Doubled haploids generated through anther culture from an elite long duration rice hybrid, CRHR32: Method optimization and molecular characterization

Abstract

An improved procedure has been developed for high frequency androgenesis in an elite long duration indica rice hybrid. The effects of cold temperature pretreatment, duration of treatment and media with different plant growth regulators on callus induction and shoot regeneration were examined for generation of doubled haploids. N6 medium supplemented with 2.0 mg l-1 2,4-D, 0.5 mg l-1 BAP and 30 g l-1 maltose was found to be most effective for callusing when compared with MS and SK1. The N6 media grown calli showed maximum green shoot regeneration frequency in MS medium supplemented with 0.5 mg l-1 NAA, 0.5 mg l-1 Kinetin, 1.5 mg l-1 BAP and 30 g l-1 sucrose after 2 week of culture. The cold temperature treatment of spike at 10°C for 2 days alongside by 8 days was found to be most suitable conditions for callusing and green shoot regeneration producing 186 green plants from indica rice hybrid, CRHR32. The ploidy status assessed on the basis of morpho-agronomic characters revealed fertile diploids at a frequency of about 81.10%; 16.10%, 2.68% and 1.08% were polyploids, haploids and mixploids respectively. Microsatellite marker analysis showed 1: 1 ratio of the alleles of CMS and restorer lines used for development of CRHR32. Homozygosity was detected for all the marker loci in 150 DHs and only one plant was identified as heterozygote. This investigation identified the favorable media composition and condition for callus induction and green plant regeneration which would further increase the knowledge and better understanding in rice hybrids for development of DHs.