Micropropagation and somaclonal variation of Doritis pulcherrima (Lindl.)

In this research, in vitro micropropagation of the economically important orchid Doritis pulcherrima was conducted because its numbers in nature are greatly decreasing in Thailand. Different types of medium (Vacin and Went (VW), Murashige and Skoog (MS), and New Dogashima Media (NDM)) with plant growth regulators (1-naphthalene acetic acid (NAA) and 6-benzyladenine (BA) at 0, 0.1, 0.5, 1.0, and 1.5 mg·L–1) were evaluated for protocorm-like body (PLB) formation and plantlet regeneration. The results indicated that after three months of culture, NDM medium produced a significantly higher survival rate (46.70 ± 0.51%), number (11.00 ± 2.94 PLBs per protocorm), and size (6.35 ± 4.31 mm) of PLBs. Additionally, all media could stimulate plantlet regeneration. However, NDM supplemented with NAA and BA at a concentration of 0.1 mg L−1 provided the best stimulation of most observed parameters, including the shoot number and leaf and root number and length. Unexpectedly, after maintaining and subculturing D. pulcherrima plantlets for two years, somaclonal variation occurred, with the clones showing a morphology and physiology that were distinct from those of the wild type. Last, a suitable medium for wild-type and cloned plantlet development was investigated, and VW and ½MS supplemented with or without 2,4-dichlorophenoxyacetic acid (2,4-D) (1.0 mg·L–1) were used. The results showed that VW supplemented with 2,4-D at a concentration of 1.0 mg L−1 led to the best plantlet development. This report presented an efficient system for the micropropagation of D. pulcherrima and showed that prolonged subculture leads to somaclonal variation.