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A Rapid and Efficient Protocol for Adventitious Shoot Regeneration and Genetic Transformation of Manchurian Ash (Fraxinus mandshurica Rupr.) using Hypocotyl Explants


 Background: Manchurian ash (Fraxinus mandshurica Rupr.) is an endangered hardwood tree species, providing both economic and medicinal benefits. However, observations such as browning of adventitious shoot buds and high rate of somatic embryo abnormality, were presented in protocols of F. mandshurica regeneration. Therefore, a rapid and high-efficiency regeneration system is demanded for mass propagation and genetic transformation of F. mandshurica.Results: We have developed an efficient regeneration system through adventitious shoot organogenesis in F. mandshurica using hypocotyl explants, with which the adventitious shoots are able to elongate and were obtained in an affordable time. Hypocotyls excised from embryos were pre-cultured in the dark on woody plant medium (WPM) supplemented with 6 g L -1 potassium citrate, and then inoculated on WPM medium supplemented with different concentrations of plant growth regulators (PGRs) to induce adventitious shoot bud formation. The induction medium supplemented with a single PGR of 1.0 mg L -1 thidiazuron (TDZ) was the best treatment, showing 86.67% shoot bud induction with a 15-day initial dark culture, followed by culture under a low light condition. The survival rate of regenerated shoot buds reached 70.97% on WPM medium supplemented with 0.025 mg L -1 TDZ and 1.0 mg L -1 gibberellic acid (GA3). Based on this regeneration system, By using the sonication plus vacuum-infiltration method,a protocol for Agrobacterium tumefaciens -mediated transformation of hypocotyls was established,the transformation rate was determined to be 3.57%. Conclusions: Key factors, such as the potassium citrate pretreatment, wound treatment on explants, variable light conditions, and significant PGR interactions, were revealed to affect the induction and elongation of adventitious shoots from F. mandshurica hypocotyls in this study. The adventitious shoots, tissue culture plantlets, and rooted plantlets were obtained at 40, 80-100, and 160 days, respectively. This regeneration system shortens the period of traditional regeneration methods, which require months to induce callus from leaves or stems, and additional several months for organ differentiation. In addition, the Agrobacterium-mediated transformation protocol established on the basis of this regeneration system provides a foundation for breeding, genetic improvement and genomic studies of F. mandshurica.