Plant Tissue Culture

Dr. David Leung – School of Biological Sciences

Principal Goal:

To introduce a plant tissue culture technique that may be used to clone plants either for enjoyment, educational, research or commercial endeavours.

Some useful resources:

An academically sound article on the web:

Lineberger R.D. The many dimensions of plant tissue culture research.http://aggie-horticulture.tamu.edu/tissuecult/pltissue/pltissue.htm1

A popular book with helpful laboratory protocols:

Kyte L., Kleyn J. 1996. Plants from test tubes: An introduction to micropropagation. 3rd edition. Timber Press, Inc. ISBN 0-88192-361-3

Wahlenbergia Stricta

A useful website with many interesting plant tissue culture links including suppliers for everything required to start plant tissue culture work:

Kitchen Culture Kits, Inc.http://www.home.turbonet.com/kitchenculture/tcinfo.htmComposition of a plant tissue culture medium for beginners:

Murashige and Skoog (MS) tissue culture medium (1962)

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College of Science Outreach

(I) Preparation of Stock Solutions

Four stock solutions are required and amounts shown are for 500 mL of solution.

1) Major salts (500 mL)

NH4NO3 8.25 g

KNO3 9.5 g

CaCl2.2H2O 2.2 g

MgSO4.7H2O 1.85 g

KH2PO4 0.85 g

Bring to volume (500 mL) with distilled water.

Store at 40C.

2) Minor salts (500 mL)

KI 0.0415 g

H3BO3 0.310 g

MnSO4.4H2O 1.115 g

ZnSO4.7H2O 0.43 g

CuSO4.5H2O 0.00125 g

CoCl2.6H2O 0.00125 g

Na2MoO4.2H2O 0.0125 g

Bring to volume (500 mL) with distilled water.

Store at 40C.

3) Organic Supplement (500 mL)

Myo-inositol 5 g

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Nicotinic acid 0.025 g

Pyridoxine-HCl 0.025 g

Thiamine-HCl 0.005 g

Glycine 0.1 g

Bring to volume (500 mL) with distilled water.

Store at 40C.

4) Iron Stock (500 mL)

Solution A: FeSO4.7H2O 1.39 g in 200 mL of distilled water

Solution B: Na2EDTA.2H2O 1.865 g in 200 mL of distilled water

Mix solutions A and B.

Adjust volume to 500 mL with distilled water.

Store in a brown bottle at 40C.

(II) Volumes of the stock solutions (the first column from the left) required for making different volumes of the MS medium (indicated in the first row)

200 mL 500 mL One Litre

Distilled water 150 mL 375 mL 750 mL

Major salts 20 mL 50 mL 100 mL

Minor salts 2 mL 5 mL 10 mL

Organic supplement 2 mL 5 mL 10 mL

Iron stock 2 mL 5 mL 10 mL

(III) Steps involved in the preparation of MS medium

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1) Mix the stock solutions and distilled water as indicated in (II).

2) Add the required amount of sucrose (for example, 3% or 3 g per 100 mL) and stir

until dissolved.

3) Adjust pH of medium to 5.6 - 5.8 and then bring to the required volume with

distilled water.

4) Dispense the medium (40 mL per autoclavable glass or polycarbonate tissue

culture jar).

5) Autoclave the medium.

Reference:

Murashige, T. and F. Skoog, 1962. A revised medium for rapid growth and bioassays

with tobacco tissue cultures. Physiologia Plantarum,15: 473-497.

Acknowledgement:

The contribution of all Dr Leung’s postgraduate research students at the University of

Canterbury in preparation of this handout is gratefully acknowledged.

Further enquiries are welcome:

Science Outreach Coordinator,College of Science University of CanterburyPrivate Bag 4800,Christchurch 8140,New Zealand

outreach@canterbury.ac.nz

www.outreach.canterbury.ac

Science Outreach: www.outreach.canterbury.ac.nz