Open LabManual2015 2
Open LabManual2015 2
Open LabManual2015 2
B.S. Dwivedi
G.D. Sharma
F.C. Amule
G.S.Tagore
A.K. Dwivedi
N.G. Mitra
Organized by
Centre of Advanced Faculty Training
Department of Soil Science & Agricultural Chemistry
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur 482 004 (M.P.)
© 2015, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur 482004 (M.P.), India
Published by
Director
Centre of Advanced Faculty Training
Department of Soil Science & Agricultural Chemistry
Jawaharlal Nehru Krishi Vishwa Vidyalaya
Jabalpur 482 004 (M.P.), India
Printed at
Fortune Graphics & Scanning Centre
Jabalpur 482 002 (M.P.), Ph.: 0761-4069025
Laboratory Manual on Soil and Plant Analysis
National Training
on
Climate Resilient Soil Management Strategies for
Sustainable Agriculture
(14th October to 3rd November, 2015)
B. S. Dwivedi
G.D. Sharma
F.C. Amule
G.S. Tagore
A.K. Dwivedi
N.G. Mitra
Sponsored by
Indian Council of Agricultural Research
Organized by
Centre of Advanced Faculty Training
Department of Soil Science and Agricultural Chemistry
Jawaharlal Nehru Krishi Vishwavidyalaya
Jabalpur–482004 (M.P.)
INDEX
1. Method of Collection of Soil and Plant Samples for Their Testing 1-4
3. Creation of spatial variability maps of soil nutrient using geo-statistical tool 9-10
14. Gamma Irradiation and its Importance for Food Preservation 30-34
20. Definitions 41
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To meet objective of soil testing is divided into 4 recorded. Under intensified cultivation sampling
phases: should be done every year at the same time. For
1. Collection of soil samples sampling of soft and moist soil, the tube auger,
2. Extraction and determination of the available spades, or khurpi is quite satisfactory for sampling.
nutrients A screw type auger is more convenient on hard/dry
3. Interpretation of analytical results soil while, post whole auger is useful for sampling
4. Providing recommendation excessive wet or rice field. If spade or khurpi is
5. Follow up of results and evaluation of the used a V shaped cut may first be made up to the
recommendations made plough layer. A uniform 15 cm thick slice taken
Procedure for soil testing out. Auger are generally useful for deep profile
The procedure for testing the soil to meet sampling.
these objectives are divided into the following How to take representative soil sample
phases: 1. Divide the field into separate units depending
(i) Collection of soil samples and its on variation in slope, colour, texture, crop
preparation growth and management.
(ii) Extraction and determination of nutrients 2. Remove the debris, rocks, gravels etc from the
and physico-chemical properties of the soil. surface before collecting soil sample.
(iii) Interpretation of analytical results 3. Make a V shape cut into the soil to a depth of
(iv) Recommendation and follow up of results sampling (0-15 cm) and obtain 2 to 3 cm thick
and evaluation of recommendations. vertical slices along the depth.
Collection of soil samples 4. Collect 10-15 samples randomly in zig-zag
Since soil is a very heterogeneous mass manner from each field.
and the greatest source of error is usually soil 5. Mix samples by quartering method and
samples itself hence, the soil sample collected approximate 500 g of sample is retained.
should be representative of the area sampled and 6. The sample must be kept in a clear cloth or
should also be uniform. Variations in slope, polythene bag.
texture, colour, crops grown, and management 7. Label it with suitable description and
levels must be taken in to account. Recently identification marks.
fertilized plot, bunds and channel, spot near tree, 8. Send the soil samples to soil testing laboratory
wells, compost pits and other non-representative along with the information sheet.
locations must be avoided while sampling. When
crops are planted in rows, samples can be drawn in Information sheet : The soil sample thus collected
between the lines. The sample should be taken in a must be furnished important information like –
zig-zag manner. A representative composite soil 1. Sample number
samples can be composed of 8 to 20 sub samples 2. Name and address of the farmers.
from a uniform field (Jones 1988). A common error 3. Details of the field and site. Local name
in soil sampling occurs when the top few cm of soil field,Khasra no etc.
are dry and are not included in the normal sample. 4. Date of sampling
Hence, the value of soil test depends on how well 5. Name of crop and variety to be sown
the sample represents a field. 6. Source of irrigation
A variety of factors such as depth of 7. Whether the crop in the subsequent season will
sampling, number of cores of composite sample, be irrigated or un-irrigated.
season, crop etc. influence the collection of soil 8. Name of crops and fertilizer used in previous
samples. For field crops a sampling depth upto 15- years.
30 is desired while, 10 cm depth is normally 9. Date of harvest of the previous crop.
sufficient for sampling for the pasture crops (Rao, 10. Any other problem observed in the field.
1995). For deep rooted crops, horticultural crops or
under dry farming conditions sample from various
depths may be preferred. For immobile nutrient,
testing of sample to tillage depth may give
satisfactory results but for testing of mobile
nutrients (NO+3-, Cl-, SO4-- etc.) sample should be
taken to a depth of 60 cm. In saline alkali soils, salt
crust (visible) on the top soil surface should be
taken separately along with the depth of sampling
DIVISION OF AREA
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Plant sampling
The particular method to be used will depend upon
the kinds of plants to be sampled determinations to
be made and over all objectives.
i) Take enough individual plant or plant parts to
overcome the factor of plant variability.
ii) If correlation with soils are to be made make
sampling distribution representative of a given
SELECTION OF SAMPLING POINTS
soil area.
iii) Collected samples should be cleared to avoid
Preparation of soil sample for testing contamination from fumes or decomposition.
1. Spread sample for drying on clean cloth, iv) Suitable methods should be employed for
plastic or brown paper sheet. grinding.
2. Remove the stone pieces, roots, leaves & other If testing is to be done for conforming
un-decomposed organic residues from the deficiency/ toxicity as displayed by visual
samples. symptoms then 100 to 200 leaves showing
3. Large lumps of moist soils should be broken. particular visual symptoms should be collected.
4. After air drying the samples should be crushed If the testing is to be used for continued
gently and sieved through a 2 mm sieve. fertilizer guidance then sampling should be done by
5. About 250 g of sieved sample should be kept dividing the whole area into units and then
in properly labeled sample bag for testing. collecting the samples from each unit. Units may
Appropriate time for soil sampling be selected on the basis of slope, texture, depth and
An ideal time for soil sampling is just after colour of soil etc. in case of orchards at least 20%
harvest of the rabi crops, of the trees should be sampled. Seven to 10 months
Precautions to be taken during collection of soil old spring cycle leaves be selected. Those leaves
sampling should be so selected so that they represent average
1. Remove all debris from surface before foliar condition.
collection of soil sample. In fact plant sampling can be modified
2. Avoid taking sample from upland and low land depending upon the purpose for which the sample
areas in the same field. is required. In studying the influence of factors as
3. Take separate sample from the areas of the stage of growth, cultural practices, manurial
different appearances. treatment or grazing on mineral composition of a
4. In row crop take sample in between rows. crop of pasture whole above ground part of the
5. Keep the sample in a clean bag. plant from selected areas will be required. When
6. A sample should not be taken from large area composition of different species of plants growing
(more than 1-2 ha). on various soil types is needed, the material sample
7. Sample for micronutrient analysis must be should be restricted to pure species at comparable
collected by steel or rust free khurpi/auger and stage of growth. To study the effect on nutrient
kept in clean polythene bag. uptake the desired investigations of soil types plant
Plant analysis should be taken from a small area typical of the soil
Plant analysis is used as a diagnostic type and a composite soil sample should also be
technique to determine the nutritional status of collected to represent the same area.
