Thesis modified

1
CHAPTER – I
INTRODUCTION
Neem (Azadirachta indica A Juss.) a well known multipurpose tree species, widely
distributed through introductions, mainly in the arid tropical and subtropical countries of Asia.
Neem is versatile tree of tropics and can be established without irrigation in hot and dry regions
with low annual rainfall of 500mm or less. It can grow on poor, shallow, stony soil and on
alkaline and low acidic soils (Rawat 1995). Neem has been credited as a tree for solving global
problems in view of its potential of improving pest control, bettering health, assisting
reforestation and perhaps checking over-population (Anon. 1992).
Neem is very important culturally, medicinally and pesticidally. It provides firewood
and products to meet the basic needs in rural household like medicines, pesticides, mosquito
repellant, fertilizers, soaps, gums, lubricants, agricultural implements, tooth paste, tooth brush
sticks, etc. (Schumutterer 1995). Being a hardy tree, neem is ideal for reforestation programmes
and for rehabilitating degraded, semi-arid and arid lands and coastal areas (Maramorosch
1999). The use of neem tree started from the vedic period of India, which began about more
than 4,000 years BC. Hindus venerate neem and use various parts of it in religious ceremonies
(Mohan Ram and Nair 1993).
Neem cake which is a residue upon extraction of neem oil from the seed can be used as
biofertilizer by which it will nourishes the plants and increase the yield of crops (Lokanadhan et
al 2012). Neem oil is ued to prevent aflatoxin which is produced by Aspergillus flavus due to
contamination of the poultry feed and the neem extract antagonizes the production of Patulin
caused by Penicillium expansium. The processed neem cake poses a good appetizer
characteristic together with wormicidal activity, which is used as poultry feed. Neem leaves
have significant amount of protein, minerals (except zinc) and digestable amounts of curde
protein and total digestible proteins, which serves a better nutrition to the poultry animals such
as, goat, sheep and cow (Girish and Bha 2008).
In recent years, neem has emerged one of the most valuable tree species for
afforestation in the arid and semi arid zones of the world. Neem has been recommended as a
species for tropical plantations, for reforestation in Hounduras, for integrated landuse systems
in Malawai and has been identified as a multipurpose tree for the Himalayan region (Burley and
Carlowitz 1984).
The species is propagated through seeds but seeds lose viability within a few days
under ordinary room conditions and it is frequently recommended that the seed should be sown
immediately after collection (Maithani et al 1989). It is also difficult to transport the seeds to
long distances without impairing their quality.

2
Neem has attracted worldwide attention due to its wide ranging capacity as a biocide
(Jattan et al 1995). Of all the compounds found in neem, azadiractin finds the maximum uses. It
is the standrized ingredient in insecticidal and pesticidal formulations. Azadiractin is found (in
maximum amounts) in the neem seed kernels. Variation in azadiractin content of the neem seed
kernel of different sources have been observed by many researchers. Appreciating the
commercial benefits of the chemicals found in the plants of neem and the very wide range of
natural variation occurring within this species, the breeding and genetic improvement of neem
assumes greater significance.
Ezumah (1986) and Maithani et al (1989) described the neem seeds as short lived.
Rapid loss of viability is a major problem in the seeds (Kumar 2013). Little success has so far
been achieved to understand the physiology of neem seed and techniques to maintain viability
for short to medium term. In Punjab, we are losing the tree biodiversity due to various reasons,
neem is an indegenous tree called as 'Miracle tree' because of its multifamous uses.
Neem is a multipurpose tree for centuries in the Indian subcontinent. Neem seeds lose
their viability rapidly (Ezumah 1986 Nagavani et al 1987) due to changes in biochemical
composition (Pukacka and Ratajczak 2007). The most important change during seed
deterioration is alternation of the cell membrane, which affects seed vigor. The plasma lemma
designates the cell inner membrane, while the term cell membrane is more generally used for
the entire membrane system of the cell. The decline in seed vigor is associated with weaking of
the cell membrane (Heydecker 1972).
The recalcitrant seeds in which water properties appear not to be linked with
desiccation tolerance (Pammenter et al 1991, 1993) no predictive models have been proposed.
The relatively high water content at which recalcitrant seeds start to lose viability is far above
that at which a glassy state can exist at room temperature and non-freezable water is lost.
Many recalcitrant tropical seeds are chilling-sensitive and rapidly lose viability even if stored at
relatively high moisture contents. Due to the active metabolism in the hydrated state, viable
recalcitrant seeds cannot be stored for long-term periods. The exact causes of recalcitrant seed
storage behaviour remain to be ascertained (Vertucci and Farrant 1992), although orthodox
seeds feature typical changes in key compounds during the acquisition of desiccation tolerance
that do not occur to the same extent in recalcitrant seeds eg. the increase in oligosaccharide
content (Horbowicz and Obendori 1994, Steadman et al 1990). The significant loss of viability
upon drying and cold storage indicates that neem seed still poses more problems than an
orthodox seed would do (Sacande et al 1998). The storage behaviour of neem seeds appears
very uncertain. There are many conflicting reports as to status of seed as recalcitrant,
intermediate or orthodox probably owing to the limited desiccation conditions to determine the
desiccation tolerance of seed and their subsequent longevity.

3
Neem is generally propagated by seeds, however, the seed have a short storage life and
loose viability rapidly, which is a major problem for tree planting programmes. The longevity
of neem seeds appears very uncertain. Neem seed of Asian origin have shown more or less
recalcitrant habit (Gamene et al 1994) while those of African provenances as orthodox
(Bellefontaine and Audinet 1993). However, behaviour of neem seed has been described as
short-lived (Ezumah 1986, Maithani et al 1989). Present study has been planned with an
objective to know the conflicting behaviour of seeds either as recalcitrant or orthodox.
The seeds of neem typically lose viability within a few days. We investigated the
biochemical seed deterioration of neem seeds and its impact on germination during storage.
Our objective were to understand and quantify the food reserves of neem seeds during storage.
The objective of the proposed research, therefore, are:
i) To study the effect of seed treatments and storage conditions on germination and
biochemical parameters of neem seeds.
ii) To study correlation among the biochemical parameters and with germination
parameters.

4
CHAPTER – II
REVIEW OF LITERATURE
The literature on neem seed germination and related issues has been reviewed and
presented here for analyzing the issue and appropriate approaches being followed to address the
problem in neem and other tree species. The available literature has been review under the
following two heading:
Reviewed under following headings:
2.1 Physiological parameters
2.2 Chemical parameters
2.1 Physiological parameters
Neem (Azadirachta indica) is an important tropical tree species with many uses, whose
seeds have been categorized as having intermediate storage longevity (Sacande et al 1996,
1998, Hong and Ellis 1998). Neem seeds are chilling sensitive at MC > 10%. Their limited
desiccation tolerance has been partially attributed to their sensitivity to imbibitional stress
below 10% MC. Survival after dehydration was improved by rehydrating the dry seeds at
elevated temperatures of around 35°C (Sacande et al 1998). This sensitivity to both chilling and
imbibitional stress has contributed to neem seed's reputation as being difficult to store.
Reduction in moisture content of seed and lowering the temperature and relative
humidity in which seed is stored extends the storage life of most seeds (Roberts 1972). The
moisture content of seed is directly affected by the relative humidity of the atmosphere around
it. Relative humidity increases the seed moisture percentage. Seeds are shed from the parent
plant at low moisture content, having undergone maturation drying and are capable of
tolerating dehydration down to 5 to 6 per cent are called orthodox seed. When dry, the viability
of these seeds can be prolonged by keeping them at the lowest temperatures and moisture.
Roberts (1973) stressed that the seeds longevity can be predicted successfully from
moisture content and storage temperature specification for most of the species. Seeds that can
be dried to moisture content of 5% are generally regarded as desiccation tolerant and known as
orthodox seeds. Longevity of orthodox seeds is increased in a specific and predictable way,
over a wide range of environment conditions by decreasing storage temperature and moisture.
Schmidt (2000) studied that for species with recalcitrants seeds, fruiting during the
early rainy season with concurrent germination and seedling establishment before the onset of
the dry season is common. Seeds must thus either be stored over the first rainy season and then
sown 2-3 months before the rainy season, or they must be shown immediately and then kept as
seedling in the nursery during the dry season. Moisture absorption is obviously not a problem
for recalcitrant seeds, but a generally short viability may make immediate sowing necessary.
The seedlings must then be maintained in the nursery through the interin dry season, which

