Chapter-3
MATERIALS AND METHODS
The present investigation entitled “Effect of stionic combinations and spacing on
pomological and physiological parameters under high density planting of apple (Malus
x domestica Borkh.)” was conducted during the cropping season of 2021 and 2022. The
detail of the material and methodologies employed during the study has been described under
the following heads and sub-heads:
3.1
GEOGRAPHICAL SITE
The studies were carried out in Experimental farm of Department of Fruit Science, Dr
YS Parmar University of Horticulture and Forestry, Nauni, Solan, Himachal Pradesh. The
experimental site is located at 30°51´49´´ North latitude and 77°9´23´´ East longitudes at an
elevation of 1279 m above mean sea level under sub-humid mid-hill agro climatic conditions.
The climate of the study area is typically sub-temperate. Annual rainfall exhibited in the
range of 125-130 cm, 80 per cent of which is received during the month of July until
September.
3.2
EXPERIMENTAL DETAILS
3.2.1 Plant material
The experimental orchard was established in the year of 2019. Budded feathered
plants were imported and planted. Pollinizer cultivars within the experimental orchard were
maintained in the ratio of 3:1 (variety: pollinizer). Three-years-old uniform trees on nine
different scion-stock combinations at two different planting densities were selected for the
experimental
study.
Velox/EMLA111,
Scion-stock
King
combinations
Roat/EMLA111,
includes
Schelet
Jeromine/EMLA111,
Spur/EMLA111,
Gala
Red
Schniga
Schnico/EMLA111 and Gala Schniga Schnico/EMLA9 spaced at 2.5 x 2.0 m with planting
density of 2000 plants/ha. Similarly, Jeromine/EMLA111, Gala Schniga Schnico/EMLA111
and Gala Schniga Schnico/EMLA9 were spaced at 2.0 x 1.5 m with planting density of 3333
plants/ha. Selected plants were kept under uniform cultural practices during the course of
investigation.
3.2.2 The edaphic conditions
The experimental soil was sandy clay loam in texture, towards neutral in soil reaction
(pH 6.92) 1:2 soil water suspension (Jackson, 1973), electrical conductivity 0.31 dS/m,
organic carbon 2.32% (Walkey and Black, 1934), 341.2 kg/ha of alkaline KMnO4
extractable-N (Subbiah and Asija, 1956), 78.64 kg/ha of available NaHCO3-extractable P
(Olsen et al., 1954) and 324.8 kg/ha of available NH4OAC-K (Merwin and Peach, 1951).
Diethylene-triamine-penta-acetic acid (DTPA)-extractable micronutrient cations buffered at
pH 7.3 ± 0.05, namely, zinc (Zn), manganese (Mn), iron (Fe) and copper (Cu) were 3.1, 8.2,
16.7 and 2.3 ppm, respectively (Lindsay and Norvell, 1978).
3.3
OBSERVATION RECORDED
3.3.1 Vegetative Growth Indices
3.3.1.1 Plant height
Plant height was measured with the help of graduated flag staff from the soil to the
top of a tree, once before the start of the experiment during the month of February and again
at the end of growing season in the month of December. The increase in height of the trees
over the growing season was calculated and expressed in centimeter (cm).
3.3.1.2 Tree girth
Tree girth was measured 15 cm above the graft union with the help of a digital vernier
caliper, once before the start of the experiment and then at the end of growing season.
Average of tree girth was taken and expressed in centimeter (cm).
3.3.1.3 Shoot growth
Five uniform and healthy shoots from the current season’s growth in all the four
directions were selected randomly for measuring shoot growth. The length of these shoots
was measured with the help of measuring tape and the values were expressed in centimeter
(cm).
3.3.1.4 Leaf area
Twenty fully expanded leaves from each experimental tree were randomly collected
from each experimental tree in the month of August. Leaf area was recorded with the help of
LI-3000C Leaf Area Meter and the values were expressed as square centimeter (cm2).
3.3.1.5 Leaf area index
Leaf area index (LAI) is the area of leaves per unit area of soil surface and was
recorded in the month of June with the help of Accu PAR PAR/LAI Ceptometer.
.3.3.1.6 Root suckering
Root suckers were counted from selected trees once at the start of experiment and
again at the end of growing season.
