| Soybean ovarian in planta transformation is one kind of in planta technology by improving pollen-tube pathway method. and it have some advantagies, such as short cycle, low cost and not restricted to genotypes, compared to the tranditional transformation techniques, but the transformation system also need to be optimized for complete the technique syatem and increase the transfer frequency. This study construct new types of minimal EPSPS gene cassettes and combined with effective selection of just-opening flowers in field to improve the ovrian in planta transformation system. A new glyphosate-dotting phenoype method was established in soybean and combining molecular indentification to identify the original transformant and transgenic progeny plants and the inheritable transgenic soybean were obtained and set up the basis for the new glyphosate-tolerant transgenic soybean cultivar breeding.The main content is as follows:1. Glyphosate tolerance determination and phytotoxicity-restorationDotting glyphosate on the hypocotyl of soybean young seedling in the early development stage can concern the half inhibitory concentration of plant height, IC50 value, and the different IC50 values among 173 cultivars from the mini core plasm showed the glyphosate-tolerance diversity; Glyphosate-tolerant transgenic soybean also would behavior the phytotoxicity symptom when treated with the high-concentration glyphosate and that would hamper the further utilization in agricultural production. Two concentrations of the three aromatic amino acids including tryptophan(Trp), phenylalanine (Phe) and tyrosine(Tyr), and two hormones, GA3 and IAA were used in glyphosate phytotoxicity-restoration experiment and the results showed that tryptophan is a kind of potential safety of transgenic soybeans.2. Optimizing the ovarian in planta transformation systemDesign the two types of T-DNA left and right border sequences from octopine and nopaline type of Agrobacterium at the end of minimal EPSPS gene cassette, named 12-1 and 12-2; further removal of extra one promoter sequence based on 12-1 and 12-2 and construct the new minimal cassettes, named M1 or M2, respectively, the minimal EPSPS gene cassette without the two kinds of left and right border sequences, named M0;Best flower optimization strategies in field transformation are formulated:The openning flowers give priority to be selected for transformation treatments on the main stem nodes from No.8-15 or thicker and stronger branches; At the inflorescence axis, flowers opened with axillary base section which is in early open flowers is gave priority to treatment; The " whole line" or " entire plant" transformation manipulation strategy, can increase the total amount of flowers treated per unit time;3. Creating the Glyphosate-tolerant transgenic soybean12-1 and 12-2 were transformed into two major varieties of huanghuai region Zhuanghuang 56, Zhanghuang57 via ovary, and a late sowing cultivar, Green 75, and harvested TO seeds, after sowing,31 of herbicide-resistant fertile soybean were identified after spraying field dose,200 ml/mu glyphosate, and which are also positive ones by PCR identification. Four plants of T1 progeny plants from 13 lines among the aboved thirty-one lines were suvived after 400 ml/mu glyphosate screening, two of them after PCR identified, showed that the segregation ratio is 3:1 following the Mendel's law of segregation, that indicated the single gene insertion.3% of T3 plants of them after glyphosate dotting showed glyphosate-tolerant, all the above results showed the glyphosate-tolerance traits of the transgenic plants from ovarian in planta transformation can be inherited into the progeny generations. |
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