Introduction CHARACTERIZATION OF TFTRES EXPRESSING PLANTS

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. TfTreS expressing lines in response to drought Leaf water retention and leaf recovery test In this experiment, leaves were detached from the plants and measured their weight at zero time followed by certain interval of times after detachment, in this experiment the time course used were: 30’, 60’, 90’, 2h and 21h. Leaf Water Loss was calculated by subtraction of the fresh weight by dry weight that was recorded after 96h incubation at 55 o C. Seven plants each of wild type and 7 TfTreS expressing lines were tested. Leaf recovery was tested by rehydrating excised leaves after 16h exposure to room temperature RT. The number of leaves with fully rehydrated after 1, 2 and 24 h was recorded. In planta TfTreS expressing plants in response to drought First experiment. TfTreS expressing lines were tested to see their capability to face drought. A week old seedlings resistant to 50 mgl kanamycin were transferred to ½ MS medium and kept for a week to eliminate the effect of kanamycin. The seedlings then were transferred to potting mix until they have established growth. Drought treatment was applied by withholding water. Observation was made at day 11 th after treatment and day 2 nd after rehydration. Second experiment. Fresh and dry weight of recover plants need to be measured to clarify the presence of TfTreS on plant in response to drought. For this reason, second experiment was set up using T3 plants. Withholding water for a week was applied to 24 TfTreS expressing plants and 9 plants of Wild type. As a control, 17 TfTreS expressing plants and 5 Wt plants were watered normally non-stressesd as the control. The number of recovered plants at the 4 th h and 24 th h after rehydration were recorded. Fresh weight were measured 2 weeks after rewatering to give a chance of those experienced to drought to establish their growth.

6.3 RESULT AND DISCUSSION

6.3.1 Growing Arabidopsis thaliana in tropical climate Arabidopsis thaliana is not normally grown in tropical area. In order to be able to characterize TfTreS expressing lines, the plants should be adapted first in tropical climate. From few conditions for trial and error that has been carried out for 14 months, it was found a condition that 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