In Vivo Polyploidy Induction Of Phalaenopsis Amabilis In A Bubble Bioreactor System Using Colchicine

Abstract

Phalaenopsis amabilis Blume var. grandiflora Bateman is economically important as cut and pot flower. Polyploidy is consideredas a valuable tool in improvement and evolution of ornamental plants. Protocorm-like bodies (PLBs) of P. amabilis were culturedon Murashige and Skoog medium containing 0.20 mg L–1IBA together with 2.00 mg L–1KIN and 1.00 g L–1activated charcoal andgrown for a period of five months. Fully-developed plantlets from in vitro grown PLBs were immersed in a bubble reactor filledwith half-strength Hoagland solution containing the antimitotic agent colchicine (0.05%, 0.10% and 0.15%, w/v) for 72 h with afew drops (1 mL of 0.1%) of octylphenoxypolyethoxyethanol or Nonidet (P-40) as a surfactant. Plantlets were aerated to preventhypoxia. Colchicine–treated and untreated plantlets were transferred to pots for a period of 60 days. Tetraploidy was successfullyinduced by 0.15% colchicine. Polyploidy levels were firstly detected using flow cytometry and then confirmed by cytological andmorphological observations. The chromosome number was 2n = 2x = 38 in diploids and 2n = 4x = 76 in tetraploid. Incubationof plantlets in liquid medium containing 0.15% colchicine induced the maximum recovered tetraploids with minimum frequencyof survival (50%). The tetraploid plants were more compact and exhibited round and thick leaves with darker green color thandiploids. Stomata size in tetraploids were larger with less density than diploids. Chloroplast number in guard cells of tetraploidswas about two times more than that of control. These results indicate that induction of tetraploids are a reliable and powerful toolfor generation of novel phenotypes with ornamental and horticultural value for genetic improvement and breeding. Producedtetraploids in current study have potential in the ornamental/floriculture trade