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Plant Material, Chemicals and Extraction of oil: The ripe neem seeds were collected
from selected tree (Tree - 1, Tree 2 and Tree - 3) in the month of August. Depulping
is a process, removal of seed coat and pulp from the neem seed. It is done by hand.
39
MATERIAL AND METHOD
The ripe neem fruits were rubbed between palms in the bucket of water and washed
the seed. Clean water used for depulping. After depulping, the neem seeds were kept
in a cool and dry place. The neem seed kernels thus obtained were ground in a mixer
grinder. The defatted neem seed kernel powder was used for subsequent extractions
employing petroleum ether as solvent. Then 10 gm seek kernels powder was put in
whatman thimble and placed in soxhlet unit of 250 ml R.B. flask containing petroleum
ether (60 - 80 C) (250 ml) in condenser. The soxhlet was kept for 6 8 hour and the
material was extracted. The extract was collected in the flask and the organic solvent
was completely removed by distillation under vacuum for 2 3 days. The remaining oil
content was weighed to get the oil yield.
40
MATERIAL AND METHOD
Transfered 2 ml the above solution with the help of pipette elute into pre wetted
(with solvent mixture) RP C18 micro column. Elute azadirachtin out of the column
by repeatedly washing into 10 ml volumetric flask till the level is up to the mark.
Preparation of sample solution:
Weighed accurately sample equivalent to 2 mg of azadirachtin into 50 ml
volumetric flask dissolve in methanol: water (90:10) shake well and keep aside to
separate the layers.
Transfered 2 ml the above solution with the help of pipette elute into pre wetted
(with solvent mixture) RP C 18 micro column. Elute azadirachtin out of the column
by repeatedly washing the column with solvent mixture. Collect elute and
washing into 10 ml volumetric flask till the level is up to the mark.
A1 M1
Azadirachtin content percent by mass = ----- x ------
A2 M2
Where,
M1 : mass in gm of sample taken for the test,
M2 : mass in gm of Azadirachtin reference standard solution
A1 : peak area of Azadirachtin in the sample solution
A2 : peak area of Azadirachtin in the reference standard solution
P : percent purity Azadirachtin content
3.1.3 Neem seed oil and Azadirachtin content: the amount of seed oil and
azadirachtin in neem seed kernel showed variation among the trees (Figure 1). The
seeds oil and azadirachtin content in all the three trees are as follows:
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MATERIAL AND METHOD
187111.73 56.3
Azadirachtin content (%) = X X 41.32
8667491.15 1000.3
= 0.05020 %
725840.38 56.3
Azadirachtin content (%) = X X 41.32
8667491.15 1502.8
= 0.129632 %
639983.28 56.3
Azadirachtin content (%) = X X 41.32
8667491.15 1502.8
= 0.11429 %
3.1.4 Seed germination test: Neem seeds were collected after maturity of fruiting
(August). Seed germination test were carried out in the poly house of AFRI, campus.
The seeds were pre-treated by soaking in water at room temperature (252 0C) for 10
min. before sowing. The seeds were sown in root trainers containing soil + FYM (5:1).
The seeds were sown at 1.50 cm depths. Seeds were watered immediately after
sowing. The numbers of germinated seeds was recorded daily from the time
germination began (Day 7) until the germination ceased (Day 30). Seed was
considered germinated when the radical emerged about 1 cm above substrate (Plate
3). The detail of germination percent of neem seed were described in Table 3.
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MATERIAL AND METHOD
Table 3: Detail of characters noted for the selected trees of Azadirachta indica
(Neem).
