Establishment and application of efficient protoplast isolation and transformation system from leaves of...

Seven widely cultivated, representative populus varieties across four taxonomic sections were employed to establish a multi-genotype protoplast isolation and transformation system, with systematic evaluation of differences in protoplast dissociation, purification, and transformation efficiency. As representatives, Populus alba (P. davidiana × P. simonii) × P. tomentosa '741' from the Sect. Leuce, Populus euramericana from the Sect. Aigeiros, Populus deltoides × P. cathayana 'Senhai 4' from the Sect. Tacamahaca, and Populus euphratica from the Sect. Turanga were selected to compare two enzyme solutions. Enzyme Solution I (1.50 % cellulase R-10 + 0.50 % macerozyme R-10) consistently yielded protoplasts with significantly higher viability than Enzyme Solution II (1.50 % cellulase R-10 + 0.50 % pectinase Y-23). Taking Populus × '741' as an example, direct washing with W5 solution outperformed sucrose density gradient centrifugation in preserving protoplast viability, demonstrating broader applicability across genotypes. Under optimized conditions (Enzyme Solution I and W5 purification), all seven genotypes produced ≥ 7 × 10⁶ cells/g, though protoplast viability and GFP transformation efficiency exhibited striking variation. Sect. Leuce varieties (e.g., Populus × '741' and Populus alba var. pyramidalis 'xinjiang') showed both high viability (93.87 %, 92.16 %) and transformation efficiency (49.55 %, 46.23 %), whereas Sect. Aigeiros, Tacamahaca, and Turanga genotypes displayed lower performance. Notably, Populus × 'Senhai 4' achieved a relatively high transformation efficiency (37.14 %) despite low viability (11.28 %), highlighting the complex interplay of factors governing transformation beyond simple viability. This study establishes a robust foundation for gene editing and molecular breeding in poplars, with implications for improving transgenic efficiency across woody plant species.