CHAPTER - III
MATERIAL AND METHODS
Present investigation on was conducted on “Genetic variability, diversity
and association studies of indeterminate tomato (solanum lycopersicum l.)
accessions in polyhouse" in Rabi season of the year 2022-2023. The materials
used, methodology followed and statistical analysis adopted during the course of
investigation are described in this chapter.
3.1. Experimental site:
The experiment was conducted in naturally ventilated polyhouse with plot
area of 600m2 during the year 2022 at Vegetable block, College of horticulture,
Sri Konda Laxman Telangana State Horticultural University, Rajendranagar,
Telangana which is situated at Latitude 18.1124 o N and Longitude 79.0193o E and
on altitude of about 536 meters above the mean sea level (MSL).
3.2. Polyhouse details
The crop was raised under multi span type naturally ventilated polyhouse of
600 m2 area with the working space of 350 m2.
3.3. Experimental details
3.3.1. Genotypes and source of collection
Thirty three (indeterminate and semi determinate types) genotypes of
tomato were collected from various sources and which are represented in Table
3.1.
3.3.2. Design and experimental layout
The experiment was designed using a Randomized Block Design (RBD),
and there were two replications. The treatments in each replication were assigned
at random using a random number table. Plate 1 shows the experiment's plan and
layout. Plates 2 show a broad perspective of the experimental plot and the
genotypes that were employed in the experiment.
3.3.3. The experimental details are as follows
Season : Rabi,2022
Name of the crop : Tomato (Solanum lycopersicum L.)
Experimental design : Randomized Block Design (RBD)
Number of genotypes : 32+1 (check variety)
Number of replications : 2
Spacing : 60 cm x 40 cm
Location : College of Horticulture, Rajendranagar
3.3.4. TREATMENT DETAILS
T1 : EC-636482 T12 : EC-688516 T23 : EC-636877
T2 : EC-690982 T13 : EC-620482 T24 : EC-631477
T3 : EC-690989 T14 : EC-631415 T25 : EC-635527
T4 : EC-620397 T15 : EC-617055 T26 : EC-631455
T5 : EC-631369 T16 : EC-631406 T27 : EC-620388
T6 : EC-635529 T17 : EC-638521 T28 : EC-615040
T7 : EC-617060 T18 : EC-631436 T29 : EC-620360
T8 : EC-638518 T19 : EC-687604 T30 : EC-631386
T9 : EC-631325 T20 : EC-687423 T31 : Pusa Ruby
T10 : EC-631373 T21 : EC-631378 T32 : MHTO-101
T11 : EC-632944 T22 : EC-620366 T33 : MHTO-100
3.4. Cultural operations
The information regarding the various cultural operations carried out during
the period of research are furnished below.
3.4.1. Preparation of experimental plot
To appropriately prepare the experimental plot, a disc plough was used two
times, followed by a tractor-drawn cultivator to bring the soil to a fine tilth. The
area was leveled after the weeds were removed. The leveled land was also
utilized to manually prepare each individual plot as needed for the plan. To
perform tasks like spraying, weeding, and other tasks, around 30 cm of space
maintained between two beds. The beds' surface is level and smooth.
3.4.2. Sowing of seeds and nursery maintenance
In 98 celled protrays using cocoa peat as the substrate, the seeds were
sowed. All prescribed cultural procedures, such as drenching, irrigation, and
spraying, was regularly carried out to raise healthy seedlings in addition to the
foliar application of nursey seedlings with 19:19:19 (3g/li) at 15 days following
sowing.
3.4.3. Transplanting
Well-developed two to four true leaf staged 30 days old healthy seedlings
were used for transplanting on the raised beds at the spacing of 60 cm row to row
and 40 cm plant to plant.
