The Construction Of Transgenic Regeneration System From Stem Tip Of Maize (Zea Mays L.)

Up to now, tissue culture technology has become an important method of Transgenic Breeding formaize. A successful and efficient regeneration transformation system is necessary for success of transgenicbreeding. The aim of this study is to establish a comprehensive regeneration of shoot apices of maizetransformation system successfully. At present, the immature embryo is still the chief material for maizetransgenic breeding. But immature embryo is limited by the season and the number. The apical meristemhas an advantage in these aspects, which make it possible that choosing the maize apical meristem asexplants to establish a renewable transformation system. However, the apical meristem is subject to strictlimit of genotype and difficult to induce callus and regenerate plants. In this study, the most important taskis to select materials with strong regeneration ability from a large number of genotypes. Then we establishan efficient regeneration-transformation system with these marterials selected. After a large number ofscreening works, the results are as the following:First,31genotypes of maize apical meristem are selected. During the screening process, according tothe experimental data of test materials in different stages, including the characters of induction of callus,subculture and differentiation, these materials are grouped. We successfully select4genotypes, Li Liaoning,IRF315, MZ17and HI-Ⅱ, which is adequate for the regeneration of apical meristem. Of the fourgenotypes, the induction rate of hybrids HI-II embryogenic callus is the highest, which reaches80.58%.The induction rates of the other three inbred line varieties are27.03%,42.97%and63.83%, respectively,which also reaches our expectation.Second, it is confirmed that the embryogenic callus induced from shoot tip can also grow well andproliferate on the subculture medium of immature embryo growth, which breaks the culture mediumrestriction of shoot tip culture and immature embryo to reduce the waste of manpower and raw materials.Transferring embryogenic callus with shoot tip to the3mg/L6-BA differentiation medium of immatureembryo makes high regeneration rates, which reaches91.5%,92.9%,75%and96.6%, respectively. Still,the rate of HI-II is the highest, which further confirms the superiority of the hybrids in maize tissue culture.Third, during the construction of apical meristem regeneration transformation system, callus biopsy results in various stages are observed and analyzed in detail. It is found that the whole callus formationprocess can be divided to three stages, the primary callus, secondary callus and embryogenic callus. Thesecondary callus is the transitional stage from primary callus to embryogenic callus. As a result, it is veryimportant to study the cultivation of it, which is directly related to the formation of embryogenic calluswith high-quality and high-volume or not. In addition, the study also found that the seeding-derivedembryogenic callus and the immature embryo-derived embryogenic callus are undifferentiated in thestructure and character of the cell.