In vitro production of doubled haploid plants in Camellia spp. and assessment of homozygosity using microsatellite markers

Abstract

In this report, in vitro doubled haploid (DH) plants were established in two tea (Camellia spp) cultivars, TV21 (Assam Type) and TV19 (Cambod Type). Androgenic globular stage haploid embryos, obtained via callusing from microspores at an early-to-late uninucleate stage in anther cultures, were diploidized by colchicine treatments at varying concentrations and durations under dark incubation at 25 ± 2 °C temperature. Thereafter, treated embryos were transferred to development medium, Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP; 1 μM) + gibberellic acid (GA₃; 0.3 μM) + L-glutamine (80 mg l^-1) + L-serine (20 mg l^-1) and incubated in diffused light. Ploidy of germinating embryos was evaluated by flow-cytometry and cytological squash preparation. High chromosome doubling, 76.89% and 67.34%, was obtained in embryos of TV21 and TV19, respectively, at 0.2% colchicine treatment for 24 h. The DH plants were further multiplied via axillary-bud proliferation on multiplication medium, MS + glucose (30 g l^-1) + BAP (5 μM) + GA₃ (0.5 μM) + IBA (0.5 μM) + L- glutamine (80 mg l^-1) + L-serine (20 mg l^-1). Rooting of shoots was achieved on ⅓ MS basal medium within 50 days of inoculation when shoots were pre-treated with IBA (175 μM) for ten days. The rooted plants were acclimatized in field. Homozygosity in diploidized plants was validated by SSR marker.

Keywords: Chromosome; Colchicine; Doubled haploid; Flow cytometry; Haploid; SSR marker.