Download Plant-Tissue-Culture

Plant Tissue Culture
Topics covered in this
presentation-History
and
scope,
preparation
and
sterilization, terms used in
tissue culture, plant tissue
culture media. Types of
culture, basic technique of
plant tissue culture.
Ppt By- Nitin Swamy
Asst. Professor,
Department of Biotecnology,
St. Aloysius College (Autonomous),
Jabalpur
What is plant tissue culture?
Plant tissue culture is a technique of growing plant
cells, tissues, organs, seeds or other plant parts in a
sterile environment on a nutrient medium.
 Tissue culture had its origins at
the beginning of the 20th
century with the work of
Gottleib Haberlandt (plants).
The Background,
 The first commercial use of
plant clonal propagation on
artificial media was in the
germination and growth of
orchid plants, in the 1920’s
 In the 1950’s and 60’s there
was a great deal of research,
but it was only after the
development of a reliable
artificial
medium
(Murashige & Skoog,
1962) that plant tissue
culture really ‘took off’
commercially
Young cymbidium orchids
WHY?
 The production of clones of plants that produce particularly good
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flowers, fruits, or have other desirable traits.
To quickly produce mature plants.
The production of multiples of plants in the absence of seeds or
necessary pollinators to produce seeds.
The regeneration of whole plants from plant cells that have
been genetically modified.
The production of plants in sterile containers reduces disease
transmission
Allows production of plants from seeds that otherwise have very low
chances of germinating and growing, i.e.: orchids and Nepenthes.
To clean particular plants of viral and other infections and to quickly
multiply these plants as 'cleaned stock' for horticulture and agriculture.
How?
Totipotency:
It is the ability of a tissue or an organ of a plant to produce the whole plant,
under the optional laboratory conditions and this is called as Totipotency.
This is the baseline over which plant tissue culture relies upon.
Plant Tissue Culture Terminology
Adventitious---Developing from unusual points of origin, such as shoot
or root tissues, from callus or embryos, from sources other than zygotes.
Agar---a polysaccharide powder derived from algae used to gel a medium.
Agar is generally used at a concentration of 6-12 g/liter.
Aseptic---Free of microorganisms.
Aseptic Technique---Procedures used to prevent the introduction of
fungi, bacteria, viruses, mycoplasma or other microorganisms into
cultures.
Callus---An unorganized, proliferate mass of differentiated plant cells, a
wound response.
Chemically Defined Medium---A nutritive solution for culturing cells
in which each component is specifiable and ideally of known chemical
structure.
Contamination---Being infested with unwanted microorganisms such as
bacteria or fungi. Culture—A plant growing in vitro.
Differentiated---Cells that maintain, in culture, all or much of the
specialized structure and function typical of the cell type in vivo.
Modifications of new cells to form tissues or organs with a specific
function.
Plant Tissue Culture Terminology
Explant---Tissue taken from its original site and transferred to an artificial
medium for growth or maintenance.
Hormones---Growth regulators, generally synthetic in occurrence, that
strongly affects growth (i.e. cytokinins, auxins, and gibberellins).
Internode---The space between two nodes on a stem
Media---Plural of medium
Medium---A nutritive solution, solid or liquid, for culturing cells.
Micropropagation---In vitro Clonal propagation of plants from shoot tips
or nodal explants, usually with an accelerated proliferation of shoots during
subcultures. Node—A part of the plant stem from which a leaf, shoot or
flower originates.
Plant Tissue Culture---The growth or maintenance of plant cells, tissues,
organs or whole plants in vitro.
Regeneration---In plant cultures, a morphogenetic response to a stimulus
that results in the products of organs, embryos, or whole plants.
Somaclones---Plants derived from any form of cell culture involving the
use of somatic plant cells.
Plant Tissue Culture Terminology
Stage I---A step in in vitro propagation characterized by the
establishment of an aseptic tissue culture of a plant.
Stage II---A step in in vitro propagation characterized by the rapid
numerical increase of organs or other structures.
Stage III---A step in in vitro propagation characterized by preparation of
propagules for successful transfer to soil, a process involving rooting of
shoot cuttings, hardening of plants, and initiating the change from the
heterotrophic to the autotropic state.