3.4.4. Application of manures and fertilizers
Well decomposed farmyard manure (FYM) was applied at the rate of 25 t
ha-1 and recommended dose of NPK fertilizers (125:120:120 kg/ha) one third of
nitrogen and entire dose of phosphorus and potash were applied in the form of
urea, single super phosphate and murate of potash, respectively before
transplanting. Remaining dose of nitrogen was applied in equal doses, i.e., 30 and
60 days after transplanting. Foliar application of micronutrients was done at
every fortnight intervals along with drenching of calcium nitrate.
3.4.5. Irrigation
After transplanting of tomato seedling irrigation was provided for better and
uniform establishment of the seedlings. The subsequent irrigation was given at 6-
8 days interval. Crop was lightly irrigated immediately after planting for
obtaining better establishment. Drip irrigation system was installed after the
experimental plot was layed out. Mainline and inline drip pipes with flow rate of
4 liters per hour was installed with dripper spacing at 30 cm.
3.4.6. Weeding
Experiment plot were kept weed-free by regular hand weeding at an interval
of 20 to 25 days.
3.4.7. Training and pruning
Plants were supported by using specially designed tomato training threads
and wires which were supported with the help of clips at bottom of stem and
hanged the threads with the help of hooks on GI wire which is stretched between
the GI pole of polyhouse. All the suckers and side branches arising from the main
stem were removed regularly.
3.4.8. Trellising
String (overhead) trellising is a technique where individual tomato plants
are wrapped around a piece of twine as they grow, with the twine being
suspended from a tall support structure above. This method is generally used for
indeterminate tomatoes that will keep on growing vertically.
3.4.9. Plant protection
Regular plant protection measures were carried out starting from preventive
application of chloropyriphos 10G granules, seedling drenching with Metalaxyl -
4% + Mancozeb - 64% (68% WP) one day before transplanting in the main field.
Throughout the crop stand, regular spraying of systemic (Spinosad 45% SC,
Imidacloprid 17.8% SL and Thiamethoxam 25% WG) and contact (lambda-
cyhalothin 5% EC, Chlorpyriphos 20% EC and Emamectin Benzoate 5% SG)
insecticides along with fungicides (Carbendazim 12% + Mancozeb 63% WP,
Streptomycin Sulphate 90% + Tetracycline Hydrochloride 10% + Metalaxyl -
4% and Mancozeb - 64% (68% WP)) were done to maintain healthy crop.
3.5. Observations recorded
Every observation, with the exception of the earliness characteristics, was
recorded on five randomly selected plants in each replication. Five plants' means
were chosen for analysis. The characters examined and the recording methods
used to document the observations are listed below.
3.5.1 Growth parameters (30,60,90 DAT)
3.5.1.1. Plant height (cm)
The plant's height was measured in centimeters from the soil line to the
plant's tip.
3.5.1.2. Number of primary branches per plant
The number of primary branches per plant emerging from the main stem
was recorded.
3.5.1.3. Leaf area (cm2)
The data was recorded using a leaf area meter (Leaf area model 211), leaf
area was measured at 30, 60, and 90 days after transplanting (DAT) and
expressed in square centimeters.
3.5.1.4. Stem girth (cm)
The girth of the main stem at 5 cm above ground level was measured using
a digital Vernier calliper.
3.5.2. Flowering parameters
3.5.2.1. Days to first flowering
The number of days taken from the transplanting date to the first bloom
opening was calculated and recorded.
3.5.2.2. Days to 50 per cent flowering
In each treatment, the number of days from transplanting to first flower
appearance in 50 per cent of the plants was recorded and the average was
computed.
3.5.2.3 Number of flowers per cluster
To calculate the average number of flowers per cluster in each entry, the
numbers of flowers in each cluster of five selected plants were tallied.
3.5.2.4. Days taken to fruit set
To calculate the average number of days taken from flower opening to fruit
set in each entry of the five selected plants was counted.
3.5.2.5. Fruit set percentage (%)
Fruit set percentage was calculated with the help of following formulae.
Total number of fruits per cluster
Fruit set percentage = X 100
Total number of flowers per cluster
3.5.3. Yield parameters
3.5.3.1. Number of fruits per cluster
Before the initial picking, the fruit in each cluster of the five chosen plants
was counted in order to determine the average quantity of fruits per cluster for
each entry.
