Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (21): 4514-4524.doi: 10.3864/j.issn.0578-1752.2021.21.003


Optimization of Cotton Mesophyll Protoplast Transient Expression System

LI Qing(),YU HaiPeng,ZHANG ZiHao,SUN ZhengWen,ZHANG Yan,ZHANG DongMei,WANG XingFen,MA ZhiYing,YAN YuanYuan()   

  1. College of Agronomy, Hebei Agricultural University/State Key Laboratory of North China Crop Improvement and Regulation/Key Laboratory for Crop Germplasm Resources of Hebei, Baoding 071001, Hebei
  • Received:2021-04-25 Accepted:2021-06-24 Online:2021-11-01 Published:2021-11-09
  • Contact: YuanYuan YAN;


【Objective】Cells of true leaves can well mimic the plant endogenous situation. It is an efficient way for expediting cotton functional study to establish an effective transient expression system using cotton protoplasts obtained from true leaves.【Method】The enzyme combination of cellulose and macerozyme were used to isolate protoplasts from true leaves of Gossypium hirsutum L. acc. TM-1. The effects of osmotic pressure, components of digestion buffer and digestion time on protoplast yield were studied and the validity of protoplasts were compared under different mannitol concentration and digestion time. To improve the transformation efficiency of cotton protoplast, the effects of mannitol and PEG concentration and buffers for protoplast culture were subsequently studied. In order to verify the optimized transient expression system, the vector 35S:LTP-GFP was constructed and transformed into protoplasts of Arabidopsis and cotton and tobacco epidermal cells followed by observation of fusion protein localization.【Result】High concentration of CaCl2 in the digestion buffer significantly inhibited the isolation of protoplast from cotton true leaves, which was opposite of that using cotyledon. 10 mmol·L -1 CaCl2 was employable for digestion buffer to isolate cotton protoplasts from true leaves. Mannitol concentration significantly affected protoplast yield that peaked under mannitol concentration of 0.5 mol·L-1, and protoplast validity decreased moiety under 0.4 mol·L-1 mannitol, suggesting that 0.5 mol·L-1 mannitol was most suitable to maintain the osmotic pressure of cotton protoplasts. Cotton protoplasts displayed suitable size when isolated from newly flattened true leaves, while protoplast enlarged and yield decreased when produced from young leaves flattened 5 days. The protoplasts dissociate slowly until being digested 9 h when the yield reached the peak. The transformation efficiency was greatly improved under isotonic condition of 40% PEG buffer. While hypotonic condition that is commonly applied to facilitate transformation was against the entrance of exogenous DNA into cotton protoplasts. After transformation, the protoplast ruptured abundantly in WI buffer,whereas the shape maintained well in W5 buffer adding 0.5 mol·L-1 mannitol. The transformation efficiency was improved to 90% using the optimized transient expression system. The subcellular location analysis results showed consistent GFP signal in protoplasts of cotton and Arabidopsis true leaf and epidermal cells of tobacco leaf.【Conclusion】Our study has optimized the cotton mesophyll protoplast transient expression system, which could produce 8.10×10 6·mL-1 fine protoplasts with validity above 95% and transformation efficiency reached to 90%. This system is applicable for analysis of subcellular location, protein interaction and research on metabolism and regulation network.

Key words: cotton, TM-1, isolation of protoplasts, transient expression

Fig. 1

Effect of CaCl2 and mannitol concentration on protoplast isolation A: Digestion effect of enzyme buffer with different CaCl2 concentration; B: Effect of mannitol concentration on protoplast yield (different letters show significant difference at P<0.05. The same as below); C: Effect of mannitol on protoplast viability"

Fig. 2

Effect of leave developmental stage on protoplast isolation A: True leaves for protoplast isolation; B: Protoplast yields using the true leaves at different developmental stages; C: Protoplasts produced from elder leaves; D: Protoplasts produced from newly flattened young leaves"

Fig. 3

Effect of digestion time on protoplast isolation A: Protoplast yield after different digestion time; B-E: Isolated protoplasts after 7-12 h digestion; F-G: Detection of protoplast viability"

Fig. 4

Optimization of transformation conditions A, C and D: Effect of protoplast culture solution, mannitol concentration and PEG concentration on transformation efficiency; B: Protoplasts after transformation under fluorescence microscope"

Fig. 5

Transient expression of cotton LTP-GFP A: Cotton mesophyll protoplast; B: Arabidopsis mesophyll protoplast; C: Tobacco epidermal cells"

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