Transgenic Plant Improvement PDF

Transgenic Science in Plant Improvement **Figure : **Decision making box by using both conventional and modern biotechnology approaches for crop breeding **ule 6 : TRANSGENIC SCIENCE AND GENETIC IMPROVEMENT** Transgenic Science in Plant Improvement  **Figure : **Steps Invol...

Transgenic Science in Plant Improvement **Figure : **Decision making box by using both conventional and modern biotechnology approaches for crop breeding **ule 6 : TRANSGENIC SCIENCE AND GENETIC IMPROVEMENT** Transgenic Science in Plant Improvement  **Figure : **Steps Involved in production of transgenic plants **Isolation and Cloning the Gene of Interest** Identification and cloning of the gene of interest is a first **limiting step **in the transgenic development process. Locating , identifying, characterizing, and cloning genes of agricultural importance requires, a huge effort both in terms of human and financial capital. One of the earliest development is the introduction of insect resistance by transgenic technology. The discovery of **Bt Genes **has revolutionized plant transgenics. Spores of the soil bacterium *Bacillus thuringiensis *(Bt) contain a **crystal (cry) protein (δ-endotoxin)**. Inside insect gut, the crystals break apart and release a toxin that binds to and creates pores in the intestinal lining. Instead of whole gene **truncated cry gene **is used in **Bt crops. **Figure 6-1.2.2.2 shows the truncated cry gene structure. **Figure : **Gene sequence showing the truncated cry gene **Mechanism of Toxicity:** ►*Bt *gene (also known as *cry *gene) was found in a gram positive bacteria *Bacillus thuringiensis *. The structure of Bt gene is shown in Figure 6-1.2.2.2 ►This *Bt *gene is used in the production of insect resistant crops (genetically modified) and biological insecticides as well. ►*Bacillus thuringiensis*, during sporulation produces a toxic protein which possesses insecticidal activity against Lepidoptera, Coleoptera, Hymenoptera, Diptera and Nematode. ►This crystal protein is called as Cry protein, encoded by cry gene present on the plasmid (non-chromosomal gene). ►As soon as the Cry protein crystals reach the digestive tract of the insect, the prevailing alkaline condition there causes denaturation of the insoluble crystals. This denaturation makes the crystal soluble and prone to proteases activity in the gut of insect. ►Proteolysis of Cry crystal leads to release of cry toxin, which forms pore in the cell membrane of the gut by inserting themselves into it. The pore causes cell lysis and ultimately death of insects. **Control of Gene Expression** ►The level of gene expression is determined by regulatory sequences such as promoters as well as 5\' UTR elements (Described in detail in module 5- lecture 5). ►**Transgene Promoters**: Most commonly used is the **CaMV 35S promoter **of cauliflower mosaic virus. It is a **constitutive **promoter (turned on all the time in all tissues), that gives high levels of expression in plants. Most commonly used terminator sequence is the nopaline synthase (*nos*) gene from *Agrobacterium tumefaciens*  **Figure 6:** Shows expression cassette of Bt gene along w **Selectable Markers** Various selectable markers are used for the selection of transgenic plants (described in detail in Lecture 4 and 5 of Module 5). **Plant Tissues Used For Transformation** The tissue must be capable of generating **callus **(undifferentiated tissue), from which the complete plant can be produced. The choice of tissue depends on the species. Some commonly used tissues are immature embryos, leaf disks, and apical meristems.  **Introduction of Gene Construct into Plant Cells (Transformation) ** The basic methods and techniques used for plant cell transformation are as follows: i)  *Agrobacterium *mediated transformation ii)  Agro-infection iii)  Chloroplast transformation iv)  Indirect gene transfer v)  Electroporation vi)  Biolistic (Gene gun) Method vii)  Chemical method of gene transfer viii)  Microinjection ix)  Pollen Transformation x)  Direct DNA uptake by mature zygotic embryos ** ** **Selection of Transformants** ►Cells/tissues in which new genes are incorporated into plant\'s DNA are identified and then grown in media containing antibiotics or herbicides. ►So during transformation, selectable marker gene (antibiotic resistant etc.) is incorporated such that, cells which are devoid of this plasmids get killed or their growth is arrested. Kanamycin, an antibiotic capable of killing plant cells. ►Since transformed plants contain kanamycin resistance gene, they only can survive. ►In the *Agrobacterium *infiltration method, the seed of the infiltrated plants are plated on agar containing kanamycin -- the plant seed containing the introduced DNA will germinate (less in number) and will grow on agar plates containing Kanamycin. **Regeneration of Transformant** ►   During regeneration, whole plants with inserted genes are developed through tissue culture. ►   Cultured transformed plant cells are regenerated under appropriate conditions. ►   Auxin/cytokinin ratio is important for plant regeneration. ►In the culture media high concentration of auxin (plant growth regulator), causes rooting of the cultured transformed cells. Further, shooting is initiated by increasing the concentration of cytokinin (plant growth regulator-phytohormones) and new plantlet develops from the culture. ►   After that plantlets are transferred to soil for proper anatomical and physiological developments, and for better acclimatization to environment. **Confirmation of transformed plants** The presence and activity of introduced gene is confirmed by observation of phenotype and by advanced methods as listed below. 