In vitro propagation and root anatomical features of Phragmipedium kovachii J.T. Atwood, Dalström & Ric.Fernández

ABSTRACT Phragmipedium kovachii is a renowned orchid for its striking flowers and ecological significance. This study aimed to develop an efficient protocol for its micropropagation and conservation. Shoots measuring 0.7-1.0 cm were used as explants and cultured on half-strength Murashige and Skoog (MS) medium with various combinations of 6-benzylaminopurine (6-BAP) and indole-3-acetic acid (IAA), with or without 100 mL L-¹ coconut water. The highest protocorm-like bodies (PLBs) formation rate (96.7%) and the maximum PLBs per explant (12.58 ± 0.33) were obtained on medium containing 2.0 mg L-¹ 6-BAP, 0.6 mg L-¹ IAA, and coconut water. During the regeneration phase, PLBs regenerated on media supplemented with either 0.5 mg L-¹ 6-BAP and coconut water, as well as those on media containing 0.5 mg L-¹ meta-Topolin (mT) and banana homogenate, produced optimal plantlets for subsequent rooting stages. Rooting was enhanced using 1.0 mg L-¹ IAA with coconut water or 0.5 mg L-¹ indole-3-butyric acid (IBA) with banana homogenate. Plantlets grown on coconut water-supplemented media developed longer roots with a pentarch stele, well-differentiated sclerenchyma tissue, and numerous metaxylem vessels, exhibiting the highest survival rate. In contrast, those cultured on banana homogenate produced thicker roots with a polyarch stele. Acclimatization was successfully achieved using substrates consisting of equal proportions of bark, perlite, and peat, as well as a mixture of soil, sand and perlite (2:1:1), ensuring high ex vitro survival rates (87 and 80%, respectively). This study provides an effective protocol for the ex situ conservation and large-scale production of P. kovachii plants.