Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná, Prague, Czech Republic.
Institut de Systématique, Évolution, Biodiversité (ISYEB), Muséum national d'Histoire naturelle, CNRS, Sorbonne Université, EPHE, Paris, France.
Ann Bot. 2019 Mar 14;123(4):625-639. doi: 10.1093/aob/mcy195.
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.
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.
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.
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.
Pyroloids 是石南科的矮小灌木，它们具有混合营养的特性，即同时利用光合作用和与其菌根真菌从环境中获取碳。Pyroloids 具有药用价值，但难以在实验室外进行培养；特别是它们的粉质种子含有未分化的、少细胞的胚胎，其萌发通常完全由真菌伙伴支持。因此，它们的萌发和早期个体发育仍然难以捉摸。
为了研究种子休眠的强度以及不同培养基和条件（包括光照、赤霉素和可溶性糖）对萌发的影响，我们开发了一个优化的 Pyroloideae 亚科五个代表种的体外培养系统。对获得的植物进行形态、解剖和组织化学发育分析。
得益于这种新型的培养方法，休眠被打破，萌发率高达 100%，我们在体外获得了所有 Pyroloideae 属代表种的绿叶芽。在所有情况下，第一个萌发后的阶段是一个未分化的结构，从中后来会出现一个根分生组织，远早于不定芽的形成。
这种培养方法可用于进一步的研究或 Pyroloideae 物种的离体保存。在打破强烈的种子休眠后，Pyroloids 的微小球状胚胎会萌发为一个中间区，该中间区在功能上与其他具有粉质种子的植物（如兰花）的原球茎趋同。与兰花原球茎一样，这个中间区产生一个单一的分生组织：但与兰花不同的是，兰花产生一个茎分生组织，而 Pyroloids 首先产生一个根分生组织。