3.3.1.7 Tree habit
Tree habit of selected plants was visually observed and classified as vigorous, semivigorous and dwarf based on their growth habit
3.3.2 Generative Traits
3.3.2.1 Trunk cross–sectional area
Trunk cross–sectional area (TCSA) was calculated according to the method suggested
by Westwood (1978) using formula, TCSA= πr2, where, r = radius of the trunk.
3.3.2.2 Canopy diameter
The canopy diameter was measured in two directions (east-west and north-south) with
before the commencement of the experiment and again after the end of growing season with
the help of measuring tape at the point. Average of two directions was taken and the values
were expressed in centimeter (cm).
3.3.2.3 Canopy area
Canopy area (CA) was measured by taking average of canopy diameter according to
Westwood (1978) using formula, CA = πr2 [radius (r) = canopy diameter/2]. The values were
expressed as square meter (m2).
3.3.2.4 Tree canopy volume
To calculate Tree canopy volume (TCV), total above ground volume of each tree was
measured from height and spread method once at the start of experiment and again at the end
of growing season during the year 2021, as per method suggested by Westwood (1978) as
given below:
i)
For the trees which were taller than its width
Tree volume = 4/3 π ab2
ii)
For the trees which were wider than its height
Tree volume = 4/3 π a2b
Where, π = 3.14
a = ½ of length of major axis (height).
b = ½ of length of minor axis (width).
Tree canopy volume was worked subsequently and the values were expressed as cubic
meter (m3).
3.3.3 FLOWERING PARAMETERS
3.3.3.1 Date of pink bud emergence
The appearance of pink bud was recorded visually from the selected trees and
accordingly the date of emergence of pink bud was recorded.
3.3.3.2 Date of first flowering
The date when 4-5 floral buds had opened was considered to be the date of first
flowering.
3.3.3.3 Date of full bloom
The date on which about 75 – 80 per cent of the flowers were open was considered to
be the date of full bloom.
3.3.3.4 Duration of flowering
Periodical observations relevant to flowering were recorded from the start of
flowering and continued till the flowering ended. The duration of flowering was worked out
which was expressed in days.
3.3.4 YIELD CONTRIBUTING TRAITS
3.3.4.1 Spur density
Four shoots of uniform length (30 cm) were selected around the tree periphery and
marked. Before flowering, number of spurs was counted from these selected shoots and spur
density were subsequently worked out in terms of number of spur per centimeter of shoot
length.
3.3.4.2 Fruit set
Total number of flowers on the selected trees was counted. Three weeks after petal
fall, the total number of fruits was counted. The final fruit set was then calculated using the
formula proposed by Westwood, 1978.
Fruit set % =
x 100
3.3.4.3 Days from full bloom to harvest
The days from the date of full bloom to the date of harvesting was considered as the
days from full bloom to harvesting and the mean value was worked out.
3.3.4.4 Number of fruits per tree
Fruit count was recorded at the time of harvest from the selected trees.
3.3.4.5 Fruit yield per tree
The final yield of fruits under different combinations and spacing was recorded at the
time of harvest by weighing the total fruits retained in particular tree. The yield was
expressed in kilogram per tree (kg/tree).
3.3.4.6 Productivity
Productivity was calculated by multiplying the average fruit yield per tree with the
total number of trees per hectare in each of the planting density and was expressed as metric
tons per hectare (MT/ha).
3.3.4.7 Yield efficiency
Yield efficiency is a measure to estimate yield potential per unit area. Yield efficiency
was calculated by dividing fruit yield kg/tree with TCSA, CA, TCV and leaf area, as per
method suggested by Westwood (1978). The values obtained were expressed as kg/cm2 of
TCSA, kg/m3 of TCV, kg/cm2 of CA and kg/cm2 of leaf area.
3.3.5 FRUIT QUALITY CHARACTERISTICS
3.3.5.1 Physical
3.3.5.1.1 Fruit dimension
Ten fruits were randomly selected and polar length of selected fruits was recorded
between calyx and styler end and diameter of the same fruits was recorded by measuring
distance between cheeks of fruits with the help of digital vernier caliper and the values were
expressed in millimeter (mm).
3.3.5.1.2 Fruit weight
Ten selected fruits taken for recording the fruit size data were weighed on electronic
top pan balance and the average fruit weight was expressed in gram (g).
