49

MATERIAL AND METHODS

TISSUE CULTURE STUDIES:

This section presents the details of material and methods which were employed

for undertaking the present study.

PLANT MATERIAL: The mother plant was collected from the Tri Shakti

farms, Sanathnagar, Hyderabad. The explants used were the Shoot Apex, Nodal

Region, and Leaf Segments.

Glassware :

All glassware used during the course of experiments like culture tubes, beakers,

pipettes and funnels were produced from m/s. Borosil India Ltd., Mumbai Luxbro

India Ltd., supplied polypropylene caps for culture tubes, while caps of bottles

were obtained from Varsha Storage Racks.

Chemicals and other media ingredients:

Chemicals required for media preparation were of analytical grade obtained from

Sigma, BDH, Hi-media and LOBA chemical companies, sugar used as a source of

sucrose was obtained from local market.

Preparation of Explants:

Shoots of 10-15 cm lengths of Stevia auxillary buds were detached and brought

from the field to the Tissue Culture Laboratory. The shoots, leaves and nodal

regions were cut into 1-2 cm. Length as shoot tips and nodal cuttings and were

50

used as explants to establish in vitro cultures. The procedure of surface

sterilization followed in given here under.

Explants were washed under running tap water with 5.1 Teepol solution

immediately they were soaked in solution of 2% Bavistin and K-Cycline which

served as fungicide and bactericide respectively. Two explants were soaked for

1hrs. Then the explants are transferred the laminar air flow cabinet for surface and

aseptic sterilization.

Explants surface sterilization:

Explants washed with sterile water.

Explants washed with 70% alcohol for 30 seconds.

Washed with sterile distilled water for 2 or 3 minutes.

The explants washed with 0.01% mercuric chloride + Tween 20 (1 or 2

days ) for 10 minutes .

Then washed with sterile distilled water four times.

First time - 4 minutes

Second Time - 4 minutes

Third Time - 4 minutes

Fourth Time - 12 minutes

Explants surface sterilization is over. Then the explants were inoculated in the

appropriate media.

51

MEDIA COMPOSITION:

A modified Murashige and Skoog (1962) medium containing 3% Sucrose

solidified with 0.8% Agar was used as the basal medium. The composition is

given in the Table.

Ingredients Salt

Concentration

of stock solution

(g/l)

Amount

taken for 1

liter

medium

(mg/1)

Final

concentration of

salt in a 1 liter

culture medium

(mg/l)

Strength of

the final

concentration

Macro-nutrients

KNO3 19 1900

NH4NO3 165 100 1650 10 x A

CaCl2.2H2O 4.4 440

MgSO4. 7H2O 3.7 170

KH2.PO4 1.7 170

Micro-nutrients

MnSO4. 4H2O 2.23 22.3

ZnSO4. 7H2O) 0.86 8.6

H3BO3 0.62 10 6.2 100 x B

CuSO4. 5H2O 0.025 0.0025

KI 0.083 0.83

Na2MoO4. 2H2O 0.025 0.25

CoCl2. 6H2O 0.0025 0.025

FeSO4. 7H2O 2.78 27.8 100 x C

Na2EDTA 3.73 10 37.3

Vitamins :

Myo-inositol 10 100

Thiamine 0.01 0.1

Nitrotinine acid 0.05 10 0.5 100 x D

Pyridoxide HCl 0.05 0.5

Glycine 0.2 2

52

Growth Regulators :

Benzylamino purine 2 mg /l

Kinetin 1 mg /l

Napthalene acetic acid 1 mg /l

2,4-Dichloro acetic acid 2 mg /l & 3 mg /l

53

Preparation of culture media:

The macro nutrients, micro nutrients and vitamins drawn from the stock

solution were mixed in the required quantity. The growth substances were added

as necessary. Sucrose as a source of carbon was dissolved at the rate of 30 g/l

(3%). The final volume of known quantity was obtained by adding double

distilled water. The pH was adjusted to 5.0 by a addition of in HCl or NaOH as

required.

Agar-Agar was added to the boiling media at a rate of 8 g/l (0.8%) slowly

and gradually with constant stirring to avoid formation of any clumps. Then the

medium was dispossed into culture vessels i.e. glass bottles (baby jars of 250 ml.

