Table 1. Frequency of putative mutants selected on kanamycin or trehalose from 40 mg seeds about 2000 seeds harvested from plants transformed with
clone 816 and 918 of TfTreS.
Selection agent Clone 816 Clone 918 control
Kanamycin 9
14 11
7 16
12 12
10 19
14 7
4 10
12 24
5 10
2 20
Average 12.2
10.8 STDEV
3.7 7.0
Trehalose 3
3 10
2 4
5 4
2 5
1 3
1 2
6 1
2 1
Average 4.2
1.2 STDEV
2.4 1.0
As control, they were also grown on ½ MS medium. It was observed that those resistant to kanamycin were also resistant to trehalose and those resistant to
trehalose were resistant to kanamycin Figure 15. Hence, both selection media are representative each other, either kanamycin or trehalose can be used for
putative mutant selection of the construct pBin1935STfTreS. While the low number of putative mutants obtained from 125 mM trehalose selection might be
due to the concentration used in this selection was too high. Normally a 100 mM trehalose is used in the laboratory. The frequency of putative mutants obtained
from clone no 918 is higher than that obtained from clone no 816. The reason behind of this phenomenon is not known yet. Both clones have 2 point mutations
at the same site but clone 918, has another point mutation. All these mutations did not alter protein sequent, hence, it should not alter the properties of the
enzyme.
clone 918 C clone 816 clone 918 C clone 816
Figure 15. Seedling T2 resistant to kanamycin grown on medium 100 mM trehalosa A vice versa B. Negative control non-transgenic,
positive control to trehalose , clon no 816 and 918 , T3 plant .
4.3.2 The presence of TfTreS within putative mutants The presence of TfTreS putative mutant was determined by PCR analysis
of T2 plants resistant to trehalosa using the same primer used for gene isolation from genomic T. fusca. The PCR product showed that all putative tested
produced a single band similar in size to TfTreS as shown by the positive control Figure 16, in this case, 2 colonies of Agrobacterium bearing TfTreS was used
as template. The negative control water and wild type Arabidopsis did not produce any band. This result suggested that TfTreS has integrated within
genomic Arabidopsis. In this experiment, they are annotated as “TfTreS Expressing Lines” or TfTreS expressing plants.
M W WT +
1 2 3 4 5 6 7 8 9 10
Figure 16. PCR product of putative mutants. A. PCR product of positive and negative control. Molecular marker M, Water temp late W, wilt type WT and positive
control +, B. PCR product of negative control, water 1, wild type 2-3, positive control 4-5 and putative plant samples 6-10
TreS gene
A B
4.3.3 Inheritability of TreS Expressing Lines The resistance of TfTreS expressing lines was observed by growing T2 and
T3 seeds on trehalose 100 mM containing medium Table 2. The result showed that some of the seeds resistant to trehalose T2 seedlings ranging from
36-100 and T3 seedlings ranging from 0-100, indicating that the resistance was inherited to the next generation. The resistance was segregated differently of
every individual line. This percentage might give the chance to obtain the desirable lines for further researches.
Table 2. Frequency of Trehalosa Resistant Seedlings of T2 dan T3
T2 T3
Plant lines
? Resistant
? Sensitive
Plant lines
? Resistant
? Sensitive
wt -
100 T3-3-1
94 32 6 2
lategermin? TreF
67 33 33 16
T3-3-2 70 40
30 17 T2-1
38 20 62 35
T3-3-3 84 26
16 5 T2-3
64 35 36 22
T3-3-4 100 47
0 0 T2-6
79 38 21 10
T3-3-5 92 22
8 2 T2-8
57 74 43 56
T3-3-6 79.5 31
10.5 8 T2-7
100 23 0 0
T3-1-1 79 19
21 5 T2-10
74 20 26 6
T3-3-6 80 28
20 7 T3-3-7
100 33 0 0
T3-3-8 85 40
15 7 T3-6-1
61.5 16 38.5 10
T3-6-2 85 17
15 3 T3-4-4
0 0 100 3
T3-3-8 85 40
15 7 T3-4-4
0 0 100 3
T3-3-8 0 0
100 2 T3-6-4
96 24 4 1
T3-6-0 83 54
17 11 IPB-Sedijani 2006
Utrecht-Schluepmann 2006 personal communication
3.4 Conclusion
