无刺枸骨原生质体制备与瞬时转化体系建立

葛剑斌; 吴兴鑫; 张军; 金小军; 周宇生; 朱雨涵; 卢心可; 金松恒; 董彬

doi:10.11983/CBB25096

植物学报 >

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DOI: https://doi.org/10.11983/CBB25096

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无刺枸骨原生质体制备与瞬时转化体系建立

葛剑斌 ,
吴兴鑫 ,
张军 ,
金小军 ,
周宇生 ,
朱雨涵 ,
卢心可 ,
金松恒 ,
董彬

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1浙江农林大学风景园林与建筑学院, 杭州 311300; 2同创工程设计有限公司, 绍兴 312000; 3浙江农林大学暨阳学院, 诸暨 311800

收稿日期: 2025-05-30

修回日期: 2025-08-21

网络出版日期: 2025-09-03

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Preparation of Protoplasts from Ilex cornuta ‘Fortunei’ Leaves and Establishment of a Transient Transformation System

GE Jian-Bin ,
WU Xin-Xin ,
ZHANG Jun ,
JIN Xiao-Jun ,
ZHOU Yu-Sheng ,
ZHU Yu-Han ,
LV Xin-Ke ,
JIN Song-Heng ,
DONG Ban

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1College of Landscape Architecture, Zhejiang Agriculture and Forestry University, Hangzhou 311300, China; 2Tongchuang Engineering Design Co. Ltd, Shaoxing 312000, China; 3Jiyang college, Zhejiang Agriculture and Forestry University, Zhuji 311800, China

Received date: 2025-05-30

Revised date: 2025-08-21

Online published: 2025-09-03

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摘要

无刺枸骨(Ilex cornuta ‘Fortunei’)在园林绿化中应用非常普遍, 也是常见的造型树种。高效简便的原生质体制备分离体系对于木本植物育种和基因研究具有重要价值。该研究以无刺枸骨幼嫩叶片为材料, 采用L9(34)正交试验、浓度梯度实验探究影响原生质体分离的影响因素, 并采用聚乙二醇(PEG-4000)介导法进行原生质体瞬时转化。结果表明, 2.5% (W/V)纤维素酶、1.0% (W/V)离析酶、0.5% (W/V)果胶酶和0.4% (W/V)蜗牛酶为最适酶浓度组合; 酶解3.5小时后, 原生质体产量最高, 达1.18×107∙g–1 FW, 活力为68.30%。通过40% (W/V) PEG-4000介导转化pORE_R4-IcFPS2质粒后, 在激光共聚焦显微镜下可观察到细胞核内的绿色荧光信号和DAPI蓝色荧光信号。研究成功构建了无刺枸骨原生质体高效分离体系, 为后续无刺枸骨的原生质融合育种及基因功能研究奠定了基础。

关键词： 无刺枸骨; 原生质体; 酶解法; 瞬时转化

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葛剑斌 , 吴兴鑫 , 张军 , 金小军 , 周宇生 , 朱雨涵 , 卢心可 , 金松恒 , 董彬 . 无刺枸骨原生质体制备与瞬时转化体系建立[J]. 植物学报, 0 : 1 -0 . DOI: 10.11983/CBB25096

Abstract

INTRODUCTION: Ilex cornuta ‘Fortunei’ is widely used in landscape gardening and is a common sculptured tree species. An efficient and simple protoplast preparation and separation system is of great value for breeding and gene research of woody plants. However, there are no relevant reports on the protoplast isolation and transformation system of I. cornuta ‘Fortunei’ so far, making it necessary to carry out this study.

RATIONALE: Woody plants face challenges in protoplast isolation and transformation due to characteristics such as perennial stem lignification and leaf leatheriness. Differences in cell wall composition and tissue structure among species require optimization of protoplast systems for specific species. To establish an efficient protoplast preparation and transient transformation system for I. cornuta ‘Fortunei’, orthogonal experiments and gradient experiments were used to explore factors affecting protoplast separation, and PEG-mediated method was applied for transient transformation.

RESULTS: Compared with other enzyme combinations, the orthogonal test showed that the optimal enzyme concentration combination was 2.5% (W/V) cellulase, 1.0% (W/V) macerozyme, 0.5% (W/V) pectinase, and 0.4% (W/V) snailase. After 3.5 h of enzymolysis, the protoplast yield was the highest (1.18×107∙g–1 FW) with a viability of 68.30%. The suitable mannitol concentration for osmotic regulation was 0.30 mol·L–1, and dark pretreatment of leaves for 12 h improved the effect. For transient transformation, 40% (W/V) PEG-4000 mediated the transformation of pORE_R4-IcFPS2-GFP plasmid into protoplasts, and green fluorescent signals in the nucleus overlapping with DAPI blue fluorescent signals were observed under a laser confocal microscope, indicating successful transformation.

CONCLUSION: In conclusion, this study successfully established an efficient protoplast isolation system for I. cornuta ‘Fortunei’ with optimal conditions including specific enzyme combination, enzymolysis time, osmotic regulator, and dark pretreatment. The PEG-mediated transient transformation system was also successfully constructed. These results provide a foundation for future protoplast fusion breeding and gene function research of I. cornuta.

Key words： Ilex cornuta ‘Fortunei’; protoplast; enzymatic hydrolysis; transient transformation

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