TREE SELCTION
Observations TREE - 1 TREE - 2 TREE - 3
GEOGRAPHICAL PARAMETERS
VEGETATIVE CHARACTERS
Beginning of leaf fall First week of February First week of February First week of February
Initiation of new leaves Last week of February Last week of February Last week of February
Second week of march to Second week of march
Initiation of flower Second week of march to April
April to April
Second week of march to Second week of march to
Flowering period Second week of march to June
June June
Initiation of fruiting After third week of June After third week of June After third week of June
Number of fruit/bunch 46 56 50
Second week of July to Second week of July to second Second week of July to
Maturity of fruiting
second week of August week of August second week of August
SEED TRAITS
100 Seed weight (g) 39.20 37.03 41.42
Viability of seed
(germination %) after
100 % 100 % 100 %
storage of 15 days in
Soil+FYM (1:5)
Viability of seed
(germination %) after
40 % 50 % 40 %
storage of 1 month in
Soil+FYM (1:5)
Seed oil content
40.92 % 41.89 % 39.73 %
(per 10 gm of seed kernal)
Azadirachtin content
0.050 % 0.1296 % 0.1143 %
(per 10 gm of seed kernal)
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MATERIAL AND METHOD
3.2.1 Climate
The climate of Jodhpur and its vicinity is mainly semi arid and dry. There are extreme
temperatures with much diurnal variations, dust storms in May and June, and
potential evaporation exceeding annual precipitation and bright sunshine with clear
visibility. The extreme temperature is a striking feature. The winter is swearing cold
and the summer intensely hot scorching. The temperature increases gradually from
February onwards. April, May and June are the hottest months. During the monsoon
season, average temperatures decrease slightly. However, the city's generally low
humidity rises and this adds to the normal discomfort from the heat. Rain fall is scanty
and about 90% is received during monsoon period (i.e. last week of June to
September), generally associated with depression from the Bay of Bengal or land
depression approaching Rajasthan from the east. During the December to February,
5% of rainfall occurs in association with the western disturbance. Meteorological data
for the year 2011 and 2012 (ANNEXURE, Source: CAZRI, Jodhpur).
Summer season: The climate of Jodhpur is typical desert type, with scorching
summers. In summer the maximum temperature is around 41 C and the minimum
temperature is around 22 C.
Monsoon or Rainy season: The monsoon season tends to fall between June and
September, with the climate quickly changing following the build up of constant heat
and the arrival of thick clouds. Monsoon offers low to medium rainfall, with varying
quantum year to year, though the temperature settles down to a more comfortable
level during monsoon season.
44
MATERIAL AND METHOD
Study was carried out at Arid Forest Research Institute, Jodhpur, Rajasthan (2440N,
7115 E) during different seasons i.e. Monsoon ( July September,2011), Winter
(November 2011 to January 2012) and Summer (April June, 2012) to find out best
season for rooting of cuttings. Various research experiments were carried out for
induction of rooting in stem cuttings collected from mature tree.
The cuttings were collected from Tree 2, which is naturally growing at Forest
Genetics and Tree Breeding Field at Jodhpur. This tree was marked as resource mother
stock for collecting stem cuttings for macropropagation studies. The selected tree was
watered at 3 week interval to increase the number of new sprouts and their further
growth. Selected Tree was also treated with fungicide and insecticide to prevent the
attack of pathogens and insects. The stem cuttings were harvested with the help of
sterile sharp secateurs in morning time. After harvesting, firstly these were screened
45
MATERIAL AND METHOD
for desired length (30 - 35cm by using scale) and diameter (ranges from 0.5 - 2.5 cm)
by using calibrated vernier calliper then kept in ice box (for prevention from damage)
for transportation to laboratory site. The lower end is cut close to the node in a
slanting manner, to increase the surface area for absorption of nutrients and to
expose more meristematic tissues (Table 4 & Plate 4). After preparation, cuttings
were given a prophylactic treatment against fungal attack using aqueous solution of
0.1% Bavistin (Carbendazim 50% WP, Systemic fungicide, BASF India Limited, Bombay)
for 15 minutes, subsequently washed with distilled water. After fungicidal treatment
they were subjected to various auxin treatments. These cuttings were prepared
according to the procedure described by Hartmann et al., (2011).
