玉米茎尖再生转化体系的建立
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摘要
迄今为止，组织培养技术已经成为玉米转基因遗传育种的重要方法。转基因育种的成功首先需要的是一个成功高效的再生转化体系，本实验致力于建立一个完善的玉米茎尖再生转化体系，并取得了成功。目前幼胚仍然是玉米转基因育种的首选材料，但幼胚的取材受时节和数量的限制，而茎尖分生组织与之相比在这些方面上则更有优势。这就为选用玉米茎尖分生组织作为外植体，建立再生转化体系提供了可能，但是茎尖分生组织受到严格的基因型限制，是比较难于诱导出愈伤组织并再生出植株的外植体。因此，本研究首要的工作是从众多的基因型中选出再生能力较强的材料，再由这些材料建立高效的再生转化体系，经过了大量的筛选工作和团队的努力工作，现将取得的研究结果汇报如下：
1、筛选了31个基因型的玉米茎尖分生组织。在筛选的过程中，根据各个阶段受试材料的实验数据，包括诱导愈伤、继代培养和分化再生的性状表现，将这些材料进行分组，并成功地筛选出了李辽宁、IRF315、MZ17和HI-Ⅱ4个适合茎尖分生组织再生的基因型，在这4个基因型中，以杂交种HI-Ⅱ的胚性愈伤诱导效率最高，达到了80.58％，其余的三个自交系品种也都分别达到了较为理想的27.03％、42.97％和63.83％。
2、证实了由茎尖诱导的胚性愈伤组织同样也可以在培养幼胚的继代培养基上生长良好，迅速增殖，突破了茎尖培养与幼胚培养在培养基上的限制，减少了人力和原料的浪费。将这些茎尖来源的胚性愈伤组织转移到添加有3mg/L6-BA的幼胚的分化培养基上后，均获得了较高的再生效率，分别达到了91.5％、92.9％、75%和96.6％，其中仍以HI-Ⅱ的再生率最高，也进一步的证实了杂交品种在玉米组织培养中的优越性。
3、在建立茎尖分生组织再生转化体系的过程中，详细地观察分析了各个阶段愈伤组织的切片结果。结果发现，整个过程中愈伤的形成需要经历初级愈伤组织、次级愈伤组织和胚性愈伤组织三个阶段，其中次级愈伤组织处在初级愈伤组织向胚性愈伤组织转型的过渡阶段，因此它的培养研究工作非常重要，直接关系到胚性愈伤组织能否高质高量的形成；另外，本研究还发现，不论是茎尖来源的胚性愈伤组织还是幼胚来源的胚性愈伤组织，它们在细胞的结构和特点上是完全相同的。
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.
1、筛选了31个基因型的玉米茎尖分生组织。在筛选的过程中，根据各个阶段受试材料的实验数据，包括诱导愈伤、继代培养和分化再生的性状表现，将这些材料进行分组，并成功地筛选出了李辽宁、IRF315、MZ17和HI-Ⅱ4个适合茎尖分生组织再生的基因型，在这4个基因型中，以杂交种HI-Ⅱ的胚性愈伤诱导效率最高，达到了80.58％，其余的三个自交系品种也都分别达到了较为理想的27.03％、42.97％和63.83％。
2、证实了由茎尖诱导的胚性愈伤组织同样也可以在培养幼胚的继代培养基上生长良好，迅速增殖，突破了茎尖培养与幼胚培养在培养基上的限制，减少了人力和原料的浪费。将这些茎尖来源的胚性愈伤组织转移到添加有3mg/L6-BA的幼胚的分化培养基上后，均获得了较高的再生效率，分别达到了91.5％、92.9％、75%和96.6％，其中仍以HI-Ⅱ的再生率最高，也进一步的证实了杂交品种在玉米组织培养中的优越性。
3、在建立茎尖分生组织再生转化体系的过程中，详细地观察分析了各个阶段愈伤组织的切片结果。结果发现，整个过程中愈伤的形成需要经历初级愈伤组织、次级愈伤组织和胚性愈伤组织三个阶段，其中次级愈伤组织处在初级愈伤组织向胚性愈伤组织转型的过渡阶段，因此它的培养研究工作非常重要，直接关系到胚性愈伤组织能否高质高量的形成；另外，本研究还发现，不论是茎尖来源的胚性愈伤组织还是幼胚来源的胚性愈伤组织，它们在细胞的结构和特点上是完全相同的。
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.
引文
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