Stage IV---A step in in vitro plant propagation characterized by the
establishment in soil of a tissue culture derived plant, either after
undergoing a Stage III pretransplant treatment, or in certain species, after
the direct transfer of plants from Stage II into soil.
Sterile--- (A) Without life. (B) Inability of an organism to produce
functional gametes. (C) A culture that is free of viable microorganisms.
Sterile Techniques---The practice of working with cultures in an
environment free from microorganisms.
Plant Tissue Culture Terminology
Subculture---See “Passage”. With plant cultures, this is the process by
which the tissue or explant is first subdivide, then transferred into fresh
culture medium.
Tissue Culture---The maintenance or growth of tissue, in vitro, in a way
that may allow differentiation and preservation of their function.
Totipotency---A cell characteristic in which the potential for forming all
the cell types in the adult organism are retained.
Undifferentiated---With plant cells, existing in a state of cell
development characterized by isodiametric cell shape, very little or no
vacuole, a large nucleus, and exemplified by cells comprising an apical
meristem or embryo.
Plant tissue Culture Basics
 Modern plant tissue culture is performed under
aseptic conditions.
 Living plant materials from the environment are
naturally contaminated on their surfaces (and
sometimes interiors) with microorganisms, so
surface sterilization of starting material (explants)
in chemical solutions (usually alcohol and sodium or
calcium hypochlorite is required).
Plant tissue Culture Basics
 Explants are then usually placed on the surface of a
solid culture medium, but are sometimes placed
directly into a liquid medium, when cell suspension
cultures are desired.
 Culture
media are generally composed of
inorganic salts plus a few organic nutrients, vitamins
and plant hormones.
Plant Tissue Culture Laboratory
A plant tissue culture laboratory, whether for research or for
commercial purpose, should provide certain basic facilities (i) washing and storage of glassware, plasticware and other
labwares,
(ii) preparation, sterilization and storage of nutrient media,
(iii) aseptic manipulation of plant material,
(iv) maintenance of cultures under controlled conditions of
temperature, light and humidity,
(v) observation of cultures and
(vi) hardening of in vitro developed plants.
The extent of sophistication in terms of equipment and
facilities depends on the need and the funds available. Therefore,
establishment of a new tissue culture facility requiring ingenuity
and careful planning.
Plant Tissue Culture Media
WHAT’S REALLY IMPORTANT?
Plant Tissue Culture owes its origin to the ideas of the German
Scientist, Haberlandt, in the beginning of the 20th century. This was just
the beginning of the tissue culture; thereafter in 70's began the
commercialization of the technology.
Synthetic and natural media:
When a medium is composed of chemically defined components, it is
referred to as a synthetic medium. On the other hand, if a medium contains
chemically undefined compounds (e.g., vegetable extract, fruit juice, plant
extract), it is regarded as a natural medium. Synthetic media have almost
replaced the natural media for tissue culture.
Expression of concentrations in media:
The concentrations of inorganic and organic constituents in culture media
are usually expressed as mass values (mg/l or ppm or mg I-1). However, as
per the recommendations of the International Association of Plant
Physiology, the concentrations of macronutrients should be expressed as
mmol/l– and micronutrients as µmol/l–.
Major Types of Media:
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White’s medium:
This is one of the earliest plant tissue culture media developed for root
culture.
MS medium:
Murashige and Skoog (MS) originally formulated a medium to induce
organogenesis, and regeneration of plants in cultured tissues. These days,
MS medium is widely used for many types of culture systems.
B5 medium:
Developed by Gamborg, B5 medium was originally designed for cell
suspension and callus cultures. At present with certain modifications, this
medium is used for protoplast culture.
N6 medium:
Chu formulated this medium and it is used for cereal anther culture, besides
other tissue cultures.
Nitsch’s medium:
This medium was developed by Nitsch and Nitsch and frequently used for
anther cultures. Among the media referred above, MS medium is most
frequently used in plant tissue culture work due to its success
with several plant species and culture systems.
Major Constituents
 Salt Mixtures
 Organic Substances
 Natural Complexes
 Inert Supportive Materials
 Growth Regulators
Macro-nutrient salts
What the ?
