An efficient and reproducible method for development of androgenic haploid plants from in vitro anther cultures of Camellia assamica ssp. assamica (Masters)

Abstract

This study describes the development of haploid plantlets through androgenesis in Camellia assamica ssp. assamica (tea). Androgenic haploid embryos were produced through callus formation from microspores during the early-to-late uninucleate stages in anther cultures. A high percentage of callus induction (96%) was obtained on Murashige and Skoog’s (MS) medium containing 6% (w/v) glucose, supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 5 μM 6-furfurylaminopurine (kinetin), 800 mg L−1 L-glutamine, and 200 mg L−1 L-serine (callus induction medium). Further proliferation of callus occurred when glucose was replaced with 3% (w/v) sucrose in the medium. Embryogenesis was achieved in 85% of the androgenic callus cultures on MS medium containing 10 μM 6-benzylaminopurine (BAP), 3 μM gibberellic acid (GA3), 800 mg L−1 L-glutamine, and 200 mg L−1 L-serine (embryo induction medium). Maturation of embryos occurred when the concentration of the growth regulators and adjuvants contained in the embryo induction medium were reduced by tenfold. Embryos germinated into complete plantlets in 65% of the cultures when the MS medium was supplemented with 10 μM BAP, 1 μM indole-3-butyric acid (IBA), 0.5 μM GA3, 80 mg L−1 L-glutamine, and 20 mg L−1 L-serine. These plantlets continued to grow when the major salt concentration in the embryo germination medium was reduced by half (1/2 MS). The chromosomal constitution of these in vitro plantlets was confirmed as 2n = X = 15 by cytological squash preparation of the root tips. Flow cytometric analysis of leaves from these in vitro plantlets confirmed the ploidy status as haploid. This study is an effort to overcome the inherent heterozygosity in tea.