Highly efficient <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis> L.) through somatic embryogenesis |
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Authors: | Guo-Qing Song A V Loskutov K C Sink |
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Institution: | (1) Plant Transformation Center, Department of Horticulture, Michigan State University, East Lansing, MI 48824, USA;(2) Center for Innovations in Medicine, The Biodesign Institute at Arizona State University, Tempe, AZ 85287-5901, USA |
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Abstract: | A protocol was developed for rapid and efficient production of transgenic celery plants via somatic embryo regeneration from
Agrobacterium tumefaciens- inoculated leaf sections, cotyledons and hypocotyls. These explants were excised from in vitro seedlings of the cvs. XP166
and XP85 and inoculated with A. tumefaciens strain 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−1 Km + 250 mg l−1 timentin 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−1 Km + 250 mg l−1 timentin 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 of gusA. 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. |
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Keywords: | β -glucuronidase Plant regeneration Somatic embryo Transgenic plant |
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