Anther and microspore culture of chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitam.)

Abstract

To investigate the possibility of producing haploid plants of chrysanthemums, anthers and microspores of six cultivars were cultured. In anther culture, callus formation was highest in anthers when sampled at the uninucleate stage. The rate of callus formation among cultivars differed little, but equally good results were obtained with the modified MS medium supplemented with NAA 0.1-0.5 mg·liter-1 and BAP 0.5-1.0 mg·liter-1, or 2,4-D 0.5 mg·liter-1 and BAP 2.0 mg·liter-1. Adventitious bud formation from calli in these media differed considerably among cultivars. We did not recognize any distinct effects of light condition and cold pretreatment on callus formation in anther culture, although starvation and high temperature pretreatments restricted callus and adventitious bud formation. Shoots elongated from adventitious buds that were initiated in the modified MS medium supplemented with NAA 0.5 mg·liter-1 and BAP 0.5 mg·liter-1 ('Aomori-kii', 'Shuho-no-chikara'), starvation pretreatment ('Mibu-wase') and continuous treatment of cold pretreatment and high temperature pre-culture ('Aomori-kii'). Histological observation of anthers under culture conditions showed that, microspores with two nuclei appeared after 3 to 5 days of culture, thereafter, connective tissue between the thecae developed callus, whereas microspores degenerated after 10 days of culture. The chromosome numbers of 76 regenerants, 2n = 54, were similar to the original plants. Isozyme analyses of the regenerants revealed zymograms exhibiting banding patterns identical to the original plants. In an isolated microspore culture, the results showed no morphological change or nuclear division of microspores.