plants and fertilizer needs. Mineral composition of
plant is influenced by many factors which are also Interpretation of the results
to be considered. Fundamental advances in Results of foliar diagnosis must be
quantitative studies at relationship between nutrient confirmed by plant analysis and concentration of
and crop yield have been attempted. In older nutrient in the soil. It will give general relationship
methods of analysis the quantities of minerals in between growth and quantity of the nutrient
the manure plant were determined but under the absorbed.
newer concept of analysis only functionally Percentage of an element in the plant
assimilating portion of plant is analyzed. Hence, alone cannot be taken as the basis of judging the
sampling of the assimilatory portion of the plant is deficiency/sufficiency of the element. However, if
important. it is used then physiological age of the leaf must be
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Vertisoils or deep black soils occur globally under They clayey soils that shrink and swell
various parent materials and environmental extensively upon changing soil moisture
conditions (Table 1). conditions. Vertisols exhibit unique morphological
Table 1. Distribution of Vertisols and associated properties such as the presence of slickensides,
soils wedge-shaped aggregates, diapir (mukara), and
Juris- Location Area % of gilgai. Shrink-swell phenomena are the dominant
Diction (m ha) Gross pedogenic processes in Vertisols and are attributed
Black to changes in interparticle and intraparticle
soils
porosity with changes in moisture content.
Continent Africa 105.0 38.7
Definition of Vertisols :
Asia & far East 70.3 25.9
(Mainly India) Taxonomically for defining Vertisols,
there must be
Australia 48.8 17.7
1. A layer 25 cm or more thick with an upper
Latin America 27.0 9.9
boundary within 100 cm of the mineral soil
North America 10.0 3.7
surface, that has either SLICKENSIDES or
Near & Middle East 5.7 2.1
WEDGE SHAPED PEDS that have their long
Europe 5.4 2.0
axes tilted 10 to 60o from the horizontal; and
TOTAL 271.4 100 2. A weighted average of 30 % or more clay in
Country India 70.3 25.9 fine earth fraction either between the mineral
Australia 48.8 17.7 soil surface and a depth of 18 cm or in Ap
Sudan 43.4 16.6 horizon, whichever is thicker and
USA 18.1 6.7 3. 30 % or more clay in fine earth fraction of all
CHAD 15.5 5.7 horizons between a depth of 18 cm and either
China 11.6 4.3 a depth of 50 cm or a densic, lithic or
Others 64.5 23.7 paralithic contact, a duripan, or a petrocalcic
( in parts) horizon if shallower and
TOTAL 271.4 100 4. Cracks that open and close periodically.
India MS 24.2 34.4 Vertisols are significant global resources
MP 21.2 30.1 that serve as the lifeline in subsistence agriculture
GUJ. 4.9 7.0 due to their high productivity.
AP 9.4 13.4 Efforts towards comprehension and
KTK. 5.8 8.2 successful utilization are imperative for continued
TN 2.6 3.7 productivity and long term sustainability of these
RAJ. 1.1 1.6 resources for current and future civilizations.
UP 1.1 1.6 Morphology of a soil is best evaluated from the in
TOTAL 70.3 100 situ examination of the soil profile. A recently dug
MP Vertisols 8.0 37.7 pit large enough for observation of a pedon is
Inceptisols 8.6 40.6 desirable. Old exposures such as road banks and
Entisols 4.2 19.8
ditches are acceptable only for preliminary studies
because morphological features often become
Alfisols 0.4 1.9
altered after prolong exposure.
TOTAL 21.2 100
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which the parent material is basic and gives rise under the influence of wet dry climate.
to the formation of 2:1 expanding lattice silicates
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Soil fertility, a dynamic natural property, fluctuates was collected, processed and analyzed for soil
nutrients in the laboratory.
throughout the growing season of each year due to
alteration in the quantity and availability of nutrients Geo-statistical analysis using GIS
by the addition of fertilizers, manure and compost. In the 1970s a new technique called
Due to the bulkiness, weight and lack of “Kriging” and its variants were widely recognized as
technologies to apply manure, it is restricted to an important spatial interpolation technique in land
fields near the settlements. This always causes over- resource inventories (Hengl et al., 2004). It is a fact
or under application of the required plant nutrients that soil properties vary from place to place even
and therefore, creates a nutrient gradient across the within the same field. As a result, the spatial
cultivated lands and leads to other undesirable structure can vary at scales that differ by several
environmental impacts. The resulting spatial orders of magnitude from a few meters to hundred
variability of soil fertility poses great challenge to kilometers. Such variation with distance can be
land management and reflects in variable yields over described well with the help of geo-statistics (Simon
farmlands. Some earlier studies showed that the et al., 2013). Because “Kriging” assumes the normal
effect of variability of soil properties on crop distribution for each estimated variable, it is
performance could be detrimental, especially when necessary to check whether the nutrient contents in
the fields were patchy. Ideally, application rates soils are approximately normally distributed or not.
should be adjusted based on estimates of the A normal distribution was estimated based on
requirements for optimum production at each skewness values and the variable datasets having a
location because there is high spatial variability of skewness ranged between -1 to 1 were considered
N, P, and K within individual fields. From the normally distributed (Ortiz et al., 2010). The spatial
literature point of view, it is revealed that high costs dependency of selected soil parameters was
spent in classical methods for collecting soil samples analyzed using semi-variogram analyses with
and the results have been restricted to only mean normalized data. Semi-variogram is computed as
values of specific classes. In the old studies latitude half the average squared difference between the soil
and longitude of sampling sites are missing. Keeping properties of data pairs and semi variance is
this in mind, the present investigation was focused estimated using equation
on generation of spatial variability maps of soil
nutrients using geo-statistical tool in Arc GIS
environment and the use of GPS receivers to map as:
the exact location of sampling sites, which can be Where N(h) is the number of data pairs within a
used as input data in GIS and run the queries to given class of distance and direction, z(xi) is the
know various properties of soil. value of the variable at the location xi and z(xi + h)
Methodology is the value of the variable at a lag of h from the
The sampling sites decided randomly location xi.
distributed over agricultural land of the study area
can be perfectly located by considering land use and
soil association maps, topography and heterogeneity
of the soil type. Field data collection and soil
sampling were carried out by using GPS by
navigating those points during off season from the
agricultural land to avoid the effect of fertilization
during crop cultivation. For each main sampling
point, 1.0 kg of representative composite soil sample Fig. 1 Semi-variogarm parameters
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Then, this was generally fitted with a which could have been derived from using the
theoretical model, such as Exponential, Spherical sample mean alone(Agterberg, 1984),
and Gaussian models (Goovarts, 1999). Choice of
the best-fitted model was based on the lowest
residual sum of square (RSS) and the largest
where z is the sample mean. If G = 100, it indicates
perfect prediction, while negative values indicate
that the predictions are less reliable than using
sample mean as the predictors (Schloeder et al.,
2001).
Case study
From Hoshangabad district, 305 soil
samples were collected using GPS and analysed for
soil properties. Then spatial variability maps of pH,
OC Zn and B using geo-statistical tool in GIS
environment was generated.
coefficient of determination (R2). Nugget is the References:
variance at distance zero, sill is the semi- variance
value at which the semi-variogram reaches the upper • Agterberg, FP 1984. Trend surface analysis. In
bound after its initial increase, and range is a value Spatial Statistics and Models (eds Gaile, G. L.
(x axis) at which one variable becomes spatially and Willmott, C. J.), Reidel, Dordrecht, The
independent. Netherlands:147–171.
