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GENETIC MANIPULATION THROUGH RECOMBINATION BREEDING COURSE
- 2. GENETIC MANIPULATION The manipulation of genetic material to produce specific results in an organism OR The use of various methods to manipulate the DNA (genetic material) of cells to change hereditary traits or produce biological products. The first organisms engineered were bacteria in 1973 and then mice in 1974. Insulin producing bacteria were commercialized in 1982 and genetically modified food has been sold since 1994.
- 3. What is Recombinant DNA? The process of forming new allelic combination in offspring by exchanges between genetic materials (as exchange of DNA sequences between DNA molecules)
- 4. Modern Genetic Modification Inserting one or few genes to achieve desired traits. GM Crops Relatively Specific Changes are Subtle Allows Flexibility Expeditious
- 5. Need for responsive crop improvement -World population growth -Climate change -Water scarcity -Land degradation -Continual risks of new disease epidemics 2009Issues in classical breeding strategy -Narrow genetic base -Tedious or inaccurate selection methods -> Slow and limited genetic progress Need for new resources and technologies Need for new technologies in plant breeding
- 6. Methods Used to Date for Plant Transformation Agrobacterium tumefaciens Gene Gun Microprojectiles (PDS) Microinjection Silica Carbide fibers Microlaser Ultrasound mediated Lipofection Electroporation
- 7. Agrobacterium tumefaciens Mediated Transformation Agrobacteria are nature plant parasites, and their natural ability to transfer genes is used for the development of genetically engineered plants. To create a suitable environment for themselves , these Agrobacteria insert their genes into plant hosts , resulting in a proliferation of plant cells near the soil level (crown gall). The genetic information for tumour growth is encoded on a mobile, circular DNA fragment (plasmid). When Agrobacteria infects a plant , it transfers this T-DNA to random site in plant genome. When used in genetic engineering the bacterial T-DNA is removed from the bacterial plasmid and replaced with the desired foreign gene.
- 8. plasmid Desired DNA Agrobacterium tumefaciens containing Wt Ti plasmid A. tumefaciens containing engineered Ti plasmid Plant cell inoculated with A. tumefaciens Plant cell containing Desired DNA Cultured plant cells Regenerant Adult plant expressing desired trait (DNA) Inserting foreign genes into plant cells. A plasmid containing DNA is cut with a restriction enzyme & DNA of desired gene (red) inserted. Desired gene then inserted into Ti (tumor-inducing) plasmid naturally found in A. tumefaciens. Plant cell inoculated with A. tumefaciens containing engineered Ti plasmid + the desired DNA transfers desired DNA + t-DNA into plant chromosomes. Plantlets with desired trait then regenerated.
- 9. BIOLISTIC METHOD In the biolistic method ,DNA is bound to tiny particles of gold or tungsten which are subsequently “shot” into plant tissue or single plant cells under high pressure. The accelerated particles penetrate both the cell wall and membranes. The DNA separates from the metal and is integrated into the plant genome inside the nucleus. This method has been applied successfully for many cultivated crop , especially monocots like wheat or maize, for which transformation using Agrobacterium tumefaciens has been less successful. The major disadvantage of this procedure is that serious damage can be done to the cellular tissue.
- 10. The Gene Gun PDS1000 Microparticle Delivery System Helium chamber Rupture disk Macrocarrier DNA coated gold particle Stopping screen Focusing device Target tissue Gene gun
- 11. Biolistic approach has advantages and potential for general applicability • It is easy to handle. • One shot can lead to multiple hits (transfer of genes into many cells). • Cells survive the intrusion of one particle. • The genes coated on the particle have biological activity. • Target cells can be as different as pollen, cell culture cells, cells in differentiated tissues and meristems. • They can be located at the surface or in deeper layers of organs. • The method depends on physical parameters only, and so on.
- 12. Microinjection • Microinjection uses micro capillaries and microscopic devices to deliver DNA into defined cells in such a way that the injected cell survives and can proliferate • In comparison with biolistics, microinjection has disadvantages, Only one cell receives DNA per injection, and handling requires more skill and instrumentation It also has advantages: 1. The quantity of DNA delivered can be optimized. 2. The experimenter can decide into which cell to deliver DNA. 3. Delivery is precise and predictable, even into the cell nucleus, and is under visual control. 4. Cells of small structures (e.g. microspores and few-celled proembryos,) which are not available in the large quantities required for the biolistic technique, can be precisely targeted. 5. Defined micro injected cells can be microcultured.
