Response Of Genotypes To Chromosome Doubling Through Treating In Vitro Microspores With Colchicine In Brassica Napus And The Ploidy Testing Of Microspore-derived Plants

The establishment of microspore culture system in Brassica napus in the late 1980s made the doubled haploid plants breeding program possible. But the spontaneous chromosome doubling frequency of microspore-derived plants is very low, 70% to 90% plants remains haploid, which flower abnormally and can not produce enough seeds, so it can not be utilized in genetic and breeding program research. Some researchers made efforts on the chromosome doubling techniques of direct colchicine treatment of isolated microspores and achieved over 90% doubling frequency in some experiments. But the doubling frequency is different between different genotypes. Microspore-derived plants consist of haploid, doubled haploid and chimera. Their ploidy level usually were identified by observing their morphological characteristics during budding and flowering. Haploid plants were dig up from soil and roots were soaked in a high concentration colchicine solution. However, this chromosome doubling method is labour intensive, time consuming, substantially retards plant growth and isn't suitable for treating large number of plants. So an effective and convenient method to identify them at early stage is required.Response of genotypes in Brassica napus to chromosome doubling was studied by treating isolated microspores with colchicine. The ploidy level of microspore-derived plants was evaluated by three methods. The results are as follows:1. The ratio of doubled haploid plants derived from treated microspores of 21 genotypes ranged from 23.94% to 99.42%. The results of deviance analysis showed that chromosome doubling rate of the similar genotypes was not significantly different, whereas significant difference was identified among some distinct genotypes.2. The relationship between chloroplast number in stomatal guard cells and the ploidy level of plants were studied using tube seedlings. The mean chloroplast number per guard cell is 7 and 12 of haploid and doubled haploid, ranged from 4 to 10 and 7 to 17 respectively . The difference in the mean chloroplast number between haploid and doubled haploid plantlets was highly significant. The results demonstrate that counting chloroplast number in stomatal guard cells is an efficient means of distinguishing a large number of plantlets for ploidy levels.3. The ploidy level of microspore-derived plants was analyzed by flow cytometry. The results show that flow cytometry can identify haploid, chimera and doubled haploid accurately, but it may not be used to identify a large number of plants forploidy level because of needing expensive equipment and drugs.4. Chimera derived from microspores were self-pollinated to observe their trait of generating seeds. Chimera from the 903 line produced 19 seeds per bagged inflorescence. This show that some chimera are useful for genetic and breeding program study when many inflorescences are bagged.