Comparative Biosafety Evaluation ofGM Canola Presented by: Saira Karimi M. Phil 2nd Plant Sciences
INTRODUCTIONWith the advent of molecular gene technology, biologists have been able tomove novel gene constructs that are coupled with novel promoters into cropplant genomes, creating transgenic plants.enables the plants to express novel compounds, including insecticidal substancesthat kill certain organisms when they feed on the transgenic crop.The toxins released by these genetically modified plants are controversial and their effecton associated weeds and soil rhizosphere is not totally explored. Therefore, the presentinvestigation is aimed on determination of adverse effects of genetically modified canolaon soil beneficial microbes and fertility, succeeding crop species as well as behavior ofgene flow from genetically modified maize and canola into surrounding interspecificweeds.
Also , the hypothesis of present investigation is that whether gene flow occurs from genetically modified maize and canola into natural plants and genetically modified canola adversely affect the associated weed species, soil rhizosphere beneficial microbes and soil nutrient status and their effects on succeeding crop species.
Objectives To analyzed the root exudates of To investigate genetically phenomenon of modified and gene flow non modified crops. Effect of To evaluate the genetically effect of modified crop genetically on soil modified crops beneficial on soil fertility microbes
Their seeds are used to produce edible oil that is fit Canola is one of two cultivars for human consumption of rapeseed or Brassica because it has lower levels of campestris erucicacid than (Brassica napus L. and B. traditional rapeseed oils and campestris L.). to produce livestock feed because it has reduced levels of the toxic glucosinolates.Fig 1: Canola blooms
Weed control and environmental benefits improved nutritional value Canola is now the third most important winter weed control options grain crop grown in Australia GM canola with effect on wheat tolerance to either critically important for glyphosate or the cropping belt. glufosinate-ammonium herbicidesCanola field in Temora, newsouth whales
Why canola?• The development of transgenic crops has especially raised some issues more especially the problem of environmental safety.• Things that aren’t immediately obvious in the environment are the vast number of microorganisms that live in the soil.• What will the impact be of GM crops on these?Soil microorganisms play a vital role in natural cycles such as the carbon cycle and the nitrogen cycle. They are a source of plant nutrition, nutrient cycling, pesticide and pollutant decomposition and the source of soil fertility.
GM canola• Herbicide-resistant GM canola is grown on about 80% of the acres in western Canada• GM canola was first introduced in 1995.• Growers reported an average 10% yield increase (3 bu/ac) for their GM canola compared to conventional canola (2001 study).• The factors that contributed to this increase included better yielding varieties, earlier seeding and better weed control.
• More experiments are needed to systematically examine the relationship among Bt toxin, soil microorganisms, and soil biochemical properties (Liu, 2009).• Transgene products have also been shown to be released directly from the plant roots either from sloughed or damaged root cells as well as through root exudation. Transgenic Bt crops was found to release a Bacillus thuringiensis insecticidal endo-toxin from its roots (Saxena and Stotzky, 2000).
• Possibility of transgene flow from engineered crops to their wild relatives with undesirable consequences was independently recognized by several scientists (e.g. Colwell et al., 1985; Ellstrand, 1988; Dale, 1992).• When genetic crossing between a transgenic plant and a non-modified strain occurs there is also some worry that the novel genetic material will disturb the entire genome, creating unknown and potentially dangerous side-effects (Mann 2002). Consumers also worry about the possibility for transgenic DNA to be toxic or easily integrated into their own genomes.
Hydroponic Experiment The root exudates will be analyzed according to method described by Sun et al (2003). A hydroponic experiment is designed using two varieties of GM canola (B.napus), named Tornado and Telfor. Plant are given regular aeration by an aeration pump. Hoagland s solution is changed weekly. I started this experiment on 1st April now it is in 4th week Two non Gm varieties are also hydroponically grown as a control named Dunkled and Pakola.
