High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material

Wasana Phlaetita; Dong Poh Chin; Valentine Otang Ntui; Ikuo Nakamura; Masahiro Mii

doi:10.5511/plantbiotechnology.15.0804a

High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material

Wasana Phlaetita, Dong Poh Chin, Valentine Otang Ntui, Ikuo Nakamura, Masahiro Mii

Center for Desert Agriculture

Research output: Contribution to journal › Article › peer-review

9 Scopus citations

Abstract

Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 gl−1 maltose, 30 mgl−1 hygromycin and 20 mgl−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

Original language	English (US)
Pages (from-to)	323-327
Number of pages	5
Journal	Plant Biotechnology
Volume	32
Issue number	4
DOIs	https://doi.org/10.5511/plantbiotechnology.15.0804a
State	Published - 2015

ASJC Scopus subject areas

Plant Science
Agronomy and Crop Science
Biotechnology

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title = "High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material",

abstract = "Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 gl−1 maltose, 30 mgl−1 hygromycin and 20 mgl−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.",

author = "Wasana Phlaetita and Chin, {Dong Poh} and Ntui, {Valentine Otang} and Ikuo Nakamura and Masahiro Mii",

note = "KAUST Repository Item: Exported on 2021-03-31 Acknowledgements: We thank Mr. Nobuyuki Asai, Asai Daikeikan Co., Ltd. to kindly provide the seeds as plant materials in this research.",

year = "2015",

doi = "10.5511/plantbiotechnology.15.0804a",

language = "English (US)",

volume = "32",

pages = "323--327",

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publisher = "Japanese Society for Plant Cell and Molecular Biology",

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T1 - High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material

AU - Phlaetita, Wasana

AU - Chin, Dong Poh

AU - Ntui, Valentine Otang

AU - Nakamura, Ikuo

AU - Mii, Masahiro

N1 - KAUST Repository Item: Exported on 2021-03-31 Acknowledgements: We thank Mr. Nobuyuki Asai, Asai Daikeikan Co., Ltd. to kindly provide the seeds as plant materials in this research.

PY - 2015

Y1 - 2015

N2 - Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 gl−1 maltose, 30 mgl−1 hygromycin and 20 mgl−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

AB - Dendrobium orchid is one of the most popular cut flower and potted plants. In this study, a protocol for efficient genetic transformation of D. nobile-type orchids was established by co-cultivating 21 day-old protocorms for 3 days with Agrobacterium tumefaciens strain EHA101 carrying pIG121Hm harboring β-glucuronidase (GUS) gene as reporter gene and hygromycin phosphotransferase (hpt) gene as selectable marker gene. After selection of the infected protocorms on New Dogashima (ND) medium containing 10 gl−1 maltose, 30 mgl−1 hygromycin and 20 mgl−1 meropenem for 3 months followed by the culture on hygromycin-free recovery medium for 1 month, secondary protocorm-like bodies (PLBs) produced on this medium were again transferred onto secondary selection (regeneration) medium. Plantlets were successfully regenerated from these secondary PLBs after the transfer. The highest transformation efficiency of 27.3% was obtained when protocomrs were inoculated with 10 times diluted Agrobacterium solution (OD600=0.1) for 300 min. Transformation of the selected plants was confirmed by GUS assay, PCR and Southern blot analysis. This protocol could be adopted to produce transgenic D. nobile-type orchids with various traits such as novel flower color and resistances to biotic and abiotic stresses.

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JO - Plant Biotechnology

JF - Plant Biotechnology

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ER -

High efficiency Agrobacterium-mediated transformation of Dendrobium orchid using protocorms as a target material

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