MATERIAL AND METHOD

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CHAPTER – 3: MATERIAL AND METHOD

The present investigation titled Clonal Propagation of Azadirachta indica A. Juss

(Neem) through Macro and Micropropagation & Field Trials was conducted to figure

out techniques, which can be utilized for the production of good planting material for

large scale plantation. The present investigation was conducted during 2011-14 in the

poly house (mist chamber) and Plant tissue culture laboratory located at Forest

Genetics and Tree Breeding Division, Arid Forest Research Institute, Jodhpur at 15°26’

north latitude, 76°07’ east longitude and an altitude of 678 m above mean sea level.

3.1 Selection of Azadirachta indica (Neem) Trees

Variant in the population exhibited on the basis of morphological/phenotypic

variations is known as population variants. Azadirachta indica (Neem) trees were

selected from neem tree population at AFRI Campus and FGTB Field on the basis of

morphological characters. Characters were noted in selected trees are Geographical

parameters, vegetative characters (General Growth, girth of the main stem at breast

level (DBH), plant height & crown diameter), reproductive characters (Regeneration

ability, initiation of leaf fall, initiation of new leaves, initiation of flowering, initiation of

fruiting, Number of fruits/bunch, fruiting period and Maturity of fruiting) and Seed

traits vis. 100 Seed weight (g), Oil percentage, Seed viability & Azadirachtin

percentage. The selected population variant was demarked as Tree – 1, Tree – 2 and

Tree – 3 (Table 3, Plate No. 1, 2 & 3, Figure 1).

3.1.1 Seed oil extraction

Requirements: Soxhlet unit, whatman paper, petroleum ether (Merck specialties

private limited, Mumbai, India), 150 ml beaker, weighing balance, certrifuge tubes (50

ml) for storing seed oil and mixer grinder.

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.

MATERIAL AND METHOD

40

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 - 80º 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.

3.1.2 Determination of azadirachtin content by HPLC methods

Principle: Azadirachtin in the sample is dissolved in methanol:water (90:10) and

analysed on HPLC at 215 nm.

Requirements: Apparatus: Pipette (2 ml), volumetric flask (10 ml & 50 ml), Sep. Pak RP

C 18 cartrige filters, beakers (100 ml) and burette stand.

Equipment: Analytical balance, high Performance Liquid chromatography (HPLC unit

equipped with ultra violet (UV) detector and data processing system) and column

(stainless steel 25 cm x 4.6 mm, 5µ particle size or equivalent packed with – C18)

Flow Rate: 1ml/min

Detector: UV (215 nm)

Injection volume: 20 µl

Reagents: Acetonitrile (HPLC grade), Water (HPLC grade), Methanol (HPLC grade),

Azadirachtin sample and Reference Azadirachtin standard (42% purity).

Procedure

Preparation of mobile phase: Mix Acetonitrile and water in the proportion of 35:65

(v/v) and pass through 0.2 µ/0.45µ membrane filter under vacuum.

Preparation of reference standard solution:

Weighed accurately 2 mg working Azadirachtin standard of known purity into a 50

ml volumetric flask dissolve in methanol: water (90:10) make up to the mark and

shake well.

MATERIAL AND METHOD

41

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.

Estimation: Injected 20 µl of the working standard solution twice followed by sample

solution twice respectively to get area reproducibility. The area of two consecutive

injections should not vary by more than two percent. Calculated of azaditachtin from

the HPLC chromatogram.

Calculation:

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:

MATERIAL AND METHOD

42

Azadirachtin content in Tree – 1

Seed oil content (%) = 40.92 % 187111.73 56.3

Azadirachtin content (%) = X X 41.32 8667491.15 1000.3

= 0.05020 %

Azadirachtin content in Tree – 2

Seed oil content (%) = 41.89 % 725840.38 56.3

Azadirachtin content (%) = X X 41.32 8667491.15 1502.8

= 0.129632 %

Azadirachtin content in Tree – 3

Seed oil content (%) = 39.73 % 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 (25±20C) 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.