 NH4NO3
 KNO3
 CaCl2 -2 H2O
 MgSO4 -7 H2O
 KH2PO4
 FeNaEDTA
 H3BO3
 MnSO4 - 4 H2O
 ZnSO4 - 7 H2O
 KI
 Na2MoO4 - 2 H2O
 CuSO4 - 5 H2O
 CoCl2 - H2O
Ammonium nitrate
Potassium nitrate
Calcium chloride (Anhydrous)
Magnesium sulfide (Epsom Salts)
Potassium hypophosphate
Fe/Na ethylene-diamine-tetra acetate
Boric Acid
Manganese sulfate
Zinc sulfate
Potassium iodide
Sodium molybdate
Cupric sulfate
Cobaltous sulfide
Macronutrient salts
 Nitrogen – Influences plant growth rate, essential in plant nucleic
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acids (DNA), proteins, chlorophyll, amino acids, and hormones.
Phosphorus – Abundant in meristematic and fast growing tissue,
essential in photosynthesis, respiration.
Potassium – Necessary for cell division, meristematic tissue, helps
in the pathways for carbohydrate, protein and chlorophyll
synthesis.
Calcium - Involved in formation of cell walls and root and leaf
development. Participates in translocation of sugars, amino acids,
and ties up oxalic acid (toxin).
Iron - Involved in respiration , chlorophyll synthesis and
photosynthesis.
FeNaEDTA = sodium salt of EDTA sequesters iron, making it
available to plants.
Micronutrient salts
 Magnesium
- Involved in photosynthetic and
respiration systems. Active in uptake of phosphate
and translocation of phosphate and starches.
 Sulfur - Involved in formation of nodules and
chlorophyll synthesis, structural component of amino
acids and enzymes.
 Manganese - Involved in regulation of enzymes and
growth hormones. Assists in photosynthesis and
respiration.
Micronutrient salts
 Molybdenum - Involved in enzymatic reduction of
nitrates to ammonia. Assists in conversion of inorganic
phosphate to organic form.
 Zinc - Involved in production of growth hormones and
chlorophyll. Active in respiration and carbohydrate
synthesis.
 Boron - Involved in production of growth hormones and
chlorophyll. Active in respiration and carbohydrate
synthesis.
 Copper -Involved in photosynthetic and respiration
systems. Assists chlorophyll synthesis and used as reaction
catalyst.
Organic Compounds
 Carbon Sources – Sucrose, sometimes Glucose or Fructose (Plants
Need Carbon)
 Vitamins –
 Adenine – part of RNA and DNA
 Inositol – part of the B complex, in phosphate form is part of cell
membranes, organelles and is not essential to growth but beneficial
 Thiamine – essential as a coenzyme in the citric acid cycle.
 Organic Acids
 Citric acid (150 mg/l) typically used with ascorbic acid (100 mg/l) as
an antioxidant.
 Can also use some of Kreb Cycle acids
 Phenolic compounds
 Phloroglucinol - Stimulates rooting of shoot sections.
Natural Complexes
 Coconut endosperm
 Protein hydrolysates
 Tomato juice
 Yeast extracts
 Malt extract
 Potato agar
 Activated
charcoal is used as a detoxifying agent.
Detoxifies wastes from plant tissues, impurities
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Impurities and absorption quality vary
Concentration normally used is 0.3 % or lower
 Charcoal for tissue culture
 acid washed and neutralized
 never reuse
Growth regulators
What is a Growth Regulator?
Plant Cell Growth regulators (e.g. Auxins, Cytokinins and Gibberellins) Plant hormones play an important role in growth and differentiation of
cultured cells and tissues. There are many classes of plant growth
regulators used in culture media involves namely: Auxins, Cytokinins,
Gibberellins, Abscisic acid, and Ethylene.
 Auxin - Roots
 Cytokinin - Shoots
 Gibberellin – Cell Enlargement
 Abscisic acid – Plant stress hormone
 Ethylene – BAD!
Auxins
 The
Auxins facilitate cell division and root
differentiation. Auxins induce cell division, cell
elongation, and formation of callus in cultures.
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Indole-3-acetic acid =IAA
Naphthalene acetic acid NAA
Indole-3-butyric acid
2,4-D
2,4,5-T
Picloram
For example, 2,4-dichlorophenoxy acetic acid
is one of the most commonly added auxins in plant
cell cultures.
Cytokinins
 The Cytokinins induce cell division and differentiation.
