| Traditional methods of genetic breeding were difficult to improve in variety of improvement and yield.With the rapid development of plant genetic engineering,transgenic technology has become a new way to cultivate high-yield and high-quality wheat varieties.The key of wheat gene engineering research was to solve the problem of somatic cell regeneration and genetic transformation.Compared with crops such as Brassica napus,Zea mays and Oryza sativa,wheat belonged to the monocotyledonous plant with typical self-pollination,and the frequency regeneration of calli was lower.What was more,monocotyledons were not the natural hosts of agrobacterium,there were many problems such as low frequency of DNA introduction and chimera plants in the process of genetic transformation.It was significance to optimize the regeneration system of wheat somatic cells and improve the efficiency of genetic transformation for molecular breeding.Spring wheat ‘Bobwhite' mature embryo was used as explant,this research analyzed on the effects of calli regeneration induced by picloram and 2,4-D,and the sensitivity of agrobacterium to calli at the different stages of culture was preliminary explored.In order to provide some references for wheat genetic engineering.The main results were as follow:1.Spring wheat mature embryo was used as explant to analysis the influence of calli regeneration induced by picloram and 2,4-D through cytology,physicochemical property and q RT-PCR.(1)The results showed that there was no significant difference in the ability to induced calli on two hormones,but the quality of calli induced by picloram were dense and yellow.The structure of calli induced by 2,4-D was porous,transparent and the texture was poor.After differentiation,regeneration frequency of calli induced by picloram(48.2%)was approximately twice higher than 2,4-d(29.7%).(2)The results of cytological showed that cells induced by picloram orderly arranged,intercellular space were small,most cells had nucleus structures,and the meristem has been formed,the cells induced by 2,4-D were disorder arrangement,osteoporosis and morphological abnormalities,only a handful of the cells had the nucleus which was consistent with the appearance of organization formed.(3)Compared with 2,4-D,calli induced by picloram during somatic embryogenesis accumulated more soluble sugar used for protein synthesis material energy metabolism to promote the rate of embryonic callus.The activity of SOD and POD were closely related to the proliferation and differentiation of callus,and the decrease activity of CAT was beneficial to the induction and differentiation of callus.(4)Due to the increase of Ta LEC1 gene,picloram was faster than 2,4-D to promote the formation of calli in the early stage of somatic embryogenesis.Alhough high expression of somatic embryogenesis of related genes(Ta BBM,Ta WUS)could improve the formation of callus,excessive expression of related genes were not conducive to callus regeneration.2.(1)Agrobacterium infected callus inducted by picloram and 2,4-D,the results showed that most of calli induced by 2,4-D which can not regeneration,and produced a lot of roots.It was proved that explant induced by picloram was more suitable for agrobacterium-mediated in wheat.(2)The results of calli at different stages of wheat mature embryo infected by agrobacterium showed that the transient expression of GUS gene and infection area of calli of differentiation culture were more than others.Further,the calli of differentiation culture had a certain frequency to obtain the transgenic plants.(3)Culture time was too long to make the explant of somatic embryo in bud,and improved the high frequency of false positive plant,only one transgenic plant was identified on this research.Therefore,the further studied about this method. |
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