IV. INTRODUCING GENE ENCODING TREHALOSE SYNTHASE INTO Arabidopsis thaliana
4.1 Introduction
In the line of crop improvement, plasmid pBin1935STfTreS containing gene encoding for trehalose synthase from T. fusca has been constructed, and
available within Agrobacterium for plant transformation Part 3. This research is aimed to introduce the gene to Arabidopsis thaliana in order to obtained
transgenic plant model via Agrobacterium mediated transformation. When the transgenic material is obtained, they will be characterized to see the influence of
the gene to plant responses to stresses. Arabidopsis was used as plant model in this experiment for some reasons: its life cycle is short 2 months, whole genome
sequence is available and mutant collections are also available. The information about Arabidopsis can be obtained from The Arabidopsis Information Resources
TAIR. The main goal of this experiment is to obtained transgenic plants
expressing gene encoding for trehalose synthase TRES. Some queries, however, are expected to be answered: whether the plants withstand on high
trehalose level, whether those resistant to trehalose are also resistant to kanamycin, whether the resistance to trehalose is inheritable to the next
generations.
4.2 Materials and Methods
4.2.1 Place and Time of Research This research was done from March-July 2005 at The Department of
Plant Molecular Physiology, Utrecht University.
4.2.2 Materials Arabidopsis thaliana
wild type, Columbia 0 Col 0, the plant expression cassette pBin1935STfTreS, and Agrobacterium LBA4404 were provided by the
Department of Plant Molecular Physiology, Utrecht University. The gene source bacteria Thermobifida fusca was purchased from DSMZ Germany. The gene
has been constructed within plasmid pBIN1935STfTreS previous work, Chapter 3.
4.2.3 Preparation Agrobacterium solution for floral dipping Starter of Agrobacterium was prepared by inoculating one colony from
stock culture into a 15 ml Luria Bertani Broth LB medium within 50 ml flask, with addition of 25 mgl riphampicin and 50 mgL kanamycin. The starter culture was
kept at 30
o
C for overnight; and was used all to inoculate 500 ml of the same medium within 2L jar, incubated at 30
C until reach optical density OD of about 4. The culture was centrifuged and re-suspended in 20 sucrose to obtain OD of
0.8, and silwet L-77 of 0.005-0.05 was added. The bacterial suspension was immediately prepared before use.
4.2.4 Floral dipping The flowering plant was prepared previously to obtain the ideal stage of
inflorescent for transformation. Each individual plant was grown in a small bucket within a little pot; hence, it can be removed from the pot easily without disturbing
the roots. The right stage of the flower was determined by the presence of 1-2 opening flowers within an inflorescent. All aerial part of plants was dipped into
the suspension OD 0.8 within 20 sucrose for about 10 seconds, and then was put back into the pot. When all plants has been dipped, they were covered with
plastic wrap and kept in dark for 24h before growing normally in growth chamber until all seeds were entirely dry.
4.2.5 Selection of transgenic lines The seeds were sterilized using chlorine gas. About 40 mg seeds about
2000 seeds were put in micro tube, then all tubes were kept in a properly closed container. A 100 ml of calcium hypochlorite within 200 ml beaker glass was put
into the container. A 5 ml of hydrogen chlorite 37 HCl was added to the beaker glass and the lid of the container was closed properly as quickly as
possible. After 4h incubation, the sterilized seeds were kept in laminar flow to remove the remaining gas before sawing on square plate contained of ½ MS
medium pH 5.8 supplemented by 50 mgL kanamycin or 125 mM trehalose. For this selection, 10 replicates were used for each treatment. After sawing the
seeds, the plates were kept in dark room at 4
o
C for 2 days, followed by light exposure in the growth chamber. Observation was done at 1-2 week after
exposure to light and the putatively transformed seedling was indicated by the
normal growth within selective medium.
.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
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