3. MATERIALS AND METHODS

Transcription

1 29 3. MATERIALS AND METHODS The present investigations were carried out at the Vegetable Research Farm of Department of Vegetable Science and Floriculture, CSKHPKV, Palampur (H.P) during Kharif season 2012 and The material used and methods employed in the present investigation are presented as here under: 3.1 Experimental material The experimental material was developed from two intervarietal crosses viz; Swarna Pratibha x Hisar Shyamal and Arka Keshav x Bhola Nath. While selecting the parents emphasis was given upon the growth habit, fruit shape, fruit colour, fruit maturity, average fruit yield and reaction to bacterial wilt disease. The main characteristics of the parents constituting the crosses are given in Table 3.1 Table 3.1 Parents used for the development of biparental progenies, their sources and chief characteristics S. No. Genotpye Source Characteristics 1 Swarna Pratibha ICAR Research Complex for Eastern Region, Research Centre Ranchi (Jharkhand) Mid maturity, erect and vigorous plant habit, Oblong shiny light purple fruits 2 Hisar Shyamal (H-8) CCS HAU, Hisar (Haryana) Early maturity, Plants are semi erect with purple green foliage, round fleshy, bright purple fruits with dark green purple pedicel. 3 Arka Keshav Division of Vegetable Crops, Indian Institute of Horticultural Research, Hessarghatta, Bangalore (Karnataka) Mid maturity, erect, long fruits with purple glossy uniform skin, borne in clusters. 4 Bhola Nath ICAR Complex for North Eastern Hill Region Tripura Centre Lambucherra, West Tripura Small purple round fruits with green tinge

2 Max., Min. temp. ( o C) and Evaporation (mm) RH I, II (%), Rainfall (mm) April May June July August September October November RH I RH II (%) Rainfall (mm) Max Temp Min Temp Evaporation per day (mm) Figure 3.1 Weather data during cropping season 2012

3 Max., Min. temp. ( o C) and Evaporation (mm) RH I, II (%), Rainfall (mm) April May June July August September October November RH I RH II (%) Rainfall (mm) Max Temp Min Temp Evaporation per day (mm) Figure 3.2 Weather data during cropping season 2013

4 32 Mating Design Biparental progenies were developed in F 2 generations of two intervarietal crosses using North Carolina Design I as suggested by Comstock and Robinson (1948 and 1952). The biparental progenies were developed by designating four F 2 plants as male parents and crossing each of these to four plants selected as females. The plants used as males and females were chosen at random for the development of biparental progenies and no seed parent was used in more than one mating. The plants used in making the biparental progenies were also selfed. Thus, the family consisted of sixteen progenies (four in each male group). Twenty F 3 families were developed by selfing (4 males and 16 females). The experiment comprised three such sets or a total of 48 biparental progenies and 60 F 3 families in each crosses. 3.2 Location The Vegetable Research Farm of CSKHPKV, Palampur is situated at an elevation of about meters above mean sea level with North latitude and East longitude, representing mid hills zone of Himachal Pradesh and has a sub-temperate climate with high rainfall during monsoon season. The soil of this zone is silt clay loam with acidic reaction. The weather data was collected from the meteorological observatory, Department of Agronomy, CSKHPKV, Palampur as presented in Appendix-I 3.3 Experimental layout and design The biparental progenies (BIP s) and F 3 progenies were grown in Randomized Block Design (RBD) with three replications, in two experiments relating to two different crosses. Each experimental plot consisted of two rows of 2.70m length for biparental and F 3 progenies with inter and intra plant distance of 60 cm and 45 cm, respectively. These progenies were arranged in three sets, each comprising sixteen BIP s and twenty F 3 progenies. The sets and progenies within the sets were randomized separately. In addition, six rows of each F 2, two

