A Stable and Efficient Genetic Transformation Method for Embryogenic Callus of Fraxinus mandshurica

Abstract

Fraxinus mandshurica is a widely used greening and ornamental tree species. However, its genetic transformation system has been hampered by problems such as low transformation efficiency, among others, which can hinder research related to molecular breeding and the analysis of functional genes. Thus, in this study, a novel genetic transformation method for efficient transformation of the embryonic callus of Fraxinus mandshurica was investigated. The method was optimized in terms of factors such as antibiotics, infection solution concentrations, co-culture time, and somatic embryo maturation. The results indicated that the optimal antibiotic concentration was 10 mg·L−1 of hygromycin (Hyg). At this point, the callus proliferation multiple was only 0.12. The highest transformation efficiency was found to be 93.93% when the absorbance of the infection solution concentration at OD600 was 0.4. Interestingly, transformation efficiency was found to be highest (77.9%) at 48 h of co-culture, with a GUS staining rate of 88.23%. The medium for somatic embryo maturation of transformed callus was half-strength MS medium (MS 1/2) containing 60 g·L−1 polyethylene glycol, 1 mg·L−1 abscisic acid, 400 mg·L−1 casein enzymatic hydrolysate (CH), 20 g·L−1 sucrose, 1 g·L−1 activated charcoal, and 5 g·L−1 gellan gum. The medium for somatic embryo germination was MS ½, containing 0.2 mg·L−1 of N-(Phenylmethyl)-9H-purin-6-amine(6-BA) and 5.0 mg·L−1 of gibberellin (GA). These results are of significance for the verification of the gene function and future genetic improvement of Fraxinus mandshurica.