Since the discovery of natural plant rooting hormones for propagation, it has
been intuitive to apply these substances to the basal end of cuttings to produce new
roots. Six concentration of aqueous rooting solutions i.e.100, 250, 500, 750, 1000 and
1500 ppm (part per million) of IBA (Indole-3-Butyric Acid, Duchefa Biochemi, Postbus,
Netherland) IAA (Indole-3-Acetic Acid, Duchefa Biochemi, Postbus, Netherland) and
NAA (-naphthalene acetic acid, Duchefa Biochemi, Postbus ,Netherland) prepared in
1N NaOH: Autoclaved distilled water (v/v). Each group of cuttings were given hormone
treatment by Basal Long Soak Methods (Kroin, 1992, 2011 and 2011a) by dipping 2/3
portion of basal ends of cuttings for 10 minute. Untreated cuttings were considered as
control and different control was taken for each rooting medium.
After auxin treatment, the cuttings were sown in root trainer (250cc)
containing different potting mixture or rooting media (Sand, vermiculite and soil). The
cuttings were planted in such a way that at least one bud remains inside the rooting
media and upper portion bear 3 5 buds. Placing the cuttings in rooting medium is
called as sticking. After sticking the upper layer of rooting medium was level with
hand. Holes were punched into the medium to allow the insertion of the cuttings
without damaging the cambium or removing the rooting hormone. After the cuttings
were stuck, the rooting medium was pressed slightly around them. Four replication of
five cutting in each were randomly assigned to each treatment. The above described
methods were followed in all season experiments.
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MATERIAL AND METHOD
The cuttings were kept under intermittent mist (misting flow for 60 second in 30
minute of interval on a root trainer) maintained at 60 80% relative humidity and 25-
30 C/15-20C day/night temperature. The cuttings were regularly watered and
treated with 0.1% Bavistin to avoid desiccation damage and attack of pathogens at
every 15 day interval. The rooting experiment was run for 60 days.
After rooting in cuttings, for hardening and acclimatization process, the rooted
cuttings were transfered from high humidity of mist to reduced humidity. This
weaning process enables the rooted cuttings become more self-sufficient in absorbing
nutrients and water through root system, in photosynthesizing, and in conditioning
new developed leaves and stem to better tolerate the stress of lower relative
humidity coupled with higher temperature and light irradiance. Then rooted cuttings
were transferred to polythene bags (16x28 cm) containing Soil + FYM (field yard
manure) (5:1) and kept in poly house for 15 to 20 days. The polythene bags were
moved daily in order to minimize misting variation. After this, polythene bags
containing rooted cutting were transferred to Agro shade house for hardening. In
agro-shade house, plants were manually irrigated by tap water once in a day. After 35
- 45 days of hardening, plants were planted in field and were manually irrigated by tap
water once in a week.
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MATERIAL AND METHOD
The planted cuttings were allowed to root for 60 days. The cuttings (five numbers per
treatment per replication) were carefully removed from the root trainer and dipped in
water to remove the medium (sand, vermiculite and soil) particles adhering to roots to
record the observations pertaining to roots viz., sprouting percent, number of sprout,
rooting percentage, number of roots and root length. Numbers of leaves recorded
after cuttings were planted in poly bags.
48
MATERIAL AND METHOD
In present investigation, explants (nodal segments) were collected from mature trees,
identified as population variants (Tree 1, Tree 2 and Tree 3).
For tissue culture studies glassware of Borosil make were used. Borosil culture
tubes without rim (25 X 150mm), conical flask (100 and 250 ml), and culture bottles
(300 and 400 ml) were used as glasswares. Culture tubes and conical flasks were
plugged using non-absorbent cotton wrapped in muslin cloth, whereas, culture bottles
were closed by screw type plastic capes. Apart from the culture vessel other
glassware like petridishes, measuring flasks, measuring cylinders, beakers (100, 500,
1000 ml), pipettes (0.1 ml, 1ml, 5ml) were used. Prior to use all the glassware were
first cleaned with laboratory grade detergent (Ases chemical work, Jodhpur) and
thoroughly washed with 0.1N HCl and were washed thoroughly 3 or 4 times in distilled
water. They were finally rinsed with distilled water and then oven dried at 80 C for 1
hr.
