开发实用的染色体加倍体系构建成烟草DH群体

Developing Practicable System of Chromosomal Doubling for the Construction of Doubled Haploid Population in Tobacco

通过对4种染色体加倍方法(烟草浸花药法、浸花培苗法,叶片再生法及浸腋芽法等)的比较研究表明:以4 g/L浓度的秋水仙碱浸泡花药法加倍率最高达47.5%～75%,其次为叶片再生法(30%～36.67%)和浸苗法(16.67%～32.35%),而浸腋芽法较低(17.78%).前两种方法加倍率虽高,但有较高畸形苗比率,叶片再生法工序较繁琐.作者认为烟草加倍单倍体产生,应以采用浸苗法为主.在使用秋水仙碱浸苗加倍时,添加DMSO可明显促进秋水仙碱的加倍效率,且促进作用随时间延长而提高;从高效、快捷和节约等原则考虑,我们开发了烟草有效的染色体加倍体系,以4g/L的秋水仙碱+20g/L DMSO溶液浸苗48h的效果最好.对两个组合烟草单倍体苗浸12h以上,其染色体加倍效率达到对照的2.30～2.93倍.本试验用较低浓度秋水仙碱添加DMSO有利于节约实验费用.通过完善染色体加倍技术程序已构建成2个DH群体,供基因定位研究.

A comparative analysis of effects of four methods on chromosome doubling in tobacco indicated that the efficiency of colchicines (4g/L) applied directly to anther excised from the flower buds of tobacco just before anther culture was the highest (47.5%~75%), followed by that of leaves pre-treated by colchicines before regeneration was 30%~36.67%; that of the haploid plantlets was soaked in colchicine was 16.67%~32.35%; and the efficiency of colchicine soaked the auxiliary bud was the lowest (17.78%). The former both the methods brought about higher score in doubling, but had the higher abnormal plantlets ratio and the more tedious working procedure. Therefore, the method of soaking young plantlets in colchicine was highly effective, quickly and saves, when considering of the construction DH population. It was obvious that the doubling haploids plantlets of tobacco increase with soaking duration, when application of 4g/L of colchicine solution mixed with 20g/L of DMSO. The highest doubling haploid plantlets were obtained in colchicines soaking for 48h. With soaking plantlets in colchicine more than 12 h, the frequency of doubling in two tobacco crosses was raised to 2.30~2.93. Also, the application of DMSO to colchicine at low concentrations could lead to the higher doubling efficiency. Through completing the technical procedure of chromosome doubling, we have constructed two DH populations for the gene localization research.