5
necessitates access to water during the dry season. This problem has been encountered for
semi-recalcitrant neem (azadirachta indica) in east Africa, where it is a limiting factor for the
cultivation of the species. To reduced the need for watering during the dry season, seedling may
be pruned or stored moist as stumps.
Oxidative stress has also been shown to take place during storage of seeds (Hendry et
al 1992) and to be involved in the viability loss of entire plants under environmental stress from
chilling, freezing, or exposure to environmental pollution (Bowler et al 1992).
Chauhan et al (2009) reported that seed germination indices as recorded after
subjecting the seeds to different storage period at room temperature and 10±2ºC. The studies
clearly reflect the lack of storage potential of neem seeds for a longer period. Low temperature
storage does not help in any way, rather de-pulped seeds stored at room temperature retain their
viability for a longer period.
Neem seeds from various locations show variable responses to desiccation, but less is
known about how maturity affects neem seed storage performance in the dry state. Seeds
collected in Burkina Faso at three different developmental stages were stored in the seed
laboratory at various temperatures (25°C in a cabinet, 4°C in a cool room and -18°C in a
freezer) and different moisture contents (36–42%, 12%, 8% and 4%). Some seeds of all three
developmental ages showed sensitivity to dehydration to the lowest moisture content, with
viability falling to around 40–70% from 80–100%. During storage for up to 6 months, highest
survival was observed for seeds from the last collection subsequently held at near full hydration
and at 25°C, and seed longevity at 36–42% ≥ 4% ≥ 7% ≥ 12% moisture. At 4°C and -18°C,
suggest that the benefits of cold dry storage over wet warm storage would become evident in
neem seeds after 6 months storage.
Varghese and Naithani (2008) reported that the seeds of Azadirachta indica were
successfully cryopreserved for 12 months with 45% survival followed drying to 0.16 g H2O g-1
dry mass (DM). Intermediate or orthodox probably owing to the limited dessication conditions
in which earlier tests were conducted. The seeds were subjected to different drying conditions
to determine the desciccation tolerance of seed and their subsequent longevity. In addition,
longevity of seed was determined at temperatures ranging from 5° to 35°C and moisture
content (MC) ranging from original 39 to 7 per-cent. Seeds tolerance desiccation to as low as
3.2% MC, through when dried to 4.6 per-cent mc over seed/silica gel ratio of 1:1 at 15°C,
showed maximum half viability period (P50) of 663 days. Seeds stored with reduced MC at
15°C exhibited maximum P50 while higher and lower temperatures and higher MC were
deleterious. This paper reports a twenty fold increase in longevity of neem seed of Asian origin.
A relatively large variation in the longevity of neem seeds has given rise to conflicting
opinions on its classification. Neem seeds of Asian origin are reported to be more or less
recalcitrant, where some African provenances have been described as orthodox. Studies have

6
been conducted by various workers to be better understand the storage life in relation to
physiology of seeds and improve the longevity of seeds. But storage physiology of neem is not
well understood and being variably classified as orthodox, intermediate or recalcitrant. Neem
seed cannot be stored at freezing temperature due to low temperature sensitivity (Maithani et al
1989 Chauhan et al 2002).
Bonner (1990) classified seed storage physiology into four classes: true orthodox, sub-
orthodox, temperate recalcitrant and tropical recalcitrant. Storage of recalcitrant seeds is still a
major objective in research. Some Shorea species suffered chilling damage at or below 14⁰C,
but they survived for about 180 days at 4⁰C, whereas, some Parashorea and Hopea species
survived for 20 days at 4⁰C but they were all dead within 30 days. The chilling damage,
dehydration, early germination and fungal attack were the biggest problems.
Depending on longevity characteristics of different species and deterioration due to
storage conditions, seeds maintain their germination until aging renders them non-viable. Pre-
sowing treatments also vary with aging and time of sowing (Sharma 2014).
Kumar et al (2007) stored seeds in open container at ambient room temperature. Seeds
were tested in the laboratory for their germination at different intervals of storage viz. Fresh 0,
15, 60, 90, 165, 230, 350, 380, 410, 440 and 470 days. Germination tests were conducted
between germination paper (BP) using four replications of 100 seeds each, incubated at
30±1°C. Fresh seeds collected from morphologically superior and inferior trees showed no
significant difference in germination test in laboratory. No significant difference was observed
with respect to mean germination time (MGT) and germination values also. Seeds sown in
nursery showed more or less similar results in germination.
Gunasena and Marambe (1995) and Msanga (1996) concluded that neem seeds lose
their viability after one to four months of storage and have consequently classified them as
recalcitrant. Recorderer and Bellefontaine (1989), Dickie and Smith (pers. Comm.) showed that
neem seeds survive progressive desiccation to a moisture content of about 4% (fresh mass
basis) and long-term storage (8 to 10 years) in hermetically-sealed containers at a temperature
of either 4°C or -20°C. These authors concluded that neem seeds are orthodox.
Gamene et al (1996) and Sacande et al (1996) showed that neem seeds are intermediate
in seed storage behaviour when compared with orthodox and recalcitrant seeds. These clear
differences in the ability of seeds of the species to retain viability might be due to factors such
as provenance, storage conditions and seed developmental stage.
Kumar (2015) reported that effect of four storage temperatures (ambient room
temperature (35±5°C), Air conditioner (25±2½C), Fridge (5±1°C) and Freezer (-4±1°C) and
four moisture levels 38%, 21%, 12%, 5.5% were studied on seed longevity of seeds under
controlled conditions. It resulted that seeds were stored at very low temperature (4°C) at low

7
moisture 5.5% were observed the best combination of temperature and moisture level for
storage of seed in controlled conditions. Studies are in accordance with Sacande et al (2000) as
they reported that the storage behaviour of neem seeds has featured that characterize it as
orthodox.
Kumar (2013) reported rapid loss in viability of neem (Azadirachta indica A. Juss.)
seed is a major problem and undertaken for developing a set pattern for assessing of viability
and vigour in seed of various mother tree ages of neem (I-06 years, II-15 years,III-25 years and
IV-＞30 years old). Various viability test viz. triphenyle tetrazolium chloride test, electrical
conductivity, excised embryo test, and germination test were performed on seeds obtained from
mother tree age classes. Inconsistency was observed with the TTC and EC test in germination
of seed in laboratory as well as nursery. While various vigour tests viz. cold test, chemical
stress test (methanol stress test), and accelerated ageing test along with ageing index,
germination test (G%, MGT and GV) and various seedling growth parameters like seedling
length (cm), number of leaves, collar diameter (cm), total biomass (g) along with mathematical
indices i.e. vigour index, sturdiness quotient, volume index, quality index, root/shoot ratio in
nursery as well have been taken for study and showed better consistency. On the basis present
study results of various viability and vigour test indicated that mother tree age class II
performed better in comparison to others and it can be recommended for seed collection.
Priestley (1986) Seed storage longevity depends on intrinsic properties of the species
and on external factors during storage such as temperature, relative humidity and to a lesser
extent, composition of the gaseous atmosphere.
To survive long-term storage in the dehydrated state, seeds have to withstand
desiccation to low water contents. A large group of so called orthodox seeds have the ability,
whereas, another group of mainly tropical seeds, designated recalcitrant, are damaged during
drying, often in the range of 0.4-1.0g H²O g-1.DW. Hong et al (1996)
Roberts et al (1972) Empirical models on the basis of moisture content and temperature
have been constructed for the storage behaviour of orthodox seeds, which can predict the
viability of a seed lot over time at a broad range of different water contents and storage
temperatures (Roberts 1972 Justice and Bass 1978 Ellis and Roberts 1980)
Dwivedi (1993) reported large variations in 100 seed weight, which varied from 12.3 to
20.1g. Germination percentage varied from nil in seeds from Dharwad and Pinjore source to
93% in Jodhpur source. Seedling height varied from 6.0cm in Ranchi to 25.1cm in Augul
source. Thus, great variations were observed in these traits in 37 provenance sources of neem in
india.
Palanisamy et al (1996) studied the engineering properties of neem nut. They reported
that the length and diameter at stem end measured at the moisture content range 7-20 per cent

8
were 14.83mm and 7.12mm, respectively. The mass of 1000 nuts increased linearly with the
increase in moisture content. Visvanathan et al (1996) also studies the physical properties of
neem nut.
An attempt can be made to determine the optimum temperature and moisture content
during the storage of neem seed which may provide a lead for researchers to resolve of seed
storage behaviour protocol of neem seed.
2.2 Chemical parameters
Rajiv and Rai (1996) collected neem fruits from 18 geographic sources in india at the
yellowish tinge stage, depulped immediately and dried overnight. The seeds were then sown in
plastic trays and polybags at the nursery of institute of Social Forestry at Allahabad (U.P).
Significant differences between provenances for germination percentage were found.
Rengasamy et al (1996) reported that neem ecotypes of India varied in azadirachtin content
depending on the climate, soil type and altitude. The azadirachtin content in the test samples
ranged from 0.14 to 1.66 per cent. Ecotypes, which grew in regions with moderate climate, red
laterite and shallow medium black soils, and altitudes less than 500m above mean sea level
were rich in azadirachtin content as compared to the ecotypes, which grew in high altitude,
alluvial soils with extreme hot and cold climates.
The method o extract of A.indica leaves shows antibacterial activity against Bacillus
subtilis, Staphylococcus aureus,Proteus vulgaris, salmonella typhi, and showed low activity on
Pseudomonas aeruginosa but it is infective against Escherichia coli. The petroleum ether and
methol extract of A.indica leaves were highly effective against candida albicans Grover et al
(2011)
The Azadirachta indica seeds poses an antibacterial activity against the bacteria that
causes eye infection (Ophthalmic infection) such as Staphylococcus aureus, Staphylococcus
pyogenes, Escherichia coli and Pseudomonas aeruginosa Vashit and Jindal (2012).
Jacobson (1981) reported that neem seeds from Indian origin contained only 1-3
percent of azadiractin, whereas seeds of African sources averaged 5-6 percent with few lots
exceeding nine percent and observed that as the neem is distributed throughout the tropical and
subtropical world, quantitative and qualitative difference were present in azadiractin content.
Morgan (1982) also studied the variation in azadiractin content isolated from various
neem ecotyopes. The results showed that seeds from Kenya, Negeria and Ghana contained
higher azadirachtin content (1.0-3.5g) than the Indian neem (0.2-0.7g) per 100gm seeds.
Singh (1987) reported a higher antifeedant activity of extracts from the hernel of neem
trees growing in arid areas in india when compared with those growing in coastal areas, which
yielded more oil. In samples from Senegal, differences in azadirachtin content could be
attributed to seasonal factors. In that country neem seeds were harvested, in some regions,
twice a year, namely june/july and during actober to December.