3.3.5.1.3 Fruit volume
The volume of fruits was computed by water displacement method. Ten fruits which
were selected for recording fruit weight were immersed in a measuring cylinder filled with
water to obtain certain graduation. The difference between initial and final readings gave the
measurement of volume of fruit samples. The results were averaged and expressed in cubic
centimeter per fruit (cm3/fruit).
3.3.5.1.4 Fruit firmness
The firmness of fruit at the time of harvest was taken with the help of penetrometer
model FT 327. Thin layer of fruit skin was peeled off with stainless steel knife at three places
on the single fruit and the penetrometer was inserted inside the fruit. The pressure was
recorded and expressed in kilograms per square centimeter (kg/cm2).
3.3.5.1.5 Shape index
Shape Index was calculated by dividing the corresponding value of length to the
diameter of the fruit.
3.3.5.1.6 Specific gravity
Specific gravity of fruits was recorded by measuring weight in air and in water using
the formula as
Specific gravity =
3.3.5.1.7 Fruit colour
After harvesting the fruits were observed visually for colour expression with the help
of colour chart of the Royal Horticultural Society, London according to Wilson (1941).
3.3.5.2 Biochemical
3.3.5.2.1 Total soluble solids
Total soluble solids (TSS) content in fruit samples was determined by Erma–Hand
Refractometer (0 to 32 °Brix). The refractometer was calibrated with distilled water before
use. A few drops of fruit juice were placed on the prism and reading was recorded. The
values of total soluble solids were expressed as °Brix (°B).
3.3.5.2.2 Titratable acidity
Titratable acidity was determined by standard method suggested by Ranganna (1995).
Twenty five gram of fruit pulp was thoroughly homogenized with distilled water in an
electric blender and volume was made to 250 ml in a volumetric flask. Out of this, 50 ml
extract was taken for the estimation of acidity and the rest used for determining the total and
reducing sugars. 50 ml of the extract as obtained above was then filtered through Whatman
No. 1 filter paper. 25 ml of this discoloured juice was then titrated against N/10 NaOH
solution, using phenolphthalein as an indicator till it gave pink colour as end point. The
titratable acidity was calculated in terms of malic acid content and expressed in percentage.
Titratable acidity (%) =
x 100
Where, T
=
Titre value
N
=
Normality of NaOH
V1
=
Volume made
E
=
Equivalent weight of malic acid
V2
=
Volume of aliquot taken
W
=
weight of sample (g)
3.3.5.2.3 Total sugars
The sugar content of the fruit samples was determined by volumetric method based on
the principle that sucrose content of fruit is quantitatively hydrolyzed to glucose and fructose
in the presence of hydrochloric acid as per the method suggested by Ranganna (1995). The
remnant of the 200 ml extract left from titratable acidity was taken in a 250 ml volumetric
flask and 5 ml of 45 per cent standard lead acetate was added. After 5-10 minutes, 5 ml of 22
per cent potassium oxalate was added to precipitate the excess of lead acetate and volume
was made 250 ml followed by the filtration of the solution. Afterwards, 50 ml of the filtrate
was taken and hydrolyzed by adding 5 ml of concentrated HCl. The solution was left
overnight for hydrolysis at room temperature. The next day, the excess of was neutralized
with saturated NaOH solution and final volume of 250 ml with distilled water was made. The
total sugars was then estimated by titrating boiling mixture of 5 ml each of Fehling A and
Fehling B against hydrolyzed solution using methylene blue as an indicator. The end point
was indicated by the appearance of brick red colour. The total sugars content expressed as
percentage of fresh weight of fruit pulp.
Total sugars (%) =
x 100
3.3.5.2.4 Reducing sugars
The remaining unhydrolyzed, deleaded and clarified solution obtained from the total
sugars estimation was titrated against a boiling solution of 5 ml each of Fehling A and
Fehling B using methylene blue as an indicator (Ranganna 1995). Reducing sugars content
was expressed as percentage of fresh pulp weight.
Reducing sugars (%) =
3.3.5.2.5 Non-reducing sugars
x 100
The amount of non-reducing sugars was calculated by subtracting the reducing sugars
from total sugars and multiplying the difference by a standard factor i.e. 0.95. The results
were expressed as per cent sugars.
3.3.5.2.6 TSS:acid ratio
The ratio was obtained by dividing the corresponding value of TSS to the malic acid
content of fruit juice.
