plant tissue culture practical note book

Plant Tissue culture(Practical Note Book)

By;

Nasir Hussain(M.Phil Roll NO#05)

Submitted To;

Dr. Ash Muhammad Dr. Iqbal Muhammad

Plant Genomics &BiotechnologyNational University of Agricultural Sciences

National Agricultural Research centre Islamabad

1

Experiment No: 01 In vitro seedling growth of tobacco

Objectives:In vitro explants source for onward experiments (in vitro multiplication, in vitro rooting,

callus induction and regeneration and hardening in green house)

Medium:

MS (plain) (Murashige and Skoog, 1962) medium was used as basal medium in present

investigation. Composition of MS media is given in table.

Composition of medium:

1. Solution A (KNO3, NH3NO3, CaCl2.2H2O),

2. Solution B (MgSO4.7H2O),

3. Solution C (KH2PO4),

4. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O,

C4SO4.5H2O, CoCl2.6H2O),

5. Solution E (FeSO4.7H2O, Na2 EDTA.2H2O)

6. Solution F (vitamins), Sucrose, Agar and distilled water.

Table: Composition of Murashige and Skoog (1962) Medium

Media Volume (1000ml)

S. No Constituents Formula Conc. in stock solutions g/l

Volume of stock/l of medium (ml)

Macronutrients

1 Magnesium sulphate MgSO4.7H2O 7.4

50

2 Calcium chloride CaCl2.2H2O 8.8

3 Potassium nitrate KNO3 38

4 Ammonium nitrate NH4NO3 33

5 Potassium di hydrogen phosphate

KH2PO4 3.4

Micronutrients

6 Manganese sulphate MnSO4. H2O 4.457 Zinc sulphate ZnSO4.H2O 1.72

2

8 Copper Sulphate CuSO4.2H2O 0.01

9 Cobalt chloride CoCl2.6H2O 0.005

10 Potassium iodide KI 1.67

11 Boric acid H3BO3 1.24

12 Sodium molybdate Na2MoO4.2H2O 0.05

Iron Source

13 Ferrous sulphate FeSO4.7H2O 5.565

14 Sodium EDTA Na2EDTA.2H2O 7.46

Organic Supplements (Vitamins)

15 Myo-inositol 20

5

16 Nicotinic acid 0.1

17 Pyridoxine-HCl 0.1

18 Thiamine-HCl 0.1

19 Glycine 0.4

Carbon Source

20 30 g /l of Sucrose was used in MS medium.

PREPARATION OF 1 LITER MS MEDIUM:

One liter medium was prepared according to the following protocol.

50 ml of macronutrients, 5 ml of micronutrients, 5 ml of vitamins and 5ml of iron source

from their stock solutions were added by; 20ml of solution A and solution B, 10ml of

solution C and solution D and vitamin solution, 5ml of solution E and 30g of sucrose 3%

(w/v) was dissolved in 100 ml of distilled water contained in a 2-litre flask and then

poured in graduated cylinder. Volume was raised up to one liter with distilled water.

pH adjustment:

To adjust pH of the media, pH meter was calibrated. First the lens of pH metre was

washed with distilled water and dry with clean washing paper. Then buffer 4 was used to

adjust the pH to 4, error was adjusted from slope button. Wash the lens with distilled

water and dry with clean washing paper. Checked with buffer 7 and adjust the pH to 7,

error was removed from buffer button. Again washed the lens with distilled water,dry it

and check with buffer 4 for conformation. Then pH of media was adjusted to 5.80.The

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error was removed by using NaOH (0.1 N) or HCl (0.1 N) in case of high or low pH of

media.

Solidifying Agent:

Agar 8.0g (w/v) was added and medium was boiled with continuous stirring for

thorough mixing of agar into medium.

Pouring:

The medium was dispensed into culture vessels i.e. 50 ml test tubes.

Approximately 10-15 ml of medium in test tubes was poured.

Plugging:

Culture vessels were plugged with test tube caps and wrapped with aluminum

foil.

Autoclaving for Sterilization:

Medium was autoclaved at 15 Ib/in2 pressure for 20 minutes at 121oC.After

sterilization the medium was kept for 24-48 hours in growth room before inoculation to

check whether it was satisfactorily sterilized.