Protocol for hydroponicsHoaglands Solution (Plant Nutrient Solution)Component Stock Solution mL Stock Solution/1L2M KNO3 202g/L 2.52M Ca(NO3)2 x 4H2O 236g/0.5L 2.5Iron (Sprint 138 iron chelate) 15g/L 1.52M MgSO4 x 7H2O 493g/L 11M NH4NO3 80g/L 1Minors: 1 H3BO3 2.86g/L MnCl2 x 4H2O 1.81g/L ZnSO4 x 7H2O 0.22g/L CuSO4 0.051g/L H3MoO4 x H2O or 0.09g/LNa2MoO4 x 2H2O 0.12g/L1M KH2PO4 (pH to 6.0 136g/L 0.5 with 3M KOH)1) Make up stock solutions and store in separate bottles with appropriate label.2) Add each component to 800mL deionized water then fill to 1L.3) After the solution is mixed, it is ready to water plants.
Effect of GMO`s on soil chemistry Pot experiments will be conducted using GM canola. As I included hydroponic also, under control conditions as well as three different soil types will be used. The soil will be Homogenized before used. The soil will be sampled at the start of experiment and also after completion
Chemical analysisThe soil will be analyzed for The soil nutrient status asbulk density organic matter affected by GM canola will content, pH, total N,P,K, be determined by the ammonium and nitrate. method of Schwab (1977)
Effect on soil chemistry of genetically modified(GM) vs. non-GM maize A study was conducted to find the effects of genetically modified (GM) maize (Zea mays L.) expressing the Bacillus thuringiensis Berliner Cry1Fa2 protein (Bt) and glyphosate herbicide tolerance on soil chemistry (organic matter, N, P, K and pH), compared with non-GM controls, were assessed in field and pot experiments Results indicate that growing GM crops instead of conventional crops may alter soil chemistry, but not greatly, and that effects will vary with both the specific genetic modification and the soil.
Soil Micro-organisms isolation andcharacterization The rhizosphere contains majority of Rhizosphere micro biota in soil microbes will be and plant-microbe under taken on the interaction in the basis of Colony rhizosphere are Counting by the among the major method of Miller factors the regulate (1972) health and growth of plants.
ISB News ReportPLANT RESEARCH NEWSA STRATEGY FOR TRANSGENE CONTAINMENTIN PLANTS BASED ON THE REPRESSION OF ASEED-LETHAL COMPONENTJohann P. Schernthaner
Gene flow The protein profile of modified vs. non modified crop specie will be The DNA of GM determined by PCR canola and non GM and SDS-PAGE. variety named Dunkled and Pakola will be extracted through CTAB method.
Protocol for SDS-PAGEMaterialsTo Pour Gels: 30% acrylamide, 10% SDS, 10% APS (make fresh each time)TEMED, 1.5 M Tris, pH 8.8 (resolving gel),1.0 M Tris, pH 6.8 (stacking gel)5x SDS Running Buffer (1 L).Tris 15 g.,Glycine 72 gSDS 5 g, Coomassie Blue Stain, 10% (v/v) acetic acid0.006% (w/v) Coomassie Blue dye90% ddH2O, Isopropanol Fixing Solution10% (v/v) acetic acid,25% (v/v) isopropanol, 65% ddH2OSDS sample loading buffer (40 ml), ddH2O 16 ml, 0.5 M Tris, pH 6.8 5 ml50% Glycerol 8 ml, 10% SDS 8 ml, 2βmercaptoethanol 2 ml (add immediately before use)bromophenol blue, 10% (v/v) acetic acid
Protocol1. Prepare polyacrylamide gel according to standard protocol.2. Load samples and run gel @ 25 mA (2 gels run @ 50 mA) in 1x SDS RunningBuffer.3. At this point, the gel can either be transferred to a membrane (see Western protocol) or stained with Coomassie (see below).4. Place gel in a plastic container. Cover with isopropanol fixing solution and shakeat room temperature. For 0.75 mm-thick gels, shake 10 to 15 min; for 1.5 mmthick gels, shake 30 to 60 min.5. Pour off fixing solution. Cover with Coomassie blue staining solution and shakeat RT for 2 hr. Apparatus used for SDS-PAGE6. Pour off staining solution. Wash gel with 10% acetic acid to destain, shaking at RT ON
Confirmation of Stable gene integrationFor the purpose to confirm thestable integration of insecticidalgene (BT cry1A and cry2A)incanola molecular analysis likePCR and Southern Blot will beperform.