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This study reports a protocol for the germination and early seedling growth of Pinus peuce Gris. using zygotic embryo culture. In order to overcome seed dormancy and optimize organogenesis, the effect of nutritional, plant growth regulatory and physical factors on in vitro germination and growth of isolated mature zygotic embryos of P. peuce were investigated.

Cytokinins are involved in plant cell proliferation leading to plant growth and morphogenesis. The size, activity and maintenance of the shoot apical meristem (SAM) are defined by a balanced rate of mitotic cell divisions and functional cell differentiation that are controlled by cytokinins. In order to investigate the effect of exogenous cytokinin on SAM, morpho-anatomical changes in the shoot apices of tobacco treated with benzyladenine (BA) were compared to those of untreated control plants.

The effects of nutritional, hormonal, and physical factors on the germination and growth of isolated mature zygotic embryos of Pinus heldreichii were studied under in vitro conditions. The optimum medium for embryo germination and adequate conversion into seedlings was basal Gresshoff and Doy (1GD) medium. All tested carbohydrates at the applied concentrations stimulated embryo growth, but only 3% sucrose increased embryo germination compared to the control, while maltose had an inhibitory effect. Among the applied plant growth regulators, only gibberelic acid (GA3) had a few benefits on the development of isolated embryos into plantlets, while the others had a negative effect. Embryos cultured on an agar-solidified medium grew better and the germination percentage was higher than in a liquid medium. Light treatment did not affect embryo germination, but generally stimulated seedling growth.

The potential for somatic embryogenesis in zygotic embryo and megagametophyte cultures of Pinus heldreichii was examined. Somatic embryogenesis was initiated from megagametophytes containing immature zygotic embryos at early stages of development. An induction frequency of up to 6.7% was obtained on Gresshoff and Doy medium in the presence of 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/l benzyladenine (BA). Formation and further proliferation of embryogenic tissue were achieved upon transfer of explants to a medium with reduced levels of growth regulators. Somatic embryos are being cultured for further development. .