49
MATERIAL AND METHOD
Stock solutions of phyto hormones i.e. auxins (IAA, IBA and NAA) and cytokinin
(BAP and Kn) were prepared by dissolving them in minimal amount of ethanol or 0.1N
NaOH as a solvent, whereas, for cytokinins, 0.1N HCl was used as a solvent. Auxins and
cytokinins were prepared at strength of 1000 ppm (1mg/ml). All the stock solutions,
vitamins, amino acids and growth regulators were stored at 4C temperature in the
refrigerator. Other than MS medium, B5, and Whites were weighed and added
individually at the time of preparation. Myo-inositol, agar agar, sucrose and
additives ascorbic acid (100 mg/l), citric acid (60 mg/l), adenine sulphate (80 mg/l),
ammonium sulphate (80 mg/l), proline (10 mg/l), cystine (10 mg/l) and glutamine (10
mg/l) were used freshly. pH of the medium was adjusted to 5.8 by 0.1 N HCl or NaOH.
For the present study 4 types of nutrient media were used. These include MS medium
(Murashige and Skoog, 1962), B5 medium (Gamborg et al., 1968) WPM medium and
Whites medium (White, 1934) (Annexure II).
Stock solutions of all major (20x), minor, iron, organic components (200x) of four
defined media used were prepared in distilled water. Iron stocks were kept in amber
colored bottles. All stocks salt solutions were kept in refrigerator at 4C. Medium was
prepared using desired volume of stock solution in required volume of distilled water,
additives namely Ascorbic acid (100 mg/l), Citric acid (60 mg/l), Proline (10 20 mg/l),
Cysteine (10 20 mg/l), Glutamine (10 - 20 mg/l), Adenine sulphate (80 mg/l) and
Ammonium sulphate (80 mg/l). Agar was used for solidifying of medium in
concentration of 0.6-0.8%, and sucrose (3%) was added as a source of carbohydrate.
pH of medium was adjusted to 5.8 prior to autoclaving. 40 ml of medium was poured
in culture tubes, 60 ml in culture flasks (250 ml). Each culture vessel was plugged with
non-absorbent fresh cotton and covered with aluminium foil. Finally media was
50
MATERIAL AND METHOD
autoclaved and sterilized at 15 lb pressure for 15 minutes at 121C (250 F). All media
were supplemented with required amount of PGRs that were stored at 4C. IAA used
was stored in amber colour bottles to avoid photo oxidation. All hormones used were
prepared from stocks of 1mg/ml solutions (ANNEXURE III, IV & V).
The culture initiation and amplification were done aseptically in laminar air flow hood.
Inoculation of explants was carried out in a laminar airflow cabinet under sterile
conditions. The floor of the chamber was thoroughly cleaned with 70% alcohol
swabbed cotton. The laminar airflow cabinet was UV sterilized prior to inoculation or
working. All the required paraphernalia i.e. forceps, scalpel, petri dishes, flasks
containing sterile distilled water, beakers etc. were sterilized. Forceps, scalpels and
other instruments used for aseptic operation were sterilized by dipping in 90% ethanol
followed by red-hot flaming and cooling. The neck and mouth of culture vessels was
also flame sterilized. These cultures were kept in culture room under controlled set of
environmental conditions. Culture were incubated on tissue culture racks in growth
room having a temperature of 262C in 16 h light and 8h dark photoperiod and 1600
lux intensity light via white cool florescent tube (Philips, India).
Explants were collected from selected Trees. Nodal segments containing axillary buds
were used as explants. Explants were washed under running tap water to remove dirt
and superficies impurities. Explants were washed in autoclaved distilled water (RO
water, Milipore RiOS5) having 2-3 drops of Tween 80 for gentile shaking for 5-10 min,
then rinsed with distilled water for 3-4 times. In aseptic condition, explants were
treated with 0.1% (w/v) Bavestin (BASF India limited, Mumbai, India) and 0.1% (w/v)
streptomycin for 8-10 min to reduce the chance of fungal contamination. After
treatment explants were rinsed with autoclaved distilled water and again treated with
HgCl2 (0.1%, w/v) for 6 - 10 min for surface sterilization and later washed with
autoclaved distilled water for 3-4 times.
