MATERIALS AND METHODS
3.1.12 Data recording
3.1.12.3 Nutritional traits
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3.1.12.2.5 Determination of Percent Moisture and Dry Matter Content in Fruit Wight of fresh fruit of each plant was taken. Fruit was pressed so that some moisture was released and it was kept in hot air oven at 80°C for 48 hours. After 48 hours, dry weight of fruit was measured and percentage of Moisture content was measured by following formula;
%Moisture Content =weight of freash fruit−Weight of oven dry fruit
Weight of freash fruit × 100
Dry Matter content was determined by following formula;
% Dry Matter Content= 100-%Moisture content
3.1.12.2.7 Determination of Na+ and K+ Content
Oven-dried (70oC) tomato plants shoot samples were ground in a Wiley Hammer Mill, passed through 40 mesh screens, mixed well and stored in plastic vials. The ground plant samples were digested by Micro-Kjeldahl method (Thomas and Nambisan, 1999). One gram oven-dried tomato plant shoot samples were taken in kjeldahl flasks. About 15 mL of diacidic mixture (HNO3 : 60% HClO4 = 2:1) were taken in a digestion tube and left to stand for 20 minutes and then transferred to digestion block and continued heating at 100oC. The temperature was increased to 365oC gradually to prevent frothing (50oC steps) and left to digest until yellowish color of the solution turned to whitish color. Then the digestion tubes were removed from the heating source and allowed to cool to room temperature. About 40 mL of de- ionized water was carefully added to the digestion tubes and the contents filtered through Whatman no. 40 filter paper into a 100 mL volumetric flask and the volume up to the mark with de-ionized water. The samples were stored at room temperature in labeled containers. Content of Na+ and K+ were determined by Flame Photometer.
38 3.1.12.3.1 Determination of Brix %
Brix percentages were measured by Portable Refractometer (ERMA, Tokyo, Japan) at room temperature. Single fruit was blend and juice was collected to measure Brix percentage. Determination of Brix percentage is shown in plate 3.
3.1.12.3.2 Determination of Vitamin C ( mg/100 g fruit)
Vitamin-C was measured by Oxidation Reduction Titration Method (Tee et al., 1988).
Determination of vitamin C is shown in plate 3.
3.1.12.3.2.1 Dye preparation
260 mg 2, 6-dichloro indophenols with 210 mg sodium bicarbonate were mixed with one litter of distilled water. It was used in burette.
3.1.12.3.2.2 5% oxalic acid preparation
50 mg oxalic acid was mixed with one litter of distilled water and it was used for washing the fruit and for the preparation of fruit juice preparation.
3.1.12.3.2.3 L-ascorbic acid preparation
10 mg of granular L-ascorbic acid was mixed with 100 ml oxalic acid solution. 5 ml was taken and volume was made up to 100 ml. from this solution, 5 ml was taken for titration against 2,6-dichloro indophenol from burette for 3 times and their mean was recorded as the required amount of dye for titrating L-ascorbic acid.
3.1.12.3.2.4 Preparation of tomato solution
Single fruit was weighted and was blend with some drops of oxalic acid solution. It was filtered through whatman filter paper and the juice was collected. Volume was made up to 100 ml with oxalic acid. 5 ml was taken from that solution and titrated against dye solution. The required amount of dye was recorded for titrating tomato solution. The amount of vitamin C was determined by following formula;
Vitamin C= 0.5×𝑑𝑦𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑓𝑜𝑟 𝑡𝑜𝑚𝑎𝑡𝑜 𝑗𝑢𝑖𝑐𝑒×100×100 𝑑𝑦𝑒 𝑟𝑒𝑞𝑢𝑖𝑟𝑒𝑑 𝑓𝑜𝑟 𝐿−𝑎𝑠𝑐𝑜𝑟𝑏𝑖𝑐 𝑎𝑐𝑖𝑑×5×𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑟𝑢𝑖𝑡
39 3.1.12.3.3 Determination of Lycopene content
Absorption determination for lycopene content was estimated following the method of Alda et al. (2009) by using T60 UV-Visible Spectrophotometer. Determination of lycopene content is shown in plate 3. Lycopene in the tomato was extracted using hexane: ethanol: acetone (2:1:1) (v/v) mixture. One gram juice of the each sample were homogenized with 25ml of hexane: ethanol: acetone, which were then placed on the orbital shaker for 30 min., adding 10 ml distilled water and was continued agitation for another two min. The solution was then left to separate into distinct polar and non- polar layers. The absorbance was measured at 472 nm and 502 nm, using hexane as a blank. The lycopene concentration was calculated using its specific extinction coefficient (E 1%, 1cm) of 3450 in hexane at 472 nm and 3150 at 502 nm.
The lycopene concentration was expressed as mg/100 g product.
At λ = 472 nm: lycopene content (mg/100g) = At λ = 502 nm: lycopene content (mg /100g) =
Where, m = the weight of the product (g) E = extinction coefficient
3.1.12.3.4 Determination of Fruit pH
Fruit pH was determined by using REX pH meter model –PHS-3C. Single fruit was blended and then it was filtered through whatman filter paper and juice was collected.
The electrode was inserted into the juice and pH was recorded.
3.1.12.3.5 Determination of Titrable acidity
Firstly 0.1 N NaOH solutions was prepared by taking 4 gm NaOH pellet into 1000 ml distilled water. It was used in burette. Single fruit was weighted and it was blended.
Fruit juice was collected by passing it through whatman filter paper. Volume was made up to 50 ml by adding distilled water. 10 ml solution was taken and 2 drops of Phenolphthalein was added. It was titrated against 0.1 N NaOH and required amount of NaOH was recorded. Titrable acidity was determined by following formula;
%Acidity = 𝑡𝑖𝑡𝑟𝑎𝑡𝑒×𝑁𝑜𝑟𝑚𝑎𝑙𝑖𝑡𝑦 𝑜𝑓 𝑎𝑙𝑘𝑎𝑙𝑖×𝑣𝑜𝑙.𝑚𝑎𝑑𝑒 𝑢𝑝×𝐸𝑞𝑢𝑖𝑣𝑎𝑙𝑒𝑛𝑡 𝑤𝑡.𝑜𝑓 𝑎𝑐𝑖𝑑×100 𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑡𝑎𝑘𝑒𝑛×𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒×1000
20 m E
3.45 . E 3.15
20 m .
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Plate 3. Nutritional analysis in lab A. Determination of Brix% B. Juice of tomato for vitamin C determination C. Titration of tomato juice with dye for vitamin C determination D. Tomato juice with solution on orbital shaker for lycopene content E.
development of layer containing lycopene after removing from orbital shaker F. Spectrophotometer reading for lycopene content determination.
A B
C D
E F
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Plate 4. Determination of pH and titrable acidity content A. preparation of tomato juice for pH determination B. Estimation of fruit pH by using REX pH meter model –PHS-3C C. Preparation of fruit juice for titratble aciity determination D. Titration for determination of % titrable acidity.
A B
D
C
42 3.1.13 Statistical analysis
Collected data were statistically analyzed using Statistix 10 program. Mean for every treatments were calculated and analysis of variance for each character was performed.
Genotype treatment interaction was also performed. Comparison among all treatments was assessed by Least Significant Difference (LSD) test at 5% level of significance (Gomez and Gomez, 1984).
3.2 Experiment 2: Genotype × stress interaction under drought condition in