Download Plant Tissue Culture:

 Plant Tissue Culture:

History;
• Schwann and Schleiden (1838) - Totipotency theory
• Haberlandt (1902) - Concept of cell culture published in first report
“Experiments on the culture of isolated plant cells”
• Skoog and Miller (1957) - Predicted hypothesis “ Regulation of initiation
of shoot and root in cultured callus by auxin and cytokinin”
• Steward (1958) and Reinert (1959) - Somatic embryogenesis in carrot
cultures
• Murashige and Skoog (1962) - MS medium
• Chilton (1983) - Production of transgenic tobacco
• 1984 – onwards - Commercial multiplication of crops, disease free crops,
release of GM crops like tomato, soybean, corn, cotton, canola, potato etc.

What is plant tissue culture?
• It is a collection of techniques used to
 maintain or grow plant cells, tissues or organs under sterile conditions
on a nutrient culture medium of known composition i.e. in vitro
• Plant tissue culture is widely used to produce clones of a plant in a method
known as micro-propagation
• Different techniques in plant tissue culture may offer certain advantages over
traditional methods of propagation, including:
 production of exact copies of plants that produce particularly good
flowers, fruits, or have other desirable traits
 quick production of mature plants
 production of multiples of plants in the absence of seeds or necessary
pollinators to produce seeds
 regeneration of whole plants from plant cells that have been genetically
modified
 The production of plants in sterile containers that allows them to be moved
with greatly reduced chances of transmitting diseases, pests, and pathogens
 The production of plants from seeds that otherwise have very low chances
of germinating and growing i.e. Orchids and Nepenthes (tropical pitcher
plants or monkey cups)
 To clean particular plants of viral and other infections and to quickly
multiply these plants as 'cleaned stock' for horticulture and agriculture
• Plant tissue culture relies on the fact that
 many plant cells have the ability to regenerate a whole plant (totipotency)
 Single cells, plant cells without cell walls (protoplasts), pieces of leaves,
stems or roots can often be used to generate a new plant on culture media
given the required nutrients and plant hormones
Fig 7.2 Alberts 5th Ed

Basis for Plant Tissue Culture:
• Two hormones affect plant differentiation:
 Auxin - stimulates root development
 Cytokinin - stimulates shoot development
• Generally, the ratio of these two hormones can determine plant
development:
 auxin ↓cytokinin - root development
 cytokinin ↓auxin - shoot development
auxin = cytokinin - callus development
Figs downloaded
 Factors Affecting Plant Tissue Culture:
•
Growth Media - Minerals, Growth factors, Carbon source
Control of in vitro culture
Cytokinin
Leaf strip
Adventitious
Shoot
Root
Callus
Auxin
• Environmental Factors - Light, Temperature, Photoperiod
• Explant Source - Usually, the younger, less differentiated the explant,
the better for tissue culture like ;
 Shoot tips
 Axillary buds
 Seeds
 Hypocotyl from germinated seed
 Leaves
 Fundamental Abilities of Plants:
• Totipotency;
 The potential or inherent capacity of a plant cell to develop into an
entire plant if suitably stimulated
 It implies that all the information necessary for growth and
reproduction of the organism is contained in the cell
•
Differentiation;
 The capacity of mature cells to return to meristematic condition and
development of a new growing point, followed by redifferentiation
which is the ability to reorganize into new organs
• Competency;
 The endogenous potential of a given cell or tissue to develop in a
particular way
 Types of in vitro Culture:
• Culture of intact plants (seed orchid culture)
•
Embryo culture (embryo rescue)
•
Organ culture
 Shoot tip culture
 Root culture
 Leaf culture
 Anther culture
• Callus culture
• Cell suspension and single cell culture
• Protoplast culture
Fig

Breeding Applications of Tissue Culture:
• Micropropagation
• Germplasm preservation
• Somaclonal variation
• Embryo culture
• Haploid and dihaploid production
• In vitro hybridization - protoplast fusion
• Plant genetic engineering
•
Micropropagation;
 Embryogenesis
o
Direct embryogenesis
o
Indirect embryogenesis
 Organogenesis

