Ecological Archives C006-092-A2
Rogelio Cruz-Reyes, Germán Ávila-Sakar, Gumersindo Sánchez-Montoya, and Mauricio Quesada. 2015. Experimental assessment of gene flow between transgenic squash and a wild relative in the center of origin of cucurbits. Ecosphere 6:248. http://dx.doi.org/10.1890/es15-00304.1
Appendix B. Details of sowing in experimental plot and molecular analysis in plants to detect the gene marker of transgenic cassette.
Plants in experimental plot
Seeds for all plants used in our study were germinated in 10 cm square pots filled with wet potting mix (Promix BX), in growth chambers (Conviron E15) at 25C, 12:12 light:dark cycle and 80% humidity. Seedlings were transplanted to an agricultural plot two weeks after initial planting.
Wild parental individuals were planted every 2 m on rows 2.5 m apart in a 40 × 80 m plot. F1, F2, and BC progeny were more compact, and therefore they were planted every 1 m on rows 1.5 m apart. A second plot of similar size was used on years 2 and 3. Weeds were removed manually every other week. Plant position on the plot was randomized with respect to type of cross and pollination treatment. Mexican legislation in Biosecurity does not allow for the cultivation of Genetically Modified Organisms, particularly for crops related to wild taxa at their center of origin, which is the case of Cucurbita spp. Permits may be granted for GM plant growth only under enclosed highly regulated facilities. We obtained one of these permits to plant transgenic parentals in a greenhouse.
Molecular Analysis for the detection of the transgene
All plants in our study (parentals, F1, F2, and backcrosses) were screened for the presence of the VRT using the expression of NPTII and the promoter 35S of the cauliflower mosaic virus (P-35S CaMV) as indicators of VRT presence and functionality (Tricoll et al. 1995). Molecular analyses were performed on DNA extracted from 0.4 g of dry leaf tissue from three true leaves of each seedling. Leaves were ground in liquid N and 1000 μl of 2X CTAB buffer. For the final PCR step, the concentration of reactants used was 10 mM of dNTPs, 25 mM Mg, 10 μl of each oligo and buffer 10X, 0.1 mg/ml of DNA.
For detection of P-35S CaMV, we used the following primers: forward GCT CCT ACA AAT GCC ATC, reverse GAT AGT AGG ATT GTG CGT CA. We conducted PCR in a thermocycler (GenAmp 9700) using the following protocol: 35 cycles of 1 min at 94°C, 45 s at 94°C, 65 s at 65°C, 45 s at 72°C with a final stretch of 10 min at 72°C. We detected the CaMV 35S promoter as a 195 bp band along on a 1.5% agarose gel stained with ethidium bromide. We detected expression of NPTII for each individual using the colorimetric test from DAS-ELISA (commercially available kit from Agdia, Elkhart USA) on 0.06 g of tissue from six leaves from each plant.