2. Materials and Methods
The research work was carried out at National Agriculture Research Center (NARC) Islamabad during April 2005-june 2009 in colleboratin with Department of Plant Sciences, Quaid-e-Azam University, Islamabad.
Different genotypes of tomato used during the present study were obtained from Ayub Agricultural Research Institute (AARI), Faisalabad and Horticulture Research institute (HRI), NARC, Islamabad. Following tomato genotypes were used in the experiments.
Avinash, 88572, NTH-242, Lyp No 1, Checo and Pakit
Pant Bahar, Riogrande, Roma, BARI, Gol and Nagina
HRI, NARC, Islamabad
2.1 Experiment No.1 Morphological and Physiological Studies of tomato genotypes under NaCl salinity.
2.1.1 Growing condition
Experiments were conducted in greenhouse under controlled environment condition (Temperature , Humidity and day length).
2.1.2 Raising of plants
Seed of tomato genotypes were sown in trays having equal ratio of sand and soil. After three weeks of germination the plants were transferred into pots (12 inches deep and 37cm in diameter) which contain 1:2 ratios of sand and soil, the plants were allowed to grow further for one week before starting the salinity treatment.
2.1.3 Salinity treatment
Prior to the experiment, the amount of water held by soils was calculated from the difference between dry and wet soil weight. After determination of field capacity, three NaCl salinity treatments (i.e. 3, 6 and 9 dS/m-1) other than control were determined by using the amount of water held by soils (Naik & Widholm, 1993). Salt solutions of different NaCl concentrations were prepared by dissolving NaCl in deionised water. Salinity was induced in the pot containing plant by adding NaCl solution in the soil till the electric conductivity (EC) reached to required concentration. The salinity levels were maintained by regular checking the EC level.
2.1.3 Growth and Biomass
The data of following parameters i.e., plant height (cm), weight of shoot and root (g), and shoot and root water content were recorded at the time of harvest. For fresh shoot weight, plants were washed with distilled water and prior to weighing; excess water was soaked with paper towels. For fresh root weight, after removing soil from roots in water, they were washed with distilled water and kept for an hour between paper towels to remove excess water and then weighed. Shoot and root dry weights were determined after drying them at 70oC for 72 hrs in air dried oven and then shoot and root water content was measured by subtracting dry weight from fresh weight.
All the treatments and control were not fed with any nutrient solutions in order to alleviate nutrient stress arising from salinity.
2.1.4 Determination of ionic contents of tomato after harvesting
22.214.171.124 Material preparation
At flowering stage (?) Days after sowing, the plants were harvested and the plant material was dried in an oven at 70 (+ 2 oC) for 72 h; it was grounded finely, so as to pass through 2mm sieve. For the determination of ionic contents this ground material (0.5 g) was digested with sulphuric acid and hydrogen peroxide according to the method of Wolf (1982).
126.96.36.199 Determination of Na+, K+, Ca2+ and Mg2+ in tomato
The dried ground material (0.5g) was placed in digestion tubes then added approximately 3 ml of concentrated H2SO4 and incubated overnight at room temperature. Then 1ml of H2O2 (35% A.R. grade extra pure) poured through the sides of the digestion tubes and placed in a digestion block and heated up to 350oC until fumes were produced and continued to heat for another 30 minutes. The digestion tubes were removed from the block and cooled for 10 minutes. Then 1 ml of H2O2 was slowly added and placed the tubes back into the digestion block until fumes were produced. The digestion tubes from the block were removed, 1ml of H2O2 was added slowly and the tubes were placed back into the digestion block. The above step was repeated until the material in the test tubes became colourless. The volume of the extract was utilized for determining Na+, Ca, K, and mg nitrogen.
The concentrations of sodium (Na+), potassium (K+), calcium (Ca2+) and magnesium (Mg2+) were analyzed by Atomic absorption photometer (Perkin Elmer). A graded series of standard of Na+, K+, Ca2+ and Mg2+ were used for standard curve and total quantities in sample were calculated.
2.1.5 Determination of Proline content of leaves
Free proline content of shoot and root of tomato plants were estimated according to Bates et al., (1973). The reagents required are detailed in appendices.