o
Organogenesis via callus formation
o
Direct adventitious organ formation
Microcutting
o
Meristem and shoot tip culture
o
Bud culture
 Somatic Embryogenesis:
o
The process of initiation and development of embryos or embryo-like
structures from somatic cells
o
The production of embryos from somatic or “non-germ” cells
o
Usually involves a callus intermediate stage which can result in
variation among seedlings
o
Not a common micro-propagation technique but is currently being
used to produce superior pine seedlings
o
Tissue culture maintains the genetic of the cell or tissue used as an
explants
o
Tissue culture conditions can be modified to cause to somatic cells to
reprogram into a bipolar structure
o
These bipolar structures behave like a true embryo - called somatic
embryos
 Organogenesis:
o
The process of initiation and development of a structure that shows
natural organ form and/or function
Figs
Somatic embryogenesis from Pro-embryonic masses (PEMs)
+ Auxin leads to high [Putrescine]
PEM
Single cells sloughed
off the surface
Development and cycling
of Pro-embryonic masses
Eg. Carrot,
Monocots, some
conifers
Remove
Auxin
Polyamine
Inter-convesions
Putrescine
to Spermidine
Spermidine
to Spermine
Secondary embryo formation - Mostly in dicots
Abundant
Secondary
Embryos
+ Cytokinin
Early embryo
+ Charcoal
+ ABA
- Cytokinin
o
The ability of non-meristematic plant tissues to form various organs de
novo
o
The production of roots, shoots or leaves
o
These organs may arise out of pre-existing meristems or out of
differentiated cells
o
This, like embryogenesis, may involve a callus intermediate but often
occurs without callus
Fig

o
Somatic embryogenesis and Organogenesis - Both of these
technologies can be used as methods of micro-propagation
o
Not always desirable because they may not always result in
populations of identical plants
o
The most beneficial use of somatic embryogenesis and organogenesis
is in the production of whole plants from a single cell or a few cells
Tissue Culture Applications:
•
Micropropagation
•
Germplasm preservation
•
Somaclonal variation
Tissue culture of Lilium speciosum (source: Chang et al., 2000)
Regeneration in Callus Cultures
Different stages of embryo formation
4- Stages of embryos
Callus Regeneration
• Synthetic/Artificial seeds
• Embryo culture
• Haploid and dihaploid production
• In vitro hybridization – protoplast fusion
• Industrial products from cell cultures
• Plant genetic engineering
•
Micropropagation:
 The art and science of plant multiplication in vitro
 Usually derived from meristems or vegetative buds) without a callus
stage
 Tends to reduce or eliminate somaclonal variation, resulting in true
clones
 Can be derived from other explant or callus but these are often
problematic
 Steps of Micropropagation are:
o Stage 0 i.e. selection and preparation of the mother plant sterilization of the plant tissue takes place
o
o
o
o
Stage I i.e. Initiation of culture - explant placed into growth media
Stage II i.e. Multiplication - explants transferred to shoot media;
shoots can be constantly divided
Stage III i.e. Rooting - explants transferred to root media
Stage IV i.e. Transfer to soil - explants returned to soil; hardened
off
Fig
 Features of Micropropagation:
o Clonal reproduction - Way of maintaining heterozygozity
o Multiplication Stage can be recycled many times to produce an
unlimited number of clones - Routinely used commercially for
many ornamental species, some vegetatively propagated crops
o Easy to manipulate production cycles - Not limited by field
seasons/environmental influences
o Disease-free plants can be produced - Has been used to eliminate
viruses from donor plants
Fig
• Germplasm Preservation:
 Two methods
In Vitro Clonal Propagation of Plants
Slow growth techniques;
i.
ii.
o
↓ Temp., ↓ Light, media supplements (osmotic inhibitors, growth
retardants), tissue dehydration
o
Medium-term storage (1 to 4 years)
Cryo-preservation;
o
Ultra low temperatures - liquid nitrogen (-196oC) in presence of
Cryoprotective agents like Glycerol, DMSO, PEG
o
Stops cell division and metabolic processes
o
Very long-term (indefinite)
• Somaclonal Variation:

A general phenomenon of all plant regeneration systems that involve a
callus phase

Two general types of Somaclonal Variation;
i. Heritable, genetic changes (alter the DNA)
ii. Stable, but non-heritable changes (alter gene expression,
epigenetic)

Done in cell suspension culture
o
Then apply physical or chemical mutagen and selection pressure to
culture
o
Regeneration of whole plants from surviving cells
Fig
o
It Important alternative of creation of additional genetic variability
in crops where tissue culture and regeneration system has been
established
- Somaclonal mutants can be enriched during in vitro culture
include
- Resistance to disease and herbicides
- Tolerance to environmental stress and chemical stress
- Increased seedling vigor in lettuce
- Joint less pedicels in tomato
- Increase production of secondary metabolites
- New varieties have been developed through somaclonal
variation in tomato, sugar cane, celery, Brassica, sorghum
•
Synthetic / Artificial Seeds;

Synthetic or artificial seeds are somatic embryos engineered for use in
the commercial propagation of plants
 These techniques have been further developed for the production of
plants from embryos developed by non-sexual methods (haploid
production discussed later)
 Very useful technology where
o
True seeds are not used or readily available for multiplication (e.g.
potato)
o
hybrid plants (e.g. hybrid rice)
o
Vegetatively propagated plants are more prone to infections (e.g.
day lily, garlic, potato, sugarcane, sweet potato, grape and mango).
 Synthetic seeds are also useful for multiplication of
o
Genetically engineered plants (transgenic plants)
o
Somatic and cytoplasmic hybrids (obtained through protoplast
fusion techniques)
o
Sterile and unstable genotypes
o
preservation of desirable elite genotypes
• Embryo Culture;
 Embryo culture developed from the need to rescue embryos (embryo
rescue) from wide crosses where fertilization occurred, but embryo
development did not occur
Somatic embryos encapsulated in gel