Capacity) or test tubes (25 x 150mm. Size) @ 40 ml. respectively. These vessels

were plugged with polypropylene caps and were then autoclaved along with other

surgical instruments required for transfer operation at 121 º C and at a pressure of

15 lbs for 20 minutes.

Stock Solution of Potassium Iodide KI (1000X):

0.0830 (83mg) of KI was dissolved in 100 ml distilled water: usage =1ml/l

Stock Solution of Na2EDTA (100X):

Dissolve 37.2mg of Na2EDTA (Ethylene di amine tetra acetic acid, di sodium

salt) in 50 ml DH20. Boil Na2EDTA solution and dissolve 27.8 mg of FeSo4.7H2o

gently by stirring and final volume is made up to 100ml. To prepare 1000 ml of

culture medium. Usage- 1ml/l.

54

Stock Solution of Calcium chloride (100X):

4.4 g of Calcium Chloride was dissolved in 80 ml of sterile distilled water and the

final volume is made up to 100 ml. Usage-10 ml/l.

Stock Solution of Myoinositol (10X):

1000 mg of myoinositol was dissolved in 100 ml distilled water. Usage-1ml /l.

ROLE OF PLANT GROWTH REGULATORS:

Some chemicals occurring naturally within plant tissue (i.e. endogenously) have a

regulatory rather than a nutritional role in growth and development. Growth as

well as differentiation of tissues in vitro is controlled by various growth regulators

Auxins, Cytokinins, Gibberellins, Ethylene, Abscisic acid etc.

Auxins :

Auxins (IAA, NAA, 2,4-D, IBA) are phytohormones that influence cell

enlargement, root initiation, adventitious bud formation. They suppress the

initiation of lateral buds.

2,4-D: (2,4 dichlorophenoxy acetic acid: C8 H6 C12 O3) is a synthetic

auxin known primarily as a weedicide. It is used for callus induction.

IAA (Indole – 3 – acetic acid: C10 H9 NO2) It is a promoter rooting of

cuttings.

IBA (indole – 3 – butyric acid:C12 H13 NO2) It is a rooting hormone and

more stable than IAA.

55

NAA ( Napthalene acetic acid : C12 H16 O2 ) :

It is also a root-inducing hormone and also promotes callus kinins.

Cytokinins: The cytokinin (KN, BAP, 2-ip and Zeatin) is adenine a derivatives

and is formerly called Kinins.

BAP (Benzyl amino Purine: C12 H11N5 ) : It is used to promote auxillary

bud growth.

2-ip (i PA N6 – (2-iso pentyl adenine: C10 N5): H13 it causes rapid cell

division and consequent irregular growth.

Kinetin (6- Furfuryl aminopurine: (C10 H9 N5 O): promote cell division.

Zeatin (6-[4-hydroxy – 3 – methyl but – 2 – enly aminopurine: C10 H13

N5 O): it is a common alternative cytokinin to 2-ip.

Cytokinins are even effective in promoting direct or indirect shoot

initiation Wickson and Thimann (1958) discovered that cytokinin could release

the lateral buds from apical dominance in an intact stem tip.

The role of cytokinin in shoot organogensis is well-established (Flick et al.

1983). Cytokinin in combination with an auxins appear essential for the onset of

growth and the induction of embryogenisis (Fujimure and Komamine 1980).

Gibberellins: Gibberellins (gibberellic acid, GA3 C19 H22 O6): Influence

cell enlargement and stem elongation.

Abscisic Acid: It is useful in embryo culture and somatic embryogenesis.

Ethylene (C2H4): It is a gas produced by plants. It is involved with fruit

ripening, flowering and leaf abscission.

56

GROWTH REGULATORS:

Plant Growth Regulators incurred in the preparation of culture medium include

6´-benzyl adenine (BAP), Kinetin (Kn) Adenine Sulphate (AS), 2,4-dichloro

phenoxy acetic acid (2,4-D),1-Napthalene acetic acid (NAA).These were prepared

in desired concentrations (to be maintained as Stock Solution) to induce plant

regeneration from explants.

6´-benzyl adenine(BAP):

6´-benzyl adenine(BAP) is a cytokinin [ A synthetic N- (Phenyl)-1H-Purine-6-

amine compound ].

10 mg of 6´-benzyl adenine(BAP) was dissolved in 1 ml of 1N NaOH was

adjusted to 8 ml with distilled water, then final volume was made up to 10 ml

with distilled water, filter sterilized and stored at 4°C.