The nugget to sill ratio was used to define • Cambardella CA, Moorman TB, Novak JM,
different classes of spatial dependence for the soil Parkin TB, Karlen DL, Turco RF and Konopka
properties. According to Cambardella et al., (1994) AE. 1994. Field scale variability of soil
nugget/sill ratio of 25%, 25-75% and >75% were properties in Central Iowa soils. Soil Sci. Soc.
classified as having strong, moderate and weak of Am. J.58:1501–1511.
spatial dependence, respectively.
• Goovaerts, P.1999. Geo-statistics in soil
Accuracy of the prediction was evaluated
science: state-of-the-art and perspectives.
through cross-validation approach
Geoderma 89:1-45.
Mean absolute error (MAE), and mean squared error
(MSE), measure the accuracy of prediction, whereas • Hengl T, Heuvelink GBM and Stein A. 2004. A
goodness of prediction (G) measures the generic framework for spatial prediction of soil
effectiveness of prediction criteria (Santra et al., variables based on regression-kriging.
2008). MAE is a measure of the sum of the residuals Geoderma. 120:75–93.
(e.g. predicted minus observed) as stated by Voltz
• Santra P.,Chopra UK. and Chakraborty D. 2008.
and Webster (1990).
Spatial variability of soil properties and its
application in predicting surface map of
hydraulic parameters in an agricultural farm.
where zˆ(xi ) is the predicted value at location i. Curr.Sci. 95,(7).
small MAE values indicate few errors. The MAE • Voltz M and Webster R.1990. A comparison of
measure, however, does not reveal the magnitude of kriging, cubic splines and classification for
error that might occur at any point and hence MSE predicting soil properties from sample
was calculated, information. J. Soil Sci. 41: 473–490.
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Electrical Conductivity :
Amount of soluble salts in a sample is pH Meter :-
expressed in terms of the electrical conductivity
(EC) and measured by a conductivity meter. The
instrument consists of an AC solubridge or
electrical resistance bridge and conductivity cell
having electrodes coated with platinum black. The
Instrument is also available as an already
calibrated assembly (Solubridge) for representing
the conductivity of solutions in dSm-1 (deci
Siemen per meter) at 250C.
Principle
A simple wheatstone bride circuit is used
to measure EC by null method. The bridge
consists of two known and fixed resistance r1, r2,
one variable-standard resistance r4 and the
unknown r3. The variable resistance r4 is adjusted
until a minimum or zero current flows through the
AC galvanometer. At equilibrium.
r1 r3 r1
r2 = r4 or = r3 r2 X r4
Reagents :
Potassium chloride: Dissolve 0.7456g dry
potassium chloride (AR) in distilled water and
make up the volume to one litre.
Procedure :
Take 20 g of soil in 100 ml beaker, add
40 ml of distilled water and shake intermittently
for 30 minutes. Determine the conductivity of the
supernatant liquid with the help of conductivity
meter. The electrical conductivity of saturation
extract (E.C.) is also determined for salinity
ratings.
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The majority of mineral surface soils range from Apparatus and Reagents :
1.2 to 3.5% organic carbon. Since soil organic 1. 500 ml conical flasks.
matter averages about 58% carbon, it follows that 2. Pipette
soils generally range from about 2 to 6 % organic 3. Burette
matter (% O.M. = %C x 1,724. The factor 1.724 = 4. Phosphoric acid 85%.
100/58). There is also a close relationship between 5. Sodium fluoride 2%.
carbon and nitrogen in soils. Most organic matter 6. Sulphuric acid 96 % containing 1.25 %
average about 5% nitrogen so that the N : C ratio Ag2SO4.
is 1:11.6. Therefore by multiplying the soil 7. Standard 1N K2Cr2O7 – 49.04 g/liter.
organic matter percentage by 0.05 an approximate 8. Standard 0.5 N Fe (NH4)2 (SO4)2. 6H2O 196 g
value for the soil nitrogen, percentage is obtained. in 800 ml water containing 20 cc H2SO4 and
In soil the chief source of some of the diluted to1 litre.
nutrients essential for plant growth is organic 9. Diphenylamine – 0.5g in 20cc water and add
matter, such nutrients are N, S and boron is also 100 ml conc. H2SO4.
largely derived from organic matter.
Procedure :
1. Weigh 1g soil sample in 500 ml conical flask.
Principle :
Add 10 ml of 1 N K2Cr2O7 and 20 ml conc.
A suitable quantity of the soil is digested
H2SO4 (containing Ag2SO4). Mix thoroughly
with chromic acid and sulphuric acid making the
and allow reaction to proceed for 30 minutes.
use of heat of dilution of sulphuric acid soil is
2. Dilute the reaction mixture with 200 ml water
digested and organic matter of the soil is oxidized.
and 10 H3PO4 add 10 ml of NaF solution and
Excess of chromic acid left over unreduced by the
2 ml of diphenylamine.
organic matter of the soil is determined by a
3. Titrate the solution with standard FAS to a
titration with standard Ferrous Ammonium
brilliant green colour. A blank without soil
sulphate solution using diphenylamine as
should be run simultaneously.
indicator.
In this exercise, chromic acid in the
Observations & Results :
presence of excess H2SO4 is to be used as an
Weight of sample - 1g
oxidizing agent for oxidizable organic matter of
Normality of K2Cr2O7 used - 1N
the soil. The heat of dilution of H2SO4 works as a
Vol. of K2Cr2O7 - 10 ml
standardized ferrous sulphate solution.
Normality of FAS - 0.5 N
10 0.003 x 100
4 Cr6+ + 3C 4Cr+++ + 3C4+ OC (%) = (Blank - Reading) X
2H2Cr2O7+ 2Cr2(SO4)3+ Blank Wt. of soil
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Calculations :
R (Titer reading - Blank reading) × Normality of acid
-1 × Atomic weight of nitrogen × Weight of one hectare of soil
Available N (kg ha ) =
Sample weight (g) x 1000
R × 0.02 × 14 × 2.24 × 106
=
5 x 1000
Factor = R x 125.44
Interpretation of results :
Available N (kg ha-1) Soil rating
< 280 : Low
280-560 : Medium
> 560 : High
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R x 0.1 x 14 x 100
= 1 x 1000
Factor = R x 0.14
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The available potassium i.e. exchangeable and NH4OAc Soln. Adjust the flame photometer
water soluble potassium is determined by reading at zero with blank (zero K) solution and at
extracting soil with neutral normal ammonium 100 for 40 mg kg-1 K solution. Take the flame
acetate solution. The estimation of potassium is photometer readings for every dilution. Plot the
carried out by flame photometer. standard curve on graph paper by taking K
concentration on X axis and flame photometer
1. Principle : reading an y axis. This will give a factor (F) of 1
flame photometer reading = 0.4 mg kg-1 K.