5 33 rows each of the original parents and F 1 s were also included in each replication for making comparisons. The F 2 seeds of two intervarietal crosses viz., Swarana Pratibha x Hisar Shyamal (SP x H-8) and Arka Keshav x Bhola Nath (AK x BN) obtained from crosses attempted during Kharif 2011 were sown during March, Material used in study before making biparental progenies was developed at the Department of Vegetable Science, CSKHPKV, Palampur. This material was used to produce seeds of biparental and F 3 progenies. The seeds of F 1 were also obtained by making fresh crosses. The final experiment was conducted during Kharif 2013 with the experimental material comprising parents (P 1, P 2 ), F 1, F 2, BIP s and F 3 generations. The seeds of these generations in respect of crosses Swarana Pratibha x Hisar Shyamal (SP x H-8) and Arka Keshav x Bhola Nath (AK x BN) were sown during April, Cultural practices The crop was transplanted in already bacterial wilt sick plots. 20 t/ha was added in the soil at the time of field preparation. The chemical fertilizers were applied in the soil before transplanting the crop as per recommended package of practices (100 kg N, 75 kg P 2 O 5 and 50 kg K 2 O / ha). One third of N and full dose of P 2 O 5 and K 2 O were applied before transplanting. Remaining two third N was top dressed in equal doses after 30 and 45 days after transplanting. The intercultural operations were carried out as per recommended package of practices. Regular weedings were carried out to keep the experimental field free from weeds and plant protection measures adopted to raise a healthy crop. 3.5 Observations recorded The observations were recorded on randomly taken five competitive plants in each entry for most of the traits except days to 50 per cent flowering and bacterial wilt incidence (plant survival) for which observations were recorded on plot basis Marketable fruit yield per plant (kg) The total weight of marketable fruits obtained from each entry was divided by the total number of randomly taken plants.

6 34 Swarna Pratibha (SP) Hisar Shyamal (H-8) Arka Keshav (AK) Bhola Nath (BN) Plate 3.1: Parents used in the study

7 Plate 3.2: General view of crossing block 35

8 Days to 50 per cent flowering The number of days taken from the date of transplanting to the date of first flower appeared in 50 per cent plants in each entry was counted and average worked out Days to first picking The number of days taken from the date of transplanting to the date of first harvesting in each entry were counted and average worked out Pedicel length (cm) Pedicel length of five randomly taken fruits each in 4 th to 7 th pickings were recorded and averaged out Fruit length (cm) Five randomly taken fruits each in fourth picking were taken for measuring length (excluding pedicle length) and average value calculated Fruit weight (g) Fruit weight was obtained by dividing total marketable fruit yield by the total number of marketable fruits in each entry Fruit diameter (cm) After measuring length in fourth picking, the diameter of the same fruits were measured from the centre of the fruits and averaged out Number of marketable fruits per plant The number of marketable fruits obtained from each entry were summed up and divided by the total number of randomly taken plants to get marketable fruits plant Number of branches per plant At the time of final picking, the number of branches were counted and averaged out.

9 Plant height (cm) Plant height was recorded from base to the top of the main branch at the time of final picking and the mean values worked out Total soluble solid ( Brix) Three randomly selected fruits were taken for determining total soluble solids by hand refractometer and average values calculated in each case in each replication. The mean values were expressed as per cent (A.O.A.C., 1970) Dry matter content (%) 100 g fresh weight of each treatment was oven dried at 65 ± 2 0 C till the constant weight was obtained. The per cent dry matter was calculated as below: Final dry weight Dry matter (%) = X 100 Initial weight Iron Content Iron content was estimated by the method proposed by Rangana (2007). a) Preparation of ash solution: The weighed amount of dried sample (2 g) is taken and put in previously dried and weighed crucibles. The samples were first incinerated over an electric hot plate followed by ashing in muffle furnance at the temperature of C for 6 h (until a pale white residue is obtained). The samples after ashing were taken out from the muffle furnance and kept in the dessicator for two hours for cooling. Dish containing sample covered with watch glass and 4-5ml dilute HCl (1:1) added with the help of pipette. The watch glass was used to prevent the spattering. Heated over the waterbath for 30 min and added another 10 ml of HCl (1:1). Filtered into 100ml volumetric flask. Made up the volume with water. The ash solution was ready for determination of iron.