3.5.3.2. Number of fruit clusters per plant
The total numbers of fruit bearing clusters manifested on individual plant
were totaled on five selected plants up to the end of crop cycle of each entry.
3.5.3.3. Days taken for first harvest
The number of days from the date of transplanting to the first fruit's
reaching marketable or horticultural maturity (turning stage) was counted.
3.5.3.4. Days taken from first harvest to last harvest
For each of the five selected plants, the days between the first and last fruit
harvest were counted in order to determine the average number of days between
harvests.
3.5.3.5. Number of fruits per plant
At each picking, the number of fruits from five tagged plants was counted.
The total quantity of fruits per plant was calculated by summing the total number
of harvests and dividing it by the number of plants.
3.5.3.6. Fruit yield per plant (g)
The total fruit weight of all the harvests from each reference plant was
added to determine the yield of fruits per plant and expressed in grams.
3.5.4. Fruit Parameters
3.5.4.1. Fruit length (cm)
At the third harvest, fruit length was measured using digital vernier calliper
from the stalk end to the blossom end, and an average of ten fruits was
calculated.
3.5.4.2. Fruit diameter (cm)
Using digital vernier callipers, the fruit's diameter was measured at the
fruit's largest bulged region during the third harvest, and an average of ten fruits
was calculated.
3.5.4.3. Number of locules per fruit
Total of ten fruits were sliced along a horizontal axis to count the number of
locules in each replication.
3.5.4.4. Pericarp thickness (mm)
Ten fruits were cut along a horizontal axis and Vernier callipers were used
to measure the thickness of the pericarp and expressed in millimeters.
3.5.4.5. Pulp to seed ratio
Pulp to seed ratio was measured with help of following formula.
Total pulp weight (g)
Pulp to seed ratio =
Total seed weight (g)
3.5.5. Screening parameters under natural conditions
3.5.5.6. Root knot nematode root gall index (%)
The number of plants exhibiting root knot nematode incidence symptoms
was counted by uprooting plants after last harvest and noted. The percentage of
plants showing the symptoms per plot was then calculated using the following
formula:
Number of infected plants
Root knot nematode root gall index (%) =
Total number of plants
3.5.5.7. Fusarium wilt incidence (%)
The number of plants exhibiting fusarium wilt incidence symptoms was
counted and noted. The percentage of plants showing the symptoms per plot was
then calculated using the following formula:
Number of infected plants
Fusarium wilt incidence (%) =
Total number of plants
3.5.6. Quality parameters
3.5.6.1 Ascorbic acid content (mg/100 g fresh weight)
3.5.6.1.1. Reagents
1. Metaphosphoric acid (HPO3) 3%: Prepared by dissolving the sticks or
pellets of HPO3 in glass distilled water.
2. Ascorbic acid standard: 100 mg of L-ascorbic acid was dissolved in 3%
HPO3 and volume made up to 100 ml. Dilute 10 ml with 3% HPO3 (1 ml =
0.1 mg of ascorbic acid).
3. Dye solution: 50 mg of the sodium salt of 2, 6-dichlorophenol-indophenol
was dissolved in approximately 150 ml hot glass distilled water containing
42 mg of sodium bicarbonate. Cooled and diluted with distilled water to 200
ml stored in a refrigerator and every day standardization was done.
3.5.6.1.2. Standardization of dye
To a standard ascorbic acid solution of 5 ml, 5 ml of HPO3 was added.
Microburrete was filled with the dye. Titrated with dye solution to a pink colour
which persisted for 15 sec. dye factor was determined i.e., milligram of ascorbic acid
per millilitre of dye, using the formula given by Ranganna (1986).
