Establishment Of High-frequency Regeneration Ability Transgenic Acceptor System From Mature Embryos Of Maize (Zea Mays L.)

Successful genetic transformation depends primarily on the excellent acceptorsystem. Compared with immature embryos, the mean explants for transgenicacceptor system of maize, mature embryos are easy to handle, available year roundand in bulk quantities. The tissue culture system established from mature embryoscould be an ideal transgenic acceptor system. But mature embryos of maize are hardto induct and regenerate, and also restricted by genotype. The study of matureembryos of maize focus on the genotype selected from a large number of genotypesthat can form embryogenic callus and germinate in high-frequency with matureembryos. We did lots of works in this area with many research methods. And themain results were:1). 71 genotypes of maize mature embryos were studied. All the genotypes wereclassified depend on the state of primary callus and pro-embryogenic callus, and twoevaluation systems for the ability of induction and subculture were established. Thenthree inbred lines CML295, CML304 and 18-599R were selected as the meanresearch genotype. The rates of embryogenic callus formation from mature embryosof them were 37.1%, 35.8% and 30.7%.2). The embryogenic callus initiated from mature embryo proliferated rapidly onsubculture medium for immature embryos. High-frequencies were got from the threegenotypes on the regeneration medium for immature embryos culture supplied with2mg/L 6-BA, the rates were 68.6%, 75.4% and 84.8%, and the No. of shoots percallus were 2.45,2.43 and 2.75.3). Paraffin sections were made with callus from mature embryos in different states.The results showed that primary callus were the explants in process ofdedifferentiation. Granular callus in the surface of pro-embryogenic callus weremeristematic tissue nodes in histology. Embryogenic callus were formed in thesurface of meristematic tissue nodes. Embryogenic callus from mature embryos andthat from immature embryos had same structure in histology.4). Transformation was carried out by particle gun with embryogenic callus frommature embryos. GUS staining frequencies were 57.9%, 62.5% and 73.1% after the transformation of vector pCAMBIA1301 which carried GUS report gene. And gfpexpression frequencies were 23.3%, 40% and 45.5% after the transformation ofvector pCAMBIA1303 which carried GFP report gene. The frequencies were similarto those of immature embryos.Creative operation combined the method of callus induction from mature embryos ofmaize and the tissue culture system of immature embryos was carried out in thisstudy. And the subculture of embryogenic callus from mature embryos wasoptimized. In this study the range of transgenic acceptors of maize was expanded bythe system of tissue culture, regeneration and transformation that established frommature embryos, and that was supported by the results of tissue slice, regenerationand transformation. And genetic improvement and functional genome research ofmaize can got an important technology support from this system.