Improvement of Tissue Culture and Agrobacterium tumefaciens Genetic Transformation System in Osmanthus fragrans

Abstract

Osmanthus fragrans is a traditional and famous woody fragrant flowering plant in China with high ornamental value, edible value, and economic value. Due to the long juvenile period, complex reproductive system, and high heterozygosity of O. fragrans , it is difficult to develop new cultivars via traditional crossbreeding methods. Therefore, molecular breeding via genetic engineering, that is, the use of genetic transformation technology, is targeted to improve the traits of O. fragrans . In this study, we established an Agrobacterium -mediated genetic transformation system of O. fragrans by optimizing tissue culture regeneration system of Osmanthus , and using stem segment of O. fragrans sterile seedling as transformation receptor. The culture medium that is optimal for seed buds emergence is Murashige and Skoog medium (MS) + 3.0 mg/L 6-Benzylaminopurine (6-BA) + 0.10 mg/L α-Naphthylacetic acid (NAA), which results on an average bud emergence percentage of 96.53%. For stem differentiation, the optimal culture medium is B5 medium (B5) + 0.5 mg/L Thidiazuron (TDZ) + 0.05 mg/L NAA, which results on an average bud emergence rate of 75.93%. The most suitable medium for shoot rooting is 1/2 MS + 2.0 mg/L NAA, which results on an average rooting rate of 31.48%. An overexpression vector was constructed with the O. fragrans flower color-related gene OfGGPPS1 as an objective gene, and Agrobacterium tumefaciens was used to transform O. fragrans histoculture seedlings. Quantitative analysis of the transformed plant bodies revealed high expression of the target gene, indicating successful transformation. Therefore, this method can be used for the study of gene function in O. fragrans and provides a reference for the exploration of gene function in other plant species.