Induction of green ash embryogenic cultures with potential for scalable somatic embryo production using suspension culture.

摘要

All North American ash (Fraxinus) species are under threat of extirpation from their native ranges by the emerald ash borer (EAB; Agrilus planipennis), an exotic wood-boring beetle that has already destroyed millions of ash trees in 15 U.S. states and Canada. We tested treatments aimed at initiating embryogenic cultures from seeds of green ash (F. pennsylvanica), with the long-term goal of using these cultures to aid in research to generate EAB-resistant ash trees for restoration. In preparation for somatic embryogenesis induction experiments, we first defined specific stage(s) of green ash zygotic embryo development using time-tracing sampling by collecting samaras of two green ash trees from May to August in 2012. Seed development was divided into seven stages according to both seed and embryo size, and the numbers of seeds and embryos in each stage were recorded for each collection date. Surprisingly, a broad range of seed and embryo developmental stages could be found in samaras collected from the same tree on the same date, in particular for the later collection dates. Using this information, single-date collections of seeds with embryos at various stages of development were made from three local Athens, GA green ash trees and one horticultural cultivar and cultured on two different basal media with different combinations of plant growth regulators (PGRs). A low percentage of zygotic embryo explants at an intermediate stage of development from all three local source trees produced proembryogenic masses (PEMs) when cultured on a modified Woody Plant Medium with 2,4-dichlorophenoxyacetic acid and benzyladenine. Although embryogenesis was also induced from explants of the horticultural cultivar, these cultures failed to produce germinable somatic embryos. Transfer of PEMs to PGR-free medium resulted in highly dense production of somatic embryos, some of which were germinated to produce somatic seedlings.