| In this study,Yunnan quince(Cydonia oblonga Mill)was chosen as material.The factors that influence adventitious buds regeneration from transverse thin cell layers(tTCLs)were examined systematically and tTCLs regeneration system in vitro were established;the effects of Pingyangmycin(PYM)and ethylmethanesulphonate(EMS)treatment on plant regeneration and mutant induction were investigated and the salt-tolerant mutants were selected from germination medium containing 1.2 %(w/v)NaCl;as to the induction of tetraploid,the proliferating shoots and the tTCLs of Yunnan quince were treated with different concentrations of colchicine,the ploidy level of the colchicine treated individuals was analysed by flow cytometry and tetraploids were obtained.The main results as follows:1.Establishment of the regeneration system from tTCLs of Yunnan quinceSeries of factors(i.e.plant growth regulators,basic media,carbon sources,darkness period)that influence adventitious buds regeneration from tTCLs were examined systematically.Optimum response to direct adventitious shoot bud induction from tTCLs was observed on MS medium containing 1.5 mg·L-1 TDZ,0.4 mg·L-1 IBA,30 g·L-1 sucrose,7.0 g·L-1 agar,and extra dark period of 7 days at the beginning of culture.The highest regeneration frequency of Yunnan quince was 35.41%,and the mean bud number per tTCL was 2.15.The regenerated shoots were rooted on 1/2 MS+0.3 mg·L-1NAA + 20 g·L-1sucrose + 7 g·L-1agar.2.Salt-tolerant mutation screening of Yunnan quinceThe tTCLs of Yunnan quince were treated with different mutagenic dose treatments of PYM and EMS.The dose at which 50% of the tTCLs recovered(RP50)after the mutagen treatment was used as the optimum dose.Explants of Yunnan quince were treated with RP50 PYM and EMS dosages,respectively,and subsequent regenerants that arose from tTCLs were transferred to germination medium containing 1.2 %(w/v)NaCl for screening salt-tolerant mutants.Under salt stress the SOD and POD activity in the mutation regeneration was higher than that induced processing plants but MDA and Proline content was lower than control plants.3.Polyploid induction and identification of Yunnan quinceProliferating shoots and the tTCLs of Yunnan quince were treated with different concentrations of colchicine,then shoots were transferred to germination medium and tTCLs were transferred to shoot regeneration medium,respectively.The ploidy level of the colchicine treated individuals was analysed by flow cytometry.2 tetraploids appeared in the shoots regenerated from tTCLs which excised from shoots of Yunnan quince treated with 0.03 %(w/v)colchicine.Under salt stress the SOD and POD activity in polyploid was higher than that induced processing plants but MDA content was lower than control plants. |