3.3.5.2.7 Anthocyanins content
Total anthocyanin present in all the samples was determined by the method suggested
by Ranganna (1997). The procedure involved extraction of the anthocyanin with ethanolicHCI and measurement of colour at the wavelength of 535 nm against blank of ethanolic-HCI
using a Nukes UV-VIS spectrophotometer. The anthocyanins were calculated and expressed
as mg per 100 mg using the formula given below:
Total OD/100mg =
x 100
Total anthocyanins content (mg/100 ml) =
Where, E (Extinction coefficient) = 98.20
3.3.6 Physiological parameters
3.3.6.1 Total Carbohydrates content
3.3.6.1.1 Collection and preparation of samples
Fully grown leaves from the current season's growth of each experimental tree were
collected. The leaf samples collected were brought directly to the laboratory thoroughly
washed, surface dried and was subsequently put into paper bags which were kept in hot air
oven at 65± 5˚C for final drying. The dried samples were ground in a pestle and mortar and
stored in butter paper bags for the estimation of total carbohydrates. Fruiting and non-fruiting
shoots were sampled 10 days before harvesting and brought to laboratory for estimation of
total carbohydrate using standard procedure. For the estimation of carbohydrate in fruits,
fully matured fruit were collected from marked tree.
3.3.6.1.2 Determination of total carbohydrates
Anthrone reagent method given by Hodge and Horfreiter (1962) was used for the
estimation of total carbohydrate content. 100 mg of dried sample was taken in a beaker and
hydrolyzed with 5 ml of 2.5 N HCl for 3 hours on a boiling water bath. After cooling, sodium
carbonate was added to neutralize the acid until the effervescence ceased. The final volume
was made to 100 ml with distilled water, followed by centrifugation. From the supernatant,
1ml aliquot was taken in a test tube to which 4 ml of anthrone reagent was added and the test
tube was kept in a boiling water bath for 8 minutes and cooled rapidly. The absorbance of
green to dark green colour developed was recorded at 630 nm on Nukes UV-VIS
spectrophotometer. Standard curve was prepared by using different concentration of glucose
and from the standard curve; the carbohydrate present in a sample was calculated using the
following formula:
Total carbohydrates =
Total carbohydrate was expressed as mg g-1 on dry weight basis.
3.3.6.2 Total Leaf Chlorophylls
The representative sample of ten fully grown leaves from the current season's growth
of each experimental tree was detached in July during morning hours, immediately placed in
ice box and brought to the laboratory (Halfacre et al. 1968). The samples were then kept in
the refrigerator below 0˚C to avoid any degradation of chlorophyll pigments.
Extraction
Leaves from each sample were washed and chopped into fine pieces under subdued
light and 100 mg of chopped material was placed in vial containing 7 ml of dimethyl
sulphoxide (DMSO). The contents of the vials were incubated at 65˚C temperature for half an
hour and then extract was transferred to graduate test tube and final volume was made to 10
ml with DMSO (Hiscox and Israelstam, 1979).
Estimation
The optical density of the extract was recorded on Nukes UV-VIS Spectrophotometer
on 645 and 663 nm wavelengths against DMSO blank solution and total chlorophyll content
was calculated by using formula:
Total chlorophylls (mg/g) =
×V
Where,
V
=
Volume of extract used
a
=
Length of light path in cell (usually 1cm)
W
=
Weight of the sample (g)
A663
=
Absorbance at 663 nm
A645
=
Absorbance at 645 nm
The results thus obtained were expressed as mg of total chlorophyll per gram of fresh weight.
3.3.6.3 Photosynthetic rate, transpiration rate and stomatal conductance
Photosynthetic rate (µmol CO2 m/2/s), transpiration rate (m mol/m2/s) and stomatal
conductance (mol H2O m/2/s) were measured between 9:00 AM-12:00 noon using LI6400XT (LI-COR) portable photosynthesis system. Reference air (CO2) (400 µmol/s) and
light intensity (1000 µmol m2 /s) were set. Measurement was performed on five mature leaves
around the periphery of tree as described by Erf and Proctor (1987).
