Flow cytometry determination of ploidy level in winged bean [Psophocarpus tetragonolobus (L.) DC] and its response to colchicine-induced mutagenesis

Abstract

Ploidy determination and mutation breeding of crop plants are inseparable twins given that mutation breeding is hinged majorly on polyploidization of crop’s chromosome number. The present research was aimed at determining the ploidy level of 20 accessions of winged bean (Psophoscarpus tetragonolobus) using known diploid standard as control and also subjecting two accessions to colchicine mutagenesis. For flow cytometric analysis, approximately 50mm² of young fresh intact leaves of 8 weeks post-seedling emergence from the 20 accessions were used. Crude isolation of nuclei was done by chopping the leaf in 1ml of ice cold buffer from DNA kit (cysteine UV Partec GmbH Germany) using a razor blade. The crude suspension was then filtered through a 30μm pore diameter nylon mesh and stained with 4,6- diaminodino-2-phenylindole (DAPI). Fluorescence intensity of isolated nuclei was measured using a partec PAII flow cytometry equipped with an argon ion laser at 488nm.For mutation induction, 20 seeds from accessionsTPt26 and TPt154 were soaked in three different concentrations of colchicine (5,10, 15mg/l) for 24, 48, and 72 hours, while the control group was soaked in water. A plot of land measuring 10X10 meters in the University of Calabar Experimental farm was used. Four beds were made and three seeds were sown in per hole using a Randomized Complete Block Design (RCBD) in a 2X3X4 factorial experiment layout with 5 replications. At the end of the field experiment, phenological, yield and yield-related traits were collected and subjected to analysis of variance using Predictive Analytical Software (PASW) version 20. Results obtained showed that there were significant effect of colchicine on phenological traits except on germination. However, there were significant increasing effects on yield and yield-related traits, which was colchicine concentration, exposure time as well as accession-specific. Exposing winged bean seeds to 15mg/l of colchicine for 24 and 48 hour duration gave encouraging results and should be considered for further tests and exploitation.

Keywords: Ploidy determination, flow cytometry, mutation breeding, colchicine, Psophoscarpus tetragonolobus