Table 30 Summary effect of overexpression of HbERF-IXc4 or HbERF-IXc5 on plant
morphology. Notes: blue significantly different to controls with lower value, orange not significantly different to controls, red significantly different to
controls with higher value and white no data
Construct Line
Height Stem
diameter Leaves
and leaflets
Total weight
Leaf weight
Stem weight
Root weight
Ratio Rtot
plant
Ratio R1tot
R
35S::HbERF-IXc4
TS17A61 TS17A79
HEV2.1::HbERF-IXc4
TS18A09 TS18A13
TS18A20 TS18A37
TS18A69
35S::HbERF-IXc5
TS19A46 TS19A90
HEV2.1::HbERF-IXc5
TS20A47 TS20A69
TS20A75
3.6 Changes in some histological parameters 3.6.1 Analysis of quantitative and qualitative parameter from various wild-type and
transgenic lines overexpressing HbERF-IXc5
A histo-cytological analysis study was undertaken to characterize the differences existing between wild-type CI07060 and transgenic plants TS19A46, TS19A90, TS20A69,
and TS20A75 for Hevea clone PB260 overexpressing HbERF-IXc5 under the control 35S CaMV and HEV2.1 promoter, respectively. The study was carried out on green stem, lignified
stem, taproot R1, and leaves on 12-month-old plants grown in greenhouse. Better knowledge of the histo-cytology is required to examine tissue structures at the microscopic
level in order to understand the physiological and anatomical functions between wild-type and transgenic plants.
3.6.1.1 Analysis of quantitative and qualitative parameters in leaves
Observations were carried out on two main parts of leaves: main nerve counting of latex cells, and lamina counting of latex cells, width measurement of cuticle, upper
epidermis, palisade parenchyma, spongy parenchyma, lower epidermis, counting of palisade cell until 1-2 mm, and counting of stomata numbers Figure 51. Statistical analyses showed
there was significantly higher number of latex cells in transgenic lines TS19A46 and TS20A69 compared to wild-type. For lower epidermis, TS20A69 line showed significantly
different values compared to wild-type and other transgenic lines. The width of lower epidermis of TS20A69 was thinner compared to others Table 31.
Table 31 The effect of lines on mean value of width from different parts collected on leaf
from wild-type and various transgenic lines overexpressing HbERF-IXc5 and the number of palisade cells and stomata
Construct Line
Latex cell No.
in main nerve
Lamina Latex cell
No. Cuticle
µm Upper
epidermis µm
Palisade parenchyma
µm Spongy
parenchyma µm
Lower epidermis
µm Palisade
cell for 1 mm No.
Palisade cell for 2
mm No. Stomata
No.
Wild-type CI07060
50.50
a
4.50
a
1.62
a
7.25
a
41.66
a
49.05
a
8.17
b
69.67
a
148.67
a
4.33
a 35S::HbERF-IXc5
TS19A46
157.00
c
5.00
a
1.93
a
8.42
a
49.20
a
52.57
a
8.14
b
76.06
a
160.11
a
6.00
a 35S::HbERF-IXc5
TS19A90
37.00
a
3.00
a
1.69
a
8.26
a
45.03
a
52.57
a
7.52
ab
78.00
a
164.00
a
2.00
a HEV2.1::HbERF-IXc5 TS20A69
113.00
bc
12.00
a
1.74
a
7.93
a
48.13
a
42.28
a
3.61
a
88.00
a
173.33
a
4.00
a HEV2.1::HbERF-IXc5 TS20A75
84.00
ab
2.50
a
1.58
a
7.05
a
49.70
a
45.29
a
6.99
ab
76.39
a
160.17
a
6.33
a
The data were analysed with XLSTAT software. Statistical analysis was performed with an ANOVA followed by Tukey 5 test. Values with the same letter are not significantly different at the 0.05 probability level.
Figure 51 Histo-cytological description of leaf cross-section from WT plants of clone
PB260. The histological sections were stained with Oil Red O. A Leaf section, B Main nerve, C Lamina. Cross-sections of leaf were annotated: MN. main
nerve; LC. latex cell. C. cuticle; L. lamina; S. stomata; UP. upper epidermis; PP. palisade parenchyma; SP. spongy parenchyma; LE. lower epidermis
Statistical analyses revealed a significant difference in the number of latex cells between the construct of 35S::HbERF-IXc5 and HEV2.1::HbERF-IXc5 compared with wild-
type. These data suggest that candidate gene of HbERF-IXc5 had more capability for producing latex cells compared to wild-type Table 32 and Figure 52.
Table 32 Effect of promoters driving the HbERF-IXc5 gene on width mean value for
different parts of leaves: number of palisade cells, and stomata
Construct Line
Latex cell No. in
main nerve
Lamina Latex cell
No. Cuticle
µm Upper
epidermis µm
Palisade parenchyma
µm Spongy
parenchyma µm
Lower epidermis
µm Palisade
cell for 1 mm No.
Palisade cell for 2
mm No. Stomata
No.
Wild-type
CI07060
50.50
a
4.50
a
1.62
a
7.25
a
41.66
a
49.05
a
8.17
a
69.67
a
148.67
a
4.33
a 35S::HbERF-IXc5
TS19A46
157.00
c
5.00
a
1.93
a
8.42
a
49.20
a
52.57
a
8.14
a
76.06
a
160.11
a
6.00
a HEV2.1::HbERF-IXc5
TS20A75
84.00
b
2.50
a
1.58
a
7.05
a
49.70
a
45.29
a
6.99
a
76.39
a
160.17
a
6.33
a
The data were analysed with XLSTAT software. Statistical analysis was performed with an ANOVA followed by Tukey 5 test. Values with the same letter are not significantly different at the 0.05 probability level.
Figure 52 Localization of latex cells in leaves. A Wild-type CI07060, B Transgenic line
TS19A46. The histological sections were stained with Oil Red O. Cross-sections of leaf were annotated: MN. main nerve; LC. latex cells; WT. wild-type
The qualitative analysis led to identify more starch reserves in main nerve compared to lamina. Starch was observed by staining periodic Acid-Schiff and Naphtol Blue Black
NBB in pink-red colour. These dyes specifically stained soluble and storage proteins, like polysaccharides starch in violet. This histological analysis allowed identifying some
compounds. Lines TS19A90 35S::HbERF-IXc5 and TS20A69 HEV2.1::HbERF-IXc5 had the highest starch content in main nerve Table 33. Starch represents the most important
carbohydrate used for nutrient. Starch accumulates mainly in the parenchyma and maybe related to growth in diameter and in length of the stem. Analysis of polyphenol showed that
polyphenol was less produced in wild-type and transgenic lines. Polyphenol can be stained by Oil Red O in the dark blue.
Table 33 Evaluation of starch and polyphenol contents in leaves of plants from wild-type and
transgenic lines. Notes: -: absence; +: rare; ++: 10-20 ; +++: 20-50 ;
++++: 50
Construct Line
Starch Polyphenol
Main nerve Lamina
Main nerve Lamina
Wild-type CI07060
+ +
+ +
35S::HbERF-IXc5 TS19A46
++ +
+ +
35S::HbERF-IXc5 TS19A90
+++ +
++ +
HEV2.1::HbERF-IXc5 TS20A69
+++ +
+ -
HEV2.1::HbERF-IXc5 TS20A75
++ +
+ +
3.6.1.2 Analysis of quantitative and qualitative parameters of green stem