Proline content of fresh leaves was determined by the method of Bates et al ;( 1973). 0.5 g of leaves was blended in 3 % aqueous sulfosalicylic acid and the homogenate was filtered through Whatman #2 filter paper. 2ml filtrate, 2ml of acid ninhydrin and 2ml of glacial acetic acid in test tubes were boiled at 100 ˚C for 60 minutes. The reaction was terminated in an ice bath and the mixture was mixed with 4 ml toluene with vigorous stirring for 15-20 sec. The organic phase was collected and the absorbance was measured at 520 nm with a spectrophotometer (Shimadzu). Proline content was calculated from calibration curve and expressed on fresh weight basis.
2.1.6 Endogenous Content of Abscisic acid (µg/g)
The plant material (samples of 1g each) were ground in 80 % methanol at 4C˚ with an antioxidant namely butylated hydroxyl toluene (BHT), and kept for 72 h with one change of the solvent. The extract was centrifuged and the supernatant reduced to its aqueous phase using a rotary film evaporator. The pH of the aqueous phase was adjusted to 2.5-3.0 and partitioned four times with ½ volume of ethyl acetate. The ethyl acetate extract was fully dried using a rotary thin film evaporator (RFE). For determination of bound ABA, the xylem sap was hydrolyzed at pH 11 (1N NaOH) for 1h at 60˚. Under these conditions ABA is liberated from ester linkages.
Quantification of ABA was performed by gas-liquid chromatography with according to Dorffling et al. (1990). The dried sample was redissolved in 1ml methanol (100%) and analyzed using HPLC with a UV detector and a C-18 column. For identifying hormones, samples filtered through 0.45 Millipore filters were injected into the column. Pure ABA was used as standard for identification and quantification of the plant hormones. These hormones were identified on the basis of retention time and peak area of the standards. Methanol, acetic acid and water (30:1:70) were used as mobile phase. (Li et al., 1994). The wave lengths used for detection were 254 nm for ABA.
2.1.7 Statistical analysis
Experiment was designed in completely randomized design (CRD) with factorial arrangements having three replications. Data was analyzed by MSTATC computer package. Multivariate analysis of morphological and physiological character was carried out by computer software Statistical version 7.
2.2 Experiment No 2. RAPD analysis of different tomato genotypes
2.2.1 Extraction of Genomic DNA
Following are the different steps used during the extraction of DNA
1. Fresh micro-prep buffer (DNA Extraction Buffer: 0.1M Tris, 5mM EDTA.Na2 and 0.35M Sorbitol; Nuclei Lysis Buffer: 0.2M Tris, 0.05M EDTA.Na2, 2M NaCl and 2% CTAB; 5% Sarkosyl; 0.1g NaBisulfite) was prepared and kept at room temperature.
2. 50-100mg (4-8 new leaflets, up to 1.5 cm long) of plant material from 1 to3 week-old seedlings was collected and placed it in 1.5 ml Eppendorf tubes.
3. 200µl of micro-prep buffer was added in the Eppendorf tube and tissues were grind with power drill having plastic bit until the mixture become fine. After that another 550µl of micro-prep buffer was added and tubes were shaked manually.
4. Prepared samples were incubated at 65°C water bath for 30 to 60 min with occasional mixing.
5. Eppendorf tube containing samples were filled with chloroform/isoamyl (24:1) and mixed well (by vortexing each tube or sandwiching tubes between two racks and inverting or shaking up and down for 50-100 times)
6. Eppendorf tube containing samples were centrifuged at 10,000 rpm for 5 min at room temperature.
7. Aqueous phase (usually 0.6ml) was poured into new tubes and then cold isopropanol was added at the rate of 2/3 to 1 times to the volume of each tube. After that tube were inverted repeatedly until DNA precipitate.
8. The tubes were spun immediately at 10,000 rpm for 5 min (no more) and isopropanol was pour off. After that DNA pellet was washed with 70% ethanol (500 to 1000µl).
9. Tubes were placed upside down on paper towels for 1 hour to dry DNA pellet by placing on sides in seed dryer for 15 min (longer if necessary).
10. DNA was re-suspended in 50 µl TE at 65°C for 15 min and spun for 10 min at 10,000 rpm in centrifuge. After that it is stored at 4°C.
