关键词: 小麦, 大龄幼胚, 破碎处理, 植株再生, 农杆菌转化

Abstract:

【Objective】 The immature embryo culture of wheat is closely related with the explant age. Thereby, it is essential to improve the regeneration efficiency of wheat immature embryos with larger size. 【Method】 Eighteen Triticum aestivum genotypes and five Triticum durum genotypes were used as material in this research, and their large immature embryos post pollination for 15-17 days were cultured or transformed with Agrobacterium after or before using cutting pieces treatment. Regeneration and transformation of the larger explants were evaluated by shoots induction rate, transient expression of histochemical staining, and PCR detection. 【Result】 The results indicated that the larger immature embryos of 1.5-1.8 mm in size gave an increased regeneration rate of 18.2% when precultured for 2 days and then cut into pieces for further culture, which was obviously higher than the control of no cutting treatment and the other two treatments with 4 days preculture or 6 days preculture, respectively, before cutting. The regeneration frequency of the larger immature embryos from different wheat genotypes varied from 16.9% to 46.7% by employing the breaking treatment, being raised by 5 to 10 times comparing with the control treatment for different cultivars, among which the larger immature embryos fragments of Bobwhite and Zhong8423 performed a regeneration rate of more than 40%. Weak light had a positive effect on the regeneration rate of the fragments of the larger immature embryos, 5.4% to 47.4% higher than the control. Being transformed by Agrobacterium, the larger immature embryos tissues showed a GUS transient expression rate of 0 to 76.7% among the genotypes tested, in which Kenong199 was the highest, Verry and Alondra were followed by 64.4% and 47.2%. Putative transgenic plants were generated from the larger immature embryos infected by Agrobacterium, and identified by PCR further. 【Conclusion】 Regeneration of wheat large immature embryos was improved greatly by treatments of cutting and weak light culture. The larger immature embryos of Kenong199, Verry and Alondra were sensitive to Agrobacterium infection, suggesting to be used in the transformation protocol. Transgenic plants can be achieved by employing the larger explants.

Key words: wheat, large immature embryos, broken treatment, plant regeneration, Agrobacterium-mediated transformation