MATERIALS & METHODS
MATERIALS AND METHODS The effect of chemical mutagens Methyl Methane Sulphonate (MMS) and Ethyl Methane Sulphonate (EMS) and heavy metals Cadmium nitrate [Cd(NO 3 ) 2 ] and Lead nitrate [Pb(NO 3 ) 2 ] has been studied on the cyto-morphological characters of Trigonella foenum graecum L. Certified, true breeding, healthy seeds of Trigonella were procured from Genetics Division, Indian Agricultural Research Institute (IARI), New Delhi. 1. MUTAGENS USED The seeds of Trigonella were treated in 0.10, 0.25, 0.50, 0.75 and 1.0% MMS and EMS and 20, 40, 60, 80 and 100ppm of Cd(NO 3 ) 2 and Pb(NO 3 ) 2 for 24 hours. These doses were standardized on the basis of LD 50 results. 2. MATERIALS REQUIRED Phosphate buffer solution, distilled water, seeds, mutagens [MMS, EMS, Cd(NO 3 ) 2 and Pb(NO 3 ) 2 ]. 3. METHOD OF CHEMICAL TREATMENT: (A) PREPARATION OF MUTAGENIC STOCK SOLUTION: The stock solution (1.0%) of MMS and EMS were prepared by adding 1.0 ml of MMS and EMS in 99 ml phosphate buffer solution maintained at ph-7. The stock solution of Cd(NO 3 ) 2 and Pb(NO 3 ) 2 were prepared by dissolving 10mg of each mutagen in 100ml of DW. The stock solutions were further diluted to required concentrations using the formula: S 1 V 1 =S 2 V 2 Where, S 1 = V 1 = S 2 = Strength of stock solution. Volume of stock solution. Strength of desired solution.
Materials and Methods 31 V 2 = Volume of desired solution. (B) PRE-SOAKING AND SOWING OF SEEDS The sets of 300 healthy seeds of Trigonella were pre-soaked in distilled water at room temperature (25±1 o C) for 12 hours. Thereafter, the seeds were subjected to treatments in each concentration of mutagens for 24 hours. One set of seeds was kept untreated and soaked in distilled water to act as control for comparison. After the treatment the seeds were washed in running tap water to remove residual mutagens adhered to the seed coat and then the treated as well as untreated seeds were sown in three replicated (100 seeds in each replicate) in the field designed in RBD (Randomized Block Design) of 2x2m plot area at a distance of 25cm apart. Some selected seedlings were sown in earthen pots also. The cyto-morphological observations were recorded from treated as well as control populations. 4. EVALUATION IN M 1 GENERATION A detailed study on the effect of different concentrations of mutagens on the following parameters was taken into consideration. (A) BIOLOGICAL DAMAGE (i) SEED GERMINATION (%) The seed germination was recorded right from the emerging of the first shoot in each treatment as well as control on every alternate day, till the maximum germination was attained and calculated on the basis of following formula: Germination percentage (%) = Number of seeds germinated Total number of seeds sown 100 (ii) INHIBITION IN SEED GERMINATION (%) Following formula was used to calculate the inhibition in germination:
Materials and Methods 32 Inhibition percentage (%) = Germination in control Germination in treated seedlings Germination in control 100 (iii) PLANT SURVIVAL (%) The survival of plants was counted at the time of maturity. The survival percentage and percent lethality was calculated by using the following formulae: Survival percentage (%) = Total number of plants survived at maturity Total number of seeds germinated 100 Lethality (%) = Control-Treated Control 100 (iv) POLLEN FERTILITY (%) Fresh and young flowers from 25-30 randomly selected plants were taken from each treatment and the control. The pollens from mature and undehisced anthers were dusted on slide containing a drop of 1% propionocarmine. Pollen grains which took stain and had a regular outline were considered as fertile, while those without stain, irregular shape and size were considered as sterile. Percentage pollen fertility and percent reduction (sterility) were estimated by following formulae: Pollen Fertility (%) = Number of fertile pollen grains Total number of pollen grains 100 Percent reduction (sterility) = Control-Treated Control 100 (B) MORPHOLOGICAL AND YIELD TRAITS The data of the following morphological and yield traits were taken in present study. (i) PLANT HEIGHT (cm) Plant height was measured at maturity in centimeters from the base up to the apex of plant.