These techniques have been further developed for the production of
plants from embryos developed by non-sexual methods (haploid
production discussed later)
 It is use to
o
rescue F1 hybrid from a wide cross
o
overcome seed dormancy, usually with addition of hormone to
media (GA)
o
overcome immaturity in seed
o
speed generations in a breeding program
o
rescue a cross or self (valuable genotype) from dead or dying plant
Fig
•
Haploid Plant Production;
 Anther culture/Microspore culture - culturing of anthers or pollen
grains (microspores) - derive a mature plant from a single microspore
Fig
 Ovule culture - culturing of unfertilized ovules (macrospores)
o
Effective for crops that do not yet have an efficient microspore
culture system. For example Melon, onion
Anther/Microspore Culture
Anther Culture
 What do you do with the haploid?
o
Weak, sterile plant
o
Usually want to double the chromosomes, creating a di-haploid
plant with normal growth and fertility
o
Chromosomes can be doubled by
a.
Colchicine treatment
b.
Spontaneous doubling
a.
Tends to occur in all haploids at varying levels
b.
Many systems rely on it, using visual observation to detect
spontaneous di-haploids
c.
Can be confirmed using flow cytometry
• Protoplast Fusion;
Figs
 It is used to
o
Combine two complete genomes - another way to create
allopolyploids
, Electrofusion, Ca
ions
o
Partial genome transfer - exchange single or few traits between
species which may or may not require ionizing radiation
o
Genetic
engineering
Agrobacterium
o
Transfer of organelles - unique to protoplast fusion i.e. the transfer
of mitochondria and/or chloroplasts between species
-
micro-injection,
electroporation,
Fig
o
•
Examples are;
-
Protoplast fusion between male sterile cabbage and
normal cabbage was done, and cybrids were selected that
contained the radish mitochondria and the cabbage
chloroplast
-
Current procedure is to irradiate the cytoplasmic donor to
eliminate nuclear DNA – routinely used in the industry to
re-create male sterile brassica crops
Industrial Products;
 Secondary metabolites produced by plants - alkaloids, terpenoids,
steroids, anthocyanins, anthraquinones, polyphenols
 Often restricted production - specific species, tissue or organ
• Plant Genetic Engineering
Possible Result of Fusion of Two Genetically Different Protoplasts
= chloroplast
= mitochondria
Fusion
= nucleus
heterokaryon
cybrid
hybrid
hybrid
cybrid
 Over All Applications:
• Plant tissue culture is used widely in the plant sciences, forestry, and in
horticulture. Applications include:
• The commercial production of plants used as potting, landscape, and florist
subjects, which uses meristem and shoot culture to produce large numbers of
identical individuals
• To conserve rare or endangered plant species
• A plant breeder may use tissue culture 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 protoplasm fusion and regeneration of
the novel hybrid
• To rapidly study the molecular basis for physiological, biochemical, and
reproductive mechanisms in plants, for example in vitro selection for stress
tolerant plants, and in vitro flowering studies.
• To cross-pollinate distantly related species and then tissue culture the
resulting embryo which would otherwise normally die (Embryo Rescue)
• For chromosome doubling and induction of polypoidy. For example
doubled haploids, tetraploids, and other forms of polypoloids. This is
usually achieved by application of antimitotic agents such
as colchicine or oryzalin
• As a tissue for transformation, followed by either short-term testing of
genetic constructs or regeneration of transgenic plants
• Certain techniques such as meristem tip culture can be used to produce
clean plant material from viruses stock, such as potatoes and many
species of soft fruit
• Production of identical sterile hybrid species can be obtained
 Cryopreservation Requirements:
• Pre-culturing
 Usually a rapid growth rate to create cells with small vacuoles and low
water content
• Cryo-protection
 Glycerol, DMSO, PEG, to protect against ice damage and alter the
form of ice crystals
• Freezing
 The most critical phase - one of two methods:
- Slow freezing allows for cytoplasmic dehydration
- Quick freezing results in fast intercellular freezing with little dehydration
• Storage
 Usually in liquid nitrogen (-196oC) to avoid changes in ice crystals that
occur above -100oC
• Thawing
 Usually rapid thawing to avoid damage from ice crystal growth
• Recovery
 Thawed cells must be washed of cryo-protectants and nursed back to
normal growth
 Avoid callus production to maintain genetic stability
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