Abstract
The present protocol was aimed at establishing a routine transformation procedure via Agrobacterium tumefaciens for an important Oncidium orchid cultivar. An expression vector containing hptII and gusA genes driven by the cauliflower mosaic virus (CaMV) 35S promoter was successfully introduced into the Oncidium orchid genome by A. tumefaciens. Protocorm-like bodies (PLBs) derived from protocorms, were the target explants for transformation. The transformation was performed through two stages of cocultivation, the first stage occurring on antibiotic-free medium for 3 days, and the subsequent stage on medium containing 100 mg/l timentin for 1 month. Among 1,000 inoculated PLBs, 108 putatively transformed PLBs were proliferated on 5 mg/l hygromycin selection medium. A total of 28 independent transgenic orchid plants were obtained, from which six transgenic lines that were positive for β-glucuronidase were randomly selected and confirmed by Southern, northern and western blot analyses. These results indicated that the foreign DNA was successfully integrated into the orchid genome and expressed transcriptionally and translationally in these orchid plants. The present transformation method reported is suitable for improving the Oncidium orchid through genetic engineering.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Abbreviations
- AS :
-
Acetosyringone
- CaMV :
-
Cauliflower mosaic virus
- GUS:
-
β-Glucuronidase
- Intron-GUS :
-
β-Glucuronidase gene with intron
- PLB :
-
Protocorm-like bodies
References
Anzai H, Ishii Y, Schichinohe M, Katsumata K, Nojiri C, Morikawa H, Tanaka M (1996) Transformation of Phalaenopsis by particle bombardment. Plant Tissue Cult Lett 13:265–271
Belarmino MM, Mii M (2000) Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Rep 19:435–442
Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606
Chan MT, Yu SM (1998) The 3′-untranslated region of a rice alpha-amylase gene functions as a sugar-dependent mRNA stability determinant. Proc Natl Acad Sci USA 95:6543–6547
Chan MT, Lee TM, Chang HH (1992) Transformation of indica rice (Oryza sativa L.) mediated by Agrobacterium tumefaciens. Plant Cell Physiol 33:577–583
Chan MT, Chang HH, Ho SL, Tong WF, Yu SM (1993) Agrobacterium-mediated production of transgenic rice plants expressing a chimeric alpha-amylase promoter/beta-glucuronidase gene. Plant Mol Biol 22:491–506
Chan MT, Chao YC, Yu SM (1994) Novel gene expression system for plant cells based on induction of alpha-amylase promoter by carbohydrate starvation. J Biol Chem 269:17635–17641
Chia TF, Chan YS, Chua NH (1994) The firefly luciferase gene as a non-invasive reporter for Dendrobium transformation. Plant J 6:441–446
Feinberg AP, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13
Hiei Y, Komari T, Kubo T (1997) Transformation of rice mediated by Agrobacterium tumefaciens. Plant Mol Biol 35:205–218
Hsieh TH, Lee JT, Charng Y, Chan M T (2002a) Tomato plants ectopically expressing Arabidopsis CBF1 show enhanced resistance to water deficit stress. Plant Physiol 130:618–626
Hsieh TH, Lee JT, Yang PT, Chiu LH, Charng Y, Wang YC, Chan MT (2002b) Heterologous expression of the Arabidopsis C-repeat/Dehydration response element binding factor 1 gene confers elevated tolerance to chilling and oxidative stresses in transgenic tomato. Plant Physiol 129:1086–1094
Knapp JE, Kausch AP, Chandlee JM (2000) Transformation of three genera of orchid using the bar gene as a selectable marker. Plant Cell Rep 19:893–898
Knudson L (1946) A new nutrient solution for germination of orchid seed. Am Orchid Soc Bull 15:214–217
Komari T, Hiei Y, Ishida Y, Kumashiro T, Kubo T (1998) Advances in cereal gene transfer. Curr Opin Plant Biol 1:161–165
Kuehnle AR, Sugii N (1992) Transformation of Dendrobium orchid using particle bombardment of protocorms. Plant Cell Rep 11:484–488
Lin YS, Chen WH, Hsieh RM, Tsai WT, Fu YM, Wu CC, Chyou MS (1995) Kanamycin sensitivity of Phalaenopsis. Rep Taiwan Sugar Res Inst 147:11–19
Nan GL, Tang CS, Kuehnle AR, Kado CI (1997) Dendrobium orchid contain an inducer of Agrobacterium virulence genes. Physiol Mol Plant Pathol 51:391–399
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
Stachel SE, Messens E, Montagu M van, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318: 624–629
Yu H, Yang SH, Goh CJ (2001) Agrobacterium-mediated transformation of a Dendrobium orchid with the class 1 knox gene DOH1. Plant Cell Rep 20: 301–305
Acknowledgements
We wish to thank Miss Fang-Fei Yeh and Julie Huang for their helpful suggestions on this manuscript. We thank Dr. Yu-Chie Wang, Department of Biology, National Taiwan Normal University, Taipei, Taiwan for providing GUS polyclonal antibody. We are also grateful to The Institute of Molecular Biology, Academia Sinica for providing experimental equipment and facilities. This work was supported by a grant from Academia Sinica and grant NSC91-2317-B-001-003 from the National Science Council of the Republic of China.
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by J. M. Widholm
Rights and permissions
About this article
Cite this article
Liau, CH., You, SJ., Prasad, V. et al. Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Rep 21, 993–998 (2003). https://doi.org/10.1007/s00299-003-0614-9
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00299-003-0614-9