Intra-varietal variation for in vitro plant regeneration in the genus Trifolium

Abstract

Trifolium repens, white clover, Trifolium pratense, red clover, Trifolium arvense, plant regeneration, somatic embryogenesis, genotypic variation . SUMMARY Seedlings of Trifolium repens showed considerable variation with regard to the morphology and growth of their calli, and their ability for in vitro differentiation of shoots . One of the lines selected for regeneration in primary callus cultures also showed shoot formation from protoplasts . Somatic embryogenesis in callus cultures of T. pratense and T. arvense occurred only in selected seedling lines . This paper highlights the importance of screening a large number of plants within a cultivar of outbreeding species to achieve reproducible plant regeneration from tissue culture .

Clovers (Trifolium spp.)

“…Transfer was required to a shoot-promoting medium (LSP, Thble 2) containing BAP (0.2 mg/l) and PIC (0.002 mg/l), and finally to a rooting medium (RL, Table 2) containing only IAA (0.2 mg/l) and having reduced levels of most mineral salts. Somewhat similar observations were made by Bhojwani et al (1984), who induced somatic embryos in callus cultures of T. pratense using an MS medium supplemented with 2,4-D (0.05 mg/I) and 2iP (0.5 mg/l). The resulting shoots were rooted on hormone-free basal medium.…”

“…This has been repeatedly demonstrated in studies involving shoot cultures (Oelck and Schieder 1983;Tomes 1982, 1984), callus, cell suspension and protoplast cultures (Phillips and Collins 1979a;Gresshoff 1980;Keyes et al 1980;Mohapatra and Gresshoff 1982;Oelck et al 1982;Bhojwani and White 1982;Ahuja et al 1983;White 1984;Bhojwani et al 1984) and embryo rescue (Williams and Verry 1981;Taylor et al 1981;Phillips et al 1982;Williams 1987). For most purposes, therefore, a prudent strategy is to select a genotype known to be responsive to the techniques intended, or to screen a range of cultivars, ecotypes or individual seedlings.…”

“…For T. pratense best results were obtained with BAP (0-10 mg/l) in combination with PIC (0.006 mg/l) (Phillips and Collins 1979a). For T. arvense, shoot growth was observed only after transfer of callus to a medium supplemented with TIBA (2 mg/l) (Bhojwani et al 1984). White (1984) found a high NHt level important in the differentiation of shoot meristemoids in suspension cultures of T. repens.…”

“…It is worth re-emphasizing at this point that the single most important factor in regeneration is genotype. For all species and cUltivars investigated to date, it has proved necessary to screen a range of seedling genotypes to find those capable of regeneration under a particular set of cultural conditions (Phillips and Collins 1979a;Keyes et al 1980;Gresshoff 1980;Phillips and Collins 1980;Mohapatra and Gresshoff 1982;Webb et al 1983;Oelck and Schieder 1983;Bhojwani et al 1984;White 1984). At the same time, however, species such as red and white clover show considerable plasticity in responses (Gresshoff 1980;Rupert and Collins 1985), so that regenerating genotypes can be selected on a range of basal media and growth-regulator regimes.…”

“…Subculture is usually necessary after bud or embryo initiation in order to obtain elongated shoots and subsequently roots. Shoot growth is promoted by high cytokinin to auxin ratios, rooting by high levels of auxins (other than 2,4-D), and somatic embryogenesis by 2,4-D (Phillips and Collins 1980;Bhojwani et al 1984).…”