GROWTH REGULATOR EFFECTS ON BUD INITIATION IN CALLUS CULTURES OF MEDICAGO SATIVA

Abstract

Influence of growth regulators on bud initiation in callus of alfalfa (Medicago sativa L.) was studied by varying levels and combinations in the first medium of a two‐medium sequence used to obtain whole plants. Callus of tetraploid clone S‐4 (cv. ‘Saranac‘) was initiated from immature ovaries on a modified Blaydes' basal medium containing all combinations of six concentrations (0–36 μM) of kinetin (K), six concentrations (0–44 μM) of naphthaleneacetic acid (NAA), and seven concentrations (0–36 μM) of 2,4‐dichlorophenoxyacetic acid (2,4‐D). After 28 days the callus was challenged to form buds by transfer to the modified Blaydes' medium containing 2.0 g/liter yeast extract and 0.57 mM inositol. No buds were produced in the absence of 2,4‐D in the first medium, and the frequency of bud formation on the second medium was directly proportional to the 2,4‐D concentration in the range 2.3–54 μM in the preceding medium. Buds were produced in the absence of kinetin in the first medium, but its presence in the range 2.3–36 μM markedly increased bud formation. NAA was not required for bud formation, and the budding frequency increased only slightly with increasing NAA concentration in the first medium. Budding of callus of two other alfalfa clones was also influenced by the 2,4‐D concentration in the initial medium. There were several indications that many of the buds were initiated on the first medium and completed development on the second medium. These included the differential effect on budding of combinations of 2,4‐D, NAA and kinetin in the callus initiation medium, the specific media sequence required, and the presence of embryoids on the callus which after transfer to the yeast extract‐inositol medium produced buds.