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Background <p>Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.</p> Results <p>To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5% (w/v) cellulase, 0.75% (w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4&#xa0;mol·L⁻<sup>1</sup> mannitol. After 5&#xa0;h of dark incubation at 25 ˚C, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10⁶ protoplasts per gram (fresh weight) and 95% viability.&#xa0;We incubated 100 μL protoplasts (2.5 × 10<sup>5</sup>·mL⁻<sup>1</sup>) with 15&#xa0;μg plasmid in the solution of 0.4&#xa0;mol·L⁻<sup>1</sup> mannitol and 40% PEG 4000 for 15&#xa0;min, ultimately achieving an optimal transient transfection efficiency of 71.47%.</p> Conclusions <p>This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation (BiFC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.</p>

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Development of an efficient and expedited cotyledon protoplast-based transient transformation system in Gossypium hirsutum L

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年8月25日

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  • Wang Zhicheng,
  • Cao Shuaiting,
  • Cai Qiuyan,
  • Guan Zhenhui,
  • Cheng Hailiang,
  • Peng Fanjia,
  • Li Yujun,
  • Zuo Dongyun,
  • Song Guoli

摘要

Background

Cotton is an important crop providing the most natural fibers all over the world. The cotton genomics community has utilized whole genome sequencing data to construct an elite gene pool in which functional genes are related to agronomic traits. However, the functional validation of these genes is hindered by time-consuming and inefficient genetic transformation methods. Thus, establishing a transient transformation system of high efficiency is necessary for cotton genomics.

Results

To improve the efficiency of transient transformation, we used the protoplasts isolated from the etiolated cotyledon as recipient. The enzymatic digestion buffer comprised 1.5% (w/v) cellulase, 0.75% (w/v) macerozyme, and 1% hemicellulase, osmotically buffered with 0.4 mol·L⁻1 mannitol. After 5 h of dark incubation at 25 ˚C, uniform cotton protoplasts were successfully isolated with a yield of 4.6 × 10⁶ protoplasts per gram (fresh weight) and 95% viability. We incubated 100 μL protoplasts (2.5 × 105·mL⁻1) with 15 μg plasmid in the solution of 0.4 mol·L⁻1 mannitol and 40% PEG 4000 for 15 min, ultimately achieving an optimal transient transfection efficiency of 71.47%.

Conclusions

This transient system demonstrated effective utility in cellular biology research through successful applications in subcellular localization analyses, bimolecular fluorescence complementation (BiFC) verification, and prime editing vector validation. Through systematic optimization, we established an efficient and expedited protoplast-based transient transformation system and successfully applied this platform to cotton functional genomics studies.