Optimisation and establishment of a high frequency regeneration system for Lolium multiflorum‘Tetragold’ and L. perenne ‘Four seasons’

Using mature seeds of the major ryegrass cultivars in Guizhou Province, annual ryegrass Tetragold and perennial ryegrass Four seasons, as explants, experiments on callus induction and plantlet regeneration were carried out. The results indicated that by peeling off the glume of mature seeds, resecting one-third of the endosperm end, and putting the treated explants of annual ryegrass Tetragold on a medium of CC+7 mg·L-1 2,4-D+0.5 mg·L-1 6-BA and those of perennial ryegrass Four seasons on a medium of CC+5 mg·L-1 2,4-D+0.5 mg·L-1 6-BA, the contamination rates of the tissue cultures were significantly decreased, and the highest callus induction rates occurred (65.52% for annual ryegrass Tetragold and 63.55% for perennial ryegrass Four seasons). Transferring the callus induced in the two cultivars on a subculture medium of MS+0.5 mg·L-1 2,4-D+0.5 mg·L-1 6-BA+1.25 mg·L-1 CuSO4+1.0 g·L-1 CH promoted the formation of type Ⅱ embryogenic callus with better quality. The plant expression vector pCAMBIA 1300 would be used in the subsequent experiment on transformation, and hygromycin was chosen as the best antibiotic selective agent (appropriate critical concentration 30 to 40 mg·L-1). On a medium of MS+2.0 mg·L-1 6-BA+0.5 mg·L-1 NAA+0.1 mg·L-1 TDZ, the highest differentiation rate was 86.37% for embryogenic callus of annual ryegrass Tetragold, and on a medium of MS+6.0 mg·L-1 6-BA+0.3 mg·L-1 NAA+5.0 mg·L-1 KT, the embryogenic callus of perennial ryegrass Four seasons produced the highest differentiation rate of 85.40%. On a rooting culture medium of 1/2MS+0.5 mg·L-1 NAA+0.5 mg·L-1 IAA, the adventitious buds of both the cultivars attained a rooting rate of 100%; thus, a large number of plantlets were produced by tissue culture. The nutrition soil which was composed of the same proportion of humus, vermiculite, and sandy loam ensured a survival rate of more than 99%. In the present study, the high frequency regeneration systems for annual ryegrass Tetragold and perennial ryegrass Four seasons were optimised and established, which would lay a reliable foundation for their genetic transformation.