High-throughput cell analysis based protocol for ploidy determination in anther-derived rice callus

Abstract

The study provides high-throughput protocol for ploidy determination which may accelerate the production of double haploids in rice. Advancements in anther-culture technique have accelerated the production of completely homozygous breeding lines in a shortened time frame. However, the success is dependent upon several factors including precise and accurate determination of ploidy status at callus stage. In the present study, we describe a protocol for nuclei isolation and performed DNA content based ploidy analysis in anther-derived rice callus. Tris-MgCl2 buffer (200 mM Tris, 4 mM MgCl2·6H2O, 0.5% (vol/vol) Triton X-100, adjusted pH to 7.5 with 1 N HCl) was used for isolating high-density nuclei suspension. The nuclei were stained with propidium iodide and fluorescence image was captured using Cellista-Acumen Software in high throughput cell analyser (HTCA). The region of interest was defined on stored image and DNA content was calculated on the basis of fluorescence intensity as well as volume. The protocol was validated using anther callus from different indica rice varieties. Thus, the adoption of high-throughput protocol for ploidy determination will accelerate the development of successful double haploids in rice.