9
Ermel (1995) analysed samples of neem seed kernels from different continents/
countries for more than four years and evaluated 256 smaples from 22 countries and the results
showed that the mean value of azadirachtin of all samples measured was 3.6mg/g kernel. There
were marked variation between samples from different countries and among samples
originating from the same country. The highest amounts od azadirachtin were determined in
samples coming South and South-East Asia e.g India, Myanmar and Thailand. Below average
azadirachtin content were recorded in samples from countries with extremely high temperatures
during the summer months, such as Sudan, Somatra, Mali, Niger, etc. An exception to this rule
was seeds from different locations in yemen which had high azadirachtin content.
Chaitanya and nithani (1994) studied that Loss of viability in desiccation-sensitive
seeds has been frequently related to oxidative injury. Reactive oxygen species (ROS) and its
derivatives have been implicated in oxidative damaged and their accumulation has been shown
to be positively correlated with desiccation-sensitivity in various seeds.
McKersie (1991) and Leprince et al (1993) suggest that activate oxygen produced
during dehydration causes severe membrane brane perturbation. It activate phospholipid
degradation, leading to irreversible formation of gel phase domains and loss of membrane
function.
Bowler et al (1992) cellular damage caused by ROS might be reduced or prevented by
protective mechanism involving free radical scavenging enzymes such as superoxide dismutase
(SOD), catalase(CA) and peroxidase(POD). SOD is generally considered a key enzyme in the
regulation of intracellular concentration of superoxide and peroxide, which can be react in the
Fenton reaction to form hydroxyl radicals.
Fridovich (1986) Catalase and peroxidase are involved in the removal of hydrogen
peroxide, a product of SOD catalysed dismutation of superoxide anion. A decrease in POD and
CAT activities could lead to accumulation of hydrogen oxide, which is cytotoxic in the
existence of metal catalysts and result in high level of toxic products such as hydroxyl radicals
and lipid peroxidation.
Benson and Bremner (2004) oxidative stress and related metabolism occur in and
detrimental to several plants and animal tissues during low and cryotemperatures. Synthesis and
accumulation of active oxygen species (AOS), which is highly toxic and reactive chemical
species, is central to all oxidativestress-related metabolism. These are apparently generated
during dessication, chilling and cryogenic treatments of plant tissue. Membrane lipids are
primary targets for active oxygen species attack and oxidized fatty acid reaction products like
conjugated dienes, hydroperoxides and malondialdehyde are normally used as markers of
oxidative stress. The sueroxide radical. Free radical damage to cellular membrane could
proceed by de-esterification of membrane phospholipids and resultant accumulation of fatty
acids would lead to reduced membrane function.

10
Niehasus (1978) lipid peroxidation as manifested by changes in fatty acid saturation or
malondialdehyde production has been assumed to be the primary mechanism by which the
putative free radical injury is imposed on plant membranes. Another mode of damage could be
brought by nucleophilic attack by superperoxide radical.
Kumar D (2014) protein is the main form of the nitrogen storage in food reserves in
seed. During storage, proteins become less soluble and are degraded into free amino acids.
Several investigation have shown that reductions in vigor and viability were closely associated
with declines in protein synthesis. The protein content of neem cake is 25.4% while 17.03% to
36% crude protein and 26% carbohydrate in neem cake.
Bewley and Black (1982) studies od seed deterioration have used many techniques to
examine stages of degeneration for a wide variety of seeds. The cause of varying vigor and
consequent loss of viability in seeds has been related to weakning of cell membranes,
mechanical damage or low metabolic activity. Increasing quantities of cellular constituents can
be leached from seeds as they deteriorate due to ageing or sudden trauma.
Varghese and Naithani(2001) reported loss of viability in azadirachta indica seeds
during cryostorage for one year. The possible cause of cryoinjury by monitoring active oxygen
species, possible associated membrane perturbations through lipid peroxidation and antioxidant
enzymes during long term (1-year) conservation of neem seeds at cryogenic temperature.
Mccomb and winstead (1964) reported that reduced levels of several amino acids in
seeds were due to fungal invasion.
Kumar D (2014) neem seeds lost its viability and vigor after 35 days when stored at
room temperature. No germination was observed in seeds obtained from any mother tree age
class after 65 days of storage. Decline in the level of enzyme activity led to rancidity of oil in
the seed, which led to cell membrane disfunction. Cell membrane permeability tests for
electrical conductivity, water soluble sugars and amino acids all showed declines with
increasing duration of storage. Reduction of cell membrane permeability and released amounts
of solute may be due to repair during imbibitions of seeds. Reduction of food (rotein, total
sugars and total starch) was recorded with increasing duration of the storage period.
Deterioration in seeds obtained from all the mother tree age classes during storage, the loss of
viability and vigor of seeds are strongly related to loss of protein, sugars, starch, indicating
weakening of cell intergrity in terms of phospholipids, peroxidase and lipid peroxidation.

11
CHAPTER – III
MATERIALS AND METHOD
The present investigation entitled, “effect of fresh and stored seeds on germination and
biochemical parameters of neem (Azadirachta indica A. JUSS) was conducted both at the
experimental farm and laboratory of the Department of Botany, PAU, Ludhiana respectively
during 2016-2017.
Seeds will be collected from healthy trees of neem during month of June 2016. Treated
seeds were kept at room temperature. Data was collected at an interval of ten days from the
treated seeds till they loses their viability.
3.1 Experiment Details
3.1.1 Location
The experimental site is located at an elevation of 247 m above mean sea level in the
central zone of Punjab and lies between 30⁰-50’N latitude and 75⁰-52’E longitude.
3.1.2 Climatic Condition
The climate of this area is sub-tropical to tropical. Average annual rainfall 47.85 mm
and relative humidity is 70.5%. The maximum temperature during summer month between the
period of May and June was 37.5⁰C whereas; minimum temperature during winter month
between the period of December and January was 19.7⁰C in 2016.
3.2 Observations
3.2.1 Physiological Observations
i) Moisture content
For the moisture content, the seeds were dried in an electric oven at 1030C for 17
hours. Moisture content of seeds was determined by the following formula given by [8].
Moisture percentage = (Fresh weight – Dry weight)/ Fresh weight X 100
ii) Germination percentage
Germination percentage was recorded at the end of 10th
day. The numbers of normal
seedlings were counted and expressed in percentage.
Germination percentage 100
keptseedsofNo.
germinatedseedsofNo.

iii) Germination value
it is measure combining speed and completeness of seed germination with a single
figure. Higher the germination value better is the seed stock.
Germination value = final MDG × PV (Czabator, 1962)
Where MDG = mean daily germination
PV = peak value

12
testofendthetosowingsinceDays
testtheofendatgerminatedseedofcentperCumulative
MDG 
Peak value is maximum mean daily germination reached at any time during the period
of test.
sowingsinceDays
centperngerminatioCumulative
PV 
100x
sownseedofnumberTotal
totalCumulative
=centperngerminatioCumulative
iv) Germination energy
In the per cent of seed in a given sample which germinate up to the time of peak
germination. Where peak germination is the highest number of germination in a particular day
(William 1985)
v) Germination index: ( G1/t1 + G2/t2 + ___________ + Gn/tn)
Where
G1, G2,……….. Gn = Germination count taken from day 1 to nth
day
t1, t2,…………..tn = Time taken in days from day 1 to nth
day
3.2.2 Biochemical Analysis
The contents of total soluble sugars, total starch content, total soluble proteins, total
free amino acids, and activities of catalase, peroxidase and α-amylase were estimated from
refrigerator stored, room temperature stored and accelerated aged seeds at the time sowing.
i) Total Soluble Sugars
Total soluble sugars were estimated as per Dubois et al (1956).
Principle: Sugars react with concentrated sulphuric acid to form dehydration product i.e.,
furfural or 5-hydroxymethyl furfural. This dehydration product then reacts with phenol, which
acts as chromophore and gives orange yellow colour.
Reagents:
A: 80 % Ethanol
B: 5 % Phenol
C: Concentrated H2SO4
Extraction: The seed (0.1g) was homogenized in 80% ethanol and then centrifuged at 5000
rpm for 10 minutes. The residue was re-extracted with 80% ethanol to ensure complete
extraction. The supernatants were pooled and the ethanol was evaporated. Aqueous solution
was diluted to a known volume with distilled water and used for estimation of total soluble
sugars.
Estimation: To 0.1 ml of seed extract, added 1 ml of 5% phenol and kept for ten minutes
followed by addition of 5 ml of concentrated sulphuric acid. The sulphuric acid was poured

13
directly in the middle of the test tube to ensure the proper mixing of the solutions. After ten
minutes, the tubes were cooled to room temperature under running water. After another 20
minutes, the absorbance was measured at 490 nm against reagent blank (1 ml 5 % phenol + 5
ml cold conc. H2SO4). The concentration of total soluble sugars was calculated from the
glucose standards (10-60µg) run simultaneously.
ii) Total Starch
Total starch was estimated as per Dubois et al (1956).
Principle: Starch is hydrolyzed with the help of perchloric acid (HClO4) to release free sugars,
which form the dehydration product with concentrated sulphuric acid. This dehydration product
then reacts with phenol, which acts as chromophore and gives orange yellow colour.
Extraction: Extraction of starch was done from the residue of sugars. To this residue, 3 ml of
52 % perchloric acid (74.28 ml perchloric acid + 25.72 ml distilled water) was added in the
centrifugation tube. The centrifugation tubes were stirred continuously for 15 minutes. The
tubes were then centrifuged for 15 minutes at 2000 rpm. The supernatant so obtained was
poured in another volumetric flask. The extraction procedure was repeated with the residue to
which 2 ml of 52 % perchloric acid was added and then centrifuged for another 15 minutes at
2000 rpm. The supernatant so obtained was pooled with the previous supernatant in the
volumetric flask. The final volume was made to 10 ml with distilled water and used for the
estimation of total starch.
Estimation: Same procedure was followed for the estimation of starch as given for the
estimation of total soluble sugars.
iii) Total soluble proteins
Total soluble proteins were estimated as per Lowry et al (1951).
Principle: Proteins (peptide bonds) in the sample react with copper tartarate complex in
alkaline solution. The protein-copper complex then reduces phosphomolybdate of folin reagent
to a blue-coloured complex having maximum absorbance at 520 nm.
Reagents:
A: Reagent I: 2 % sodium carbonate in 0.1N sodium hydroxide
B: Reagent II: 0.5 % copper sulphate in 1 % solution of sodium potassium tartarate.
C: Reagent III: Reagent mixture was prepared immediately before use by mixing
reagent (I) and reagent (II) in 50: 1 ratio.
D: Reagent IV: Folin Ciocalteau phenol reagent (1N).
E: TCA: 20 %.
Extraction: Seeds (0.1g) were homogenized in 10 ml of 0.1N NaOH followed by
centrifugation at 6000 rpm for 15 minutes at 4⁰
C. The supernatant was collected. From this, 2
ml of supernatant was taken and treated with 2 ml of 20 % TCA and kept at 4o
C for 24 hours.
This extract was later centrifuged for 15 minutes at 6000 rpm and precipitates so obtained were

