An Improvement Technique in in vitro Propagation of Vanda curvifolia, a Rare Orchid Species

Abstract

Background and Objectives: Thailand is widely recognized as a globally significant epicenter of biological resources and a biodiversity hotspot. This ecological prominence is attributed to its strategic geographical location at the confluence of three major phytogeographical regions: the Indo-Chinese, Indo-Malayan, and Indo-Burmese regions. Such a unique positioning facilitates a remarkable richness in natural resources, with the Orchidaceae family representing one of the most ecologically and economically significant groups. Among these, Vanda curvifolia (Lindl.) L.M. Gardiner, an epiphytic orchid known for its striking floral characteristics, is currently facing a precarious survival status. It is classified as a rare species in Thailand, with scientific reports indicating a rapid and alarming decline in wild populations. The primary drivers of this decline include the intensifying effects of climate change, extensive habitat encroachment due to land development, and the persistent pressure of illegal poaching for the horticultural trade. Given the slow natural regeneration rate of this species, establishing a method for mass propagation within a short timeframe is imperative for its survival. This study was therefore designed to systematically evaluate several critical factors affecting the in vitro propagation of V. curvifolia seedlings. The research objectives focused on: 1) evaluating the efficiency of different basal media formulations; 2) investigating the physiological effects of plant growth regulators within the auxin and cytokinin groups; and 3) examining the growth-stimulating potential of chitosan on seedling proliferation. As there is currently a total absence of prior comprehensive reports regarding these specific developmental factors for V. curvifolia, this research provides a critical scientific foundation for the genetic conservation and sustainable management of this rare orchid species.

Methodology: The experimental plant materials consisted of V. curvifolia seedlings previously obtained through asymbiotic seed germination under sterile in vitro conditions. The research was divided into four distinct experimental phases to ensure a comprehensive analysis of growth factors. Experiment 1 focused on the influence of basal media; four distinct formulations VW, ½VW, MS, and ½MS were compared to identify the optimal nutrient base for vegetative growth. Experiment 2utilized the superior basal medium identified in the first phase, supplementing it with various auxins, namely NAA, IAA, and IBA. These were tested at concentrations of 0, 0.1, 0.5, 1.0, and 2.0 mg/L to determine their efficacy in inducing organogenesis. Experiment 3 followed a similar concentration gradient (0–2.0 mg/L) but focused on the cytokinin group, including BA, Kinetin, and TDZ, to evaluate shoot multiplication. Finally, Experiment 4 investigated the biostimulatory effects of chitosan. Chitosan was added to the optimal basal medium at concentrations ranging from 0 to 100 mg/L (at 10 mg/L intervals) to pinpoint the most effective concentration for enhancing seedling quality and quantity. All experiments were conducted under controlled environmental conditions with regular monitoring of growth parameters.

Main Results: Following a rigorous 12-week culture period, the data revealed significant variations across treatments. In the initial assessment of basal media, the VW medium emerged as the most effective formulation. It induced the highest shoot formation rate at 13.33%, with an average of 1.23 shoots per explant. This result was statistically significant (p≤0.05) when compared to the MS and ½MS formulations, which showed lower performance. Furthermore, seedlings cultured on VW medium exhibited superior vegetative development, characterized by the highest average number of leaves (4.46 leaves per explant) and the greatest average seedling height (0.94 cm). Regarding the application of exogenous hormones, the results from Experiment 2 indicated that auxin supplementation significantly influenced developmental pathways. Specifically, VW medium supplemented with 0.5 mg/L IAA yielded the most favorable outcomes, inducing a 40% shoot formation rate and an average of 1.70 shoots per explant. This treatment also stimulated the highest callus formation rate (20%) and the highest protocorm-like body (PLB) formation (25%), with an average of 2.45 protocorms per explant, showing clear statistical superiority over NAA and IBA treatments. In the cytokinin trials (Experiment 3), BA proved to be the most effective cytokinin; the addition of 0.5 mg/L BA resulted in a 35% shoot formation rate and a maximum average of 5.35 leaves per explant. One of the most notable findings of this study was the impact of chitosan (Experiment 4). The addition of 100 mg/L chitosan to the VW medium significantly outperformed other concentrations, resulting in a 35% shoot formation rate. More importantly, it produced the highest average number of shoots at 3.50 per explant and the most robust root system, with an average of 4.95 roots per explant. It also maintained a high rate of protocorm proliferation (2.10 per explant). These results suggest that chitosan acts as a powerful growth enhancer for V. curvifolia, providing a viable alternative or supplement to traditional plant growth regulators.

Conclusions: This research successfully established a highly efficient propagation protocol for V. curvifolia using VW medium supplemented with 100 mg/L chitosan. This method enables the rapid production of high-quality seedlings in large quantities. This protocol serves as a vital tool for ex-situ conservation, facilitating plant production for forest restoration or sustainable commercial trade, which can ultimately reduce the illegal poaching of V. curvifolia from the wild and promote the sustainable conservation of orchid biodiversity.