Establishment of Agrobacterium rhizogenes-mediated genetic transformation system for Packham pear

cy of 95.66% using leaf explants, significantly exceeding rates reported for other European pear cultivars. Second, the visual RUBY reporter enables non- destructive screening, reducing labor and cost while allowing direct observation with the naked eye. Additionally, this study introduces basal callus as a novel, time-efficient explant for pear transformation, which can be isolated in just 14 days compared to the 2- 3 weeks required for traditional explants. This system enables the generation of transgenic hairy roots within 28 days, providing a rapid platform for studying critical root traits, including nutrient uptake, disease resistance, and abiotic stress tolerance, which are vital for enhancing yield and crop sustainability. Furthermore, the system serves as an effective pre-screening tool for CRISPR/Cas9 genome editing, allowing researchers to assess editing efficiency and off-target effects in hairy roots before undertaking resource- intensive whole- plant transformation. Future studies could optimize basal callus transformation efficiency by adjusting co-cultivation hormone ratios or testing different A. rhizogenes strains. Overall, this transformation technique can accelerate functional genomics in a recalcitrant pear cultivar and support the development of improved varieties through molecular biotechnology.【Objective】The European pear (Pyrus communis L.) cultivar Packham is globally recognized for its exceptional fruit attributes, including crisp texture, sweet-tart flavor, and long postharvest shelf life. However, its genetic improvement is constrained by prolonged juvenility, transformation recalcitrance, and hybridization barriers, which collectively hinder the introduction of traits such as disease resistance and improved fruit quality. Its genetic improvement is constrained by three interrelated biological and technical bottlenecks. First, long juvenility phase requires 5-8 years to reach reproductive maturity. Second, there exists a genotype-specific recalcitrance in pear transformation, while existing established protocols function for Asian pear cultivars (Pyrus pyrifolia Nakai), they consistently fail to yield stable transgene integration and expression in European germplasm, including Packham. Third, interspecific hybridization barriers restrict the efficient introgression of desirable traits, such as resistant genes against fire blight (Erwinia amylovora) or enhanced tolerance to drought stress. These limitations have severely delayed the development of Packham varieties with enhanced biotic/abiotic stress resistance, improved fruit quality, and adapted agronomic traits. Against this backdrop, this study aims to establish a stable, efficient Agrobacterium rhizogenes -mediated genetic transformation system specifically tailored to Packham pear. Unlike traditional transformation systems that target whole plants (which are time- consuming and inefficient for woody species), the proposed system focuses on generating transgenic hairy roots—an approach that offers unique advantages for pre- breeding research. Hairy roots, induced by A. rhizogenes, exhibit rapid growth, stable transgene expression, and physiological similarity to natural roots, making them an ideal model for (1) validating the function of root- related genes (e.g., genes regulating phosphate uptake under low-nutrient conditions, or genes encoding resistance proteins against soil-borne pathogens like Phytophthora cactorum); (2) optimizing trait improvement strategies, such as testing the efficacy of candidate genes before full plant transformation; (3) accelerating pre-breeding research by reducing the time required to evaluate gene function from years to months. Furthermore, this work seeks to fill a critical gap in pear biotechnology: while numerous transformation protocols exist for Asian pears and a few European pear cultivars, no dedicated system has been reported for Packham, a cultivar with distinct physiological characteristics that affect explant responsiveness and transformation efficiency. By developing this system, the study aims to provide a technical reference for researchers working on other recalcitrant pear cultivars, thereby facilitating the broader application of modern biotechnologies in pear genetic enhancement. Ultimately, the established system will serve as a foundational tool for unlocking Packham’s genetic potential, enabling the development of improved varieties that meet the evolving demands of growers, processors, and consumers. 【Methods】In this study, four types of culture media (proliferation, callus, liquid, and rooting) were employed for different developmental stages of Packham pear explants. Each medium was formulated with a targeted nutrient composition to meet the specific physiological requirements of Packham pear explants at different culture stages, ensuring effective support for explant proliferation, callus formation, and root development. A. rhizogenes strain K599 was selected as the transforming strain for its proven efficiency in woody plants, which is crucial for achieving efficient transformation in Packham pear. For explant preparation, annual branches of Packham pear were used as the initial material, undergoing hydroponic culture first to promote new shoot germination. A two-step sterilization procedure was implemented: 30-second surface sterilization with 75% ethanol, followed by 8-minute disinfection with 2% sodium hypochlorite, with multiple sterile water rinses between and after treatments. Then they were cultivated on the proliferation medium. After new shoots emerged, the branches with new shoots were cut into 2-3 cm stem segment explants and cultivated on the proliferation or callus medium. The explants were infected with A. rhizogenes (OD600 = 0.6-0.8) and then co-cultured on the proliferation medium in darkness for two days. Subsequently, they were transferred to screening medium for hairy root induction. The induction rates were recorded at 7, 14, 21, and 28 days post-infection.【Results】Based on the A. rhizogenes-mediated transformation method, hairy roots were successfully induced in all tissue types of Packham pear. Although induction efficiency varied among tissues, the differences were not significant, with callus showing the best hairy root induction effect. The hairy root induction rates for leaves and callus, all exceeded 86.36%, and each explant type achieved an induction rate above 76%. The transformation efficiencies were 95.66% for leaves, 91.33% for callus, and 71.11% for basal callus, indicating that leaves performed most effectively while basal callus showed the lowest efficiency. Temporal observation of leaf explants revealed that no red hairy roots were visible at 7 days post-transformation; a limited number emerged by 14 days but grew slowly; root number increased and growth accelerated by 21 days; and the highest proliferation was observed by 28 days. Additionally, all the red roots detected were positive and the white roots were negative by PCR identification.【Conclusions】In summary, this study successfully establishes an efficient, economical, and rapid A. rhizogenes- mediated transformation system for Packham pear. First, key achievements include a high transformation efficien-