CHAPTER-III

MATERIAL AND METHODS

The present investigation entitled “Evaluation of F1 hybrids of Cashew (Anacardium

occidentale L.) for growth, yield and quality” was carried out at Cashew Research Station, Bapatla

during 2012-2013 with the objective of evaluating F1 hybrids of cashew for growth, yield and quality

parameters and to assess the extent of heterosis exhibited by them.

The details of the experimental techniques followed and the material used are described in this

chapter.

3.1 Location

The experiments were conducted at cashew Research Station Bapatla. The Research Station

Bapatla is situated at an altitude of 5.49 m above mean sea level with 800 28 eastern longitude and 150 54

Northern latitude.

3.2 Experimental Details

Title of the experiment:

“Evaluation of f1 hybrids of Cashew (Anacardium occidentale L) for growth, yield and quality”

Crop : Cashew

Design : RBD

Replications : 3

Treatments : the experiment was conducted on three sets of hybrids (grouped

according to year planting)

Set Year of planting F1s Parents

 Set-I 1998 H 77 T.NO 1 × T.NO 273
H 85 BPP-8 × T.NO 228
H 94 Priyanka × VRI-2
H 95 T.NO 273 × T.NO 2/22
H 104 T.NO 228 × T.NO 30/1
H 112 T.No40 × BPP-6
H 116 BPP5 × T.No 2/22
H 117 BPP8 × Priyanka

Set-II 2000 H 176 BPP5 × T.NO 2/22

H 180 BPP6 × T.NO 2/22
H 187 T.NO 228 × T.NO 273
H 193 BPP9 × T.NO 273
H 194 T.No 228 × F.No 5
H 200 BPP8 × T.No 228
H 203 T.No 30/1 × T.No 228
H 206 T.No 228×BPP8

Set-III 2005 H 292 BPP6 × Ullal-3

H 298 BPP6 × sel-2
H 303 BPP6 × sel-1
H 306 BPP8 × T.No 228
H 313 BPP8 × Ullal-2
H 319 BPP6 × Sel-2
H 328 BPP8 × Ullal-4
H 338 BPP6 × Ullal-4
Year of study : 2012-13
Location : Cashew Research Station, Bapatla

3.3 Cultural practices

 The standard package of practices was followed throughout the studies.

3.4 Observations recorded

3.4.1 VEGETATIVE PARAMETERS

Observations on morphological and yield parameters, descriptive as well as parametric, were

recorded and genotypes categorized, wherever applicable, as per the standard descriptors of cashew

(IBPGR, 1986; Swamy et al., 1998).

3.4.1.1 Tree height

The height of the tree was measured vertically from the ground to the tip of the tree in meters and

recorded as tree height in meters.

3.4.1.2 Trunk girth

The circumference of the tree trunk was measured at 30 cm above from the base and recorded as

tree girth in centimeters.

3.4.1.3 Canopy spread

The diametric length of the ground space occupied by the tree was measured in two directions

and the canopy spread was recorded in meters as “North-South Spread” and “East- West Spread” for all

genotypes

3.4.1.4 Branching pattern

Based on the growth habit of branches, the genotypes were grouped into extensive and intensive

pattern based on the descriptors of cashew.

3.4.2 REPRODUCTIVE PARMETERS

3.4.2.1 Season of flowering

Time of flowering was recorded by noting down the date of emergence of first 10-15 panicles per
tree canopy. Based on this, the genotypes were identified as “early” (November– December), “mid
season” (December – January) and “late” (January – February) types.
3.4.2.2 Date of flower initiation
 The date of appearance of panicle was recorded on each tree in four directions and the mean date

was calculated for each replication. Depending on the mean date of flowering of each genotype, they were

classified in to three groups as below, as outlined by descriptor list (IBPGR, 1986).