0.5
Dye factor =
Titre value
0.5 = 0.5mg of ascorbic acid in 5ml of 100ppm standard ascorbic acid solution
Titre = Volume of dye used to neutralize 5ml of 100ppm standard ascorbic acid
solution along with 5ml of metaphosphoric acid
3.5.6.1.3. Preparation of sample
Ten grams of grounded fruit sample was blended with 3% metaphosphoric
acid (HPO3) and volume made up to 100ml with 3% HPO 3. The contents after
shaking well were filtered through Whatman No. 1 filter paper
3.5.6.1.4. Assay of extract
Ten grams of fruit pulp was grounded and blended with 3 per cent
metaphosphoric acid (HPO3) and volume made up to 100 ml with 3 per cent
HPO3. The contents after shaking well were filtered through Whatman No.1
filter paper. Ten ml of the filtrate was titrated against 2, 6- Dichlorophenol-
Indophenol dye until the light pink color persisted for at least 15 seconds.
3.5.6.1.5. Calculation
The ascorbic acid content was estimated using the given formula and
expressed as mg 100 g-1 (Ranganna, 1986).
Ascorbic acid (Titre value x Dye factor x Volume made up)
(mg/100g) = (Aliquot of extra taken for estimation x Volume of sample for X 100
estimation)
Titre value = Volume of dye used to titrate the aliquot of extract of a given
sample.
3.5.6.2. Lycopene (mg/100g)
3.5.6.2.1. Reagents
1. Acetone
2. Petroleum ether
3. Anhydrous sodium sulphate
3.5.6.2.2. Procedure
Take 5 to 10 g of sample and crush repeatedly in acetone in pestle and
mortar until the residue is colourless. Transfer the acetone extracts to a
separatory funnel containing 10 to 15 ml of petroleum ether. Mix gently to take
up the pigments into the petroleum ether phase. Transfer the lower (acetone)
phase to a 100 ml volumetric flask and extract it repeatedly with petroleum ether
until colourless. Combine the petroleum ether extracts and dry over a small
quantity of anhydrous sodium sulphate. Make up to 100 ml with petroleum ether
and measure the O.D. of the solution at 503 nm using petroleum ether as blank.
3.5.6.2.3. Calculation
Milligrams of lycopene per 100gm sample, using the formula given by R.P.
Srivastava and Sanjeev kumar (2002).
O.D. of 1.0 = 3.106 μg of lycopene / ml
Lycopene (mg/100gm) =
3.5.6.3. Shelf-life days
Fruits harvested at red ripe stage were kept in room temperature and
observed for days till the consumption stage was over and shelf-life in days was
recorded.
3.5.6.4. Total soluble solids (°Brix)
By dropping a small amount of the filtered juice onto the refractometer
prism, the percentage of the total soluble solids was calculated using a hand
refractometer. Distilled water was used to check the refractometer for errors
before getting the reading (Ranganna, 1986).
3.5.6.5. Titrable acidity (%)
By diluting tomato juice to a known amount (25 ml) from a known volume
(2 ml), the titrable acidity of the juice was obtained. Using phenolphthalein (1%)
as an indicator, a 5 ml aliquot was collected and titrated against a reference
solution of 0.1 N NaOH. The end point was defined as the appearance of light
pink color. The value was given as a percentage of the juice's titrable acidity in
terms of citric acid (Anon., 1984).
Titre value × volume made up × equivalent weight
of acid
Titrable acidity (%) =
Aliquat taken × weight or volume of sample ×
1000
3.6. Statistical analysis
The experimental data was compiled by taking the mean value of the thirty-
three genotypes of tomato for yield and its component traits from all the two
replications. Then it was subjected to the following statistical analysis:
1. Analysis of variance for R.B.D
2. Estimation of coefficient of variation
3. Estimation of heritability
4. Genetic advance in per cent of mean
5. Assessment of correlation
6. Assessment of path coefficient analysis
7. Estimation of genetic divergence.
3.6.1 Analysis of variance:
The mean value of the data obtained for the recorded characters were
evaluated statistically, and the F- test was used to perform analysis of variance.
The significance among the genotypes was suggested by a probability level of 5
per cent.