14
dissolved in 10 ml of 0.1N NaOH. This served as protein extract.
Estimation: To 0.1 ml of protein extract, 0.9 ml of distilled water was added. To this, 5 ml of
reagent C was added and kept at room temperature for 10 minutes after proper mixing. To this,
0.5 ml of Folin’s reagent was added and kept at room temperature for 30 minutes. The
absorbance of blue colour so obtained was measured at 520 nm against a blank. Protein was
quantified from standard curve prepared by using Bovine serum albumin (BSA) in the
concentration range of 20-100 µg.
iv) Total free amino acids
Total free amino acids were estimated as per Lee and Takahashi (1956).
Principle: The amino-group in the amino-acid reduces ninhydrin, thus giving rise to ammonia,
carbon-dioxide and an aldehyde. This reduced ninhydrin further reacts with the oxidized
ninhydrin, which produces purple-violet coloured complex having maximum absorbance at 570
nm.
Reagents
A. Ethanol: 80 %.
B. Citrate buffer, (pH5.5) : 0.5 M
C. Standard amino-acid: Glycine
D. Glycerol: 100 %
E. Ninhydrin reagent: Prepared fresh by mixing 1 g ninhydrin dissolved in 100 ml of
citrate buffer + pure glycerol + Citrate buffer and pH was adjusted to 5.5.
Extraction: Same procedure was followed for the extraction of total free amino acids as given
for the extraction of total soluble proteins. At the last step of protein extraction, the supernatant
obtained was used as amino acid extract.
Estimation: To 0.2 ml of amino acid extract, 5 ml of ninhydrin reagent was added in a test
tube. The test tubes were then heated in water bath for one hour till the purple-violet colour
appeared. They were then cooled under running water. The absorbance was measured at 570
nm in a spectrophotometer.
PARAMETERS FROM GERMINATED SEEDS:
v) Catalase
The Catalase enzyme activity was estimated as per Goth (1991).
Catalase activity was measured by an assay of hydrogen peroxide based on the
formation of its stable compound with ammonium molybdate.
Extraction
The enzyme was extracted with 60 mM sodium phosphate buffer (pH 7.5) containing
1% P.V.P.
Reagents:
A: 65 mM H2O2

15
B: 60 mM sodium phosphate buffer (pH 7.5)
C: 32.4 mM ammonium molybdate
Procedure: 0.2 ml of seed extract was incubated in 1 ml reaction mixture containing 65 mM
H2O2 in 60 mM sodium phosphate buffer (pH 7.5) at 25°C for 4 minutes. The enzymatic
reaction was stopped with 1 ml of 32.4 mM ammonium molybdate and the concentration of
yellow complex of molybdate and H2O2 was measured at 405 nm.
271
3)(blankA2)(blankA
1)(blankA(sample)A
activityCAT 



vi)  - amylase
The α- amylase enzyme activity was estimated as per Murata et al (1968).
Reagents:
A. Stock standard maltose solution: 100 mg of maltose was dissolved in 100 ml distilled
water.
B. Working standard maltose solution: 1.0 ml of stock solution was made upto 10 ml with
distilled water.
C. 0.1M phosphate buffer (pH 7): 11.9 gm of monobasic sodium phosphate was taken in
500 ml of distilled water (Solution A). 17.7 g of dibasic sodium phosphate was taken in
500 ml distilled water (Solution B). 97.5 ml of solution A was made upto 250 ml with
solution B. Then this mixture was made up to 500ml with distilled water.
D. Soluble starch 1%: 1g starch was dissolve in cold distilled water to make the solution
100 ml in beaker. Then the solution was fully boiled and used for estimation when
cooled.
E. DNS Reagent (Dinitrosalicyclic acid): 1g of DNS in 20 ml of 2 N sodium hydroxide
and 30 g of sodium potassium tartarate was made upto 100 ml with distilled water.
F. Sodium chloride (NaCl) 1%: 1g of NaCl was dissolved in 100 ml distilled water in
beaker.
G. 1N Sodium hydroxide (NaOH): 8g of NaOH was dissolved in 100 ml distilled water in
beaker.
Extraction: 0.1 g seeds were homogenized in pestle mortar and made a fine paste of it by
adding 5 ml of phosphate buffer. Then centrifuged the mixture at 3000 rpm for 10 minutes.
Supernatant was pooled and took 1ml of the supernatant and diluted upto 10 ml with phosphate
buffer.
Estimation: To 1.5 ml of phosphate buffer, 1.5 ml of 1% soluble starch and 1.5 ml of 1% NaCl
was added in the test tube and pre-incubated for 10 minutes in rectangular water bath. 0.5 ml of
enzyme extract was then added to the test-tube and again incubated for 15 minutes. Then 0.5 ml
of NaOH and 0.5 ml of DNS reagent was added and kept in boiling water for 10 minutes,
cooled and made upto 10 ml with distilled water. After the development colour, optical

16
absorbance was taken at 540 nm using the spectrophotometer.
vii) Peroxidase
The peroxidase enzyme activity was estimated as per Shannon et al (1966).
Peroxidase detoxifies H2O2 in the cytosolic part of the cell. They are non-specific in
utilizing electron donor for oxidation of H2O2.
Guaiacol + H2O2 H2O + Tetrahydroguaiacol
Extraction: The enzyme was extracted from 0.1 g seeds in 0.1 M potassium phosphate buffer
(pH 6.5) containing 1.0% PVP,1 mM EDTA and 10 mM β– mercaptoethanol. The extract were
passed through a muslin cloth and centrifuged at 10,000 g for 10 min.
Reagents:
A: 0.05 M guaiacol prepared in 0.1 M potassium phosphate buffer. (pH 6.5)
B: 0.8 M H2O2
Assay: The reaction mixture contained 3 ml of 0.05 M guaiacol prepared in 0.1 M potassium
phosphate buffer (pH 6.5), 0.1 ml of enzyme extract and 0.1 ml of 0.8 M H2O2 . The reaction
mixture without H2O2 was measured as a blank. The reaction was initiated by adding H2O2 and
rate of change in absorbance was recorded at 470 nm for 3 minutes at an interval of 30 seconds.
Peroxidase activity has been defined as change in absorbance/min/g of tissue.
Statistical Analysis: Statistical analysis was done for analysis of variance by completely
randomized block design. Correlations among germination parameter and biochemical traits
were computed by using statistical analysis system software (CPCS 1).

17
CHAPTER – IV
RESULT AND DISCUSSION
The present investigation on “Effect of fresh and stored seeds on germination and
biochemical parameters of neem” was carried out during the year 2016-17 in the fields
department of botany to know about the germination behavior of neem seeds in fresh and stored
conditions. Results are discussed under following headings and discussed in light of literature
available for this species and also with other species.
4.1 Germination parameters
4.2 Biochemical studies
4.3 Correlation among germination and biochemical parameters
4.1 Germination Parameters
The physical characters of collected seeds and drupes are presented in table 1 the seed
length was 1.23± 0.03 cm whereas the drupe length was recorded to be 1.68 ± 0.02 cm was
calculated from freshly collected seeds. The width of collected neem seeds was 0.63±0.03cm
while drupe width was 1.25±0.02 cm. The seed and drupe weight was recorded to be 1.19±0.02
g and 1.65±0.03 respectively. Treated seeds were kept at room temperature.
Table 1: Physical characters of neem seeds and drupes
Physical characters Seed Drupe
Length (cm) 1.24±0.03 1.68±0.02
Width (cm) 0.63±0.03 1.25±0.02
Weight (g) 1.19±0.02 1.65±0.03
4.1.1 Germination percentage
Data presented in table 2 reveals that the values for germination per cent are non
significant for fresh seeds in all treatments. High values for germination percentage were
recorded for fresh seeds under all treatments which range from (96.66-91.66%). Reduction
percentage range from (12.06-3.63%) in all treatments with maximum reduction in case of
temperature 20⁰C (12.06%).
However, after ten days seed storage, significant variation was observed within all the
treatments. The higher values after ten days storage were recorded in wax coating (90%) and
temperature 10⁰C (90%), these values were at par with clay coating (86.66%) and low
temperature (85%) treatments. Lowest values were recorded in sun drying 12 hours and 24
hours i.e 78.33% and 75.00% respectively. Maximum reduction in germination per cent over
the fresh seed germination in case of sun drying 12 hours (16.66%) and minimum in case of
gum coating(3.63%).

