| Pollen-tube pathway transformation method is one kind of in planta techniques that is tissue culture-independent. It is also a promising method that can replace the conventional Agrobacterium-mediated transformation and particle bombardment methods in the transformation of recalcitrant crops. However, the low repeatability and transformation frequency still prevents its application in soybean. Thus, it is of great significance to identify the factors on transformation frequency and optimize the key factors of this transformation method and to standardize the manipulation of the protocol in soybean. Low grain yield in legumes has been found to be attributed to abortion of flowers and pods, and abscission rates of 43 to 81% have been reported for different genotype soybean varieties. At the same time, the environmental cues, such as water deficiency, light insufficiency, inadequate nutrition and high temperature-would-enhance the abscission of reproductive organs. The stress hormone, ethylene, serves as a key signaling molecule and plays a critical role in response to the various abiotic stresses.1-aminocyclopropane-l-carboxylate synthase (ACC synthase, EC 4.4.114) is the rate-limiting enzyme in ethylene biosynthesis pathway. Its expression regulates the release of ethylene in plants; hence it might be a good attempt to transform soybean with ACC synthase antisense gene to improve the yield in soybean through the reverse genetics pathway and investigate the relationship between the ethylene release and yield-related traits.The aims and results of this study were as follow:1. To determine whether the length of the passageway through which foreign DNA enters the embryo sac is a key factor to the success of pollen-tube pathway transformation, we compared the transformation efficiencies of two different lengths of the passageway including cutting off 1/3 stigma or cutting near the ovary, using the plasmid pBI121 as the foreign DNA. Different lengths of the passageway were found to produce significant different transformation efficiencies,1.6% and 3.1%.2. By reference to the mechanism of T-DNA borders in Agrobacterium-mediated transformation, we designed the minimal cassette, which only contains the 35S promoter, gus reporter gene, and nos terminator, and both ends of the cassette were flanked with T-DNA borders and obtained them by Polymerase Chain Reaction (PCR). Transformation of this minimal cassette into transcient expression system of tobacco leaves resulted in three positive expression of the reporter gene among all thirty leaves, thus pointing to the feasibility of its design.3. To optimize the transformation passageway, we compared the effects of four types of of passageway lengths on the transformation, and the optimized length of the passageway was further tested in more soybean cultivars to investigate the effects of genotypes. A maximum transformation frequency of 11.0% was obtained in soybean cultivar Liaodou 14 with their styles extensively removed, whereas removal of the stigma, partial style cutting and partial ovary cutting contributed to a transformation frequency of 0%, 1.0% and 2.0%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (Liaodou14, Liaodou13, and Tiefeng29) were transformed by this optimized method. No significant difference in the transformation frequencies was observed among the different cultivars, indicating no genotype specificity. This also supported the feasibility for standardizing the manipulation through optimizing the transformation pathway, and hence, the modified protocol along with using minimal cassettes was designated as ovarian in planta transformation.4. To investigate whether the natural antisense transcript (NATs) of ACC synthase gene exists in soybean, the expression of the non-template strand was analyzed by RT-PCR. The NATs of ACC synthase gene (designated as ASACS2) was detected in six soybean cultivars by RT-PCR and sequence analysis showed it and its sense counterpart, SACS2 belonged to cis-NATs, the ratios of SACS2 to SACS2, as measured by real-time RT-PCR were constant among the various soybean cultivars, indicating the presence of cultivars specificity. NATs might be a new regulation pathway governing the release of ethylene in soybean.5. To improve the soybean yield, the ovarian in planta transformation of minimal ACC synthase antisense gene cassettes were carried out. One cDNA of wound-induced ACC synthase, SACS2, was cloned from soybean leave and inversely inserted into the plasmid pBI121 to give the construct pBI121-SACS2. A linear minimal cassette composed of 35S promoter, ACC synthase antisense gene, and nos terminator and the flanked T-DNA border sequences was amplified from pBI121-SACS2 by PCR. The integration of the foreign ACC synthase antisense DNA into the soybean genome and its subsequent inheritance by the progenies were confirmed by PCR and Southern blotting analysis. The activity of ACC synthase was not reduced according to the theory expectation of antisense gene, but increased for 17.6%-23.4%, indicating there must be a complex regulation system to ethylene release, and the total quantity of ethylene released has first increased and then decrased in the young etiolated transgenic plants compared to the wild type. In field trials, all transgenic plants (To-T2) had one prolonged vegetative growth stage, the lasting seven days longer than that of wild type and result into a delayed reproductive stage. Average pod-bearing number and average one-hundred-grain weight of the partial transgenic lines had increased significantly compared to wildtype ones, that showed the traits of increasing yield, and on the plant external morphology, the plant height had decreased, the node number had increased, and the average distance of stem-stem was significantly shortened in all detected transgenic lines, showed the traits of the decreased ethylene release.In conclusion, the ovarian in planta transformation in soybean with the linear minimal cassette and the optimal transformation passageway is a new prospective transformation method in yield and quality improvement. |
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