flavr-savr-tomato-golden-rice-bt-cotton.pdf

Flavr Savr Tomato, Golden Rice, Bt Cotton Mitesh Shrestha  Flavr Savr Tomato The first FDA approved genetically modified food Licensed in 1994 Ripening causes production of an enzyme Polygalacturonase in a gradual increasing level, which is responsib...

Flavr Savr Tomato, Golden Rice, Bt Cotton Mitesh Shrestha  Flavr Savr Tomato The first FDA approved genetically modified food Licensed in 1994 Ripening causes production of an enzyme Polygalacturonase in a gradual increasing level, which is responsible for softening of the tomato and which becomes the cause of rottening Calgene introduced a gene in plant which synthesize a complementary mRNA to PG gene and inhibiting the synthesis of PG enzyme.  Antisense RNA Antisense RNA (asRNA) is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Put PG gene sequences in the reverse orientation into the vector Transformation Isolate the nuclei used for in vitro transcription Isolate the RNA Hybridize RNA Measure PG activity Lycopene determination  Flavr Savr Tomatoes Constructs used: pCGN1436, pCGN1547, pCGN1548, pCGN1549, pCGN1557, pCGN1558, pCGN1559, pCGN1578 or pCGN4109 LB T-Tml NptII P-35S T-Tml pg P-35S RB Components of construct pCGN4109: tml-termination sequences from plasmid pTiA6 plasmid, npt-neomycin phosphotransferase; pg- polygalacturonase, 35S- CaMV 35S promoter, LB- left border, RB-right border  Flavr Savr Tomatoes LB P-mas NptII T-mas P-35S pg T-tml RB Components of construct pCGN14369:mas-mannopine synthase gene from A. tumefaciens, tml-termination sequences from plasmid pTiA6 plasmid, npt-neomycin phosphotransferase; pg- polygalacturonase, 35S- CaMV 35S promoter, LB- left border, RB-right border  Effects of Malnutrition Symptoms of vitamin A deficiency (VAD) include; night blindness, increased susceptibility to infection and cancer, anemia (lack of red blood cells or hemoglobin), deterioration of the eye tissue, and cardiovascular disease Nearly 9 million children die from malnutrition each year. A large proportion of those children die from common illnesses that could have been avoided through adequate nutrition The reduced immune competence increases the morbidity and mortality rates of children  Who Began the Golden Rice Project? Started in 1982 by Ingo Potrykus-Professor emeritus of the Institute for Plant Sciences Peter Beyer-Professor of Centre for Applied Biosciences, Uni. Of Freiburg, Germany Funded by the Rockefeller Foundation, the Swiss Federal Institute of Technology, and Syngenta, a crop protection company. Golden Rice Humanitarian Board-responsible for the global development, introduction and free distribution of Golden Rice to target countries.  Why Rice? Global staple food. Cultivated for over 10,000 years Rice provides as much as 80 percent or more of the daily caloric intake of 3 billion people, which is half the world’s population Other plants, such as sweet potatoes have varieties that are either rich (orange-fleshed) or poor (white fleshed) in pro-vitamin A Carrots were originally white or purple in the 1600’s. A Dutch horticulturist mutated the carrot to produce carotenes to symbolize the color of the Dutch Royal House of Orange  Golden rice Golden rice is a variety of Oryza sativa rice produced through genetic engineering to biosynthesize beta- carotene, a precursor of vitamin A, in the edible parts of rice. Biofortification - The creation of plants that make or accumulate micronutrients The research was conducted with the goal of producing a fortified food to be grown and consumed in areas with a shortage of dietary vitamin A, which is estimated to kill 670,000 children under 5 each year. Golden rice differs from its parental strain by the addition of three beta-carotene biosynthesis gene  Golden Rice Orange/golden colour due to synthesis of proVitamin A in the kernels Developed at the Swiss Federal Institute of Technology (ETH) Zurich Provitamin A pathway is an extension of lycopene pathway Rice embryos can produce Geranyl Geranyl pyrophosphate, but