Download Lecture Notes 9-2012: Plant Tissue Culture

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Plant Tissue Culture
Text Pages: 643 – 649; 659 – 673.
Objectives:
1. Be able to describe and explain terms involved in plant tissue culture.
2. Be able to describe and explain different types of plant tissue culture as they relate
to the type of plant part(s) regenerated.
3. Be able to distinguish and identify which type or technique of plant tissue culture
should be used based on the type of propagation desired.
I.
PLANT TISSUE CULTURE
A. Terms:
1. Tissue culture - is the science of growing plant cells, tissues, or
a. Biotechnology – what is it?
b. Biotechnology is the use of
2. Aseptic - free from microorganisms:
a. Tissue culture is completed under
b. Microbes may or may not
3. Explant – the plant part that
a. An explant can be a single cell, tissue,
b. The explant should also be free from
4. Micropropagation - is the production of plants from
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a. Micropropagation involves the use of single cells, a tissue, or
b. For this class, we are interested in multiplying whole plants
5. Somatic - cells or tissues that are
6. Germ - cells or tissue that are involved in
7. Embryo - is a rudimentary plant
8. Propagule - tissue that is divided and used
9. Plantlets - small complete plants that were
B. Definitions for Plant Tissue Culture - Complete List:
Adventitious - in reference to a bud, embryo, root or shoot that arises in tissues and
locations that are not the normal origin in the plant.
Aseptic - free from bacteria, fungi or other microorganisms.
Autoclave - equipment that provides heat under high steam pressure for purposes of
sterilization.
Axillary bud or shoot - a bud or shoot that arises from the axil of leaves or normal
origin.
Callus - tissue that develops as a response to injury caused by physical or chemical
means; cells may be differentiated but are unorganized.
Cell - a structural and physiological unit of a living organism (plant).
DNA (Deoxyribonucleic acid) - composed of organic chemicals and is the genetic
material that ultimately determines an organism's characteristics.
Embryo - a rudimentary plant
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Epigenetic changes - persistent changes in phenotype that involve the expression of
particular genes.
Explant - the plant part that is put into tissue culture.
Gene - specific sequence of DNA that codes for a specific trait.
Genotype - the sum total of all genes present in an organism (plant).
Germ - cells or tissues that are involved in reproduction and have one-half of the
genetic material of somatic cells or tissues.
Habituation - tissue cultures losing their requirements for a supply of exogenous
growth regulators.
In vitro - isolated from the living organism and artificially maintained.
Micropropagation - the production of plants from plant parts used in tissue culture.
Mutant - an organism (plant) that has a mutation.
Mutation - change in the DNA sequence that is different from the original sequence.
Organ - a distinct and visibly differentiated part of an organism.
Organogenesis - refers to forming organs, in particular both roots and shoots from
callus cells or other types of cells (e.g., parenchyma cells).
Phenotype - actual appearance and behavior of an organism (plant).
Plantlets - small complete plants that were produced via tissue culture.
Propagules - tissue that is divided and used for further multiplication.
Somaclonal Variation - variation (usually in phenotype and/or perhaps genotype)
induced in cells by the tissue culture process.
Somatic - cells or tissues that are vegetative and contain the complete genetic material
of the organism (plant).
Subculture - divide a propagule and transfer individual parts into other culture vessels.
Tissue - a group of cells organized into a structural and functional unit.
Tissue culture - is the science of growing plant cells, tissues, or organs under artificial
conditions.
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C. Types of Tissue Culture – Based on Type of Regenerated Plant or Part
1. Many types of tissue culture can be used to reproduce plants,
a. The techniques described will be based on
b. Almost all techniques could be used to regenerate whole plants, but some
2. Seedling formation -
a. Aseptic seed culture - used for plants, such as
i.
culture medium keeps seeds alive
ii. in nature, orchid seeds develop with the help of
b. Embryo culture - involves excising an
i.
embryo rescue - the technique is used to rescue or save embryos
ii. ovule culture and ovary culture are also methods of embryo culture
3. Plantlet formation
a. A plantlet is
b. Plantlet formation is divided into two categories:
c. Axillary shoot formation
i.
where is the axil?
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ii. two types of axillary shoots formation will be covered:
iii. meristem culture - uses the meristem tip
aa. the apical meristem is removed (excised) from
bb. the explant usually is less than
cc. this method is usually used to obtain
dd. viruses and other pathogens are absent from the newest cell layers
and
ee. Rule to Remember: The smaller the amount of tissue taken,
ff. meristem culture is successful for
iv. Axillary shoot cultures - are techniques that involve stem
aa. these techniques are usually referred to as micropropagation and
used extensively in
bb. because the apical meristems are already formed, these
techniques provide reliable
cc. with these techniques, lateral (axillary) buds break and
dd. large numbers of lateral shoots often grow, allowing
ee. the techniques designated as axillary shoot cultures include:
i.
axillary branching - many shoots form from
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ii. nodal cultures - single nodes are cut from
iii. 'stool' shoots - a shoot with several nodes in laid at an angle
aa. what causes numerous stems to grow from a axil?
bb. answer:
d. Adventitious shoot formation – can lead to
i.
shoots are considered organs, so adventitious shoots are
aa. direct shoot formation – shoots form directly
bb. indirect shoot formation – callus forms first on explant and
ii. roots also form via organogenesis
iii. species, explant selection, and growth regulators used can
iv. explants include almost any plant part:
v. high rates of regeneration can be obtained
vi. increased numbers of shoots with different or
e. Regenerate haploid plants -
i.
plant part characteristic:
ii. two techniques:
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iii. either an anther or immature pollen is isolated from flowers
4. Callus formation –
a. Stationary callus cultures –
i.
callus – response from
ii. callus (wound cells) grow/proliferate and
iii. high auxin concentrations are often used to promote callus formation,
but
iv. callus can be used for organogenesis
b. Cell suspension culture - is callus tissue placed in liquid culture medium,
i.
whole plants can be regenerated
ii. this technique is usually used for research
c. Protoplast culture - are plant cells without
i.
cell walls are
ii. this technique is usually used for research,
5. Somatic embryogenesis
a. Embryogenesis - refers to forming an underdeveloped or
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b. Somatic - refers to the embryo forming from a
c. Somatic embryos can be formed by
d. To be considered somatic embryos, the developing embryos must go
through
C. Summary
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Micropropagation of Plants
Text Pages: 650 – 659; 699 – 702; 712 – 723.
