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<p>Dahlia (<i>Dahlia</i> sp.) is commonly cultivated in China but encounters challenges in breeding due to limited cross-breeding success and extended breeding timelines. Genetic engineering offers a promising solution; however, the lack of an optimized protocol for protoplast isolation hinders progress. This study presents an improved enzymatic digestion approach for efficiently isolating leaf mesophyll and petal protoplasts from various dahlia cultivars. Petal protoplasts were isolated through enzymatic digestion with 0.5% macerozyme, 1.0% cellulase, and 0.4% pectinase in 1.0&#xa0;mol/L mannitol for 10&#xa0;h, yielding 5.46 × 10<sup>6</sup> protoplasts per gram of fresh petal tissue with 68.69% viability. The same enzyme mixture in 0.8&#xa0;mol/L mannitol was applied for 4&#xa0;h for leaf protoplasts, yielding 6.13 × 10<sup>6</sup> protoplasts per gram of fresh leaf tissue with 75.89% viability. Both protocols incorporated a 12-h dark pretreatment at 4°C and a 30-min vacuum infiltration. Transformation efficiency using the pGBin-EGFP construct ranged from 32.57 to 60.67%, with optimal conditions being a 50&#xa0;ng/µL plasmid concentration, 20% PEG, and a 20-min transformation duration. This optimized protocol provides a valuable tool for molecular research in dahlia and establishes a foundation for future genetic engineering applications.</p>

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Efficient protoplast isolation and transient gene expression in dahlia (Dahlia sp.)

  • 期刊论文

  • 发表日期：

    2025年4月13日

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  • Jing Zhou,
  • Dong Yang,
  • YangBo Tian,
  • Zhen Tian,
  • Qing Duan,
  • Mohamed A. A. Ahmed,
  • LiHua Wang,
  • XueWei Wu

摘要

Dahlia (Dahlia sp.) is commonly cultivated in China but encounters challenges in breeding due to limited cross-breeding success and extended breeding timelines. Genetic engineering offers a promising solution; however, the lack of an optimized protocol for protoplast isolation hinders progress. This study presents an improved enzymatic digestion approach for efficiently isolating leaf mesophyll and petal protoplasts from various dahlia cultivars. Petal protoplasts were isolated through enzymatic digestion with 0.5% macerozyme, 1.0% cellulase, and 0.4% pectinase in 1.0 mol/L mannitol for 10 h, yielding 5.46 × 106 protoplasts per gram of fresh petal tissue with 68.69% viability. The same enzyme mixture in 0.8 mol/L mannitol was applied for 4 h for leaf protoplasts, yielding 6.13 × 106 protoplasts per gram of fresh leaf tissue with 75.89% viability. Both protocols incorporated a 12-h dark pretreatment at 4°C and a 30-min vacuum infiltration. Transformation efficiency using the pGBin-EGFP construct ranged from 32.57 to 60.67%, with optimal conditions being a 50 ng/µL plasmid concentration, 20% PEG, and a 20-min transformation duration. This optimized protocol provides a valuable tool for molecular research in dahlia and establishes a foundation for future genetic engineering applications.