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香石竹毛状根诱导、离体培养及其植株再生
引用本文:施和平,朱远锋,王蓓,孙蒋兵,黄胜琴. 香石竹毛状根诱导、离体培养及其植株再生[J]. 生物工程学报, 2014, 30(11): 1742-1750
作者姓名:施和平  朱远锋  王蓓  孙蒋兵  黄胜琴
作者单位:华南师范大学生命科学学院 广东省植物发育生物工程重点实验室,广东 广州 510631;华南师范大学生命科学学院 广东省植物发育生物工程重点实验室,广东 广州 510631;华南师范大学生命科学学院 广东省植物发育生物工程重点实验室,广东 广州 510631;华南师范大学生命科学学院 广东省植物发育生物工程重点实验室,广东 广州 510631;华南师范大学生命科学学院 广东省植物发育生物工程重点实验室,广东 广州 510631
基金项目:广东省科技计划项目 (No. 2011B020304006) 资助。
摘    要:为了探讨利用发根农杆菌遗传转化所产生的毛状根来创新香石竹种质的可能性,本文采用叶盘法,建立了发根农杆菌Agrobacterium rhizogenes对香石竹Dianthus caryophyllus L.叶片外植体的遗传转化及其植株再生体系。结果表明,发根农杆菌ATCC15834感染香石竹幼嫩叶片外植体12 d后,从叶片外植体切口中脉处产生白色毛状根,21 d后约90%的叶片外植体产生毛状根。所获得的无菌毛状根能在无外源激素的MS固体和液体培养基中快速自主生长。PCR扩增和硅胶薄层层析结果显示发根农杆菌Ri质粒的rol B和rol C基因以及冠瘿碱合成酶基因已在香石竹毛状根基因组中整合并得到表达。将毛状根置于MS+6-BA 1.0-3.0 mg/L+NAA 0.1-0.2 mg/L中培养15 d后产生淡黄绿色的疏松愈伤组织。愈伤组织不定芽分化的最适培养基为MS+6-BA 2.0 mg/L+NAA 0.02 mg/L,培养6周后不定芽分化率为100%;平均每个愈伤组织产生30-40个不定芽;将不定芽转至1/2 MS或1/2 MS+0.5 mg/L NAA的培养基中10 d后产生不定根,发育成再生植株。再生植株移植于栽培基质中20 d后,成活率达95%以上。

关 键 词:香石竹  发根农杆菌  毛状根  植株再生
收稿时间:2014-01-25

Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration
Heping Shi,Yuanfeng Zhu,Bei Wang,Jiangbing Sun and Shengqin Huang. Induction and in vitro culture of hairy roots of Dianthus caryophyllus and its plant regeneration[J]. Chinese journal of biotechnology, 2014, 30(11): 1742-1750
Authors:Heping Shi  Yuanfeng Zhu  Bei Wang  Jiangbing Sun  Shengqin Huang
Affiliation:Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, Guangdong, China;Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, Guangdong, China;Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, Guangdong, China;Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, Guangdong, China;Guangdong Provincial Key Laboratory of Biotechnology for Plant Development, College of Life Science, South China Normal University, Guangzhou 510631, Guangdong, China
Abstract:To use Agrobacterium rhizogenes-induced hairy roots to create new germplasm of Dianthus caryophyllus, we transformed D. caryophyllus with A. rhizogenes by leaf disc for plant regeneration from hairy roots. The white hairy roots could be induced from the basal surface of leaf explants of D. caryophyllus 12 days after inoculation with A. rhizogenes ATCC15834. The percentage of the rooting leaf explants was about 90% 21 days after inoculation. The hairy roots could grow rapidly and autonomously in liquid or solid phytohormone-free MS medium. The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and silica gel thin-layer chromatography of opines from D. caryophyllus hairy roots. Hairy roots could form light green callus after cultured on MS+6-BA 1.0?3.0 mg/L +NAA 0.1?0.2 mg/L for 15 days. The optimum medium for adventitious shoots formation was MS + 6-BA 2.0 mg/L +NAA 0.02 mg/L, where the rate of adventitious shoot induction was 100% after cultured for 6 weeks. The mean number of adventitious shoot per callus was 30?40. The adventitious shoots can form roots when cultured on phytohormone-free 1/2 MS or 1/2 MS +0.5 mg/L NAA for 10 days. When the rooted plantlets transplanted in the substrate mixed with perlite sand and peat (volume ratio of 1:2), the survival rate was above 95%.
Keywords:Dianthus caryophyllus   Agrobacterium rhizogenes   hairy roots   plant regeneration
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