Kinetin (Kn):

Kinetin (Kn) is a cytokinin [ a synthetic N-(2- furanyl methyl) 1H –Purine -6-

amine compound].

10 mg of Kinetin was dissolved in 1 ml of 1N NaOH, volume was adjusted top

8ml with distilled water, then final volume was made up to 10 ml with distilled

water, filter sterilized and stored at 4°C.

Adenine Sulphate (AS):

Adenine Sulphate (AS) is a synthetic cytokinin, and its formula is

(C5H5N5)2H2SO4).

57

10 mg of Adenin Sulphate was dissolved in 1 ml of 1 N NaOH, volume was

adjusted to 8 ml with distilled water. Then final volume was made up to 10 ml

with distilled water, filter sterilized and stored at 4°C.

2,4-Dichloro phenoxy acetic acid (2,4-D):

10 mg of 2,4-D was dissolved in 1 ml of 1N NaOH, the volume was adjusted to 8

ml with distilled water. Then final volume was made up to 10 ml with distilled

water, filter sterilized and stored at 4°C.

Naphthalene acetic acid (NAA):

10 mg of NAA was dissolved in 1 ml of 1 N NaOH, volume was adjusted to 8 ml

with distilled water. Then final volume was made up to 10 ml with distilled water,

filter sterilized and stored at 4°C.

Indole butyric acid (IBA): [a synthetic 4 –(indole-3-yl) butyric acid

compound].

10 mg of IBA was dissolved in 1 ml of 1N NaOH, the volume was

adjusted to 8 ml with distilled water, then final volume was made up to 10 ml

with distilled water, filter sterilized and stored at 40° C.

Preparation of transfer area for aseptic culture:

Aseptic culture works like final surface sterilization of plant material,

preparation and inoculation of explants and further sub culturing of in vitro

cultures were carried out in a laminar Air flow cabinet. Initially before the use of

the cabinet, the working surface was sterilized by swabbing the surface of the

58

cabinet with 70% ethyl alcohol. Later the cabinet was ensured sterile by switching

on UV light 2500 Aº for about 15 minutes before use. Then the sterile airflow was

switched on and left for at least 10 minutes before use. During the course of

transfer, between each transfer of explants to culture bottles, or tubes, the surgical

instruments were dipped in absolute ethyl alcohol followed by dipping them in

glass head sterilization for 15-20 seconds, where the temperature was maintained

at 250 ºC and cooled before use. After the completion of sterile transfer operation,

the laminar airflow was cleaned and sprayed with 70% ethyl alcohol and kept

closed.

Incubation room:

The culture was incubated in an air-conditioned room at the

temperature of 25 ± 2ºC under a micro propagation region of 16 hours light and 8

hours dark cycle.

Growth room:

Each growth room is fitted with mobile culture incubation racks fitted

with 40 watts cool day white fluorescent tube lights for providing light for

photosynthesis of tissues. Growth room is maintained clean was of CL 1,00,000

and temperature of 25± 27ºC for temperature crops 16 hours photoperiod and

8hours darkness are provided in each growth room. The photoperiod and

temperature is maintained.

59

MAINTENANCE OF CULTURES IN LABORATORY:

The in vitro cultures of Stevia rebaudiana Bertonii or all the

experiments were maintained at 25±2°C and 3000 Lux Illumination comprising a

16 hour photoperiod provided by cool flourescent light and with a relative

humidity of 50±20%.

INITIATION OF CULTURES :

After Surface Sterilization the explants were cut into a specific size with a

scalpel on pre sterilized petri plates and embedded on to the media with various

concentrations of hormones. The cultures were maintained for 4 weeks and after

proper callusing they were Sub cultured on different media.

All the experiments were repeated 3 times and 12 replications (Test

Tubes/culture vessels) for each treatment were used each time. The culture were

observed regularly to watch the growth and regeneration and recorded the data

for various parameters in each experiment.

MICRO PROPAGATION:

Micro propagation became one of the most ways to produce crops

that are difficult to propagate by conventional methods like cuttings or seeds. The

application of tissue culture technique to produce virus free plants initially led to

subsequent micro propagation of a great diversity of plants in vitro.

