RESULT AND DISCUSSION CHARACTERIZATION OF TFTRES EXPRESSING PLANTS
Arabidopsis can be grown on soil mixture of soil and husk ash that has
been supplemented with manure at ratio of 1:1. The mixture was autoclaved well before use. Vigorous growth Figure 21 was obtained
when the plants were kept out door, under shelter, with enough light and avoided from rain and excessive light. It is most suitable grown in dry
season. Hence, further characterization of TfTreS expressing lines can be done in tropical climate.
Figure 21. Grown Arabidopsis in tropical area. Grown for leaves harvest A and grown
for seed production B.
In order to provide seeds for characterization of TfTreS expressing lines, plants were grown in the condition as mentioned before. When seeds of
the plants were entirely dry, they were harvested and selected on trehalose containing medium Figure 22. The most obvious character of
seedling resistance to trehalose is the capability of the seedlings to develop roots. As mentioned in Chapter 2, the presence of high trehalose level induces
starch accumulation in chloroplast hence, sugar is not mobilized to sink Wingler et al
. 2000, including roots consequently stunted growth is resulted. It was observed that none of Wt was able to develop roots on 100 mM trehalose containing
A B
Figure 22. T3 plants selected on trehalose. A = Wt plants and TfTreS expressing
plants grown on ½ MS medium as control if the seed is still viable. B = Wt plants and TfTreS expressing plants grown on 100 mM trehalose. Root
length is clearly shown. Wt and the gene inverted orientation seeds did not show any growth.
medium, while in ½ MS medium, roots were develop normally as well as their
shoots Figure 22. This might indicate that the gene is expressed to the next generation T3 of transgenic plants. Among the three lines tested, it was shown
that all lines have similar ratio between the resistant and the sensitive ones 1:1.
wt TfTreS
Inverted orientation
B
TfTreS
A
Wt Inverted clone
Wt
TfTreS
From the above mentioned, it can be suggested that Arabidopsis thaliana can be grown in tropical climate with certain care.
Table 4: Number of T3 seedlings resistant to 100 mM trehalose Lines
Resistant Sensitive
Total Number Wt
95 95
020 85
85 TfTreS
101 16
15 31
TfTreS 102
29 31
60 TfTreS
103 30
25 55
6.3.2 Trehalose content and enzyme activity of Wt and TfTreS expressing Plants, 101, 102 and 103 annotate the clone number
Trehalose level and enzyme activity are presented in Figure 23. Trehalose content among 12 wild type plans ranged from 0 – 85.31 µmol gr
-1
leaf fresh weights FW, with 3 plants were at undetectable level, and 9 plants 75
were contained trehalose at various level. Among 23 TfTreS lines tested, the content ranged from 0 - 180 µmol gr-
1
leaf FW, with 11 plants were at undetectable level and 13 plants 54 contained trehalose at various level.
Box Plot Analysis was used to screen the out layers. It was revealed that the highest 4 out of 23 data trehalose content of TfTreS plants are out layers. The
rest of the data were then subjected to T-Test statistical analysis which revealed that there was no significant different in term of tehalose content from the Wt
plants at P = 0.05. This is only significantly different at P = 0.18 Appendix 6. While the out layers data were highly significantly different at P = 0.005 in
comparison to the content of Wt plants or at P = 0.007 in comparison to the content of the remaining TfTreS plants. This suggests that there is a tendency
that TfTreS plants contain more trehalose than Wt P = 18, and there is a possibility 17.4 to obtain TfTreS plants contained high trehalose level.
These out layers of trehalose level ranged from 68 – 180 µ
M 25 – 65 mg g
-1
FW. These values are much higher than trehalose content of transgenic plants that have been reported such as Table 5 adopted from Zhang et al 2005.
In term of ratio between the content of Wt and TfTreS plants, Garg et al. 2002 reported that trehalose level increased 10-20 folds in transgenic rice bearing
fused gene encoding for trehalose synthases OtsA-OtsB from E. coli. Zhang et
al 2006 found that transgenic sugarcane bearing TSase from Grifola frondosa
contained trehalose level ranged from 9-13 mg gr
-1
FW, with no detectable level in Wt plants that mean that it has very high ration 8. While in this work it ratio
was 0-8.5 folds from the Wt average, that might be considered that the increase is still lower than these two examples, but it is about similar or higher to others
shown on Table 5.
