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The
improvement and the optimization of desirable characteristics of soybean, were attempted using various
transformation techniques. The efficiency of one of the few successful methods, the cotyledonary-node (cot-node) soybean transformation system, which is based on Agrobacterium-mediated transferred DNA (T-DNA) delivery of genes into regenerable cells in the axillary meristems of the cotyledonary node, is low, due to poor T-DNA delivery to cells in the cotyledonary-node axillary meristem (cot-node cells), inefficient selection of transgenic cells giving rise to shoot meristems, and low rates of transgenic shoot regeneration and plant establishments. It is also associated with chimerism, and recovery of transgenic plants has not been very successful, even with soybean genotypes that are highly susceptible to Agrobacterium infection. This is the first report of a system of embryonic tip regeneration from mature soybean seeds and its use in high-efficiency Agrobacterium-mediated transformation with Chinese cultivars Hefeng 35, Hefeng 39 and Dongnong 42. The embryonic tip system has the highest frequency of regeneration (88.6%), followed by the hypocotyl segment system (56.4%) and then the cot-node system (40.3%). For generation of fertile transgenic soybeans using an organogenesis regeneration system, it is necessary that the shoots regenerate in a relatively short time. The embryonic tip system regenerated adventitious roots with more uniform length than the other two systems, and also formed multiple shoots more frequently. The embryonic tip system showed faster growth of explants and the multiple shoots. The explants grew up to 3 to 4 folds after culturing for 2 days, and the plantlets grew normally in the greenhouse. As an effective regeneration system should form multiple shoots in a relatively short period of time, the time each system took to generate shoots from explants were monitored. It was found that the embryonic tip system formed shoots more quickly than the other two systems. Taking into account the time required for germination, the cot-node and hypocotyl segment systems are relatively slow and complicated in comparison to the embryonic tip system. The embryonic tip system was more sensitive to antibiotic selection than the other two systems as it was unable to form any shoots when treated with 20 mg/l kanamycin or more but the cot-node system was able to regenerate shoots even in medium supplemented with 40 mg/l kanamycin or levels of cefotaxime as high as 500 mg/l. Embryonic tips infected with A. tumefaciens strain EHA105, which harbors the standard binary vector pCAMBIA2301, grew normally when suspended in the liquid infection medium for up to 20 h. GUS staining was carried out to determine whether transient gus
expression was increased with extended suspension time. More frequent and stronger gus expression was associated with longer suspension times. Transient GUS expression was very obvious in regions characterized by strong division ability, such as the embryonic and root tip meristem. The stable GUS expression efficiency was of infected explants cultured for 5 days in resting medium and 1 week in selection medium with the highest frequency of GUS expression (78.2%) in embryonic tips suspended with A. tumefaciens for 20 h. The adventitious shoots stably expressed the gus gene, suggesting that the epidermis was not involved in the formation of adventitious shoots under these conditions. The high-efficiency embryonic tip transformation required 5–7 days culture on resting medium after co-culture as the explants became green on the resting medium. When the kanamycin-resistant plants capable of rooting on medium containing 25 mg/ l kanamycin were PCR-screened for the presence of the gus gene, the PCR results were confirmed with histochemical GUS assays, the data indicated that the embryonic tip system yielded transformation efficiencies of 8.0, 11.7 and 15.8% on three different China cultivars; the transgenic plants grew and matured as well as the uninfected control plants. Southern blot analysis of HindIII-digested genomic DNA using an -32P-labeled gus probe confirmed gus gene integration in the T0 transgenic plants. Southern blot analysis of T1 transgenic plants (nptII PCR-positive plants) with an nptII probe confirmed that the T-DNA was singly integrated in lines 6 and 7. However, the embryonic tip system described here, monitored by transient GUS expression, did not show increased T-DNA transfer in the cot-node system with l-cysteine treatment possibly because (i) the embryonic tip explants are more sensitive to A. tumefaciens because they contain promeristems and procambium; (ii) the long suspension time might improve the T-DNA transfer efficiency; and (iii) the long duration of co-cultivation might have the same effect.