EXPLANTS PREPARATION:

The tobacco seed explant were sterilized by soaking in sodium hypo chlorate for 10mins

and then rinsed with distal water. Size of explant is very important factor as if the size of

explant was smaller than required, it produced small amount of callus and if the size of

explant was larger than required then more microorganisms were likely to enter the

culturing medium.

DATE OF INITIATION: 19TH OCT, 2008

INOCULATION PROCEDURE:

1. Explants were transferred under Laminar Flow Cabinet with HEPA (High

Efficiency Particulate Air Filters).Following procedure was used.

2. Laminar Flow Cabinet was turned on and all floors and walls were swabbed down

with 70 % ethyl alcohol carefully.

3. Autoclaved test tubes with culturing media, Petri-plates, distilled water; forceps,

cutters, mask, cap, gloves and match box were place inside Cabinet. Flask

4

containing 0.1% HgCl2 wiped with 70 % ethyl alcohol and Benson burner was

also placed in Laminar Flow Cabinet.

4. Transfer room was treated with UV rays (Peak emission of 2537A0) for 15

minutes before use.

5. UV lamp was turned of and hands were sterilized with spirit.

6. For surface sterilization, explant were dipped in 0.1% Hg Cl2 (w / v) solution.

After one minute they were rinsed with autoclaved distilled water for three times

to remove any traces of Mercuric chloride.

7. Forceps and cutter were dipped in pure ethyl alcohol then incinerated under flame

of Benson burner to ensure any likely contamination. Then cooled in distilled

water and dried with filter paper.

8. Explant were chopped to prepare for inoculation i.e. stem, leaf and shoot tips as

given above, then transferred to test tubes containing media in them near flame.

9. After each inoculation in a test tube the manipulated tools were dipped in 70%

ethyl alcohol then incinerated under flame, cooled and reused.

INCUBATION OF CULTURES:

Culturing test tubes were then placed in growth chamber for incubation. Standard

conditions were prevailed in culture room. Through out the temperature of growth room

was 25±10C for present research. Photoperiod was 16/8 hours light/dark cycle. Light

intensity was 1000 lux and relative humidity was 75 ±5%. Experiments conducted by

Murashige (1974) with Asparagus, Gerbera, Saxifraga, and bromeliads indicated an

optimum light intensity of 1,000 lux during culture initiation and shoot proliferation

. Observations:

After one week, the test tubes were examined thoroughly for the germination and

if any contamination and following observations were noted.

Date of observation: 26th Oct 2008

5

In vitro seedling growth of tobacco

Test Tube No No of cultures (Replicates) Responded Contaminated Type of response1 5 4 0 Seedling growth2 2 2 0 Seedling growth

3 3 3 0 Seedling growth






















24 76 59 0

6

Experiment No: 02 Callus induction

Objectives:

Optimization of medium and explants for callus induction of tobacco plant

Medium: MS + 2.4.D (1ml/L).

Composition of medium:

7. Solution A (KNO3, NH3NO3, CaCl2.2H2O),







Preparation:

400ml of distilled water were taken in a beaker. 20ml of solution A and solution

B, 10ml of solution C and solution D and vitamin solution, 5ml of solution E and 30g of

sucrose were added to the beaker. Then 1ml of 2.4.D solution was added and the beaker

contents were mixed on an electrical stirrer. Afterwards the pH of the mixture was

adjusted at 5.8. Then 6g of agar were added in the above mixture and the mixture was

heated for about 5mins in an oven. The mixture was then diluted with distilled water up

to 1000ml and was again heated for about 6mins in oven. After that 15-20ml of liquid

media was poured into 5 glass jars under sterilized conditions and the mouths of jars were

covered with plastic sheets by means of rubber bands. The glass jars were then wrapped

with paper and were then subjected to the autoclave at 121ºC for about 20mins in order to

maintain sterilized conditions in glass jars.

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Date of initiation: 03 Dec 2008.

