Highly efficient protoplast isolation, fusion and PEG-mediated transient transformation system of pear

Isolation of protoplasts from tissues of woody plants has always been a major technical bottleneck, protoplast based approaches enable gene transfer but are poorly developed in pear ( Pyrus ). Here, we established an efficient protoplast isolation, fusion, and transient transformation platform using stems of tissue cultured seedlings of two elite Chinese pear cultivars, Pyrus pyrifolia ‘Cuiguan’ and Pyrus ussuriensis ‘Qiuzi’. Cultivar specific enzymatic digestion (0.45∼0.65 M mannitol, 1.0∼1.5 % (w/v) Cellulase R-10, 0.2 % (w/v) Macerozyme R-10, 6∼7 h) yielded high yields (4.78∼5.44×10⁷ g⁻¹ FW) and >92 % viability. A hybrid fusion protocol combining AC pre aggregation (15 V/cm, 34.41 % aggregation efficiency) with 30 % PEG4000 achieved 5.13 % fusion efficiency and 3.38 % pairwise fusion efficiency. PEG-Ca²⁺ mediated transformation using 5 μg plasmid DNA and 40 % PEG8000 for 20 min reached 51.99 % protoplast transformation efficiency. This integrated platform accomplishes the entire workflow from high quality protoplast isolation to directed fusion and transient expression in pear for the first time. It provides a powerful multifunctional toolkit for somatic hybridization, high throughput functional genomics, and genetic improvement, thereby significantly accelerating molecular breeding of perennial woody fruit crops.