Figure 23. Trehalose concentration and enzyme activity of TfTreS expressing
lines versus control plants. Scatered data of trehalose content A; and sactered data of enzyme activity B.
Enzyme assay showed that among 12 plants of Wt tested the activity ranged from 0-10.9 µmol h
-1
gr-
1
FW, 5 plants did not show enzyme activity and 7 plants 57.5 showed activities 0.12 – 10.9 µmol h
-1
gr-
1
FW. Among 22 plants of TfTreS line tested, the enzyme activity the plants ranged from 0 – 65 µmol h
-1
gr
-1
FW, 6 were undetected and 16 plants 73 showed the activity spanned from 0.23 – 65 µmol h
-1
gr
-1
FW. T-Test Analysis reveals that there was highly Scatered data of trehalose content of TfTreS
expressing lines
50 100
150 200
5 10
15 20
25 30
Member of the group mmol g-1
Wt TreS
A
Trehalose synthase activity
0.00 10.00
20.00 30.00
40.00 50.00
60.00
5 10
15 20
25 Member of the group
mol h
-1
g-1 Wt
TreS
B
significant different P = 0.003 between enzyme activity of transgenic plants from Wt plants. Therefore, it can be mentioned that TfTreS plants have a higher
enzyme activity than the Wt plants. This activity indicates that the gene is expressed and the TRES is functionally active.
Table 5: Trehalose level of some reported transgenic plants
Trehalose contents in transgenic plants
Transgenic plants Transgene
Trehalose production mggFW
Reference Arabidopsis
THermobifida fusca TreS 0-65
Present study sugarcane
TSase grifola frondosa 9-13 mggr FW
Zhang et al 2006 Tobacco
TSase 2.156-2.556
Zhang et al 2005 Rice
E.coli otsAotsB fusion 0.30-1.076
Jang et al 2003 Tobacco
E.coli otsA 0.0147
Dai et al 2001 Tobacco
E.coli otsA and otsB
0.038-0.04 Pilon Smith et al 1998
Tobacco E.coli otsA
and otsB 0.005-0.07
Godijn et al 1997 Tobacco
E.coli otsA 0.02-0.11
Godijn et al 1997 Potato
E.coli otsA and otsB
0.003-0.02 Godijn et al 1997
Tobacco Yeast TPS1
0.16-0.64 Holmstrom et al 1996
Tobacco Yeast TPS1
0.17 Romero et al 1997
Adopted from Zhang et al 2005. Present study, trehalose measuremet was done by using enzymatic measurement.
6.3.3 TfTreS expressing lines in response to drought Leaf Water Retention
There are several ways to indicate water status under water-stress, such as leaf relative water content LRWC Blum and Johnson 1993; Teulat et al.
1997; Dhanda and Sethi 1998, relative water loss RWL Golestani Araghi and Assad 1998, excised-leaves water retention ELWR Sharma and Sethi 1998;
Kumar and Sharma 2007. Drought resistant wheat cultivars loss water more slowly than those less resistant cultivars Bayles et al 1937; Shandu 1958.
Better-excised leaf water retention had been shown on hardy wheat cultivars than on less hardy cultivars and it was well correlated with the order of hardiness observed in
drought and heat test Salim et al 1969; Clark et al 1982. The capability of excised leaf water retention is heritable Clark and McCaig 1982; Clark and Twenley-Smith
1986; Permachandra and Shimada 1988 and this feature is governed by dominant gene Dedio 1975. Excised leaf water retention and plant water retention capability
differentiate drought tolerance Sharma and Sethi 1998. Kumar and Sharma 2007
study the genetic effect of excised leaf water retention and relative water content in order to obtain the improved trait of bead wheat.
