Improvement of Anther Culture to integrate Doubled Haploid Technology in Temperate Rice (Oryza sativa L.) Breeding

Abstract

Doubled haploid (DH) plant production, such as anther culture (AC), is an effective tool used in modern rice breeding programs. The improved efficient protocols applied can shorten the process of breeding. The effect of combinations of plant growth regulators (2.5 mg/L NAA, 1 mg/L 2,4-D and 0.5 mg/L kinetin; 2 mg/L 2,4-D and 0.5 mg/L BAP) in the induction medium were compared in AC for five rice breeding materials and combinations. Induction of calli ranged from 264.6 ± 67.07 to 468.8 ± 123.2 calli/100 anthers in AC of rice genotypes. Two basal media (MS and N6) and two combinations of growth regulators (1 mg/L NAA, 1 mg/L BAP and 1 mg/L kinetin; 1.5 mg/L BAP, 0.5 mg/L NAA and 0.5 mg/L kinetin) were used as regeneration media. The in vitro green plant production was the highest with the application of the N6NDK induction medium (NAA, 2,4-D and kinetin) and the MS-based regeneration medium (1 mg/L NAA, 1 mg/BAP and 1 mg/L kinetin) in anther culture of the ‘1009’ genotype (95.2 green plantlets/100 anthers). The mean of five genotypes was 24.48 green plantlets/100 anthers for the best treatment. Flow cytometric analyses conducted identified the microspore origin of the haploid calli produced in AC, while the uniformity of spontaneous DH plants was checked in the DH1 and DH2 generations. Spontaneous chromosome doubling ranged from 38.1% to 57.9% (mean 42.1%), depending on the breeding source. The generated and selected DH lines were tested in micro- and small-plot field experiments to identify promising lines for a pedigree breeding program. The improved AC method was integrated in a Hungarian temperate rice pedigree breeding program.