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

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.