Construction of the Regeneration and Transformation System of Chrysanthemum Nannong Danxin

INTRODUCTION: Chrysanthemum (Chrysanthemum × morifolium Ramat.) is one of the most economically important ornamental crops worldwide, with broad applications in cut flower production, landscaping and potted plant cultivation. The independently bred chrysanthemum cultivar ‘Nannong Danxin’ (variety authorization number: CNA20201000874) exhibits excellent horticultural traits including vibrant flower color, compact plant architecture and strong stress resistance, showing great market potential. However, the lack of an efficient regeneration and genetic transformation system has become a major bottleneck restricting its molecular breeding and functional gene research. This study aims to establish a stable and high-efficiency regeneration and genetic transformation system for ‘Nannong Danxin’ to provide technical support for its large-scale tissue culture propagation and genetic improvement. RATIONALE: Plant regeneration efficiency and transformation frequency in chrysanthemum are highly genotype-dependent, and the optimal hormone combinations and screening agent concentrations vary significantly among different cultivars. In this study, leaf discs of ‘Nannong Danxin’ were used as explants to systematically optimize two key factors affecting the establishment of the transformation system: firstly, the concentration ratio of plant growth regulators 6-benzyladenine (6-BA) and 1-naphthaleneacetic acid (NAA) for adventitious bud differentiation; secondly, the appropriate screening concentrations of three commonly used resistance selection agents, kanamycin (Km), hygromycin (Hyg) and glufosinate (Basta), for both adventitious bud differentiation stage and rooting stage. On this basis, we further verified the feasibility of the established system by introducing the target gene CtA3’5’GT into ‘Nannong Danxin’ through Agrobacterium-mediated transformation. RESULTS: (1) The optimal medium for adventitious bud differentiation from ‘Nannong Danxin’ leaf discs was determined as Murashige and Skoog (MS) medium supplemented with 1.0 mg•L-1 6-BA and 0.8 mg•L-1 NAA. Under this condition, the adventitious bud differentiation rate reached 95%, with robust bud growth and low abnormal bud rate. (2) For the adventitious bud differentiation stage, the suitable screening concentrations were 30 mg•L-1 for Km, 8 mg•L-1 for Hyg and 0.03 mg•L-1 for Basta, which could effectively inhibit the growth of non-transformed explants without causing excessive damage to transformed cells. For the rooting stage, the corresponding optimal screening concentrations were 20 mg•L-1 for Km, 8 mg•L-1 for Hyg and 0.15 mg•L-1 for Basta, which could completely inhibit the rooting of non-transgenic plantlets while having no significant effect on the rooting of positive transformants. (3) Based on the established regeneration system of ‘Nannong Danxin’, its genetic transformation system was initially established. Leaf discs were transformed via Agrobacterium-mediated transformation. The target gene CtA3’5’GT was identified through Km resistance screening, PCR identification, and semi-quantitative RT-PCR. A total of 2 chrysanthemum seedlings with positive PCR results were obtained. CONCLUSION: This study successfully established a highly efficient and stable regeneration and genetic transformation system for the excellent chrysanthemum cultivar ‘Nannong Danxin’. The optimal hormone combination for adventitious bud differentiation and the appropriate screening concentrations of three commonly used resistance agents were clarified, and the feasibility of the system was verified by obtaining transgenic lines carrying the CtA3’5’GT gene. This technical system provides a solid foundation for the tissue culture rapid propagation, functional gene verification and molecular breeding of new varieties with improved traits for ‘Nannong Danxin’, and also offers a reference for the establishment of transformation systems for other chrysanthemum cultivars.