Accumulation pattern and identification of seed storage proteins in zygotic embryos of Pinus strobus and in somatic embryos from different maturation treatments

Abstract

Somatic embryogenesis (SE) of Pinus strobus L. has been greatly improved over the last few years with respect to both the initiation frequencies from a number of seed families and production of mature somatic embryos that readily convert to plants. However, there are no data on biochemical characterization of somatic embryos in relation to zygotic embryos of eastern white pine and on the optimal duration of the maturation stage. It is believed that somatic embryos closely resembling zygotic embryos not only morphologically but biochemically would display more vigorous growth. Hence, in this study the accumulation pattern of the most abundant seed storage proteins in zygotic and somatic embryos were characterized by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and identified by amino acid sequencing and tandem mass spectrometry (MS/MS). This showed that somatic embryos accumulated storage proteins in a similar manner to zygotic embryos and that the most abundant were the buffer‐insoluble 11S‐ globulins MW 59.6 kDa, which dissociated under reduced conditions to 38.2–40.0 and 22.5–23.5 kDa range polypeptides, and buffer‐soluble 7S vicilin‐like proteins MW 46.0–49.0 kDa, which did not separate under reduced conditions. Other relatively abundant soluble proteins were in the ranges of 25–27 and 27–29 kDa. The only group of proteins that showed different migration profiles in the presence of β‐mercaptoethanol (ME) were the low molecular mass proteins of 14.6–16.5 kDa. Somatic embryos that matured for 9 weeks on medium with 6% sucrose accumulated more storage proteins than those matured on medium with 3% sucrose and the extension of the maturation period to 12 weeks resulted in significant reduction of the storage proteins on both media. As expected, somatic embryos matured on medium with 6% sucrose had lower water potential (Ψ) than those from medium with 3% sucrose. Nonetheless, the somatic embryos matured under the best of tested conditions (6% sucrose for 9 weeks) had slightly higher water content; 1.35 ± 0.28 g H2O g−1 DM (mean ± sd) than the mature non‐dried zygotic embryos; (1.16 ± 0.09 g H2O g−1 DM), and accumulated less storage proteins, whose amounts were either similar to (7S‐vicilins) or below (11S‐globulins) those found in the immature zygotic embryos collected 2 weeks prior to the usual cone collection. The implications of these results for further research and development of viable artificial seed is discussed.