Materials and Methods 33 (ii) NUMBER OF BRANCHES PER PLANT Branches were counted emerging out of main stem and branches of the control and treated plants. (iii) NUMBER OF PODS PER PLANT Numbers of pods per plant were counted at maturity and noted as the number of pods born on the whole plant. (iv) LENGTH PER POD Length per pods was recorded in treated plants as well as in control. (v) NUMBER OF SEEDS PER POD: Number of seeds per pod was estimated in control as well as in treated populations. (vi) 1000 SEEDS WEIGHT (g) It was the weight of a random sample of 1000 seeds from the harvested plants to get the test weight. (vii) YIELD PER PLANT (g) The yield per plant was the weight of total number of seeds harvested per plant. (viii) FREQUENCY OF MORPHOLOGICAL VARIATIONS The morphological variations were scored on the basis of characters in control plants and their deviations in treated populations. Variation frequency was calculated using following formula: Variation/Mutation frequency (%) = No. of plants with varied/mutant characters Total number of plants survived 100 (C) CYTOLOGICAL STUDIES (i) FIXATION OF FLOWER BUDS Cytological studies were carried out on pollen mother cells by fixing young flower buds from each treatment as well as control. For meiotic studies the flower buds of
Materials and Methods 34 appropriate size were collected separately from each treatment between 8:30 to 11:00 a.m. and fixed in Carnoy s fluid (6 part alcohol : 3 part chloroform : 1 part glacial acetic acid) for 1 hour or until complete dissolution of chlorophyll and then transferred to propionic acid saturated with ferric acetate for 12 hours. The material was finally washed and preserved in 70% alcohol for further study. (ii) STAINING AND SLIDE PREPARATION Anthers were squashed in 0.5% propionocarmine stain (Swaminathan et al., 1954), dehydrated in normal butyl alcohol series (Bhaduri and Ghosh, 1954), mounted on Canada balsam and dried in incubator at 45 C for 3-5 days. (iii) MEIOTIC OBSERVATIONS: Cytological observations and photomicrographs were taken from temporary as well as permanent slides under light microscope with the aid of Olympus photomicrographic unit. Meiotic abnormalities were scored on the basis of structure and behavior of chromosomes. Any deviation from normal configuration was considered as abnormality like reduction in chiasma frequency, increase in the frequency of univalents, multivalents, stickiness, laggards, bridges fragments, micronuclei etc. 5. EVALUATION IN M 2 GENERATION Seeds from each M 1 plant were harvested separately in treated as well as control plants. For raising M 2 generation, from each plant were selected in each treatment as well as control then sown in three replicate following complete randomized block design field. A. OBSERATION RECORDED IN M 2 GENERATION Observations were recorded on 30 plants of each progeny from treated as well as control populations. The progenies segregating for macro mutations were not used
Materials and Methods 35 and all morphological, yields as well as cytological characters were thoroughly studied as in M 1 generation. (i) MUTAGENIC EFFECTIVENESS & EFFICIENCY Mutagenic effectiveness is a measure of the frequency of mutations induced by unit dose of a mutagen (time x concentration), while mutagenic efficiency represents the proportion of mutations in relation to biological damage. The formulae suggested by Konzak et al. (1965) were used to evaluate mutagenic effectiveness and efficiency of the mutagens. (1) Mutagenic effectiveness = Percentage of mutated plant progenies (Mp) Concentrations of mutagen duration of treatments (hrs) (2) Mutagenic efficiency = Percentage of mutated plant progenies (Mp) Biological damage BIOLOGICAL DAMAGE Two different criteria were used for calculating biological damage: (a) INJURY (I): Inhibition in seed germination. (b) STERILITY (S): Percentage reduction in Pollen fertility or sterility. (ii) VIABLE MUTATIONS The mutagen treated as well as control plant of M 2 generation were carefully screened for scoring viable mutations throughout the life span of the plant in the field. Viable mutations affecting different morphological characters either in general architecture of the plants, leaf size and shape, plant height, branches per plant, number of pods per plant, length per pod, size and colour of seeds in M 2 generation were recorded. The spectrum and frequency of viable mutations in M 2 generation was calculated.
