Double-Haploid Plant Production Through Anther and Ovule Culture of Wild Cyclamen Persicum Mill. and Melody F1 Cyclamen Cultivar

Abstract

In vitro anther and ovule culture has been mostly used in haploidization studies of annual and perennial plants to shorten the process of breeding. Cyclamen genus is one of the major perennial geophytes widely used as an ornamental plant. The aim of this study was to develop an efficient haploid plant regeneration protocol via anther and ovule culture for wild Cyclamen persicum and commercial F1 Melody cultivar. The uninuclear stage of microspore was determined with DAPI (4 ',6-diamidino- 2-phenylindole dihydrochloride) dye for C. persicum Mill. and commercial cultivar. Cold pre-treatment ( 4 degrees C) was applied to the buds for two d before in vitro ovule and anther cultures. Anthers were cultured on B5 medium combined with different dosages of 1-naphthaleneacetic acid (NAA), 135.0 g L- 1 maltose, silver nitrate ( AgNO3), and activated charcoal (AC). Ovules were cultured on Murashige and Skoog (MS) medium including the varied amount of 2,4-dichlorophenoxyacetic acid (2,4-D) and N6-( 2-isopentenyladenine) (2iP) and sucrose. Embryos were maturated and germinated on the M2 medium (MS containing 0.2 mg L- 1 giberellic acid (GA3), 0.1 mg L- 1 6-benzylaminopurine (BA), 1.0 g L- 1 proline, and 0.05 mg L- 1 spermine) for anther culture and MS medium without plant growth regulator (PGR) for ovule culture. Haploid embryos were obtained from B5 medium, including 1.0 mg L- 1 NAA for C. persicum Mill. (100%). An efficient ovule culture protocol was determined for C. persicum Mill. as 2.0 mg L- 1 2,4-D and 0.8 mg L- 1 2iP; and 2.0 mg L- 1 2,4-D and 0.5 mg L- 1 2iP for Melody F1 cultivar as 100%. Spontaneous double haploidization was detected on C. persicum Mill. via flow cytometric analysis. Plants were transferred to the soil, and blooming was observed 4 mo after acclimatization.