Materials:

Sterilized Tobacco seeds (sterilized by soaking in sodium hypo chlorate for

10mins), Safety cabinet (Laminar Flow Unit), 70% ethanol, Spatula, Sterilized glass jars

containing MS + 2.4.D media, A control jar containing plane MS media, Metal racks,

Cotton plugs, Plastic covers, Rubber bands, Markers, Ethanol spray bottle, Benzene

burner and Petri plates.

Stem Explant:

Stem explants were also excised from plant young stems by cutting the stem

transversely into pieces of 0.5 cm .These were also cut longitudinally to produce

maximum cuts on the surface that would explant touch the surface of the medium.

Methodology:

The work was carried out in the inoculation room. Before starting, the hands and

safety cabinet were sterilized with 70% ethanol to avoid contamination. In safety cabinet

under the presence of benzene burner, first the tobacco seedlings grown in the previous

experiments were pulled out of the test tubes with help of sterilized spatula and were

placed in a Petri plate. The rooting potion of each seedling was then cut with the help of a

blade. These seedlings without the rooting portions are our explants. 3 to 4 of these

explants were inoculated in each of the glass jars containing the MS + 2.4.D media and

the control jar containing the plane MS media. The inoculation tools, i.e. spatula and

blade were sterilized with ethanol after each inoculation to minimize the chance of

contamination. All the work was done very carefully and attentively. The inoculated glass

jars were then labeled with marker and were placed in the growth room under optimum

conditions for growth.

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Observations:

After one week, the glass jars were examined thoroughly for the germination and


Date of observation: 17th Dec 2008

Glass jars No No of cultures (Replicates) Responded Contaminated Type of responseControl 5 5 0 Seedlings

1 4 4 0 Callus2 4 4 0 Callus3 5 5 0 Callus4 5 5 0 Callus5 3 3 0 Callus6 26 26 0

Experiment No: 03

9

In Vitro Multiplication of Tobacco

Objectives: Mass scale plant production

Medium: MS+BAP (1mg/l)

MATERIALS AND METHODS

PROCUREMENT OF MATERIALS:The present research work was carried out in Plant Tissue Culture laboratory in

Plant Biotechnology program at NARC, Islamabad. (NWFP) Pakistan. A brief account

of the materials and methods used and the procedures adopted is given below.

GLASS WARE:

In tissue culture techniques, the glass Ware used include Erlenmeyer flasks(1000

ml, 500 ml, 250 ml, test tubes (50 ml ), beakers (1000 ml, 500 ml, 250 ml, 100 ml ),

pipettes ( 25 ml,10 ml, etc), micropipettes (0.1-1.0 ml) Graduated cylinder (1000 ml, 500

ml ) and Petri-plates. New glassware may release chemicals that are toxic to the cultured

tissues. Additional information can be obtained from Street (1973) and Biondi and

Thorpe (1981). All the glassware used in the study was made up of Pyrex and

Borosilicate.

EQUIPMENT:

The equipment used in tissue culture techniques include Electrical balance (GF-

300), Burner, pH meter (Jenway 3305), Autoclave (KP-30L, ALP Tokyo, Japan),

Laminar Flow Transfer Cabinet (ESCO), Magnetic Heating Stirrer (Guohua electric

appliance Co.LTD) and Water Distillation Plant.

STERILIZATION TECHNIQUES:

All type of glass ware like Erlenmeyer flasks, pipettes, Petri-plates, beakers and

test tubes were washed with commercial detergent (lemon Max liquid).After scrubbing

with a brush, the glassware is rinses repeatedly with tap water, and then given two or

three rinses in distilled water until all traces of dirt were removed. Washed test tubes and

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flasks were plugged after pouring Growth Agar Media in it to avoid entrance of any

traces of contamination. Contaminated cultures and discarded culture were first

autoclaved to kill all types of microbes and fungal spores as well as to liquefy the agar

that made it easy to wash.

Sterilization of Inoculation Area:

Transfer room was cleaned with a commercial detergent on monthly basis and

sprayed with methylated spirit before start of work. Before using Laminar Flow Transfer

Cabinet, all floors and walls were swabbed down with 70 % ethyl alcohol carefully.

Transfer room was treated with UV rays (Peak emission of 2537A0) for 15 minutes

before each use. Aseptic transfer of tissues was carried out in a Laminar Flow Transfer

cabinet fitted with a HEPA filter.