60

Days taken For the Establishment Of Explants in vitro:

Murashige and Skoog medium (1962) along with different concentrations of

cytokinin BAP (0.25mg/l, 0.5mg/l, 1.0mg/l, 1.5mg/l, 2.0mg/l, 2.5mg/l) were used.

The following data were collected during this experiment.

1. Average number of days taken for the initiation of shoot bud, first

leaf, and second leaf:

The mean time taken for establishment i.e., the initiation of shoot bud, first

leaf, second leaf varied with different concentrations of growth regulators. The

response of Initiation of Shoot bud, first leaf, second leaf was early i.e., 15 days.

In the medium supplemented with different concentrations of BAP.

The mean number of days taken for the emergence of first leaf varied

with different concentrations of growth regulators in the basal media. The early

initiation of first leaf was observed within 5 days in medium supplemented with

three different concentrations of BAP.

MULTIPLE SHOOTS:

Multiple shoots formation was observed in MS medium with different

concentration of BAP. Maximum numbers of multiple shoots were obtained in

media supplemented with 1.0 mg/l and significant difference was not found in

low concentration of 0.5 mg/l. The mean number of days taken for the multiple

shoots induction was recorded. Frequency of multiple shoots can be measured by

using the formula.

61

No. of explants that responded

(by producing multiple shoots)

Frequency of Multiple Shoots = --------------------------------------- × 100

Total No. of explants inoculated

1. Shoot length in cms

The shoot length of the plantlet was recorded after the 90 days of initiation and

the tip of the top most leaf formed to the initiation point of roots were taken into

consideration.

2. Root length in cms

The root length of the plantlet was recorded by measuring the longest root of the

plantlet this was taken into records before the plantlet bottles were shifted to

green house for acclimatization after 90 days of initiation.

3. Number of leaves

The total number of freshly surviving healthy green leaves both long and short

were counted before the cultures bottles were shifted to green house 90 days for

further process of acclimatization the leaf at the tip of the plantlet to the lower

point near the rooting area were taken into consideration.

4. Number of roots

The total number of roots per each plantlet after 90 days was taken into account

before shifting them to green house all the roots were thoroughly washed with

water and roots were cleaned from medium in which it was inoculated.

No. of rooted shoots

Frequency of Rooting = -------------------------------------------- ×100

Total No. of inoculated explants

62

5. Fresh weight

The in vitro rooted plantlets after 90 days were removed from the culture vessels,

and the agar was removed with tissue paper. And fresh weight was recorded in

(mg).

6. Percentage of survival during Hardening of the plantlets

In vitro rooted plantlets from different treatments after 90 days were taken to

green house from the laboratory for acclimatization. The plantlets removed from

the culture bottles, washed off the agar and planted in portray having sterilized

potting mixture of decomposed coco peat + vermiculite at 1:1 ratio. After placing

the portrays with plants transferred to high Relative humidity sheds in the green

house for 30 days at 80 -90 % (RH) Relative humidity and temperature of

30ºC±2ºC. After 30 days plants transferred to shade house and taken the data on

percentage of survival.

No. of surviving plants

in the shade house

Frequency percentage of survival in shade house =-------------------------------×100

Total No. of transferred plants

ORGANOGENESIS:

In the present study the full strength MS media was supplemented with two plant

growth regulators for the further growth of the plant material before its

acclimatized. It was found that BAP 0.5 mg/l + KN 0.4 mg/l showed more results

of organogenesis.

63

CALLUS INDUCTION:

In the present study full strength MS medium has been used. The response of

Initiation and callus formation was early i.e., 15 days. In the medium

supplemented with different concentrations of BAP, 2,4-D mg/l.

No. of explants showing response

Callus Induction Frequency = --------------------------------------------- ×100

Total No. of explants inoculated

1. Mean days taken for callus initiation

The mean number of days taken for the initiation of callus i.e. formation of

undifferentiated tissue in the cultured vessels/bottles of the experiment

2. Fresh weight of the callus

Fresh weight of the callus was recorded after 60 days of callus initiation.

3. Nature of the callus

Nature of the callus, colour of the callus was recorded in different treatments i.e.

globular, smooth, green, light green, brown.

GROWTH REGULATORS:

The growth regulator used in callus initiation was BAP, and 2,4-D

with different concentrations (0.25 mg/l, 0.5 mg/l, 1.0 mg/l, 1.5 mg/l, 2.0 mg/l,

2.5 mg/l, 3.0 mg/l) for both the explants (i.e.) nodal region and leaf segment. The

shoots developed from the embryos of the explants were transferred to rooting

medium of MS medium with 1mg NAA along with activated charcoal for rooting

of plantlets.

