Agrobacterium-Mediated Transformation Of Maize Elite Inbred Lines And High Frequency Plant Regeneration From Mature Embryos Of Maize

Huang Xueqing (Genetics ) Directed by Prof. Wei ZhimingMaize (Zea Mays L.) is a widely grown cereal crop and the most important fodder crop inthe world today. It is very important for engineered breeding for maize to establish anAgrobacterium-mediated transformation system for maize. In the dissertation, an efficienttransformation system was developed for maize (Zea mays L.) elite inbred lines usingAgrobacterium-mediated gene transfer by identifying important factors that affectedtransformation efficiency. The results showed that the immature embryos of 1.0–2.0 mm inlength were optimal transformation explants. The hypervirulent Agrobacteriumtumefaciens strain EHA105 proved to be more infectious. Inclusion of acetosyringone (200μmol/L ) and ascorbatic acid (50 mg/L) in both infection medium and cocultivationmedium led to a significantly increase in the transformation efficiency. Improvedtransformation efficiencies were obtained when immature embryos were incubated withAgrobacterium suspension cells (optical density, OD600=0.8) for 20 min in the presence of0.1% (v/v) of a surfactant (Tween20) in the infection medium. Optimized cocultivationwas performed in the acidic medium (pH5.4) at 22℃ in the dark for 3 days. However,high osmotic treatment the explants before inoculation didn't improve transformationefficiency. Delaying selection was beneficial to the survival of resistant calli. Using theoptimized transformation procedure, 42 PCR-positive transgenic plants were obtained fromthe 4 elite inbred lines and the frequency of PCR-positive plant ranged from 2.35% to5.26%. Stable integration, expression, and inheritance of the transgenes were confirmed bymolecular and genetic analysis. T1 plants were analysed and transmission of transgenes to 4农杆菌介导的玉米优良自交系遗传转化研究及成熟胚再生系统的建立the T1 generation in a Mendelian fashion was verified. This system should facilitate theintroduction of agronomically important genes into commercial genotypes.Immature embryos have been frequently used as an explant source in maize tissue cultureand genetic transformation, but it is usually difficult to obtain immature embryosthroughout the year and their suitable stage for culture is also strictly limited. This is incontrast to ready availability and abundance of mature embryos from seeds. However,mature embryos are considered more recalcitrant to tissue cultures than immature embryos.In the paper, an efficient maize regeneration system was developed using mature embryosas explants. Some factors affected plant regeneration from mature embryos wereinvestigated. The results showed that inclusion higher concentration of 2,4-Dichlorophenoxyacetic acid (2,4-D, 4.0 mg/L) into induction medium was a criticalfactor for producing primary calli. Supplement with optimal level of 2,4-D (2.0 mg/L),6-Benzylaminopurine (BA, 0.2 mg/L) and silver nitrate (10 mg/L) in the subculturemedium significantly promoted the formation of embryogenic callus. And the addition 0.5mg/L of BA into regeneration medium has significantly increased the frequency of plantregeneration. Using this system, plantlets were regenerated from seven elite maize inbredlines. The frequency of forming green shoots ranged from 19.8% to 32.4%. This efficientregeneration system provides a solid basis for genetic transformation of maize.