Somatic embryogenesis and plantlet regeneration in Pinus elliottiiand its hybrids

Objective  To define the conditions suitable for somatic embryogenesis of Pinus elliottii and its hybrids, and develop a mature protocol for somatic embryogenesis maturation and germination.

Method  The immature zygotic embryos (with endosperm) of two P. elliottii families (EE1, EE2) and two P. elliottii hybrid (EC, EH) were sampled in June 2016. Three series of medium from induction, proliferation maturation to germination were set to compare the effects of explant collection date, family and basal medium on embryogenic callus induction. The embryogenic callus were identified through microscopic observation, and choosen for proliferation, maturation and germination culture. Finally the regenerated plantlets were developed.

Result  The zygotic embryo development processes of P. elliottii and hybrids were divided into eight stages. The immature embryos at stage Ⅱ and Ⅲ were suitable for somatic embryogenesis. The embryos at stage Ⅲ first appeared in EC. The embryogenic callus with embryonal suspensor mass (ESM) could be further proliferated. The induction mediums (T1, T2 and T3) played vital roles in callus formation and embryogenic callus proportion. T1 medium had the highest callus induction efficiency (49.0%), and T2 medium had the highest embryogenic callus proportion (22.4%).The mediums had genotype-specific induction rates. The overall induction rate of T1 medium was low, and T2 medium had the highest induction rate (5.82%) for EE1. T3 medium was suitable for all tested materials, with the highest mean induction rate of 3.75%. The somatic embryo induction rates of EE1 and EH gradually increased with the sampling time extension, while the somatic embryo induction rates of EE2 and EC gradually decreased. The results of somatic embryo induction rate were basically corresponded with zygote development stages. The stage Ⅲ zygotic embryo appeared late, and its induction rate was low in early stage. After the embryogenic callus were subcultured for 24 times, the embryo activities progressively decreased. The maturation medium T1S and T3S could accomplish embryo maturation. Their average ripening efficiencies in per gram of mature medium were 23.3 and 15.9 cotyledons, respectively. The germination rate was 32.1% and the transplanting rate was 47.8%.

Conclusion  All tested materials can induce somatic embryo in T3 medium. T3 medium is widely applicable, and all materials have high induction rates when zygotic embryo appears at stage Ⅲ, illustrating that stage Ⅲ may be the optimal for somatic embryo induction. The experiment establishes the protocol for somatic embryogenesis and plantlet regeneration.