64

SURFACE STERILIZATION:

The Callus was removed from the culture bottles and placed on sterile

paper. Dried callus, hard callus and dead tissue and if any multiple clamps were

removed by using sterile forceps and blades. Then the callus were placed on the

fresh media if it is in the shoot development stage for further growth of the

plantlets from callus media comprising of 1 mg/l BAP.

The following observations were made during the experiment.

After the first sub culture of the callus obtained from the nodal

region weight about 2.0 mg in 2, 4-D 1.5 mg/l. The colour of the callus was Light

green smooth in nature whereas the callus obtained from the leaf segment weight

about 1.8 mg in 1.5 mg/l 2,4-D here also the colour of the callus was Light green

and smooth in nature.

SUB CULTURE OF PLANTLETS FROM GROWN CALLUS

The plantlets were removed from the culture bottles and placed on sterile

paper. Dried leaves, hard callus and dead tissue and if any multiple clamps were

removed by using sterile forceps and blades. Then the callus were placed on the

fresh media if it is in the shoot development stage and later on transferred to the

rooting medium comprising of 1mg/l NAA. The survivals of plantlets during

acclimatization after transferring all the fully developed plantlets to field were

monitored.

To study the effect of different growth regulators on callus initiation and

regeneration through somatic embryogenesis from the callus of nodal and Leaf of

Stevia rebaudiana Bertonii.

65

In the present study the full strength MS medium was used along with two

different plant growth regulators (i.e.) 2,4-D, and 2-ip with different

concentrations.

The growth regulators along with basal medium used for the Initiation of

Somatic embryos takes about 3 months of time. The concentrations used for both

the callus obtained from nodal and leaf callus were 2,4-D (0.01mg/l,0.25mg/l,

0.5mg/l, 1.0mg/l and 2ip (0.01mg/l, 0.05mg/l, 1.0mg/l,1.5mg/l). The number of

days taken for the initiation of somatic embryos is 90 days and it was found that

2,4-D with 1.0mg/l and 2-ip with 1.5mg/l showed more number of somatic

embryos. (as evidenced by the development of globular callus on the nodal callus)

was recorded.

Nature of the callus:

Nature of the callus, colour of the callus was recorded in different

treatments i.e. single spherical cell, early globular shape with conspicuous

suspensor, typical globular shape, elongated shape, early heart shape, typical heart

shape, torpedo shape ( as evidenced by SEM photos).

MATURATION OF SOMATIC EMBRYOS:

After four weeks of induction, calli bearing somatic embryos were shifted

to the full strength MS media was supplemented with two plant growth regulators

for the further growth. It was found that BAP 0.5 mg/l + KN 0.4 mg/l showed

more results.

66

The cultures were maintained at 25±2°C and 3000 Lux Illumination

comprising a 16 hour Photoperiod provided by cool fluorescent light and with a

relative humidity of 50±20%. After four weeks the frequency of maturation of

somatic embryos (as evidenced by the transition of globular stage to torpedo stage

of embryo) was recorded.

Mean days taken for the organogenesis from the somatic embryos:

During the above mentioned maturation stage of somatic embryos. The

mean days taken for the organogenesis from somatic embryos were recorded.

Number of embryos formed, Percentage of somatic embryos and shoot formation

was taken into consideration.

Germination of somatic embryos:

The somatic embryos after attaining to the torpedo stage of development

on maturation medium were transferred to the rooting medium comprising of MS

medium supplemented with NAA for both the 2,4-D and 2-ip grown somatic

embryo genetically grown plants for germination.

The cultures were incubated for 2 weeks at 25±2°C and 3000 Lux

Illumination comprising a 16 hour Photoperiod provided by cool fluorescent light

and with a relative humidity of 50±20%.

1. Shoot length in cms

The shoot length of the plantlet was recorded after the 90 days of initiation and

the tip of the top most leaf formed to the initiation point of roots were taken into

consideration.

67

2. Root length in cms

The root length of the plantlet was recorded by measuring the longest root of the

plantlet this was taken into records before the plantlet bottles were shifted to

green house for acclimatization after 90 days of initiation.