1\.   **Southern blot: **In Southern blotting separated DNA fragments obtained after electrophoresis, are transferred to a filter membrane and subsequent fragment detection is accompanied by probe hybridization. 2\.   **Northern blot: **It is used to study gene expression by detection of mRNA (or isolated mRNA) in a plant sample. 3\.   **Western blot: **It is used to study the gene expression by detection of the protein produced by the transformed regenerated plants. ** Evaluation of Transformed Plants** ►   Plant is evaluated for the presence and activity of introduced gene. ►   The effect of various environmental factors on the transgenic plant is also evaluated. ►   Evaluation for food or feed safety. ►  Evaluation of basic containment level is also very important. **Improvement of Plant Traits** Plastid genome can be used to engineer many agronomic traits such as herbicide resistance, insect resistance, and antibiotic resistance. **Biotic Stress** Expression of insect resistance genes such as *cry* genes can be well performed in the plastid genome without modifications of codon usage or other sequence manipulations. **Abiotic Stress** Chloroplast engineering can be executed for the successful development of plants suffering from abiotic stresses like salinity, high or low temperature, drought etc. Some examples are listed below- - The over-expression of enzymes required for Glycine betaine (GlyBet) biosynthesis in transgenic plants improve tolerance to various abiotic stresses.  - Chloroplast transformation has been used to transfer choline monooxygenase (BvCMO), an enzyme that catalyzes the conversion of choline into betaine aldehyde from beet (*Beta vulgaris) *into the plastid genome of tobacco.  - Transplastomic carrot plants expressing betaine aldehyde dehydrogenase (BADH) gene have also been developed using chloroplast engineering showing highest level of salt tolerance. **Production of biopharmaceuticals** - Chloroplast engineering can also be employed for the production of biopharmaceutical proteins, antigens listed in - **Table : **Depicts biopharmaceutical proteins expressed via chloroplast transformation **Biopharmaceutical Proteins** **Gene** **Site  of Integration** **Promoter** **5\'/3\' Regulatory Elements** -------------------------------- ------------- -------------------------- --------------- --------------------------------- **Elastin Derived Polymer** *EG 121* *tm\|/trnA* *Prrn* *ggagg/TpsbA* **Antimicrobial Peptide** *MSI-99* *tm\|/trnA* *Prrn* *ggagg/TpsbA* **Insulin-like growth factor** *IGF-1* *tm\|/trnA* *Prrn* *PpsbA/TpsbA* **Interferon alpha 5** *INFα5* *tm\|/trnA* *Prrn* *PpsbA/TpsbA* **Interferon alpha 2b** *INFα2b* *tm\|/trnA* *Prrn* *PpsbA/TpsbA* **Human serum Albumin** *hsa* *tm\|/trnA* *Prrn, PpabA* *ggagga,TpsbA /TpsbA* **Monoclonal Antibodies** *Guy\'s 13* *tm\|/trnA* *Prrn* *ggagg/TpsbA* **Interferon gamma** *IFN-g* *rbcL/accD* *PpsbA* *PpsbA/TpsbA* **Human somatotropin** *hST* *trnV/rps 12/7* *Prrn, PpabA* *T7gene 10psbA/ trps16* - **Table** Depicts vaccine antigens expressed via chloroplast transformation ---------------------------------------------------------------------------------------------------------------------------------------- **Vaccine Antigens** **Gene** **Site of Integration** **Promoter** **5\'/3\' Regulatory**\ **Elements** -------------------------------- ----------------------------------- ------------------------- -------------- -------------------------- **Cholera toxin** *ctxB* *trnI/trnA* *Prrn* *ggagg/ TpsbA* **Canine Parvovirus (CPV)** *ctxB-2L21  gfp-2L21* *trnI/trnA* *Prrn* *PpsbA/ TpsbA* **Anthrax Protective antigen** *pag* *trnI/trnA* *Prrn* *PpsbA/ TpsbA* **Plague Vaccine** *caF1\~LcrV* *trnI/trnA* *Prrn* *PpsbA/ TpsbA* **Tetanus toxin** *tetC  (bacterial and synthetic)* *trnV/rps 12/7* *Prrn* *T7 gene 10, atpB/Trbcl* ---------------------------------------------------------------------------------------------------------------------------------------- **Metabolic Pathway Engineering** - Chloroplast engineering represents an attractive alternative to conventional nuclear transgene expression for metabolic engineering. The strong transgene containment and possibility to stack multiple transgenes by linking them in operon makes this process widely exploited in chloroplast genome. \ \ Synthesis of polyhydroxybutyrate (PHB), a bioplastic has been achieved by introduction of the most complex metabolic pathway into the chloroplast genome so far. Three enzymes of PHB biosynthesis are co-transcribed into the tobacco plastid genome. Significant accumulation of PHB in chloroplasts causes male sterility and severe growth retardation. The application of plastid transformation to metabolic pathway engineering is still restricted to few model species like agrobacterium.  - Metabolic engineering has emerged as a promising technology in food crops. Recently, plastid transformation has been performed in tomato to alter carotenoid biosynthesis for the production of fruits with elevated contents of provitamin A (β- carotene), an important antioxidant and essential vitamin for human nutrition. **Examples of GM (Genetically modified) Crops** **or transgenic plants**