The principle underlying this is that a
large number of elements when excited in a flame,
5. Procedure :
emit radiation of characteristic wave length. The
excitation cause one of the outer electron of Take 5g soil in 100 ml conical flask and
neutral atoms to move to an outer orbit of higher add 25 ml of 1N NH4OAc Soln. Shake the content
energy level or the atoms may be excited for 5 minutes and then filter through Whatman
sufficiently to lose an electron completely from No.1 filter paper. Potassium extract is measured
the attractive force of the nucleus where excited by flame photometer after calibration.
atom return to lower energy level, light at
characteristic wave length is emitted. Excited 6. Calculation :
atoms or ions give line radiation at very definite RxFx25x100x20x1.121
wave length and thus K gives at 404.4 and 767 Available K (kg ha-1) =
mµ. The flame photometer employees a relatively 5 x 1000
low temperature excitation and measures with a = R x F x 11.217.
photocell the emission intensity which is
proportional and to concentration in selected wave Limits of available K in soil :
length (767 mµ) and for this red filter is used. : Less than 200 K kg ha-1
Very low
2. Apparatus and reagents: Low : 200 – 250 K kg ha-1
a) Flame photometer with red filter, Medium : 250 – 400 K kg ha-1
b) Pipette, volumetric flasks and conical flask High : 400 – 600 K kg ha-1
(100 ml). Very high : More than 600 k kg ha-1
3. Reagents : 8. Precaution :
(a) Neutral Normal Ammonium Acetate :
Add 58 ml of glacial acetic acid to about a) These should not be any turbidity or
600 ml H2O and then add 70 ml of concentrated suspended particles in extract, it will chock
ammonia (sp. gr 0.90) Dilute the solution to one the capillary feeding tube .
litre. Then adjust pH of solution at 7.0 with the b) The gas and air pressure should be constant.
help of ammonia or acetic Acid or this can be c) If sample reading goes beyond 100 then
prepared by dissolving ammo. Acetate dilute the extract.
(CH3COONH4) (77.08 eq.wt.) directly in H2O and
volume to be made one litre and then adjust the 9. Determination of k in plant sample :
pH 7.0 . (a) Wet digestion :
(b) Standard Potassium Solution : Place 1-2g of ground plant sample in
Dissolve 1.9066 g of dried KCl (AR) in 100ml digestion flask. Add 20-25 ml of acid
distilled water and dilute to one litre. This is 1000 mixture Acid mixture 750 ml conc. HNO3 + 150
mg kg-1 K solution. 100 ml of this solution diluted ml conc H2SO4 + 300 ml of HClO4 and mix the
to 1 lit. to make 100 ppm K solution. contents of the flask by swirling well. Heat the
4. Preparation of the standard curve : flask at a low temp and then slowly increase the
Take 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 ml flame or temp. of hot plate. Completion of
of 100 mg kg-1 K solution in different 25 ml digestion is confirmed when liquid is colorless.
volumetric flasks. Make up the volume with 1N After cooling, add 20-25 ml H2O and filter
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References :
Black, C.A. (1965) Methods of soil analysis Part I
Am. Soc. Agron. Inc. Publi. Madison
Wisconsin USA.
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designed and operated in such a way that the lines simultaneous complexing of Zn, Cu, Fe and Mn,
to be measured are sharp, of stable intensity and Cd, Co, Ni and Pb (Lindsay and Norvell, 1978).
free from background. Buffering of extractant in a slightly alkaline pH
range (7.3) by including soluble Ca2+, avoids the
(b) Atomizer-Burner assembly :
dissolution of CaCO3 with the release of occluded
A means of producing atomic vapours of
micronutrients due to CO2 partial pressure of
the element to be analyzed. The solution to be
approximately 10 times that in atmosphere, as the
analyzed is drawn by capillary and converted into
soil contains slightly higher CO2 levels than found
stream of compressed air to a fine spray which
in the atmosphere.
after condensation of larger droplets is mixed with
(a) Extracting solution : (0.005 M DTPA)
the fuel gas acetylene and burnt in a long flame (at
Dissolve 1.9679g of DTPA (Diethylene tri
2100-2400oC) in a stainless steel burner.
amine penta acetic acid) + 13.3 ml TEA
(c) A Monochromator : (Triethanol amine) + 1.47g CaCl2 .2H2O in
It isolates the absorbing resonance lines 200 ml distilled water, dilute to 900 ml, adjust
from other non absorbing lines. When the light pH 7.3 with 6N HCl while stirring and then
coming from the HCL, after traversing the flam, make upto 1 liter and mix thoroughly.
enters the monochromator which is already set at (b) Apparatus required : Shaker (Horizontal or
the wavelength of the resonance lines of the Rotatory), iodine value flasks (100 ml
desired element, the monochromator performs its capacity) or conical flasks with glass stoppers,
function. funnels, filter paper whatman No.1, plastic
(d) A Detector : storage bottles and Atomic absorption
It measures the magnitude of absorption spectrophotometer.
of the characteristic radiation. (c) Stock Standard Solutions : The standard
(e) A Photomultiplier Tube : solutions of different micro-nutrients should
It amplifies the absorption signal and preferably be prepared by using their wires.
converts the light radiation into electrical energy. Dissolve 1g wire in a minimum volume of 1:1
(f) A readout system : nitric acid and dilute to 1000ml with distilled
It measures the absorbance in volts. It is water to obtain 1000 µg/ml solution of micro-
normally a strip chart recorder, a digital display, a nutrient, or take salts of metals as follows:
meter or printer. The presently available AAS Zn- 4.398g l-1 ZnSO4,7H2O
have features like automatic calibration with one Cu- 3.929g l-1 CuSO4,5H2O
or more standards, automatic curve corrections, Fe- 4.977g l-1 FeSO4,7H2O
automatic and foolproof gas switching and Mn- 3.598g l-1 MnSO4,H2O.
calculation of average and standard deviations in The prepared standards are also available
repetitive runs. in the market. Out of these standards, prepare
Collection and preparation of soil and plant working solution of 50 ppm. Then a series of
samples : To avoid contamination, soil samples standard solution of 0.5, 1.0, 1.5, 2.0 and 2.5
are to be collected in plastic tub, using rust free ppm may be prepared for each metal.
instrument or wood and kept in polythene lined (d) Background correction : The reading of a
cloth bags. Samples are prepared with the help of spectral line always includes any
wooden mortar and pestle and sieved through contribution from the flame and sample
2mm nylon screen/mosquito net cloth or stainless matrix. Failure to correct properly for the
steel sieve. background reading can be a source of
Similarly plant samples (leaves, grains or serious error. Although the need for fast
straw) should be washed with 0.01N HCl, rinsed background correction is most obvious with
with glass distilled water dried in oven at 65°C graphite furnace work, it is also a
and crushed with the help of stainless steel consideration with flame atomic absorption.
scissors. The most common method of background
correction in atomic absorption spectrometry
Soil extraction : DTPA offers the most favorable involves the use of a continuum source such as a
combination of stability constants for the deuterium lamp to measure the background. The
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 25
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Boron occurs as anion in soils and is required by Irrigation water containing Boron
plants in very small quantity. Water soluble B between 0.3 to 0.6 mg kg-1 can be used safely,
makes the estimate of its availability to plants. whereas, irrigating soils with water containing 1 to
Total boron in soils varies from 20 to 200 mg kg-1 3 mg kg-1 B causes toxicity of B in plants.
and available (water soluble) boron in soils ranges
from 0.03 to 12 mg kg-1 respectively. The Boron determination (Azomethine H Method) :
threshold value ranging from 0.1 to 0.5 mg kg-1 Azomethine H forms coloured complex
(water soluble B) depends upon the soil type, with H3BO3 in aqueous media. Over a
crops, and other factors, below which the response concentration range of 0.5 to 10 µg B/ml the
to applied boron may be expected. Some sensitive complex is stable at pH 5.1. Maximum absorbance
crops to boron deficiency are listed in table 1. Its occur at 420 nm with little or no interference from
availability is affected by soil pH as under: a wide variety of salts. This technique is rapid,
• Deficiency of B is generally observed in old reliable and more convenient to use than
acid leached soils. traditional procedures employing carmin,
• Availability increased with the rise in soil pH curcumin or quinalizarin (John et al., 1975).
having significant positive correlation with pH
Apparatus:
rising from 4.7 to 6.7.