3. Number of leaves

The total number of freshly surviving healthy green leaves both long and short

were counted before the cultures bottles were shifted to green house 90 days for

further process of acclimatization the leaf at the tip of the plantlet to the lower

point near the rooting area were taken into consideration.

4. Number of roots

The total number of roots per each plantlet after 90 days was taken into account

before shifting them to green house all the roots were thoroughly washed with

water and roots were cleaned from medium in which it was inoculated.

No. of rooted shoots

Frequency of Rooting = -------------------------------------------- ×100

Total No. of inoculated explants

5. Fresh weight

The in vitro rooted plantlets after 90 days were removed from the culture vessels,

and the agar was removed with tissue paper. And fresh weight was recorded in

(mg).

6. Percentage of survival during hardening of the plantlets

In vitro rooted plantlets from different treatments after 90 days were taken to

green house from the laboratory for acclimatization. The plantlets removed from

the culture bottles, washed off the agar and planted in portray having sterilized

68

potting mixture of decomposed coco peat + vermiculite at 1:1 ratio. After placing

the portrays with plants transferred to high Relative humidity sheds in the green

house for 30 days at 80 -90 % (RH) Relative humidity and temperature of

30ºC±2ºC. After 30 days plants transferred to shade house and taken the data on

% ge of survival.

No. of surviving plants

in the shade house

Frequency percentage of survival in shade house = ------------------------------×100

Total No. of transferred plants

SEM (Scanning electron microscope) :

The embryogenic callus at different stages was used for the SEM studies.

The callus samples were processed by fixing 4% (v/v) glutaraldehyde in 0.1 M

phosphate buffer (pH6.8) for about 3 hours of duration. The samples were washed

thoroughly in distilled water 3 times. Then the samples were dehydrated by

passing through acetone series from low to high concentrations

[20,40,60,80,100% (w/v)] each lasting about 30 minutes of duration .The samples

were dried to critical point for gold coating, they were mounted onto the stubs

using “JEOL – JFC – 1100E/JEOL – 100 CX –BALZAR‟S - 4 CD – Ion

Sputtering device”. After gold coating, the material was observed under” JEOL –

JSM – 5200/JEOL – 100CX – Scanning electron microscope” and different stages

of somatic embryogenesis were photographed.

The fine structure of somatic embryos was clearly identified during the

following sequential developmental stages: single spherical cell, early globular

69

shape with conspicuous suspensor, typical globular shape, elongated shape, early

heart shape, typical heart shape, and torpedo shape.

High performance liquid chromatography (HPLC) analysis:

Material and Method:

Dried leaves of Stevia plant [ex vitro grown plant]- 1 g

Dried leaves of Stevia plant [in vitro grown plant]-1g

Stevioside standard solution [mg/ml]

Acetonitrile [Mercks company]

Water.

Equipments used:

Soxhlet extractor apparatus.

High Performance Liquid Chromatography (Preparative HPLC Waters Auto

PurificationTM

System).

SAMPLE PREPARATION:

1g leaves of each of ex vitro and in vitro grown plants were dried and

ground to fine powder using a dry blender. The sample of dried Stevia leaves

were put into extraction thimble and place sample into Soxhlet extractor.

Extraction was made using a solvent [Dichloromethane and methanol (1:1)].

[HPLC grade Merck].

70

STANDARD SOLUTION PREPARATION:

The 2.5 mg of each two standards solution were prepared by diluting

with 80% methanol in water into volumetric flask respectively in prior to sonicate

them for 15 minutes.

The separation of stevioside from the leaves was carried out by

preparatory HPLC [Waters HPLC purification System]. The HPLC operating

conditions and parameters are as follows: The column used was C18, with

dimensions of 250 X 4.6mm, 5μ. The mobile phase used was Acetonitrile : water

[80:20 v/v] the Wavelength of detection was at 254nm.

HPLC analysis of S. rebaudiana crude extract was carried out to

determine the stevioside content found in the leaves of S. rebaudiana in

comparison with a reference standard. Acetonitrile - water (80:20 v/v) was used

as the mobile phase. Stevioside was separated by means of a reversed phase

Agilent technologies HPLC system comprised of quaternary pump, a column

oven, sample freeze and UV detector. A ZORBAX Eclipse XDB-C18 column

(4.6x150 mm, 5 µm) was employed, at 30ºC. Separation was made in isocratic

mode, using Acetonitrile : water (80:20 v/v) at a flow rate of 1ml/min with 20 µl

injection volume; detector and column temperature were set at 30ºC. The

detection wave length was 210 nm (Ahmed and Smith, 2002).

