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黄萱, 徐子勤, 郝建国, 李晶. 基因枪介导小麦遗传转化的几个重要影响因素的研究[J]. 植物科学学报, 2004, 22(2): 111-115.
引用本文: 黄萱, 徐子勤, 郝建国, 李晶. 基因枪介导小麦遗传转化的几个重要影响因素的研究[J]. 植物科学学报, 2004, 22(2): 111-115.
HUANG Xuan, XU Zi-Qin, HAO Jian-Guo, LI Jing. Factors Affecting Wheat(Triticum aestivum L.)Transformation Mediated by Biolistic Bombardment[J]. Plant Science Journal, 2004, 22(2): 111-115.
Citation: HUANG Xuan, XU Zi-Qin, HAO Jian-Guo, LI Jing. Factors Affecting Wheat(Triticum aestivum L.)Transformation Mediated by Biolistic Bombardment[J]. Plant Science Journal, 2004, 22(2): 111-115.

基因枪介导小麦遗传转化的几个重要影响因素的研究

Factors Affecting Wheat(Triticum aestivum L.)Transformation Mediated by Biolistic Bombardment

  • 摘要: 用基因枪将携带gus-bar双标记基因的质粒pDB1及携带RC24几丁质酶基因的质粒pARN6、pBAB3、pYAO24(pYAO24还带有nptⅡ选择标记基因)以pARN6+pDB1、pBAB3+pDB1及pYAO24三种组合方式转入8个小麦品种的未成熟胚盾片组织,经选择培养获得转基因植株。对基因枪介导小麦遗传转化的主要影响因素,如基因型、材料、金粒制备和轰击参数进行分析,发现西农88是理想的转基因受体基因型,开花两周后的小麦幼胚为理想的轰击材料,制备子弹时合适的金粒含量为30~50μg/次,充分混匀金粒悬浮液可以减少幼胚损伤和提高转化频率。GUS染色发现以蔗糖为渗透剂所产生的蓝色斑点比以甘露醇为渗透剂所产生的蓝色斑点大。针对载体上的nptⅡ筛选标记基因,150mg/L的硫酸卡那霉素为较理想的选择剂浓度;针对载体上的bar筛选标记基因,PPT的浓度为2mg/L易出现假阳性植株,因此应将浓度增加到3~5mg/L。

     

    Abstract: The plasmid pDB1 with gus-bar as double marker gene,and the plamsid pARN6、pBAB3、pYAO24 with RC24 gene encoding rice basic chitinase were co-transferred to immature embryos of 8 wheat genotypes in combination of pDB1+pARN6,pDB1+pBAB3 or pYAO24 via particle bombardment.pYAO24 has nptⅡ as selective gene.Transgenic plants were regenerated after selective culture.Factors influencing wheat transformation via particle bombardment,such as genotype,gold particle preparation,parameters of bombardment,were analyzed.It is found that Xinong 88 is a very ideal genotype of genetic transformation,immature embryos two weeks post-pollination were suitable wheat materials of bombardment,30-50 μg gold particle per bombardment was appropriate,mixing the gold particle suspension thoroughly could reduce the injury to immature embryos and enhance the transformation frequency.GUS histochemical staining revealed that blue spots produced by sucrose as osmoticum were bigger than that of mannitol as osmoticum.150 mg/L kanamycin was adopted when nptⅡ was used as selective gene.It can produce false positive plants easily that the phosphinothricin(PPT) concentration was 2 mg/L when bar was used as selective gene.We should enhance the concentration form 2 mg/L to 3-5 mg/L in the future.

     

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