光果柱花草转基因毛状根及其原生质体制备体系的建立

doi:10.11733/j.issn.1007-0435.2024.05.028

草地学报 ›› 2024, Vol. 32 ›› Issue (5): 1583-1591.DOI: 10.11733/j.issn.1007-0435.2024.05.028

• 技术研发 • 上一篇

光果柱花草转基因毛状根及其原生质体制备体系的建立

孙昊^1,2,3, 张建禹^1,2,3, 罗丽娟^1,2, 刘攀道³

1. 海南大学热带农林学院, 海南海口 570228;
2. 海南大学三亚南繁研究院, 海南三亚 572025;
3. 中国热带农业科学院热带作物品种资源研究所, 农业农村部华南作物基因资源与种质创制重点实验室 & 海南省热带作物资源遗传改良与创新重点实验室, 海南海口 571101

收稿日期:2024-01-13 修回日期:2024-03-21 发布日期:2024-06-01
通讯作者: 罗丽娟，E-mail:liupandao@foxmail.com;刘攀道，E-mail:luoljd@126.com
作者简介:孙昊(1999-)，男，汉族，河南安阳人，硕士研究生，主要从事热带牧草转基因技术研究，E-mail：sunh0713@163.cm
基金资助:
海南省自然科学基金项目(No. 323CXTD387;No. 322RC771)；国家自然科学基金面上项目(No. 32371769)；中央级公益性科研院所基本科研业务费专项(No. 1630032022023)资助

Development of Transgenic Hairy Root Induction and Protoplast Preparation Systems for Stylosanthes leiocarpa

SUN Hao^1,2,3, ZHANG Jian-yu^1,2,3, LUO Li-juan^1,2, LIU Pan-dao³

1. School of Tropical Agriculture and Forestry, Hainan University, Haikou, Hainan Province 570228, China;
2. Sanya Institute Breeding and Multiplication, Hainan University, Sanya, Hainan Province 572025, China;
3. Tropical Crops Genetic Resources Institute, Chinese Academy of Tropical Agricultural Sciences & Key Laboratory of Crop Gene Resources and Germplasm Enhancement in Southern China, Ministry of Agriculture and Rual Affairs & Key Laboratory of Tropical Crops Germplasm Resources Genetic Improvement and Innovation of Hainan Province, Haikou, Hainan Province 571101, China

Received:2024-01-13 Revised:2024-03-21 Published:2024-06-01

摘要/Abstract

摘要： 光果柱花草(Stylosanthes leiocarpa)是柱花草属(Stylosanthes Sw.)中可用于林果园间作的一个野生种。截至目前，光果柱花草的转基因体系尚未建立。本研究以光果柱花草的子叶节为外植体，比较不同的发根农杆菌菌株与潮霉素B浓度对毛状根诱导率及转基因阳性率的影响。结果发现，以K599为诱导菌株且培养基添加8 mg·L^-1潮霉素B为筛选压，是最优的毛状根诱导条件，诱导率达23.33%，阳性率为100%。在此基础上，利用转基因毛状根制备原生质体，结果表明，添加2.5%纤维素酶和2%离析酶，以及8 h的酶解时间为最优条件，原生质体的产量达2.745×10⁵个·g^-1，活力为91.02%，制备的原生质体100%为转基因细胞。本研究成功建立了一种发根农杆菌介导的光果柱花草转基因毛状根诱导体系及原生质体制备体系，为后续光果柱花草的基因功能研究奠定了基础。

关键词: 光果柱花草, 发根农杆菌, 转基因毛状根, 原生质体制备

Abstract: The wild species Stylosanthes leiocarpa,belonging to the genus Stylosanthes Sw. is suitable for intercropping in agroforestry systems. As of now,a transgenic system for Stylosanthes leiocarpa has not been established. In this study,cotyledonary nodes of Stylosanthes leiocarpa were used as explants to investigate the effects of different Agrobacterium rhizogenes strains and concentrations of hygromycin B on hairy root induction efficiency and transgenic positivity. The results showed that induction using strain K599 with the addition of 8 mg·L^-1 hygromycin B as the selection pressure was the optimal condition,resulting in a hairy root induction efficiency of 23.33% and 100% transgenic positivity. Subsequently,transgenic hairy roots were used to prepare protoplasts,and the optimal conditions for this process were the addition of 2.5% cellulase R-10 and 2% macerozyme R-10,with an enzymatic digestion time of 8 hours. The protoplast yield reached 2.745×10⁵ cells·g^-1 and a viability of 91.02%. Notably,all obtained protoplasts were derived from transgenic cells. This study successfully established an Agrobacterium rhizogenes-mediated transgenic hairy root induction system and a protoplast preparation system for Stylosanthes leiocarpa,laying the foundation for further investigation of gene function in this species.

Key words: Stylosanthes leiocarpa, Agrobacterium rhizogenes, Transgenic hairy roots, Protoplast preparation

中图分类号:

Q789

孙昊, 张建禹, 罗丽娟, 刘攀道. 光果柱花草转基因毛状根及其原生质体制备体系的建立[J]. 草地学报, 2024, 32(5): 1583-1591.

SUN Hao, ZHANG Jian-yu, LUO Li-juan, LIU Pan-dao. Development of Transgenic Hairy Root Induction and Protoplast Preparation Systems for Stylosanthes leiocarpa[J]. Acta Agrestia Sinica, 2024, 32(5): 1583-1591.

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光果柱花草转基因毛状根及其原生质体制备体系的建立

Development of Transgenic Hairy Root Induction and Protoplast Preparation Systems for Stylosanthes leiocarpa

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