Development of Somatic Hybridization Protocol for Taro (Colocasia esculeneta esculeneta (L) Schott. and Colocasia antiquorum (L) Schott.) to Address Conventional Breeding Constrain.

Abstract

Taro (Collocasia esculenta and C. antiquorum) locally known as ‘Nduma’is a root crop belonging to family Araceae. It rarely flowers and thus conventional breeding is hindered. The aim of our study was to determine amenability of taro to somatic hybridization as an alternative to conventional breeding. Protoplast was isolated from friable embryogenic calli and young leaves obtained from in vitro grown C. esculenta and C. antiquorum plants. Prior to protoplasts isolation, best explant (leaf or microcorm) for production of embryogenic calli was determined using 1/2 MS containing 2, 4-D (1-8mg/L) in combination with TDZ (0.5 mg/L). Protoplasts was isolated using cellulase R-10 (1.0% w/v) combined with pectinase R-10 (0.15% w/v) and yield assessed after 1-6 h of incubation. Fusion frequency of calli- and leaf-derived protoplast using PEG 6000 (0-30% w/v), CaCl₂ (0-0.15M) and NaNO₃ (0-4% w/v) as fusogens for 10-30 min was evaluated. Results obtained indicate that microcorm was the best explant for production of embryogenic calli. On the other hand, high numbers of viable protoplast were obtained from leaf explants unlike embryogenic calli. Highest viable protoplasts yield (6.86×10⁶ protoplasts/ml) was obtained on C. esculenta leaves after 2 h enzyme incubation, while incubation in PEG (20 w/v) for 20 min produced highest fusion frequency. However, higher rates of heterokaryons viability were obtained on 20% PEG after 10 min. The capacity of heterokaryons to form cell colonies was higher in PEG (10 or 20%) after 10 or 20 min incubation. An inverse relationship between heterokaryon frequency and concentration of fusogen was observed. Overall, leaf and PEG (10 or 20 %) were optimal explant for isolation of viable protoplast and fusogen, respectively. Our study demonstrates that optimized protoplast fusion and coupled with plant regeneration is a viable alternative to conventional breeding for taro.