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.

COMMONLY USED DISINFECTANTS Sterilization procedures may be enhanced

FOR PLANT TISSUE CULTURE by:
Concentratio Exposure
Disinfectant Product No.
n (%) (min) 1.Placing the material in a 70% ethyl alcohol
Calcium
21,138-9 9-10 5-30 solution prior to treatment with another
hypochlorite
disinfectant solution. The use of a two-step (two-
Sodium source) sterilization procedure has proven
42,504-4 0.5-5 5-30
hypochlorite*
beneficial with certain species.
Hydrogen
H 1009 3-12 5-15
peroxide
2.Using a wetting agent, such as Tween 20 or 80
Ethyl alcohol E7148 70-95 0.1-5.0
(Product Nos. P 1379 or P 1754, respectively),
Silver nitrate S 7276 1 5-30
can be added to the disinfectants to reduce
Mercuric surface tension and allow better surface contact.
M 1136 0.1-1.0 2-10
chloride
Benzalkoniu
m chloride
B 1383 0.01-0.1 5-20 3.Conducting the sterilization process under
vacuum. This results in the removal of air
*Commercial bleach contains about 5% sodium hypochlorite, and thus bubbles and provides a more efficient
may be used at a concentration of 10-20%, which is equivalent to 0.5- sterilization process.
1.0% sodium hypochlorite.
 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 Murashige and Skoog basal salt mixture (MS)
M 0654 Murashige and Skoog basal salt macronutrient solution
M 0529 Murashige and Skoog basal salt micronutrient solution
M 0153 Murashige and Skoog modified basal salt mixture
M 2909 Murashige and Skoog modified basal salt mixture
M 8900 Murashige and Skoog modified basal salt mixture
M 8280 Murashige and Skoog modified basal salt mixture

Component (mg/L) M 5524 M 0654 M 0529 M 0153 M 2909 M 8900 M 8280

Aluminum chloride • 6H2O

Ammonium nitrate 1650.0 1650.0 825.0 825.0
Ammonium phosphate monobasic
Ammonium sulfate
Boric acid 6.2 6.2 6.2 6.2 6.2 6.2
Calcium chloride anhydrous 332.2 332.2 166.1 332.2 332.2 332.2
Calcium nitrate
Calcium phosphate tribasic
Cobalt chloride • 6H2O 0.025 0.025 0.025 0.025 0.025 0.025
Cupric sulfate • 5H2O 0.025 0.025 0.025 0.025 0.025 0.025
Na2-EDTA 37.26 37.3 18.63 37.26 37.26 37.26
ZnNa2-EDTA
Ferric chloride • 6H2O
Ferric citrate
Ferric sulfate
Ferric tartrate • 2H2O
Ferrous sulfate • 7H2O 27.8 27.8 13.9 27.8 27.8
Magnesium sulfate 180.7 180.7 90.35 180.7 180.7 180.7
Manganese chloride • 4H2O
Manganese sulfate • H2O 16.9 16.9 16.9 16.9 16.9 16.9
Molybdenum trioxide
Molybdic acid (sodium salt) • 2H2O 0.25 0.25 0.25 0.25 0.25 0.25
Nickel chloride • 6H2O
Nickel sulfate • 6H2O
Potassium chloride
Potassium iodide 0.83 0.83 0.83 0.83 0.83 0.83
Potassium nitrate 1900.0 1900.0 950.0 1900.0 950.0 1900.0
Potassium phosphate monobasic 170.0 170.0 85.0 170.0 170.0 170.0
Potassium sulfate
Sodium nitrate 1751.0
Sodium phosphate monobasic
Sodium sulfate
Zinc nitrate • 6H2O
Zinc sulfate • 7H2O 8.6 8.6 8.6 8.6 8.6 8.6
 Classic Plant Media
C 1416 Chu (N6) basal salt mixture
D 6162 DKW/Juglans basal salt mixture
G 5768 Gamborg's B-5 basal salt mixture
G 5893 Gamborg's B-5 basal salt mixture with minimal organics
H 2395 Hoagland's No. 2 basal salt mixture
M 6774 McCown's woody plant basal salt mixture
M 5524 Murashige and Skoog basal salt mixture (MS)
Q 3376 Quoirin and Lepoivre basal salt mixture
S 6765 Schenk and Hildebrandt basal salt mixture
W 0876 White's basal salt mixture

Component (mg/L) C 1416 D 6162 G 5768 G 5893 H 2395 M 6774 M 5524 Q 3376 S 6765 W 0876
Aluminum chloride • 6H2O
Ammonium nitrate 1416.0 400.0 1650.0 400.0
Ammonium phosphate monobasic 115.03 300.0
Ammonium sulfate 463.0 134.0 134.0
Boric acid 1.6 4.8 3.0 3.0 2.86 6.2 6.2 6.2 5.0 1.5
Calcium chloride anhydrous 125.33 112.5 113.24 113.24 72.5 332.2 151.0
Calcium nitrate 1367.0 656.4 386.0 833.77 200.0
Calcium phosphate tribasic
Cobalt chloride • 6H2O 0.025 0.025 0.025 0.025 0.1
Cupric sulfate • 5H2O 0.25 0.025 0.025 0.08 0.25 0.025 0.025 0.2
Na2-EDTA 37.25 45.4 37.3 37.25 37.3 37.26 37.3 20.0
ZnNa2-EDTA
Ferric chloride • 6H2O
Ferric citrate
Ferric sulfate 2.5
Ferric tartrate • 2H2O 5.32
Ferrous sulfate • 7H2O 27.85 33.8 27.8 27.85 27.8 27.8 15.0
Magnesium sulfate 90.37 361.49 122.09 122.09 240.76 180.7 180.7 175.79 195.4 360.0
Manganese chloride • 4H2O 1.81
Manganese sulfate • H2O 3.33 33.5 10.0 10.0 22.3 16.9 0.76 10.0 5.04
Molybdenum trioxide 0.016
Molybdic acid (sodium salt) • 2H2O 0.39 0.25 0.25 0.25 0.25 0.25 0.1
Nickel chloride • 6H2O
Nickel sulfate • 6H2O 0.005
Potassium chloride 65.0
Potassium iodide 0.8 0.75 0.75 0.83 0.08 1.0 0.75
Potassium nitrate 2830.0 2500.0 2500.0 606.6 1900.0 1800.0 2500.0 80.0
Potassium phosphate monobasic 400.0 265.0 170.0 170.0 270.0
Potassium sulfate 1559.0 990.0
Sodium nitrate
Sodium phosphate monobasic 130.5 130.5 16.5
Sodium sulfate 200.0
Zinc nitrate • 6H2O 17.0
Zinc sulfate • 7H2O 1.5 2.0 2.0 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