Production of transgenic diploid Cucumis melo plants
Production of transgenic diploid Cucumis melo plants
复制标题
DOI：
10.1007/s11240-017-1227-2
复制
发表时间：
2017-08-01
影响因子：
3
通讯作者：
Burgos, L.
中科院分区：
文献类型：
--
作者：
Garcia-Almodovar, R. C.;Gosalvez, B.;Burgos, L.
甜瓜（Cucumis梅洛L.）被认为是遗传转化的一个重要物种。此外，许多研究已经观察到再生的转基因植物通常是多倍体。本研究对甜瓜遗传转化的几个方面进行了研究，旨在提高转化效率，获得二倍体转化植株。该方案是基于使用子叶外植体从静止的种子，保留分生组织细胞，这有利于转化的二倍体甜瓜植物的再生。在这项研究中，我们评估了使用两种不同的外植体类型的甜瓜种子近端部分的再生植株的倍性状态（通过流式细胞仪评估）的影响。我们还确定了这些类型的外植体从四个不同的基因型获得的转化效率。从所有外植体类型获得再生。使用静止的种子产生的二倍体植株的百分比范围从85.2%至94.1%，取决于外植体的类型。另一方面，只有一半的老种子子叶（2或3天）再生的植物是二倍体。根据外植体和使用四种甜瓜基因型中的哪一种，产生具有可变转化效率的转基因植物。具有最佳行为的外植体产生具有最高效率的转基因植物，这两种效率在表达视觉标记转基因的植物（范围从4.5%至15.4%）和在选择性培养基中生根的植物的数量（范围从1.3%至3.8%）方面都是有史以来公布的。虽然转化效率仍然相对较低，但它们对于所测试的四种非常不同的甜瓜基因型是一致的。此外，至少85%的植物是二倍体。
Melon (Cucumis melo L.) is considered to be a recalcitrant species for genetic transformation. Additionally, many studies have observed that regenerated transgenic plants are frequently polyploids. Here we have studied several aspects of melon transformation with the aim of improving transformation efficiency and producing diploid transformed plants. The protocol was based on using cotyledon explants from quiescent seeds that retain meristematic cells, which facilitated the regeneration of transformed diploid melon plants. In this study we evaluated the effect of using two different explant types from the proximal portion of melon seeds on the ploidy status (evaluated by flow cytometry) of regenerated plants. We also determined the transformation efficiencies obtained with these types of explants from four different genotypes. Regeneration was obtained from all explant types. Using quiescent seeds the percentage of diploid plants produced ranged from 85.2 to 94.1%, depending on the type of explant. On the other hand, only half of the plants regenerated from older-seed cotyledons (2- or 3-day-old) were diploids. Transgenic plants were produced with variable transformation efficiencies depending on the explant and which of the four melon genotypes was used. The explants with the best behavior produced transgenic plants with the highest efficiencies ever published both, in terms of plants expressing the visual marker transgenes (ranging from 4.5 to 15.4%) and the number of rooted plants in selective medium (ranging from 1.3 to 3.8%). Although the transformation efficiencies were still relatively low, they were consistent for the four very different melon genotypes tested. Furthermore, at least 85% of plants produced were diploid.