Intergeneric hybridization of two endangered orchids, Vanda stangeana and Phalaenopsis hygrochila, and molecular confirmation of hybridity using SSR and SCoT markers

Abstract

Vanda stangeana Rchb. f. is an endemic species of Manipur, India. Its flowers are greenish-yellow, 5 cm in size with a pink mid-lobe, tessellated in chestnut brown color and tough texture with a long shelf life. Phalaenopsis hygrochila J.M.H. Shaw is a large-sized, hot to cool growing, epiphytic orchid with sturdy, 20 to 40 cm, more or less flattened stem with elliptic-oblong leaves. The flowers are fragrant, pale greenish yellow, more or less dotted with brown or brown-red, with a whitish or white base, waxy, long-lasting, up to 6 cm in diameter, column white, labellum slightly purplish purple, paler towards the margins. Both plants are included under CITES' Appendix II. The current study concentrates on a hybridization program involving two vandaceous orchid genera, namely Vanda stangeana and Phalaenopsis hygrochila to investigate their cross-ability and molecular confirmation of the synthesized hybrid using SSR and SCoT marker system. MS medium at half-strength enriched with 0.5 mg/L NAA + 0.5 mg/L Kn was the optimal medium for the development of the synthesized hybrid seedlings with the longest shoot (2.6 cm), the maximum number of leaves (4.5), leaf length (2.5 cm) and highest number of root (4.4) after 150 days of culture on the medium. In total, 65 amplified bands were revealed by seven SCoT primers (SCoT4, SCoT6, SCoT9, SCoT10, SCoT34, SCoT35 and SCoT36), of which 38 (58.46%) were polymorphic with the percentage ranging from 22.22% to 85.75%, thereby convincingly demonstrated the hybridity. Out of 8 pairs of SSR primers used, 3 primers (C106, MOK26 and C268) showed complementary banding patterns. Our study demonstrated that an intergeneric hybrid can be successfully produced by the hybridization of vandaceous orchids belonging to two different genera.