The Study On The Genetic Stability Repeatedly Subcultured In Vitro Of Dendrobium Huoshanense

Dendrobium huoshanense is a kind of precious traditional Chinese medicine in Anhui Province,and is protected by government for its medicinal value.Whether D.huoshanense seedling after subculturing several times can maintain genetic stability concerns its industrialization.The genetic stability of D.huoshanense repeatedly subcultured in vitro for one to four times with three different original materials of mature seed,stem segment,and callus,was examined by using RAPD,ISSR,DALP and SRAP,and a rapid propagation way which can maintain D.huoshanense genetic stability is hoped to find out,thus promoting its industrialization.1.In the study,the genomic DNA of D.huoshanense repeatedly subcultured in vitro extracted by advanced CTAB method could be fully used for all kinds of molecular marker method,furthermore,the method explored was applicable,economical,simple and rapid.2.To gain a suitable system for various molecular marker method,the experimental profile and parameters were optimized.SRAP is a newly developed molecular marker method,which is stable,reliable,effective and reproductive.The optimized reaction system was as follows:25μL reaction system containing 30ng template,2.5mmol/L Mg²⁺,0.8μmol/L primers,0.2mmol/L dNTPs and Taq polymerase 1U.The optimal amplification program was as follows:5min at 94℃,followed by 94℃for 1min,35℃for 1min,72℃for 1min,5 cycles,1min at 94℃,50℃for 1min,72℃for 1min,35 cycles,and final extension time of 72℃for 7min.3.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed by RAPD,and the results showed that some weakening bands at approximately 1000bp were found within the third and the fourth descend,when D.huoshanense repeatedly subcultured in vitro with materials of stem segments were detected by primer S22;Besides,some weakening bands were found at 1000bp and 500bp within the fourth descend,when D.huoshanense repeatedly subcultured in vitro with materials of callus were detected by primer S22 and S25.4.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed by ISSR,the results indicated that weakening bands of primer UBC-820 were found at approximately 2250bp,primer UBC-870 at approximately 1500bp,within the four descends of D.huoshanense repeatedly subcultured in vitro with materials of callus;Some bands weakened clearly at approximately 750bp,when D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments were detected by primer UBC-807.5.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analyzed through DALP,the results indicated that every band of the fourth descend weakened clearly,which were 2250,1500,1000,750bp;Some bands at approximately 3000bp gradually weakened from the second descend,when D.huoshanense repeatedly subcultured in vitro with materials of callus were detected by primer DALP241-DALPR.6.D.huoshanense seedling repeatedly subcultured in vitro for one to four times was analysis through SRAP,the results showed that weakening bands of primer m8-em5 were found at 800bp within the four descends of D.huoshanense repeatedly subcultured in vitro with materials of callus.The four molecular marker methods showed D.huoshanense seedlings repeatedly subcultured in vitro maintained genetic stability basically the same.No variation was detected within the descends of D.huoshanense repeatedly subcultured in vitro with materials of mature seed,maintaining highly genetic stability;Some bands weakening were detected,but no variation spot was found within the descends of D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments.With the comparison of the three subculturing methods,it indicated that some variations were found within the descends of D.huoshanense repeatedly subcultured in vitro with materials of callus and stem segments,and callus varied more than stem segments.Repeatedly subculturing with the materials of mature seed was better rapid propagation way to maintain the genetic stability of D.huoshanense.