role as transcription factor which correlated to latex production and also stress response in H. brasiliensis Duan et al. 2010; Putranto et al. 2015a. However, analysis of specific function
for these transcription factors had not been done. This thesis was the first study which led to characterize the function of HbERF-IXc4 and HbERF-IXc5 using genetic modification
strategy. The results of this study were expected to provide some new biological knowledge for better understanding of the effect of overexpression of these candidate genes, HbERF-
IXc4 and HbERF-IXc5 and to provide deep understanding of the functional analysis of genes. 4.2 Effect of promoters and candidate genes at different stages of the genetic
modification process
This study was conducted on four constructs combining two genes HbERF-IXc4 and HbERF-IXc5 and two promoters CaMV and HEV2.1. Several changes were observed for
these four constructs. Interestingly, some changes in terms of callus proliferation, embryo production, plant regeneration, and plant morphology can lead us to determine specific
effects of promoters and studied genes.
4.2.1 Effects of promoters
Analysis the effects of promoters for each candidate genes had been done in this research. HbERF-IXc4 gene has a lower callus proliferation was observed with the 35S
CaMV promoter compared to HEV2.1. For instance, HbERF-IXc4 gene with 35S CaMV need more subcultures than with HEV2.1 to get a sufficient quantity of callus for further plant
regeneration and callus cryopreservation. All lines could produce abundant yellow callus. At the beginning of culture, the callus appeared creamy in colour and then gradually became
yellow or dark yellow. Finer 1988 has been classified the cotyledon callus based on the colour as green, yellow, white, brown, and red. Only yellow callus could yield embryogenic
subcultures Finer 1988. Some studies verified that the constitutive promoter 35S CaMV is a weak promoter for transgene expression in young olive somatic embryos. This promoter was
more active in an organized tissue of mature alfalfa somatic embryos than in the less- organised tissues of young embryos Perez-Barranco et al. 2009; Tian et al. 2000.
The number of total embryos was similar for the two promoters 35S CaMV and HEV2.1. A high percentage of abnormal embryos were also recorded for all promoters. The
rate of conversion of embryos into plantlets was lower for 35S CaMV than HEV2.1. Finally, that led to produce a low quantity of plantlets for lines harbouring 35S::HbERF-IXc4
compared to lines harbouring HEV2.1::HbERF-IXc4. Because of this low quantity of plants especially for 35S::HbERF-IXc4 lines, promoter effects could not be analysed during plant
development for the HbERF-IXc4 gene.
Lines of HbERF-IXc5 gene had a good callus proliferation, embryo, and plant regeneration capacity for both promoters. By contrast, promoter effects were observed during
plant development. Histological analysis effectively characterized differences of specific tissue structure between wild-type and transgenic plants overexpressing HbERF-IXc5 under
two different promoters 35S CaMV and HEV2.1. For the 35S CaMV promoter, a larger number of latex cells and thicker xylem were measured in leaves and stem, respectively,
compared to HEV2.1.
For the HEV2.1 promoter, a thicker cambium was measured in plants green stem and taproot compared to 35S CaMV promoter. Activation of cell division and differentiation in
the cambium led to the thickening of stem and roots that related to the increasing of biomass Miyashima et al. 2013. Cambium width of construct HEV2.1::HbERF-IXc5 were threefold
higher than construct 35S::HbERF-IXc5. This data indicated HEV2.1 play a role in promotes
in differentiation of active cells in cambium. HEV2.1 has been reported as being specifically expressed in latex cells according to in situ hybridisation data Montoro et al. 2008. Inner
soft bark of Hevea contains productive and continuous latex vessels differentiated from vascular cambium. Latex vessels are more concentrated in the region near cambium
Prekumari and Panikkar 1992.
Application of abiotic stresses in this study showed that lines harbouring HEV2.1 had a better response to cold stress compared to 35S CaMV it showed by the FvFm value, P.
Index, and SPAD value. Analysis of number of leaflets described that all promoters could raise the adaptability of transgenic plant against senescence due to cold stress.
In salinity stress analysis, average value of FvFm and P. Index value showed 35S CaMV promoter was higher than HEV2.1. The increase of FvFm and P. Index was a
consequence of salt-stressed plants. FvFm described a maximum efficiency of light absorbed by the light-harvesting antennae of PSII. Plants exposed to salinity appear to have a more
efficient PSII. Consequently, the long-term saline environment could produce an adaptation process in the plants.
Line under control of 35S CaMV promoter could have delayed water deficit status revealed by FTSW value 0.2. It was suggested that this promoter had better capability to
avoid water deficit. Water deficit refers to ability of plant to maintain high water status even when water is scarce, for example by growing long roots to reach deep soil moisture, or
reducing water loss by restricting the aperture of the stomata on leaf surfaces. In fact, stomata play a major role in plant adaptation to stress Cominelli and Tonelli 2010.
4.2.2 Effects of candidate genes
