In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae)

  • Published source details Yamazaki J. & Miyoshi K. (2006) In vitro asymbiotic germination of immature seed and formation of protocorm by Cephalanthera falcata (Orchidaceae). Annals of Botany, 98, 1197-1206.


Many orchid species are threatened due to factors such as habitat loss and over-collection for horticultural purposes. Generally, artificial propagation from seeds has proven difficult for most terrestrial orchids native to temperate regions. For many orchid species, higher frequencies of germination have been achieved by culturing immature seeds rather than mature seeds, although the reasons for this remain unclear, it is perhaps due to dormancy mechanisms which develop as seeds mature. The genus Cephalanthera are distributed across Europe, Asia and North America. C.falcata, a temperate species of East Asia, is an endangered species in Japan where it is classified as 'vulnerable'. In its natural habitat, C.falcata capsules full of fertile seeds are rarely observed; observations over three seasons revealed few opportunities for pollination by insects, as well as damage of pollinated ovaries during development by insects. Therefore, effects of hand-pollination and bagging treatment of ovaries (to try and prevent insect damage) were examined.

Successful propagation from C. falcata seeds has not been reported before; a method was developed as part of this study (only briefly summarised here) for seed production in situ and propagation using immature seeds in asymbiotic culture in vitro (see original paper for full details).

Study site: Fifty-five C.falcata stems, growing naturally in Quercus forest at Tamagawa University campus, Tokyo, Japan, were used in the experimental hand pollination trials.

Hand pollination: Hand pollination and bagging were conducted at the full bloom period from 17 to 24 April, 2002. The pollinia were transferred onto the column of the same flower to achieve self-pollination. Only flowers with two intact pollinia were used as they were considered to have not been previously visited by insect pollinators. The number of hand-pollinated flowers was limited to three per plant to avoid inhibition of capsule development caused by excess capsule set. After pollination, remaining unpollinated flowers or buds on the same stem were removed, and on 38 stems (those confirmed as pollinated), sepals and petals of 80 flowers were removed with forceps in an attempt to reduce contamination of expected fruits by micro-organisms.

Bagging: The effect bagging of ovaries using two bag materials i.e. aluminium foil and parchment paper, on the production of capsules was evaluated. Bagging treatment was conducted just after pollination by placing either aluminium foil bags (approx. 5 x 10 cm made by folding foil of 12 x 12 cm), bags of parchment paper (5 cm wide x 10–13 cm long), or no bags (control). Stems were staked to support the additional weight of the bags.
On the 17 stems, 106 flowers that were confirmed to be unpollinated were used as controls (flowers without hand-pollination or bagging treatment). Paper bags were occasionally replaced when damaged by rain or attacked by snails or crows.

Seed culture: Young capsules were collected every 10 d from 50 d after pollination until 120 d after pollination. Immature seeds from these capsules were cultured asymbiotically on modified Kano medium and ND medium. Seed viability was examined and histological observations were made on viable seeds at each collection stage.

Effects of hand pollination and ovary bagging: Hand-pollination greatly enhanced capsule formation (by 92.5%) with or without bagging. Capsule formation was 100 % 2 weeks after hand-pollination, but only 7.5 % without hand-pollination. After 5 months, all unbagged capsules had been lost due to being eaten by larvae of one unidentified fly species. In contrast, 74.5% of capsules that had been bagged with aluminium foil survived; about 20 % of capsules were lost to decay over the 5 months subsequent to hand-pollination. For those bagged with paper, 35.3% of capsules survived.

The surviving capsules fully matured in autumn and contained 12–49 mg (average 31 mg estimated on 20 capsules) seeds per capsule; approx. 90 % of seeds contained fully developed embryos.

Seed germination: The highest frequency (39.8 %) of seed germination was obtained with seeds harvested 70 d after pollination. Germination success declined with progress of seed maturity on the plant. Minimal germination (<1%) was observed with seeds harvested 100 d or later after pollination.

Note: If using or referring to this published study, please read and quote the original paper, this can be viewed at:


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