Cytokinins promote RNA synthesis and stimulate
protein and enzyme activities in tissues.
 -Enhances adventitious shoot formation
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benzyl adenine
2iP
Kinetin
Zeatin
For example, Kinetin and benzyl-aminopurine are the
most frequently used cytokinins in plant cell cultures
The ratio of auxins and cytokinins play an important role in the morphogenesis of
culture systems. When the ratio of auxins to cytokinins is high, embryogenesis,
callus initiation, and root initiation occur. For axillary proliferation and shoot
proliferation, the ratio of auxins to cytokinins is kept low.
Gibberellin
Abscisic Acid
 Not generally used in tissue
 Primarily a growth inhibitor
culture
 Tends
to suppress root
formation and adventitious
embryo formation.
but enables more normal
development of embryos,
both zygotic and adventitious.
Ethylene
 Question is not how much to add but how to get rid of it in-vitro
 Natural substance produced by tissue cultures at fairly high levels
especially when cells are under stress
 Enhances senescense
 Supresses embryogenesis and development in general.
Hormone Combinations
 Callus development
 Adventitious embryogenesis
 Rooting of shoot cuttings
 Adventitious shoot and root formation
Culturing (Micropropagating) Plant Tissue
The Steps, I
• Selection of the plant
tissue (explant) from a
healthy
vigorous
‘mother plant’ - this is
often the apical bud,
but can be other tissue.
• This tissue must be
sterilized to remove
microbial
contaminants.
The Steps, II
 Establishment of the
explant in a culture
medium. The medium
sustains the plant cells
and
encourages
cell
division. It can be solid
or liquid
 Each plant species
(and
sometimes
the
variety within a species)
has particular medium
requirements that must
be established by trial
and error.
The Steps, III
 Multiplication-
Dividing shoots
The
explant gives rise to a
callus (a mass of loosely
arranged cells) which is
manipulated by varying
sugar
concentrations
and the auxin (low):
cytokinin
(high)
ratios
to
form
multiple shoots.
 The
Warmth and good light are essential
callus may be
subdivided a number of
times.
The Steps, IV
 Root
formation The
shoots
are
transferred to a
growth
medium
with
relatively
higher
auxin:
cytokinin ratios
The bottles on these racks are young
banana plants and are growing roots.
The Steps, V
 The
rooted shoots are
potted up (deflasked) and
‘hardened off’ by gradually
decreasing the humidity
 This is necessary as many
young tissue culture plants
have no waxy cuticle to
prevent water loss
Tissue culture plants sold to
a nursery & then potted up.
Transfer to soil
Plant Tissue Culture Applications
 The commercial production of plants used as potting, landscape,
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and florist subjects
To conserve rare or endangered plant species.
To screen cells rather than plants for advantageous characters,
e.g. herbicide resistance/tolerance.
Large-scale growth of plant cells in liquid culture in bioreactors for
production of valuable compounds, like plant-derived secondary
metabolites and recombinant proteins used as biopharmaceuticals.
To cross distantly related species by protoplast fusion and
regeneration of the novel hybrid.
To produce clean plant material from stock infected by viruses or
other pathogens.
Production of identical sterile hybrid species can be obtained.
Plant Tissue Culture Types
Types of Plant tissue culture
Callus culture:
This involves the culture of differentiated tissue from explant which dedifferentiates
in vitro to form callus.
Organ culture:
Culture of isolated plant organs is referred to as organ culture. The organ used may
be embryo, seed, root, endosperm, anther, ovary, ovule, meristem (shoot tip) or
nucellus. The organ culture may be organized or unorganized.
Organized organ culture:
When a well-organized structure of a plant (seed, embryo) is used in culture, it is
referred to as organized culture. In this type of culture, the characteristic individual
organ structure is maintained and the progeny formed is similar in structure as that
of the original organ.
Unorganized organ culture:
This involves the isolation of cells or tissues of a part of the organ, and their culture
in vitro. Unorganized culture results in the formation of callus. The callus can be
dispersed into aggregates of cells and/or single cells to give a suspension culture.
Cell culture:
The culture of isolated individual cells, obtained from an explant tissue or callus is
regarded as cell culture. These cultures are carried out in dispension medium and
are referred to as cell suspension cultures.
