Analysis of callus proliferation in floating anther culture of rice

Although it is well known that a larger number of calli are induced by culturing rice anthers on liquid medium than on solid medium, their actual rate of increase has not been elucidated. We have demonstrated here that the number of calli increased by the division of calli on liquid medium as well as by induction from pollen grains and estimated the ratio of division-derived calli to the total number of calli. A Japonica rice cultivar, Kita-ake was used. Anthers including mid- to late uninucleate pollen grains were collected from the spikelets at restricted positions of an ear on the main culm of plants growing densely in pots, and 6 anthers from each spikelet were cultured in each plastic Petri dish containing liquid medium. The number of calli was counted under a microscope every 7 days during the culture period of 35 days. After scoring, all the calli were removed from the liquid medium and then the anthers were continuously cultured in dishes sealed with Parafilm. The calli scored in this way were considered to be those directly derived from the pollen grains during the 7 day period immediately preceding the examination day. The number of pollen-derived calli was examined by using 557 anthers from 95 spikelets. The pollen-derived calli began to be induced during the second week after the start of anther culture and new calli were induced repeatedly every week. In the second experiment, a single callus was transplanted into a plastic Petri dish containing liquid medium and the number of calli was counted every 7 days. The rate of increase of such division-derived calli was determined by culturing 36 calli with a diameter of 0.1-0.9mm. The average number of division-derived calli increased to 1.6 times one week after and to 4.8 times three weeks after callus transplantation. Based on these results, the ratio of division-derived calli to the total number of calli was estimated to be 46.7% at 35 days after the start of anther culture. It remains to be determined whether the genotype of the division-derived calli is the same as that of the pollen-derived ones in the float-ing anther culture method in the near future.