are not capable of synthesis of either lycopene or  carotene as they lack the required enzymes  Golden rice was designed to produce beta-carotene, a precursor of vitamin A, in the edible part of rice, the endosperm. The rice plant can naturally produce beta-carotene in its leaves, where it is involved in photosynthesis. However, the plant does not normally produce the pigment in the endosperm, where photosynthesis does not occur. Golden rice was created by transforming rice with two beta-carotene biosynthesis genes: 1.psy (phytoene synthase) from daffodil (Narcissus pseudonarcissus) 2.crtI from the soil bacterium Erwinia uredovora  The psy and crtI genes were transformed into the rice nuclear genome and placed under the control of an endosperm-specific promoter, so they are only expressed in the endosperm. The exogenous lyc gene has a transit peptide sequence attached so it is targeted to the plastid, where geranylgeranyl diphosphate formation occurs. The bacterial crtI gene was an important inclusion to complete the pathway, since it can catalyze multiple steps in the synthesis of carotenoids up to lycopene, while these steps require more than one enzyme in plants. ] The end product of the engineered pathway is lycopene, but if the plant accumulated lycopene, the rice would be red. Recent analysis has shown the plant's endogenous enzymes process the lycopene to beta-carotene in the endosperm, giving the rice the distinctive yellow color for which it is named.  Golden Rice Conversion of phytoene to  carotene requires four enzymes: Phytoene synthase, phytoene desaturase, carotene desaturase and lycopene cyclase to convert phytoene to  carotene Early transformation experiments with psy (phytoene synthase) from daffodil fused to a rice endosperm specific promoter resulted in accumulation of phytoene in rice endosperm – opened new possibilities for manipulation of pro-vitamin A synthesis in rice grains  Golden Rice But the rice plant still needed three more enzymes to convert phytoene to  carotene: phytoene desaturase, carotene desaturase and lycopene cyclase The first two desaturases introduce double bonds in phytoene to produce lycopene and the lycopene cyclase forms rings in to  carotene A carotene desaturase from a bacteria Erwinina uredovora can substitute for these two desaturases Carotenoid biosynthesis pathway in golden rice Geranyl geranyl pp Phyotene synthase Phytoene + pyrophosphate Crt1 Zeta carotene Crt2 Lycopene Lycopene cyclase Alpha carotene beta carotene Golden Rice  Golden Rice Comparison of Rice (left), Golden rice1 (middle) and Golden rice 2 (right). Source: Paine et al., 2005  Golden Rice LB GT1-p psy nos 35S-p ctp crt1 nos RB LB 35S 3’ aphIV 35S-p 35S 3’ lcy Gt1-p RB Constructs for Golden rice1: Initially the rice plant was co-transformed with two constructs. The first construct pZPsC consists of phytoene synthase (psy) gene from daffodil (Narcissus pseudonarcissus) driven by the rice glutelin promoter (Gt1) in tandem with a carotene desaturase gene (crt1) from Erwinina uredovora driven by the 35S promoter. The second construct pZLcyH contains a daffodil lycopene cyclase (lcy) gene driven by the rice glutelin promoter in tandem with the hygromycin- resistace selectable marker gene (aphIV) driven by CaMV 35S promoter. Source: Slater et al, 2008. p252.  Golden Rice DNA construct present in Golden Rice 2 (Paine and others 2005). Key to source of DNA elements: Glu: rice glutelin Glut01 (Glu) promoter (nucleotides 1568--2406) SSUcrtl: functional fusion of the pea RUBISCO small subunit plastid transit peptide with Erwinia uredovora crtI (D90087; Misawa and others 1993) Terminator regions of A. tumefaciens nos (nucleotides 1848–2100, V00087). Zea mays phytoene synthase (psy) Zea Mays polyubiquitin Ubi–1 promoter with intron E. coli phospho-mannose isomerase (PMI) selectable marker. http://onlinelibrary.wiley.com/doi/10.1111/j.1541-4337.2007.00029_7.x/pdf Differences between Golden rice 1 and 2 35 μg of carotinoids per gram of dry Golden rice 2 instead of 1.6 μg of carotinoids per gram of dry Golden rice 1. More efficient phy gene introduced. Removal of CaMV 35S by polyubiquitin gene. Incorporation of phosphomannose-isomerase sugar-based selection system instead of antibiotic selection system.  