代谢互补抑制剂碘乙酰胺和罗丹明对3种豆科牧草原生质体活力和再长的影响

EFFECT OF IOA AND R-6G ON VIABILITY AND REGENERATIVE CAPACITY OF PROTOPLASTS FROM THREE LEGUME FORAGES

研究了不同浓度代谢互补抑制剂碘乙酰胺(IOA)和罗丹明6G(R-6G)处理与里奥百脉根(Lotuscorniculatus L.cv.Leon)、扁蓿豆(Melilotoides ruthenica(L.)Sojak)及清水紫花苜蓿(Medicago sativa L.cv.Qingshui)3种豆科牧草愈伤组织原生质体活力和再生力之间的相关性。结果表明,3～10mmol/L的IOA和40～70μg/ml的R-6G分别处理10min,可使3种豆科牧草原生质体的活力及植板率明显下降,各处理浓度与原生质体活力和植板率之间均存在极显著的负相关(p<0.01)。培养第7天,所有处理均可见再生的小细胞团;培养30～40d,抑制剂临界浓度下原生质体丧失形成愈伤组织的能力。IOA对原生质活力的抑制作用更明显,R-6G对植板率的影响更显著,并可使原生质体发出红色荧光,有利于细胞融合时亲本原生质体的标识。适宜3种豆科牧草的抑制剂及临界浓度分别是:百脉根5mmol/L IOA或50μg/ml R-6G;扁蓿豆7mmol/L IOA或70μg/ml R-6G;清水紫花苜蓿3mmol/L IOA或40μg/mL R-6G。

A study was conducted to investigate correlations among the viability,regenerative capacity of protoplasts isolated from three legume forages（Lotus corniculatus L.cv.Leon,Melilotoides ruthenica（L.） Sojak and Medicago sativa L.cv.Qingshui） after treated with metabolic complement inhibitors of IOA and R-6G.Results showed that the viability and plate rate of protoplasts from three species were obviously reduced under treatments of IOA（3~10mmol/L） and R-6G（40~70μg/ml） for 10min.The viability and plate rate of three species were significantly（p〈0.01） negative correlated with effects of IOA and R-6G of different concentrations.All protoplasts regenerated small aggregated cell clusters under the influence of IOA and R-6G after culture for 7 days.The development of these protoplasts was inhibited and they could not form calli after culture for 30 to 40 days under the IOA and R-6G treatments at a critical concentration.Compared with R-6G,IOA could apparently suppress the viability of protoplasts of the tested materials,while R-6G could efficiently impact the plate rate of protoplasts.The protoplasts emit red fluorescence,which could be used to identify parent protoplasts in the fusion process.The suitable inhibitor and critical concentrations were 5mmol/L IOA or 50μg/ml R-6G for L.corniculatus,7mmol/L IOA or 70μg/ml R-6G for M.ruthenica and 3mmol/L IOA or 40μg/ml R-6G for M.sativa.The above results showed that this technique was extremely useful in the screening process for selection of suitable inhibitor to inactivate protoplasts of three legume forages and R-6G could mark protoplasts under fluorescent light.