干燥体细胞胚用作转基因苜蓿人工种子的研究

Desiccation Somatic Embryos of Transgenic Alfalfa

为探明干化的体细胞胚作为转基因苜蓿(Medicago sativa)人工种子的可行性,采用农杆菌菌株GV3101感染苜蓿叶柄的遗传转化方法,获得转化苜蓿植株并诱导转化出苜蓿植株的体细胞胚。将成熟的子叶期体细胞胚先置于含10mM脱落酸的胚形成培养基BOi2Y中培养14d,然后将胚置于一系列的6种干燥剂中缓慢干燥7d,最后将胚转到1/2MSO萌发培养基中,直到茎叶萌发和根形成。分析不同转基因株系干化人工种子的萌发率和再生植株中的GUS表达,并利用Southern杂交分析转基因干化人工种子再生植株的遗传变异。结果表明:干化后的转基因人工种子萌发率达到74.3%,产生的植株从形态上相似于没有干化处理的体细胞胚再生植株。在随机的3个转基因株系中,Southern杂交结果表明再生植株仍能稳定表达GUS,每个株系基因整合位点一致。因此,干化体细胞胚用作转基因植物种质保藏的方法是有效可行的。

In order to explore the feasibility of using desiccated somatic embryos as the artificial seeds of transgenic alfalfa,transformed alfalfa plantlets were obtained by means of alfalfa genetic transformation methods to induce somatic embryos.Mature cotyledon-stage somatic embryos were cultured on BOi2Y medium containing 10 mM abscisic acid for 14 days.Then,somatic embryos were desiccated in a series of desiccators for 7 days of slow drying.Finally,the desiccated somatic embryos were transferred to 1/2 MSO medium for germination and transgenic plantlet development.Germination rates of artificial seeds were analyzed in different transgenic alfalfa lines and GUS expression was detected in regeneration plantlets by histochemical analysis.Hereditary variations in regeneration plants of artificial seeds were analyzed by southern blotting.Results showed that the germination rate of desiccated transgenic somatic embryos was 74.3%.The morphology of plants produced from desiccated somatic embryos was similar to that from originals.Southern hybridization confirmed that the regeneration plants of desiccated somatic embryos induced from transgenic alfalfa still had stable GUS expression and the integration site of gene was consistent in tested alfalfa transformation lines.Findings show this method to be feasible for using desiccated somatic embryos as germplasm preservation of transgenic plants.