大花蕙兰‘黄金小神童’胚性愈伤组织诱导及植株再生研究

Embryogenic callus induction and plant regeneration of Cymbidium Golden Elf‘Sundust’

为解决大花蕙兰园艺品种‘黄金小神童’(Cymbidium Golden Elf‘Sundust’)人工繁育周期长、系数低等问题,该研究以其侧芽茎尖为外植体,在1/2MS+1.0 mg·L^(-1)NAA+50 g·L^(-1)香蕉泥+15 g·L^(-1)蔗糖中培养60 d后,以获得的愈伤组织与原球茎混合物为材料,通过L_(9)(3)^(4)正交与完全组合实验研究不同因素及组合对‘黄金小神童’胚性愈伤组织和原球茎发生及增殖的影响,进而建立‘黄金小神童’高效、稳定的丛芽增殖和植株再生体系。结果表明:(1)在MS+2.0 mg·L^(-1)6-BA+150 mL·L^(-1)椰汁+20 g·L^(-1)蔗糖中,培养70 d后增殖系数达8.13,同时可获得桑葚状原球茎团。(2)原球茎团在MS+1.0 mg·L^(-1)6-BA+1.0 mg·L^(-1)NAA中培养70 d后,原球茎发育为幼芽,丛芽发生系数可达5.36;此时,将由原球茎诱导得到的簇状丛芽转接至MS+1.0 mg·L^(-1)6-BA+1.0 mg·L^(-1)NAA中,以“芽繁芽”方式增殖,其增殖系数也达到4.28,此时可建立起稳定的增殖体系。(3)无菌苗生根则在MS+1.0 mg·L^(-1)NAA+150 g·L^(-1)香蕉泥中进行,培养60 d可得到具4~7片真叶、高度为8~10 cm的健壮生根苗,生根率达96.5%;再生苗经露苗后移栽到松树皮和山基土体积比为3∶2的基质中,成活率在85%以上。通过愈伤组织与原球茎-胚性愈伤组织与原球茎-原球茎-丛芽-再生植株途径最终建立了‘黄金小神童’高效、稳定的丛芽快速繁殖体系,该研究结果为进一步开展其人工繁育和遗传转化提供了实验依据,也为兰科其他物种的无性快速繁殖提供了参考。

In order to solve the obstacle of a long artificial breeding cycle and low coefficient of Cymbidium Golden Elf‘Sundust’,the stem tip of the lateral bud was used as the explant for the initial culture in 1/2MS with 1.0 mg·L^(-1)NAA,50 g·L^(-1)banana puree and 15 g·L^(-1)sucrose.After 60 d culture,the mixture of callus and protocorm was used as the materials for investigating the effects of different factors and combinations on the embryonic callus and protocorm occurrence,proliferation via L_(9)(3)^(4) orthogonal and complete combination experiments.Finally,an efficient and stable proliferation system of Cymbidium Golden Elf‘Sundust’was established in the present study.The results were as follows:(1)Proliferation coefficient was 8.13 and the protocorm masses similar to mulberry could be obtained on the MS medium containing 2.0 mg·L^(-1)6-BA,150 mL·L^(-1)coconut juice and 20 g·L^(-1)sucrose for 70 d.(2)The protocorm was cultured in MS medium containing 1.0 mg·L^(-1)6-BA and 1.0 mg·L^(-1)NAA for 70 d,the protocorm developed into shoots with a 5.36 bud proliferation coefficient;At this time,cluster buds induced from protocorm were transferred into MS medium with 1.0 mg·L^(-1)6-BA and 1.0 mg·L^(-1)NAA to proliferate via the proliferation mode of bud to bud,and the proliferation coefficient reached 4.28,and the stable proliferation system could be established.(3)The rooting rate was up to 96.5%in MS medium equipped with 1.0 mg·L^(-1)NAA and 150 g·L^(-1)banana puree,and the healthy seedlings with 4-7 true leaves and a height of 8-10 cm could be obtained after 60 d culture;The survival rate of seedlings was more than 85%when they were transplanted into polyethylene basin with a volume ratio of pine bark to mountain soil of 3∶2.In this study,the efficient and rapid propagation system of cluster bud of Cymbidium Golden Elf‘Sundust’was established by the pathway of callus and protocorm to embryonic callus and protocorm to protocorm to cluster bud to regeneration plant,which provides an experimental basis for further artificial breeding and genetic transformation.Meanwhile,this protocol also provides the reference for asexual rapid propagation of other Orchidaceae species.