Factors affecting agrobacterium-mediated genetic transformation of marine microalga Nannochloropsis Sp.

Willy Y., . and Cha T. S., . and Aziz A., . Factors affecting agrobacterium-mediated genetic transformation of marine microalga Nannochloropsis Sp. pp. 153-163. ISSN 1823-8556

The optimization condition of an Agrobacterium tumefaciens-mediated transformation method for the marine microalgae Nannochloropsis sp. (UMT-M3) is reported using a binary vector harbouring the green fluorescent protein and -glucuronidase (gfp-gusA) as reporter genes and a hygromycin phosphotransferase (hpt) as a selection marker. For an efficient Agrobacterium mediated genetic transformation of marine microalgae Nannochloropsis sp. parameters namely pre-culture duration inoculum density co-cultivation temperature co-cultivation duration pH of co-cultivation medium and the concentration of acetosyringone (AS) were evaluated in this study by monitoring -glucuronidase (gusA) expression two days post-infection. Results showed that 5 days of pre-culture 3 days of co-cultivation at 24C medium pH 5.5 bacterial density of OD‚‚‚1.0 and 50M AS are optional in the transformation ogf Nannochloropsis sp. The use of combined optimized parameters resulted in an average transformation frequency of 24.5. The transgenic nature of transformants was confirmed by polymerase chain reaction with gfp-gusA and hpt-specific primers on colonies resistant to 18mg L hygromycin. The developed transformation method will enable manipulation of important biochemical pathways in Nannochloropsis sp. And facilitate the genetic improvement of this commercially-important microalga.

Full text available from: Official URL: http://jssm.umt.edu.my/files/2012/11/7.pdf

Abstract

The optimization condition of an Agrobacterium tumefaciens-mediated transformation method for the marine microalgae Nannochloropsis sp. (UMT-M3) is reported using a binary vector harbouring the green fluorescent protein and -glucuronidase (gfp-gusA) as reporter genes and a hygromycin phosphotransferase (hpt) as a selection marker. For an efficient Agrobacterium mediated genetic transformation of marine microalgae Nannochloropsis sp. parameters namely pre-culture duration inoculum density co-cultivation temperature co-cultivation duration pH of co-cultivation medium and the concentration of acetosyringone (AS) were evaluated in this study by monitoring -glucuronidase (gusA) expression two days post-infection. Results showed that 5 days of pre-culture 3 days of co-cultivation at 24C medium pH 5.5 bacterial density of OD‚‚‚1.0 and 50M AS are optional in the transformation ogf Nannochloropsis sp. The use of combined optimized parameters resulted in an average transformation frequency of 24.5. The transgenic nature of transformants was confirmed by polymerase chain reaction with gfp-gusA and hpt-specific primers on colonies resistant to 18mg L hygromycin. The developed transformation method will enable manipulation of important biochemical pathways in Nannochloropsis sp. And facilitate the genetic improvement of this commercially-important microalga.

[error in script]

Item Type:	Article
AGROVOC Term:	Algae
AGROVOC Term:	Aquatic plants
AGROVOC Term:	Agrobacterium
AGROVOC Term:	Agrobacterium tumefaciens
AGROVOC Term:	Genetic transformation
AGROVOC Term:	Genes
AGROVOC Term:	Gene transfer
AGROVOC Term:	Gene expression
AGROVOC Term:	Beta glucosidase
AGROVOC Term:	Hygromycin
Depositing User:	Mr. AFANDI ABDUL MALEK
Last Modified:	24 Apr 2025 00:54
URI:	http://webagris.upm.edu.my/id/eprint/8717

Actions (login required)

View Item