多花黑麦草特高和多年生黑麦草四季高频组培再生体系的优化与建立

摘要

对在贵州地区广泛种植的多花黑麦草特高(Lolium multiflorum''Tetragold'')和多年生黑麦草四季(L.perenne''Four seasons'')的成熟种子进行了愈伤组织诱导及植株再生的研究,建立了两个品种的胚性愈伤组织高频诱导与再生体系.结果表明,剥去成熟种子的颖壳,切除1/3胚乳端,将特高接种于CC+7 mg·L-1 2,4-D+0.5 mg·L-1 6-BA、四季接种于CC+5 mg·L-1 2,4-D+0.5 mg·L-1 6-BA的培养基中,在明显降低组培污染率的同时,能分别得到65.52%和63.55%的最高愈伤组织诱导率;将两个品种诱导出的愈伤组织转移在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的继代培养基上,能促进质量较好的Ⅱ型胚性愈伤组织的形成;根据后续转化试验所选用的植物表达载体pCAMBIA 1300的抗性特点,30~40 mg·L-1的潮霉素是两个品种愈伤组织最佳的筛选剂和临界浓度;特高的胚性愈伤组织接种在MS+2.0 mg·L-1 6-BA+0.5 mg·L-1 NAA+0.1 mg·L-1 TDZ的培养基上,可以产生86.37%的最高分化率,四季的胚性愈伤组织接种在MS+6.0 mg·L-1 6-BA+0.3 mg·L-1 NAA+5.0 mg·L-1 KT的培养基上,能得到85.40%的最高分化率;生根培养基1/2MS+0.5 mg·L-1 NAA+0.5 mg·L-1 IAA能让两个品种分化出的不定芽形成100%的生根率;以腐殖土∶蛭石∶砂壤土=1∶1∶1的混合材料作为营养土,能保证组培再生苗达到99%以上的成活率.优化建立的高频组培再生体系为下一步的遗传转化奠定了基础.%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.