- 13. Electroporation Involves a pulse of high voltage applied to protoplasts /cells/tissues to make temporary pores in the plasma membrane which facilitates the uptake of foreign DNA. The cells are placed in a solution containing DNA and the subjected to electrical shocks to cause holes in the membranes. The foreign DNA fragments enter though the holes into the cytoplasm and then to nucleus.
- 14. •Maize and tobacco tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells • DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA • Maize cells were treated with a plasmid carrying both the BAR gene, whose product confers resistance to the herbicide BASTA, and a gene encoding fl- glucuronidase (GUS). • Tobacco cells were treated with two plasmids to co-transfer genes encoding neomycin phosphotransferase (NPTI1) and GUS from the respective plasmids Silica Carbide fibers
- 15. Microlaser • A microlaser beam focused into the light path of a microscope can be used to bum holes into cell walls and membranes •There are no conclusive data available on DNA uptake, and there are problems with DNA adsorption to cell wall material even before it could be taken up •As microinjection and biolistics definitely transport DNA into walled plant cells, the microlaser could offer advantages only in very specific cases where those techniques would not be applicable •It was hoped that incubation of perforated cells in DNA solutions could serve as a basis for vector independent gene transfer into walled cells
- 16. ULTRASOUND MEDIATED DNA TRANSFORMATION Ultrasound is used described for stimulating uptake of foreign DNA by plant protoplasts and leaf segments of tobacco. Procedure involves immersion of explant in sonication buffer containing plasmid DNA and sonication with an Ultrasonic pulse generator at an acoustic intensity of o.5 w/cm square for 30min. Samples are rinsed in a buffer solution and then cultured for growth and differentiation. This technique has the advantages of being simple ,inexpensive and multifunctional.
- 17. LIPOSOME MEDIATED GENE TRANSFER OR LIPOFECTION Liposome are circular lipid molecules with an aqueous interior that can carry nucleic acid . Liposome encapsulate the DNA fragments and then adhere to cell membranes and fuse with them to transfer DNA fragments. Thus ,the DNA enters cell and then to the nucleus. Liposome is a very efficient technique used to transfer gene in bacterial ,animal and plant cells.
- 18. Biological Aspects There seem to be at least four major objectives being pursued at this time in crop plant genetic engineering research. These are: •To improve biological protection of crops against insects, weeds and fungi by inserting genes for the natural production of an insecticide (Feder, 1996) or for resistance to fungi or an herbicide (Hinchee et al, 1988). •To elevate levels of important nutrients (e.g. methionine levels in soybeans - Beardsley, 1996) so as to make crops more nutritious. • To obtain better control of ripening and post-harvest storage life to assure that produce are in peak condition when taken to market (Maryanski, 1995). •To specifically modify genomes to produce a specific product (e.g. a caffeine-less coffee bean, edible vaccines in potatoes - Pollack, 2000).
- 19. Possible dangers of genetic engineering Genetic engineering has numerous potential benefits, some of which have been discussed. However any new scientific discoveries offer the possibility of both beneficial and destructive effects. Some of the possible dangers due to genetic engineering might be as follows: 1. Due to manipulation of genes might, by accident, result in the origin of various new kinds of diseases or organisms containing fatal genetic element. 2. There is a risk of creation of drug resistance germs and out-break diseases against which there is no known prevention. 3. Any accidental escape of laboratory strains may create havoc on earth. It may contaminate a large population. 4. Introduction of gene like that of viral cancer into bacteria through plasmids may involve the risk of introducing these harmful genes into man when these bacteria infect human. 5. Hybrid genomes may create some serious ecological problems, the nature of which is still unknown. Naturally all these harmful hybrids possibly raise many moral, ethical and legal questions.