18
After twenty days storage, the values further decreased with the highest germination
recorded in all temperature i.e. 88.33%, 86.66, 81.66 respectively for 10⁰C, 15⁰C, 20⁰C and
lowest value recorded for sun drying (12 hours and 24 hours) i.e. 63.33% and 58.33%
respectively.
After thirty days storage, seeds stored at 15⁰C showed significant value for germination
68.33% and minimum value were recorded sun drying 24 hours i.e. 36.66%.
After fourty days very high reduction in germination was recorded over the
germination of fresh seeds in sun drying (24hours) seeds 75.93% and minimum reduction
recorded in temperature 10⁰C i.e. 55.17%. However, there was insignificant for fresh, storage
condition germination values are found to be highly significant. It was observed that there
decrease in germination with storage for all the treatments but this decreases was maximum for
sun drying 24 hours (21.66%) seeds after fourty days. Maximum reduction is in sundrying
75.93 with germination per cent value of 21.66% after 40days. However no germination
recorded after 40days in all the treatments.
Results are in accordance with the work reported by Kumar (2015) and chauhan et al
(2009). Chauhan et al (2009) also reported that the reduction was significant they stored seeds
at room temperature under seven storage condition and observed maximum germination even
after 60 days (20.88%) in the neem seeds stored in earthern containers. The average
germination values were at par when the seeds were stored either in gunny bags or coated with
clay and Kumar (2015) reported that seeds stored at high temperature lost viability very soon
whereas seeds stored at lower temperature maintained viability well and exhibited highest mean
germination percentage. Temperature treatments in present study shows good germination in
accordance with sacande et al (1999). Gairola et al (2011) revealed that lower temperature
resulted in significant increase in germination percentage. Neem seeds lose their viability
rapidly due to changes in biochemical composition.

19
Table 2: Effect of different treatments on germination percentage in neem seeds
Treatments Fresh 10 Days Reduction
(%)
20 Days Reduction
(%)
30 Days Reduction
(%)
40 Days Reduction
(%)
Control (Room temp.) 93.33 86.66 7.14 66.66 28.57 41.66 55.36 28.33 69.64
Gum Coating 91.66 88.33 3.63 78.33 14.54 56.66 38.18 41.66 54.54
Wax Coating 95 90.00 5.26 75.00 21.05 53.33 43.86 36.66 61.14
Clay Coating 96.66 86.66 10.34 76.66 20.69 51.66 46.55 31.66 67.24
Wet Storage 91.66 86.66 5.45 78.33 14.54 60.00 34.54 38.66 57.82
Sun Drying 12 Hrs 88.33 78.33 11.32 63.33 28.30 40.00 54.71 25.00 71.69
Sun Drying 24 Hrs 90.00 75.00 16.66 58.33 35.18 36.66 59.92 21.66 75.93
Temp. 10⁰C 96.66 90.00 6.89 88.33 8.61 66.66 31.03 43.33 55.17
Temp. 15⁰C 95.00 85.00 10.52 86.66 8.77 68.33 28.07 41.66 56.14
Temp. 20⁰C 96.66 85.00 12.06 81.66 15.51 61.66 36.20 40.00 58.61
Mean 93.50 85.16 75.33 53.66 36.64
CD (5%) N.S. 9.19 7.12 10.9 7.50

20
Fig. 1: Variation in reduction percentage for different pre sowing treatment
4.1.2 Moisture content
The moisture content of seed is directly affected by the relative humidity of the
atmosphere around it. When dry, the viability of orthodox seeds can be prolonged by keeping
them at low temperatures (Roberts 1972). The data for variation in moisture content under
different pre-sowing treatments is presented in table 3. Data revealed that values for moisture
content are non significant for fresh in all the treatments with maximum range from 25.33% -
18.73%.
After ten days there is decrease in moisture content with increasing storage duration.
Higher moisture content recorded in temperature 10⁰C(25.33%) followed by clay coating
(24.09%) and minimum were recorded in sun drying 12 hours and 24 hours i.e. 18.73% &
19.85% respectively.
After twenty days storage maximum moisture content were recorded is in temperature
10⁰C (22.52%) and clay coating (22.59%) followed by wet storage (21.59%) and minimum
were recorded in sun drying 24 hours (14.41%).
After thirty days storage moisture content further decreases with maximum were
recorded in temperature 10⁰C (22.95%) and minimum recorded in sun drying 24 hours
(8.79%).
After fourty days, significant maximum moisture content was exhibited by the low
temperature (10⁰C, 15⁰C, 20⁰C) pre-treated seeds (22.95%, 24.11% & 26.70% respectively) as
compared to other treatments. Minimum moisture content was reported in sun-drying 12hours
& 24hours seeds i.e. 5.97% & 5.80% respectively. Rest treatment showed significantly poorer
moisture content as compared to low temperature treatments.
After 40days higher reduction per cent in moisture content was recorded over fresh is
in sun drying 24 hours (76.77%) and minimum were recorded in wet storage (21.35%).
0
10
20
30
40
50
60
70
80
90
reduction % 10 days
reduction % 20 days
reduction % 30 days
reduction % 40 days

21
Bewley and black (1994) that dry seeds there is practically no metabolism, the seed is
alive without any measurable life manifestation. In desiccation sensitive(recalcitrant) seeds
moisture content is always high and the seeds currently metabolically active.
Berjeck and pammenter (1996) reported that seeds continue to accumulate dry weight
up to the time of dispersal and germination events from more or less continuum of the
maturation process. Hence, recalcitrant seeds are metabolically active when shed and remain so
throughout storage, but the rate of metabolism can usually be reduced by storing at reduced
temperature and moisture content.
Chaisurisri et al (1986) observed inferior germination in neem seeds after longer
duration of sun drying.
Thompest (1982) found that the rate of drying was of no importance for the storage
behaviour of Araucaria hunsteinii the effect of drying rate on most other recalcitrant seeds still
needs to be revealed.

22
Table 3 : Effect of different treatments on moisture content in Neem Seeds (Azadirachta indica A. JUSS)
%
20 Days Reduction
%
30 Days Reduction
%
40
days
Reduction
%
Control 25.16 19.20 23.68 15.07 40.10 10.78 57.15 6.74 73.21
Gum Coating 25.17 21.66 13.94 18.91 24.82 11.69 53.55 10.76 57.25
Wax Coating 24.64 21.81 11.48 17.96 27.11 12.04 51.13 11.81 52.06
Clay Coating 24.81 24.09 2.90 22.59 8.94 19.08 23.09 18.74 24.46
Wet Storage 23.46 22.28 5.02 21.59 7.97 19.25 17.94 18.45 21.35
Sun Drying 12 Hrs 23.00 18.73 18.56 14.41 37.34 10.35 55 5.97 74.04
Sun Drying 24 Hrs 21.87 19.85 9.23 13.79 36.94 8.79 59.80 5.08 76.77
Temp. 10⁰C 25.48 25.33 0.58 22.52 11.61 22.95 9.97 19.63 22.95
Temp. 15⁰C 23.89 22.88 4.22 20.62 13.68 21.10 11.67 18.13 24.11
Temp. 20⁰C 23.70 22.39 5.52 20.24 14.59 21.29 14.38 17.37 26.70
Mean 24.11 21.92 19.07 15.59 13.92
CD(5%) N.S. 2.14 1.34 1.59 1.87

23
4.1.3 Mean daily germination
Data presented in table 4 revealed that mean daily germination are non significant for
fresh. High mean daily germination recorded for fresh which range from (6.72-5.72).
The value for mean daily germination percentage are shown significantly variable after
ten days. High values for mean daily germination were recorded for clay coating (5.93) and
followed by temperature 10⁰C (5.92) under all treatments and minimum recorded in control
(4.75).
However, after twenty days storage significantly variable has been observed within all
treatments. The higher mean daily germination after twenty days storage were recorded in
temperature 15⁰C (4.95) which were at par with temperature 10⁰C (4.46) and followed by clay
coating and wet storage. Lowest values recorded in control (untreated seeds) (3.36) which were
followed by sun drying treatments 12 hours and 24 hours i.e. 3.85 & 3.44 respectively.
After thirty days storage the values further decreases with highest mean daily
germination were recorded in temperature 10⁰C (3.86) followed by temperature 15⁰C and 20⁰C
(3.65 and 3.56 respectively). Minimum were recorded in control as untreated seeds (2.27) and
sun drying 24hours (2.25).
After forty days highest decrease shown in sun drying 12 hours and 24 hours (2.1 and
1.98 respectively) and minimum decrease values were recorded in 10⁰C and 15⁰C (3.25 and
3.09 respectively). However storage conditions germination values found to be highly
significant. It was observed that decrease in mean daily germination with storage for all
treatments but this decrease maximum for sun drying seeds after forty days. No mean daily
germination were recorded after forty days.
Gairola et al reported the maximum mean daily germination in temperature 37⁰C
(5.43±1.30) and minimum were recorded in 20⁰C (1.18±0.23) . Present study revealed that the
optimum temperature 10⁰C- 20⁰C favored seed germination in Jatropha curcas Linn. Billah et
-10
0
10
20
30
40
50
60
70
80
90
reduction % 10 days
reduction % 20 days
reduction % 30 days
reduction % 40 days

