Asymbiotic Seed Germination and In Vitro Seedling Development of the Endangered Orchid Species Cypripedium guttatum

Abstract

Cypripedium guttatum is a highly restricted terrestrial orchid that faces increasing endangerment owing to its habitat destruction and illegal collection. Compared to epiphytic orchids, terrestrial orchids such as C. guttatum have harder seed coats and more demanding in vitro germination conditions. This study aimed to develop an effective in vitro propagation system for C. guttatum to aid in its conservation. Seeds from mature capsules were subjected to various conditions, including sterilization using 1% sodium hypochlorite (NaOCl) and different light conditions, culture media, hormones, and organic supplements, to assess germination and early seedling development in vitro. Sterilization with 1% NaOCl significantly improved the germination rate, especially under dark conditions. Germination initiation occurred at 2 and 3 months in orchid seed sowing medium (OSM) and Murashige and Skoog (MS) medium, respectively. The addition of 1 mg/L naphthaleneacetic acid (NAA) further enhanced germination. However, the inclusion of organic supplements, such as apple and banana homogenates, in the culture medium led to substantial growth inhibition after 12 months. Notably, orchid maintenance medium (OMM) without organic additives proved to be the most suitable for seedling growth. The results of this study show that sterilization, appropriate light, and optimal NAA concentrations are beneficial for seed germination.

Keywords: Cypripedium guttatum; culture media; germination; light condition; organic supplementation; seeding growth.

Figure 1

(a) Cypripedium guttatum plants, (b) flower, (c) maturing capsules, (d) typical habitat of Cypripedium guttatum on Hambaek mountain.

Figure 2

In vitro asymbiotic germination of Cypripedium guttatum. (a) Stage 0 “no germination”; (b) Stage 1, “pregermination”; (c) Stage 2, “germination” (tr = testa ruptured); (d) Stage 3, “protocorms”; (e) Stage 4 “appearance of protomeristem” (pm = protomeristem); (f) Stage 5 “pointed shoot apex and rhizoids” (rh = rhizoids); (g) Stage 6 “elongation of root” (s = shoot, r = root). Bars: 0.5 mm (a–c); 1 mm (d–g).

Figure 3

Comparative effects of culture media and growth regulators on the in vitro seedling development stage of Cypripedium guttatum after 3 (a,b) and 5 (c,d) months of asymbiotic culture. OSM: orchid seed sowing medium (PhytoTechnology, P723); 1/2MS: half-strength Murashige and Skoog medium; BA: 6-benzyladenopurine; NAA: α-naphthalene acetic acid.

Figure 4

Effect of apple and banana homogenates on Cypripedium guttatum seedling growth. (a) Growth differences in seedlings were determined seven months after sowing; (b) Fresh weight and (c) root length were measured. Scale bar = 5 cm. MS: full-strength Murashige and Skoog medium; OMM: orchid maintenance medium; A: apple homogenate; B: banana homogenate. The data are presented as the mean ± SD of the values obtained from triplicate experiments. * p ≤ 0.05.