STOCK SOLUTION PREPARATION:

Different stock solutions prepared for M.S media include macronutrient stock,

micronutrient stock, vitamin stock, iron stock and hormonal stock(BAP, 2,4-D) in

concentration of 1mg/1ml.BAP stock(0.1g/100ml) was stored in freezer at -20c and 2,4-

D(0.1g/100ml) was stored in refrigerator at 4c.

Composition of medium:13. Solution A (KNO3, NH3NO3, CaCl2.2H2O),







11

DATE OF INITIATION:31/.12/2008

INOCULATION PROCEDURE:

10. Explants were transferred under Laminar Flow Cabinet with HEPA (High

Efficiency Particulate Air Filters).Following procedure was used.

11. Laminar Flow Cabinet was turned on and all floors and walls were swabbed down

with 70 % ethyl alcohol carefully.

12. Autoclaved test tubes with culturing media, Petri-plates, distilled water; forceps,

cutters, mask, cap, gloves and match box were place inside Cabinet. Flask

containing 0.1% HgCl2 wiped with 70 % ethyl alcohol and Benson burner was

also placed in Laminar Flow Cabinet.

13. Transfer room was treated with UV rays (Peak emission of 2537A0) for 15

minutes before use.

14. UV lamp was turned of and hands were sterilized with spirit.

15. For surface sterilization, explant were dipped in 0.1% Hg Cl2 (w / v) solution.

After one minute they were rinsed with autoclaved distilled water for three times

to remove any traces of Mercuric chloride.

16. Forceps and cutter were dipped in pure ethyl alcohol then incinerated under flame

of Benson burner to ensure any likely contamination. Then cooled in distilled

water and dried with filter paper.

17. Explant were chopped to prepare for inoculation i.e. stem, leaf and shoot tips as

given above, then transferred to test tubes containing media in them near flame.

18. After each inoculation in a test tube the manipulated tools were dipped in 70%

ethyl alcohol then incinerated under flame, cooled and reused.

INCUBATION OF CULTURES:

Culturing test tubes were then placed in growth chamber for incubation. Standard

conditions were prevailed in culture room. Through out the temperature of growth room

was 25±10C for present research. Photoperiod was 16/8 hours light/dark cycle. Light

intensity was 1000 lux and relative humidity was 75 ±5%. Experiments conducted by

12

Murashige (1974) with Asparagus, Gerbera, Saxifraga, and bromeliads indicated an

optimum light intensity of 1,000 lux during culture initiation and shoot proliferation

Observations:

After one week, the test tubes were examined thoroughly for the germination and


Date of observation: 14th Jan; 2009

S/No of Jars No. of culture(replicates)

Respond Contaminated Type of Response

1 4 4 0 yes2 4 4 0 yes3 4 4 0 yes4 4 4 0 yes5 4 4 0 yes6(control) 4 4 0 Weak Seedlings

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Experiment No: 04 CALLUS REGENERATION

Objective:

To produce disease free plants Medium: MS + 0.1ul/ltr NAA, 0.5ul/ltr BAP

Date of initiation: 31-12-2008

Materials and Methods:

Preparation of medium:

First of all MS medium was prepared by using six stock solutions A, B ,C, D, E and a stock solution containing vitamins. A beaker was taken and was filled with distilled water up to about 600 ml. Then the following compositions of solutions was addedStock A 20ml/ltrStock B 20ml/ltrStock C 10ml/ltrStock D 10ml/ltrStock E 5ml/ltrAnd 10ml/ltr of stock solutions containing vitamins were added to beaker. The vitamins solution contain the following ingredients,1) Nicotinic acid 0.5ug/ltr2) Pyridoxine 0.5mg/ltr3) Thymine HCL 0.1mg/ltr4) Myoenositol 0.1mg/ltrAnd 0.1ul of NAA and 0.5ul of BAP were taken with the help of a micropipette and added to the beaker.30 g sugar (sucrose) was weighed on an electric balance and was added to the beaker. Then the sugar was dissolved with a stirrer and PH was measured with the PH meter. PH was adjusted at 5.70 by adding few drops of NAOH. Then 6 g agar was added to the beaker and the volume of the solution was made 1000 ml by adding the required volume of distilled water. Then the beaker containing the solution was kept in microwave oven for 6-8 minutes and boiled to dissolve the agar. After the dissolution of agar, beaker was taken out of the oven and solution was stirred. Then the solution was immediately added to the glass bottles. About 250 ml of the solution was added to each glass bottle. These glass bottles were then subjected to steam sterilization in an autoclave for 15-20 minutes at 15 lbs and 121C.