- 20. Food Properties of the genetically modified variety Modification Percent Modified in US Percent Modified in world Soybeans Resistant to glyphosate or glufosinate herbicides Herbicide resistant gene taken from bacteria inserted into soybean 93% 77% Corn, field (Maize) Resistant to glyphosate or glufosinate herbicides. Insect resistance via producing Bt proteins, some previously used as pesticides in organic crop production. Vitamin-enriched corn derived from South African white corn variety M37W has bright orange kernels, with 169x increase in beta carotene, 6x the vitamin C and 2x folate. New genes, some from the bacterium Bacillus thuringiensis, added/transferred into plant genome. 86% 26% Cotton (cottonseed oil) Pest-resistant cotton Bt crystal protein gene added/transferred into plant genome 93% 49% Alfalfa Resistant to glyphosate or glufosinate herbicides New genes added/transferred into plant genome. Planted in the US from 2005– 2007; banned until January 2011 and presently deregulated
- 21. Hawaiian papaya Variety is resistant to the papaya ringspot virus.[ New gene added/transferred into plant genome 80% Tomatoes Variety in which the production of the enzyme polygalacturonase (PG) is suppressed, retarding fruit softening after harvesting.[ A reverse copy (an antisense gene) of the gene responsible for the production of PG enzyme added into plant genome Taken off the market due to commercial failure. Small quantities grown in China Canola Resistance to herbicides (glyphosate or glufosinate), high laurate canola. New genes added/transferred into plant genome 93% 21% Sugar cane Resistance to certain pesticides, high sucrose content. New genes added/transferred into plant genome Sugar beet Resistance to glyphosate, glufosinate herbicides New genes added/transferred into plant genome 95% (2010); planting in 2011 under controlled conditions 9% Rice Golden Rice: genetically modified to contain beta-carotene (a source of vitamin A) Current version of Golden Rice under development contains genes from maize and a common soil microorganism.[ Previous prototype version contained three new genes: two from daffodils and the third from a bacterium Forecast to be on the market in 2013
- 22. Squash (Zucchini) Resistance to watermelon, cucumber and zucchini yellow mosaic viruses. Contains coat protein genes of viruses. 13% Sweet Peppers Resistance to virus Contains coat protein genes of the virus. Small quantities grown in China
- 23. TRAITS THAT HAVE BEEN INTRODUCED INTO PLANTS USING GENETIC ENGINEERING Viral Resistant Plants Herbicide Resistant Plants Insect Resistant Plants “Golden Rice” Vitamin A Enhanced
- 24. Beta-carotene gives carrots their orange colour and is the reason why genetically modified rice is golden. For the golden rice to make beta- carotene three new genes are implanted: two from daffodils and the third from a bacterium. Golden rice Golden rice is genetically modified rice that now contains a large amount of A-vitamins. Or more correctly, the rice contains the element beta-carotene which is converted in the body into Vitamin-A. So when you eat golden rice, you get more vitamin A.
- 25. Advantages: •The rice can be considered a particular advantage to poor people in underdeveloped countries. They eat only an extremely limited diet lacking in the essential bodily vitamins. The consequences of this restricted diet causes many people to die or become blind. This is particularly true in areas of Asia, where most of the population live on rice from morning to evening. Disadvantages: •Critics fear that poor people in underdeveloped countries are becoming too dependent on the rich western world. Usually, it is the large private companies in the West that have the means to develop genetically modified plants. By making the plants sterile these large companies can prevent farmers from growing plant-seed for the following year - forcing them to buy new rice from the companies. •Some opposers of genetic modification see the "golden rice" as a method of making genetic engineering more widely accepted. Opponents fear that companies will go on to develop other genetically modified plants from which they can make a profit. A situation could develop where the large companies own the rights to all the good crops.