24
al (2015) reported mean daily germination varied in different days in different treatments for
tectona grandis seeds. The highest mean daily germination was observed 3.21 in T4 (27th
days
after sowing).
Table 4: Mean daily germination effected by treatments and storage period
Treatments Fresh 10
days
20
days
30
days
40
days
Control (untreated seeds) 5.72 4.75 3.36 2.27 2.05
Gum coating 5.88 5.66 3.92 2.97 2.36
Paraffin wax 6.14 5.58 3.87 2.67 2.26
Clay coating 6.21 5.93 4.1 3.31 2.92
Wet storage 6.13 4.98 4.04 2.99 2.76
Sun drying 12hours 6.02 5.01 3.85 2.56 2.1
Sundrying 24hours 5.83 5.9 3.44 2.25 1.98
Temperature 10⁰C 6.27 5.92 4.46 3.86 3.25
Temperature 15⁰C 6.35 5.82 4.95 3.65 3.09
Temperature 20⁰C 6.25 5.76 4.04 3.56 2.99
Mean 6.18 5.57 4.00 3.00 2.6
CD 5% n.s 0.32 0.91 0.80 0.23
4.1.4 Germination value
Data presented in Table 5 revealed that the values for germination value are non
significant for fresh. High value were recorded for fresh seeds under all treatments range from
(5.36-4.88).
After ten days storage seeds higher values for germination value were recorded for ten
days storage under all treatments which ranges from (4.4-5.11). however, higher value recorded
in clay coating (5.05) and temperature (5.11) respectively these values at par with control and
wet storage followed by gum coating and paraffin wax coating. Lowest values recorded in
temperature 10⁰C (4.4) and sun drying 12 hours and 24 hours (4.42 and 4.4 respectively).
After twenty days storage, the values further decrease with high germination value
recorded in temperature 20⁰C (3.82) and followed by clay coating (3.72) respectively these
values at par with each other. Minimum were recorded in sun drying 24 hours (2.98) and
followed by gum coating (3.01) and control as untreated seeds (3.04).
After thirty days, seeds stored at 10⁰C and 15⁰C showed significant germination value
(2.81 and 2.55) and minimum germination value shown in sun drying 12 hours and 24 hours
(1.48 and 1.26 respectively) followed by paraffin wax coating (1.62).

25
After forty days storage, maximum germination value were recorded in 10⁰C (2.03)
followed by 15⁰C, 20⁰C, clay coating (1.95, 1.92, 1.91 respectively) and least germination were
recorded in sun drying treatments 12 hours and 24 hours (1.1 and 0.93 respectively).
Sharma R (2014) reported the maximum germination value (0.93) among the different
pre sowing treatments drupes of melia azedarach. Sahoo K U and Lilabati L (2015) reported
the effect of pre-treatments of emblica officinalis germination value which lies between (0-
4.63) same trend of germination value is shown in current experiment. Gairola et al reported
maximum germination value at temperature 37⁰C (28.40±5.80) and minimum were recorded in
20⁰C (0.02±0.02).
Table 5: Germination value effected by treatments and storage period
Treatments Fresh 10
days
20
days
30
days
40
days
Gum coating 4.88 4.55 3.01 2.13 1.85
Paraffin wax 5.25 4.48 3.07 1.62 1.35
Clay coating 5.31 5.05 3.72 2.28 1.91
Wet storage 5.01 4.99 3.17 1.94 1.28
Sun drying 12hours 4.88 4.42 3.08 1.48 1.1
Sundrying 24hours 4.98 4.4 2.98 1.26 0.93
Temperature 10⁰C 5.06 4.4 3.05 2.81 2.03
Temperature 15⁰C 5.23 4.46 3.20 2.55 1.95
Temperature 20⁰C 5.36 5.11 3.82 2.51 1.92
Mean 5.10 4.64 3.21 2.04 1.48
CD 5% n.s 0.31 0.11 0.80 0.07
4.1.5 Germination index
Data presented in table 6 values for germination index are non significant for fresh
which range from (4.50-3.99). after ten days seed storage high values for germination index
were recorded for ten days storage which ranges from (4.37-3.6) and maximum value were
recorded in paraffin wax (4.37) which is followed by gum coating (4.21) and temperature 15⁰C
(4.24).
After twenty days storage values further decreases , maximum germination index were
recorded in temperature 15⁰C and paraffin wax coating (4.1 in each) and lowest values were
recorded in sun drying 24 hours (3.02) which was followed by sun drying 12 hours (3.220 and
control as untreated seed ( 3.14).

26
After thirty days seeds stored at 10⁰C and 15⁰C showed the maximum germination
index (3.78 and 3.84 respectively) and minimum were recorded in sun drying treatments 12
hours and 24 hours (2.81 and 2.66 respectively).
After forty days maximum value were shown by temperature treatments 10⁰C, 15⁰C
(2.27 and 2.18 respectively followed by clay coating (2.15) and minimum were recorded in sun
drying 12 hours and 24 hours (1.81 and 1.75 respectively).
Table 6: Germination index effected by treatments and storage period
Treatments Fresh 10
days
20
days
30
days
40
days
Gum coating 4.50 4.21 4.1 3.66 2.05
Paraffin wax 4.45 4.37 3.97 3.35 2.01
Clay coating 4.24 4.02 3.87 3.1 2.15
Wet storage 3.99 3.69 3.44 3.2 1.98
Sun drying 12hours 4.01 3.46 3.22 2.81 1.81
Sundrying 24hours 4.22 3.6 3.02 2.66 1.75
Temperature 10⁰C 4.45 4.01 3.99 3.78 2.27
Temperature 15⁰C 4.34 4.24 4.1 3.84 2.18
Temperature 20⁰C 4.36 4.05 3.98 3.69 2.04
Mean 4.36 3.93 3.68 3.30 2.01
CD 5% n.s 0.63 0.81 0.13 0.53
4.1.4 Peak value
Data presented in table 7 reveals that the peak values are non significant for fresh under
all the treatments which range from (1-0.92). Maximum recorded in gum coating and paraffin
wax coating which wereat par with each other.
After ten days seed storage the peak value are non significant ranges from (0.99-0.8).
maximum peak value shown by temperature 15⁰C (0.91) paraffin wax (0.90) and gum coating
(0.90) these value were at par with each other and also with temperature 10⁰C (0.89).
After twenty days the peak value decrease with highest peak value shown in
temperature 10⁰C & 15⁰C (0.80 in each) and minimum value were recorded in control (0.61)
and temperature 20⁰C (0.66) respectively.
After thirty days storage maximum value shown in temperature 10⁰C (0.77) and (0.72)
in gum coating and minimum shown in control (0.40) which were followed by sun drying 12
hours and 24 hours (0.58 & 0.56 respectively).

27
After forty days the maximum value were recorded in temperature 10⁰C (0.60) which
were followed by 15⁰C (0.52), 20⁰C (0.55), gum coating (0.56) and minimum values were
recorded in sun drying 12 hours & 24 hours (0.17 and 0.14 respectively).
Haseeb et al (2014) reported highest peak value (2.46) was seen in dharali site of abies
pindrow
Gairola et al (2011) reported peak value maximum at temperature 37⁰C (4.94±1.92)
and minimum at temperature 20⁰C (0.15±0.09) . The results are accordance with our results.
Table 7: Peak value effected by treatments and storage period
Treatments Fresh 10
days
20
days
30
days
40
days
Gum coating 1 0.81 0.76 0.72 0.56
Paraffin wax 1 0.90 0.72 0.61 0.37
Clay coating 0.92 0.90 0.78 0.69 0.38
Wet storage 0.97 0.87 0.73 0.65 0.31
Sun drying 12hours 0.99 0.80 0.80 0.58 0.17
Sundrying 24hours 0.98 0.81 0.76 0.56 0.14
Temperature 10⁰C 0.92 0.89 0.80 0.77 0.60
Temperature 15⁰C 0.95 0.91 0.80 0.70 0.52
Temperature 20⁰C 0.96 0.82 0.66 0.61 0.55
Mean 0.96 0.85 0.74 0.62 0.38
CD 5% n.s n.s 0.41 0.11 0.43
4.1.5 Cumulative germination (%)
Data presented in table 8 values for cumulative germination are non significant for
fresh seeds in all the treatments. High value for cumulative germination were recorded for fresh
which range from (96.65-92.45%).
After ten days high values for cumulative germination were recorded for ten days seed
storage under all treatments shows significant variation which ranges from (91.2-80.3%). The
higher value shown by temperature 10⁰C (91.2%) which is followed by gum coating (90.96%)
which were at par with temperature 15⁰C and wet storage (89.92 and 89.7% ) respectively.
After twenty days storage the values decreases with the highest cumulative germination
were recorded for wet storage (84.96) which are at par with temperature 15⁰C (84.29). The
minimum values were recorded in sun drying 24 hours (68.6%) and control (70.63%).
After thirty days storage , seed stored at 10⁰C and 15⁰C (80.94 and 82.15%) showed
the maximum cumulative germination and lowest value were recorded in sun drying 12 hours
and 24 hours (57.5 and 51.9%) which was followed by control (59.3%).