14

Inoculation of the explants for multiplication:

Hands were sterilized with the 70% alcohol to ensure aseptic conditions. Callus that were growing in the glass tubes were taken out. The calli were excised with the blade and were carefully cultured in the glass bottles containing the medium for callus regeneration.4-5 explants were cultured per bottle. All this inoculation work was done under aseptic conditions provided by the laminar air flow cabinet.

DATA RECRDED

Date of observation; 13-01-2008

No of observation

No of cultures Responded Contaminated Type of response

1

3

3

0

Callus become regenerated to plants

2 3 2 0

Calli become brown and one regenerated

3 3 3 0Callus become regenerated

4 3 2 0

Callus become regenerated

5 3 3 0

Calli become regenerated to green plants

15

Experiment No: 05

ROOT INITIATION OF INVITRO MULTIPLICATED SHOOTS

Objective:

To produce roots of in-vitro multiplicated shoots from single shoots

Medium: plain MS media was used




First of all plain MS medium was prepared by using six stock solutions A, B ,C, D, E and a stock solution containing vitamins. A beaker was taken and was filled with distilled water up to about 600 ml. Then the following compositions of solutions was addedStock A 20ml/ltrStock B 20ml/ltrStock C 10ml/ltrStock D 10ml/ltrStock E 5ml/ltr

And 10ml/ltr of stock solutions containing vitamins were added to beaker. The vitamins solution contain the following ingredients,

1) Nicotinic acid 0.5ug/ltr2) Pyridoxine 0.5mg/ltr3) Thymine HCL 0.1mg/ltr4) Myoenositol 0.1mg/ltr30 g sugar (sucrose) was weighed on an electric balance and was added to the

beaker. Then the sugar was dissolved with a stirrer and PH was measured with the PH meter. PH was adjusted at 5.80 by adding few drops of NAOH. Then 6 g agar was added to the beaker and the volume of the solution was made 1000 ml by adding the required volume of distilled water. Then the beaker containing the solution was kept in microwave oven for 6-8 minutes and boiled to dissolve the agar. After the dissolution of agar, beaker was taken out of the oven and solution was stirred. Then the solution was immediately added to the glass bottles. About 250 ml of the solution was added to each glass bottle. These glass bottles were then subjected to steam sterilization in an autoclave for 15-20 minutes at 15 lbs and 121C.

16


Hands were sterilized with the 70% alcohol to ensure aseptic conditions. Multiplicated shoots growing in glass flasks were taken out. The larger shoots were excised with the blade into smaller pieces and were carefully cultured in the glass bottles containing the medium for root initiation.2-3 explants were cultured per bottle. All this inoculation work was done under aseptic conditions provided by the laminar air flow cabinet.

DATA RECRDED


No of observation


1 2 2 0 Rooting started to all plants

2 2 2 0 Rooting started



5 2 NO YES Nill

17

Experiment No: 06

CELL CULTURE IN LIQUID MEDIA Objective:

To maintain media for cell suspension culture so those to obtain disease free plants from single cells. Medium: MS + 5mg/ ltr 2,4 D +1mg/ ltr BAP




First of all MS medium was prepared by using six stock solutions A, B ,C, D, E and a stock solution containing vitamins. A beaker was taken and was filled with distilled water up to about 600 ml. Then the following compositions of solutions was added