MATERIAL AND METHOD

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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

Mapping of the area Arid Forest Research

Campus, Jodhpur.

Forest Genetics and Tree Breeding Division Field,

Jodhpur.

Arid Forest Research Campus, Jodhpur.

Topography Flat Flat Flat

GPS latitude N 26˚14΄01.1" N 26˚13΄57.1" N 26˚13΄43.5"

GPS longitude E 073˚02΄19.6" E 073˚01΄50.4" E 073˚01΄54.8"

Altitude 226 m/t m 226 m/t m 226 m/t m

VEGETATIVE CHARACTERS

General growth Good Good Good

Height 3.1 m 5.5 m 5.1 m

DBH (cm) 60 cm 120 cm 110 cm

Crown diameter (m) 3.4 m 6.5 m 4.5 m

Regeneration ability (origin of new shoots)

Good Good Good

REPRODUCTIVE 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

Initiation of flower Second week of march to

April Second week of march to April

Second week of march to April

Flowering period Second week of march to

June Second week of march to June

Second week of march to 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

Maturity of fruiting Second week of July to second week of August

Second week of July to second week of August

Second week of July to second week of August

SEED TRAITS

100 Seed weight (g) 39.20 37.03 41.42

Viability of seed (germination %) after storage of 15 days in

Soil+FYM (1:5)

100 % 100 % 100 %

Viability of seed (germination %) after storage of 1 month in

Soil+FYM (1:5)

40 % 50 % 40 %

Seed oil content (per 10 gm of seed kernal)

40.92 % 41.89 % 39.73 %

Azadirachtin content (per 10 gm of seed kernal)

0.050 % 0.1296 % 0.1143 %

MATERIAL AND METHOD

44

3.2 Macropropagation of Azadirachta indica A. Juss (Neem)

Vegetative propagation is an important tool for tree improvement programme. It aims

at the formation of adventitious roots on stems for successful plant regeneration. The

details of the materials used, techniques employed and observation noted during the

course of this study are described below.

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.

Winter season: The winter month’s temperature varies from 10° C to 36° C. January is

the coolest month of the winter.

MATERIAL AND METHOD

45

3.2.2 General characterization of experiments

Study was carried out at Arid Forest Research Institute, Jodhpur, Rajasthan (24°40’N,

71°15’ 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.

In present investigation, an attempt was made to study the rooting ability in

different types of cuttings viz. hard wood (diameter 0.5 - 2.5 cm), semi hard wood

(diameter 0.5 - 2.5 cm) and mini cuttings by the influence of auxins i.e. IBA (Indole-3-

Butyric Acid), IAA (Indole-3-Acetic Acid) and NAA (α-naphthalene acetic acid) and their

different concentration in three rooting media i.e. sand, vermiculite and soil. Six

concentrations of IBA, IAA and NAA (100, 250, 500,750, 1000 & 1500ppm) used for

hard wood, semi hard wood and mini cuttings respectively. The studies conducted are

explained and discussed under following sub-headings:

Preparation of different types of cuttings (hardwood, semi-hard wood and mini

cuttings) collected in season (monsoon, winter & summer), dip treatment of

different auxin (IBA, IAA and NAA) and planted in three rooting media (sand,

vermiculite & soil) for adventitious rooting.

Hardening and acclimatization of plants.

Field transfer of plants.

Evaluation of growth performance of macropropagated plants at field stage.

Data analysis.

3.2.3 Preparation of cuttings

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

MATERIAL AND METHOD

46

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 (α-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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3.3.4 Environmental conditions

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°-20°C 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.

Table 4: Different types of cuttings used.

Cutting types Description

Hardwood cutting Hardwood cutting is one which is taken from a completely mature stem (Mature, dormant, quiescent stem)

Semi – Hardwood cutting

Semi hardwood cuttings are usually prepared from partially-matured wood of current season's growth a short while after a flush of growth.

