Gene cloning in agriculture

gene cloning in agri

1. Gene Cloning in Agriculture Jannat Iftikhar B11-16 6 th semester 1

2. Contents Gene addition Gene subtraction Problem with genetically modified crops 2

3. Agriculture: World ‘s oldest biotechnology Human have constantly searched for improved varieties of their crop plants. Better nutritional qualities, higher yields. Gene cloning provides a new dimension to crop breeding. Enable directed changes to be made to genotype of a plant. 3

4. Two general strategies Gene addition: Cloning is used to alter characteristics of a plant by providing it by one or more new genes. Gene subtraction: Gene engineering techniques are used to inactivate one or more of plant’s existing genes. 4

5. Gene addition approach Gene addition: Use of cloning techniques to introduce into a plant one or more new genes coding for a useful characteristic that plant lacks. A good example : Development of plants that resist insect attack by synthesizing insecticides coded by cloned genes. A number of projects are being carried out around the world. 5

6. Plants that make their own insecticides Most conventional insecticides e.g. Pyrethroids & organophosphates. Relative non-specific poisons that kill a broad spectrum of insects. High toxicity-some also have potentially harmful side effects for other members of local biosphere. Exacerbate need to apply them to plants’ surfaces by spraying. Subsequent movement of them in ecosystem cannot be controlled. 6

7. The δ- endotoxins of Bacillus thuringiensis Intracellular crystalline bodies contain an insecticidal protein ( δ- endotoxins ). Highly poisonous to insects. More toxic than organophosphates (80,000X ). Relatively selective. Different strains of bacterium synthesizing proteins effective against larvae of different groups of insects. 7

8. Mode of action of δ- endotoxins 8

9. Cloning a δ- endotoxin gene in maize A major pest: European corn borer ( Ostrinia nubilialis ). 1 st attempt at countering this pest by engineering maize plants was made in 1993, with CryǀA version of protein. Cry protein is 1155 amino acid in length. Toxic activity residing in 29-607 amino acids. Rather than isolating the natural gene, a shortened version containing the first 648 codons was made by artificial gene synthesis . Introduction into the maize embryos. 9

10. Procedure used to obtain GM maize plants expressing an artificial δ- endotoxin 10

11. Cloning a δ- endotoxin gene in maize Immunological test Amount of δ- endotoxin varies from about 250ng to 1750ng. Difference due to positional effect. 11

12. Cloning δ-endotoxin genes in chloroplasts Tobacco: CryIIA (a2) gene A broader toxicity spectrum: two-winged fly as well as Lepidopterans . Fig. CryIIA (a2) operon, One advantage: chloroplasts like bacteria is able to express all genes in an operon. 12

13. Countering insect resistance to δ-endotoxin crop Crops synthesizing δ-endotoxin might become ineffective after a few seasons. Resistance among insect populations. Various strategies have been proposed to prevent the develop­ment of o-endotoxin resistant insects. 1 st , to develop crops expressing both the CryI and CryII genes. 2 nd , to engineer toxin production in such a way that synthesis occurs only in those parts of the plant that need protection. 3 rd , to mix GM plants with non-GM ones . 13

14. Herbicide resistant crops Most important transgenic plants: Those have been engineered to withstand herbicide glyphosate. Widely used by farmers & horticulturists. Environmentally friendly: non-toxic to insects & animals; a short residence time in soils; breaking down over a period of a few days into harmless products. Glyphosate kills all plants (both weeds & crops ). 14

15. ‘Roundup ready crops’ 1 st engineered crop for glyphosate, by Monsanto Co. called ‘roundup ready’. 15

16. A new generation of glyphosate resistant crops Recently a few report Organisms can actively degrade glyphosate. Relatively common among genus Bacillus . Possess an enzyme: glyphosate N- acetyltransferase (GAT). Detoxify glyphosate is by adding an acetyl group. Most active detoxifier: a strain of Bacillus licheniformis . Rates are too low to be of value if transferred to a GM crop. 16

17. Multigene shuffling; a type of directed evolution Bacterium possesses 3 related genes. Take parts of each member of a multigene family & reassembling these parts to create new gene variants. Most active genes are identified. Clone all variants in E. Coli & assay recombinant colonies for GAT activity. As substrates for next round of shuffling. 11 rounds: a gene specifies a GAT with 10,000X activity. GM maize: 6X in glyphosate tolerance. w ithout any reduction in productivity of plant. 17

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19. Other gene addition projects 19

20. Gene subtraction Misnomer modification does not involve actual removal of a gene, merely its inactivation. Several strategies Most successful, antisense technology 20

21. Antisense technology The gene to be clone is ligated into the vector in reverse orientation. When the cloned gene is transcribed, the RNA that is synthesized is the reverse compliment of the mRNA, sometimes abbreviated to asRNA . 21

22. Antisense RNA & engineering of fruit ripening in tomato GM tomato by antisense technology. Fruit ripening process is slowed down. Leave fruits on plant until they ripen to stage where flavor has fully developed. 22

23. Timescale for development of a fruit 23

24. Using antisense RNA to inactivate polygalacturonase gene Partial inactivation of polygalacturonase 730 bp restriction fragment Orientation was reversed Cauliflower mosaic virus promoter Plant poly(A) signal Ti plasmid pBIN19 Agrobacterium tumefaciens 24

25. Using antisense RNA to inactivate polygalacturonase gene 25

26. Using antisense RNA to inactivate ethylene synthesis Ethylene : a gas, acts as a hormone Switch on gene involved in later stage of tomato ripening 2nd way: delaying plant ripening Engineer plant: not synthesize ethylene Unable to complete ripening process Artificial ripening Spraying tomatoes with ethylene 26

27. Other gene subtraction projects 27

28. Problems with genetically modified plants Safety concerns with selectable markers The terminator technology 28

29. The terminator technology 29

30. Conclusion Gene cloning has revolutionized the agricultural practices. With the help of gene cloning we can introduce desired characteristers into the plants. There are many aspect yet to be discovered. 30

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