Culture Establishment

Induction of embryo suspensor masses (ESMs) has been reported from immature embryos, mature embryos, hypocotyls, cotyledons, and explants of somatic and zygotic seedlings of Norway spruce (Picea abies).4 Recently, ESM induction has also been reported from explants of 10-20 year old trees of Pinus radiata,5 Pinus pinaster and Picea abies.4 At Weyerhaeuser, we have initiated ESM from immature embryos (pre-dome and dome stages before development of cotyledons) of Douglas fir (Pseudotsuga menziesii) and loblolly pine (Pinus taeda). ESM cultures were initiated onto solid medium with auxin and cytokinin, details of which have been published earlier.6 There is no callus stage in ESM induction from immature embryos. ESM form directly from heads of early stage embryos through cleavage polyembryony. Cleavage polyembryony is natural in several conifer species, and multiple embryos develop inside the megagametophyte through this process. Cleavage polyembryony continues in vitro in the presence of auxin and cytokinin in the medium.

After initiation, ESM cultures are transferred to maintenance medium for continuation of true-to-conifer type cleavage poly-embryony under lower concentrations of auxin and cytokinin.6 At this time, osmolality is increased from 90-100 to 190-200 mM/ kg, with 5 g/l myo-inositol and 30 g/l maltose (compared with 0.1 g/l myo-inositol and 15 g/l sucrose in the initiation medium). We have found that the type of sugar is very important for subsequent embryo development on plates. Early stage embryos were able to fully mature only when grown in the maltose maintenance medium.7 Without increased osmolal-ity treatment, which leads to larger embryonal heads of early stage embryos, many genotypes of Douglas fir and loblolly pine did not develop good quality cotyledonary embryos.8

ESM multiplication is faster in liquid than gelled medium. We have established ESM cultures of Douglas fir and loblolly pine in liquid suspension in 1 liter Erlenmeyer flasks and maintained them by weekly subculture. Each flask contains over 10,000 early stage embryos which may double or triple in number weekly.

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