Establishment of a System for Preparation and Transient Transformation of Protoplasts of Pinus massoniana

doi:10.7525/j.issn.1673-5102.2025.02.006

Abstract

Abstract:

This study aimed to establish a system of the protoplast preparation in Pinus massoniana for the transient transformation of genes， so as to promote the study of gene function and the development of genetic transformation system in P. massoniana. The explants were selected from different developmental periods of P. massoniana， including needle leaf， hypocotyls and callus tissues. The effects of different enzymatic conditions on the isolation efficiency of protoplasts were explored. The protoplasts preparation process involving different types of tissues， different osmotic pressures and enzymatic hydrolysis time were optimized. The results showed that pine needle tissue in 0.5 mol⋅L^-1 mannitol osmotic solution for 6 h was the optimal condition for protoplasts extraction. Based on this， a transient transformation system of exogenous genes expression in P. massoniana protoplasts was constructed by comparing different transformation conditions. The optimized protoplast preparation and transformation system could significantly improve the quality and transformation efficiency of protoplasts， and the transformation efficiency of protoplasts was as high as 47.83% when using a polyethylene glycol （PEG）-mediated transformation method in P. massoniana. In summary， this study successfully constructed an effective protoplast isolation system for P. massoniana， which provides a reliable transient expression platform for analyzing gene functions in coniferous species.

Key words: Pinus massoniana, protoplast, transient expression, gene function

CLC Number:

Q813

Yanshuo LI, Minyan WANG, Diankuan LIU, Wei LI, Hengfu YIN. Establishment of a System for Preparation and Transient Transformation of Protoplasts of Pinus massoniana[J]. Bulletin of Botanical Research, 2025, 45(2): 202-210.

Figures/Tables 7

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处理组 Treatment group	组织部位 Tissue part	PEG 4000转化液质量分数 PEG 4000 transformation solution mass fraction/%	细胞活力 Cell viability/%	转化效率 Transformation efficiency/%
1	针叶	20	76.00	42.11
2	针叶	30	80.77	42.86
3	针叶	40	88.46	47.83
4	下胚轴	20	75.86	36.36
5	下胚轴	30	86.21	40.00
6	下胚轴	40	84.00	38.10
7	愈伤组织	20	63.33	31.58
8	愈伤组织	30	50.00	33.33
9	愈伤组织	40	51.72	26.67

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