3.6.1.2 Analysis of quantitative and qualitative parameters of green stem
Width of bark, cambium, xylem, pith, and the percentage of pith have been measured and calculated, respectively in green stem Figure 53. Transgenic line TS20A69 had wider
cambium compared to wild-type and other transgenic lines. This result showed that TS20A69 had bigger the activity of cambium, because it might be due to transgene position effect. This
effect probably influenced the cambium activity.
The width of pith of TS20A75 was significantly different compared to wild-type and others transgenic lines. The pith or medulla is a tissue in the stem of vascular plants which
store and transport nutrients throughout the plant. The size of pith is probably related with the capability of plant to grow and develop. Statistical analysis showed that TS19A46 had
significant difference in total radius of green stem. The averages of total radius of green stem were 3237 µm for TS19A46. There was not any difference in percentage of pith between
wild-type and transgenic lines Table 34.
Table 34 Effect of lines on mean value of width from parts collected on green stem from
various wild-type and transgenic lines overexpressing HbERF-IXc5
Construct Line
Bark µm
Cambium µm
Xylem µm
Pith µm
Total radius of green stem
µm Percentage
of pith Wild-type
CI07060 293
a
25
a
790
a
844
a
1953
a
42.4
a
35S::HbERF-IXc5 TS19A46
538
a
27
a
1041
a
1631
ab
3237
b
50.1
a
35S::HbERF-IXc5 TS19A90
435
a
27
a
642
a
1246
ab
2349
ab
53.0
a
HEV2.1::HbERF-IXc5 TS20A69
435
a
62
b
1135
a
1280
ab
2911
ab
44.0
a
HEV2.1::HbERF-IXc5 TS20A75
397
a
32
a
572
a
1833
b
2834
ab
65.0
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.
Statistical analysis of effect of construct used 3 replicates for wild-type and 3 replicates for each construct. Pith size increased dramatically in transgenic plants compared
to wild-type 42.4 of total radius and this difference become significant for construct HEV2.1::HbERF-IXc5 65.0. Construct 35S::HbERF-IXc5 had a significant difference in
bark. Construct HEV2.1::HbERF-IXc5 had a significant difference in percentage of pith, and for both construct 35S::HbERF-IXc5 and HEV2.1::HbERF-IXc5 had significant difference in
radius of pith and total radius of green stem. These data suggested that both constructs promoted the capability of transgenic plants to grow more vigorously and became taller
Table 35.
Table 35 Effect of promoters driving the HbERF-IXc5 gene on mean value of width from
parts collected on green stem from wild-type line CI07060 and various transgenic lines
Construct Line
Bark µm
Cambium µm
Xylem µm
Pith µm
Total radius of green stem
µm Percentage
of pith Wild-type
CI07060 293
a
25
a
790
a
844
a
1953
a
42.4
a
35S::HbERF-IXc5 TS19A46
538
b
27
a
1041
a
1631
b
3237
b
50.1
ab
HEV2.1::HbERF-IXc5 TS20A75
397
ab
32
a
572
a
1833
b
2834
b
65.0
b
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.
Starch content was found in all parts of green stem section bark, xylem, and pith with almost the same percentage in wild-type and transgenic plants. Likewise, the content of
polyphenol was found with lower percentage in all parts of tissue. Primary latex cell PLC and secondary laticifer SL was found in the leaves for all lines. It was interesting to notice
the number of PLC and SL for TS19A46 35S::HbERF-IXc5 was higher compared the other followed by TS20A75 HEV2.1::HbERF-IXc5 Table 36. Laticifer differentiation from
vascular cambium in Hevea stems are good model for observing cell differentiation from the cambium. On the green stem, SL has not yet performed the ring of laticifer. The number of
SL was high but they are still separated or in a group, not yet formed the anastomoses. Laticifer cells had thick cell wall, non-transparent, elastic cytoplasm, and stained in pink-red.
Most of SL is distributed randomly Figure 53.
Table 36 Evaluation of starch, polyphenol contents, primary latex cells PLC, and secondary
laticifer SL on green stem from various wild-type and transgenic lines. Notes: -: absence; +: rare; ++: 10-20 ; +++: 20-50 ; ++++: 50
Construct Line
Starch Polyphenol
PLC SL
Bark Xylem
Pith Bark Xylem Pith
Wild-type CI07060
+ +
+ +
+ -
+ +
35S::HbERF-IXc5 TS19A46
+ +
+ ++
+ +
+++ +++
35S::HbERF-IXc5 TS19A90
+++ ++
+++ ++
+ ++
+ +
HEV2.1::HbERF-IXc5 TS20A69
+++ ++
++ +
+ +
+ +
HEV2.1::HbERF-IXc5 TS20A75
+ ++
+ +
+ +
++ ++
Figure 53 Histo-cytological descriptions of latex cells on green stem. A wild-type clone PB260, B transgenic line TS19A46. The histological sections were stained
with Oil Red O. Cross-sections of green stem were annotated: B. bark; PLC. primary latex cells; SL. secondary laticifer
3.6.1.3 Analysis of quantitative and qualitative parameter of lignified stem
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