Figure 30 Acclimatization of in vitro plants in greenhouse. A Plantlet; B Transfer plantlet
to substrate; C Transferred plants into pot; D Plants 2 months 2.8 Acclimatization and plant morphology measurements
Plantlets were transferred into 120 mL paper pots Jiffy pot, Ohio, USA and were covered by special plastic box. After 2 months of first acclimatization, plants are transferred
into 2-L plastic pots Figure 30. Several parameters were measured on plants: before acclimatization month 0: height of root, diameter of root, height of stem, diameter of stem,
number of leaves, number of leaflets, and number of lateral roots from in vitro plantlets; at 2 and 6 months after acclimatization: height of plant, diameter of stem at the collar, number of
leaves, and number of leaflets; and then for 12 months after acclimatization: diameter of stem, height of stem, number of leaves, number of leaflets, weight of leaves, weight of stem,
weight of total root, and weight of the main root. 2.9 Histo-cytological analysis
2.9.1 Plant material
Leaf, green stem, lignified stem, and taproot R1 were collected from one year plants of wild-type WT line CI07060 and transgenic lines TS19A46, TS19A90, TS20A69, and
TS20A75 for Hevea clone PB260 Figure 31. All parts of samples were cut in small parts for longitudinal and transversal sections. Leaves were cut in square 1 x 1 cm
2
including main nerve and lamina; green stem were cut in cylindrical slice 0.5 x 0.5 x 0.5 cm
3
; lignified stem were cut in cylindrical slice 0.5 x 0.5 x 1 cm
3
and if possible cut in a half part; after washing with water root were cut in cylindrical slice 0.5 x 0.5 x 1 cm
3
and cut in a half part when it was possible like for lignified stem. After cutting, all samples were directly kept in the
fixative solution in a small sample bottle 40 mL one by one. Each bottle was filled up to the half part of the bottle approx. volume with fixative solution. The composition of fixative
solution is detailed in Table 3. Samples in fixative solution were kept in vacuum minimum five hours and after they remained in cold room at 8-10
o
C for three days. After this time, the fixative solution was changed gradually by ethanol 50 then ethanol 70. During each bath
the samples were kept under vacuum for two hours. For long storage, ethanol 70 must be changed by new ethanol 70 and samples must be kept in cold room at 8-.10 °C. All
methods adapted from PHIV platform CIRAD, Montpellier, France. All the process for preparing histology samples can be seen in Figure 32.
Figure 31 Part of collected samples from leaf 1, green stem 2, lignified stem 3, and
taproot 4
Table 3 Composition of stock solutions
Final solution Composition
Quantity of stock solution
Solution A Anhydrous NaH
2
PO
4
MW 120 2.4 g
Distilled H
2
Up to 100 mL Solution B
Anhydrous NaH
2
PO
4
MW 142 2.84 g
Distilled H
2
Up to 100 mL Buffer phosphate pH 7.2; 0.2 M
Solution A 28 mL
Solution B 72 mL
Fixative solution Buffer phosphate pH 7.2; 0.2 M
50 mL Paraformaldehyde 20
10 mL Glutaraldehyde 50
2 mL Caffein
1 g Distilled H
2
Up to 38 mL
2.9.2 Softening procedure
