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Plant tissue culturea.k.a. micropropagation

Plant tissue culture• Broadly refers to technique of growing plant cells, tissues,

organs, seeds or other plant parts in a sterile environment on a nutrient medium

• The first commercial use of plant propagation on artificial media was in the germination and growth of orchid plants in the 1920’s

• It was only after the development of a reliable artificial medium by Murashige & Skoog in 1962 that plant tissue culture really took off commercially

Why do plant tissue culture?• Fast commercial propagation of new cultivars

– Cost of culture (orchid price decrease)• Agriculture

– Fast selection for crop improvement – nutritional value, pest control, hardiness

– Cultivation virus free plants• Pharmaceuticals – ginseng and taxol• Cloning of rare and endangered plants• Plant cultures in approved media are easier and safer to

export

History• First attempted by Haberlandt (1902) - grew palisade cells

from leaves of various plants but they did not divide• 1934 - White generated continuously growing culture of

meristematic cells of tomato on medium containing salts,yeast extract and sucrose and 3 vit B (pyridoxine, thiamine, nicotinic acid) – established the importance of additives

• 1953 Miller and Skoog, University of Wisconsin – Madison discovered kinetin, a cytokine that plays an active role in organogenesis

Further progress• 1958-60 Morel cultured orchids and dahlias freed them

from a viral disease• 1962 Murashige and Skoog published recipe for M&S

medium• 60’s and 70’s Murashige cloned plants in vitro

– raised haploid plants from pollen grains– used protoplast fusion to hybridize 2 species of tobacco

into one plant contained 4N• 70’s and 80’S beginning of genetic engineering

Basis for the cell culture• Plant cells are totipotent• Have the ability to develop into whole plants or plant

organs in vitro when given the correct conditions• Not all plant cells are totipotent. However, there are a

sufficient number of totipotent cells in the plant (e.g. in the pith)

• Differentiated cells have to be dedifferentiated into callus and redifferentiated back to somatic embryo that will regenerate the entire plant

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Callus• Is a natural response of the plant tissue to wounding• A mass of actively dividing undifferentiated cells produced

by plant tissue explant • Cells are totipotent• Callus can be

– Resuspended in liquid media to create a susupensionculture of single totipotent cells

– Or differentiated into plant with the appropriate manipulations of culture conditions

Culturing (micropropagating) plant tissue - the steps

• Selection of the plant tissue (explant) from a healthy vigorous ‘mother plant’ - this is often the apical bud, but can be other tissue

• Establishment of the explant in a culture medium - the medium sustains plant cells growth and encourages cell division– Each plant species (and sometimes the variety within a

species) has particular medium requirements that must be established by trial and error

• Multiplication - the explant gives rise to a callus• Differentiation and organogenesis

What is needed?• Explant (some tissues culture better than others)• A suitable growth medium • Aseptic conditions, as microorganisms grow much more

quickly than plant and animal tissue • Growth regulators - in plants, both auxins & cytokinins• Frequent subculturing to ensure adequate nutrition and to

avoid the build up of waste metabolites

Plant tissue culture media• Mineralals (17 essential elements)• Energy and carbon source - sucrose is preferred • Growth regulators – auxins and cytokinins

(phytohormones)• Vitamins• Organic compounds• Water

• Usually 5.0-5.7

Vitamins• Thiamine • Pyridoxin• Nicotinic acid• Biotin• Citric acid• Ascorbic acid• Inositol

Growth regulatorsAuxins and cytokinins• Auxins stimulate cell division in explants

– Used for callus induction and growth• High concentration suppresses the organized growth and

promotes formation of meristem-like cells• IAA, indole-3-acetic acid a light sensitive, natural auxin

• Cytokinins• Promote cell division and regeneration possibly through

the effects on protein synthesis• Zeatin – natural cytokine that induces morphogenesis

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Homework• In your notebook write a detailed content of a selected

plant culture medium. It has to have concentrations of all ingredients!

• Make sure you understand what is in it and why. Again, there will be a question about it on the first exam.

• Find a name of the plant that is grown on that medium