An Improved Protocol for Asymbiotic Seed Germination and Seedling Development of Paphiopedilum tigrinum

Linling Yao,Shibao Zhang,Jialin Huang

doi:10.3390/horticulturae7090298

Abstract

Paphiopedilum tigrinum is an endangered orchid with high ornamental value. However, seed germination and seedling regeneration in P. tigrinum is very difficult in vitro. Little is known about why P. tigrinum seedlings are difficult to propagate or how to improve the seed germination and seedling rates of this species. In this study, we investigated the developmental process of P. tigrinum from asymbiotic seed germination to seedling rooting by comparing it with P. appletoniantum, a much easier species for germination and seedling formation. We found that asymbiotic seed germination in P. tigrinum is limited by severe browning of the protocorm at the seed germination stage, and protocorm rooting at the differentiation stage was also proved to be difficult. The optimal medium for seed germination of P. tigrinum was a modified Harvais (mHa) medium supplemented with 0.5 mg·L−1 kinetin (Kin), 0.1 g·L−1 activated charcoal (AC) and 100 mL·L−1 coconut water (CW). At the protocorm differentiation stage, seedlings with 1–2 leaves were obtained on a 1/4 MS medium supplemented with 1.0 mg·L−1 6-benzylaminopurin (BA), 0.3 g·L−1 AC and 50–100 mL·L−1 CW after culturing for 120 day. At the seedling subculture stage, a 1/2 MS medium supplemented with 0.5–1.5 g·L−1 AC and 100 mL·L−1 CW was better for leaf and root growth of P. tigrinum. At the rooting stage, a 1/2 MS medium supplemented with 1.0 g·L−1 AC, 0.5 g·L−1 dolomite flour, 15 g·L−1 potato homogenate and 30 g·L−1 banana homogenate was most suitable for the growth and rooting of seedlings. This study has established an effective protocol for seed germination and seedling regeneration of P. tigrinum.

Highlights

Members of the genus Paphiopedilum, commonly referred to as slipper orchids, are world-famous potted flowers
P. tigrinum germinated in 19 days (Figure 1c), whereas P. appletoniantum germinated in 30 days
The germination rate of P. tigrinum was significantly higher on modified Harvais (mHa) media than on 1/2 MS or 1/4 MS medium, mostly because seeds cultured on 1/2 MS and 1/4 MS media underwent serious browning

Summary

Introduction

Members of the genus Paphiopedilum, commonly referred to as slipper orchids, are world-famous potted flowers. Species and hybrids in this genus are very popular in floriculture because of their attractive colors, large flowers and peculiar shapes and have been extensively cultivated and produce a wide range of attractive varieties, cultivars or hybrids as a result of intensive breeding [1,2,3]. Paphiopedilum tigrinum is a very endangered orchid. It usually grows on trees or mossy rocks or rocky places in open forests at elevations between 1200 and 2200 m, and it flowers from May to August in the wild (Figure 1a) [4]. P. tigrinum has been listed in Appendix I of the Convention on International Natural populations of P. tigrinum have been drastically reduced in recent decades due to overcollection and habitat loss [5].

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