- 26. Long-lasting tomatoes Long-lasting, genetically modified tomatoes came on to the market in 1994 and were the first genetically modified food available to consumers. The genetically modified tomato produces less of the substance that causes tomatoes to rot, so remains firm and fresh for a long time. •Because the GM tomatoes can remain fresh longer they can be allowed to ripen in the sun before picking - resulting in a better tasting tomato. •GM tomatoes can tolerate a lengthier transport time. This means that market gardens can avoid picking tomatoes while they are green in order that they will tolerate the transport. •. Advantages:
- 27. Disadvantages: Scientists today can genetically modify tomatoes without inserting genes for antibiotic resistance. However the first genetically modified tomatoes contained genes that made them resistant to antibiotics. Doctors and vets use antibiotics to fight infections. These genes spread to animals and people, doctors would have difficulties fighting infectious diseases. Strawberries, pineapples, sweet peppers and bananas have all been genetically modified by scientists to remain fresh for longer. • The producers also have the advantage that all the tomatoes can be harvested simultaneously
- 28. Insecticide sweet corn Scientists have genetically modified sweet corn so that it produces a poison which kills harmful insects. This means the farmer no longer needs to fight insects with insecticides. The genetically modified corn is called Bt-corn, because the insect-killing gene in the plant comes from the bacteria Bacillus thuringiensis.
- 29. Advantages: • The farmer no longer has to use insecticide to kill insects, so the surrounding environment is no longer exposed to large amounts of harmful insecticide. • The farmer no longer needs to walk around with a drum of toxic spray wearing a mask and protective clothing. Disadvantages: • This type of genetically modified corn will poison the insects over a longer period than the farmer who would spray the crops once or twice. In this way the insects can become accustomed (or resistant) to the poison. If that happens both crop spraying and the use of genetically modified Bt-corn become ineffective. • A variety of insects are at risk of being killed. It might be predatory insects that eat the harmful ones or, perhaps attractive insects such as butterflies. In the USA, where Bt-corn is used a great deal there is much debate over the harmful effects of Bt-corn on the beautiful Monarch butterfly. Cotton and potatoes are other examples of plants that scientists have , genetically modified to produce insecticide.
- 30. Pesticide resistant rape plants Scientists have transferred a gene to the rape plant which enables the plant to resist a certain pesticide. When the farmer sprays his genetically modified rape crop with pesticides, he or she can destroy most of the pests without killing the rape plants. Advantages: • The farmer can grow a larger crop because it is easier to fight pests. • In some cases the farmer can use a more environmentally friendly crop spray. • The farmer can also protect the environment by using less crop spray.
- 31. Disadvantages: • Genes from the genetically modified rape crop could be transferred to the pests. The pests then become resistant to the crop spray and the crop spraying becomes useless. • Rape plants can pollinate weeds - for example navew which is found in rape fields. When rape plants pollinate the navew their genes are transferred. The navew then acquires pesticide resistance. Corn, soya beans and sugar cane have also been genetically modified by scientists so they are able to tolerate crop spray.
- 32. Hawaii's Papaya and the Ringspot Virus • Papayas are Hawaii's second largest crop but are subject to infection by the ring spot virus. When this virus appeared on the island with the most papaya farms, nothing would control it and plants infected simply died. • Salvation came from Cornell University genetic engineers Dennis and Carol Gonsalves • They copied the gene for the ring spot virus' coat protein into the genome of a papaya. Coat protein, without the accompanying virus DNA, is harmless, but the modified papayas produce the coat protein and the plant's own immune system becomes primed to fight it.
- 33. • Despite the current uncertainty over GM crops, one thing remains clear. This technology, with its potential to create economically important crop varieties, is simply too valuable to ignore. There are, however, some valid concerns. If these issues are to be resolved, decisions must be based on credible, science- based information. •Finally, given the importance people place on the food they eat, policies regarding GM crops will have to be based on an open and honest debate involving a wide cross-section of society.
