COMBINING ABILITY ANALYSIS OF IN-VITRO CALLUS FORMATION AND PLANT-REGENERATION IN RED-CLOVER

Red clover (Trifolium pratense L.) is an important forage species grow n in temperate climates around the world. Most genetic transformation methods require regeneration of whole plants from single undifferentia ted cells, but one of the major problems encountered in red clover tis sue culture is low frequency of plant regeneration. Nine genotypes of red clover, with a range in the level of callus formation and plant re generation [low (01L, 041, 30L), intermediate (23I, 64I, 74I), and hig h (34H, 54H, 71H)] on a B5 basal salts medium protocol, were used in a combining ability study of in vitro callus formation and plant regene ration. Hypocotyl sections from 40 progeny from each of 36 F-1 crosses were evaluated on a B5 medium protocol for callus formation at 4 wk o f culture and plant regeneration at 12 wk of culture. Analysis of vari ance showed significant effects of crosses, general combining ability (GCA), and specific combining ability (SCA). Results obtained from dia llel analysis (Griffing's Method 4, Model I) showed that crosses with 34H, 54H, or 71H as one parent were among the combinations showing the highest callus diameter and regeneration capacity. Those crosses with 01L, 04L, or 30L as one parent were among the lowest in callus diamet er and regeneration response. A highly significant positive relationsh ip (r = 0.91, df = 34) was found to exist between callus diameter and regeneration capacity. Although both general and specific combining ab ility (SCA) were significant sources of variation for callus diameter and plant regeneration, SCA effects were significant for only a few cr osses. The results are in agreement with our previous research showing that red clover regeneration from tissue culture can be improved usin g recurrent selection methods, which exploit additive genetic effects. Initial selection for large callus size should result in improved reg eneration capacity.