10 38 b) Determination of iron: Into a stoppered measuring cylinder, pipette 5 ml of sample ash solution. Added 0.5 ml conc. H 2 SO 4 to this. Then added 1ml potassium persulphate. Finally added 2ml of potassium thiocyanate. Made up the final volume to 15ml with water. After 10 min. measured the colour at 480nm setting the blank at 100% transmission. Prepared a standard curve using different concentration of standard iron solution which was prepared using ferrous ammonium sulphate. Calculation: O.D. of sample x 0.1 x total volume of ash solution x 100 Fe (mg/100g) = O.D. of standard x 5 x weight of sample taken for ashing Phenol content Phenol content was estimated by the procedure adopted by Thimmaiah (1999). Weighed exactly 0.5 to 1 g of the sample and ground it with a pestle mortar in 10 time volume of 80% ethanol. Centrifuged the homogenate at 10,000 rpm for 20 min, saved the supernatant. Re-extracted the residue with five times the volume of 80% ethanol, centrifuged and pooled the supernatants. Evaporated the supernatants to dryness. Dissolved the residue in known volume of distilled water (5 ml). Pipetted out different aliquots (0.2 to 2 ml) into test tubes. Made up the volume in each tube to 3 ml with water. Added 0.5 ml of Folin-Ciocalteu reagent. After 3 min, added 2 ml of 20% Na 2 CO 3 solution to each tube. Mixed thoroughly. Placed the tubes in boiling water for exactly one min, cooled and measured the absorbance at 650 nm against a reagent blank. Prepared a standard curve using different concentration of catechol. From the standard curve worked out the concentration of phenols in the test sample and expressed as mg phenol/100g material Bacterial wilt incidence (plant survival) Total number of plants in each entry which survived, were counted and percentage worked out. Plant survival was calculated as below: Number of plants surviving in each entry Plant survival = 100 Total number of plants in the entry

11 39 Plants were scored in relation to their resistance to bacterial wilt disease following the methodology of Hartman et al. (1996) as detailed below : Score Mortality (%) Categories/grouping 1. 0% plant mortality Resistant 2. >10 to 20% mortality Moderately resistant 3. >30 to 70% mortality Moderately susceptible 4. >70 to 100% mortality Susceptible 3.6 STATISTICAL ANALYSIS The replication wise mean values of the entries for different traits were subjected to the following statistical analysis: Estimation of components of genetic variance and average degree of dominance in biparental progenies The method of analysis of variance followed was as proposed by Comstock and Robinson (1948 and 1952). Analysis of variance (NCD-I) Source of variation Degree of freedom Mean squares (MS) Expectations of MS Sets (s-1) Replications in sets s (r-1) Males in sets s (m-1) M r 2 f + rn 2 m Females in males in sets sm (f-1) M r 2 f Remainder among plots s (mf-1) (r-1) M 13 2

12 40 Where, s = number of sets r = number of replications m = number of males in a set f = number of females per male 2 = error variance among plots of the same progeny 2 f = progeny variance arising from genetic differences among female parents 2 m = progeny variance arising from genetic differences among male parents By using appropriate mean squares, obtained from the analysis of variance, 2 m and 2 f were estimated as follows: M 11 M 12 2 m = rf M 12 M 13 2 f = r The estimates of additive genetic variance ( 2 A) and dominance variance ( 2 D) were calculated from variances due to males and females, using the following relationship: 2 m = ¼ 2 A or 2 A = 4 2 m 2 f = ¼ 2 A + ¼ 2 D or 2 D = 4 ( 2 f - 2 m) The average degree of dominance was calculated as the square root of dominance variance to additive genetic variance = 2 D 2 A

13 41 The standard errors of 2 m and 2 f were calculated as follows by the formula proposed by Moll et al. (1960). 2 Mi 2 SE = C 2 i di+2 In which: M i = the i th mean squares in the function, d i = the degrees of freedom associated with the i th mean square C = the divisor of the function of mean squares. Hence, 2 M 11 2 M 13 2 SE 2 m = (rf) 2 s (m-1) +2 s (mf-1) (r-1) M 11 2 M 13 2 SE 2 f = r 2 sm (f-1) + 2 s (mf-1) (r-1) + 2 The standard errors of 2 A and 2 D were calculated as followed by the method proposed by Panse and Sukhatme (1984). SE 2 A = 4 var. 2 m SE 2 D = 4 var. 2 f + var. 2 m

14 42 The test of significance for the estimated value was done with the help of respective standard errors of the estimates Heritability Heritability (h 2 ) estimates in the narrow sense were calculated by using the following formula: 2 A h 2 (%) = x A + 2 D Estimation of predicted genetic advance Expected gains from full-sib family selection ( means of the procedure outlined by Robinson et al. (1949). An approximate procedure was used by Goodman (1965) to estimate the expected gains from units tested are: 2 m (2.06) 2 m + 2 f + 2 / 2 where, 2 m (2.06) 2 m + 2 f is the error variance or the random plot to plot variation of the experiment. The formulae for mass selection resulted in estimates which are more conservative than are those for full-sib family selection, since 10 2 overestimates the sum of the environmental variance and within plot genetic variance.

