Plant Tissue culture

(Practical Note Book)

By; Nasir Hussain (M.Phil Roll NO#05)

Submitted To;  Dr. Ash Muhammad  Dr. Iqbal Muhammad

Plant Genomics &Biotechnology National University of Agricultural Sciences National Agricultural Research centre Islamabad

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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 Volume of stock/l solutions g/l of medium (ml)

Macronutrients 1 Magnesium sulphate MgSO4.7H2O 2 3 4 5 Calcium chloride Potassium nitrate Ammonium nitrate CaCl2.2H2O KNO3 NH4NO3

7.4 8.8 38 33 3.4 50

Potassium di KH2PO4 hydrogen phosphate

Micronutrients

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6 7 8 9 10 11 12

Manganese sulphate Zinc sulphate Copper Sulphate Cobalt chloride Potassium iodide Boric acid Sodium molybdate

MnSO4. H2O ZnSO4.H2O CuSO4.2H2O CoCl2.6H2O KI H3BO3 Na2MoO4.2H2O FeSO4.7H2O Na2EDTA.2H2O

4.4 1.72 0.01 0.005 1.67 1.24 0.05 5.56 7.46 20 0.1 0.1 0.1 0.4 5 5

Iron Source 13 Ferrous sulphate 14 Sodium EDTA

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Organic Supplements (Vitamins) 15 Myo-inositol 16 17 18 19 Nicotinic acid Pyridoxine-HCl Thiamine-HCl Glycine

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 3

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: 19
TH

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

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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 Cl 2 (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

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In vitro seedling growth of tobacco Test Tube No 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 24 No of cultures (Replicates) 5 2 3 3 2 3 3 3 2 4 3 2 3 4 3 4 3 3 5 5 3 2 3 3 76 Responded 4 2 3 3 2 2 2 3 1 2 3 1 2 2 2 4 2 2 3 5 3 2 2 2 59 Contaminated 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Type of response Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth Seedling growth

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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), 8. Solution B (MgSO4.7H2O), 9. Solution C (KH2PO4), 10. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O, C4SO4.5H2O, CoCl2.6H2O), 11. Solution E (FeSO4.7H2O, Na2 EDTA.2H2O) 12. Solution F (vitamins), Sucrose, Agar and distilled water. 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 if any contamination and following observations were noted. Date of observation: 17th Dec 2008 Glass jars No Control 1 2 3 4 5 6 No of cultures (Replicates) 5 4 4 5 5 3 26 Responded 5 4 4 5 5 3 26 Contaminated 0 0 0 0 0 0 0 Type of response Seedlings Callus Callus Callus Callus Callus

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 (GF300), 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 2,4D(0.1g/100ml) was stored in refrigerator at 4c. Composition of medium: 13. Solution A (KNO3, NH3NO3, CaCl2.2H2O), 14. Solution B (MgSO4.7H2O), 15. Solution C (KH2PO4), 16. Solution D (MnSO4.4H2O2, H3BO3, ZnSO4.4H2O, KI, Na2MoO4.2H2O, C4SO4.5H2O, CoCl2.6H2O), 17. Solution E (FeSO4.7H2O, Na2 EDTA.2H2O) 18. Solution F (vitamins), Sucrose, Agar and distilled water.

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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 Cl 2 (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

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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: 14th Jan; 2009
S/No of Jars 1 2 3 4 5 6(control) No. of culture (replicates) 4 4 4 4 4 4 Respond 4 4 4 4 4 4 Contaminated 0 0 0 0 0 0 Type of Response yes yes yes yes yes Weak Seedlings

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Experiment No: 04 CALLUS REGENERATION Objective: To produce disease free plants Medium: Date of initiation: MS + 0.1ul/ltr NAA, 0.5ul/ltr BAP 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 added Stock A 20ml/ltr Stock B 20ml/ltr Stock C 10ml/ltr Stock D 10ml/ltr Stock 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/ltr 2) Pyridoxine 0.5mg/ltr 3) Thymine HCL 0.1mg/ltr 4) Myoenositol 0.1mg/ltr And 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.

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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 1 2 3 4 5 No of cultures 3 3 3 3 3 Responded 3 2 3 2 3 Contaminated 0 0 0 0 0 Type of response Callus become regenerated to plants Calli become brown and one regenerated Callus become regenerated Callus become regenerated Calli become regenerated to green plants

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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 Date of initiation: 21-01-2008 Materials and Methods: Preparation of medium: 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 added Stock A 20ml/ltr Stock B 20ml/ltr Stock C 10ml/ltr Stock D 10ml/ltr Stock 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/ltr 2) Pyridoxine 0.5mg/ltr 3) Thymine HCL 0.1mg/ltr 4) Myoenositol 0.1mg/ltr 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 5.80 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.

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Inoculation of the explants for multiplication: 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 Date of observation; 27-01-2008 No of observation 1 2 3 4 5 No of cultures 2 2 2 2 2 Responded 2 2 2 2 NO Contaminated 0 0 0 0 YES Type of response Rooting started to all plants Rooting started Rooting started Rooting started Nill

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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 Date of initiation: 27-12-2008 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 added Stock A 20ml/ltr Stock B 20ml/ltr Stock C 10ml/ltr Stock D 10ml/ltr Stock 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/ltr 2) Pyridoxine 0.5mg/ltr 3) Thymine HCL 0.1mg/ltr 4) Myoenositol 0.1mg/ltr And 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

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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 Date of observation; 27-01-2008 No of observation 1 No of cultures Responded Contaminated Type of response Calli clumps broken to smaller pieces and remain alive. Calli clumps broken down but calli become dead

3

3

0

2

3

2

0

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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 Date of initiation: 04-02-2008 Materials and Methods: Preparation of medium: 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 added Stock A 20ml/ltr Stock B 20ml/ltr Stock C 10ml/ltr Stock D 10ml/ltr Stock 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/ltr 2) Pyridoxine 0.5mg/ltr 3) Thymine HCL 0.1mg/ltr 4) Myoenositol 0.1mg/ltr And 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.

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Inoculation of the explants for multiplication: 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 Date of observation; 24-02-2008 No of observation 1 2 3 4 5 No of cultures 3 3 3 3 3 Responded 3 3 3 3 3 Contaminated 0 0 0 0 0 Type of response Rooting started to all plants Rooting started Rooting started Rooting started Rooting started

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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 Date of initiation: 24-02-2008 Materials and Methods: Preparation of medium: 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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