Genetic transformation of table grape via organogenesis and field evaluation of DefH9-iaaM transgenic plants

Transcription

1 Genetic transformation of table grape via organogenesis and field evaluation of DefH9-iaaM transgenic plants Mezzetti B., Silvestroni O., Costantini E. Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Università Politecnica delle Marche, Ancona b.mezzetti@univpm.it Spena A., Pandolfini T. Dipartimento Scientifico Tecnologico, University of Verona, Strada Le Grazie, Verona (IT) angelo.spena@univr.it MIUR - FIRB (RBAU01JTHS)

2 The use of genetic engineering for grape improvement permits the introduction of useful agronomic traits without altering the features of the cultivar. Embryogenic calli from anther filaments are widely used for genetic transformation in grape. An alternative method useful for both propagation and Agrobacterium-mediated genetic transformation of table grape (V. vinifera) was recently described (Mezzetti et al., 2002). MIUR - FIRB (RBAU01JTHS)

3 The method is based on the formation of meristematic bulk (MB) tissue with a high regenerative capacity, using adventitious shoots as a starting material. Meristematic Bulk from proliferating shoots Competent Cells Regenerating Adventitious Shoots Propagation Genetic Manipulation

4 DefH9-IaaM Gene (Spena UniVR) LB Terminator IaaM coding DefH9 Promoter RB EcoRI HindIII P. savastanoi KpnI BamHI EcoRI EcoRI Antirrhinum majus It confers expression within the placenta and the ovules and it has been used to confer parthenocarpic fruit development to several plant species and varieties (Rotino et al., 1997) From regeneration and selection to the open field (2001)

5 Why the DefH9-IaaM Gene The developing ovules are a source of plant growth regulators (e.g. auxin) sustaining fruit growth (Gustafson, 1942). Factors affecting the biosynthesis and degradation of this hormone are therefore expected to influence all stages of fruit development including fruit ripening and quality. MIUR - FIRB (RBAU01JTHS)

6 Why the DefH9-IaaM Gene The effect of growth regulators on these important process occurring in grape berry are up to now widely studied by the exogenous application of synthetic phytohormones (mainly auxin, ABA and gibberellins), and are focused to alter the ripening processes. The DefH9-iaaM chimeric gene resulted as an important tool to study the effect of increased auxin content in flower buds on sustaining parthenocarpic fruit development and/or on improving fruit production and quality to several horticultural species (Ficcadenti et al., 1999; Donzella et al., 2000; Pandolfini et al., 2002; Mezzetti et al., 2004). MIUR - FIRB (RBAU01JTHS)

7 DefH9 iaam Table Grape (Genomic DNA digested with HindIII) Schematic drawings of the constructs used for transformation of Silcora (right) and Thompson Seedless (left) plants and probes indicated with grey boxes. Only restriction sites relevant for Southern analysis are indicated. GM Cont GM Cont GM GM Thompson Silcora Silcora Cont

8 DefH9 iaam expression in Table Grape RT-PCR analysis performed with single strand cdna synthesized from mrna extracted from young flower initials of Thompson Seedless and Silcora control and transgenic plants. The amplification product of 266 bp corresponds to the 5' end of the spliced DefH9- iaam mrna. C GM Thompson C GM GM Silcora Flower initials transgenic for the DefH9-iaaM gene had an IAA content higher than controls (data not reported). The DefH9-iaaM auxin-synthesizing gene does not however inhibit grape fruit ripening.

9 Open field transgenic trials Biotechnology Commission Minister of Health (CE 2001/18) (Table Grape, strawberry and raspberry) Notification N. B/IT/99/24 Thanks: NOE - Ancona Thanks: NOE - Ancona

10 The experimental trial: Thompson (TH) Control and GM line: 32 plants each. Silcora (SI) Control and GM line :16 plants each. Vines were spaced at 2.5 x 1.5 m, trained with the double guyot system.

11 DefH9-iaaM - Table grape Effect on shoot fruitfulness (Duncan s Multiple Range Test; p < 0.01) 2003 Bud Fertility and Position - Laboratory Field fruitfulness % Buds Buds Total bud % Buds with CLONES Total number of without fertility with shoots fertile shoots bunches per shoot fertile shoot position TH - CT TH - GM * 0.26* SI - CT , SI - GM * 0.46* Thompson seedless Control Thompson seedless DefH9-iaaM Laboratory Field fruitfulness

12 DefH9-iaaM - Table grape Effect on shoot fruitfulness Inflorescence on a shoot arose from a basal bud of DefH9- iaam Thompson seedless, unusual for this variety. Field fruitfulness

13 DefH9-iaaM - Table grape Effect on cluster morphology berry size and quality (Duncan s Multiple Range Test; p < 0.01) 2003 Clones Cluster Weight (g) Cluster Morphology N berry per cluster Berry Weight (g) Sol.Sol. (%) ph Fruit quality Tritatable Acidity (g/l) TH - CT TH GM 94.06* * 0.55* 22.16* * SI CT SI GM * * Bunches from the DefH9-iaaM clones of both cultivars were heavier than the corresponding controls. This positive modification was related to different changes of cluster characteristics depending on the cultivar. The increase of productivity promoted by the expression of DefH9-iaaM gene corresponded to a slight reduction of berry quality in comparison with the control, but always remaining at high standard level for the two cultivars

14 DefH9-iaaM - Table grape Effect on yield capacity (0ne plot production) TH Control TH GM SI Control SI GM The effect of the DefH9-iaaM in increasing shoot fruitfulness in table grape is related to the genotype, and it can represent an important tool for increasing vines productivity particularly for cultivars with low natural shoot fruitfulness.

15 Conclusions DefH9-iaaM gene confirmed to be able to improve productivity in perennial plants including table grapes In table grapes a specific effect of the gene expression depending on cultivars was observed. The two cultivars showed different changes on shoot fruitfulness and bunch size (number of berries and weight) probably related to the natural mechanisms controlling flowers differentiation as well as fruit set and development. The expression of DefH9-iaaM gene in Thompson seedless lead to an enhanced number of berries per bunch and to an unexpected increase in shoot fruitfulness. The expression of DefH9-iaaM gene in Silcora evidenced also the potential effect of the same gene in promoting larger berry size.