Research On Mutants Of Chinese Cabbage By EMS Mutagenic Buds Combining With Microspore Culture

Chinese cabbage (Brassica campestris ssp. Pekinensis), originated from China, is one ofimportant vegetable crops and occupies an important position in the supply of wintervegetables. Lack of breeding material is the key issue currently in Chinese cabbage breedingand creating breeding materials with rich genetic basis is the study focus for Chinese cabbagebreeders. The artificial mutation allows the natural mutation rate increased by more than athousand times, but artificial mutation is generally recessive mutation. A genotype ofhomozygous mutant can be quickly obtained combining artificial mutation combined withmicrospore culture.In this study, ten different genotypes of Chinese cabbage ‘A01’、‘A06’、‘23P02’、‘85-1’、‘12-2’、‘12-7’、‘A03’、‘A12’、‘A16’and‘A19’were used as test materials. Using EMS as amutagen, seed mutagenesis, bud mutation combined with isolated microspore culture methodwere conducted. By determining the appropriate EMS concentration and processing time ofdifferent treatments and by performing field morphological identification andmolecular-assisted identification on the microspores plants of seed mutagenesis offspringsand bud mutations, different variations of materials were obtained. The main findings are listedas follows:1. Seeds of seven different genotypes of Chinese cabbage were used to study the seedgermination rate and seedling rate after soaking with different EMS concentration and time.The results showed that the germination rate and seedling rate decreased with the increasingof the concentration of EMS in the concentration ranging of0to0.8%. EMS mutagenizedconcentration ranging of0.4%to0.6%and processing time ranging of4to6h were initiallyindentified to be suitable for mutagenizing seeds of Chinese cabbage.2. By morphological and molecular identification on the seed mutagenesis M2generation plants of ‘A01’,‘A03’,‘A06’ and ‘23P02’, in total we identified43mutantsincluding6leaf mutants and1late bolting mutant from170plants of A01,2leaf mutantsfrom83plants of ’A06’,7package ball mutants and1corolla mutant from76plants of‘23P02’,1bud mutant,9cotyledon mutants,7seedling leaf mutants and9flower mutantsfrom285plants of A03.3. Using ‘85-1’、‘12-2’、‘12-7’、‘A03’ and‘A19’as test materials, bud mutagenesis combined with isolated microspore culture were conducted by designing mutagenesisconcentration and time gradient. Seventy four homozygous mutants in leaf shape, leaf color,corolla size, flower color, bolting resistance and downy mildew resistance were found, withmutation frequency of15.6%. Comprehensively taking into account the microspore embryorate, seedling rate and the number of effective mutant, EMS appropriate concentration rangeof0.03to0.1%, the processing time of5to10min were initially identified for the technologyof EMS mutagenic buds combined with microspore culture.4. Investigating the ploidy level of the microspore culture plantlets after EMS budmutagenesis, the proportion of diploid plants decreased with the decreasing concentration ofEMS, while the haploid proportion increased. It suggested that the EMS has inhibitory effecton chromosome doubling in the process of mutagenesis.5. Through comparison of two mutagenesis ways of EMS seed mutagenesis and budsmutagenesis combined with microspore culture in Chinese cabbage, it proved that the newapproach of EMS bud mutation combined with microspore culture is feasible, and thismutagenesis has advantages to seeds mutagenesis and microspore culture method. And thetypes of variation were relatively abundant. It opened up new avenues for EMS mutagenesistechniques in innovating Chinese cabbage germplasm.6. Through HRM detection on microspore culture plantlets after EMS bud mutagenesis,four flower color mutant were found. And through comparison with directly sequencingfragments, it proved that HRM method is feasible for EMS screening mutant, simpleoperation, low cost, high throughput.