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体细胞胚胎发生与人工种子生产——一种用于观赏球根植物基氏德氏草(Drimiopsis kirkii Baker)的类型纯正繁殖和离体保存的生物技术方法

Somatic embryogenesis and synthetic seed production--a biotechnological approach for true-to-type propagation and in vitro conservation of an ornamental bulbaceous plant Drimiopsis kirkii Baker.

作者信息

Haque Sk Moquammel, Ghosh Biswajit

机构信息

Plant Biotechnology Laboratory, Department of Botany, Ramakrishna Mission Vivekananda Centenary College, Rahara, Kolkata, 700118, India,

出版信息

Appl Biochem Biotechnol. 2014 Apr;172(8):4013-24. doi: 10.1007/s12010-014-0817-2. Epub 2014 Mar 7.

Abstract

An efficient plant regeneration protocol through indirect somatic embryogenesis pathway via callus had been developed from the leaf explant of an ornamental bulbaceous plant Drimiopsis kirkii. Optimum friable calli were induced on Murashige and Skoog (MS) basal medium supplemented with 3.0 mg/l of 2,4-dichlorophenoxyacetic acid and 1.0 mg/l of α-naphthalene acetic acid (NAA). On subculturing the callus on MS medium supplemented with 2.5 mg/l of thidiazuron (TDZ), 73.3 % of the cultures responded with 20.4 ± 0.3 somatic embryos (SEs) per 500 mg callus at different stages of development after 6 weeks of culture. The highest response of 86.7 % with 28.3 ± 0.5 embryos per 500 mg callus was observed on MS medium supplemented with 2.5 mg/l TDZ and 1.0 mg/l NAA. SEs were encapsulated in calcium alginate beads for the production of synthetic seeds (SSs) and their storability was investigated. The highest SS germination (93.3 %) was observed in 1.0 % sodium alginate followed by 86.7 % germination with 2.5 % sodium alginate. The SSs were stored at three different temperatures (4, 15, and 24 ºC) up to 6 months. The SSs kept at 15 °C showed 64.4 % germinability even after 4 months of storage. Both nonencapsulated and encapsulated SE-derived plants were successfully transferred to soil with 93.3 and 88.3 % survival rate accordingly. Randomly amplified polymorphic DNA (RAPD) analysis revealed that there were no somaclonal variations among the plants produced via somatic embryogenesis and they are true-to-type to their parental plant. These results confirmed the most reliable methods, which can be further used for genetic transformation studies as well as for mass propagation of ornamental D. kirkii at a commercial level.

摘要

通过愈伤组织经间接体细胞胚胎发生途径,已从观赏球根植物基氏知母的叶片外植体建立了高效的植株再生体系。在添加3.0毫克/升2,4 - 二氯苯氧乙酸和1.0毫克/升α - 萘乙酸(NAA)的Murashige和Skoog(MS)基本培养基上诱导出了最佳的易碎愈伤组织。将愈伤组织继代培养在添加2.5毫克/升噻二唑素(TDZ)的MS培养基上,培养6周后,73.3%的培养物产生了不同发育阶段的体细胞胚(SEs),每500毫克愈伤组织有20.4±0.3个。在添加2.5毫克/升TDZ和1.0毫克/升NAA的MS培养基上,观察到最高响应率为86.7%,每500毫克愈伤组织有28.3±0.5个胚。将体细胞胚包埋在海藻酸钙珠中用于生产人工种子(SSs),并研究了它们的贮藏性。在1.0%海藻酸钠中观察到最高的人工种子萌发率(93.3%),其次是在2.5%海藻酸钠中萌发率为86.7%。将人工种子在三个不同温度(4、15和24℃)下保存6个月。保存在15℃的人工种子即使在保存4个月后仍有64.4%的发芽率。未包埋和包埋的体细胞胚衍生植株均成功移栽到土壤中,成活率分别为93.3%和88.3%。随机扩增多态性DNA(RAPD)分析表明,通过体细胞胚胎发生产生的植株之间没有体细胞克隆变异,它们与亲本植株类型一致。这些结果证实了该最可靠的方法,可进一步用于遗传转化研究以及在商业水平上大规模繁殖观赏植物基氏知母。

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