28
After 40 days storage maximum values for cumulative germination were recorded in
temperature 10⁰C (73.4) which was followed by temperature 15⁰C and 20⁰C (69.04 and 64.77
respectively) and minimum values were recorded in sun drying 12 hours and 24 hours (57.5 &
51.9 respectively).
Billah et al reported higher cumulative germination percent of T4-pit method was
always highest comparison to other treatments in Tectona grandis. Pre-sowing treatments
showed both higher mean daily germination and cumulative germination percent in comparison
to control seeds.
Table 7: cumulative germination effected by treatments and storage period
Treatments Fresh 10
days
20
days
30
days
40
days
Gum coating 95.33 90.96 83.30 70.30 61.13
Paraffin wax 92.45 88.33 80.94 71.87 63.12
Clay coating 96.55 87.42 81.20 72.91 60.47
Wet storage 96.00 89.70 84.96 72.84 63.87
Sun drying 12hours 94.25 82.30 71.87 63.40 57.50
Sundrying 24hours 94.33 80.94 68.60 59.40 51.90
Temperature 10⁰C 93.33 91.20 85.14 80.94 73.40
Temperature 15⁰C 92.45 89.92 84.29 82.15 69.04
Temperature 20⁰C 95.96 89.02 83.30 78.20 64.77
Mean 94.25 87.00 79.42 71.77 62.45
CD 5% n.s 1.35 3.78 2.77 1.81
0
20
40
60
80
100
120
fresh
10days
20days
30days
40days

29
4.1.6 Germination energy
Data presented in table 8 the germination energy show non-significant results for fresh
values range from (66.33-58.33). Maximum recorded in temperature 15⁰C (66.33) and
minimum were recorded in sun drying 24 hours (58.33).
However, after ten days value are non-significant. Maximum value were recorded in
10⁰C (60) which is followed by temperature 15⁰C (58.33), paraffin wax coating, clay coating
these values at par with each other and minimum value recorded in sun drying 12 hours & 24
hours (50 & 46.66 respectively).
After twenty days storage the values further decrease with the highest germination
energy recorded in wet storage (48.33) and 10⁰C (48.33) and minimum values were recorded in
sun drying 12 hours and 24 hours (35 &33.33 respectively).
After thirty days storage maximum value were recorded in temp 10⁰C (41.66) which
were followed by temperature 15⁰C, wet storage these values at par with each other and
minimum values are recorded in sun drying 24 hours (25) and control as untreated seeds
(28.33).
After forty days germination energy is maximum over ten days is in temperature 10⁰C
i.e. 26.66% and minimum was recorded in sun drying 12hours and 24hours i.e. (13.33% &
8.33% respectively).
Willan (1985) concluded that germination energy is measure of speed of germination, it
gives an idea of the vigour of the seed. The result strongly supports the current experiment
results having higher seed germination had better seed growth. In current experiment it has
been concluded that higher germination energy was seen in low temperature treatments even
after forty days and lower was recorded in sun drying treatments.
Sahoo K U and Lilabati L (2015) reported the effect of pre-treatments of emblica
officinalis germination energy in which maximum were recorded in T1-tap water for 24 hours
(84.00) and minimum were recorded in T2-thiourea for 24 hours (3.20) same trend of
germination energy is shown in current experiment.
Haseeb et al (2014) reported highest germination energy (25.00) and highest
germination value (2.66) was seen in dharali site of abies pindrow.

30
Table 8: Germination energy effected by treatments and storage days
Treatments Fresh 10
days
20
days
30
days
40
days
Control (untreated seeds) 60 51.66 36.66 28.33 16.66
Gum coating 61.66 55 41.66 33.33 18.33
Paraffin wax 63.33 56.66 45 36.66 21.66
Clay coating 58.33 53.33 46.66 38.33 23.33
Wet storage 59.66 55 48.33 40 21.66
Sun drying 12hours 63.33 50 35 31.66 13.33
Sundrying 24hours 58.33 46.66 33.33 25 8.33
Temperature 10⁰C 65 60 48.33 41.66 26.66
Temperature 15⁰C 63.33 58.33 46.66 40 25
Temperature 20⁰C 66.33 51.66 40 36.66 20
Mean 62.29 53.81 42.16 35.16 19.49
CD 5% n.s n.s 6.61 8.07 6.02
4.2 Biochemical parameters
4.2.1 Total soluble sugars
Data presented in table revealed that values are non significant for fresh under all
treatments which ranges from (22.95-21.96). After ten days storage, the maximum value were
recorded in temp 15⁰C (21.83) which were followed by clay coating (21.77), gum coating
(21.60) at were par with each other and minimum were recorded in sun drying 24 hours (17.21)
followed by control (19.77) and sun drying 12 hours (19.61).
After twenty days storage, the value further decrease with maximum value are recorded
in temperature 10⁰C (20.08) followed by temperature 15⁰C (19.2), gum coating (19.34), clay
coating (19.75) these values are at par and minimum were recorded in sun drying 24 hours
0
10
20
30
40
50
60
70
FRESH
10DAYS
20DAYS
30DAYS
40DAYS

31
(12.91) followed by control (15.10). Higher reduction per-cent shown in sun drying 24 hours
(43.74%).
After thirty days storage, maximum value were recorded in temperature 10⁰C ((17.10)
followed by temperature 15⁰C (16.08), temperature 20⁰C (15.19) and minimum were recorded
in sun drying 24 hours (6.73) followed by control (7.07). Higher reduction per-cent shown in
sun drying 24 hours (70.67%).
After forty days storage maximum sugar content were recorded in temperature 10⁰C
(16.75) followed by 15⁰C (6.02) and clay coating (16.00) and minimum were recorded in sun
drying 12 hours (4.05), control (3.69). Higher reduction pre-cent shown in sun drying 24 hours
(92.03%).
The neem seeds lose their viability and vigour after 35days when stored at room
temperature. There is reduction of food with increasing duration of storage period. Loss of
germination viability is due to this reason (Kumar & Mishra 2014). The data of total soluble
sugars of pre-treated neem seeds is presented in table 3. When seeds are fresh, there is non-
significant difference in total soluble sugar content in all the pre-germination treated seeds w.r.t
(control) untreated seeds. There is decrease in total soluble sugar as we increase in the storage
condition. The results are in accordance with a report made by Kumar and Mishra (2014). They
reported a gradual decrease in total soluble sugars content with increase storage duration in
neem seeds of all mother classes. Our results reported that the low temperature pre-treated
seeds show significantly a minimum decrease in total soluble sugars content. Sun dried seeds
showed maximum decrease in total soluble sugars after 40days with 82.03% (12hours) and
92.03% (24hours) decrease over 30days.
0
5
10
15
20
25
Control
GumCoating
WaxCoating
ClayCoating
WetStorage
SunDrying12Hrs
SunDrying24Hrs
Temp.10⁰C
Temp.15⁰C
Temp.20⁰C
Mean
TotalSolubleSugars(mg/gDW)
Treatments
Fresh
10 Days
20 Days
30 Days

32
Table 8: Total soluble sugars effected by treatments and storage days
Total Soluble Sugars (mg/g DW)
%
20 Days Reduction
%
30 Days Reduction
%
40days Reduction
%
Control 22.07 19.77 10.42 15.10 31.58 7.07 67.96 3.69 83.28
Gum Coating 22.47 21.60 3.87 19.34 13.92 14.35 36.13 13.54 39.74
Wax Coating 21.96 20.28 7.65 18.15 17.34 13.32 39.34 12.88 41.34
Clay Coating 22.19 21.77 1.89 19.75 10.99 16.12 27.35 16.00 27.89
Wet Storage 22.75 20.13 11.51 17.35 23.73 13.04 42.68 12.99 42.90
Sun Drying 12 Hrs 22.54 19.61 12.99 17.95 20.36 13.02 42.23 4.05 82.03
Sun Drying 24 Hrs 22.95 17.21 25.01 12.91 43.74 6.73 70.67 1.83 92.03
Temp. 10⁰C 22.72 22.04 2.99 20.08 11.61 17.10 24.73 16.57 27.06
Temp. 15⁰C 22.55 21.83 3.19 19.21 14.81 16.08 28.69 16.02 28.95
Temp. 20⁰C 22.45 20.12 10.37 18.43 17.90 15.19 32.33 15.14 32.56
Mean 22.46 20.43 17.83 13.40 11.35
CD(5%) N.S. 3.51 1.05 2.21 0.71

33
4.2.2. Total starch
Data presented in table revealed that values are non significant for fresh under all
treatments which ranges from (21.04-20.20).
After ten day storage, values are non significant under all treatments maximum sugar
content were recorded in temperature 10⁰C (19.36) followed by 15⁰C (19.32) and minimum
were recorded in sun drying 24 hours (16.60). Higher reduction per-cent shown by sun drying
24 hours (19.18%).
After twenty days storage, the values are significant under all the treatments. The
maximum value were recorded in temperature 10⁰C (17.30) and minimum were recorded in sun
drying 24 hours (12.25) followed by sun drying 12 hours (12.89) , control (13.05) these values
are at par. Higher reduction per-cent recorded in sun drying 24 hours (40.36%).
After thirty days storage the maximum value were recorded in temperature 10⁰C
(14.36) followed by temperature 15⁰C (13.01) wax coating (12.96) these values are at par and
minimum were recorded in sun drying 24 hours (6.13) followed by control (6.40). the
maximum per-cent reduction recorded in sun drying 24 hours (70.15%).
After forty days storage the maximum values were recorded in temperature 10⁰C
(12.92) followed by clay coating (12.07) and minimum were recorded in sun drying 24 hours
(2.29). Higher reduction per-cent recorded in control (89.14%) over fresh.
Total starch content in all pre-germination treated seeds. Food reserves in plants are
stored in the form of starch. There was a non-significant decrease in total starch content after
10days. However, there was significantly fastest decrease in total starch content with increasing
storage duration and was minimum after 40days. Reduction in total starch content may be a
cause for loss of viability and seed vigor. However, seeds pre-treated with gum coating, wax
coating, clay coating, wet storage and low temperature (10⁰C, 15⁰C, 20⁰C) had 50% starch
content even after 40days and this may be the possible reason for 40% germination of such
treated seeds have low starch content. The untreated seeds were not able to germinate because
they had a minimum starch content (2.25mg/g DW) after 40days. The results corroborate with
the findings of singh et al(1996) for Jatropha curcas and tripathi et al 1996 for neem seeds.
They reposted sharp reduction in starch content in untreated seeds.

