LfMADS1基因对新铁炮百合‘Raizen No.1’的遗传转化

生物技术通报 ›› 2014, Vol. 0 ›› Issue (3): 86-93.

LfMADS1基因对新铁炮百合‘Raizen No.1’的遗传转化

李云华, 刘青, 刘青林

(中国农业大学观赏园艺与园林系，北京 100193)

收稿日期:2013-10-18 出版日期:2014-03-29 发布日期:2014-03-31
作者简介:李云华，女，硕士研究生，研究方向:百合种质资源与生物技术；E-mail:liyunhua@cau.edu.cn；刘青同为本文第一作者通讯作者:刘青林，男，博士，副教授，研究方向:园林植物种质资源与遗传育种；E-mail:liuql@cau.edu.cn
基金资助:
国家高技术发展计划(“863”计划)(2006AA10Z187)，公益性行业(农业)科研专项(201203071)

Study on Genetic Transformation of LfMADS1 Gene into Lilium ** formosanum' Raizen No.1'

Li Yunhua, Liu Qing, Liu Qinglin

(Department of Ornamental Horticulture and Landscape Architecture，China Agricultural University，Beijing 100193)

Received:2013-10-18 Published:2014-03-29 Online:2014-03-31

摘要/Abstract

摘要： 为了获得无花粉、重瓣百合新种质，以新铁炮百合‘Raizen No.1’的离体小鳞片为受体，探讨了不同水平的抗生素和抑菌素对小鳞片再生能力的影响；利用正交试验对预培养时间、菌液浓度、侵染时间和共培养时间等4个因素的不同水平进行了筛选，优化了遗传转化体系；进行了百合花器官特性基因 LfMADS1的转化，并对转基因植株进行了分子生物学检测。结果表明，新铁炮百合小鳞片选择培养时抗生素卡那霉素(Kan)浓度为100 mg/L，抑菌素为羧苄青霉素(Carb)浓度为500 mg/L。正交试验结果表明共培养时间是遗传体系中的主要影响因子；小鳞片转化的最佳条件是预培养1 d、侵染10 min、共培养5 d、菌液OD 600值0.8。PCR检测的结果，得到4株转 LfMADS1反义基因的新铁炮百合株系，其中1株已通过PCR-Southern检测。

关键词: 小鳞片, 农杆菌介导转化, 卡那霉素, 羧苄青霉素, 正交试验

Abstract: In order to create pollen-free double flowers, the in vitro bulblet scales of Lilium × formosanum ‘Raizen No. 1’were taken as explants, the effects of Kan and Carb on regeneration from bulblet scales were studied at first. Then a orthogonal test were conducted to screening such factors as pre-culture time, bacterial concentration, infection time and co-culture time, and in this way the transformation system was optimized. Through this system, LfMADS1 gene was transferred into bulblet scales mediated by Agrobacterium, and the transgenic plants were molecularly identified. The results indicated that Kan 100 mg/L was optimal to screen adventitious buds of Kan-resistant；Carb 500 mg/L was able to control growth of Agrobacterium. There are some key points include pre-culture for 1d, immersion of explants into bacterial suspension(OD 600 0.8)for 10 min, and co-cultivate for 5 d in the optimized transformation system. Four transgenic lines were obtained through this system identified by PCR, in which the exogenous gene had successfully integrated into genome of one line confirmed by PCR-Southern. These transformed lilies can be a new germplasm for the breeding of pollen-free or double flower lilies.

Key words: Bulblet scales, Agrobacterium-mediated transformation, Kanamycin, Carbenicillin, Orthogonal test

李云华, 刘青, 刘青林. LfMADS1基因对新铁炮百合‘Raizen No.1’的遗传转化[J]. 生物技术通报, 2014, 0(3): 86-93.

Li Yunhua, Liu Qing, Liu Qinglin. Study on Genetic Transformation of LfMADS1 Gene into Lilium ** formosanum' Raizen No.1'[J]. Biotechnology Bulletin, 2014, 0(3): 86-93.

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