BACKGROUND AND AIMS: Pyroloids, forest sub-shrubs of the Ericaceae family, are an important model for their mixotrophic nutrition, which mixes carbon from photosynthesis and from their mycorrhizal fungi. They have medical uses but are difficult to cultivate ex situ; in particular, their dust seeds contain undifferentiated, few-celled embryos, whose germination is normally fully supported by fungal partners. Their germination and early ontogenesis thus remain elusive. METHODS: An optimized in vitro cultivation system of five representatives from the subfamily Pyroloideae was developed to study the strength of seed dormancy and the effect of different media and conditions (including light, gibberellins and soluble saccharides) on germination. The obtained plants were analysed for morphological, anatomical and histochemical development. KEY RESULTS: Thanks to this novel cultivation method, which breaks dormancy and achieved up to 100 % germination, leafy shoots were obtained in vitro for representatives of all pyroloid genera (Moneses, Orthilia, Pyrola and Chimaphila). In all cases, the first post-germination stage is an undifferentiated structure, from which a root meristem later emerges, well before formation of an adventive shoot. CONCLUSIONS: This cultivation method can be used for further research or for ex situ conservation of pyroloid species. After strong seed dormancy is broken, the tiny globular embryo of pyroloids germinates into an intermediary zone, which is functionally convergent with the protocorm of other plants with dust seeds such as orchids. Like the orchid protocorm, this intermediary zone produces a single meristem: however, unlike orchids, which produce a shoot meristem, pyroloids first generate a root meristem.
Plant-derived smoke and smoke-isolated compounds stimulate germination in seeds from over 80 genera. It has also been reported that smoke affects overall plant vigour and has a stimulatory effect on pollen growth. The effect of smoke on orchid seeds, however, has not been assessed. In South Africa, orchid seeds from several genera may be exposed to smoke when they are released from their seedpods. It is therefore possible that smoke may affect their germination and growth. Therefore, the effects of smoke [applied as smoke-water (SW)] and two smoke-derived compounds, karrikinolide (KAR1 ) and trimethylbutenolide (TMB), were investigated on the germination and growth of orchid seeds in vitro. The effect of SW, KAR1 and TMB were investigated on the endangered epiphytic orchid, Ansellia africana, which is indigenous to tropical areas of Africa. Smoke-water, KAR1 and TMB were infused in half-strength MS medium. The number of germinated seeds and number of seeds and protocorm bodies to reach predetermined developmental stages were recorded on a weekly basis using a dissecting microscope for a 13-week period. Infusing SW 1:250 (v:v) into half-strength MS medium significantly increased the germination rate index (GRI) and the development rate index (DRI) of the A. africana seeds. All the SW treatments significantly increased the number of large protocorm bodies at the final stage of development. Infusing KAR1 into the growing medium had no significant effect on germination or development of the seeds. The TMB treatment, however, significantly reduced the GRI and DRI of A. africana seeds.