34
Table 9: Total starch effected by treatments and storage days
Total Starch (mg/g DW)
%
20 Days Reduction
%
30 Days Reduction
%
40days Reduction
%
Control 20.72 18.18 12.25 13.05 37.01 6.40 69.11 2.25 89.14
Gum Coating 20.73 18.20 12.20 14.76 28.79 12.56 39.41 11.21 45.92
Wax Coating 20.81 18.20 12.54 14.19 31.81 12.69 39.01 10.96 47.33
Clay Coating 20.92 18.69 10.65 14.81 29.20 12.30 41.20 12.07 42.30
Wet Storage 21.04 17.90 14.92 14.03 33.31 12.88 38.78 11.69 44.43
Sun Drying 12 Hrs 20.99 17.10 18.53 12.89 38.58 9.75 53.54 3.19 84.40
Sun Drying 24 Hrs 20.54 16.60 19.18 12.25 40.36 6.13 70.15 2.29 88.85
Temp. 10⁰C 20.76 19.36 6.74 17.30 16.66 14.36 30.82 12.92 37.76
Temp. 15⁰C 20.20 19.32 4.35 15.48 23.36 13.01 35.59 11.39 43.61
Temp. 20⁰C 20.33 18.77 7.67 14.33 29.51 11.43 43.77 11.12 45.30
Mean 20.70 18.23 14.61 11.15 8.91
CD(5%) N.S. N.S 1.83 2.06 0.79

35
0
5
10
15
20
25
Control Gum
Coating
Wax
Coating
Clay
Coating
Wet
Storage
Sun Drying
12 Hrs
Sun Drying
24 Hrs
Temp. 10?CTemp. 15?CTemp. 20?C
Fresh
10 Days
20 Days
30 Days
40days

36
4.2.3. Total soluble proteins
Data presented in table for proteins show non-significant results for fresh under all
treatments which ranges from (183.53-171.33).
After ten days storage maximum protein content were recorded in temperature 10⁰C
(170.19) followed by temperature 15⁰C (169.57), temperature 20⁰C (169.20) and minimum
were recorded in control (151.90). Higher reduction per-cent were recorded in control
(17.23%).
After twenty days storage values further decreases with maximum value were recorded
in temperature 10⁰C (157.61) followed by temperature 15⁰C (154.28), temperature 20⁰C
(157.20), clay coating (150.68) these value are at par and minimum were recorded in sun
drying 24 hours (123.58) followed by sun drying 12 hours (128.71)
Table 5 shows total soluble protein content in all the pre-germination treated seeds with
respect to untreated seeds. Protein is the main form of nitrogen storage in food reserves in
seeds. During storage, proteins become less soluble and are degraded into free amino
acids(Anderson 1946). Several reports are suggested that reduction in vigor and viability of a
seed were closely related with decrease in protein synthesis (Roberts et al 1973, ghose and
chandhani 1984). The results reported a gradual decline in total protein content in untreated
seeds. Suszka(1975) also reported reduction in protein synthesis and oxygen uptake that
paralleled declines in germination of stored seeds. However, low temperature pre-treated seeds
showed significantly higher protein content as compare to other treatments and some seeds also
germinated well even after 40days. This shows that low temperature pre-germination treatment
is the best option to maintain viability and vigor of neem seeds. Sun dried seeds contained
significant minimum protein content which suggest that it is poor option to maintain viability as
the biochemical deterioration occurs due to heat stress. Thus, loss of viability is strongly related
to loss of proteins.
0
20
40
60
80
100
120
140
160
180
200
Control Gum
Coating
Wax
Coating
Clay
Coating
Wet
Storage
Sun
Drying
12 Hrs
Sun
Drying
24 Hrs
Temp.
10⁰C
Temp.
15⁰C
Temp.
20⁰C
Fresh
10 Days
20 Days
30 Days
40days

37
Table 10: Total soluble proteins effected by treatments and storage days
Total soluble proteins (mg/g DW)
%
20 Days Reduction
%
30 Days Reduction
%
40days Reduction
%
Control 183.53 151.90 17.23 126.21 16.91 120.83 4.26 108.26 10.40
Gum Coating 178.80 160.65 10.15 133.32 17.01 129.15 3.12 121.77 5.71
Wax Coating 175.66 165.76 5.63 139.87 15.61 134.26 4.01 129.74 3.36
Clay Coating 174.06 166.50 4.34 150.68 9.50 138.02 8.40 128.51 6.89
Wet Storage 173.97 165.51 4.86 142.64 13.81 120.87 15.26 117.40 2.87
Sun Drying 12 Hrs 173.42 154.75 10.76 128.71 16.82 116.66 9.36 105.47 9.59
Sun Drying 24 Hrs 171.33 152.27 11.12 123.58 18.84 111.66 9.64 101.34 9.24
Temp. 10⁰C 174.33 170.19 2.37 157.61 7.39 151.05 4.16 143.87 4.75
Temp. 15⁰C 175.40 169.57 3.32 154.78 8.72 150.12 3.01 144.62 3.66
Temp. 20⁰C 178.61 169.20 5.26 151.20 10.63 148.51 1.77 140.61 5.31
Mean 175.91 162.55 140.86 132.11 124.16
CD(5%) N.S. 3.28 10.33 7.84 8.54

38
4.2.4. Free amino acids
Free amino acids of pre germination treated neem seeds is presented table 6. The data
presented in the table reveals that there is a liner relationship between protein content and free
amino acid content. Free amino acid content of untreated seeds declined significantly as storage
duration increase as so the germination percentage decline with duration of storage. These
results are accordance with the findings of Agrawal (1977) for paddy seeds. Mccomb and
winstead (1964) documented reduction in several amino acids in seeds due to fungal invasion
during storage. Further, our results showed that the sun dried seeds had minimum amino acid
content as compare to untreated seeds. This might be due to the deterioration caused during
heat stress. The significant maximum free amino acids content was reported in low temperature
pre-treated seeds. It was also reported that these seeds don’t show a sharp decline in free amino
acids content even after 40days. This trend reveals that a decline in amino acids content is
related to decreased germination percentage.
0
1
2
3
4
5
6
Control Gum
Coating
Wax
Coating
Clay
Coating
Wet
Storage
Sun
Drying
12 Hrs
Sun
Drying
24 Hrs
Temp.
10?C
Temp.
15?C
Temp.
20?C
Fresh
10 Days
20 Days
30 Days
40days

39
Table 11: Free Amino Acids effected by treatments and storage days
Free Amino Acids (mg/g DW)
%
20 Days Reduction
%
30 Days Reduction
%
40days Reduction
%
Control 5.37 5.12 4.65 4.91 8.56 4.22 21.14 4.14 22.90
Gum Coating 5.24 5.16 1.52 4.92 6.10 4.55 13.16 4.32 17.55
Wax Coating 5.25 5.08 3.23 4.89 6.85 4.68 10.85 4.53 13.71
Clay Coating 5.33 5.15 3.37 5.01 6.00 4.39 17.63 4.22 20.82
Wet Storage 5.21 5.06 2.87 4.76 8.63 4.08 21.68 4.01 23.03
Sun Drying 12 Hrs 5.24 5.10 2.76 4.37 16.60 4.00 23.66 3.79 27.67
Sun Drying 24 Hrs 5.13 5.08 0.97 4.41 14.03 3.93 23.39 3.71 27.68
Temp. 10⁰C 5.28 5.17 2.08 5.10 3.40 4.79 9.28 4.38 17.04
Temp. 15⁰C 5.14 5.08 1.16 5.04 1.94 4.56 11.28 4.32 13.93
Temp. 20⁰C 5.14 5.09 0.97 5.03 2.14 4.68 8.94 4.43 13.81
Mean 5.23 5.15 4.85 4.38 4.18
CD(5%) N.S. N.S 0.36 0.47 .17

40
4.2.5 α-amylase
α-amylase enzymes help plants develop as the seeds germinate, sprout and root. Plants
are able to store energy from the sun by creating sugars. Amylase assists in the initial
development of the plant, before it is able use the energy from photosynthesis.
In a study of the germination of cereal seeds, α-amylase were found in the aleurone
layer. The amylase works to hydrolyse the endosperm starch into usable sugars. These sugars
provide the necessary energy for root growth and act as reserve food storage. It has been
observed in the current experiment that there is a significant decrease in α-amylase activity with
increasing storage duration (table 8). However low temperature treated seeds had minimum
reduction percentage in this enzyme significantly from other treatments and thus these seeds
germinated well even after 40days. Sun dried seeds has highest reduction percentage in α-
amylase activity and signicantly minimum α-amylase activity (0.54 and 0.52 in 12hours& 24
hours respectively) after 40days. As enzymes react to temperature. Temperatures that are too
high will typically cause the enzymes stop functioning. For this reason sun-dried seeds were not
able to sprout.

41
Table 12: Free Amino Acids effected by treatments and storage days
α-amylase activity
%
20 Days Reduction
%
30 Days Reduction
%
40days Reduction
%
Control 0 .93 0.80 13.97 0.75 6.25 0.66 12 0.58 12.12
Gum Coating 0.94 0.91 3.19 0.86 5.49 0.83 3.48 0.72 13.25
Wax Coating 0.96 0.90 6.25 0.82 8.88 0.79 3.65 0.70 11.39
Clay Coating 0.97 0.92 5.15 0.85 7.60 0.84 1.17 0.72 14.28
Wet Storage 0.95 0.89 6.31 0.88 1.12 0.82 6.81 0.73 10.97
Sun Drying 12 Hrs 0.94 0.79 15.95 0.70 11.39 0.66 5.71 0.54 18.18
Sun Drying 24 Hrs 0.94 0.82 12.76 0.72 12.19 0.64 11.11 0.52 18.75
Temp. 10⁰C 0.95 0.92 3.15 0.88 4.00 0.84 4.54 0.80 4.76
Temp. 15⁰C 0.95 0.91 4.21 0.89 2.19 0.87 2.24 0.80 8.04
Temp. 20⁰C 0.95 0.93 2.10 0.90 3.22 0.85 5.55 0.79 7.05
Mean 0.95 0.88 0.82 0.78 0.69
CD(5%) NS 0.68 0.41 0.36 0.50

42
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