Development of an alternative system for the evaluation of transgenes and regulatory elements from Vitis vinifera L.
Burger, A L
University of Stellenbosch
Manuel, C M
Van Kervel, E J
Several putative transgenic strawberry plants were regenerated from leaf disks transformed with the grapevine mrip1 promoter fused to the reporter gene, GFP (green fluorescent protein). The promoter of the CaMV 35S was used as positive control, while a promoterless construct was used as negative control. The first two putative transgenic plants transformed with the mrip1:GFP fusion gene were hardened off in January 2003 and are expected to bear fruit in the 2003 season. Functionality of the mrip1 promoter has been verified in transgenic tobacco (refer project IPB 4/4). Transgenic strawberry fruit expressing the mrip1:GFP fusion gene will be the first fruit tissue in which a promoter from grapevine will be evaluated.
A significant proportion of the strawberry material entered into the programme in the period April to June 2002 was lost due to Agrobacterium overgrowth and poor regeneration. The problem of bacterial overgrowth was resolved by optimising some of the procedures involved in the genetic transformation protocol. Mother plant material used for genetic transformation are glasshouse-grown. The condition of this material was identified as most critical for strawberry regeneration. The use of in vitro material is currently investigated.
Other transgenes to be evaluated in ripening strawberries include: 1) two promoters from grapevine (one early ripening-regulated; the other constitutively regulated during berry ripening), and 2) Pyrophosphate dependant Phosphofructokinase (PFP). It is proposed that the over-expression of PFP would alter the sugar and acid metabolism of ripening grape berries. The two grapevine promoters were fused to GFP. The two fusion genes and PFP were cloned into plasmids for plant genetic transformation and introduced to Agrobacterium.
Groenewald, J H, Botha, F C, Burger, A L, Venter, M, Hiten, F, Stander, C and Zwiegelaar, J P. 2004. Presentation at the Wine Biotechnology and IPB Institute Wine Biotechnology Open Day. 4 June, Stellenbosch, South Africa.
Burger, A L, Watts, L and Botha, F C. 2004. Isolation, characterisation and functional analysis of a post-veraison ripening-related promoter element from Vitis vinifera cv. Merlot. Paper presented at the 7th International Symposium on Grapevine Physiology and Biotechnology. 21-25 June, UC Davis, California, United States.
Burger, A L. 2005. Isolation, characterisation and functional analysis of a ripening-related promoter from Vitis Vinifera cv. Merlot. Paper presented at the 31st Conference of the South African Association of Botanists. 10-14 January, University of the Orange Free State, Bloemfontein, South Africa.
Burger, A L. 2005. Proving more versatile than just ‘strawberries and cream’? The use of strawberries in the genetic manipulation of grapevine fruit metabolism. WineLand, Mnth May (189) (p. 82-84)