广丰千金薯离体快繁及其气孔观察、染色体倍数FCM分析和DNA变异ISSR检测

Rapid Propagation in vitro of Dioscorea opposita ‘Guangfeng'and Its Stomatal Observation,FCM Analysis of Chromosome Ploidy and ISSR Detection of DNA Mutation

目的：对广丰千金薯的离体快繁进行研究,并对其移栽驯化苗和盆栽苗的气孔、染色体倍数和DNA变异进行观察比较、FCM分析和ISSR检测,旨在为广丰千金薯种苗规模化生产提供技术基础。方法：采用植物组织培养的方法,建立和优化广丰千金薯离体快繁技术体系,并用透明胶带粘取法观察和比较移栽驯化苗和盆栽苗的气孔参数,用FCM分析移栽驯化苗和盆栽苗的染色体倍数,用ISSR检测分子标记检测移栽驯化苗和盆栽苗的DNA变异。结果：广丰千金薯离体快繁技术体系为：选取带芽的稍木质化茎段,经70%酒精消毒1 min、无菌水冲洗3次、0.1%氯化汞消毒12 min、无菌水冲洗3次,然后接种到MS＋KT 1 mg/L＋NAA 0.2 mg/L固体培养基上置于温度为（25±2）℃、光照强度为1 500～2 000 Lx、光照时间为14 h/d的条件下培养。待到新芽长至2～3 cm,便将其切下接种于MS＋KT 2 mg/L＋NAA 0.5 mg/L液体培养基中继续置于上述培养条件下培养。培养90 d左右便可形成完整植株。移栽时将封口膜打开,依旧置于上述培养条件下培养2～3 d,然后取出完整植株洗净培养基后放入盛有浅层MS基本培养液的容器中进行室内移栽驯化,待到新芽从植株上长出,即可取出驯化苗移植于户外盛有沙土（1∶1,40%甲醛消毒）的盆中,每天早晚各浇水1次,成活率达100%。气孔观察、FCM分析和ISSR检测结果显示,移栽驯化苗和盆栽苗的气孔参数和染色体倍数无显著性差异,两者的ISSR扩增谱带也无特异性条带产生。结论：建立和优化了广丰千金薯离体快繁技术体系,再生植株没有发生遗传性变异,可以保证其遗传稳定性。

Objective： To study the rapid propagation in vitro of Dioscorea opposita‘Guangfeng＇,and to observe the stomas of the transplanting plantlets and potted seedlings,to test chromosome ploidy by FCM,and to detect DNA mutation by ISSR,in order to provide the technical basis for the large-scale production of Dioscorea opposita ‘Guangfeng＇plantlets. Methods： The technique system of Dioscorea opposita ‘Guangfeng＇rapid propagation in vitro was established and optimized by plant tissue culture method. The parameters of transplanting plantlets and potted seedlings were studied as follows,the stomatal parameters were observed by transparent adhesive tape method,chromosome ploidy were analyzed by FCM,and DNA mutation were detected by ISSR molecular marker. Results： The technique system of Dioscorea opposita‘Guangfeng＇rapid propagation in vitro was as follows,slightly woody stem segment with a bud were selected and inoculated onto MS ＋ KT 1 mg / L ＋ NAA 0. 2 mg / L solid culture medium and cultured in the photoperiod of 14 h / d（ the temperature was（ 25 ± 2） ℃ and light intensity was 1 500～2 000 Lx） after disinfected for 1 min in 70% alcohol prior to sterilized for 12 min with 0. 1% Hg Cl2,the materials were washed with sterile water for 3 times,repectively. The new bud was cut off when it grew to 2～3cm and inoculated into MS ＋ KT 2 mg / L ＋ NAA 0. 5 mg / L liquid culture medium and continued to culture in above culture conditions. The whole plant was formed after cultured for about 90 d. The sealing membrane was opened in transplanting,and the plantlets was still placed in above culture conditions and cultured for 2～3 d,and then the whole plant was taken out,and the culture medium washed off and then transferred into the vessel with shallow liquid MS basic culture medium and domesticated indoor. The acclimated plantlets were taken out and transplanted in the outdoor pots with the sandy soil when the new shoots grew out,and watered one time with tap water in the morning and evening per day,the survival rate reached 100%. The results of stomatal observation,FCM analysis and ISSR detection of transplanting plantlets and potted seedlings showed that the stomatal parameters,chromosome ploidy and DNA mutation of plantlets and potted seedlings had no variation. Conclusion： The results reveal that the establishment and optimization of the technique system of Dioscorea opposita ‘Guangfeng＇rapid propagation in vitro is feasible,and the regenerated plants do not have genetic variation which can ensure the stability of the genetic.