Plant material
Investigations were carried out on Vitis vinifera L. using the flower abscission-sensitive ‘Gewurztraminer’ (GW) and the non-sensitive ‘Pinot Noir’ (PN) varieties. Dormant cuttings, which had been cane-pruned were collected from the INRA vineyard in Bergheim, France (Mullins and Rajasekaran, 1981
).
Cuttings were collected at the proximal part of the cane and limited to three nodes, successively named N0 (the proximal), N1 and N2. A key point in the protocol is the sampling of the cane fragments generating the cuttings. The physiology of this fragment, in particular the amount of reserves in the wood, is important for obtaining predictable results. Harvesting fragments from any part of the annual cane provides cuttings with different sugar contents that vary in reproduction. Similarly, fragments from the distal part of the cane have low carbohydrate reserves and flower less (data not shown).
Cuttings were treated with cryptonol (2 % v/v) to prevent contamination, stored in the dark and forced for at least 2 weeks at 4 °C. After 16 h of hydration at 25–30 °C, N0 and N1 were removed and the proximal extremity of the cutting immerged in indole-3-butyric acid (IBA) at 1 g L–1 for 30 s to promote rhizogenesis. This protocol was developed from Mullins (1966) and Mullins and Rajasekaran (1981) with IBA favouring rhizogenesis and thus eliminating the need for a 4-week pre-treatment in which the cuttings are warmed at the base. The IBA-treated cuttings developed within 1 week instead of 4 weeks.
Cuttings were planted in 0·5-L pots containing perlite : sand (1 : 1) and transferred to a greenhouse at 20/30 °C (night/day), with the pots placed on a 30 °C warming blanket. The photoperiod was 16 h using natural daylight or artificial 400 W ‘Saudiclaude’ lamps supplying 1000 µE m–2 s–1, and a relative humidity of 50 %. Each pot was watered daily with 100 mL of Coïc and Lesaint medium (Coïc and Lesaint, 1971). Leaves were removed until the first inflorescence appeared (each cuttings was limited to four leaves).
Inflorescences were collected from vineyard and glasshouse cuttings (Eichhorn and Lorenz, 1977), from the visible cluster (stage 12) up to fruit set (stage 27). Since meiosis occurs in these cultivars between stages 15 and 17 in vineyards (Lebon et al., 2004), this period was subdivided (15 + 2 d and 15 + 8 d). Development was quicker in the cuttings and the intermediate stages were defined as 15 + 1 d and 15 + 3 d.
Each developmental stage occurred when at least half of the cuttings were at this stage. The numbers of flowers, berries and seeds per fruit were counted on each cutting (n = 10).
Microscopy
Samples were fixed in 2 % glutaraldehyde (v/v in a 0·1 M phosphate buffer) at pH 7·2 in 2 % sucrose (w/v) and 1 % Tween 20 (v/v) for 24 h and agitated at room temperature. After three rinses (5 min) in buffer, the flowers were post-fixed with 1 % osmium tetroxide (w/v) in the buffer without Tween 20 for 4 h. Flowers were then rinsed three times (5 min) in the buffer, dehydrated in an alcohol series, transferred to acetone, and embedded in Araldite.
For starch, 1 µm sections were collected on glass slides, bleached for 30 min with hydrogen peroxide (H2O2 10 volumes), and the periodic acid Schiff (PAS) polysaccharide-specific reaction carried out according to Clément et al. (1994). Sections were plunged in 1 % periodic acid (w/v) for 4 h, in Schiff's reagent without rinsing for 16 h, and in 5 % sodium metabisulfite (w/v) for 20 min. Sections were rinsed in distilled water, air-dried, and mounted in Eukitt®. For each stage, ten flowers from five inflorescences were used, each inflorescence sampling from a different vine or cutting.
Statistical analysis
For the number of flowers, berries and seeds per fruit, ten inflorescences were used, each inflorescence sampling from a different vine or cutting. Statistical analyses were carried out using Student's t-test. A 5 % probability was considered significant. For morphology and starch content, ten flowers from five different inflorescences per stage were sampled, each inflorescence sampling from a different vine or cutting; for these parameters, no statistical analysis was performed because observations from the ten flowers considered were similar.
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