(1) Spectrophotometer
• In neutral, saline and calcareous soils the B
(2) Poly-propylene tubes 10 ml capacity.
availability again decreases with the rise in soil
pH having significant negative correlation with Reagents :
the rise in pH from 7.1 to 8.1. In calcareous 1. Distilled water
soils B fixation occurs with the condensation 2. Buffer solution : Dissolve 250 g of
of borate radical into long chains in the ammonium acetate (NH4OAc) and 15 g of
presence of Ca. ethylene diamine tetra acetic acid (EDTA
disodium salt) in 400 ml of distilled water.
Table 1 : Sensitivity of crop to B deficiency Slowly add 125 ml of glacial acetic acid and
Sensitive Medium Low mix.
Alfalfa Apple Barley 3. Azomethine H reagent : Dissolve 0.45 g of
Cauliflower Cabbage Beans azomethine H in 100 ml of 1% L ascorbic
Rape seed Carrot Corn acid solution. Fresh reagent should be
Conifers Clover Grasses prepared weekly and stored in a refrigerator.
Peanuts Cotton Oat 4. Calcium hydroxide suspension : Add 0.4g
Sugarbeet Onion Ca(OH)2 to 100 ml distilled water.
Turnip Pea 5. 0.1 N HCl : Add 8.3 ml conc. HCl to 900 ml
Potato distilled water, mix, cool to room temperature
and make up the volume to 1000 ml.
Soybean
6. Calcium chloride 0.01 M Dissolve 1.11 g of
Wheat
anhydrous CaCl2 in 900 ml distilled water and
Rice
make up the volume to 1000 ml.
• In alkaline soils the availability of B is high
7. Boron standard solution : Dissolve 0.114g of
and may be even toxic for plant growth.
Boric acid (H3BO3) in distilled water and
Besides this the low moisture availability
adjust the volume to 1000 ml. Each ml
also causes B deficiency.
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contains 20 µg B. Dilute 10, 20, 30, 40 and For analysis of B pipette 1 ml of the aliquot
50ml of the stock solution to 100 ml with and proceed as for the standard curve.
distilled water to have solution with B 2. Plant digest : Take 0.5 g plant sample in
concentration of 2,4,6,8 and 10 µg of B/ml porcelain/platinum dishes Add 0.5 g Ca(OH)2.
respectively. Include a distilled water sample Ignite the sample in the muffle furnace at
for the 0.0 µg of B/ml standard solution. 550°C for 4 hours to obtain white grey ash.
Cool the dishes and moist the ash carefully
Procedure :
with little distilled water and then add 5 ml
Take 1 ml of aliquot of blank and diluted
0.1N HCl. Transfer the content in to 25 ml
B standards into a 10 ml polypropylene tube, add
volumetric flask mix and make up the volume
2 ml of buffer solution and mix. Add 2 ml of
to 25 ml with distilled water. For analysis of B
azomethine H reagent, mix and after 30 minutes
take 1 ml of the aliquot and proceed as for the
read the absorbance at 420 nm on
standard curve.
spetrophotometer. With the help of absorbance
readings of standard solutions of different 3. Water analysis : Take suitable quantity of
concentration of B the standard curve is drawn and water sample (containing 0.2 to 5.0 µg B) in
a factor for concentration of B for 1 absorbance is porcelain dishes add 2 ml Ca(OH)2 and
calculated which is utilized to calculate B in the proceed as described for soil extract. It is
soils, plant or water sample. important to keep a definite volume of aliquot
i.e. 1 ml of either soil, plant or water in final
Preparation of Extracts :
step of B determination.
1. Soil extracts : The hot water soluble extraction
procedure of Berger and Truog (1939) is being References:
used widely with slight modification of adding • Berger, K.C. and Truog, E. (1939). Boron
dilute electrolyte (0.01 M CaCl2) instead of determination in soils and plants. Ind. Eng.
water only. This provides clear, colourless Chem. Anal. Ed. 11 : 540-545.
extract which eliminates the need of charcoal • Bighman, F.T. (1982). Boron p. 501-508. In
for decolourzation. Beside this a negative error, A.L. Page (ed.) Methods of Soil Analysis.
associated with B adsorption by charcoal, is Part II Agron. Monogr. 9 ASA and SSSA,
also removed. Mandison, W.I.
Place 20 g air dry soil in 250 ml low B • Jackson, M.L. (Ed.) (1958). Boron
flat bottom flasks and add 40 ml of 0.01 M CaCl2 determination for soil and plant tissues. In
solution. Attach water cooled reflux condenser to Soil Chemical Analysis page 370-387.
the flask. Heat the flasks for 5 minutes and then • John, M.K., Chuah, H.H. and Neufld, J.H.
cool and filter the suspension in plastic bottles. (1975). Application of improved azomethine
Transfer 20 ml aliquot to evaporating H method for the determination of boron in
dish, add 2 ml Ca(OH)2) suspension and evaporate soil and plants. Anal. Lett. 8 : 559-568.
the solution to dryness. Heat the evaporating
dishes gently to destroy organic matter, cool to
room temperature, add 5 ml 0.1N HCl. Triturate
the residue with rubber policeman to ensure the
complete dissolution of the residue (Bingham,
1982).
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Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Gamma irradiation has been extensively used are perished every year. The reasons for such
losses are seasonal nature of fruits and vegetables
for food irradiation and sterilisation, killing of
production. The long distance between
fungus and micro organisms, sterilisation of
production and consumption centers and also
medical accessories and surgical equipments,
rising gap between demand and supply. The hot
high energy radiation chemistry, seed irradiation
and humid climate in the country is also quite
and semiconductor irradiation. Gamma Chamber
favorable for the growth of numerous insects and
can also be used in many other research
micro organisms that destroy stored crops and
applications which require irradiation of
cause spoilage of food every year. The spoilage
materials with ionizing radiations to varying
also occurs due to chemical and physiological
doses.
changes in stored foods. To preserve the food and
The radiation processing of food involve
food products, technologies such as freezing
the controlled application of energy from
caning sun drying pickling fermentation have
ionizing radiation such as gamma rays, electrons
been recommended by researchers but, each of
and X rays for food preservation. The gamma
these methods have its own merits and limitation.
rays and X-rays are short wavelength radiation of
The search for an alternative newer economical
electromagnetic spectrum, which includes radio
methods to preserve food and causes least
waves, microwaves, infrared, visible, and a violet
changes in sensory quality have been under taken
light. Radioisotopes such as cobalt 60 and
since long back, and has been observed that
caesium-137 emit the gamma rays, while
radiation processing of food is one of the latest
machines using electricity generate electrons and
method developed for food preservation.
