An <i>in vitro</i> chromosome doubling method for clovers (<i>Trifolium</i> spp.)

This research reports a new technique for chromosome doubling of clover (Trifolium sp.) axillary meristems via in vitro colchicine application. Plant material utilized included T. pratense (red clover) cv. Kenstar clones, and three interspecific hybrids: T. ambiguum (kura clover) × T. repens (white clover); T. alpestre × T. pratense; and T. sarosiense × T. pratense. Vegetative axillary meristems were excised from plants, surface sterilized, and trimmed to a length of 0.5–1 mm. Meristems were placed on the surface of a shoot proliferation medium (ML8) containing colchicine (0.1%) for 48 or 72 h and then transferred back to ML8. Alternative treatments were to preculture meristems on ML8 for 7 days prior to colchicine treatment. Plantlets with two or three trifoliolate leaves were induced to root on CR2 or RL rooting media. Preculturing of meristems on ML8 prior to colchicine exposure resulted in the highest chromosome doubling frequencies among the different genotypes, although there was apparent genotype × treatment interaction. Chromosome doubling frequencies were as high as 81 and 44% for initial root tips and mature shoots, respectively. To make rapid assessments of ploidy level of flowering plants, pollen shape was examined. Chromosome doubling increased the pollen stainability of the T. ambiguum × T. repens hybrid from 2.5 to 33.6%, but did not result in fertility in the other two interspecific hybrids.Key words: Trifolium, colchicine, chromosome doubling, interspecific hybrids.