Materials and Methods 36 6. STUDIES IN M 3 GENERATION From each treatment the mutants in M 2 progeny were selected which showed significant deviation in mean values in the positive/negative direction, particularly for the yield component under study in M 2 generation. M 3 generation was raised from bulked M 2 seeds for each treatment along with their respective control. The marked elite mutants screened and selected from the M 2 generation were sown in individual rows separately as well as in earthen pots to study their breeding behavior and mutant nature. A Random sample of each selected progeny of treatments as well as control was sown as M 2 progeny rows to raise the M 3 generation. Observations were recorded on all eight quantitative traits and cytological characters as in M 1 and M 2 generation were also continued in M 3 generation. A. PROTEIN ESTIMATION Protein estimation of selected mutant s seeds was carried out in M 3 generation (Lowery et al., 1951). REAGENTS USED (a) REAGENT A 2% of sodium carbonate in 0.1N NaOH (1:1) ratio (b) REAGENT B 0.5% CuSO 4 in 1% sodium tartarate (1:1) ratio (c) REAGENT C Alkaline CuSO 4 in solution obtained by mixing 50ml of reagent A with 1ml of reagent B (d) REAGENT D Carbonate copper sulphate same as C except for omission of NaOH
Materials and Methods 37 (e) REAGENT E Folin s phenol reagent Folin s phenol reagent was made after diluting it with DDW in the ratio of 1:2 (f) REAGENT F 1 N NaOH B. SEED PROTEIN ESTIMATION METHOD Seed protein content of the mutants isolated in M 3 generation was estimated following the method of Lowery et al., (1951). For extraction of soluble and insoluble protein, seed powder was kept in an oven at 80 0 C overnight. Then it was cooled and 50 mg sample was transferred to a mortar and ground by a pestle with 5ml of DDW. The ground material was collected in a centrifuge tube. The tube was centrifuged at 4000 rpm. The supernatant was collected in a 25 ml volumetric flask using 2-3 washings with DDW. Volume was made up to the mark with DW and kept for estimation of soluble protein. The residue was used for the estimation of insoluble protein. (i) INSOLUBLE PROTEIN ESTIMATION To the residue, 5ml of 5% trichloroacetic acid (TCA) was added. The solution was shaken thoroughly and allowed to stand at room temperature for 30 minutes. It was then centrifuged at 4000 rpm for 10 minutes and supernatant was discarded. 5 ml of 1 N sodium hydroxide was added to the residue and mixed well and kept for 30 minutes. The residue was allowed to stand in a water bath at 80 0 C for 30 minutes. Then it was cooled and centrifuged at 4000 rpm. The supernatant together with three washings with 1N sodium hydroxide was collected in a 25 ml volumetric flask. The volume was made up to the mark with 1N sodium hydroxide. For the estimation of
Materials and Methods 38 seed insoluble protein, 1 ml of sodium hydroxide extract was transferred to a 10 ml test tube and 5 ml of reagent D was added and allowed to stand for 10 minutes. 0.5 ml of reagent E was added rapidly with immediate mixing. After 30 minutes, the solution turned blue. The optical density (O.D) of the sodium was read at 660 nm on Spectronic-20 D, Milton Roy, USA, Colorimeter. A blank was run with each sample. The optical density of this solution was compared with standard curve, used for soluble protein. (ii) SOLUBLE PROTEIN ESTIMATION For the estimation of soluble protein, 1ml of water extract from supernatant was transferred to a 10 ml test tube. 5ml of reagent C was added. The solution was mixed and allowed to stand for 10 minutes at room temperature. 