Mini cuttings Mini cuttings prepared Juvenile axillary sprouts of the plants (current year new sprouts or new tender shoots).

3.3.5 Hardening and acclimatization

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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48

3.3.6 Recording of observations

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.

3.3.7 Experimental design and statistical analysis

The experimental unit is stem cutting, which can be assigned at random to a

treatment group. In present studies, completely randomized design (CRD) was used

for statistical analysis. For field trials of Azadirachta indica cutting raised plants,

randomized block design (RBD) was used for statistical analysis. Resultant data were

analyzed through General liner Model (GLM) multi variance factor analysis using

Statistical Packages for Social Sciences Software (SPSS 14.0) for all the studied

parameters i.e. Sprouting percent, number of sprout, rooting percentage, root

number, root length and leaves number. The main effects and their interactions were

studied for the test of significance. Means of control, significant factors and their

interactions were compared using Duncan Multiple Range Test (DMRT) at 0.05%

probability was used to compare the means from main effects. Minimum of 4

replicates with 5 samples (one cutting) were taken per treatment with repetition of

experiments thrice. Degree of variations was shown by Mean and standard error.

Data given in percentages were subjected to arcsine √X) transformation (Snedecor and

Cochran, 1967) before statistical analysis.

3.3 Micro propagation of Azadirachta indica A. Juss (Neem)

The micropropagation studies of Azadirachta indica A. Juss through axillary shoot

proliferation from nodal segment were studied. The details of the experimental site,

materials used, techniques employed and observation noted during the course of this

study are decribed below.

MATERIAL AND METHOD

49

3.3.1 Plant material and explants source

In present investigation, explants (nodal segments) were collected from mature trees,

identified as population variants (Tree – 1, Tree – 2 and Tree – 3).

3.3.2 Chemicals and Glassware

Standard analytical grade chemicals of Hi–media Laboratories, India were used as a

source of micro and macro nutrients, myo-inositol, gelling agent and energy source,

mercuric chloride, sodium hypochloride, streptomycin and Sodium

dichloroisocyanurate. Whereas, vitamins, amino acids, additives and Plant growth

regulators were of Sigma Chemical Company, USA and Duchefa biochemie, Postbus,

Netherlands.

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 glassware‘s. 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 80º C for 1

hr.

3.3.3 Preparation of stock solution

In order to maintain accuracy and uniformity of nutrient level in the medium,

Murashige and Skoog (1962) medium stock solutions were prepared. Based on the

solubility and stability, MS medium stocks were categorized as stock A, B, C, D, E and F

(Annexure-I). All stocks were prepared in autoclaved distilled water. Chemicals of all

the stocks (except, stock F) were weighed depending upon the volume (500 or

1000ml) prepared and dissolved in distilled water by continuous stirring. Stock F was

prepared by dissolving Na-EDTA and FeSO4.7H2O in separate containers with

MATERIAL AND METHOD

50

measured volumes of water. Solution of FeSO4.7H2O was slowly poured in solution of

Na-EDTA, with continuous stirring. Stirring was done for 2-3 hours at 60°C

temperature, till it turned to pale yellow color solution. Stock solutions of vitamins

viz. thiamine HCl, nicotinic acid and pyridoxine HCl and glycine were prepared at a

concentration of 1000 ppm (1mg/ml) in distilled water.

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 4°C temperature in the

refrigerator. Other than MS medium, B5, and White‘s 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).

3.3.4 Media Preparation

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 4ºC. 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

MATERIAL AND METHOD

51

autoclaved and sterilized at 15 lb pressure for 15 minutes at 121ºC (250º F). All media

were supplemented with required amount of PGR’s that were stored at 4ºC. 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).

3.3.5 Inoculation and Culture Conditions

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 26±2°C in 16 h light and 8h dark photoperiod and 1600

lux intensity light via white cool florescent tube (Philips, India).

