Beruflich Dokumente
Kultur Dokumente
This section presents the details of material and methods which were employed
PLANT MATERIAL: The mother plant was collected from the Tri Shakti
farms, Sanathnagar, Hyderabad. The explants used were the Shoot Apex, Nodal
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
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
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
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used as explants to establish in vitro cultures. The procedure of surface
Explants were washed under running tap water with 5.1 Teepol solution
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 is over. Then the explants were inoculated in the
appropriate media.
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MEDIA COMPOSITION:
solidified with 0.8% Agar was used as the basal medium. The composition is
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Growth Regulators :
Benzylamino purine 2 mg /l
Kinetin 1 mg /l
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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
(3%). The final volume of known quantity was obtained by adding double
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
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Stock Solution of Calcium chloride (100X):
4.4 g of Calcium Chloride was dissolved in 80 ml of sterile distilled water and the
Some chemicals occurring naturally within plant tissue (i.e. endogenously) have a
Auxins :
Auxins (IAA, NAA, 2,4-D, IBA) are phytohormones that influence cell
cuttings.
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NAA ( Napthalene acetic acid : C12 H16 O2 ) :
Cytokinins: The cytokinin (KN, BAP, 2-ip and Zeatin) is adenine a derivatives
bud growth.
2-ip (i PA N6 – (2-iso pentyl adenine: C10 N5): H13 it causes rapid cell
initiation Wickson and Thimann (1958) discovered that cytokinin could release
1983). Cytokinin in combination with an auxins appear essential for the onset of
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GROWTH REGULATORS:
6´-benzyl adenine(BAP):
amine compound ].
Kinetin (Kn):
amine compound].
8ml with distilled water, then final volume was made up to 10 ml with distilled
(C5H5N5)2H2SO4).
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10 mg of Adenin Sulphate was dissolved in 1 ml of 1 N NaOH, volume was
ml with distilled water. Then final volume was made up to 10 ml with distilled
with distilled water. Then final volume was made up to 10 ml with distilled water,
compound].
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
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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
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:
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
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.
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MAINTENANCE OF CULTURES IN LABORATORY:
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
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
MICRO PROPAGATION:
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
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Days taken For the Establishment Of Explants in vitro:
cytokinin BAP (0.25mg/l, 0.5mg/l, 1.0mg/l, 1.5mg/l, 2.0mg/l, 2.5mg/l) were used.
1. Average number of days taken for the initiation of shoot bud, first
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.
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
MULTIPLE SHOOTS:
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
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No. of explants that responded
(by producing multiple shoots)
Frequency of Multiple Shoots = --------------------------------------- × 100
Total No. of explants inoculated
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.
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
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
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.
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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).
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
30ºC±2ºC. After 30 days plants transferred to shade house and taken the data on
percentage of survival.
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.
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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
The mean number of days taken for the initiation of callus i.e. formation of
Fresh weight of the callus was recorded after 60 days of callus initiation.
Nature of the callus, colour of the callus was recorded in different treatments i.e.
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.
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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
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
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
acclimatization after transferring all the fully developed plantlets to field were
monitored.
regeneration through somatic embryogenesis from the callus of nodal and Leaf of
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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,
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.
treatments i.e. single spherical cell, early globular shape with conspicuous
suspensor, typical globular shape, elongated shape, early heart shape, typical heart
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.
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The cultures were maintained at 25±2°C and 3000 Lux Illumination
somatic embryos (as evidenced by the transition of globular stage to torpedo stage
Mean days taken for the organogenesis from the somatic embryos:
mean days taken for the organogenesis from somatic embryos were recorded.
medium supplemented with NAA for both the 2,4-D and 2-ip grown somatic
The cultures were incubated for 2 weeks at 25±2°C and 3000 Lux
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.
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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
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
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.
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).
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
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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
30ºC±2ºC. After 30 days plants transferred to shade house and taken the data on
% ge of survival.
The embryogenic callus at different stages was used for the SEM studies.
phosphate buffer (pH6.8) for about 3 hours of duration. The samples were washed
were dried to critical point for gold coating, they were mounted onto the stubs
Sputtering device”. After gold coating, the material was observed under” JEOL –
The fine structure of somatic embryos was clearly identified during the
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shape with conspicuous suspensor, typical globular shape, elongated shape, early
Water.
Equipments used:
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.
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STANDARD SOLUTION PREPARATION:
with 80% methanol in water into volumetric flask respectively in prior to sonicate
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
comparison with a reference standard. Acetonitrile - water (80:20 v/v) was used
(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
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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
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
Extraction Buffer:
mercaptoethanol)
♦ Grind the leaf samples (2 to 5 gm) in liquid Nitrogen and add into
preheated buffer
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♦ The contents were mixed gently by swirling and inverting the tube and
♦ 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
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.
and adjust pH to 8.0 with 1N HCl and final vol7ume made to 100ml.
distilled water and adjust pH to 8.0, adding NaOH pellets and final volume made
to 100ml.
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5M NaCl (100ml): 29.22g of NaCl was dissolved in 70ml of sterile distilled
about 250ml sterile distilled water and final volume make up to 500ml.
Genomic DNA was isolated from the field grown plant and regenerated
when compared with DNA and was found to be ranging from 200-700ng/l. Four
RAPD analysis :
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
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from four Samples. No polymorphism was detected after amplification by PCR
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