15 CORRELATION COEFFICIENTS The phenotypic correlations were worked out by employing the variancecovariance matrix in which the total variability has been split into that due to replications, treatments and error as given below. The treatments correspond to biparental progenies and F 3 progenies Source Df Mean sum of product Expected mean sum of product Replication (r-1) Mr xy e xy + t r xy Treatments (t-1) Mt xy e xy + r t xy Error (r-1) (t-1) Me xy e xy Genotypic co-variance ( g xy ) = (Mt xy Me xy ) Phenotypic co-variance ( p xy ) = g xy + e xy r r The phenotypic coefficient of correlation were computed as suggested by Al-Jibouri et al. (1958) Phenotypic coefficient of correlation where, p xy rp xy = p x x 2 p y p xy = Phenotypic covariance between two characters X and Y p 2 x = Phenotypic variance of character X p 2 y = Phenotypic variance of character Y The significance of coefficients of correlation were tested against r values as given by Fisher and Yates (1963) at n-2 degree of freedom.

16 Path-coefficient analysis Path coefficient is a standard partial regression coefficient. It permits the partitioning of coefficients of correlation into direct and indirect effects. The path coefficients at phenotypic level were calculated by employing the method suggested by Dewey and Lu (1959). The path coefficients were obtained by solving the following simultaneous equations, which expresses the basic relationship between correlation coefficients and path coefficients. P y1 + P y2 r 12 + P y3 r P yn r 1n = r y1. P y1 r 12 + P y2 + P y3 r P yn r 2n = r y2. P y1 r 1n + P y2 r 2n + P y3 r 3n + + P yn = r yn where, P y1, P y2, P y3, P yn are the direct path coefficients of 1, 2, 3., n variables on the dependent variable Y; r 12, r 13 r 1n, r (n-1)n are the possible coefficients of correlation between various independent variables, and r y1, r y2,.., r yn are the coefficients of independent variables with the dependent variable Y. The variation in the dependent variable, which remained undetermined by including the ten variables, was assumed to be due to variable/variables not included in the present investigation. The degree of determination (P 2 xr) of such variable/variables on the dependent variable was calculated as follows: Residual effect (P xr ) = (1-R 2 ) 1/2 where, R 2 = P y1 r y1 + P y2 r y P yn r yn = is the square multiple correlation coefficient and is the amount of variation in yield that can be accounted for by the yield component characters.

17 Comparison of means The characterwise means of the biparental progenies (BIP s) were compared with the means of F 3 progenies. The significance of difference between means was tested using t test. x - y t = S 1/n 1 + 1/n 2 (n 1-1)S (n 2-1) S 2 2 S= n 1 + n 2-2 where, x and y are the means of the same character in two populations; n 1 and n 2 were the number of BIP s and F 3 progenies, respectively. On the basis of which the characterwise means were found out; S was the pooled standard deviation and S 1 and S 2 were standard deviations of the two populations Comparison of phenotypic correlations in biparental and F 3 progenies Phenotypic correlations were calculated for the biparental progenies (BIP s) and F 3 progenies by using the formula: Cov xy r xy = (var x) (var y) The significance of the differences between any two corresponding correlation coefficients in BIP s and F 3 progenies were tested by using Z test following the method of Snedecor and Cochran (1967). If r was the correlation coefficient between character x and y in BIP s based on observations over n 1 progenies and similarly r 2 was the correlation coefficient between same pair of characters in F 3 progenies based on observations over n 2 progenies, the significance of differences between r 1 and r 2 was calculated by transforming r values to Z as given below:

18 46 Let, Z 1 = ½ log e [(1 + r 1 ) / (1-r 1 )] Z 2 = ½ log e [(1+r 2 ) / (1 r 2 )] Z 1 - Z 2 is distributed as a normal deviate as shown below: Z 1 - Z / (n 1 3) + 1/ (n 2 3) where, 1/ (n 1 3) + 1/ (n 2 3) is the SE of Z 1 Z Plant breeding comparisons The top 5% biparental and F 3 progenies were taken and their fruit yield per plant averaged. These values were then compared among themselves and with the mean values for fruit yield of the corresponding parents, F 1 s and F 2 s.