Controlled environment experiments

To contrast the vineyard trials, four experiments were carried out in a controlled environment cabinet using potted Vitis vinifera L. cv. Sauvignon blanc in 2011 and 2012. Potted vines used in these experiments were grown and prepared according to methods reported by Mullins and Rajasekaran (Mullins & Rajasekaran 1981).

3.2.1

Potted vines

Fruiting cuttings were collected and grown essentially according to the methods described by Mullins and Rajasekaran (1981). Uniform hardwood cuttings (consisting of 4/5 nodes) were collected from well-ripened dormant canes in late winter in 2010 and 2011. The canes were obtained from three rows of Vitis vinifera L. cv. Sauvignon blanc vines in the Lincoln University research vineyard. The cuttings were stored damp in plastic bags at 2°C in a cold room until required.

To initiate root growth, dormant canes were cut transversally just below the level of the lowest bud and placed in a tray with pumice (1-4 mm grade), so the bottom (8-10 cm) of the cuttings were buried in the substrate. Trays with cuttings (60 canes per tray) were then placed in a shade-room on electric heat-pads that maintained the pumice in the trays at between 20-25°C during day and night. Under such conditions root initiation was promoted in 3-4 weeks before bud burst was retarded. The cuttings were regularly watered and monitored for approximately 4 weeks. Then cuttings with roots of 5-10 cm were transferred into pots filled with potting mixture. Each 1 m3 _{of the potting mixture contained 800}

litres of composted bark and 200 litres of pumice. Nutrients were supplemented with fertilisers consisting of 2 kg of osmocote, 1 kg of agricultural lime and 1 kg of hydraflo (Scotts Australia Pty Ltd, NSW, Australia). Potted cuttings were then moved into a glasshouse and grown from November to March without artificial light supplementation. The temperature in the glasshouse was maintained at 22-25°C during the day and 16-18°C during the night.

After the bud burst, the top two buds containing inflorescences were left to develop and remaining buds were removed. All leaves and shoots adjacent to inflorescences were removed as soon as they became accessible. Later, the shoot tips were excised leaving inflorescences in terminal positions. In 15-20 days after the bud burst, the weaker of the two inflorescences was removed, and a lateral bud below the stronger inflorescence was allowed to develop into a shoot with leaves to support further growth and development of the bunch. The plants were regularly watered and prevented from overgrowing until being transferred into the controlled environment cabinet for UV-B radiation experiments. The development of berries were monitored by the total soluble solids (°Brix) of berry measured using a handheld refractometer (Atago, Tokyo, Japan). The veraison occurred at approximately 12-13 weeks from bud burst and ripe berries were produced in 16-18 weeks.

3.2.2

Controlled environments

Figure 3.3 shows the controlled environment (CE) cabinet and potted vines used for the CE experiments. The CE cabinet was designed as 1.37 m × 2.45 m. Temperature was controlled by a computer programme at 22°C and 18°C during the day and night period, respectively. The light photoperiod was controlled as 12 hours per day (7.30 am – 7.30 pm). Photosynthetically Active Radiation (PAR) was provided by Osram warm-white fluorescent tubes (New Zealand Lighting System Ltd., New Zealand). PAR value was maintained at 300 µmol/m2_{/s measured by a LI-COR LI-188B}

Quantum Radiometer (LI-COR Biosciences – Biotechnology, USA). UV-B radiation was provided by UVB- 313 UV fluorescent tubes (Q-Lab Company, USA). Fluence rates of UV-B (280–313 nm) were measured by a UVB Biometer model 501 radiometer (Solar Light Company, USA). UV-B fluence was controlled at UV Index 12 and 8 hours per day (9.30 am – 5.30 pm).

Before UV-B radiation treatment was started, the CE cabinet was divided into two separate spaces by a UV-excluding polycarbonate screen (Dotmar Universal Plastics, Christchurch, New Zealand) set up in the middle of the cabinet. In the space with UV-B radiation, six visible light tubes as well as six UV-B tubes were provided. While in the control space, six visible light lamps were provided but six UV-B lamps were not switched on. Potted vines were transferred into the CE cabinet at least one week before the UV-B radiation treatment was started to allow the vines to adjust to the microenvironment. The vines were watered regularly and the humidity of the cabinet was controlled at 70-80%. After one- week adjustment, UV-B tubes were turned on and the vines in the UV-B treatment were exposed to PAR+UV-B radiation (UVI 12), while the control vines were exposed to PAR without UV-B radiation.

Figure 3.3 Potted vines in the controlled environment cabinet and glasshouse for controlled environment trials

(a) Potted vines transferred into the controlled environment cabinet for UV-B radiation trials; (b) Potted vine with berries grown up in a glasshouse. Berries in the vine are at 17 weeks post bud burst (around 4-5 weeks post- veraison).

3.2.3

Sample collection

The UV-B radiation experiments were carried out on potted vines at four developmental stages: 10 weeks post bud burst (2-3 weeks pre-veraison, ˚Brix 3.5-4.0), 12 weeks post bud burst (around veraison, ˚Brix 5.6-8.9), 14 weeks post bud burst (1-2 weeks post-veraison, ˚Brix 10.9-12.4) and 17 weeks post bud burst (4-5 weeks post-veraison, ˚Brix 13-16). For each stage, 60 vines (each vine had one bunch of approximately 20 berries) were transferred into the CE cabinet at least one week earlier to allow the vines to adjust to the micro-environment. These vines were divided into two groups: 30 vines for the control treatment and 30 vines for the UV-B treatment. Sampling time points were selected from 4 hours, 1 day (8 hours), 1.5 day (12 hours), and 2, 3, 4, 5 to 7 days of UV-B exposure. At each time point, 3 vines from the control treatment and 3 vines from the UV-B treatment were removed from the CE cabinet. Berries were immediately harvested and frozen in liquid nitrogen and stored at - 80°C for future analysis.