Proliferation and harvesting of secondary protocorms as a novel means for improving propagation of terrestrial orchids

Abstract

This study investigated optimisation of media and primary-protocorm development stages to enhance secondary-protocorm production as a novel means for propagation of terrestrial orchids, including taxa of conservation concern. Seeds of Caladenia latifolia were germinated asymbiotically on ½-strength Murashige and Skoog (MS) medium fortified with 5% (v/v) coconut water. Resulting protocorms at 3, 5 and 7 weeks of growth were subcultured to protocorm-proliferation media treatments consisting of ½-strength MS basal-salts medium with 6-benzylaminopurine (BA) and α- naphthaleneacetic acid (NAA) singly or in combination. Conversion of seeds to primary protocorms was high (87–92%). The highest percentage of secondary-protocorm proliferation was 40.1%, using 5-week-old protocorms (early Stage 4 of protocorm development) as explants and cultured on ½-strength MS with a combination of 5 µM NAA + 2 µM BA. Half-strength MS media containing a single plant-growth regulator (BA or NAA) were substantially less effective (<10% protocorm proliferation). The present study has provided a novel approach to sequential protocorm production that will be of value particularly for threatened orchids with limited seed availability. Protocorm proliferation in vitro enables a renewable supply of protocorms with which to conduct propagation, cryostorage and pilot restoration programs.