Tissue Culture
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Plant Tissue Culture – Basics and Applications Learning objectives:
(1) Learn basic introductory and practical information concerning the in vitro culture of plants and plant seeds (2) Learn some of the major applications of plant tissue culture
Plant Tissue Culture Definition – All types of sterile plant culture procedures pertaining to the growth of plant cells, tissues, organs, embryos and plantlets
Modes of Plant Regeneration (1) Embryo culture (2) Somatic embryogenesis (3) Organogenesis
Explant Surface Sterilization 1. Wash explants in a mild detergent before treatment with the disinfecting solution. (Herbaceous material may not require this step). 2. Rinse explants thoroughly under running tap water for 10-30 minutes. 3. Submerge explants into the disinfectant solution. Seal bottle and gently agitate. 4. Under sterile conditions, decant the solution and rinse explants several times with sterile distilled water.
Sterilization procedures may be enhanced by:
COMMONLY USED DISINFECTANTS FOR PLANT TISSUE CULTURE Product No.
Concentratio n (%)
Exposure (min)
Calcium hypochlorite
21,138-9
9-10
5-30
Sodium hypochlorite*
42,504-4
0.5-5
5-30
Hydrogen peroxide
H 1009
3-12
5-15
Ethyl alcohol
E7148
70-95
0.1-5.0
Silver nitrate
S 7276
1
5-30
Mercuric chloride
M 1136
0.1-1.0
2-10
Benzalkoniu m chloride
B 1383
0.01-0.1
5-20
Disinfectant
*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10-20%, which is equivalent to 0.51.0% sodium hypochlorite.
1.Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (twosource) sterilization procedure has proven beneficial with certain species. 2.Using a wetting agent, such as Tween 20 or 80 (Product Nos. P 1379 or P 1754, respectively), can be added to the disinfectants to reduce surface tension and allow better surface contact. 3.Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.
Nutrient Medium Factors Culture Media components are classified into 6 groups: 1. Major inorganic nutrients (macronutrients) -Needed for plant nutrition and cell maintenance (K, P, Ca, Mg, S, N) 2. Trace elements (Micronutrients) -Most are needed as co-factors (Mn, Co, Bn, Md, Cu, Zn, I2) 3. Iron source 4. Carbon Source 5. Organic supplements in the form of vitamins and myoinositol 6. Organic supplements in the form of plant growth regulators (PGRs)
Types of Media Choice depends on plant species, specific explant, and goal Media most commonly used for plant cell/tissue culture work: Murasghige and Skoog (MS) - For solid culture media – high quality agar is added (6-10 g/litre) - pH is adjusted before autoclaving (5.5-6.0)
Murashige and Skoog Media Variations Basal Salts Mixtures M 5524 M 0654 M 0529 M 0153 M 2909 M 8900 M 8280
Murashige and Skoog basal salt mixture (MS) Murashige and Skoog basal salt macronutrient solution Murashige and Skoog basal salt micronutrient solution Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture
Component (mg/L) Aluminum chloride • 6H2O Ammonium nitrate Ammonium phosphate monobasic Ammonium sulfate Boric acid Calcium chloride anhydrous Calcium nitrate Calcium phosphate tribasic Cobalt chloride • 6H2O Cupric sulfate • 5H2O Na2-EDTA ZnNa2-EDTA Ferric chloride • 6H2O Ferric citrate Ferric sulfate Ferric tartrate • 2H2O Ferrous sulfate • 7H2O Magnesium sulfate Manganese chloride • 4H2O Manganese sulfate • H2O Molybdenum trioxide Molybdic acid (sodium salt) • 2H2O Nickel chloride • 6H2O Nickel sulfate • 6H2O Potassium chloride Potassium iodide Potassium nitrate Potassium phosphate monobasic Potassium sulfate Sodium nitrate Sodium phosphate monobasic Sodium sulfate Zinc nitrate • 6H2O Zinc sulfate • 7H2O
M 5524
M 0654
1650.0
1650.0
6.2 332.2
332.2
0.025 0.025 37.26
27.8 180.7
M 0529
M 0153
M 2909
825.0
M 8900
M 8280
825.0
6.2
6.2 166.1
6.2 332.2
6.2 332.2
6.2 332.2
0.025 0.025 37.3
0.025 0.025 18.63
0.025 0.025 37.26
0.025 0.025 37.26