X-rays. The gamma rays and electrons are
distinguished from other form of radiation by Irradiation by Gamma Chamber 5000 :
their ionizing ability. (That they are able to break
Gamma Chamber 5000 is a compact self
chemical bond when absorbed by material). The
shielded cobalt-60 gamma irradiator providing an
product of ionizing radiation may be electrically
irradiation volume of approximately 5000cc. The
charged ions) or neutral (free radicals). These
material for irradiation is placed in an irradiation
there further react to cause change in an
chamber located in the vertical drawer inside the
irradiated material known as the process of
lead flask. This drawer can be moved up and
radiolysis. It is this reaction that causes the death
down with the help of a system of motorized
of micro- organism, insect and parasites during
drive which enables precise positioning of the
food irradiation.
irradiation chamber at the centre of the radiation
The conservation and preservation of
field.
food is a prerequisite for food security. It
Radiation field is provided by a set of
provides self-reliance to nation. The Indian Food
stationary cobalt-60 sources placed in a
Industries contributes about 25-28% towards
cylindrical cage. The sources are doubly
GDP. The food processing sector provides 60-
encapsulated in corrosion resistant stainless steel
65% employment with a turnover of in US$ 36.1
pencils and are tested in accordance with
billion of which US$ 27.8 billion in organized
international standards. Two access holes of 8
sector, any change or any stagnation in
mm diameter are provided in the vertical drawer
technology will inevitably have very large impact
for introduction of service sleeves for gases,
throughout the economy. India is a potential
thermocouple, etc. A mechanism for
producer of fruits and vegetables live stock and
rotating/stirring samples during irradiation is also
marine products. India has a tremendous potential
incorporated. The lead shield provided around the
as the world largest food factory.
source is adequate to keep the external radiation
It has been estimated that about 30-35%
field well within permissible limits. The Gamma
of fruit and vegetables of worth Rs 3000/- corers
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 30
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Chamber 5000 unit can be installed in a room or high temperature by circulating liquid
measuring 4 metres x 4 metres x 4 metres. nitrogen or hot air. These can be
introduced through the service sleeves
GAMMA CHAMBER 5000
provided in the vertical drawer. The
irradiation temperature is sensed by a
thermocouple and displayed on the panel.
• Remote operation: An additional table top
control panel is provided for remote
operation in addition to the normal one
provided on the unit.
• Dose uniformity: Stationary source pencils,
symmetrically placed in a cylindrical
cage ensure good uniformity of radiation
field in the sample chamber. In addition a
mechanism is also provided for
rotating/stirring samples during
irradiation.
• Easy loading and unloading of samples:
Sample chamber extends to a convenient
height for easy loading and unloading of
samples.
• Safety assurance: The design of Gamm
Chamber conforms to American National
Standards, ANSI-N433.1-1977 for safe
design and use of self-contained dry
source storage gamma irradiators
(Category I). It also meets the
requirements of type B(U) package for
safety in transport of radioactive
materials as per AERB code No.SC/TR-
1, 1986 of Atomic Energy Regulatory
Board of INDIA.
Applications :
Features : Gamma Chamber is a versatile equipment for
• Safe and self-shielded: The shielding research studies in many fields such as:
provided is adequate to limit the radiation • Radiobiology
field on the external surface of the unit, • Preservation of tissue grafts
well within the permissible levels. No • Mutation breeding
additional shielding is required for its • Food preservation
installation and use. • Sterile male insect technique
• Automatic control of irradiation time: Built- • Biological and genetic effects of
in timer provides accurate control of radiations
irradiation time from 6 seconds onwards. • Radiation chemistry
The unit can also be operated manually. • Radiation effects on materials
Solid state programmable controls have • Radiation sterilization
been provided. In the event of power • Modification of properties of materials
failure battery backup displays the
Food Preservation by Gamma Radiation
programmes.
• Manual control of irradiation temperature: The radiation processing of food is
It is possible to irradiate samples at low carried out inside an irradiation chamber shielded
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Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
by 1.5 - 1.8 meter thick concrete walls. Food energy of the ray. At a density of 1000 kg m-3 half
either pre-packed or in bulk placed in suitable of the rays are absorbed in 11 cm. Halving the
containers is sent into the irradiation chamber with density approximately double the depth of
the help of an automatic conveyor. The conveyor penetration. The uniformity of dose distribution
goes through a concrete wall labyrinth, which can be expressed as a ratio of D max : D min. For
prevents radiation from reaching the work area sensitive food such as chicken the ratio should be
and operator room. When the facility is not in use as low as possible1.5.
the radiation source is stored under 6 meter deep
Potential Applications of Gamma Radiation:
water. The water shield does not allow radiation to
escape in to the irradiation chamber, thus The radiation dose administrated to a
permitting free access to personnel to carry out food depends on the resistance of the organisms
plant maintenance. For treating food, the source is present and the objective of the treatment. The
brought to the irradiation position above the water maximum recommended dose is 15 kGy, with
level after activation of all safety devices. The average dose not exceeding 100 kGy. Various
goods in aluminum carriers or tote boxes are application of this technology are as under:
mechanically positioned around the source rack
1. Sterilization ( or radappertisation) :
and are turned round their own axis, so that It is possible to sterilize meat and other
contents are irradiated on both the sides. The product, the dose required exceed the current
absorbed dose is determined by the residence time
limit of 10 kGy. A dose of 48 kGy is needed
of the carrier or tote box in irradiation position. for 12 D reduction of Cl. butulinum. High
Measurement of radiation dose : dose makes the product organoleptically un
acceptable.
Placing dosimeters at various positions in
a tote box or carrier we can check the absorbed 2. Reduction of pathogens (radicidation):
dose. The dosimeters are made from a material Food poisoning bacteria such as
including photographic film, Perspex and cobalt
salmonella typhimurium are less resistant to
glasses. The poly vinyl chloride (PVC) dosimeters radiation than Cl. Botulinum, and doses of 3-
are impregnated with a dye. The Hydrogen 10 kGy are sufficient for destruction.
chloride is released from the PVC by irradiation
and it produces a qualitative or quantitative 3. Prolonging shelf life (or radurisation) :
change in the colour of the dye to indicate the dose Relatively low doses are needed to
received. destroy yeast, moulds and non-spore forming
Dose distribution : bacteria. This process is used to increase shelf
The penetration of gamma radiation life by an overall reduction of vegetative
depends on the density of the food as well as the cells.
Table 1: List of radiation processing facilities available in the world :
S. No. Country No. of Food Commodities
irradiators
1. Algeria 1 Potato
2. Argentina 1 Spices, spinach, cocoa powder
3. Bangladesh 1 Spices, onion, dried fish
4. Belgium 1 Spices, dehydrasted vegetables, deep frozen foods
5. Brazil 3 Spices, dehydrasted vegetables, fruits, vegetables, grain
6. Canada 1 Spices
7. Chile 1 Spices, dehydrasted vegetables, onion, potato, poultry meat
8. China 11 Spices and vegetable seasonings, Chinese sausage, garlic, apple,
potato, onion, dehydrated vegetables, souses, rice, tomatoes
9. Croatia 1 Spices, food ingredients, dried beef noodles
10. Czech. Repulic 1 Spices, dry food ingredients
11. Cuba 1 Potato, onion, beans
12. Denmark 1 Spices
13. Finland 1 Spices
14. France 5 Spices, vegetable seasonings, herbs, poultry (frozen boneless
chicken, dried fruit, frozen frog legs, shrimp)
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Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
13.Calculate the mean weight diameter (MWD) of aggregates in mm and report the results as MWD
and percent aggregation.
Observations
I II
A. Air dry weight of sample (g) = 25 25
B. Moisture percent in soil (%) =
C. Frequency of oscillation (min-1) =
D. Stroke length (mm) =
E. Oven-dry weight of particles: a) aggregated (g): =
b) Unaggregated (g): =
F. Oven-dry weight of sample (g) =
Calculation
S. Size Range Weight of particles retained on Wt. of aggregated % aggregation Accumulated
No. of the sieve (g) particles percentage
aggregates Before After I Sample II Sample I Sample II Sample I Sample II Sample
(mm) dispersion dispersion
I II I II c-d (e x100)/ F
Sample Sample Sample Sample
a b c d e f g
1. 8.0-5.0
2. 5.0-2.0
3. 2.0-1.0
4. 1.0-0.5
5. 0.5-0.25
6. 0.25-0.10
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Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Density is an expression of mass of the oven-dry 3. Place the core sampler over the selected
soil per unit volume. Two types of densities are spots and insert the core in to the soil at desired
recognized - the bulk density and the particle depth by hammering on the top of the handle.
density. In bulk density total soil volume includes
4. Tilt the sampler a little forward and
the volume of solids and pore spaces, whereas in
backward to partly detach from the soil mass
particle density the volume of solids alone is
below and carefully lift the sample without any
considered.
jerk and disturbance of natural structure. In
Bulk Density: Bulk density is the ratio of oven-
certain soil fractions, a shovel may be needed to
dry mass of soil to its total volume before drying.
cut from sides and under the sampler to remove
Knowledge of bulk density is useful in calculating
the sampler without disturbance.
moisture content by volume, porosity using
particle density, volume of a known weight of 5. Unscrew the lid over the outer core
soil, and weight of a furrow-slice. Bulk density is cylinder and carefully remove the sample
a direct measure of soil compaction. holder.
