In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development

Abstract

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.

Keywords: Chimaphila; Moneses; Monotropa; Pyrola; in vitro culture; Ericaceae; convergent evolution; mixotrophy; orchid; protocorm; seed dormancy; seed germination.

Fig. 1.

Effect of different disinfection treatments on seed germination of (A) Pyrola minor, (B) Moneses uniflora, (C) Chimaphila umbellata and (D) Orthilia secunda. In vitro cultivation on Knudson C medium was evaluated 3 months after the cold stratification. Median values are given (n = 7). Different letters indicate a significant difference according to the ANOVA followed by Tukey HSD post-hoc test for multiple comparison.

Fig. 2.

Effect of different media on germination of (A) Pyrola minor, (B) Moneses uniflora and (C) Orthilia secunda, and growth of (D) Pyrola minor, (E) Moneses uniflora and (F) Orthilia secunda. Both germination and the total length of seedlings were measured 3 months after cold stratification. Different letters indicate a significant difference. Results of Tukey HSD post-hoc test for multiple comparisons are indicated by upper case letters and results of pairwise Wilcoxon rank sum test by lower case letters.

Fig. 3.

Representatives of in vitro grown seedlings of species used in this study. (A) Pyrola minor cultivated in the dark, (B) Pyrola minor cultivated in the light, and (C) Moneses uniflora, (D) Orthilia secunda, (E) Chimaphila umbellata and (F) Monotropa uniflora cultivated in the dark. Scale bars = 0.5 mm.

Fig. 4.

Effect of selected soluble saccharides on germination of Pyrola minor (A) and Orthilia secunda (B). In vitro cultivation on Knudson C medium where sucrose was replaced by saccharide is indicated. Different letters indicate a significant difference. Results of Tukey HSD post-hoc test for multiple comparisons are indicated by upper case letters and results of pairwise Wilcoxon rank sum test by lower case letters; bars indicate the first and third quantiles. Pairwise comparisons were calculated separately for each month.

Fig. 5.

Germination and early development of Pyrola minor. (A) A mature seed with globular embryo. (B) Germination starts with enlargement of the embryo (arrows indicate the former embryo). (C) A seedling develops further into a root through the less organized intermediary zone with a very heterogenic surface cell pattern. (D) Detail of the cell surface pattern of the intermediary zone. (E) A seedling 1–1.5 mm in size with an already established root meristem. (F) The meristematic zone is established very early after germination. (G) A seedling <1 mm in size with an established meristem and differentiated vascular tissues in the central part of the intermediary zone close to the former embryo. (H) A seedling approx. 3 mm in size with an already established lateral root primordium (white arrow) and differentiated vascular tissues (in detail). (I) The root structure is obvious at 1.5 cm from the position of the former embryo in a 3 cm long seedling. Abbreviations: en, endodermis; x, protoxylem vessels of the radial vascular bundle; c, cortex; rh, rhizodermis. (J) The root tip of a 3 cm long seedling. Abbreviations: lrc, lateral root cap; p, protoderm; qc, quiescent centre. (A–E) whole mounts, DIC; (C) whole mount, confocal image; (D) whole mount, UV; (F–H) paraplast sections, Safranin O + Fast Green FCF; (I) hand section, UV; (J), paraplast section, Lugol + Orange G. Black arrows indicate the position of the former embryo; scale bars = 50 μm.

Fig. 6.

Transition to shoot formation in P. minor seedlings. (A) Nine-month-old seedling with an established root system prior to the onset of shoot growth. (B) Nine-month-old seedling with an emerging shoot (arrow). (C) The shoot bud emerges at the first root branching site close to the position of the former embryo (r, root; sh, shoot bud; iz, intermediary zone connecting the former embryo; x, xylem), section stained with Safranin O and Fast Green FCF. (D) Later, additional shoot buds emerge along the root axis (arrows). (E) Detail of the first shoot bud, whole mount preparation. (F) Fully established in vitro plant. (G) Adventitious roots emerging at the stem node (sh, shoot). Scale bars = 0.5 cm, except (C) and (E) = 200 μm.

Fig. 7.

Comparison of germination and subsequent development of orchids (left) and pyroloids (right). The embryo is highlighted in red, the protocorm in blue, shoots in green and roots in brown. Abbreviations: a, adventive root; br, branching roots; e, embryo; end, endosperm; h, hilum; iz, intermediary zone; p, protocorm; ram, root meristem; sh, shoot; sam, shoot meristem; t, testa; x, xylem. The thin bar is 100 μm, the dotted bar is 1 mm and the thick bar is 0.5 cm.