Objectives:
1. Be able to describe and explain the advantages and liabilities of micropropagation.
2. Be able to name and identify the four stages of micropropagation.
3. Be able to describe and explain the factors that affect establishment of explants in
tissue culture (Stage I).
4. Be able to describe and explain the factors that affect shoot multiplication during
micropropagation (Stage II).
5. Be able to describe and explain the various manipulations used to promote rooting
during Stage III of micropropagation.
6. Be able to describe and explain how a plantlet changes during Stage IV of
micropropagation.
7. Be able to predict how various factors involved in the different stages can affect the
success of micropropagation.
II.
MICROPROPAGATION
A. Introduction
1. Micropropagation is an important propagation technique for
2. Microshoots, in vitro grown shoots, are used in micropropagation and
a. Axillary buds –
b. Organogensis or adventitious shoot regeneration –
3. Micropropagation is the term used for in vitro production of plants,
4. For this course, micropropagation involves axillary shoot cultures
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B. Characteristics of Micropropagation
1. Advantages of micropropagation
a. Mass production of specific clones
i.
very large numbers of plants can be produced
ii. plantlets take much less space
iii. rapid multiplication is important for bringing large numbers of new
b. Year-round plant (nursery) production
c. Production of pathogen-free plants
i.
micropropagation can be used to eliminate
ii. aseptic conditions must be
d. Clonal propagation of clonal stock for seed production
e. Germplasm preservation
2. Disadvantages or problems with micropropagation
a. Facilities required are costly
b. Workers must have certain skills and
c. Multiply errors: plant identity, introduction of a pathogen or
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d. Genetic or epigenetic changes caused by
III.
STAGES OF MICROPROPAGATION
A. Introduction - Four Stages of Micropropagation (Axillary Shoot Proliferation)
1. Establishment and Stabilization – of
2. Multiplication – of
3. Root Formation (also considered Transplant preparations)
4. Acclimatization –
5. The importance of these stages varies
B. Stage I - Establishment and Stabilization of the Explant
1. Explant disinfestation – surface sterilize an explant
a. The explant must be free from all
b. External surfaces and internal tissues must be
c. Ideally the tissue from which the explant is removed should be in
d. Keeping the foliage, including stems, dry
2. Explant selection is very important
a. Ontogenetic state –
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b. The plant part used as the explant should be selected to
c. Plants resulting from micropropagation should be true-to-type without
d. Repeated subculturing – may help explant shoots and propagules to
become
3. Culture medium requirements –
a. The basic medium contains
i.
woody plants were at first difficult to culture because
ii. a medium lower in
b. Growth regulators must be applied
i.
usually concerned with
ii. these plant growth regulations must be applied in
4. Environmental parameters during culturing must be regulated too
a. Light levels and
b. Temperature should be kept between
5. Exudation – endogenous plant substances
a. Some of these substances can be washed away
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b. Ways to limit explants from producing these inhibitors is to use
antioxidants
6. Stabilization – explants should be stable in vitro, meaning microshoot growth
a. The establishment stage usually lasts
b. Woody plants often take longer to stabilize than
C. Stage II - Multiplication of Propagules
1.
The objective of this stage is to increase
a. Subculturing - means taking in vitro cells, tissues, or organs, microshoots
in the case of micropropagation, dividing them, and putting
b. Take home message: consider both the number of subcultures and the
shoot
2. Multiplication of shoots
a. Propagation ratio -
i.
number of shoot produced can vary
ii. optimize the nutrient medium to obtain
b. Growth regulator concentrations
i.
usually use a high cytokinin to auxin ratio (e.g., 100:1)
ii. the ratio is not absolute and must be
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3. Frequency of transfer (subculturing)
a. The time needed between transfers (subculturing) may vary
b. If transfer (subculturing) is delayed, the propagules may
c. Example:
D. Stage III - Root Formation: preparing the microshoot for
1. Quality of the microshoots
a. Quality and health are influenced by
b. Strong, healthy microshoots should be
c. Stage III can be skipped if
2. Growth regulators are shifted to
a. Cytokinin concentration is
b. Auxin level should be regulated
c. A high cytokinin concentration used in Stage II can inhibit
3. Other medium components are also shifted, including
a. Salts or minerals may
b. Agar content may be increased to stiffen
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4. During or after root formation, plantlets must be "toughened-up"
a. Plantlets are susceptible to problems due to
b. Problems include:
E. Stage IV - Acclimatization: transplanting the plantlet into
1. The "toughened" plant is transferred from
2. The plant must now support itself or
a. Under tissue culture conditions, the propagule depended
b. Green leaves on microshoots may or may not
3. The plantlets must adjust
a. Plantlets must become
b. The plantlet must also acclimate to
4. Stomata on leaves of tissue cultured plantlets
a. Stomata stay open so that plantlets transpire at high rates
b. The type of cytokinin used
c. Therefore, growth regulator uses during micropropagation can (1) affect
explant
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5. Plantlets should be protected from pathogens during Stage IV since
a. Proper sanitation must be used including
b. Minimize plant stress
F. Summary
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