2.2.2 RAPD PCR Analysis
A modified RAPD method based on Williams et al (1990) was used with a model 9700 thermal cycler (Applied Biosystems, USA). To establish RAPD protocols for Tomato, PCR analysis was performed by changing and checking the concentrations of total genomic DNA from 5~50ng/20µl reaction volume, MgCl2 from 1.5~3.0mM, dNTPs mixture from 100~400µM each, random primer from 0.1~1.0µM and Taq DNA polymerase from 0.2~1.25 units. After standardization of PCR, 20µl reaction mixture containing 1x PCR buffer [10mM Tris HCl (pH 8.3), 50mM KCl], 1.5mM MgCl2, 200µM each deoxynucleotide triphosphate (dNTP), 0.4µM of 10-mer primer (Operon Technologies Inc., Alameda, CA), 1 unit AmpliTaq Gold DNA polymerase and approximately 20ng of template DNA was found optimum for the amplification of rice genomic DNA (Table 2.2). Taq DNA polymerase and reaction buffer were purchased from Applied Biosystems, Japan. DNA amplification was performed in a DNA thermal cycler (Perkin Elmer Cetus, Norwalk, USA). The thermal cycler was programmed to 1 cycle of 5 minutes at 94oC for initial strand separation. This was followed by 45 cycles of 1 minute at 94oC for denaturation, 1 minute at 36oC for annealing and 2 minutes at 72oC for primer extension. Finally, 1 cycle of 7 minutes at 72oC was used for final extension, followed by soaking at 40oC (Table 2.3). The reproducibility of the amplification products was checked twice for each experiment.
2.2.3 Primer Selection
Initially, three cultivars (Roma, Riogrande and Avinash) was used to optimize the RAPD protocols and select the suitable primers which exhibit polymorphisms between the three cultivars. Altogether, thirty arbitrary decamer oligonucleotides, belonging to kit OPA, OPB and OPC from Operon Technologies Inc. (Alameda, California, USA), were tested as single primers to identify the most promising ones for detecting polymorphism. After an initial screen, fifteen primers were ultimately chosen for further use on the basis of their ability to detect the polymorphism and produce the reliable and easily scorable banding patterns in tomto genotypes. Among them, eight primers could not amplify the DNA from some of the genotypes used. Therefore, finally the data of seven primers were used and compiled to examine the genetic diversity and relationship among twelve tomato genotypes.
2.2.4 Electrophoresis of Amplified Products
After amplification, 3µl of gel loading dye buffer (0.02% Bromophenol blue, 0.02% xylene cyanol FF, 50% glycerol and 1% SDS) were added directly to the reaction tubes and spun for few seconds in a micro centrifuge after mixing with the entire reaction mixtures. Aliquots of 15µl of amplification products plus loading dye were then loaded in 1.5% agarose gels for electrophoresis in 1 x TBE (10mM Tris-Borate, 1mM EDTA) buffer and run at 100V for 40 minutes to separate the amplified products. The 1Kb DNA ladder was used as a molecular weight marker. After electrophoresis, the gels were photographed under UV light using black and white film # 667 (Polaroid, Cambridge, Mass., USA).
2.2.5 Data Analysis
Photographs from ethidium bromide stained agarose gels were used to score the data for RAPD analysis. Each DNA fragment amplified by a given primer was treated as a unit character and the RAPD fragments were scored as present (1) or absent (0) for each of the primer-genotype combinations. Bands were scored from the top of the gel (band number 1) to the bottom. The left lane of the gel was considered as lane-1. Since DNA samples consisted of a bulk sample of DNA extracted from 4 to 8 young leaves, a low intensity for any particular fragment may be explained by the lesser representation of that specific sequence in the bulk sample of DNA. Therefore, the intensity of the bands was not taken into account and the fragments with the identical mobility were considered to be the identical fragments. Only major bands were scored and faint bands were not considered. The molecular size of the amplification products was calculated from a standard curve based on the known size of DNA fragments of a 1Kb molecular weight marker. The presence and absence of the bands was scored in a binary data matrix. Pair-wise comparisons of the cultivars based on the presence or absence of unique and shared amplification products were used to generate similarity coefficients. Estimates of genetic similarity (F) were calculated between all pairs of the cultivars by the Dice algorithm. The Dice algorithm is identical to that of Nei and Li (1979) as follows:
Similarity (F) = 2Nab/ (Na + Nb)
Where Na = the number of scored fragments of individual ‘a',
Nb = the number of scored fragments detected in individual ‘b' and
Nab = the number of shared fragments between individuals ‘a' and ‘b'.
The resulting similarity coefficients were used to evaluate the relationships among tomato genotypes with a cluster analysis using an un-weighted pair-group method with arithmetic averages (UPGMA) and then plotted in the form of a dendrogram. All computations were carried out using the computer program NTSYS, version 2.1 (Applied Biostatistics Inc., USA).