3.4.2.3 Number of panicle bearing branches /m2 canopy

One square meter wooden frame was hand-held on tree canopy and all the shoots, flowering as

well as non-flowering ones, falling within the wooden frame were counted. Such counts were taken in all

the four directions and mean of them is expressed as the “number of laterals per m2 canopy” in each

genotype.

3.4.2.4 Panicle Length

The length of flower panicle was measured vertically from base to the tip of inflorescence and

recorded in centimeters. Twenty panicles, five in each direction, were measured per tree and mean panicle

length was worked out.

3.4.2.5 Panicle width

Width of flower panicle was measured horizontally at the maximum width of the inflorescence

and recorded in centimeters. Twenty panicles, five in each direction, were measured per tree and mean

panicle length was worked out.

3.4.2.6 Number of primary branches per panicle

Twenty fully developed inflorescences, five in each direction, were selected randomly and

branches were counted to express the mean number of branches per inflorescence.

3.4.2.7 Duration of flowering phases

Four flowering panicles per tree, one in each direction, were tagged at random before flowering.

The panicles were tagged in all the accessions, as and when emerged, depending on the time of flowering

in each accession. The tagged panicles were used for recording of date from the day of first flower

opening to the day of last flower opening in each panicle. Four trees were selected in each replication for
studying flowering duration and other flowering characters. Depending on the flower counts (male and

hermaphrodite) different flowering phases were observed with regard to the proportion of staminate and

hermaphrodite flowers. A phase is considered as male phase up to which the number of staminate flowers

continued to be higher than that of hermaphrodite flowers. When the number of hermaphrodite flowers

becomes more than that of staminate flowers. The phase was noted as mixed phase. With this principle,

the initial and final dates of each phase were worked out on the basis of the data obtained from the four

panicles per tree, totaling to twenty panicles per treatment in each replication. Respective percentages of

staminate and hermaphrodite flowers, in each phase and the duration in number of days of each phase

were calculated.

3.4.2.8 Number of male flowers per panicle

The number of male flowers opened were counted and removed every day from the tagged

panicles described above, till the end of flowering period. Counted flowers were removed without

damaging the other growing flower buds.

3.4.2.9 Number of hermaphrodite flowers per panicle

The number of hermaphrodite flowers opened were counted and removed every day from the

tagged panicles described above, till the end of flowering period. Counted flowers were removed without

damaging the other growing flower buds.

3.4.2.10 Sex ratio (hermaphrodite flowers/male flowers)

The number of male and hermaphrodite flowers opened were counted and removed every day

from the tagged panicles described above, till the end of flowering period. Counted flowers were removed

without damaging the other growing flower buds. The ratio of hermaphrodite flowers to staminate

flowers was worked in each hybrid and expressed as “sex ratio” for comparison. The hybrids were

grouped having sex ratio – Low - <0.06 per cent; Medium- 0.06 to 0.13 per cent; High - >0.13 per cent.
3.4.2.11 Number of fruits set per panicle:

Four panicles, one in each direction were tagged at uniform height on all trees, numbering twenty

panicles per treatment in each replication. They were allowed to set fruits under natural pollination

conditions. Once in two days, the numbers of fruits set in each panicle were recorded. At the end, the total

number of fruits set per panicle and mean number of fruits set per panicle were worked out.

3.4.2.12 Percent Fruit set

Four panicles, one in each direction were tagged at uniform height on all trees, numbering twenty
panicles per treatment in each replication. They were allowed to set fruits under natural pollination
conditions. Once in two days, the numbers of fruits set in each panicle were recorded. At the end, the total
number of fruits set per panicle and mean number of fruits set per panicle were worked out. Mean
numbers of fruits set per panicle were divided by the respective mean numbers of hermaphrodite flowers
per panicle and thus mean initial fruit set percentages were worked out.

3.4.2.13 Percent Fruit retention

The fruits and nuts on the tagged panicles were allowed to mature. At harvesting period, the

number of nuts that reached maturity, which is equal to the number of attached apples was recorded for

three times in respective harvests and summed up. Mean numbers of fruits retained per panicle were

worked out. They were expressed as percentages over the respective mean number of fruits set per

panicle.