Protoplast culture:
Plant protoplasts (i.e., cells devoid of cell walls) are also used in the laboratory for
culture.
Basic Technique of Plant Tissue Culture:
The general procedure adopted for isolation and culture of plant tissues is depicted
in Fig
•The requisite explants (buds, stem, seeds) are trimmed and then
subjected to sterilization in a detergent solution.
•After washing in sterile distilled water, the explants are placed in a
suitable culture medium (liquid or semisolid form) and incubated.
This results in the establishment of culture.
•The mother cultures can be subdivided, as frequently as needed, to
give daughter cultures.
•The most important aspect of in vitro culture technique is to carry out
all the operations under aseptic conditions.
•Bacteria and fungi are the most common contaminants in plant tissue
culture. They grow much faster in culture and often kill the plant
tissue.
•Further, the contaminants also produce certain compounds which are
toxic to the plant tissue.
•Therefore, it is absolutely essential that aseptic conditions are
maintained throughout the tissue culture operations. Some of the
culture techniques are described here while a few others are discussed
at appropriate places.
Callus Culture:
Callus is the undifferentiated and
unorganized mass of plant cells. It is
basically a tumor tissue which usually forms
on wounds of differentiated tissues or
organs.
Callus cells are parenchymatous in nature
although not truly homogenous. On careful
examination, callus is found to contain some
quantity of differentiated tissue, besides the
bulk of non-differentiated tissue.
Callus formation in vivo is frequently
observed as a result of wounds at the cut
edges of stems or roots.
Invasion of microorganisms or damage by
insect feeding usually occurs through callus.
An outline of technique used for callus
culture, and initiation of suspension culture
is depicted in Fig. 42.5.
Explants for callus culture:
The starting materials (explates) for callus culture may be the
differentiated tissue from any part of the plant (root, stem, leaf, anther,
flower etc.).
The selected explant tissues may be at different stages of cell division,
cell proliferation and organization into different distinct specialized
structures. If the explant used possesses meristematic cells, then the cell
division and multiplication will be rapid.
Factors Affecting Callus Culture:
Many factors are known to influence callus formation in vitro culture.
These include the source of the explant and its genotype, composition of
the medium (MS medium most commonly used), physical factors
(temperature, light etc.) and growth factors.
Other important factors affecting callus culture are — age of the plant,
location of explant, physiology and growth conditions of the plant.
Physical factors:
A temperature in the range of 22-28°C is suitable for adequate callus
formation. As regards the effect of light on callus, it is largely
dependent on the plant species-light may be essential for some plants
while darkness is required by others.
Growth regulators:
The growth regulators to the medium strongly influence callus
formation. Based on the nature of the explant and its genotype, and the
endogenous content of the hormone, the requirements of growth
regulators may be categorized into 3 groups
1. Auxin alone
2. Cytokinin alone
3. Both auxin and cytokinin.
Suspension culture from callus:
Suspension cultures can be initiated by transferring friable callus to
liquid nutrient medium (Fig. 42.5). As the medium is liquid in nature, the
pieces of callus remain submerged. This creates anaerobic condition and
ultimately the cells may die. For this reason, suspension cultures have to
be agitated by a rotary shaker. Due to agitation, the cells gets dispersed,
besides their exposure to aeration.
Applications of Callus Cultures:
Callus cultures are slow-growth plant culture systems in static medium.
This enables to conduct several studies related to many aspects of plants
(growth, differentiation and metabolism) as listed below.
i. Nutritional requirements of plants.
ii. Cell and organ differentiation.
iii. Development of suspension and protoplast cultures.
iv. Somaclonal variations.
v. Genetic transformations.
vi. Production of secondary metabolites and their regulation.
Cell Culture:
The first attempt to culture single cells (obtained from leaves of flowering
plants) was made in as early as 1902 by Haberlandt. Although he was
unsuccessful to achieve cell division in vitro, his work gave a stimulus to
several researchers. In later years, good success was achieved not only for
cell division but also to raise complete plants from single cell cultures.
Applications of Cell Cultures:
Cultured cells have a wide range of applications in biology.
1. Elucidation of the pathways of cellular metabolism.
2. Serve as good targets for mutation and selection of desirable mutants.
3. Production of secondary metabolites of commercial interest.
4. Good potential for crop improvement.