2How To Make A Transgenic Plant? Prepare tissue for transformationLeaf, germinating seed, immature embryosTissue must be capable of developing into normal plantsIntroduce DNALocate genes for plant traitsIntroduction into cellCulture plant tissueDevelop shootsDevelop RootsField test the plantsMultiple sites, multiple years
3Introducing the Gene or Developing Transgenics StepsCreate transformation cassetteIntroduce and select for transformants
4Locating Genes for Plant Traits Identifying and locating genes for agriculturally important traits is the most limiting step.Little is known about the specific genes required to enhance yield potential, improve stress tolerance, modify chemical properties of the harvested product, or otherwise affect plant characters. For a gene to be used as transgene, following must be knownHow the gene is regulation in the cellWhat other effects it might have on the plant, andHow it interacts with other genes active in the same biochemical pathway.
5Designing Genes for Insertion Once a gene has been isolated and 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 and expression.
6Transformation Cassettes Contains1. Gene of interestThe coding region and its controlling elements2. Selectable markerDistinguishes transformed/untransformed plants3. Insertion sequencesAids Agrobacterium insertion
7Delivering the Gene to the Plant Two major delivery methodsAgrobacteriumTissue culturerequired to generatetransgenic plantsGene Gun
8Transforming Plants Agrobacterium method Use bacterial strain Agrobacterium tumificiens (soil-dwelling bacteria that has the ability to infect plant cells with a piece of its DNA)Successful transformation in tomato, cotton, tobaccoPreferable to the gene gun, because of the greater frequency of single-site insertions of the foreign DNA, making it easier to monitor.Gene Gun method(microprojectile bombardment or biolistics).Useful in transforming monocot species like corn and rice.
9Agrobacterium mediated infection Cell contains chromosome & a Ti (tumor-inducing) plasmid.The Ti plasmid containsa stretch of DNA termed T-DNA (~20 kb long) that is transferred to the plant cell in the infection process.a series of vir (virulence) genes that direct the infection process.A tumefaciens can only infect a plant through wounds. In response to wound signals, the vir genes of become activated and direct the transfer of the T-DNA from the Ti plasmid to the plant's chromosome.Transgene is inserted betweenthe T-DNA border regions
12Selection and Regeneration 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.When grown on selective media, onlyplant tissues that have successfullyintegrated the transgene construct willsurvive.
13Regeneration 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.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.
15The Next Test Is The Field Herbicide ResistanceNon-transgenicsTransgenics
16Some Achievements Improved Nutritional Quality Rice with beta-carotene, a precursor to vitamin AInsect ResistanceBacillus thuringiensis is a bacterium that is pathogenic for a number of insect pests.Its lethal effect is mediated by a protein toxin it produces.The toxin gene can be introduced directly into the genome of the plant where it is expressed and provides protection against insect pests of the plant.Disease ResistanceGenes that provide resistance against plant viruses have been successfully introduced into such crop plants as tobacco, tomatoes, and potatoes.
17Next Generation of Ag Biotech Products Golden Rice – increased Vitamin A content(but not without controversy)transgene = three pathway enzymesGolden rice is enriched for vitamin A. The controversy surrounds the actual utility of the product. Some data suggest the child would have to significantly increase their rice intact of rice for vitamin deficiencies to be alleviated. Because of this knowledge the biotech industry is being accused of promoting a consumer-friendly product only for publicity purposes. The white mold disease is associated with elevated levels of oxalic acid. Resistance is provided by inserting a gene whose product breaks down oxalic acid.Sunflower – white mold resistancetransgene = oxalate oxidase from wheat
20Some Achievements Cont… Herbicide Resistance.Genes for resistance to some of the newer herbicides have been introduced into some crop plants and enable them to thrive even when exposed to the weed killer.Effect of the herbicide bromoxynil on tobacco plants transformed witha bacterial gene whose product breaks down bromoxynil.
22Some Achievements Cont… Salt ToleranceA large fraction of the world's irrigated crop land is so laden with salt that it cannot be used to grow most important crops.Transgenic tomatoes have been developed that grow well in saline soils. The transgene was a highly-expressed sodium/proton antiport pump that sequestered excess sodium in the vacuole of leaf cells.There was no sodium buildup in the fruit.Transgenes Encoding Antisense RNA (e.g. the Flavr Savr tomato)Transgenic transcribes into an antisense RNA complementary to the mRNA for an enzyme involved in ethylene production.These tomatoes make only 10% of the normal amount of the enzyme.The goal of this work was to provide supermarket tomatoes with something closer to the appearance and taste of tomatoes harvested when ripe.
23Some Achievements Cont… "Terminator" GenesThis term is used (by opponents of the practice) for transgenes introduced into crop plants to make them produce sterile seeds.BiopharmaceuticalsThe genes for proteins to be used in human (and animal) medicine can be inserted into plants and expressed by them.Advantages:Glycoproteins can be made (bacteria like E. coli cannot do this).Virtually unlimited amounts can be grown in the field rather than in expensive fermentation tanks.There is no danger from using mammalian cells and tissue culture medium that might be contaminated with infectious agents.Purification is often easier.
24Some Achievements Cont… Some of the proteins that are being produced by transgenic crop plants:human growth hormonehumanized antibodies against such infectious agents asHIVrespiratory syncytial virus (RSV)herpes simplex virus, HSVEdible VaccinesWorks like any vaccineA transgenic plant with a pathogen protein gene is developedPotato, banana, and tomato are targetsHumans eat the plantThe body produces antibodies against pathogen proteinHumans are “immunized” against the pathogenExamples:Diarrhea, Hepatitis B, Measles
25ControversiesThe introduction of transgenic plants into agriculture has been vigorously opposed by some. There are a number of issues that worry the opponents.Examples:A gene for herbicide resistance in, e.g. corn, escaping into a weed species could make control of the weed far more difficult.The gene for Bt toxin expressed in pollen might endanger pollinators like honeybees.To date, field studies on Bt cotton and maize (corn) show that the numbers of some non-target insects are reduced somewhat but not as much as in fields treated with insecticides.Another worry is the inadvertent mixing of transgenic crops with non-transgenic food crops. Although this has occurred periodically, there is absolutely no evidence of a threat to human health.Despite the controversies, farmers are embracing transgenic crops.
26Future Developments in Transgenic Technology More efficient transformation, that is, a higher percentage of plant cells will successfully incorporate the transgene.Better marker genes to replace the use of antibiotic resistance genes.Better control of gene expression through more specific promoters, so that the inserted gene will be active only when and where needed.Transfer of multi-gene DNA fragments to modify more complex traits.