3.3.6 Explants Preparation and Surface Sterilization

Explants were collected from selected Tree’s. 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.

3.3.7 General Characterization of Experiment

The studies conducted are explained under following sub-headings:

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52

In vitro regeneration through axillary shoot proliferation

In vitro shoot multiplication

In vitro rooting

Hardening and acclimatization of tissue culture plants

Field transfer of tissue culture plants

Evaluation of growth performance of micropropagated plants at field stage.

Data analysis

The steps of procedures followed were as given below:

Axillary shoot proliferation

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).

In vitro shoot multiplication

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, agar–agar 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

MATERIAL AND METHOD

53

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).

3.3.8 Hardening and acclimatization

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 (RH80–90%, temperature 26±2◦ 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.

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3.3.9 Recording of observations

In case of micropropagation studies, observations were recorded on axillary shoot

proliferation, number of shoot regenerated, shoot length of regenerated shoot (cm),

rooting percent, number of roots and root length (cm) in regular intervals as described

in each experiments.

3.3.10 Experimental Design and Statistical Analyses

The experimental unit is explants, which can be assigned at random to a treatment

group. Thus in present studies, completely randomized design (CRD) were used for

statistical analysis. For field trials of Azadirachta indica tissue culture raised plants,

randomize block design (RBD) was used for statistical analysis. Resultant data were

analyzed through General liner Model (GLM) multi variance factor analysis using

Statistical Packages for Social Sciences Software (SPSS 14.0) for all the studied

parameters i.e. shoot proliferation percent, number of shoot regenerated, length of

regenerated shoots, rooting percentage, root number, root length, height and collar

diameter. The main effects and their interactions were studied for the test of

significance. Means of control, significant factors and their interactions were

compared using Duncan Multiple Range Test (DMRT) at 0.05% probability was used to

compare the means from main effects. Minimum of 4 replicates with 5 samples (one

nodal explants or in vitro regenerated single shoot for multiplication and rooting) were

taken per treatment and with repetition of experiments four times. Degree of

variations was shown by Mean and standard error. Data given in percentages were

subjected to arcsine √X transformation (Snedecor and Cochran, 1967) before

statistical analysis.

3.4 Field plantation of plants produced from macro and micro

propagation techniques

The plants were successfully hardened and acclimatized in poly house and then in

agro-shade house. Field plantation of micro and macro propagated raised Azadirachta

indica (Neem) plants were established for evaluation of growth performance in field.

60 Plants produced from macropropagation and micropropagation was planted in

field in September, 2013 at Forest Genetics and Tree Breeding Field, Jodhpur. Plant

MATERIAL AND METHOD

55

were planted in pit (size 1.5x1.5ft.) having distance of 3x3 meter at FGTB experimental

field, Jodhpur in the field area of 540 m2 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.

20 Plants produced from macropropagation and micropropagation were planted in

field of Basan Research Centre, Gandhinagar on August, 2013. PlantS were planted in

pit (size 1.5x1.5ft.) having distance of 5X5 at BRC Gandhinagar in the field area of 500

m2 with geographical position at an S-GPS-Latitude of N 23° 12' 06.8" and S-GPS-

Longitude E 072° 40' 44.6". The plantation (BRC, Gandhinagar) comprised of 5 plants

produced from hard wood cuttings (series – A), 5 plants produced from semi - hard

wood cuttings (series – B), 5 produced from mini cuttings (series – E), 5 produced from

plant tissue culture (series – AG/PTC). Initially watering was done on a weekly basis for

one month. After one month watering was done on a monthly basis. The detail layouts

of field plantation of Azadirachta indica plant are given below.

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Field plantation of Azadirachta indica produced from macro propagation and

micropropagation at FGTB field, Jodhpur

Field plantation of Azadirachta indica produced from macro propagation and

micropropagation at Basan Research Center, Gandhinagar.