The general procedures to obtain pl

The general procedures to obtain plant material and the experimental
design and treatments were previously reported in Pontin
et al. (2010).
5.1. Plant material
Plant material was obtained from a virus-free vineyard of Vitis
vinifera L. cv. Malbec; three-node wood cuttings were collected
and treated with 100 mg L1 indole-3-butyric acid (IBA, Sigma
Chem. Co., St. Louis, MO, USA) in order to promote root development.
Then explants were planted in 2.5 L plastic pots filled with
hydrated-perlite and maintained under greenhouse conditions.
From three-month old plants, uninodal cuttings were taken to obtain
in vitro cultured plants. Shoots were surface sterilized by
immersion in EtOH–H2O (7:3, v/v) for 3 min followed by treatment
with 1.5% NaOCl for 10 min with occasional swirling, and washing
three times with sterilized distilled H2O for 5 min. Explants
(1–1.5 cm length) with one axillary bud were cultivated in glass
tubes containing 20 mL of solid MS medium plus salts and vitamins,
30 g L1 sucrose, 1 mg L1 6-benzylaminopurine (BAP), and
7.5 g L1 agar. In vitro shoot tips were sub-cultured to fresh medium
at 6 weeks intervals into half-strength MS micro- and
macro-nutrients, excluding FeEDTA (0.1 mM Na2EDTA + 0.1 mM
FeSO4) and supplemented with full-strength MS vitamins,
30 g L1 sucrose, 0.5 lM 1-naphthaleneacetic acid and 7.5 g L1
agar. Explants (one per glass flask, 12 cm height  6.5 cm diameter)
were incubated at 25 ± 2 C and a photosynthetically active
radiation (PAR) of 80 lmol m2 s1 provided by cool fluorescent
tubes with a 16/8 h photoperiod. Flask tops were covered with
low-density polyethylene, which transmitted most of the PAR.
5.2. UV-B treatment
UV-B treatments were carried out with 45 d old in vitro grown
plants, having six fully expanded leaves, and in the same controlled
growth chambers described above. For different light treatments,supplemental UV-B was given using a TL 100W/01 tube (311 and
313 nm spectrum peaking; Philips, Eindhoven, The Netherlands)
suspended 40 cm above the flasks. A 4.75 kJ m2 d1 total effective
dose of UV-B normalized at 311 nm was provided in two different
irradiation treatments: ‘‘low UV-B’’ (8.25 lWcm2 irradiance during
the 16 h per day photoperiod) and ‘‘high UV-B’’ (33 lWcm2
irradiance during the last 4 h of the 16 h per day photoperiod).
For controls, UV-B was filtered by a clear polyester (100 lm, Oeste
Aislante, Buenos Aires, Argentina), which absorbs more than 95% of
UV-B, and reduced 15% of PAR. PAR was measured with a Li-250
light meter with a Li-190 quantum sensor (Li-COR Inc., Lincoln,
NE, USA) and UV-B irradiance was controlled at the top of the flask
with a PMA2200 radiometer with a PMA2102 UV-B detector (Solar
Light Company Inc., Glenside, PA, USA). After the treatments, three
fully expanded apical leaves (young) and two basal leaves (mature)
were harvested, weighed and used for the different analytical
determinations. Three independent biological replicates (n = 3)
were used for terpene analysis and TPS activity assays.