3.4.2.14 Length and width of apple at weekly interval

Peak length and maximum diameter were recorded for all the ten samples and the mean apple

length and the width were calculated for each treatment at weekly intervals even from after fruit set.

3.4.2.15 Length and width of nut at weekly interval

Peak length and maximum diameter were recorded for all the ten samples and the mean nut

length and width were calculated for each treatment at weekly intervals.
3.4.3 APPLE PARAMETERS

3.4.3.1 Apple shape

Cylindrical Conical to obovate Round Pyriform

The shape of the cashew apple was recorded as per the Cashew Descriptors (IBPGR, 1986;

Swamy, et al., 1998), and recorded as detailed below.

1. Cylindrical

2. Conical to obovate

3. Round

4. Pyriform

3.4.3.2 Apple weight (g)

Weight of twenty randomly selected individual matured cashew apples was recorded on weight

basis and the individual fruit weight was determined and expressed in grams. (DCR 1986)

3.4.3.3 Apple volume (cm3)

 The average of ten randomly selected fruits was measured for fruit volume by using water

displacement method.

3.4.3.4 Apple color

The fruit color of cashew apple was recorded as per the Cashew Descriptors (IBPGR, 1986; Swamy,

et al., 1998), and recorded as detailed below.

1. Yellow

2. Red

3. Yellow red

3.4.3.5 Juice recovery (%)

After recording the apple weight as described above, ten cashew apples were squeezed to extract

the juice content. The juice content of each apple was measured by volume and mean per cent juice

content on volume by weight basis, was computed and recorded for each genotype.

The average of juice extracted from the ten randomly selected fruits were recorded on weight basis

and using the formula and expressed in percentage (%).

Weight of juice per fruit

Juice recovery percentage (%) = -----------------------------x 100
Weight of fruit

Tannins (mg/100ml)

The tannin content of fresh juice, clarified juice and product was determined by Folin Denis reagent

method (A.O.A.C., 1975) and expressed in mg ml-1.

Preparations of Folin Denis Reagent

Twenty grams of sodium tungstates, 4 g of phosphomolybdic acid, 10 ml phosphoric acid and 150 ml of

distilled water were taken in 500 ml round bottom flask. This mixture was refluxed for 2 hours using water

condenser. After cooling volume was made up to 200 ml with distilled water and used in the tannin estimation.

Preparation of Saturated Sodium Carbonate Solution

Thirty five grams of sodium carbonate was dissolved in small quantity of distilled water at 70 o C and kept

overnight. Next day this super saturated solution seeded with crystals of sodium carbonate and filtered through

glass wool.

Preparation of standard tannic acid solution

In a 100 ml of volumetric flask 100 mg of tannic acid was dissolved in a small quantity of distilled water

and volume was made up. From this 5 ml was taken and diluted to 100 ml volumetric flask. 1ml = 50 µg of tannic

acid.

Procedure

For preparing standard curve 0, 1, 2, 3...10 ml of standard tannic acid solution was pipetted into a series of

clean test tubes and volume was made to 8.5 ml by adding required quantity of distilled water. Then 0.5 ml Folin

Denis reagent and 1ml of saturated sodium carbonate solution were added and left for 30 minutes for colour

development. The absorbance was measured at 760 nm against a blank prepared through the same procedure.

Samples were diluted suitably and 1 ml of diluted sample was taken in a test tube and 7.5 ml distilled water was

added. Similarly, a blank was prepared without sample and carried through the procedure described above. The

tannin values were calculated by comparing the absorbance to that of standard curve.

