meet of the public concern about bio safety for it is human, environment and biologically friendly, as well as indicated the increase of the capability of
transgenic plants withstand stresses. The presence of validamycine A in combination with trehalose increases selectivity of the selection system to obtain
transgenic plant. Trehalose 100 mM is a tight selection agent for Arabidopsis thaliana
beraing the gene.
VI. CHARACTERIZATION OF TFTRES EXPRESSING PLANTS
6.1 Introduction
Organisms may always be exposed to environmental stresses, such as drought, salt, heat, cold and excessive of light. Oxidative stress can normally be
induced by any environmental stresses which may have diverse damaging effects on plant that affect diverse array of processes. On the other hand, organisms have
always been evolved to survive in such unfavorable conditions. One of the mechanisms of the organisms to withstand stresses is by producing compatible
solutes that functioning as protective agents, e.g. simple sugars. Among the compatible solutes, trehalose is known as the most effective in protecting biological
compounds during the stresses Crowe et al 2001; Albein et al 2003. This experiment was aimed to characterize transgenic plant
Arabidopsis thaliana bearing TfTreS encoding trehalose synthase TRES from Thermobifida fusca, in response to stresses. Trehalose content,
trehalose synthase activity and the capability of the plants withstand drought
were assessed.
6.2. Material and method 6.2.1 Place and time of research
This research was carried out in the laboratory of Plant Molecular Physiology, Utrech University and in the laboratory of Indonesian Center for
Biodiversity and Biotechnology ICBB started at July 2004-December 2006. 6.2.2 Material of research
Seeds of some TfTreS expressing lines T2 generated from clone 916 obtained from previous work Chapter 4 were brought from Utrecht
University. In this experiment T3 generated from the T2 plants were used. 6.2.3. Characterization of TfTreS expressing lines
Growing Arabidopsis thaliana Seeds were sawn on top of sterilized potting mix that has been put
within pot soil : ash husk-manure = 1:1. The pots then were kept outdoor, under shelter to avoid rain and excess light.
Enzymatic trehalose determination About 200 mg of leaves was powdered within liquid nitrogen and
diluted with 900 µ
L 0.1M of phosphate buffer, pH 6.5 then was centrifuged at 10.000 x 1g for 10 minutes. Trehalose content within the
extract was digested using trehalase. A 50 µ
l of plant extract was added to 100
µ l of 0.025 M acetate buffer. A 0.1 Unit of trehalase or water as
control was added and the final volume was adjusted to 200 µ
L using aquabidest. The glucose content was detected using glucose
measurement kit. The mixture then was subjected to 525 nm wavelength on spectrophometer. Trehalose content was calculated as glucose
equivalent after subtraction of the control reaction mixture without trehalase.
Determination of trehalose synthase activity Protein was extracted as described by Avonce et al. 2004. A 200
mg of leaves was powdered within liquid nitrogen and 900 µ
l of phosphate buffer pH 6.5 then stored on freezer before used. A 300 µL protein extract
was mixed with 300 µL of 30 maltose solution in 0.1 M buffer phosphate pH 6.5 and incubated at 25
o
C for 23 h. Trehalose produced was determined Avonce et al. 2004. Trehalose within the mixture was
digested using trehalase with reaction mixture: 50 µ
L sample, 50 µ
L aquabides, 100
µ L of 25 mM acetate buffer and 0.1 Unit of trehalase.
Glucose background within plant extract was determined by the same reaction without addition of trehalase. The mixture was then incubated at
30
o
C for 5 h followed by boiling for 5 minutes to stop the reaction. The mixture was then mixed well and centrifuged for a couple of minutes
Blazqueez et al. 1994. The glucose resulted from the digestion mixture was determined using glucose determination kit. A 25 ?
µ L of digestion
mixture or 0.1 Acetate buffer: aqua bides = 3:1 as a blank reaction was added to 75
µ L deproteinase mix well followed by addition of 900
µ L of
enzyme glycosidase. The pink color shows the glucose presence. The mixture then was subjected to spectrophotometer at 525 nm wavelength.
The glucose content resulted from digestion of trehalose was the glucose content resulted from digestion mixture subtracted by glucose background
within the plant extract.
