Study On Ploidy Level Plant Regeneration From Microspore Of Chinese Cabbage

Chinese cabbage originating from China, is a subspecies of Brassica genus that could form leaf head. Isolated microspore culture could provide new solutions for improvement of Chinese cabbage breeding now. It is about 1-2 years to get homozygous Doubled Haploid(DH)regeneration plants using isolated microspore culture techeniques. Their ploidy level usually were identified by observing their morphological characteristics during budding and flowering. However, this chromosome doubling method is labour intensive, time consuming,and isn't suitable for treating large number of plants. DNA methylation is a primary way of epigenetic modification in genomic DNA level,methylcytosine usually occurs in plants. DNA methylation is known to play an important role in the regulation of gene expresstion, cell differentiation and phylogenesis, effectting florescence,fertility,flower and the form of blade. Combining Methylation-Sensitive Restriction Endonuclease PCR(MS-RE PCR), Sequence- Related Amplified Polymorphism(SRAP) and Methylation-Sensitive Amplified Polymorphism (MSAP) molecular techniques, with morphologic identification and root tip chromosome preparation to identify ploidy level of microspore cultured regeneration plants from Chinese cabbage.The main results are as follows:1. Isolated microspore culture from Chinese cabbage, getting 9 regeneration plants including 2 haploids and 7 diploids. The spontaneous doubling rate was up to 77.7%. 2 haploids and 2 diploids were selected to construct their respective bulk. Combining Methylation-Sensitive Restriction Endonuclease-PCR method with SRAP marker technique, we gained 9 mutated sites showing polymorphism screened from 768 pairs of primers. It indicated that the ploidy level of microspore cultured regeneration plants from Chinese cabbage may related to DNA methylation in plant genomics.2. Isolated microspore culture from different variety Chinese cabbage,and construct their respective bulk. Combining Methylation-Sensitive Restriction Endonuclease-PCR method with SRAP marker technique, we gained 0 mutated sites showeing polymorphism screened from 1928 pairs of primers. It indicated that this way can not apply to the bulk.3. Chinese cabbage(Z-16)have a high spontaneous chromosome doubling rate. Isolated microspore culture from it, getting 17 regeneration plants including 3 haploids and 14 diploids. The spontaneous doubling rate was up to 82.4%. We gained 2 mutated sites showing polymorphism screened from 64 pairs of primers by MSAP. It indicated that MSAP is a way to study the relationship between ploidy level of microspore cultured regeneration plants from Chinese cabbage adn DNA methylation in plant genomics.