Bulk density is highly dependent on any
6. Trim the excess soil from each end of the
kind of physical manipulation, degree of wetness,
sample holder with a straight-edged knife so
and kind and arrangement of soil particles. For
that the sample volume is the same as the
this reason, preservation of original structure in
volume of the sample holder.
the sample is most essential aspect of the
methodology for determination of bulk density. 7. Transfer the sample to a moisture box and
Common methods of determining soil bulk seal it for transportation to the laboratory.
density use soil cores or clods in their natural
8. Draw samples in duplicate from the desired
state.
depths.
Principle: The core method involves pressing
uniformly a metal core sampler of 0.05 to 0.10 m 9. In the laboratory, remove the seal, weigh
diameter into the soil to a desired depth and the sample, uncap, and place in oven at 105 0C
removing carefully. The sample is dried to for drying until constant weight.
constant weight at 105 0C and weighed, and the
bulk density is expressed as oven-dry mass 10. Report bulk density along with moisture
divided by the field volume of the sample. The content in the sample.
core method is unsuitable in stony soils. Observations
Apparatus and Accessories: Core sampler with A. Date -------------------------------------------------
removable sample cylinder, shovel, straight edged B. Field number or name-----------------------------
knife, moisture box, cellophane tape, balance and C. Present crop -------------------------, previous c
oven. D. Volume of the sample holder ---------------------
Procedure Calculation and Results
oven dry weight of soil sample
1. Select two grass-free, and crack-free A. Bulk density =
smooth soil surface for duplicate sampling. volume of sample
2. Apply a heavy lubricant on the inside wall = 1. ---------------, 2. ---------------, Av. --------------g/ cm3
of the core sample cylinder.
B. Water content =..................................
S.No. Sampling Weight of Weight of Weight of moisture Oven dry Water content
depth (m) moisture moisture box box + dry sample wt. Of soil in the sample
box (g) + fresh (g) (g) (g/g)
sample (g)
1.
2.
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Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Ascorbic acid (vitamin C) is a sugar acid and an 3. Stock standard solution: Dissolve 100 mg
antiascobutic. It is required for normal formation ascorbic acid in 100 ml of 4% oxalic acid
of connective tissue collagen, specifically for solution in a volumetric flask. The
hydroxylation of certain proline and lysine concentration of stock standard solution is 1
residues. It is also required for normal iron mg ml-1.
metabolism and a strong reducing agent losing 4. Working standard: Dilute 10 ml of the stock
hydrogen atom readily to become dehydroascorbic solution to 100 ml with 4% oxalic acid. The
acid which has also vitamin C. Ascorbic acid is concentration of working standard is 100 µg
water soluble, heat labile vitamin and acts as an ml-1.
antioxidant to protect the cell membrane from the Procedure:
toxic action of powerful oxidizing agents.
Generally it is present in all fresh vegetables and 1. Weigh 0.5-5 g fresh sample kept in a mortar,
fruits, found abundantly in berries, citrus, guavas, crush the sample with 4% oxalic acid using a
chillies and green leafy vegetables. Estimation of pestle and make up to a known volume of
ascorbic acid content in fruits and vegetables by extract (100 ml) and filter or centrifuge.
volumetric method is described below: 2. Pipette out 5 ml supernatant or aliquot into
conical flask, add 10 ml of 4% oxalic acid and
Principle: titrate against the standard dye solution (V2
Ascorbic acid reduces the 2, 6- ml) to end point appearance of pink colour.
dichlorophenol indophenols dye to a colourless 3. Repeat the procedure with a blank solution
leuco-base. The ascorbic acid gets oxidized to omitting the sample.
dehydroascorbic acid. Though the dye is a blue 4. Similarly, pipette out 5 ml ascorbic acid
coloured compound, the end point is the working standard solution into 100 ml conical
appearance of pink colour. The dye is pink flask, add 10 ml of 4% oxalic acid and titrate
coloured in acid medium. Oxalic acid is used as against the standard dye solution (V1 ml).
the titrating medium. 5. The end point is the appearance of pink
colour which persists for a few minutes. The
L-Ascorbic acid + 2, 6-Dichlorophenol indophenol amount of the dye consumed is equivalent to
(oxidized) the amount of ascorbic acid.
2, 6-Dichlorophenol indophenols 6. Calculate the ascorbic acid content of fresh
(reduced) sample of tissue as mg per 100 g.
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 39
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Chlorophylls are the essential components for mortar and pestle thoroughly with 80 %
photosynthesis, and occur in chloroplasts as green acetone and collect the clear washing in to the
pigments in all photosynthetic plant tissues. They volumetric flask.
are bound loosely to proteins but are readily 5. Make up the volume to 100 ml with 80 %
extracted in organic solvents such as acetone or acetone.
ether. Chemically, each chlorophyll molecule 6. Measure the absorbance of the solution
contains a porphyrin (tetrapyrole) nucleus with a with spectrophotometer at 645, 652 and
chelated magnesium atom at the centre and a long- 663 nm against the solvent (80 %
chain hydrocarbon (phytol) side chain attached acetone) as blank.
through a carboxylic acid group. There are at least Calculation:
five types of chlorophylls in plants. Chlorophylls a
and b occur in higher plants, ferns and mosses. Calculate the amount of chlorophyll
Chlorophylls c, d and e are only found in algae present in the extract (mg chlorophyll per g
and in certain bacteria. Estimation of chlorophylls fresh tissue) using the following equations:
content in vegetable and fruit plant materials
spectrophotometrically and their method as mg chlorophyll a g-1 tissue V
describe below: =12.7 (A663) – 2.69 (A645) ×
1000 × W
Principle:
Chlorophyll is extracted in 80 % acetone mg chlorophyll b g-1 tissue V
=22.9 (A645) – 4.68 (A663) ×
and the absorbances at 663 nm and 645 nm are 1000 × W
read in a spectrophotometer. Using the absorption
coefficients, the amount of chlorophyll is mg total chlorophyll g-1 tissue V
calculated. =20.2 (A645) + 8.02 (A663) ×
1000 × W
Equipments and glass wares:
Balance, Spectrophotometer, Centrifuge,
Mortar, Pestle, Buchner funnel, Filter paper and Where, A= Absorbance at specific wavelengths
Volumetric flask. V= Final volume of chlorophyll extract in
80 % acetone
Reagents: W=Fresh weight of tissue extracted
1. Analytical grade 80 % acetone (prechilled) References:
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 40
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Carotenoids are tetraterpenoid (C40) compounds 5. Evaporate the combined ether layers under
widely distributed in plants. They function as reduced pressure at 35 ºC in a Buchii type
accessory pigments in photosynthesis and as rotatory evaporator or in hot water bath and
colouring matters in flowers and fruits. Some of dissolve the residue in minimum quantity of
the commonly occurring carotenoids are simple ethanol.
unsaturated hydrocarbons based on lycopene and 6. Add 60% aqueous KOH at the rate of 1 ml for
their oxygenated derivatives known as every 10 ml of the ethanol extract to saponify
xanthophylls. β-Carotene is the most common the residue.
pigment in this group found in higher plants. A 7. Boil the mixture with add an equal volume of
method to extract total caretenoids from fresh water and partition the mixture twice with
materials of vegetable and fruit plants in acetone petroleum ether.
and petroleum ether, respectively and their 8. Evaporate the combined ether layers as before
determination is described below: and dissolve the residue in minimum volume
Principle: of ethanol.
