Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos |
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| Authors: | Xiaofei Lin Wenbo Zhang Katsuaki Takechi Susumu Takio Kanji Ono Hiroyoshi Takano |
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| Institution: | (1) Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan;(2) Center for Marine Environment Studies, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan;(3) Department of Biological Science, Faculty of Science, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan |
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| Abstract: | We report a new protocol for the stable transformation of Larix gmelinii. Thirty mature zygotic embryos precultured for 3 days on solid medium supplemented with benzyladenine were bombarded with plasmids pUC-GHG (GUS, HPT, and GFP genes) or pBI221-HPT (HPT and GUS genes). After a 2-month culture on selection medium, hygromycin-resistant calli appeared on the surfaces of the necrotic embryos. The frequencies of embryos with resistant calli were 18.4% and 17.4% in the transformations with pUC-GHG and pBI221-HPT DNA, respectively. More than 20 adventitious shoots formed from each of the transgenic calli. Of 17 elongated shoots selected for culturing on a rooting medium, five shoots rooted after 2 months. Expression of the GFP and GUS genes was detected in the resistant tissues by microscopic observations and by a histological GUS activity assay, respectively. PCR and Southern analysis confirmed the stable insertion of the introduced DNA into the genome. |
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| Keywords: | Hygromycin-resistant tissue Larch Organogenesis Particle bombardment Transgenic plant |
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