3.6.1.1. ANOVA for the experiment:
Source of Degree of Sum of Mean sum of F ratio
variation freedom squares square
Replication r-1 RSS MSR MSR/MSE
Treatments t-1 TrSS MST MST/MSE
Error (r-1) (t-1) ESS MSE
Total (rt-1) TSS
Where,
r= Number of replications
t= Number of treatments
RSS = Sum of squares due to replications
TSS = Sum of squares due to treatments
ESS = Sum of squares due to error
MSR = Mean square due to replications
MST = Mean square due to treatments
MSE = Mean square due to error
The mean square due to replications and treatments were tested against
corresponding error mean square and the calculated ‘F’ value was compared with
table value of ‘F’ at P= 0.005 and P= 0.01.
The mean, standard error, critical difference and coefficients of variation
were calculated as follows:
3.6.1.2. Mean:
The mean value of each character was worked out by dividing the totals
by corresponding number of observations.
Mean (X)=
Where,
Xij = Any observation in ith genotype and jth replication,
N = Total number of observations.
3.6.1.3. Standard Error (S.E.):
The standard errors (S.E. (m)±) for genotypes were calculated with the
help of mean square due to error from the analysis of variance table by the
following formula:
SE (m) ± =
Where,
MSE = mean of square due to error
r = number of replications
3.6.1.4. Critical difference:
Critical difference was calculated to find out the superiority of one variety
over the other by following formula.
CD= t value at 5 % and1 % error d. f.
Where,
SEd =
t = Table value of ‘t’ distribution at error d. f. on P < 0.05 and 0.01.
3.6.1.5. Coefficient of variation (C.V.):
CV % =
Where,
X = General mean
3.6.1.6. Range:
Lower and upper limits of mean values for each character were arranged to
measure the range of variation for the characters.
3.6.2. Estimation of coefficient of variation
3.6.2.1. Genotypic, phenotypic and environmental variances
The variance due to genotype, phenotype and environment were computed
as follows,
2
Genotypic variance ( g )
2
Phenotypic variance( p ) = Total variance observed among the genotype
2
Environmental variance ( e ) = Error mean sum of squares
2 2 2
Phenotypic variance ( p ) = g + e (MS due to error)
Where, ‘r’ is number of replications.
3.6.2.2. Genotypic and phenotypic coefficient of variation
Genotypic and phenotypic coefficients of variance were estimated
according to Burton and Devane (1953) based on estimate of genotypic and
phenotypic variance.
Genotypic co-efficient of variation (GCV)
GCV (%) = ×100
Phenotypic co-efficient of variation (PCV)
PCV (%) = × 100
Where,
= General mean
2
g = Genotypic variance
2
p = Phenotypic variance
GCV and PCV were classified by Burton and Devane (1953) is as follows,
0-10 % : Low
10-20 % : Moderate
20 % and above : High
Estimation of coefficient of variation:
The genotypic coefficient of variation (GCV) and phenotypic coefficient
of variation (PCV) were computed following Burton and Devane (1953).
GCV =
PCV =
3.6.3. Estimation of heritability (h2):
Heritability in broad sense (h2bs) was calculated using the formula suggested
by Burton and Devane (1953).
Where,
h2bs= ×100
h2 = Heritability
σ2p = phenotypic variance
σ2g = Genotypic variance
The heritability percentage was categorized as low, moderate and high as given
by Robinson et al. (1949)
0-30 % : Low
30.1-60% : Moderate
60.1% and above : High
3.6.4. Genetic advance in per cent of mean:
Genetic advance (GA) was estimated by the method suggested by Johnson
et al. (1955).
Genetic advance as per cent over mean (GAM) = × 100
Where,
GA = Genetic advance
GA = h 2 × σ p × k
k = selection differential (2.06) at 5 per cent selection intensity
h2 = Heritability in broad sense
σp = Phenotypic standard deviation
x = General mean
3.6.5. Estimation of correlation:
The correlations between different characters at genotypic (g) and
phenotypic (p) levels were worked out between characters as suggested by Searle
(1961).
i) Phenotypic correlation coefficient between characters X and Y
rxy (p) =
ii) Genotypic correlation between characters X and Y
rxy (g) =
Where,
rxy = Correlation coefficients between X and Y.
