Stable genetic transformation of Larix gmelinii L. by particle bombardment of zygotic embryos.
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.
Lin
Xiaofei
X
Graduate School of Science and Technology, Kumamoto University, Kurokami, Kumamoto 860-8555, Japan. 031d9307@gsst.stud.kumamoto-u.ac.jp
Zhang
Wenbo
W
Takechi
Katsuaki
K
Takio
Susumu
S
Ono
Kanji
K
Takano
Hiroyoshi
H
D019470:Biolistics / Q000379:methods* / Q000639:trends; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D018744:DNA, Plant / Q000235:genetics; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D018745:Genome, Plant; D028221:Larix / Q000196:embryology* / Q000235:genetics*; D018520:Plant Shoots / Q000235:genetics / Q000254:growth & development; D030821:Plants, Genetically Modified / Q000235:genetics / Q000254:growth & development; D012639:Seeds / Q000235:genetics* / Q000254:growth & development*; D014170:Transformation, Genetic / Q000235:genetics*
D003470:Culture Media; D018744:DNA, Plant