3.4.3.6 Vitamin-C (mg/100ml)

The ascorbic acid was determined by 2, 6-dichlorophenol indophenol`s visual titration method Ranganna,

(1986) and expressed in mg 100 g-1

2, 6 diclorophenol indophenols dye solution

In a beaker 50 mg of sodium salt of 2, 6 diclorophenol indophenols dye and 42 mg of sodium bicarbonate

were taken and dissolved in 150 ml hot distilled water. The volume was made up to 200 ml of distilled water.
3% metaphosphoric acid

Thirty grams of metaphosphoric acid was dissolved in a small quantity of distilled water and the volume

made up to 1000 ml.

Standard ascorbic acid

Hundred milligrams of L- ascorbic acid was dissolved in a small quantity of 3% metaphosphoric acid in

100 ml volumetric flask and dilute to volume. From this 100 ml was taken in another 100 ml volumetric flask and

volume was made up with 3% metaphosphoric acid.

Preparation of sample

The sample was prepared by taking 10 ml of sample in a100 ml volumetric flask was made up with 3%

metaphosphoric acid for its observation.

Procedure

Five millilitres of 3% metaphosphoric acid and extract of sample was taken in a conical flask and titrated

with standard dye. The end point was pink colour which existed for 15 seconds. The ascorbic acid was calculated by

using formula.

Titre value x Dye factor x Volume made up x 100

-------------------------------------------------------------------------------
Adequate of extract x Weight or volume of taken for estimation

3.4.3.7 Total soluble solids (oBrix):

The total soluble solids were determined by using hand refractometer and expressed as ᴼBrix as followed

by Ranganna, (1986).

3.4.4 NUT PARAMETERS

3.4.4.1 Nut weight (g)

Individual weight of thirty sun-dried nuts, collected from the peak period of season, was recorded

in grams and mean weight computed was expressed as nut weight in grams. Genotypes were classified

into those having nuts of low – <5g, medium - 5- 7 g and high > 7g weight.
3.4.4.2 Nut – apple ratio

The ratio between nut and apple weight and it is expressed in ratio.

3.4.4.3 Nut volume (cm3)

The average of ten randomly selected nuts was measured for fruit volume by using water

displacement method.

3.4.4.3 Nut shape

Kidney-shaped Oblong-ellipsoid

the shape of the nut was recorded as per the Cashew Descriptors

(IBPGR, 1986), and recorded as detailed below.

1. Kidney-shaped

2. Oblong-ellipsoid

3.4.4.5 Nut Yield per tree

Total weight of raw nuts collected from each of three trees during the entire season, was recorded

in kilograms and mean weight was expressed as nut yield per tree in kilograms.
3.4.4.6 Shelling percentage

Seed weight divided by dry net weight multiplied by 100.

Low (16-18%)

Intermediate (22-24%)

High (28-30%)

3.4.5 KERNEL PARAMETERS

3.4.5.1 Kernel weight (g)

Individual weight of thirty kernels, obtained after cutting the nuts used for above parameters, was

recorded in grams. The mean kernel weight was computed and expressed in grams. Based on nut weight

genotypes categorized into those having nut size of low-<1.2g, medium- 1.2 – 2.5 g, high > 7g size.

3.4.5.2 Kernel to nut ratio

The ratio between kernel weight and nut weight and it is expressed in ratio

3.4.5.3 Kernel shape

The shape of the kernel was recorded as per the Cashew Descriptors (IBPGR, 1986), and

recorded as detailed below.

1. Kidney-shaped

2. Oblong-ellipsoid

3.4.5.4 Kernel volume (cm3)

 The average of ten randomly selected kernels was measured for fruit volume by using water

displacement method.

3.4.6 STATISTICAL ANALYSIS

The data obtained in respect of all the characters have been subjected to the
following statistical analysis.
3.4.6.1 Analysis of variance (ANOVA):
Data were analyzed by the methods outlined by Panse and Sukhatme (1985) using
the mean values of random plants in each replication from all genotypes to find out the
significance of genotypes effect.
The data for different characters were statistically analysed on the basis of the
model suggested by Cochran and Cox (1950) for RBD.

