Isolation and characterization of a molecule stimulatory to growth of somatic embryos from early stage female gametophyte tissue of loblolly pine.

Loblolly pine (LP, Pinus taeda) is the primary commercial species in southern forests of the US. Somatic embryogenesis (SE) is an effective technique to implement clonal tree production of high-value genotypes from breeding and genetic engineering programs. Unlike angiosperm embryos with attached cotyledons as seed storage organs, the diploid conifer embryo is surrounded by the unattached haploid female gametophyte (FG). The FG is not present in culture. This presents a dilemma if the FG produces necessary or regulatory compounds for embryo growth, since in culture these important compounds would be missing and would have to be added as supplements. We report here the direct evidence that extracts from early-stage FG indeed stimulate early-stage somatic embryo (SME) growth and multiplication, whereas extracts from late-stage FG inhibit early-stage SME growth. Furthermore, we have now isolated this stimulatory substance from early-stage FG tissue, and identified this substance as citric acid on the basis of NMR and mass spectrometry. We then demonstrated that topical application of citric acid to SMEs stimulates embryo colony growth at P = 0.05. Moreover, we find that there is a good correlation between the amount of citric acid isolated from FG tissue (65 nmoles per stage 2-3 FG) and the amount of citric acid that stimulates colony growth (25-50 nmoles) when applied topically to SMEs. This approach of isolating and characterizing a molecule from plant tissue, and investigating its role on SE processes can provide valuable information leading to further applications of these molecules to improve LP SE protocols.