Study of Breeding of Mulberry by Tissue Culture

以原生质体融合为重点的组织培养桑葚新品种选育，通过一些基础试验进行了验证。首先，我们研究了愈伤组织样品的保存方法。然后，证实了预处理后的冷藏方法（st 5 ℃）使得保存长达20周成为可能。在本冷藏试验中，我们发现冷态细胞存在类似休眠的现象。很明显，这种现象在3周后开始，在8周后变得最显著，但在18周后，它们从这种现象中醒来，并进入休眠状态，这是由外场温度条件引起的。除难休眠的桑树（如冲绳桑葚等）外，其余休眠桑葚愈伤组织均具有此特性。没有。用琼脂糖凝胶电泳法对运输期的愈伤组织进行酶和蛋白质分析。低温期酶带的消长与培养基中添加赤霉素和二硫苏糖醇打破休眠的类似现象有一定的相似性。在长期冷冻保存的基础上，通过对预处理药物和方法的研究，开发出了比普通液氮法更简便的冷冻保存方法-接着，通过使用生长调节物质研究样品细胞的胶体分离方法。通过对上述细胞进行原生质体电融合，确认桑葚品种间存在融合困难。在尝试将它们与外来植物融合的过程中，它们几乎获得了成功，但其中一些被高压击穿。对常规培养基的组成进行了改进，增加了从不定胚到不定根的诱导生长。我们现在正在饲养它们。根据这些结果，我们可以报告达到了我们原计划的目的。

New breeding of mulberry used a tissue culture laying stress on a protoplast fusion was tested by some basic experiments. At first, we investigated a preservation method of callus sample. Then, it was confirmed that a cold storage method (st 5^ﾟC) after pretreatment made the preservation possible up to 20 weeks. In this cold storage test, we found of exist a similar phenomenon as dormancy in the cold staged cellus. It became clear that this phenomenon began after 3 weeks and became the most marked after 8 weeks, but after 18 weeks they awaked from this phenomena and moved to the state just as dormancy, caused by the condition of the outer field temperature. It appeared that all dormant mulberry callus had this character but the mulberry with difficult dormancy (for example Okinawa mulberry etc.) had not. Enzyme and protein of callus at shipping were analyzed by the electrophoresic method. The rise and fall of their bands in period of the cold stage had something in common with that of the similar phenomena as dormancy, which broken down by adding gibberellin and dithiothreitol to the culture medium. With a freezing preservation for long time storage, further, we develod more simple freezing preservation method by a refregiration than an ordinary liquid nitrogen method by our investigation of pretreatment drug and method. Next, the collosion isolation method of sample cell was studied by using the growth regulatory substances. By protoplast electric fusion of the abovementioned cell, it is recognized to exist the difficult fusion between mulberry varieties. With attempting the fusions between them and the foreign plant, they were almost succeeded, but some of them were broken by high voltage. We improved the ordinary composition of culture medium and atta ined tothe inducible growth from adventitious embryo to adventitious root. We are rearing them now. From these results, we can report to attain our purpose of the original plan.