Controversy Against “Fool’s Gold” Health – May cause allergies or fail to perform desired effect – Supply does not provide a substantial quantity as the recommended daily intake Environment – Loss of Biodiversity. May become a gregarious weed and endanger the existence of natural rice plants – Genetic contamination of natural, global staple foods Culture – Some people prefer to cultivate and eat only white rice based on traditional values and spiritual beliefs  What is Bt cotton? Genetically modified to produce insecticidal toxins derived from the bacterium Bacillus thuringiensis. Toxins are crystalline proteins (Cry- proteins) that target specific pests. How is it produced? Table: Various cry genes encoding for the proteins with varying degree of specificity to different insect groups Source: Karthikiyen et al., 2012  Mechanism Action of Bt toxin Solubilization in the mid gut after ingestion by insect larva Toxin Protein converted to protoxins (133-138 Kda) with three domain(Domain І, ІІ and ІІІ) Protoxins then cleaved by midgut proteases into two І- involved in pore halves, with N- terminal half having toxic property formation ІІ- Receptor binding (toxin selectivity) Such fragment bind to midgut epithelial membrane (Receptor Binding) ІІІ- protection to the toxin from proteases Domain І insert into the membrane leading to pore formation The disturbance in osmotic equilibrium and cell lysis lead to insect paralysis and death  Advantages & Limitations Advantages Limitations – High insect specificity Control crop damage and disease vectors – Nontoxic to non-target – Susceptible to species resistance – Biodegradable – Reduction of other – High seed cost insecticides 94.5 million kg (19.4%) from 1996 to 2005 for cotton – Yield increases  Other insecticidal proteins: Transgenic plant expressing PIs, α- amylase inhibitor & lectins 1. Proteinase inhibitor (PIs) Plant defence protein present mainly in seeds and tubers Example : CowpeaTrypsin inhibitor (cpTi), showing resistance against Brunchid Beetle. Mode of action: Such cpTi have expressed in Inhibit gut proteinase of insect transgenic plant and showed resistance to tobacco Bud worm (Hilder et al, No protein digestion 1987) Deficiency of essential amino acids Following Cowpea Ti, several PIs (including potato serine PI (PPI-II), Exert physiological stress on the tomato serine PI (TI-II), rice cystein PI insect (OC-1), Mustard trypsin inhibitor (MTI- 2)) have been expressed in transgenic Growth Retardation plant.  2. Plant lectins Proteins having affinity for specific carbohydrates (Carbohydrates binding protein). Bind to glycoprotein in the peritrophic matrix lining of the insect midgut to disrupt digestion process and nutrient assimilation. Examples includes:  transgenic tobacco and potato expressing a lectins from snowdrop (Galanthus nivalis) found toxic to aphids. Transgenic rice (engineered with lectins) showed resistance against Brown plant hopper and Green leaf hopper. Wheat germ agglutinin, peas lectins, jacalins & rice lectins have been expressed in tobacco, maize and potato mainly against aphids. Some lectins are toxic or allergenic to mammals  3. α- amylase inhibitors (α-AI)  α-AI forms complex with certain insect amylases & is supposed to play a role in plant defence against insect. For example  Expression of bean α-AI gene in pea confer resistance to the Burchid beetles 4. Chitinases  Chitin: forms Insoluble polysaccharide, found in exoskeleton & gut lining of insects and protect them from water loss and abrasive agents. Because of critical functions chitin is potential target. Expression of protein that interfere with chitin metabolism is likely to have serious effect on the growth of insect Use if chitinases in conjugation with Bt toxin would enhance more effective control of insect pests