserted Resistance to Vector Achievement 1) Potato PLRV-R PVX, PVY, PLRV Agrobacterium tumefacience First transgenic potato in world by – Monsanto Ltd. U.S.A. 2000 2) Cumcumber GUS and Markar gene NPT II CMV, ZYMV, WMV 2 Agrobacterium tumefacience First resistance variety Beit alpha MR 3) Faba bean Chitinase gene from Seratica marcescence Chocolate spot disease Plasmid First resistance variety in 2002 4) Banana FR gene Panama wilt Plasmid First resistance variety isCavendish 5) Papaya PRVR gene Papaya ring spot virus Plasmid First resistance variety is Sunup and second variety is Rainbow in 1999 Hawai .

25. Transgenic breeding for Herbicide Resistance Crop Gene transfer resistance to Vector Achivement 1.Rice Bar chimeric gene Hygromycine Plasmid Oryza sativa cv. IR 72 2.Nilgiri (Eucalyptus) Cry 3A Glufosinate ammonium(Liberty@6l/ha) Agrobacterium tumefacience Variety- Ecofriend 3.Sugarcane PPTR( phospinothricine resistant gene) Phosphinothricine Agrobacterium tumefacience I st herbicide resistant sugarcane was developed in USA . 4.Cotton Phosphinothric acetyl transeferase All non selective herbicides Particle gun bombardment method I st variety was releasedIn year 2000.

26. Crop Gene transferred Resistance to Vector/ Transferred by Achievement 5.Pineapple GUS (B- glucuronidase ) and Bialphos resistant (Bar) gene BASTA Microprojectile bombardment method Var - Bialphos Ready 6.Sugarbeet Rr gene ( roundup resistant gene) Round up Microprojectile bombardment method Variety-H7-1 Released by Monsanto Ltd. in 2008 in USA. 7.Soybean Rr gene ( roundup resistant gene) Round up Microprojectile bombardment method I st Roundup resistant var. in soybean was released in 1997 in USA. (Source –www.google.co.in)

27. Transgenic breeding for adding Male sterility Crop Gene inserted Source of gene 1.Rapeseed mustard msi (male sterility iducing gene) Bacillus amyloliquefacience Source – Essentials of Plant Breeding , Transgenic breeding for drought resistance 1.Drought resistance in sorghum Gene transformed mlt D gene Vector Agrobacterium tumefacience Developed at CRIDA, Hyderabad Number of transgenics released 14.

28. 2. Drought resistance in Wheat 1. Crop American bread wheat 2.Genes transformed mlt D and HVA 1. 3.Source of gene Osmotin gene complex from Atriplex numularia 4. By Microprojectile bombardment system Transgenic breeding for changing morphological character 1. Crop Petunia 2. Character modified Orange flower colour 3.Gene transferred Dihydroflavanal 4 reductase (FR) gene. 4.Source of gene Zea mays L. (Corn) Original Petunia Modified Petunia

29. Increasing grain yield and fodder yield. Creation of disease resistant, pest resistant, adding quality characters, inducing male sterility, production of herbicide resistant, metal toxicity resistant crops . Reduces cost of cultivation . Reduction in air pollution caused by use of the pesticides. An ecofriendly method. Source – Essentials of Plant Breeding Advantages of Transgenic Breeding

30. Disadvantages of Transgenic Breeding High cost of seed production. Require high skill . Require high cost facilities for laboratory work. Screening of large number of protoplasts is essential. Occurance of minor pests increases. Production of the resistant generations of pests. Transfer of only monogenic character is possible at a time. Unstable performance at several locations. Monopoly of private seed companies Source – Essentials of Plant Breeding

31. Thank You!