The total carotenoids are extracted and 9. Measure the absorbance of the solution at 450
partitioned in organic solvents (acetone or nm with spectrophotometer and calculate the
methanol) on the basis of their solubility. total carotenoids content (mg 100 g-1) in the
Carotenoids that are bound as esters are sample using a calibration curve prepared
hydrolyzed using aqueous 60% KOH. The amount against a high purity β-carotene.
of carotenoids present in sample is estimated Make suitable dilution of the acetone or
spectrophotometrically at 450 nm using β-carotene methanol extract of the plant tissues and
as a standard. measure the absorbance at 450 and 670 nm in
a spectrophotometer. If A450 is 10 times that
Equipments: of A670, the total carotenoids present in the
Balance, Spectrophotometer, Rotatory extract may be estimated from the A450 values
evaporator or Water bath, Mortar, Pestle, using the following formula:
Separatory funnel, Buchner funnel, Filter paper,
Conical flak and Volumetric flak. C= D × V × f × 10
Reagents: 2500
Where,
1. Acetone or methanol (distilled)
2. Peroxide-free petroleum ether C= Total amount of carotenoids (mg)
3. Ethanol D= Absorbance at 450 nm in a 1.0 cm cell
4. Aqueous KOH (60%) V= Volume of the original extract in ml
5. Standard β-carotene solution F= Dilution factor and
2500= Average extinction coefficient of the pigments
Procedure:
References:
1. Cut the fresh sample of vegetable or fruit • Jenson, A. 1978. Chlorophylls and
tissue and grind a known amount (2 g) in a Carotenoids. In: Hellebust, A and J. S.
mortar with 20 ml of either distilled acetone Crargie (eds.) Handbook of Phytological
or methanol. methods, Cambridge university press,
2. Filter the extract on a Buchner funnel through London, pp. 59-70.
Whatman No. 42 filter paper.
• Mahadevan, A. and Sridhar, R. 1986. In:
3. Repeat the extraction until the tissue was free
Methods in Physiological Plant Pathology
from pigments.
(3rd edn.), Sivakami Publications, Chennai,
4. Pool the filtrates and partition thrice with
pp. 9-11.
equal volume of peroxide free petroleumether
• Thimmaiah, S. K. 1999. Standard methods of
using a reparatory funnel (add water if
biochemical analysis. Kalyani Publishers,
necessary to produce two layers during initial
New Delhi, pp. 304-305.
ether extraction).
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 41
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Definitions
Atomic Weight : Atomic weight of an element is the relative weight of the atom on the basis of
oxygen as 16 e.g. atomic weight of sodium is 23.
Molecular Weight: The sum of the atomic weights of all the atoms in a molecule is its molecular
weight.
Equivalent Weight : Equivalent Weight of a substance is the number of grams of the substance
required to react with, replace to react with, replace or furnish one mole of H2O+or OH-
The equivalent weight of an acid is the weight that contains one atomic weight of acidic
hydrogen i.e., the hydrogen that reacts during neutralization of acid with base.
For example, the equivalent weight of H2SO4 is 49. Since H2SO4 contains two replaceable
hydrogen, so that equivalent weight is molecular weight/2 i.e., 98/2=49
Percent solution (W/v): One percent solution of a substance contains one gram of the substance in
100ml of the solvent.
Molar Solution (M): One molar solution of a substance contains one mole Or one gram molecular
weight of the substance in one litre of solution.
Normal Solution (N): One normal solution of a substance contains one equivalent of one gram
equivalent weight of the substance in one litre of solution (i.e. molecular weight divided by the
hydrogen equivalent of the substance) e.g. In H2SO4 contains 49g H2SO4 in one litre solution.
Buffer: A solution containing both a weak acid and its conjugate weak base whose pH changes only
slightly on the addition of acid or alkali.
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 42
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Preparation of Glassware
Aim: Preparation of glassware’s.
Requirements:
1. Glassware:- test tubes, Beaker, conical flask, measuring cylinders.
2. Chemicals:- 70% alcohol, potassium dichromate, Conc.H2SO4
3. Others:- balance, Detergent ,washing tub , brush.
Procedure:-
1. Wash the glassware thoroughly with the detergent under tap water.
3. Now rinse the glassware’s thoroughly with Chromic acid and washes under tap water till the
Chromic acid is removed.
4. Again rinse the glassware with distilled water and dry them.
5. Keep them in oven at 110 OC for biochemical, microbiological and other routine work for one
or two hour and then it is ready for use.
6. For microbiology and biotechnology purpose rinse the glassware’s with a piece of cotton
dipped in 70% alcohol dry the glassware’s in air , wrap them in paper tie with thread and put
them in autoclave.
Precautions
1. After rinsing the glassware’s with chromic acid, it should be washed under tap water
thoroughly to remove chromic acid.
2. The glassware’s should be dried before wiping them with cotton dipped in 70% alcohol.
Interpretation:
The glassware’s must be washed properly as to make them free of dirt and dust. Proper
washing of the glassware’s is an essential step to be performed before conducting any biochemical,
microbiological or biotechnological experiments.
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 43
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Appendix I
Conversion factors :
me. mg/eq.wt
P X 2.29 P2O5
P2O5 0.44 X P
K X 1.20 K2O
K2O X 0.83 K
SX3 SO4
N X 1.12 NH3
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 44
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Appendix II
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 45
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Appendix III
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 46
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Note: In wheat, paddy, mustard and maize the 50% of urea to be applied as basal doses. Rest 50% may be applies in 2 to
3 split doses as top dressing.
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 47
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Appendix IV
Zinc Zinc sulphate (25-50 kg ha-1) (25 kg ha-1 0.5% zinc sulphate + 0.25%lime
for light soils and 50 kg for heavy soils)
Iron Ferrous sulphate (25-50 kg ha-1) 1-2% ferrous sulphate + half of lime
Copper Copper sulphate (10 kg ha-1) 0.1% copper sulphate + 0.05% lime
Appendix V
Rating of Nutrients:
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 48
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Appendix VI
I. General Information :
Farmers Name
Age
Male/Female
Education
Address
Name of the Area (ha) Survey Owned Leased Irrigated/ rainfed Soil
field No in/out type
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 49
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 50
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Name of laboratory :
Date of Sampling :
Date of Sampling :
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 51
Climate Resilient Soil Management Strategies for Sustainable Agriculture from14th October to 3rd November, 2015
Recommendation :
Biofertilizers
• Azosprillum
• Rhizobium
• Phospho bacteria
Other details :
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur – 482004 (M.P.) “Healthy Soils for a Healthy Life” 52
Centre of Advanced Faculty Training
Department of Soil Science & Agricultural Chemistry
Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur 482004 (M.P.)
Phone: 0761-2681119; Fax: 0761-2681119
E-mail: [email protected]; [email protected]
Web: www.jnkvv.nic.in