Table 6: Water loss at pointed time of Total Water Loss
Lines 30 W 30T 60W 60T 90W 90T 2hW 2hT 21W
21T 45hW 45hT
1 11.04 10.04 4.18 2.95 2.99 3.35 3.58 2.76 76.72 66.14 1.49 14.76
2 11.61 9.21 9.74 5.95 4.87 4.99 5.24 4.41 68.16 73.70 0.37 1.73
3 10.85 3.10 5.66 3.62 4.25 2.33 3.77 2.33 75.47 77.52 0.00 11.11
4 9.34
7.79 4.52 3.66 3.92 3.02 2.71 2.86 71.08 63.75 8.43 18.92 5
12.60 6.24 6.17 9.83 4.56 5.28 4.29 5.04 69.71 72.66 2.68 0.96 6
9.70 8.25 7.27 7.31 5.30 5.90 4.85 5.90 72.88 71.46 0.00 1.18
7 10.57 6.99 6.34 3.16 5.71 2.66 5.50 3.57 71.88 22.63 0.00 22.63
Ave 10.82 7.37 6.27 5.21 4.51 3.93 4.28 3.84 72.27 64.86 1.85 10.18
Std 1.11
2.28 1.86 2.60 0.91 1.42 0.99 12.13 3.04 16.57 3.07 9.04
Note: T = Transgenic, W = Wild type, ‘ = minutes and h = hours
Figure 24. Excised leaf water loss average at pointed time. Water loss after a given time until achieved dry weight is considered as water retention by the
given time. In this experiment ELWR was used, the appearance of leaves 21h after
detached is presented in Figure 25. The figure shows that almost all wild type leaves were shrunk, while some of TfTreS expressing lines showed less shrinkage than the
Wt leaves. Table 6 showed that the rate of water lost is declining along with the time of incubation. All along the stage of incubation, the Wt plants released slightly more
water than the TfTreS expressing plants. The TfTreS expressing lines, however, showed varying in water lost rate among individual plant, particularly the water lost
after 21h before they were entirely dry. The amount of leaf water loss after 21h up to dry weight was achieved is considered as leaf water retention after 21h. T he average
of leaf water retention of wild typeplants was 1.1 of total water loss. Four 57 out of 7 leaves of transgenic lines TfTreS102-2, TfTreS102-6, TfTreS102-8, TfTreS102-
10 20
30 40
50 60
70 80
30 60
90 2h
21h 45h
Time of Total water loss
Wt TreS
13 were 14.8, 11.1, 19.0 and 23 respectively table 6. These water contents were in agreement with the leaf appearance, where which those that
contained more water showed less shrinkage than the rest after 21h detachment.
Figure 25. Drought tolerance test using leaf water retention. Leaves performance detached at zero time A and at 21h after keeping at RT B. Leaf water retention
scattered data after 21h at RT C and average of leaf water retention of Wt and TfTreS
leaves D. Almost all of the wild type plants did not retain water after 21h detached, while 4 out of 7 TfTreS expressing leaves performed better and
retained water between 11-23 of the total water content TWC.
A B
C
D C
Wt TfTreS
Leaf water retention after 21h detached TWC
5 10
15 20
25
2 4
6 8
Member of the group Wt
TreS
1.8 ± 7.8
10.2 ± 7.9
2 4
6 8
10 12
TreS Wt
Leaft water retention of TWC
One-Way Anova Analysis suggested that the water lost during the first 30 minutes was significantly higher in Wt leaves than that of TfTreS leaves at P =
0.004 Appendix 8A. Water released after 21h until dry weight was achieved, was significantly higher in transgenic plants, with P = 0.04. This shown that water
retained at 21h incubation was higher in transgenic plant than in Wt. While the 2
nd
interval up to 21h, they were not significantly different statistically, although higher values were shown by TfTreS leaves Table 6. Similar trend was
observed on Total Water Lost TWL where which TfTreS plants showed total water lost that was slightly less than Wt leaves Table 7, however, it is
statistically significant different at P = 0.03 Appendix 8G.
The leaf dry weight of TfTreS plants showed higher than that of Wt plants, and statistically, there was significant different in comparison to that
of wild type at P = 0.03 Appendix 12. This result is in agreement with Garg et al 2002; Jang et al 2003, Zhang et al 2005 and Zhang et al 2006
that reported their transgenic plants show higher yield than the Wt plants.
Table 7: Detached Leaf Total Water Loss and Leaf Dry Weight
No. Total Water Loss Dry Weight
Wt TreS
Wt TreS
1 91.03
91.04 3.3
5 2
92.39 90.61
2.2 5.4
3 91.71
91.82 2.2
4.1 4
92.56 90.26
3 5.6
5 91.79
90.02 3
4.5 6
91.31 91.20
5.9 4.7
Average 91.80