0.5 ml of reagent E was added rapidly with immediate mixing. After 30 minutes, the blue coloured solution was transferred to a colorimetric tube and its intensity was measured by reading its optical density at 660nm, using a Spectronic-20D colorimeter. A blank was run simultaneously. The soluble protein content was estimated by comparing the optical density of each sample with a calibration curve plotted by taking known dilutions of a standard solution of egg albumin. (iii) STANDARD FOR PROTEIN 40 mg of egg albumin was taken in a 100 ml volumetric flask, to which 1-2 ml of 0.1N NaOH was added. The flask was rotated carefully placed on a water bath for a short period (5-10 minutes) for heating. After the albumin became solubilized, the volume of the flask was made upto the mark by double distilled water. From this solution a range of 10 volumes i.e. 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 and 1.0 ml was pipetted out to ten different test tubes. The solution in each test tube was diluted
Materials and Methods 39 to 1 ml by adding 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 and 0.0 ml of double distilled water, respectively. In each test tube 5ml of reagent C was mixed and allowed to stand for 10 minutes at room temperature. 0.5 ml of reagent E was then added rapidly with immediate mixing. The optical density of the solution was read at 660nm using a Spectronic- 20D colorimeter. A blank was also run simultaneously and a calibration curve was plotted. The soluble protein content was estimated by comparing the optical density of each sample with a calibration curve plotted by taking known dilution of a standard solution of egg albumin. (iv) TOTAL PROTEIN ESTIMATION The total protein content of the seeds was obtained by adding the value for the soluble and insoluble protein. REAGENT PREPARATION FOR PROTEIN PROFILING (a) RE-SUSPENSION BUFFER Tris (P H 6.8) -0.50mM DDW-100mM 2% Sodium dodecyl sulphate (SDS) (b) RESOLVING GEL (10% GEL) Distilled water -12.3ml 1.5M Tris HCl (ph 8.8) -7.5ml 20% Sodium dodecyl sulphate (SDS)-0.15ml 30% Acrylamide solution -9.9ml 10% Ammonium per-sulphate (APS) -0.15ml TEMED-0.015ml
Materials and Methods 40 (c) STACKING GEL (4% GEL) Distilled water-3.075ml 0.5M Tris HCl (ph 6.8) -1.25ml 20% Sodium dodecyl sulphate (SDS)-0.025ml 30% Acrylamide solution -0.67ml 10% Ammonium per-sulphate (APS) -0.25ml TEMED-0.015ml (d) 5X RUNNING BUFFER Tris base -15g Glycine -72g Sodium dodecyl sulphate (SDS) -5g Distilled H 2 O-make volume upto 1 litre (Diluted to 1x before use) (e) STAINING Washing solution - 1ml formaldehyde + 40ml methanol + 60ml distilled water Sodium thio-sulphate -200mg in 1 litre water Silver nitrate solution -0.10% Developer -Sodium carbonate (3g) in 80 ml water + sodium thio-sulphate solution (1ml) and formaldehyde (1ml) make the volume 100ml with water Stopper- acetic acid solution (5%) (f) MOLECULAR MARKERS A small amount of commercially available molecular marker mixture, covering a wide range of molecular weights (3-80 KDa and 10-108 KDa) was used.
