Establishment of an Efficient Hairy Root Induction System from Pumpkin Leaves via Agrobacterium rhizogenes Without Tissue Culture

Pumpkin (Cucurbita moschata Duchesne) is an economically important vegetable crop in the family Cucurbitaceae. However, existing genetic transformation systems for pumpkin are limited by cultivar dependence, low efficiency, lengthy regeneration, and poor stability. In this study, we developed and optimized an Agrobacterium rhizogenes-mediated hairy root induction system for pumpkin leaves under non-tissue culture conditions, using the cultivar ‘Jintian No. 1’ as the experimental material. Systematic comparisons were performed across multiple parameters, including infection method, duration, bacterial strain, bacterial concentration, seedling age, and leaf position. The optimized transformation conditions were as follows: the second fully expanded leaf of 30-day-old seedlings, A. rhizogenes strain K599 at an optical density (OD600) of 0.8, and direct immersion for 15 min. Under these optimized conditions, the positive hairy root induction rate reached 89.58%. Moreover, the system successfully induced positive hairy roots in all tested pumpkin cultivars. In summary, this study establishes a highly efficient and practical hairy root induction system for pumpkin leaves, providing a robust platform for root-specific gene functional analysis and rapid validation of candidate genes related to root development and stress responses in pumpkin.