Highly efficient Agrobacterium tumefaciens-mediated transformation of celery (Apium graveolens L.) through somatic embryogenesis
Highly efficient Agrobacterium tumefaciens-mediated transformation of celery (Apium graveolens L.) through somatic embryogenesis
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DOI：
10.1007/s11240-006-9190-3
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发表时间：
2007-01
期刊：
影响因子：
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通讯作者：
Guo Qing Song;A. Loskutov;A. Loskutov;K. Sink
中科院分区：
文献类型：
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作者：
Guo Qing Song;A. Loskutov;A. Loskutov;K. Sink
开发了一种通过从接种根癌农杆菌的叶切片、子叶和下胚轴再生体细胞胚来快速高效生产转基因芹菜植物的方案。这些外植体是从CV的体外幼苗中切下的。 XP166 和 XP85 并接种 A.含有二元载体pBISN1的tumefaciensstrain EHA105。 PBISN1 具有新霉素磷酸转移酶基因 (nptII) 和内含子中断的 β-葡萄糖醛酸酶 (GUS) 报告基因 (gusA)。在黑暗中在愈伤组织诱导培养基 (CIM) 上共培养 4 天：Gamborg B5 + 2.79 μM 激动素 + 2.26 μM 2,4-二氯苯氧基乙酸 (2,4-D)，补充有 100 μM 乙酰丁香酮。 12 周后，对卡那霉素 (Km) 具有抗性的胚性愈伤组织在 CIM + 25 mg l−1Km + 250 mg l−1timentin 上恢复。随后，通过在 Gamborg B5 + 4.92 μM 6 (γ,γ-二甲基烯丙氨基)-嘌呤 (2iP) + 1.93 μM α-萘乙酸上进行体细胞胚胎发生，再生出大量 Km 抗性和 GUS 阳性转化体，每个外植体数十至数百个。 (NAA) + 25 mg l−1Km + 8 周后 250 mg l−1timentin。使用该方案，叶切片的转化频率为5.0%和5.0%，子叶的转化频率为17.8%和18.3%，cv的下胚轴外植体的转化频率为15.9%和16.7%。分别是XP85和XP166。通过 gusA 的 Southern 印迹分析，在所有十个随机选择的转基因事件中证实了具有 1-3 个拷贝数的模型转基因的稳定整合。通过组织化学 GUS 测定进行的后代分析显示转基因具有稳定的孟德尔遗传。因此，A.根瘤菌介导的子叶或下胚轴转化为转基因芹菜植物的大规模生产提供了有效且可重复的方案。
A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration fromAgrobacterium tumefaciens-inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166 and XP85 and inoculated withA. tumefaciensstrain EHA105 containing the binary vector pBISN1. PBISN1 has the neomycin phosphotransferase gene (nptII) and an intron interrupted β-glucuronidase (GUS) reporter gene (gusA). Co-cultivation was carried out for 4 d in the dark on callus induction medium (CIM): Gamborg B5 + 2.79 μM kinetin + 2.26 μM 2,4-dichlorophenoxyacetic acid (2,4-D) supplemented with 100 μM acetosyringone. Embryogenic calluses resistant to kanamycin (Km) were then recovered on CIM + 25 mg l−1Km + 250 mg l−1timentin after 12 weeks. Subsequently, a large number of Km-resistant and GUS-positive transformants, tens to hundreds per explant were regenerated via somatic embryogenesis on Gamborg B5 + 4.92 μM 6 (γ,γ-dimethylallylamino)-purine (2iP) + 1.93 μM α-naphthaleneacetic acid (NAA) + 25 mg l−1Km + 250 mg l−1timentin after 8 weeks. Using this protocol, the transformation frequency was 5.0% and 5.0% for leaf sections, 17.8% and 18.3% for cotyledons, and 15.9% and 16.7% for hypocotyl explants of cvs. XP85 and XP166, respectively. Stable integration of the model transgenes with 1–3 copy numbers was confirmed in all ten randomly selected transgenic events by Southern blot analysis ofgusA. Progeny analysis by histochemical GUS assay showed stable Mendelian inheritance of the transgenes. Thus,A. tumefaciens-mediated transformation of cotyledons or hypocotyls provides an effective and reproducible protocol for large-scale production of transgenic celery plants.