0.025 0.025 37.26
27.8
13.9 90.35
180.7
27.8 180.7
27.8 180.7
180.7
16.9
16.9
16.9
16.9
16.9
16.9
0.25
0.25
0.25
0.25
0.25
0.25
0.83
0.83 950.0 85.0
0.83 1900.0 170.0
0.83 950.0 170.0
0.83 1900.0 170.0
0.83 1900.0 170.0
1900.0 170.0
1751.0
8.6
8.6
8.6
8.6
8.6
8.6
Classic Plant Media C 1416 D 6162 G 5768 G 5893 H 2395 M 6774 M 5524 Q 3376 S 6765 W 0876
Component (mg/L) Aluminum chloride • 6H2O Ammonium nitrate Ammonium phosphate monobasic Ammonium sulfate Boric acid Calcium chloride anhydrous Calcium nitrate Calcium phosphate tribasic Cobalt chloride • 6H2O Cupric sulfate • 5H2O Na2-EDTA ZnNa2-EDTA Ferric chloride • 6H2O Ferric citrate Ferric sulfate Ferric tartrate • 2H2O Ferrous sulfate • 7H2O Magnesium sulfate Manganese chloride • 4H2O Manganese sulfate • H2O Molybdenum trioxide Molybdic acid (sodium salt) • 2H2O Nickel chloride • 6H2O Nickel sulfate • 6H2O Potassium chloride Potassium iodide Potassium nitrate Potassium phosphate monobasic Potassium sulfate Sodium nitrate Sodium phosphate monobasic Sodium sulfate Zinc nitrate • 6H2O Zinc sulfate • 7H2O
C 1416
D 6162
Chu (N6) basal salt mixture DKW/Juglans basal salt mixture Gamborg's B-5 basal salt mixture Gamborg's B-5 basal salt mixture with minimal organics Hoagland's No. 2 basal salt mixture McCown's woody plant basal salt mixture Murashige and Skoog basal salt mixture (MS) Quoirin and Lepoivre basal salt mixture Schenk and Hildebrandt basal salt mixture White's basal salt mixture
G 5768
G 5893
H 2395
1416.0
M 6774
M 5524
Q 3376
400.0
1650.0
400.0
115.03 463.0 1.6 125.33
37.25
4.8 112.5 1367.0
0.25 45.4
134.0 3.0 113.24
134.0 3.0 113.24
0.025 0.025 37.3
0.025 0.025 37.25
2.86 656.4
0.08
S 6765
W 0876
300.0 6.2 72.5 386.0
0.25 37.3
6.2 332.2
6.2
0.025 0.025 37.26
0.025 0.025 37.3
5.0 151.0
833.77
1.5 200.0
0.1 0.2 20.0
2.5 5.32 27.85 90.37
33.8 361.49
27.8 122.09
27.85 122.09
3.33
33.5
10.0
10.0
0.39
0.25
0.25
0.75 2500.0
0.75 2500.0
240.76 1.81
180.7
27.8 180.7
27.8 175.79
15.0 195.4
360.0
22.3
16.9
0.76
10.0
5.04
0.25
0.25
0.25
0.1
0.83 1900.0 170.0
0.08 1800.0 270.0
1.0 2500.0
0.016
0.005 0.8 2830.0 400.0
606.6
265.0 1559.0
170.0 990.0 130.5
130.5
2.0
2.0
65.0 0.75 80.0
16.5 200.0
17.0 1.5
0.22
8.6
8.6
8.6
1.0
2.67
Modes of Regeneration
(1) Embryo Culture: Aseptic culture of a zygotic (2N) embryo Flower development Æ pollination Æ fertilization Æ seed develops (2N embryo plus surrounding seed tissues) - Embryo is excised from seed/ovule and placed on a nutrient medium - Under certain conditions, embryo development occurs as if it would within the seed - Transfer to germination medium to obtain plantlet
Modes of Regeneration Continued
(2) Somatic Embryogenesis -Production of embryo-like bipolar structures from somatic cells: “embryoids” -Embryos can further develop and germinate into plantlets (very much like zygotic embryos)
Modes of Regeneration Continued
(3) Organogenesis Formation and outgrowths of shoots (usually from callus) and their subsequent adventitious rooting Auxin to Cytokinin Ratios: 10:1 Æ Callus 4:1 Æ Shoots 100:1Æ Roots
(1) Learn basic introductory and practical information concerning the in vitro culture of plants and plant seeds (2) Learn some of the major applications of plant tissue culture
Plant Tissue Culture Definition – All types of sterile plant culture procedures pertaining to the growth of plant cells, tissues, organs, embryos and plantlets
Modes of Plant Regeneration (1) Embryo culture (2) Somatic embryogenesis (3) Organogenesis
Explant Surface Sterilization 1. Wash explants in a mild detergent before treatment with the disinfecting solution. (Herbaceous material may not require this step). 2. Rinse explants thoroughly under running tap water for 10-30 minutes. 3. Submerge explants into the disinfectant solution. Seal bottle and gently agitate. 4. Under sterile conditions, decant the solution and rinse explants several times with sterile distilled water.
