籼稻愈伤组织分化调控及机理初探
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摘要
水稻愈伤组织诱导及分化的研究在粳稻上已经取得了很大的进展,但是籼稻的研究进展却相对缓慢,至今未能形成有效的抑制愈伤组织老化的研究体系,尤其是籼稻愈伤组织的诱导、增殖及分化能力低,更加制约了该研究在籼稻上的发展。本实验室创建的愈伤组织培养方法可以使籼稻愈伤组织在室温下保存一年以上仍然具有较高的分化率,且可以对愈伤组织的分化进行人工调控。
本研究针对这种籼稻愈伤组织的保存方法展开实验。首先选用重复性好的成熟胚为材料进行培养,连续诱导愈伤组织并将一部分进行处理,调查分析愈伤组织增殖及分化情况,另外对组培苗的性状进行调查,分析变异情况。结果发现愈伤组织在处理时保持增殖状态,不分化也不会随培养时间的延长而老化,在经过约6~7次继代后其分化率趋于稳定,保持在50%左右;将持续增殖的愈伤组织转到分化培养基上后,与新诱导的愈伤组织一样可以在1周左右变绿,2周出现芽的分化,3~4周出苗,且绿苗率高,达99.8%;通过对组培苗的观察及农艺性状的调查发现,经过长期保存的愈伤组织分化出的组培苗没有出现明显的不良性状变异。另外以不同品种的花药及成熟胚培养诱导产生的愈伤组织为材料,结果表明不同外植体和不同品种得到的愈伤组织经该处理后均保持增殖状态而不分化,但转到分化培养基后能很快分化出苗。同时采用SDS-聚丙烯酰胺凝胶电泳对所得籼稻成熟胚愈伤组织全蛋白进行分析,结果发现处理的愈伤组织全蛋白和对照的相比,存在3条稳定的差异条带,其分子量大小分别约为48kd、42kd和40kd。
Establishment of high efficiency transformation has greatly facilitated the widespread application of transformation in japonica rice. In contrast, there have been very few successful cases reported for indica rice. Recently, through the seeds-derived calluses of indica rice, an efficient callus preservation system has been successfully established in Zhaoming Pan’s laboratory.
The utilization and molecular mechanism of this method will be uncovered in this study. It was found that the frequency of callus differentiation going a stable state along the time, which was high, feasibility of long-term callus persevered was carried out. Up to now, embryogenesis callus have been cultured for 15 months, which were light yellow, drying, compact and with high differentiation rate compared with those of the control. The callus would start green in a short time after transferred to regeneration media, bud in two weeks and regenerate quantity of caespitose seedlings, which rate was high to 99.8%. And there was no obvious ill varitiation in the development of the seedings. In addition, different varieties of anther and seed were used for callus induction to validate the applicability on callus from different explants and genotype. It turned out that it was not the behavior of the callus from special explant or genotype, but the treat method induced the long-term preservation of callus. Synchronously, the proteins of the callus were analyzed by SDS-PAGE, some special proteins were separated from the callus, and the molecular weight of which were about 40KD, 42KD and 48KD.
本研究针对这种籼稻愈伤组织的保存方法展开实验。首先选用重复性好的成熟胚为材料进行培养,连续诱导愈伤组织并将一部分进行处理,调查分析愈伤组织增殖及分化情况,另外对组培苗的性状进行调查,分析变异情况。结果发现愈伤组织在处理时保持增殖状态,不分化也不会随培养时间的延长而老化,在经过约6~7次继代后其分化率趋于稳定,保持在50%左右;将持续增殖的愈伤组织转到分化培养基上后,与新诱导的愈伤组织一样可以在1周左右变绿,2周出现芽的分化,3~4周出苗,且绿苗率高,达99.8%;通过对组培苗的观察及农艺性状的调查发现,经过长期保存的愈伤组织分化出的组培苗没有出现明显的不良性状变异。另外以不同品种的花药及成熟胚培养诱导产生的愈伤组织为材料,结果表明不同外植体和不同品种得到的愈伤组织经该处理后均保持增殖状态而不分化,但转到分化培养基后能很快分化出苗。同时采用SDS-聚丙烯酰胺凝胶电泳对所得籼稻成熟胚愈伤组织全蛋白进行分析,结果发现处理的愈伤组织全蛋白和对照的相比,存在3条稳定的差异条带,其分子量大小分别约为48kd、42kd和40kd。
Establishment of high efficiency transformation has greatly facilitated the widespread application of transformation in japonica rice. In contrast, there have been very few successful cases reported for indica rice. Recently, through the seeds-derived calluses of indica rice, an efficient callus preservation system has been successfully established in Zhaoming Pan’s laboratory.
The utilization and molecular mechanism of this method will be uncovered in this study. It was found that the frequency of callus differentiation going a stable state along the time, which was high, feasibility of long-term callus persevered was carried out. Up to now, embryogenesis callus have been cultured for 15 months, which were light yellow, drying, compact and with high differentiation rate compared with those of the control. The callus would start green in a short time after transferred to regeneration media, bud in two weeks and regenerate quantity of caespitose seedlings, which rate was high to 99.8%. And there was no obvious ill varitiation in the development of the seedings. In addition, different varieties of anther and seed were used for callus induction to validate the applicability on callus from different explants and genotype. It turned out that it was not the behavior of the callus from special explant or genotype, but the treat method induced the long-term preservation of callus. Synchronously, the proteins of the callus were analyzed by SDS-PAGE, some special proteins were separated from the callus, and the molecular weight of which were about 40KD, 42KD and 48KD.
引文
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