Genetic transformation of Vitis vinifera via organogenesis

Abstract

Background: Efficient transformation and regeneration methods are a priority for successful application of genetic engineering to vegetative propagated plants such as grape. The current methods for the production of transgenic grape plants are based on Agrobacterium-mediated transformation followed by regeneration from embryogenic callus. However, grape embryogenic calli are laborious to establish and the phenotype of the regenerated plants can be altered.

Results: Transgenic grape plants (V. vinifera, table-grape cultivars Silcora and Thompson Seedless) were produced using a method based on regeneration via organogenesis. In vitro proliferating shoots were cultured in the presence of increasing concentrations of N6-benzyl adenine. The apical dome of the shoot was removed at each transplantation which, after three months, produced meristematic bulk tissue characterized by a strong capacity to differentiate adventitious shoots. Slices prepared from the meristematic bulk were used for Agrobacterium-mediated transformation of grape plants with the gene DefH9-iaaM. After rooting on kanamycin containing media and greenhouse acclimatization, transgenic plants were transferred to the field. At the end of the first year of field cultivation, DefH9-iaaM grape plants were phenotypically homogeneous and did not show any morphological alterations in vegetative growth. The expression of DefH9-iaaM gene was detected in transgenic flower buds of both cultivars.

Conclusions: The phenotypic homogeneity of the regenerated plants highlights the validity of this method for both propagation and genetic transformation of table grape cultivars. Expression of the DefH9-iaaM gene takes place in young flower buds of transgenic plants from both grape cultivars.

Figure 1

Grape micropropagation and genetic transformation: schematic representation of the in vitro processes. BA, benzyl adenine; MB, meristematic bulk.

Figure 2

Meristematic bulk tissue (MB) generated from in vitro-proliferated grape shoots: (A) standard proliferation stage; (B) Internal section (×40) of the MB showing hyper-trophic parenchymatous cells with highly vascularized bands; many initiation nodules (×40) are also visible (C). The nodules generate adventitious buds (×5) (D).

Figure 3

Grape regeneration via organogenesis: (A) slices (approx. 1 cm², 2 mm thick) prepared from the MB and used for propagation or genetic transformation; (B) shoot regeneration obtained after 30 days of culture (×5).

Figure 4

Regenerating transgenic lines on the selection medium supplemented with 50 mg/l kanamycin 60 days after infection.

Figure 5

Southern blot analysis of grape plants transgenic for DefH9-iaaM gene. (A) Genomic DNA (digested with HindIII) from control untransformed Silcora plants (lanes 4, 7), three independent transgenic Silcora lines (plants #6, #29, #35, lanes 3, 5, 6, respectively), control untransformed Thompson Seedless plants (lane 2) and transgenic Thompson Seedless line (plant #4, lane 1). (B) Schematic drawings of the constructs used for transformation of Silcora (right) and Thompson Seedless (left) plants are reported. The probes are indicated with grey boxes. Only restriction sites relevant for Southern analysis are indicated. LB, left border; R, right border.

Figure 6

RT-PCR analysis of flower buds from transgenic grape plants. Analysis was performed with single strand cDNA synthesized from mRNA extracted from young flower buds of Silcora control and transgenic plants #6 and #35 (lanes 3, 4 and 5, respectively) and Thompson Seedless control and transgenic plants (lanes 1 and 2, control and #4, respectively). The amplification product of 266 bp corresponds to the 5' end of the spliced DefH9-iaaM mRNA.

Figure 7

First expanded leaves of transgenic and control lines of Thompson Seedless (A. left: control; right: line #4) and Silcora (B left: line #6; centre: control; right: line #35).

Figure 8

Adult leaves (collected from the 6^th to 12^th position on the branch) of transgenic and control lines of Thompson Seedless (A: control; B: line # 4) and Silcora (C: control; D: Line # 6; E: Line # 35).