4 Yij = µ + bi + tj + eij

Where,
Yij = Performance of the jth genotype in the ith block
µ = general mean
bi = true effect of ith block
tj = true effect of jth genotype
eij = random error associated with ith block and jth genotype.
The analysis of variance for each character was carried out as indicated below:

Sources of variation Df SS MSS F ratio

Replications r-1 RSS RMSS RMSS/EMSS
Treatments t-1 TrSS TrMSS TrMSS/EMSS
Error (r-1) (t-1) ESS EMSS
Total (rt-1) TSS
Where,
r = Number of replications
 t = Number of genotypes or treatments
df = degrees of freedom
SS = sum of squares
MSS = Mean sum of squares
RSS = Replication sum of squares
TrSS = Treatment sum of squares
ESS = Error sum of squares
TSS = Total sum of squares
RMSS = Mean squares due to replications
TrMSS = Mean squares due to treatments
EMSS = Mean squares due to error
The test of significance was carried out by ‘F’ table values given by Fisher and
Yates (1963).
Components of variance:
Genotype MSS-Error MSS
Genotypic variance (Vg or σ2g) = -----------------------------------------
r

Error MSS
Environmental variance (Ve or σ2e) = -----------------------
r
Where,
r = number of replications
Phenotypic variance (Vp or σ2p) = Vg+ Ve
Significance of treatment MSS was assessed with reference to F table values at 1
and 5 percent probabilities. Further, computation was carried out only when the treatment
effects were significant.
3.4.6.2 Estimation of genetic parameters
 The genetic parameters such as genotypic coefficient of variation (GCV),
phenotypic coefficient of variation (PCV), heritability in broad sense and genetic advance
for different characters were worked out by following the standard procedures for all the
genotypes under study.
Genotypic and phenotypic coefficient of variation
Genotypic and phenotypic coefficients of variation were estimated according to
Burton and Devane (1953) by using the following formulae.

σp2
PCV = --------- x 100
X

σg2
GCV = --------- x 100
X
Where,
σg2 = Genotypic variance
σe2 = Environment variance
σp2 = Phenotypic variance
X = General mean
PCV and GCV were classified as shown below (Sivasubramanian and Menon
1973).
Less than 10% = Low
10-20% = Moderate
More than 20 % = High

3.4.6.3 Heritability in Broad sense [ h2 (b) ]

 Heritability in broad sense was estimated as per the formulae suggested by Allard
(1960).
σg2
h2 (b) = ------ x 100
σp2
Where,
h2 (b) = Heritability estimates in broad sense
σg2 = Genotypic variance
σp2 = Phenotypic variance
The heritability (h2 (b)) was categorised as suggested by Johnson et al. (1955).
0-30% = Low
31-60% = Medium
61% and above = High
3.4.6.4 Genetic advance (GA)
This was estimated as per formula proposed by Allard (1960)
GA = Kx σp x h2 (b)
Where,
K = Selection differential at 5 per cent selection intensity which accounts
to a constant value 2.06
h2 (b) = Heritability in broad sense
σp = Phenotypic standard deviation

3.5.6.5 Genetic advance as per cent of mean (GAM)

Genetic advance over mean (GAM) was calculated using the following formula
and was expressed in percentage.
GA
GAM = ----------- x 100
X
Where,
GA = genetic advance
X = general mean of the character
The genetic advance as per cent over mean was categorized as mentioned below
(Johnson et al., 1955).
Less than 10% = Low
10-20% = Moderate
More than 20 % = High

3.4.6.6 Correlation studies

Phenotypic and genotypic correlations were worked out by using formula
suggested by Falconer (1964).
Phenotypic coefficient of correlation (rp )
COV (xi.xj)p
r(xi.xj)p = --------------------------
V (xi)p . V (xj)p
Where,
r(xi.xj)p = Phenotypic correlation between ith and jth character.
COV (xi.xj)p = Phenotypic covariance between ith and jth character.
V (xi)p = Phenotypic variance of ith character.
V (xj)p = Phenotypic variance of jth character.