Materials and Methods 41 C. SODIUM DODECYL SULPHATE-POLYACRYLAMIDE GEL ELECTROPHORESIS (i) PROTEIN EXTRACTION Seeds of the mutants isolated in M 3 generation and control were ground into fine powder using mortar pestle. 50mg fine power was weighted into a fresh Eppendorf. Powder was dissolved in 600µl of reverse distilled water. Sample P H was adjust to 11 using 0.1N NaOH and made up the volume to 1µl using distilled water. Sample was incubated at room temperature for 1h on a rocker and centrifuged at 12000 rpm for 5 min. Supernatant was transferred into a fresh Eppendorf. P H was gradually bought down to 4.5 using 1M HCl, with proper mixing at each step. Sample was centrifuged at 12000 rpm for 2 minute. Supernatant was discarded and the pellet was dissolved in 200ml of re-suspension buffer with act as the total protein sample for SDS-PAGE. The protein was extracted by following method of Alsohaimy et al., (2007). (ii) ELECTROPHORESIS SDS Polyacrylamide gel electrophoresis of total proteins extracted from seed samples was performed by using the method of Sadasivam and Manickam, (2008). Dry and cleaned SDS-PAGE apparatus were placed in a proper position for casting using clips. Then 10ml resolving gel mixture was poured between the plates till the level was 2cm below from the top edge of the plate. In order to smooth the upper surface of the resolving gel 1ml butanol solution was poured at top to ensure the even surface. After the gel settled down (with normally took 30-45minute) it was washed properly to remove un-polymerized acrylamide. The comb was inserted by leaving 1cm gap between resolving gel and comb. After that 2.5 ml stacking gel mixture was poured on the top level of the resolving gel directly. Once the stacking gel was set,
Materials and Methods 42 comb was removed very carefully without disturbing the wells. Wash these wells with distilled water to remove un-polymerized acrylamide. Fixed the glass plate into the running tray carefully and filled the reservoir with 1x running buffer. Took 2µl total protein sample with 8µl distilled water and 5µl loading dye (Bromophenol blue) and carefully injected into each well. A protein molecular weight marker (GENEI, Bangalore) was also incorporated into gel as reference to direct molecular weight of the bands and then connected with power supply and run the gel at 45 ma till the dye reaches to the bottom of the glass plate. Remove the glass plates and separated them carefully using a spatula. (iii) STAINING After electrophoresis stacking gel was removed and the resolving gel was washed in a washing solution in a clean plastic container with slow shaking for 10 minutes. Wash solution was discarded and gel was rinsed with plenty of water for 2 minutes. Then sodium thio-sulphate solution was used to soak the gel for 1-2 minutes. The gel washed again twice by water and then stained with silver nitrate solution to the plastic container and shacked till yellow to dark brown colour bands appeared and when the bands develops the reaction was stopped by adding acetic acid solution and photographed. D) STATISTICAL ANALYSIS The data recorded for different characters in M 1, M 2 & M 3 generations from different treatments, together with control, were subjected to statistical analysis with a view to find individual and comparative effects of inter-mutagenic and intramutagenic treatments.
Materials and Methods 43 (i) MEAN (X ) It is a measure of central tendency and was computed by taking the sum of the number of observations divided by the total number of observations recorded. Where, X = (X 1 + X 2 + X 3...X n) N X = Ʃ X N X 1 + X 2 + X 3...X n = Observations N = Total No. of observations recorded. (ii) STANDARD DEVIATION (S.D. σ): Standard deviation is the positive square root of the average of sum of squares of deviations of all observations from their means. It is calculated by the following formula. S.D. = (X 1 -X ) 2 + (X 2 -X ) 2 +... (X n -X ) 2 / N X = Mean of observations involved X 1 = Individual observations N= No. of observations. (iii) COEFFICIENT OF VARIATIONS (C.V.) It measures the relative magnitude of variations present in observation relative to the magnitude of their arithmetic mean. It is defined as Rate of standard deviation to arithmetic mean, expressed as percentage. CV = Standard Deviation Mean 100
Materials and Methods 44 (iv) LEAST SIGNIFICANT DIFFERENCE (L.S.D.) The least significant difference was applied and calculated using the software SPSS-16.0 version. The t test was applied to assist the significance of the data at 5% and 1% level of probability. If the difference between any two samples means exceeds the L.S.D. values obtained at 5% and 1% level, the difference between two means is said to be significant at 5% and/or 1% level respectively.