Sterilization procedures may be enhanced by:
COMMONLY USED DISINFECTANTS FOR PLANT TISSUE CULTURE Product No.
Concentratio n (%)
Exposure (min)
Calcium hypochlorite
21,138-9
9-10
5-30
Sodium hypochlorite*
42,504-4
0.5-5
5-30
Hydrogen peroxide
H 1009
3-12
5-15
Ethyl alcohol
E7148
70-95
0.1-5.0
Silver nitrate
S 7276
1
5-30
Mercuric chloride
M 1136
0.1-1.0
2-10
Benzalkoniu m chloride
B 1383
0.01-0.1
5-20
Disinfectant
*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10-20%, which is equivalent to 0.51.0% sodium hypochlorite.
1.Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (twosource) sterilization procedure has proven beneficial with certain species. 2.Using a wetting agent, such as Tween 20 or 80 (Product Nos. P 1379 or P 1754, respectively), can be added to the disinfectants to reduce surface tension and allow better surface contact. 3.Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.
Nutrient Medium Factors Culture Media components are classified into 6 groups: 1. Major inorganic nutrients (macronutrients) -Needed for plant nutrition and cell maintenance (K, P, Ca, Mg, S, N) 2. Trace elements (Micronutrients) -Most are needed as co-factors (Mn, Co, Bn, Md, Cu, Zn, I2) 3. Iron source 4. Carbon Source 5. Organic supplements in the form of vitamins and myoinositol 6. Organic supplements in the form of plant growth regulators (PGRs)
Types of Media Choice depends on plant species, specific explant, and goal Media most commonly used for plant cell/tissue culture work: Murasghige and Skoog (MS) - For solid culture media – high quality agar is added (6-10 g/litre) - pH is adjusted before autoclaving (5.5-6.0)
Murashige and Skoog Media Variations Basal Salts Mixtures M 5524 M 0654 M 0529 M 0153 M 2909 M 8900 M 8280
Murashige and Skoog basal salt mixture (MS) Murashige and Skoog basal salt macronutrient solution Murashige and Skoog basal salt micronutrient solution Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture Murashige and Skoog modified basal salt mixture
Component (mg/L) Aluminum chloride • 6H2O Ammonium nitrate Ammonium phosphate monobasic Ammonium sulfate Boric acid Calcium chloride anhydrous Calcium nitrate Calcium phosphate tribasic Cobalt chloride • 6H2O Cupric sulfate • 5H2O Na2-EDTA ZnNa2-EDTA Ferric chloride • 6H2O Ferric citrate Ferric sulfate Ferric tartrate • 2H2O Ferrous sulfate • 7H2O Magnesium sulfate Manganese chloride • 4H2O Manganese sulfate • H2O Molybdenum trioxide Molybdic acid (sodium salt) • 2H2O Nickel chloride • 6H2O Nickel sulfate • 6H2O Potassium chloride Potassium iodide Potassium nitrate Potassium phosphate monobasic Potassium sulfate Sodium nitrate Sodium phosphate monobasic Sodium sulfate Zinc nitrate • 6H2O Zinc sulfate • 7H2O
M 5524
M 0654
1650.0
1650.0
6.2 332.2
332.2
0.025 0.025 37.26
27.8 180.7
M 0529
M 0153
M 2909
825.0
M 8900
M 8280
825.0
6.2
6.2 166.1
6.2 332.2
6.2 332.2
6.2 332.2
0.025 0.025 37.3
0.025 0.025 18.63
0.025 0.025 37.26
0.025 0.025 37.26
0.025 0.025 37.26
27.8
13.9 90.35
180.7
27.8 180.7
27.8 180.7
180.7
16.9