Notes de l'éditeur
- Hawaii's Papaya and the RiRebecca Grumet Michigan State University Coat-Protein-Mediated Virus Resistance in Plants In 1986, Powell-Abel et al. (30) produced transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus (TMV) and found that they were more resistant to infection by TMV than were the nontransgenic controls. This initial demonstration of the feasibility of CP-mediated protection was followed by a host of examples; by the end of 1994, there were more than 50 published reports of genetically engineered CP-mediated plant virus resistance in various systems. The different viruses for which resistance has been demonstrated represent at least 13 different groups and include positive sense, negative sense, single- and double-stranded RNA viruses and at least one DNA virus. The type and extent of protection conferred by a given coat protein gene is variable depending on the type of virus and the individual transgenic line. There even can be variation among different individuals or families derived from the same line and possessing the same gene at the same location in the genome (e.g., 34). In general, upon infection with the virus from which the gene was derived, the inoculated leaves of the transgenic plants show fewer viral lesions that do control plants, and/or systemic spread of infection is prevented, delayed, or reduced. There are also examples where plants transformed with a coat protein gene initially become infected and then later recover (e.g, 7, 16). In the majority of cases, virus accumulation is reduced or absent. The theoretical basis for the use of coat protein genes as possible virus resistance genes originally came form two directions, classical cross protection and pathogen-derived resistance. In classical cross protection, it is possible to protect a plant from the effects of infection by a severe virus by preinoculating the plant with a mild strain or mutant of the virus (5, 29). Although the mechanism of cross protection is still not understood clearly, one hypothesis suggests that the coat protein of the first virus interferes with an early stage in the life cycle of the second virus, such as attachment, entry, or uncoating. Pathogen-derived resistance, states that it should be possible to disrupt the normal pathogenic cycle by causing the host to express a pathogen gene at the wrong time, in the wrong amount, or in a counterfunctional form (33). Native or altered viral-derived genes might be used to interfere with various stages in the viral life cycle such as uncoating, translation, replication, cell-to-cell or long-distance movement, or vector-mediated transmission. In the case of coat proteins, which have many roles in the life cycle of the virus, there is good evidence to indicate that the mechanism of protection is not the same in every virus-CP-host combination. One feature of CP-mediated resistance that might reflect mechanism is the relationship between the extent of protection and the level of CP expression. One might predict that higher levels of CP would result in greater protection, and in several experiments the level of protection observed was directly correlated to the amount of CP present [e.g., alfalfa mosaic virus (AlMV) 10, 17; potato virus X (PVX) 9, 11; rice stripe virus, 8]. For AlMV and TMV, 18 transformation with translationally defective CP genes that could produce RNA but not protein, showed that it was the coat protein molecule and not the coat protein mRNA that conferred resistance (31, 43). In several systems, however, particularly for members of the potyvirus, luteovirus, and tospovirus groups, the protection conferred by the CP gene appears to be partially or completely due to the viral-derived RNA rather than the CP. This type of resistance is not within the scope of this summary and will be discussed in a later section by Dr. W. Dougherty. In some cases, such as tomato spotted wilt virus (TSWV) both RNA and protein may be involved in conferring protection (27, 39). For TMV and AlMV there is good evidence to suggest that interference with uncoating is a key component to the protection. If transgenic TMV- or AlMV-CP expressing plants or TMV CP expressing protoplasts are inoculated with whole virions they are protected against infection, but if they are inoculated with naked viral RNA, they are not protected (24, 42). On the other hand, transgenic plants expressing the coat proteins of some potex-, carla-, and nepoviruses (1, 2, 9, 19) were protected against infection even when inoculated with viral RNA. In these cases some step other than, or in addition to, interference with uncoating must be affected. In the case of cucumber mosaic virus (CMV), whole plants were protected against systemic infection by both virions and RNA, but protoplasts were protected only against virions and not against RNA (25). These observations suggest that more than one mechanism may be operating, one at the cellular level likely to involve interference with uncoating, and another at the cell-to-cell movement or whole plant level. Similarly, Wisniewski et al. (44) found limited spread of TMV after initial infection with RNA. Interestingly, with AlMV there are transgenic lines that are not protected against RNA, but there are other lines that are protected against RNA (36), again suggesting that more than one mechanism may be involved. Several hypotheses for the mechanism of CP action, such as interference with uncoating, translation, or replication, depend on interaction between the transgene-expressed CP and the viral RNA. There are some instances, however, where the ability to interact with the RNA may not be sufficient to confer