Genotypic coefficient of correlation (rg)

COV (xi.xj)g
r(xi.xj)g = ----------------------
V (xi)g . V (xj)g
Where,
r(xi.xj)g = Genotypic correlation between ith and jth character.
COV (xi.xj)g = Genotypic covariance between ith and jth character.
V (xi)g = genotypic variance of ith character.
V (xj)g = genotypic variance of jth character.
Test of significance
Significance of correlation coefficients was tested by comparing phenotypic
correlation coefficients with the table values (Fisher and Yates, 1963) at (n-2) degrees of
freedom at 5 % and 1 % level where ‘n' denotes the total number of pairs of observations
used in the calculation.
r
t= ------------- n-2
1- r2
Where,
t = test of significance
r = correlation coefficient
n = number of paired observations
3.4.6.7 Path coefficient analysis
The direct and indirect contribution of various characters to yield were calculated
through path coefficient analysis as suggested by Wright (1921) and elaborated by
Dewey and Lu (1959). The following simultaneous equations were formed and solved for
estimating various direct and indirect effects.
Path coefficients were obtained by solving the following simultaneous equations.
rly = Ply + r12P2y + r13 P3y + ……… + rlk Pky
Where,
rly = Simple correlation coefficient between x1 and y, the dependent character
Ply = Direct effect of x1 on y, the dependent character
r12P2y = Indirect effect of x1 on y through x2.
r12 = Correlation coefficient between x1 and x2.
rlkPky = Indirect effect of x1 only through kth variable.
In the same way, equations for r2y, r3y, r4y, upto rky were obtained. Besides the
direct and indirect effects, the residual effect was computed by using the formula.
Residual effect (Pry) = 1-R2
Where, R2 = Plyrly + P2yr2y + P3yr3y + …………. Piyriy
Pry = Residual effect
Ply = Direct effect of x1 on y.
r1y = Correlation coefficient of x1 and y
P2y = Direct effect of x2 on y
r2y = Correlation coefficient of x2 and y.
P3y = Direct effect of x3 on y
r3y = Correlation coefficient of x3 and y
Piy = Direct effect of xi on y
rjy = Correlation coefficient of xi and y

Pry = 1  P1 y r1 y  P2 y r2 y  ....... Pky rky

Where Pry = residual effect

Ply = direct effect of x1 only
rly = correlation coefficient of x1 only
Scales for path coefficients
Values of direct (or) indirect Rate (or) scale
effects
0.00 to 0.09 Negligible
0.10 to 0.19 Low
0.20 to 0.29 Moderate
0.30 to 0.99 High
> 1.00 Very high

3.4.6.8 Mean
 On the basis of individual plant observations, the mean for each character in all the
populations was computed as follows.
_ n
y = 1/n ( ∑yi )
i=1

Where,
_
y = Population mean
yi = Individual value
n = Number of observations
3.4.6.9 Range
The minimum and maximum values on the basis of individual plant observations were
used to indicate the range for a given character

3.4.6.10 HETEROSIS
The magnitude of heterosis in relation to mid parent better parent and check values were
worked out. These were calculated as percentage increase or decrease of F1’s over the mid
parent (MP), better parent (BP) and check (CK) values.
(a) Heterosis over mid parent (MP)
_ __
F1 – MP
Mid parent heterosis (%) = -------------------×100
MP
__ _ _
Where, MP = P1 + P2
(b) Heterosis over better parent (BP)

_ __
F1 – BP
Better parent heterosis (%) = ------------------ × 100
BP
(c) Heterosis over standard check
_ __
F1 – SC
Standard check heterosis (%) = -----------× 100
SC

Where,
_
F1 = Mean of F1
__
MP = Mean of two parents
__
BP = Mean of better parents
__
SC = Mean of standard check
Mid parental value (MP)
For each character, the arithmetic average value of two parents involved in each cross was taken.
Better parental value (BP)
For each character, the superior value between the parents in each cross was taken.