In vitro culture of orchids - Overview
From seeds to plants
Orchids produce capsules with great numbers of tiny and lightweight seeds which appear dust-like to the eye. Seed lengths vary from 0.05 (Anoectochilus imitans) to 6.00 mm (Epidendrum secundum; HALLÉ 1977). Orchid seeds are characterized by a balloon-like seed coat, the lack of the endosperm and a reduced embryo which consists of only a few cells (BURGEFF 1936). This morphology allows dispersal by floating in the air as well as on water for longer periods. In nature, colonization by mycorrhizal fungi is usually required for germination. However, asymbiotic germination is possible in vitro, using a culture medium which provides the nutrients needed for development. During germination, cells of the zygotic embryo divide, rupture the seed coat and form a globular structure known as protocorm (WEATHERHEAD ET AL. 1986). After 3-6 month, depending on species and culture conditions, the protocorm becomes polarized and develops into small plantlets.
Phase 1: Sowing
Dephyte offers low salt media developed for in vitro sowing of terrestrial and epiphytic orchids (see list below). It is possible to sow mature (capsule has opened) as well as immature seeds (green capsule). However, it has been shown for many orchid species that immature seeds result in higher germination rates. Several factors may account for the increased germinability, e.g. higher water permeability of the testa and lower concentrations of germination inhibitors (VAN WAES AND DEBERGH 1986; VAN DER KINDEREN 1987). Besides, it is much easier to sterilize green capsules than single seeds.
Phase 2: Replating
When the protocorm starts to differentiate they will form small roots and leafs. In a process called replating, the tiny seedlings should be reflasked from the germinating flask to a growing medium. The medium is adjusted to the needs of the developing seedling which could include the provision of plant growth regulators. Our replate medium for terrestrial orchids (see list below) contains benzyl adenine (BAP) necessary for a proper development.
Phase 3: Acclimatization
The acclimatization is a crucial phase in orchid microporpagation, because the young plantlets being transferred to ex vitro conditions are exposed to abiotic (e.g. high light intensity and low humidity) and biotic stress (e.g. soil microflora). Plantlets should be slowly acclimatized to the ex vitro environment which will allow the plant to reach a state of autotrophic growing (TAXEIRA DA SILVA ET AL. 2005). Culture vessels with ventilated lids can lower humidity and start the adaption of the plantlets to the ex vitro conditions. Wash off all traces of culture media before transferring the plants to pots.
BURGEFF H. (1936): Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen. Verlag von Gustav Fischer, Jena
HALLÉ N. (1977): Flore de la Nouvelle-Cale'donie et dependances. 8. Orchidace'es. Paris, France: Museum National D'Histoire Naturelle
TEIXEIRA DA SILVA J.; GIANG D.; TANAKA M. (2005): In Vitro Acclimatization of banana and Cymbidium. International Journal of Botany 1 (1): 41-49
VAN DER KINDEREN G. (1987): Abscisic acid in terrestrial orchid seeds: a possible impact on their germination. Lindleyana 2 (2): 84-87
VAN WAES J. M; DEBERGH P. C. (1986): In vitro germination of some Western European orchids. Physiologia Plantarum 67 (2): 253-261
WEATHERHEAD M. A.; ZEE S. Y., BARRETTO G. (1986): Some observations on the early stages of development of Eulophia yushuiana. Memoirs of the Hong Kong Natural History Society 17: 85-90