resistance. Although the CPs of two strains of tobacco rattle virus (TRV) are capable of reciprocal encapsidation, the CPs only confer protection against the homologous strain (41). Other experiments suggest that an amino terminal portion of the protein that is not essential for viral assembly is critical for CP-mediated protection; possibly via interaction with a host factor. Although changing the second amino acid of the AlMV CP did not alter the ability of the CP to bind AlMV RNA in vitro, the mutant CP no longer conferred resistance to infection (38). Further, although binding and assembly functions of potyviruses are thought to reside in the trypsin-resistant core portion of the CP, transgenic melon plants expressing only the core of the zucchini yellow mosaic virus (ZYMV) CP gene were only partially resistant to ZYMV infection, whereas those expressing the full length CP were completely resistant (4). Similarly, removal of the amino terminus of the CP of the potexvirus potato acuba mosaic virus eliminated the ability to confer protection against virus infection, while on the other hand, mutation in a domain thought to be essential for viral assembly did not reduce the level of resistance observed relative to the full length CP (15). 19 It should be noted than in many cases it has been possible to overcome the CP-mediated resistance with increasing concentrations of viral inocula [e.g., TMV, 30; AlMV, 37; soybean mosaic virus, 35; TSWV, 18; watermelon mosaic virus (WMV) 22]. On the other hand, comparable levels of resistance were observed over a range of inoculum levels for several viruses including CMV (3), potato leaf roll virus (12), potato virus S (19, 20), and arabis mosaic virus (1). From an applied point of view, greater levels of protection would be most valuable, but perhaps the most critical factor is whether the levels of resistance are sufficient for inoculum concentrations likely to be encountered in the field via vector mediated transmission. Where it has been tested, CP-mediated protection has been effective against insectvectored [aphid (6, 12, 14, 32, 40), leafhopper (8) and whitefly (13)] transmission. In contrast, the CP of TRV conferred protection against rub inoculation, but not against nematode transmission, possibly due to the large number of virus particles injected during nematode feeding (28). As with naturally occurring virus resistance genes, strain specificity and breadth of protection are important questions, and different genes vary in their specificity. As a general rule, the plants are best protected against the virus (or strain) from which the CP gene was derived, but in many cases, the transgenic plants also were protected against additional virus strains and/or related heterologous viruses. Although percent homology is clearly not the only factor involved, there is often a general correlation between the extent of protection and the relatedness between the challenge virus and the virus from which the CP gene was derived (21, 23, 26, 41). In other cases specific strains are capable of overcoming resistance. Interestingly, the ZYMV strains that have been found to overcome ZYMV-CP mediated resistance in melon also can systemically infect Nicotiana benthamiana, a species that is not normally a systemic host for this virus (Grumet et al. unpublished). As a final comment, a critical question--and one that makes this workshop relevant--is what is the performance of these materials in the field? The possible ecological implications of viral recombination only become a serious issue if we are dealing with large-scale agricultural production of transgenic viral gene-expressing plants. The majority of the field experiments (ca. 75% of the USDA permits issued from 1987-1993) have been performed by industry. Although little has been published regarding field trial performance, those reports that have been published have been encouraging. Perhaps more importantly, Asgrow Seed currently has transgenic CPexpressing ZYMV- and WMV-resistant squash cultivars approved and ready for market. Other virus-resistant crops are likely to become available in the near future. From here, only time will determine the real effectiveness and long-term stability of this strategy for conferring commercially valuable levels of resistance in large-scale production systems.ngspot Virus Papayas are Hawaii's second largest crop but are subject to infection by the ring spot virus. When this virus appeared on the island with the most papaya farms, nothing would control it and plants infected simply died. At first, the problem was confined to the one island, so growers responded by raising papayas on another Hawaiian island. But the ring spot virus appeared there as well. Soon all the Hawaiian islands were affected. Agronomists expected the Hawaiian papaya industry to disappear completely in a short time. Salvation came from Cornell University genetic engineers Dennis and Carol Gonsalves. They copied the gene for the ring spot virus' coat protein into the genome of a papaya. Coat protein, without the accompanying virus DNA, is harmless, but the modified papayas produce the coat protein and the plant's own immune system becomes primed to fight it. It's the same idea as we humans use to protect ourselves by vaccination. Drs. Gonsalves' transgenic papayas have thrived and are now the large majority of papayas grown in Hawaii. Now, because most of the islands' papaya plants are virus resistant, there are fewer viruses around. This has made it possible to again grow traditional papaya varieties, whose fruit can be exported to Japan. The Japanese have not yet allowed the import of transgenic papayas.