12-month-old wild-type CI07060 and transgenic plants of lines TS18A09, TS18A13, TS18A37, TS19A90, TS20A69, and TS20A75 were used for testing effects of
environmental stresses Table 7. Table 7 Number of wild type and transgenic plants for each environmental stress experiment
Line Number of plants used for each experiment
Drought Ethephon
Cold Salinity
Biotic
CI07060 10
18 21
64 2
TS18A09 10
18 6
7 TS18A13
7 12
6 7
4 TS18A37
12 6
7 2
TS19A46 2
TS19A90 10
12 6
7 2
TS20A47 2
TS20A69 10
12 6
7 4
TS20A75 10
12 6
7 2
2.10.1 Drought treatment
The experiment was performed in a greenhouse cell, under controlled conditions with a mean temperature of 28.4
o
C and 43.6 relative humidity. The daylight period in the the cell was 12 h, and the photosynthetic active radiation flux was an average of 600 µmol m
-2
s
-1
above the canopy. 12-month-old plants were cut and placed in pots with the same weight of soil EGO 140 substrate, Tref group, Netherlands. Plants from the transgenic control line
CI07060 and transgenic lines were subjected to a controlled water deficit.
Drought stress was imposed by holding water from the pots. At the onset of soil dry- down, the surfaces of the pots were sealed with cellophane to prevent soil evaporation. In this
way, it was possible to calculate both the dynamics of soil water depletion and plant transpiration from gravimetric observations. Soil water status was monitored using the
fraction of transpirable soil water FTSW Luquet et al. 2008. In order to estimate the FTSW value of each pot, full watering of all the pots the day before the start of measurements
was followed by one night of drainage. On the next morning, the initial pot water capacity was determined by weighing all the pots. FTSW was estimated as the ratio of actual
transpirable soil water ATSW to total transpirable soil water TTSW, ATSW being the mass difference between daily and final pot weight. TTSW was calculated as the difference
between initial pot capacity and the final pot weight after soil desiccation. The experiment ended when the transpiration rate of each stress pot was less than 10 of that of the fully
watered pots Sinclair and Ludlow 1986. Its value matched 1 when the plants were well watered. Drought stress continued up to FTSW= 0.1.
2.10.2 Ethephon treatment
Ethephon 2-chloro-ethylphosphoric acid solutions were prepared at various concentrations by adding gelatine in water, and then the solution was heated and left to cool.
After, the ethephon was put in gelatine solution Table 8.
Table 8 Ethephon solution with various concentrations
Final concentration of ethephon
Volume of ethephon from stock solution mL
Quantity for 0.1 gelatine mg
H
2
O mL
50 50
1 1.04
50 50
2.5 2.6
50 50
5 5.2
50 50
Leaf and stem parts of plants were covered with various solutions of ethephon using a
soft brush. Plants were kept for one week in greenhouse under standard growth conditions. Number of dropped leaves and leaflet senescence were observed until six days after
treatment. The control plants received non-ethephon treatment. 2.10.3 Cold treatment
Twenty-one 8-month-old plants from wild type CI07060 and six transgenic lines TS18A09, TS18A13, TS18A37, TS19A90, TS20A69, and TS20A75 were used for cold
treatment. All plants in 2 L pots were put inside a climatic room at 10 °C for 96 hours with a photoperiod of 8h16h. On day 4
th
, the temperature was set up to 20 °C. One day after, all plants were transferred to greenhouse and temperature was set at 28 °C. Morphological and
eco-physiological measurements were conducted every 2 days for 10 days after treatment. Morphological parameters consist of number of leaves, leaflets, senescent leaves, burned
leaves, abscission leaves, wilting leaves, and wilting-burned leaves. Eco-physiological parameters were FvFm, Performance Index P. Index, and SPAD.
2.10.4 Salinity treatment
Sixty-four 10-month-old plants from wild type CI07060 and seven transgenic lines TS18A09, TS18A13, TS18A37, TS19A46, TS19A90, TS20A69, and TS20A75 were used
for salinity treatment. Morphology parameters number of leaves, leaflets, leaves senescence, burned leaves, abscission leaves, wilting leaves, wilting-burned leaves, and colour of leaves
and eco physiology parameters FvFm, P. Index, and SPAD were measured until 28 days. Pots of plant were watered by 1 L of sodium chloride [500 mM] on the third day.
Morphology and eco physiology were observed every two days until four weeks in greenhouse. Each pot was washed by water during a week to prevent the increase in osmotic
potential from salt. 2.10.5 Biotic treatment
Hevea brasiliensis leaves from clone PB260, wild type CI07060 and 7 transgenic lines TS18A13, TSA18A37, TS19A46, TS19A90, TS20A47, TS20A69, TS20A75 were
used on the biotic stress experiment. The morphogenetic leaves stage C were detached and placed on water-soaked filter paper in large Petri, plates, abaxial side up. The lower
epidermis was gently scarified over 1 mm2 using a scalpel blade and one drop of the toxin cassiicoline Cas1 and of the filtrate CCP 15 µl was placed on the scarified spot. The plates
were maintained for 1 day to 7 days at 25 °C dark until symptoms were clearly visible. The observation was conducted until 8 days.
3 RESULTS
3.1 Establishment and cryopreservation of transgenic callus lines and plant regeneration
