Cyclic secondary somatic embryogenesis and efficient plant regeneration in mountain ash (Sorbus pohuashanensis)

摘要

An efficient protocol for secondary somatic embryogenesis in mountain ash is reported. Secondary somatic embryos (SSEs), initially obtained from primary embryos, were proliferated and maintained for more than 2 years via cyclic secondary somatic embryogenesis. SSEs were produced on the surfaces of cotyledons and radicles of maternal somatic embryos. Histological observations of the various stages of SSE development revealed four typical stages: globular, heart-shaped, torpedo, and cotyledon. Addition of a low concentration of naphthaleneacetic acid (NAA) to Murashige and Skoog (MS) medium resulted in the induction of SSEs, but addition of 2,4-dichlorophenoxyacetic acid (2,4-D) to MS medium decreased SSE formation. Addition of casein hydrolysate (CH) to MS medium promoted induction of SSEs. Cotyledonary SSEs were cultured on MS medium with 20-60 g L-1 sucrose under light for 1 month until maturation. After transferred to MS medium containing either 0.06 micro M NAA or 0.15 micro M indole-3-butyric acid in the light, over 50% of the mature SSEs developed into plantlets. Addition of 1.0 g L-1 activated charcoal was beneficial for SSE germination (over 60%). At 18 degrees C, over 90% of the germinated SSEs converted to plantlets on 1/2 MS (half-strength of MS macroelements) with 1.8 micro M NAA under light. Plantlets acclimatized successfully to ex vitro conditions and field plants developed with normal phenotypes.