3.3.7 Leaf Nutrient Analysis
3.3.7.1 Collection and preparation of samples
Leaves including petioles from mid terminal shoots of current season's growth
(Kenworthy, 1964) were collected from the marked trees. The leaf samples collected were
brought directly to the laboratory, thoroughly washed first under tap water, followed by 0.1 N
HCl and distilled water as suggested by Chapman (1964). The washed leaf samples were
spread on filter paper sheets for surface drying and were subsequently put into paper bags
which were kept in hot air oven at 65± 5˚C for 48 hours for final drying. Mature fruits were
also sampled from the selected trees for nutrient analysis. The fruit samples collected were
brought directly to the laboratory, thoroughly washed first under tap water followed by
distilled water. For determination of nutrients in flesh whole fruit was kept into paper bags
which were kept in hot air oven for final drying while for skin nutrient analysis, only peel
were kept in petri dishes which further were kept in hot air oven. The dried samples were
ground in a pestle and mortar and stored in butter paper bags for the estimation of various
nutrient elements.
3.3.7.2 Digestion of samples
The digestion of samples (1 g) for estimation of total nitrogen was carried out in
concentrated H2SO4 in the presence of digestion mixture of different chemicals viz.,
Potassium sulphate (400 parts), Copper sulphate (20 parts), Mercuric oxide (3 parts) and
Selenium (1 part). For the estimation of P, K, Ca, Mg and micronutrients (Fe, Cu, Zn and
Mn), 0.5 g of the sample was digested in di-acid mixture of HNO3 and perchloric acid in the
ratio of 4:1 as suggested by Piper (1966).
3.3.8 Determination of Nutrient element
3.3.8.1 Nitrogen
Distillation and titration:
Nitrogen (N) content of leaf and fruit samples (peel and flesh) was determined by
micro-Kjeldhal’s method. Five ml aliquot (digested) was taken in micro-Kjeldhal apparatus
and 5 ml NaOH (40%) was added slowly to it. Ammonia liberated was collected in 25 ml
boric acid (4%) solution containing mixed indicator prepared from bromocresol green and
methyl red. The boric acid changed its colour to bluish green as soon as it came in contact
with ammonia. It was titrated against standard hydrochloric acid until wine-red colour was
obtained and results were expressed in per cent on dry weight basis.
3.3.8.2 Phosphorus
Phosphorus (P) content was estimated by vanado-molybdate phosphoric yellow
colour method. Five ml of aliquot (digested) was pipette out in a 25 ml volumetric flask and 5
ml of vanado-molybdate reagent was added. Solution was then diluted to 25 ml with distilled
water and allowed to develop colour for half an hour. After the development of colour,
concentration of P in the solution was recorded on Nukes UV-VIS Spectrophotometer at 470
nm wavelengths and a blank was run simultaneously to adjust zero absorbance. Leaf and fruit
(peel and flesh) phosphorus was expressed in per cent on dry weight basis.
3.3.8.3 Potassium, Calcium and Magnesium
Leaf and fruit (peel and flesh) K, Ca and Mg content was estimated using Agilent
5110 ICP-OES and the results were expressed in per cent on dry weight basis.
3.3.8.4 Micronutrients (Fe, Mn, Zn and Cu)
Total leaf Fe, Mn, Zn and Cu content were determined by using Agilent 5110 ICPOES at 238.20, 257.61, 213.86 and 327.40 wavelengths, respectively and expressed in ppm
on dry weight basis.
3.3.9 Fruit nutrient ratios
3.3.9.1 Potassium to calcium (K/Ca)
K/Ca ratio was computed by dividing the corresponding value of peel K to the peel
Ca and similarly this ratio was calculated for fruit flesh.
3.3.9.2 (K+Mg)/Ca
(K+Mg)/Ca ratio was computed by dividing the corresponding value of peel K+Mg to
the peel Ca and similarly this ratio was calculated for fruit flesh.
3.4 Statistical Analysis
The experiment was laid out in Randomized Block Design (RBD). Six stionic
combinations (treatments) were taken and replicated four times both at spacing 2.5 m × 2.0 m and
three stionic combinations were taken and were replicated seven times at spacing 2.0 m × 1.5 m.
Mean of all the treatments shall be analyzed statistically by ANOVA using OPSTAT and
MS-Excel. The significance of variation among the treatments shall be calculated by applying
‘F’ test and critical difference at 5 per cent level will be calculated to compare the mean
values of treatments for all the parameters studies according to Panse and Sukhatme (1989).