71

Sample preparation for HPLC analysis:

Silica (25 g) was added to a column. The column was conditioned with

hexane (80 ml) and not allowed to dry. The crude extract (250 mg) dissolved in

methanol (20 ml), was mixed with silica (5 g) and dried under vacuum. Silica

adsorbed concentrated extract was then applied to the column. The analytes were

eluted with chloroform: ethyl acetate: water (65:25:4, 100 ml) (Wagner and Bladt

1996). The eluate was concentrated almost to dryness under vacuum. The residue

was re-dissolved in methanol (20 ml). A light yellow solution was obtained and

then decolorized using activated charcoal. Determination of the content of

stevioside in plant material was performed by external standard method. A stock

solution of standard stevioside (Menge Germany) was used and solutions with

concentrations of 100, 200, 300, 500 and 800 mg/l were used to draw calibration

curve. Triplicate determinations were carried out and the average taken in

drawing the calibration curve.

ISOLATION OF GENOMIC DNA:

Extraction Buffer:

2% CTAB, 100mM Tris, 20mM EDTA and 1.4 M NaCl

♦ Preheat 15 ml of CTAB buffer at 600C (Add 50mg PVP and 200 ul β-

mercaptoethanol)

♦ Grind the leaf samples (2 to 5 gm) in liquid Nitrogen and add into

preheated buffer

72

♦ The contents were mixed gently by swirling and inverting the tube and

incubated at 600C water bath for 45 min. with occasional mixing.

♦ Keep the samples at room temperature, and add equal volume of

chloroform-Isoamyl alcohol (24:1)

♦ Mix the contents by inversion for 10 minutes and centrifuged at 8000 rpm

for 10 min.

♦ Transfer the clear aqueous upper layer into a new centrifuge tube, add two

volumes of cold isopropanal and mixed by inversion and placed in -200C

for 30 min.

♦ Pellet the genomic DNA by centrifuging at 10000 rpm for 20 min at 100C

♦ Wash the pellet with 70% ethanol twice and air dry the DNA pellet

♦ Dissolve the DNA in T10E1 and use 25 – 50 ng/ul DNA for PCR analysis.

1M Tris (100ml):12.11g of Tris was dissolved in 70ml of sterile distilled water

and adjust pH to 8.0 with 1N HCl and final vol7ume made to 100ml.

0.5M EDTA (100ml): 18.612g of EDTA was dissolved in 70ml of sterile

distilled water and adjust pH to 8.0, adding NaOH pellets and final volume made

to 100ml.

73

5M NaCl (100ml): 29.22g of NaCl was dissolved in 70ml of sterile distilled

water and final volume made to 100ml and autoclaved.

CTAB Buffer (2%;500ml): 50ml of 1M TRIS, 20ml of 0.5 M EDTA,140ml of

5M NaCl,10g of CTAB,10g of PVP,10ml of beta mercapto ethanol were added in

about 250ml sterile distilled water and final volume make up to 500ml.

Testing of Clonal fidelity in tissue cultured plants using RAPDs

Isolation and quantification of genomic DNA :

Genomic DNA was isolated from the field grown plant and regenerated

plants on MS medium supplemented growth regulators. The quantification of

extracted DNA was done by Spectrophotometer and through comparison with

defined concentrations of DNA. The quantity of DNA in different samples

when compared with DNA and was found to be ranging from 200-700ng/l. Four

plant samples were taken for variability assessment.

RAPD analysis :

The results were scored as patterns of bands obtained from in vitro

regenerated plants grown on MS medium supplemented growth regulators. 20

OPA RAPD primers tested produced amplification products that were

monomorphic across all regenerants. The size of the monomorphic DNA

fragments, produced by these primers was shown. For each primer major bands

were scored and the size of the amplification products ranged between 500 bp-2.5

kb. A total of 92 bands were scored from PCR amplification of genomic DNA

74

from four Samples. No polymorphism was detected after amplification by PCR

among regenerated plants grown on different hormones. Thus, all regenerated

plants showed 100% similarity with the field grown plants.