Plant regeneration via callus culture and subsequent in vitro flowering of Dendrobium huoshanense

Abstract

A protocol for regenerating and subsequent in vitro flowering of an economical important and endangered medicinal orchid, Dendrobium huoshanense, was established mainly via indirect protocorm-like body (PLB) formation. A four-step method was developed to induce successful plant regeneration on 1/2 MS medium supplemented with suitable plant growth regulators (PGRs). Step 1 (callus induction): the root tip explants (1 cm long) were cultured at 1 mg l⁻¹ 2,4-D + 1 mg l⁻¹ TDZ for 3 months. Step 2 (callus proliferation): the calli were subcultured with a 1-month interval at 1 mg l⁻¹ 2,4-D + 1 mg l⁻¹ TDZ. Step 3 (PLB induction): the calli were cultured at 2 mg l⁻¹ NAA + 1 mg l⁻¹ BA for 2 months. Step 4 (plantlet conversion): the 2-month-old PLBs were cultured at 0.1 mg l⁻¹ IBA for 4 months. It took at least 6 months to produce well-rooted regenerated plantlets with an average of 3.2 roots and 3.6 leaves from the initial callus. The 6-month-old rooted plantlets were transferred onto PGR-free 1/2 MS medium for 6 months, and then potted with Sphagnum moss for acclimatization. After 2 month of culture, the survival rate was 100 %. The in vitro flowers were obtained on the 8-month-old plantlets at 1 mg l⁻¹ IBA, 5 mg l⁻¹ IBA and 0.1 mg l⁻¹ NAA, but the flowers showed a lack of the gynandrium. The abnormity was overcome by the aid of 5 mg l⁻¹ TDZ, and subsequently, the capsules formed without artificial pollination. This protocol provides the basis for further investigation on cell suspension, micropropagation, in vitro flowering and breeding programs in Dendrobium huoshanense.