51
MATERIAL AND METHOD
In order to achieve high frequency multiple shoot induction and subsequent shoot
growth, experiments were conducted to study the effect of plant growth regulators,
nutrient media, types of explants (mature, semi mature, young and apical shoot tips),
period/season of collection of explants (January to December) and different A. indica
trees (population variants). Observations were recorded after an interval of 4 weeks.
For axillary shoot proliferation, media were standardized by modification in
concentration of constitute of medium and addition of additives likes adenine
sulphate, ammonium sulphate and amino acids (proline, cystine and glutamine)
(ANNEXURE III).
Axillary shoots were excised from explants and sub-cultured on fresh medium with
suitable plant growth regulator for further in vitro shoot multiplication. In order to
achieve high rate of in vitro shoot multiplication and subsequent shoot growth, the
experiments were conducted to study on the effect of plant growth regulators,
nutrient media, strength of MS medium, carbohydrate, pH, semi solid and liquid MS
medium, sucrose concentration, agaragar concentration and different A. indica trees
(population variants). Observations were recorded after an interval of 6 weeks. The
number of propagules cultured and number of propagules derived at the end of
subculture gave the multiplication fold. For shoot multiplication, media were
standardized by modification in concentration of constitute of medium and addition of
52
MATERIAL AND METHOD
additives likes adenine sulphate, ammonium sulphate and amino acids (proline,
cystine and glutamine) (ANNEXURE IV).
In vitro rooting
The major step in tissue culture plant production is in vitro rooting of in vitro
developed shoots. Single in vitro regenerated micro shoots were harvested from the
shoot clumps and transferred on to MS medium for rooting. In order to achieve high
rate of in vitro rooting, the experiments were conducted to study on the effect of
plant growth regulators, tryptophan, intraction of tryptophan & auxin, nutrient media,
sucrose concentrations and different A. indica trees (population variants).
Observations were recorded after an interval of 4 weeks. For axillary shoot
proliferation, media were standardized by modification in concentration of constitute
of medium and addition of additives likes adenine sulphate, ammonium sulphate and
amino acids (proline, cystine and glutamine) (ANNEXURE V).
In vitro rooted plantlets were removed from culture vessels and washed with
autoclaved water to remove adhered nutrient agar. These were carefully transferred
to bottles containing autoclaved vermiculite and soilrite moistened with one-fourth
strength of MS salts. The bottles were capped and kept in the green house initially,
near pad section (RH8090%, temperature 262 C) in order to harden the plantlets
from high humidity of mist to reduce humidity. This weaning process enables the in
vitro regenerated plantlets to become more self-sufficient in absorbing nutrients and
water through root system, in photosynthesizing, and in conditioning new developed
leaves and stem to better tolerate the stress of lower relative humidity coupled with
higher temperature and light irradiance. Then plantlets were transferred to polythene
bags (16x28 cm) containing Soil + FYM (field yard manure) (5:1) and kept in poly house
for 15 to 20 days. The polythene bags were moved daily in order to minimize misting
variation. After this, polythene bags containing rooted cutting were transferred to
Agro shade house for hardening. In agro-shade house, plants were manually irrigated
by tap water once in a day. After 35 -45 days of hardening, plants were planted in field
and were manually irrigated by tap water once in a week.
53
MATERIAL AND METHOD
54
MATERIAL AND METHOD
were planted in pit (size 1.5x1.5ft.) having distance of 3x3 meter at FGTB experimental
2
field, Jodhpur in the field area of 540 m with geographical position at an S-GPS-
Latitude of N 26 14' 00.1" and S-GPS-Longitude E 073 02' 19.3". The plantation (FGTB
field) comprised of 15 plants produced from hard wood cuttings (series A), 10 plants
produced from semi - hard wood cuttings (series B), 10 plants produced from mini
cuttings (series E), 20 plants produced from plant tissue culture (series AG/PTC)
and 5 plants from seeding for comparative growth performance. Initially watering was
done on a weekly basis for one month. After one month watering was done on a
monthly basis. Growth data (height and collar diameter) was collected at regular three
month intervals.
55
MATERIAL AND METHOD
56