tissue culture

47
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

Upload: bandulascolaya

Post on 15-Nov-2014

24 views

Category:

Documents


4 download

DESCRIPTION

good book in tissue culture

TRANSCRIPT

Page 1: Tissue Culture

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

Page 2: 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

Page 3: Tissue Culture
Page 4: Tissue Culture
Page 5: Tissue Culture
Page 6: Tissue Culture
Page 7: Tissue Culture
Page 8: Tissue Culture
Page 9: Tissue Culture
Page 10: Tissue Culture
Page 11: Tissue Culture
Page 12: Tissue Culture
Page 13: Tissue Culture
Page 14: Tissue Culture

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 DISINFECTANTSFOR PLANT TISSUE CULTURE

Disinfectant Product No. Concentration (%)

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

Benzalkonium chloride B 1383 0.01-0.1 5-20

*Commercial bleach contains about 5% sodium hypochlorite, and thus may be used at a concentration of 10-20%, which is equivalent to 0.5-1.0% sodium hypochlorite.

Sterilization procedures may be enhanced by:

1.Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two-step (two-source) sterilization procedure has proven beneficial with certain species.

2.Using a wetting agent, such as Tween 20 or 80 (Product Nos. P 1379 P 1754or , 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.

Page 15: Tissue Culture
Page 16: Tissue Culture
Page 17: Tissue Culture
Page 18: Tissue Culture
Page 19: Tissue Culture
Page 20: Tissue Culture
Page 21: Tissue Culture

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)

Page 22: Tissue Culture

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)

Page 23: Tissue Culture

Murashige and Skoog Media VariationsBasal Salts Mixtures

Murashige and Skoog basal salt mixture (MS)Murashige and Skoog basal salt macronutrient solutionMurashige and Skoog basal salt micronutrient solutionMurashige and Skoog modified basal salt mixtureMurashige and Skoog modified basal salt mixtureMurashige and Skoog modified basal salt mixtureMurashige and Skoog modified basal salt mixture

M 5524M 0654M 0529M 0153M 2909M 8900M 8280

M 5524 M 0654 M 0529 M 0153 M 2909 M 8900 M 8280

1650.0 1650.0 825.0 825.0

6.2 6.2 6.2 6.2 6.2 6.2332.2 332.2 166.1 332.2 332.2 332.2

0.025 0.025 0.025 0.025 0.025 0.0250.025 0.025 0.025 0.025 0.025 0.02537.26 37.3 18.63 37.26 37.26 37.26

27.8 27.8 13.9 27.8 27.8180.7 180.7 90.35 180.7 180.7 180.7

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 0.83 0.83 0.83 0.831900.0 1900.0 950.0 1900.0 950.0 1900.0170.0 170.0 85.0 170.0 170.0 170.0

1751.0

8.6 8.6 8.6 8.6 8.6 8.6

Component (mg/L)

Aluminum chloride • 6H2OAmmonium nitrateAmmonium phosphate monobasicAmmonium sulfateBoric acidCalcium chloride anhydrousCalcium nitrateCalcium phosphate tribasicCobalt chloride • 6H2OCupric sulfate • 5H2ONa2-EDTAZnNa2-EDTAFerric chloride • 6H2OFerric citrateFerric sulfateFerric tartrate • 2H2OFerrous sulfate • 7H2OMagnesium sulfateManganese chloride • 4H2OManganese sulfate • H2OMolybdenum trioxideMolybdic acid (sodium salt) • 2H2ONickel chloride • 6H2ONickel sulfate • 6H2OPotassium chloridePotassium iodidePotassium nitratePotassium phosphate monobasicPotassium sulfateSodium nitrateSodium phosphate monobasicSodium sulfateZinc nitrate • 6H2OZinc sulfate • 7H2O

Page 24: Tissue Culture

Classic Plant MediaChu (N6) basal salt mixtureDKW/Juglans basal salt mixtureGamborg's B-5 basal salt mixtureGamborg's B-5 basal salt mixture with minimal organicsHoagland's No. 2 basal salt mixtureMcCown's woody plant basal salt mixtureMurashige and Skoog basal salt mixture (MS)Quoirin and Lepoivre basal salt mixtureSchenk and Hildebrandt basal salt mixtureWhite's basal salt mixture

C 1416D 6162G 5768G 5893H 2395M 6774M 5524Q 3376S 6765W 0876

C 1416 D 6162 G 5768 G 5893 H 2395 M 6774 M 5524 Q 3376 S 6765 W 0876

1416.0 400.0 1650.0 400.0115.03 300.0

463.0 134.0 134.01.6 4.8 3.0 3.0 2.86 6.2 6.2 6.2 5.0 1.5125.33 112.5 113.24 113.24 72.5 332.2 151.0

1367.0 656.4 386.0 833.77 200.0

0.025 0.025 0.025 0.025 0.10.25 0.025 0.025 0.08 0.25 0.025 0.025 0.2

37.25 45.4 37.3 37.25 37.3 37.26 37.3 20.0

2.55.32

27.85 33.8 27.8 27.85 27.8 27.8 15.090.37 361.49 122.09 122.09 240.76 180.7 180.7 175.79 195.4 360.0

1.813.33 33.5 10.0 10.0 22.3 16.9 0.76 10.0 5.04

0.0160.39 0.25 0.25 0.25 0.25 0.25 0.1

0.00565.0

0.8 0.75 0.75 0.83 0.08 1.0 0.752830.0 2500.0 2500.0 606.6 1900.0 1800.0 2500.0 80.0400.0 265.0 170.0 170.0 270.0

1559.0 990.0

130.5 130.5 16.5200.0

17.01.5 2.0 2.0 0.22 8.6 8.6 8.6 1.0 2.67

Component (mg/L)Aluminum chloride • 6H2OAmmonium nitrateAmmonium phosphate monobasicAmmonium sulfateBoric acidCalcium chloride anhydrousCalcium nitrateCalcium phosphate tribasicCobalt chloride • 6H2OCupric sulfate • 5H2ONa2-EDTAZnNa2-EDTAFerric chloride • 6H2OFerric citrateFerric sulfateFerric tartrate • 2H2OFerrous sulfate • 7H2OMagnesium sulfateManganese chloride • 4H2OManganese sulfate • H2OMolybdenum trioxideMolybdic acid (sodium salt) • 2H2ONickel chloride • 6H2ONickel sulfate • 6H2OPotassium chloridePotassium iodidePotassium nitratePotassium phosphate monobasicPotassium sulfateSodium nitrateSodium phosphate monobasicSodium sulfateZinc nitrate • 6H2OZinc sulfate • 7H2O

Page 25: Tissue Culture
Page 26: Tissue Culture
Page 27: Tissue Culture
Page 28: Tissue Culture
Page 29: Tissue Culture
Page 30: Tissue Culture
Page 31: Tissue Culture
Page 32: Tissue Culture

Modes of Regeneration

(1) Embryo Culture: Aseptic culture of a zygotic (2N) embryo

Flower development pollination fertilization 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

Page 33: Tissue Culture

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)

Page 34: Tissue Culture
Page 35: Tissue Culture
Page 36: Tissue Culture

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 Callus4:1 Shoots100:1 Roots

Page 37: Tissue Culture
Page 38: Tissue Culture
Page 39: Tissue Culture
Page 40: Tissue Culture
Page 41: Tissue Culture
Page 42: Tissue Culture
Page 43: Tissue Culture
Page 44: Tissue Culture
Page 45: Tissue Culture
Page 46: Tissue Culture
Page 47: Tissue Culture