黑果枸杞原生质体培养及植株再生  

Protoplast Culture and Plant Regeneration of Lycium ruthenicum

作　　者：宁晓春 高思丹 杨莉娜[4] 陈生蓉 何涛 NING Xiaochun;GAO Sidan;YANG Li'na;CHEN Shengrong;HE Tao(School of Eco-Environment Engineering, Qinghai University, Xining 810016, China;2Key Laboratory of Landscape Plants of Qinghai Province, Qinghai University, Xining 810016, China;3State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University, Xining 810016, China;4College of Agriculture and Animal Husbandry, Qinghai University, Xining 810016, China)

机构地区：[1]青海大学生态环境工程学院,西宁810016 [2]青海省园林植物与观赏园艺重点实验室,西宁810016 [3]省部共建三江源生态与高原农牧业国家重点实验室,西宁810016 [4]青海大学农牧学院,西宁810016

出　　处：《西北植物学报》2021年第11期1825-1833,共9页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(31660217);青海大学青年科研基金(2020-QNY-7)。

摘　　要：该研究以黑果枸杞(Lycium ruthenicum)无菌苗为材料,建立了愈伤组织来源的原生质体再生体系,采用ISSR和FCM技术对再生植株进行了遗传稳定性分析。结果表明:(1)黑果枸杞叶片愈伤组织是产生原生质体的最好材料,在含0.5 mg·mL-1甘露醇的酶液中,继代1次的叶片愈伤组织中原生质体产量为7.77×10^(6)个·g^(-1),活力为92%。(2)改良MS培养基-固体液体双层培养(MS_(2)-固液双层)是培养原生质体的最好方式,培养10 d的原生质体分裂频率为45.9%,培养20 d的细胞团形成频率为22.9%。(3)在1.5 mg·mL^(-1)6-BA+0.1 mg·mL^(-1) IBA+MS培养基中,叶片愈伤组织产生的原生质体可分化获得再生植株。(4)ISSR分析显示,再生植株的平均遗传相似系数为0.88;FCM显示再生植株为二倍体,与亲本植株一致。该研究结果为进一步研究枸杞体细胞杂交技术转移野生植物抗逆遗传性状提供科学依据,为枸杞优良品种的选育奠定了基础。An efficient protocol for plant regeneration was developed from protoplasts isolated from calli of Lycium ruthenicum,and the genetic stability of the regenerated plants was analyzed by ISSR and FCM techniques.The results showed that:(1)leaf calli were the best material for producing protoplasts,and in the enzyme solution containing 0.5 mg·mL^(-1) mannitol,the yield of protoplasts of leaf calli subcultured once was 7.77×10^(6) pieces·g^(-1),vitality was 92%.(2)Improved MS medium-Solid-Liquid Double layer culture(MS2-Solid-Liquid Double layer)was the best way to culture protoplasts.The frequency of protoplast division was 45.9%after 10 days of culture,and the frequency of cell mass formation was 22.9%after 20 days of culture.(3)In 1.5 mg·mL-16-BA+0.1 mg·mL-1 IBA+MS medium,the protoplasts produced by leaf calli could differentiate into regenerated plants.(4)ISSR showed that the average genetic similarity coefficient of the regenerated plants was 0.88;FCM showed that both the regenerated plants and the parent plants were diploid.The results of this study provide a scientific basis for further research on the somatic hybridization technology of L.ruthenicum to transfer the genetic traits of stress resistance of wild plants,and lay a foundation for the selection and breeding of excellent varieties of L.ruthenicum.

关 键 词：黑果枸杞 原生质体 植株再生 遗传稳定性 

分 类 号：Q813.11[生物学—生物工程] S567.239[农业科学—中草药栽培]