Potential application of transgenic Phalaenopsis orchid

Tang-Wung Hsiau P., . and Yih-Shyan L., . and Wen-Huei C., . (2005) Potential application of transgenic Phalaenopsis orchid. [Proceedings Paper]

To improve the conventional breeding programme of orchids at Taiwan Sugar Research Institute genetic transformation systems of Phalaenopsis orchid were developed by using microprojectile bombardment and Agrobacterium-mediated transformation. For both methods protocorm-like-bodies PLB were used as explants for transformation. The PLBs were excised and cultured for 5-13 days in regeneration medium before being bombarded with gene-coated gold particles or infected with A.tumefaciens. The bombarded or Agrobacterium-infected PLBs were then subjected to kanamycin or hygromycin selection and regenerated into new PLBs and subsequently into transgenic plantlets. For microprojectile bombardment a cDNA encoding coat protein of the Cymbidium mosaic virus CyMV was bombarded into the excised PLBs. Expression of the cDNA is controlled by the CaMV 35S romoter. Screening of 25 transformants which survived kanamycin selection by PCR and Southern blot analysis revealed that two tranformants contained the cDNA encoding CyMV coat protein. Expression of the transgene in transgenic plants was determined and evaluated for enhanced resistance against CyMV. In addition a bacterial soft rot resistant gene AP1 has also been transferred into Phalaenopsis orchid. For Agrobacterium-mediated transformation the gene encoding for glucuronidase GUS was used as a reporter gene driven by 35S promoter. The excised PLBs were infected with various strains of A. tumefaciens. Observation by scanning electron microscope and analyses of GUS transient expression indicated that all strains tested could infect PLBs in the presence of acetosyringone with the highest infection rate for strain EHA105 pMT1. About 30-50 of infected PLBs survived after kanamycin selection for 30 days. Most of the infected PLBs and their regenerated new PLBs contained GUS activity.

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Abstract

To improve the conventional breeding programme of orchids at Taiwan Sugar Research Institute genetic transformation systems of Phalaenopsis orchid were developed by using microprojectile bombardment and Agrobacterium-mediated transformation. For both methods protocorm-like-bodies PLB were used as explants for transformation. The PLBs were excised and cultured for 5-13 days in regeneration medium before being bombarded with gene-coated gold particles or infected with A.tumefaciens. The bombarded or Agrobacterium-infected PLBs were then subjected to kanamycin or hygromycin selection and regenerated into new PLBs and subsequently into transgenic plantlets. For microprojectile bombardment a cDNA encoding coat protein of the Cymbidium mosaic virus CyMV was bombarded into the excised PLBs. Expression of the cDNA is controlled by the CaMV 35S romoter. Screening of 25 transformants which survived kanamycin selection by PCR and Southern blot analysis revealed that two tranformants contained the cDNA encoding CyMV coat protein. Expression of the transgene in transgenic plants was determined and evaluated for enhanced resistance against CyMV. In addition a bacterial soft rot resistant gene AP1 has also been transferred into Phalaenopsis orchid. For Agrobacterium-mediated transformation the gene encoding for glucuronidase GUS was used as a reporter gene driven by 35S promoter. The excised PLBs were infected with various strains of A. tumefaciens. Observation by scanning electron microscope and analyses of GUS transient expression indicated that all strains tested could infect PLBs in the presence of acetosyringone with the highest infection rate for strain EHA105 pMT1. About 30-50 of infected PLBs survived after kanamycin selection for 30 days. Most of the infected PLBs and their regenerated new PLBs contained GUS activity.

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Item Type:	Proceedings Paper
Additional Information:	Summary En
AGROVOC Term:	Phalaenopsis
AGROVOC Term:	Orchidaceae
AGROVOC Term:	Transgenic plants
AGROVOC Term:	Micropropagation
AGROVOC Term:	Genetic transformation
Geographical Term:	MALAYSIA
Depositing User:	Ms. Norfaezah Khomsan
Last Modified:	24 Apr 2025 05:28
URI:	http://webagris.upm.edu.my/id/eprint/16653

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