Asymbiotic seed germination and in vitro propagation of Brasiliorchis picta

Abstract

Seed storage method for in vitro germination and propagation from leaves of Brasiliorchis picta was developed. Seeds were harvested and stored at -20 and -80°C for 1, 3, 6, and 12 months and were germinated on Knudson C (KC), Murashige and Skoog (MS), half-strength MS (½ MS macro- and micro-nutrients), and woody plant medium (WPM). Seeds stored at -20°C, the recommended temperature for seed banks, had a high germination rate (76.0%) when cultivated in WPM after 12 months of storage. WPM is the best medium for seed germination and seedling development for both harvested and stored seeds, regardless of storage time and storage temperature. Whole leaf and leaf transversal thin cell layers (tTCL) from 3-month-old in vitro grown protocorms were cultured in ½ MS supplemented with 6-benzyladenine (BA; 2.5, 5.0 and 10.0 μM) and thidiazuron (TDZ; 3.0, 6.0 and 9.0 μM) for 12 weeks. The highest frequency of regenerated protocorm-like bodies (PLBs) from explants (70.0%) occurred when whole leaves were cultured in medium containing 5.0 μM BA, whereas the best response for leaf TCL was with the basal section in medium containing 9 μM TDZ, in which PLBs developed in all regions of leaves. Plantlets were successfully acclimatized (with a survival rate of 97%) when vermiculite was used as a substrate.

Key words: Endangered species, conservation, germination rate, leaf explant, culture medium, micropropagation, Orchidaceae, thin cell layer.