刺槐同源四倍体种子胚变异及生活力分析

VARIATION AND VITALITY OF SEED EMBRYOS FROM TETRAPLOID Robinia pseudoacacia

为了阐明刺槐同源四倍体种子胚变异状况及充分利用刺槐同源四倍体有性过程创造的变异资源对四倍体刺槐进行遗传改良,研究了刺槐同源四倍体种子胚类型及其生活力特征,主要结论如下:刺槐同源四倍体种子胚按照子叶数量、颜色及饱满程度,可以划分出5个类型:黄二(YVT)、黄三(YMT)、黄四(YRF)、绿二(GNT)及白二(WNT),所有类型种子胚混合平均单胚重量接近二倍体的1/2。流式细胞仪检测证明,YVT、YMT、YRF类型样本种子胚染色体数目仍然是四倍体。刺槐同源四倍体种子胚可溶性蛋白含量、过氧化物酶活性明显低于二倍体相应指标,且YVT、YMT、YRF类型种子胚明显高于GNT、WNT类型。以刺槐同源四倍体种子胚愈伤组织诱导率作为种子胚生活力强弱的评定指标表明:上述2种指标越高的种子胚,其生活力就越高,上述2种指标均小于二倍体相应指标的30%是刺槐同源四倍体种子胚生活力丧失的临界指标;而YVT、YMT、YRF类型种子胚均高于二倍体相应指标的30%,组织培养时均有一定的愈伤组织诱导率,具一定的生活力,其中YVT类种子胚的愈伤组织诱导率最高(平均57.8%)、生活力最强,已培育出此3类胚的实生苗;GNT、WNT类型种子胚上述2种指标均低于二倍体相应指标的30%,组织培养时愈伤组织诱导率为0,胚细胞均失去了生命力。较好的种子胚愈伤组织诱导培养基综合条件为:MS基本培养基附加2,4-D 2.0mg/L和6-BA 0.5mg/L。

In order to explain clearly the variation status of embryo from tetraploid Robinia pseudoacacia （TRP） and fully use the variation resource initiated in the sexual reproduction of TRP in the further improvement of TRP, The classification and vitalities of seed embryos from tetraploid Robinia pseudoacacia （TRP） were studied. According to the cotyledon number, color and plumpness, the seed embryos of TRP could be divided into 5 types：YVT （yellow, very replete, 2 cotyledons）, YMT（ yellow, more replete,3 cotyledons ）, YRF （ yellow, replete,4 cotyledons ）, GNT （ green, no-replete, 2 cotyledons ） and WNT （ white, no-replete, 2 cotyledons ）. The average individual embryo weight of 5 types was close to half of seed embryo weight of diploid Robinia pseudoacacia （DRP）. The results of flow cytometry（FCM）show that YVT, YMT and YRF sampled embryos were still tetraploid. The content of soluble protein （SP） and activity of peroxidase （AFP） of TPR seed embryos were remarkably lower than DRP seed embryos but higher than GNT and WNT type seed embryos. By the evaluation index of seed embryo vitality based on the rate of callus induction of TPR embryos, the higher the SP and AFP of seed embryo were the higher their vitality. While both SP and AFP of TRP seed embryos were lower than 30 % of corresponding index of DRP seed embryos, the seed embryos of TRP would lose their vitality and therefore＇ lower than 30 % was critical index for seed embryo of TRP to keep living. For both SP and AFP of YVT, YMT and YRF embryo were higher than 30 % of corresponding index of DRP seed embryos, callus was induced from all of them when they were cultured on medium; and thereby they were living. Among them the callus-induce-rate of YVT seed embryo was the highest （average：57.8%）, as a result, its vitality was the most vigorous. And so far seedlings from the three types of seed embryo had been raised. In contrast, for both SP and AFP of DNT and WNT embryo were lower than 30 % of corresponding index of DRP seed embryos, no callus was induced from all of them; and this means that their cell vitality had been lost overall. The better callus-induce medium for seed embryos was MS complemented with 2,4-D 2.0mg/L and 6-BA 0.5mg/L.