Stock A 20ml/ltrStock B 20ml/ltrStock C 10ml/ltrStock D 10ml/ltrStock E 5ml/ltr

And 10ml/ltr of stock solutions containing vitamins were added to beaker. The vitamins solution contain the following ingredients,1) Nicotinic acid 0.5ug/ltr2) Pyridoxine 0.5mg/ltr3) Thymine HCL 0.1mg/ltr4) Myoenositol 0.1mg/ltrAnd 5mg/ltr 2,4Dand 1 mg/ltr BAP were taken with the help of a micropipette and added to the beaker.30 g sugar (sucrose) was weighed on an electric balance and was added to the beaker. Then the sugar was dissolved with a stirrer and PH was measured with the PH meter. PH was adjusted at 5.70 by adding few drops of NAOH. Here no agar was added and the volume of the solution was made 1000 ml by adding the required volume of distilled water. Then the beaker containing the solution was kept in microwave oven for 6-8 minutes and boiled to dissolve the agar. After the dissolution of agar, beaker was taken out of the oven and solution was stirred. Then the solution was immediately added to the glass bottles. About 250 ml of the solution was added to each glass bottle. These glass bottles were then subjected to steam sterilization in an autoclave for 15-20 minutes at 15 lbs and 121C.Only 50 Ml Media Was Taken In Each Flask

18

Maintenance of media:

Hands were sterilized with the 70% alcohol to ensure aseptic conditions. callus that were growing in the glass tubes were taken out. The calli were excised into smaller pieces with the blade and were carefully cultured in the glass bottles containing the medium.2-3 explants were cultured per bottle. All this inoculation work was done under aseptic conditions provided by the laminar air flow cabinet. Then media bottles were kept on shaker to break clumps of calli into tiny pieces so this media should be ready for suspension culture.

DATA RECRDED


No of observation


1 3 3 0

Calli clumps broken to smaller pieces and remain alive.

2 3 2 0Calli clumps broken down but calli become dead

19

Experiment No: 07

IN-VITRO ROOTING MEDIA

Objective:

To produce roots of in-vitro multiplicated shoots from single shoots

Medium: MS+vit+IAA 1mg/ltr




First of all plain MS medium was prepared by using six stock solutions A, B ,C, D, E and a stock solution containing vitamins. A beaker was taken and was filled with distilled water up to about 600 ml. Then the following compositions of solutions was addedStock A 20ml/ltrStock B 20ml/ltrStock C 10ml/ltrStock D 10ml/ltrStock E 5ml/ltrAnd 10ml/ltr of stock solutions containing vitamins were added to beaker. The vitamins solution contain the following ingredients,1) Nicotinic acid 0.5ug/ltr2) Pyridoxine 0.5mg/ltr3) Thymine HCL 0.1mg/ltr4) Myoenositol 0.1mg/ltrAnd 1mg/ltr of IAA added to solution for root induction30 g sugar (sucrose) was weighed on an electric balance and was added to the beaker. Then the sugar was dissolved with a stirrer and PH was measured with the PH meter. PH was adjusted at 5.80 by adding few drops of NAOH. Then 6 g agar was added to the beaker and the volume of the solution was made 1000 ml by adding the required volume of distilled water. Then the beaker containing the solution was kept in microwave oven for 6-8 minutes and boiled to dissolve the agar. After the dissolution of agar, beaker was taken out of the oven and solution was stirred. Then the solution was immediately added to the glass bottles. About 250 ml of the solution was added to each glass bottle. These glass bottles were then subjected to steam sterilization in an autoclave for 15-20 minutes at 15 lbs and 121C.

20


Hands were sterilized with the 70% alcohol to ensure aseptic conditions. Multiplicated shoots growing in glass flasks were taken out. The larger shoots were excised with the blade into smaller pieces and were carefully cultured in the glass bottles containing the medium for root initiation.2-3 explants were cultured per bottle. All this inoculation work was done under aseptic conditions provided by the laminar air flow cabinet.

DATA RECRDED


No of observation


1 3 3 0 Rooting started to all plants





21

Experiment No: 08

ACCLIMITIZATION

Objective:

To acclimatize the cultured plants in growth room so that they may able to grow in field/green house

Medium: peat moss clay




First of all peat moss soil containing plenty of organic compounds was taken into plastic bags.

Transplantation The plants growing in growth room in jars were taken out and roots were washed

to clean the growing media on them. Then plantation was done into plastic bags and some water was poured into bags and were kept in growth room where proper system of aeration and light was maintained. This was aimed to harden and acclimatize the plants son that they may able to grow in green house and subsequently to field.

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plant tissue culture practical note book

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