农杆菌侵染条件对柱花草遗传转化效率的影响

doi:10.11686/cyxb2015532

草业学报 ›› 2016, Vol. 25 ›› Issue (6): 102-108.DOI: 10.11686/cyxb2015532

农杆菌侵染条件对柱花草遗传转化效率的影响

陈彩虹¹, 钏秀娟¹, 王荟¹, 贾艳星¹, 陈志坚², 刘国道², 罗丽娟^{1*, *}

1.海南大学园艺园林学院,海南海口 570228;
2.中国热带农业科学院热带作物品种资源研究所,海南儋州 571737

收稿日期:2015-11-26 修回日期:2016-01-26 出版日期:2016-06-20 发布日期:2016-06-20
通讯作者: E-mail:Luoljd@126.com
作者简介:陈彩虹(1992-), 女, 海南文昌人, 在读硕士。E-mail:331202062@qq.com
基金资助:
国家自然科学基金(31360575), 国家牧草产业技术体系热带牧草育种(CARS-35-03), 海南大学优秀研究生学位论文培育计划(M8K3124001001003002)资助

Factors affecting genetic transformation efficiency for stylo (Stylosanthes guianensis) with Agrobacterium tumefaciens

CHEN Cai-Hong¹, CHUAN Xiu-Juan¹, WANG Hui¹, JIA Yan-Xing¹, CHEN Zhi-Jian², LIU Guo-Dao², LUO Li-Juan^{1, *}

1.Horticulture and Landscape of Hainan University, Haikou 570228, China;
2.Tropical Crop Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences, Danzhou 571737, China

Received:2015-11-26 Revised:2016-01-26 Online:2016-06-20 Published:2016-06-20

摘要/Abstract

摘要： 在前期建立的柱花草高效组织再生体系基础上,进一步以柱花草热研5号的下胚轴为受体材料,以 GUS基因为报告基因,研究了农杆菌介导的柱花草遗传转化的几个影响因素,包括根癌农杆菌菌液浓度、浸染时间和共培养时间等对农杆菌介导的柱花草遗传转化效率的影响。结果表明,以柱花草下胚轴为外植体,在农杆菌菌液浓度(OD₆₀₀)为0.4~0.6,农杆菌浸染时间为15 min和共培养3 d的条件下,愈伤的转化效率为72%。愈伤组织进一步经过分化、生根和炼苗等过程后,对转化植株进行GUS染色和PCR检测,结果表明外源基因已成功整合到柱花草基因组中。本研究结果对柱花草遗传转化和关键基因功能研究具有重要的参考价值。

Abstract: The objective of this study was to investigate effects of Agrobacterium concentration, infection time and co-culture duration on the genetic transformation efficiency for stylo (Stylosanthes guianensis) using a high-efficiency regeneration system that had been developed previously. Hypocotyls from stylo seedlings were used as plant material and the β-glucuronidase (GUS) gene was used as the reporter gene. The callus transformation efficiency was evaluated by GUS staining. The callus transformation efficiency reached 72% under the following conditions: Agrobacterium at a concentration of OD₆₀₀=0.4-0.6, with 15 mins infection and 3 days of co-cultivation. The exogenous gene had been successfully introduced into the stylo genome as indicated by GUS staining and PCR detection after callus selection, shoot differentiation and rooting. The results indicate that this A. tumefaciens-mediated transformation system is suitable for genetic improvement and functional gene analysis of stylo.

陈彩虹, 钏秀娟, 王荟, 贾艳星, 陈志坚, 刘国道, 罗丽娟. 农杆菌侵染条件对柱花草遗传转化效率的影响[J]. 草业学报, 2016, 25(6): 102-108.

CHEN Cai-Hong, CHUAN Xiu-Juan, WANG Hui, JIA Yan-Xing, CHEN Zhi-Jian, LIU Guo-Dao, LUO Li-Juan. Factors affecting genetic transformation efficiency for stylo (Stylosanthes guianensis) with Agrobacterium tumefaciens[J]. Acta Prataculturae Sinica, 2016, 25(6): 102-108.

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农杆菌侵染条件对柱花草遗传转化效率的影响

Factors affecting genetic transformation efficiency for stylo (Stylosanthes guianensis) with Agrobacterium tumefaciens

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