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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 in vitro propagation of F. chinensis Franch. In vitro and in vivo, the stomata, chromosome multiples and DNA variations of domestication seedlings and potted seedlings were observed, compared and analyzed by FCM and ISSR. Large-scale production of seedlings provide the technical basis. Methods: Plant tissue culture method was used to establish and optimize the rapid propagation system of Chrysanthemum chinense in vitro. The stomatal parameters of transplanted domesticated seedlings and potted seedlings were observed and compared with scotch tape adhesion method. Chromosome multiplication of potted seedling was detected by ISSR detection of molecular markers of transplanted domestication and potted seedling DNA variation. Results: The system of fast-propagation of Chrysanthemum chinense in vitro was as follows: A slightly lignified stem with buds was selected, disinfected with 70% alcohol for 1 min, rinsed with sterile water three times, disinfected with 0.1% mercuric chloride for 12 min, sterilized Water for 3 times, and then inoculated into MS + KT 1 mg / L + NAA 0.2 mg / L solid medium at a temperature of (25 ± 2) ℃, light intensity of 1500 ~ 2000 Lx, light time of 14 h / d under the conditions of culture. When the shoots grow to 2 ~ 3 cm in length, they are cut and inoculated into liquid medium MS + KT 2 mg / L + NAA 0.5 mg / L and cultured in the above culture conditions. Cultivation of about 90 d can form a complete plant. Transplanting the seal membrane to open, still placed in the above culture conditions for 2 ~ 3 d, and then remove the entire plant wash medium into shallow MS basal culture medium containers for indoor transplanting domestication, to be To the sprout grows from the plant, you can remove the domestication seedlings transplanted outdoors in sand (1: 1, 40% formaldehyde disinfection) pots, watering every morning and evening 1 time, the survival rate of 100%. Stomatal observation, FCM analysis and ISSR test showed that there was no significant difference in stomatal parameters and chromosomal fold between transplanted domestication seedlings and potted seedlings. There was also no specific bands in both bands of ISSR amplification. Conclusion: The system of in vitro propagation of Chrysanthemum ficollma was established and optimized. No genetic variation occurred in the regenerated plants, which could ensure its genetic stability.