16.9
16.9
16.9
16.9
16.9
0.25
0.25
0.25
0.25
0.25
0.25
0.83
0.83 950.0 85.0
0.83 1900.0 170.0
0.83 950.0 170.0
0.83 1900.0 170.0
0.83 1900.0 170.0
1900.0 170.0
1751.0
8.6
8.6
8.6
8.6
8.6
8.6
Classic Plant Media C 1416 D 6162 G 5768 G 5893 H 2395 M 6774 M 5524 Q 3376 S 6765 W 0876
Component (mg/L) Aluminum chloride • 6H2O Ammonium nitrate Ammonium phosphate monobasic Ammonium sulfate Boric acid Calcium chloride anhydrous Calcium nitrate Calcium phosphate tribasic Cobalt chloride • 6H2O Cupric sulfate • 5H2O Na2-EDTA ZnNa2-EDTA Ferric chloride • 6H2O Ferric citrate Ferric sulfate Ferric tartrate • 2H2O Ferrous sulfate • 7H2O Magnesium sulfate Manganese chloride • 4H2O Manganese sulfate • H2O Molybdenum trioxide Molybdic acid (sodium salt) • 2H2O Nickel chloride • 6H2O Nickel sulfate • 6H2O Potassium chloride Potassium iodide Potassium nitrate Potassium phosphate monobasic Potassium sulfate Sodium nitrate Sodium phosphate monobasic Sodium sulfate Zinc nitrate • 6H2O Zinc sulfate • 7H2O
C 1416
D 6162
Chu (N6) basal salt mixture DKW/Juglans basal salt mixture Gamborg's B-5 basal salt mixture Gamborg's B-5 basal salt mixture with minimal organics Hoagland's No. 2 basal salt mixture McCown's woody plant basal salt mixture Murashige and Skoog basal salt mixture (MS) Quoirin and Lepoivre basal salt mixture Schenk and Hildebrandt basal salt mixture White's basal salt mixture
G 5768
G 5893
H 2395
1416.0
M 6774
M 5524
Q 3376
400.0
1650.0
400.0
115.03 463.0 1.6 125.33
37.25
4.8 112.5 1367.0
0.25 45.4
134.0 3.0 113.24
134.0 3.0 113.24
0.025 0.025 37.3
0.025 0.025 37.25
2.86 656.4
0.08
S 6765
W 0876
300.0 6.2 72.5 386.0
0.25 37.3
6.2 332.2
6.2
0.025 0.025 37.26
0.025 0.025 37.3
5.0 151.0
833.77
1.5 200.0
0.1 0.2 20.0
2.5 5.32 27.85 90.37
33.8 361.49
27.8 122.09
27.85 122.09
3.33
33.5
10.0
10.0
0.39
0.25
0.25
0.75 2500.0
0.75 2500.0
240.76 1.81
180.7
27.8 180.7
27.8 175.79
15.0 195.4
360.0
22.3
16.9
0.76
10.0
5.04
0.25
0.25
0.25
0.1
0.83 1900.0 170.0
0.08 1800.0 270.0
1.0 2500.0
0.016
0.005 0.8 2830.0 400.0
606.6
265.0 1559.0
170.0 990.0 130.5
130.5
2.0
2.0
65.0 0.75 80.0
16.5 200.0
17.0 1.5
0.22
8.6
8.6
8.6
1.0
2.67
Modes of Regeneration
(1) Embryo Culture: Aseptic culture of a zygotic (2N) embryo Flower development Æ pollination Æ fertilization Æ seed develops (2N embryo plus surrounding seed tissues) - Embryo is excised from seed/ovule and placed on a nutrient medium - Under certain conditions, embryo development occurs as if it would within the seed - Transfer to germination medium to obtain plantlet
Modes of Regeneration Continued
(2) Somatic Embryogenesis -Production of embryo-like bipolar structures from somatic cells: “embryoids” -Embryos can further develop and germinate into plantlets (very much like zygotic embryos)
Modes of Regeneration Continued
(3) Organogenesis Formation and outgrowths of shoots (usually from callus) and their subsequent adventitious rooting Auxin to Cytokinin Ratios: 10:1 Æ Callus 4:1 Æ Shoots 100:1Æ Roots
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