Covariance XY= Co-variance between characters X and Y
Var. X= Variance for X character
Var. Y= Variance for Y character
The significance of phenotypic correlation coefficients was tested against
(n-2) degrees of freedom at 5 % and 1 % probability level.
Where,
n is the number of germplasm on which the observations were recorded.
3.6.6. Path-coefficient analysis:
To determine both the direct and indirect effects of morphological
characteristics on plant yield, the path co-efficient analysis method proposed by
Wright (1921) and Dewey and Lu (1959) was used. For evaluating various direct
and indirect effects, the simultaneous equations below were created and solved.
r1y = a + r12b + r13c + ……………….... + r1Ii
r2y = a + r21a + b + r23c + …………….. + r2Ii
r3y = r31a + r32b + c + ………………….. + r3Ii
r1y = r11a + r12b + r13c + …………….. + I
Where,
r1y to 11y = Co-efficient of correlation between causal factors 1 to I with
dependent characters y.
r12 to r1I = Co-efficient of correlation among causal factors
a, b, c…….i = Direct effects of characters ‘a’ to ‘I’ on the dependent
character ‘y’
Residual effect (R) was computed as follows.
Residual effect (R) = 1 - a2 + b2 + c2 + ………i2 + 2abr12 + 2acr13 + ….
3.6.7. Genetic divergence
The genetic divergence of thirty-three genotypes of tomato was worked
out by using Mahalanobis (1936) D2 statistics. The eighteen quantitative
characters in tomato were included for these analyses.
The calculation of D2 values involved following steps:
I. A set of uncorrelated linear combinations (y, s) was obtained by pivotal
condensation of the common dispersion matrix of set of correlated variables
(x, s). The common dispersion matrix was arranged with the help of error
mean squares and mean sum of products.
II. Using the relationship between y, s and x, s the mean values of different
characters were transferred in to the mean values of a set of uncorrelated
linear combinations in tomato.
III. The ‘D2’ values between ith and jth genotypes for kth characters in calculated
as under.
D2 = K (Yit- Yit)
Where,
t =1
The K components were calculated separately and added to get D2ij.
IV. The ‘K’ components and ‘D2ij for each combination were ranked in
descending order of magnitude.
V. These ranks were added up for each component D2ij over combinations of i
and j the total ranks were obtained.
3.6.7.1 Group constellation:
The D2 values were arranged in an increasing order of magnitude. The
grouping of the strains in to different clusters was done using Tocher’s method.
The two most closely associated groups were chosen and third group was found
which had the smaller average D2 value from the first two. Similarly, the fourth
was chosen to have the smallest average D 2 from the first three and so on. The D 2
value did not fit within with the former group and was, therefore, taken as
another cluster.
3.6.7.2 Intra and inter-cluster distance:
The intra-cluster D2 was calculated as the sum of n (n-1)/2 genotypes within
a cluster divided by total number of combinations. All possible D 2 values
between the groups of two clusters were added and then divided by n 1×n2 for
computing inter-cluster distance.
Where,
n1 and n2 = the number of genotypes in two clusters.
Cluster mean:
The cluster mean for the particular character is the summation of mean
values of the strains included in a cluster divided by number of strains in the
cluster.
3.6.7.3 Testing of the significance of D2 values:
The D2 values obtained for a pair of population were taken as the calculated
value of x2 and is tested against tabulated value of x for ‘P’ degree of freedom.
Where,
P = number of characters considered
3.6.7.4 Per cent contribution of each character towards genetic divergence
was calculated as below:
Percentage contribution of character X =
Where,
N= Number of genotype combinations which was ranked first for
character X.
C= All possible combinations of genotypes involved in D2 analysis.
