.4.2.6 Inheritability of TfTreS Expressing Lines Inheriability of TfTreS expressing lines was assessed by growing T2 and
T3 seeds on ½ MS supplemented with 125 mM trehalose. Number of seedling with normal growth was recorded one week after light exposure. Normal growth
means growth of seedling with long roots and expanded leaves. 4.2.7 PCR analysis of TfTreS expressing lines
Genomic DNA of plant material was isolated using method of Cheung et al
1993 with modification. About 50 mg of plant material was ground in 100 µ
L of extraction buffer NaCl 2M; Tris-HCl 200 mM, pH 8; EDTA 70 mM and 20 mM
Na
2
S
2
O
5
until smooth. All material was transferred into a clean micro tube then was centrifuged at 13.000 x 1g for 10 minutes. The supernatant was transferred
into a clean tube followed by adding of 2 volumes absolute ethanol, and incubated in room temperature RT. Centrifugation was done at 13.000 x 1g for
10 minutes, the pellet was washed using 70 ethanol and centrifuge for a minute. Supernatant was discharged and the pellet was air dried before adding 50
µ L
1XTE and stored at frozen before used. DNA obtained from above protocol was used as template for PCR to
amplify TfTreS inserted within plant genome. PCR mixture was made at final volume of 20
µ L within a 200
µ L PCR tube. PCR was run with program as follow:
94
o
C, 5’ once followed by 40 cycles of 94
o
C, 30”; 53
o
C, 30” and 72
o
C, 2’ then machine was allowed to complete elongation process 72
o
C for 10 minute followed by 4
o
C forever until the tube was remove from the machine.
4.3 Result and discussion
4.3.1 Frequency of putative mutants It was observed that on kanamycin containing medium, all the wild
seedlings were very small and leaves were bleached. While from the putative mutants plates, some of the seedlings develop normal roots and shoots with
green leaves. On trehalose containing medium, they wild type seedling were very stunted, no root growth. They were very small and showed dark green in color as
an indication of stress. Some of the putative transformed seedlings showed the normal shoot growth with long roots Figure 14.
The frequency of the putative mutant obtained from both selections medium, kanamycin or trehalose is presented in Table 1. The Table showed that
the frequency of putative transformed seedling was 12 0.6 and 11 5.5 plants for clone 816 and 918 respectively selected on kanamycin medium, and
was 4.2 0.21 and 1.2 0.06 plants of clone 816 and 918 respectively selected on 125 mM trehalose, from about 40 mg seeds about 2000 seeds per
plate. While from wild type seeds, it was no seedling 0 found to be resistant neither to kanamycin nor to trehalose. These results indicated that the selection
system worked as expected and showed that TfTreS and the gene that encode enzyme which alters kanamycin have been actively expressed and functioning in
the putative mutants that resistant to trehalose or kanamycin. The frequence of putative mutants between the clones was not significantly different when selected
on kanamycin, while that selelcted on 125 mM trehalose, clone 916 was significantly produced more putative mutants than clone 816 appendix 13.
Figure 14. One-week-old Putative TfTreS transformed seedlings selected on 50 mgL kanamycin A, on 125 mM trehalose B and a 10 days old seedling
on 125 mM trehalose C. Individual plant resistant to kanamycin , individual plant resistant to trehalose and otherwise for the sensitive
plant .
C A
B
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
