Factors Affecting Seed Germination and Seedling Growth of Terrestrial Orchids Cultured in Vitro

Terrestrial orchids (Orchidaceae) have become objects of concern to conservationists due to the plants' high sensitivity to changes in its environment (Rasmussen, 1995). Surface sterilization of mature seeds before germination eliminates barriers to germination; hypochlorites are most often used for this (Lindén, 1992). The barriers include an almost waterproof testa, and embryo integuments containing suberin or abscisic acid-type (ABA) endogenous inhibitors. Embryo suberinization is species-specific and depends on the degree of seed maturity (van Waes and Debergh, 1986a). Nitrogen is supplied to medium in inorganic form as the anion NO3 or the cation NH4 , and in organic form as polypeptides or free amino acids. Some species cannot use inorganic nitrogen, which can be explained by mycorrhizal symbiosis, when the requirements for amino compounds are most probably satisfied by transport of these substances with a symbiotic fungus (Dijk and Eck, 1995). Malmgren (1996) found that the ammoniated form of nitrogen is more appropriate than the nitrate form, and reported the fastest growth of Dactylorhiza seedlings at 50–100 mg l NH4NO3. Mitra (1989) verified that most amino acids act as inhibitors or do not increase the growth of seedlings. Only arginine and aspartic acid support growth like NH4NO3. As for growth regulators, auxins and cytokinins are most frequently used for nutrient media, to increase the percentage of germination or to stimulate protocorm and seedling development. According to De Pauw et al. (1995), their importance in germination is limited but increases during the development of protocorms. As reported by Mitra (1989), cytokinins as such or in combination with auxin indoleacetic acid (IAA) stimulate germination. On the other hand, Hadley (1970) found that 1–10 ppm kinetin with or without auxin IAA can inhibit seed germination in Dactylorhiza purpurella. Gibberellins are used as media additives only marginally. According to Hadley (1970), gibberellic acid induced abnormal elongation of protocorms and etiolation of leaves initiated after formation of shoots in Dactylorhiza purpurella. In Dactylorhiza maculata and Listera ovata, the use of gibberellic acid caused a decrease in the germination percentage (van Waes and Debergh, 1986b). This work examined factors that influence in vitro germination and seedling growth of terrestrial orchids. Mature seeds of the critically endangered species Dactylorhiza incarnata subsp. serotina (Hausskn.) Soó et D.M. Moore (tax.), Dactylorhiza