Studies On Shoots Regeneration Capacity Of Mature Embryos Of Maize Inbred

Tissue culture is the basis of maize genetic transformation.Immature embryos is used as the main explant in maize tissue culture because it is easy to induce embryogenic callus and regenerate shoots.However,the sampling of immature embryos is seriously limited by the growth season.Comparised with immature embryos,the mature embryo can be easy sampled at any time.So,it is also an excellent potential explant for maize genetic transformation.In this study six maize inbred lines(CML493,CIMBL8,Cy72,JY01,Ye478 and K12)based on the previous experimental results were randomly selected,and harvested mature embryos as explants for the further study.To optimize and establish an efficient regeneration system of mature embryo of maize,some factors including medium type,embryo cutting type,plant hormone concentration were analyzed.The results shows that the introduction efficiency of primary callus on MS medium was higher than that on N6 medium.The hypocotyl is the best explant to induce primary callus among three type explants,and the primary callus induction rate can reach 100%.Embryogenic callus could be induced on MS medium supplemented with 1.5mg L-12,4-D and 6mg L-1Dicamba.CML493 induction rate of embryogenic callus was 88.89%.Meanwhile,morphological observation of embryogenic callus showed that embryogenic callus was yellow,loose and dry in surface,and had prominent spherical granular protuberance structure.The analysis of tissue sections showed that the cells of embryogenic callus were arranged neatly,and these cells displayed obvious features of dividing cells.Then,after being dyed with 0.5% Sudanese red,the embryogenic callus become dark red.We also detected the expression levels of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KIN,BABY BOOM and LEAFY COTYLEDON2 genes in embryogenic callus and nonembryonic callus.The addition of mannitol(20g L-1)decreased browning frequency of callus to 19.44%.Taken together,it is suggested that MS regeneration medium supplemented with 2 mg L-1TDZ,6mg L-1ZT is best for the high frequency of shoot differentiation,and JY01 can be used as good explant for the shoots regeneration(43.33%)in this study.In brief,the efficient regeneration system of mature embryo provides a good data for the construction of excellent genetic transformation system in maize.