Transcript 식물조직배양 및 형질전환실험

Plant Tissue Culture &
Genetic Transformation
(식물조직배양 및 형질전환실험)
What is Plant Tissue Culture?
Plant tissue culture is a practice used to propagate plants under sterile
conditions, often to produce clones of a plant. Different techniques in plant
tissue culture may offer certain advantages over traditional methods of
propagation, including:
-The production of exact copies of plants that produce particularly good 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 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.
-To clean particular plant 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, or (less commonly) roots can often be used to
generate a new plant on culture media given the required nutrients and plant
What is Plant Tissue Culture?
Plant cells can be grown in isolation from intact plants in tissue culture
systems. The cells have the characteristics of callus cells, rather than other
plant cell types. These are the cells that appear on cut surfaces when a plant
is wounded and which gradually cover and seal the damaged area.
Pieces of plant tissue will slowly divide and grow into a colourless mass of
cells if they are kept in special conditions. These are:
- initiated from the most appropriate plant tissue for the particular plant variety
-presence of a high concentration of auxin and cytokinin growth regulators in
the growth media
- a growth medium containing organic and inorganic compounds to sustain
the cells aseptic conditions during culture to exclude competition from
microorganisms
callus
Shoot formation
Plant formation
Initiating plantlets
Regenerated African violets
Plantlets are young or small plant s used as propagules.
a propagule is any plant material used for the purpose of plant propagation .
What is Plant Tissue Culture?
The plant cells can grow on a solid surface as friable, pale-brown lumps (called
callus), or as individual or small clusters of cells in a liquid medium called a
suspension culture. These cells can be maintained indefinitely provided they
are sub-cultured regularly into fresh growth medium.
Tissue culture cells generally lack the distinctive features of most plant cells.
They have a small vacuole, lack chloroplasts and photosynthetic pathways and
the structural or chemical features that distinguish so many cell types within the
intact plant are absent. They are most similar to the undifferentiated cells found
in meristematic regions which become fated to develop into each cell type as
the plant grows. Tissue cultured cells can also be induced to re-differentiate
into whole plants by alterations to the growth media.
Plant tissue cultures can be initiated from almost any part of a plant.
Tunica-Corpus model of the apical meristem
(growing tip). The epidermal (L1) and
subepidermal (L2) layers form the outer layers
called the tunica. The inner L3 layer is called the
corpus. Cells in the L1 and L2 layers divide in a
sideways fashion which keeps these layers
distinct, while the L3 layer divides in a more
random fashion.
10x microscope image of root tip with meristem
1 - quiescent center
2 - calyptrogen (live rootcap cells)
3 - rootcap
4 - sloughed off dead rootcap cells
5 - procambium
Media - Plants in nature can synthesize their own food material. In contrast,
plants growing in vitro are heterotrophic, Le., they cannot synthesize
their own food material. Plant tissue culture media therefore require all
essential minerals plus a carbohydrate source usually added in the form
of sucrose and also other growth hormones (regulators and vitamins).
Growth and morphogenesis of plant tissues in vitro are largely governed
by the composition of the culture media. Although the basic
requirements of cultured plant tissues are similar to those of whole
plants, in practice nutritional components promoting optimal growth of a
tissue under laboratory conditions may vary with respect to the
particular species. Media compositions are thus formulated considering
specific requirements of a particular culture system. 'For example, some
tissues show better response on a solid medium while others prefer a
liquid medium.
Considerable progress has been made during the past two decades on the
development of media for growing plant cells, tissues and organs
aseptically in culture. A significant contribution to formulation of a defined
growth medium suitable for a wide range of applications was made by
Murashige and Skoog (1962), In their work to adapt tobacco callus
cultures for use as a hormone bioassay system, they evaluated many
medium constituents to achieve optimal growth of calluses. In so doing,
they improved upon existing types of plant tissue culture media to such an
extent that their medium (the MS medium) has since proved to be one of
the most widely used in plant tissue culture work.
A Heterotroph (Greek
ἕτερος heteros = another
and τροφή trophe =
nutrition) is an organism
that uses organic carbon
for growth.[1] This
contrasts with autotrophs,
such as plants, which are
able to directly use
sources of energy, such
as light to produce
organic substrates from
inorganic carbon dioxide
Plant tissue
Whole plant
Plant hormones (also known as phytohormones)
Plant hormones are chemicals that regulate plant growth. In the UK,
the term 'hormone' is not acceptable. There they are called 'plant
growth substances'. Plant hormones are signal molecules produced
within the plant, and occur in extremely low concentrations.
Hormones regulate cellular processes in targeted cells locally and
when moved to other locations, in other locations of the plant.
Hormones also determine the formation of flowers, stems, leaves, the
shedding of leaves, and the development and ripening of fruit. Plants,
unlike animals, lack glands that produce and secrete hormones. Plant
hormones shape the plant, affecting seed growth, time of flowering,
the sex of flowers, senescence of leaves and fruits. They affect which
tissues grow upward and which grow downward, leaf formation and
stem growth, fruit development and ripening, plant longevity and even
plant death. Hormones are vital to plant growth and lacking them,
plants would be mostly a mass of undifferentiated cells.
Abscisic acid
Abscisic acid also called ABA, was discovered and researched under two different
names before its chemical properties were fully known, it was called dormin and
abscicin II. Once it was determined that the two latter named compounds were the
same, it was named abscisic acid. The name "abscisic acid" was given because it was
found in high concentrations in newly-abscissed or freshly-fallen leaves.
This class of PGR is composed of one chemical compound normally produced in the
leaves of plants, originating from chloroplasts, especially when plants are under stress.
In general, it acts as an inhibitory chemical compound that affects bud growth, seed
and bud dormancy. It mediates changes within the apical meristem causing bud
dormancy and the alteration of the last set of leaves into protective bud covers. Since it
was found in freshly-abscissed leaves, it was thought to play a role in the processes of
natural leaf drop but further research has disproven this. In plant species from
temperate parts of the world it plays a role in leaf and seed dormancy by inhibiting
growth, but, as it is dissipated from seeds or buds, growth begins. In other plants, as
ABA levels decrease, growth then commences as gibberellin levels increase. Without
ABA, buds and seeds would start to grow during warm periods in winter and be killed
when it froze again. Since ABA dissipates slowly from the tissues and its effects take
time to be offset by other plant hormones, there is a delay in physiological pathways
that provide some protection from premature growth. It accumulates within seeds during
fruit maturation, preventing seed germination within the fruit, or seed germination before
winter. Abscisic acid's effects are degraded within plant tissues during cold
temperatures or by its removal by water washing in out of the tissues, releasing the
seeds and buds from dormancy.[5]
In plants under water stress ABA plays a role in closing the stomata. Soon after
plants are water stressed and the roots are deficient in water, a signal moves up
to the leaves causing the formation of ABA precursors there which then move to
the roots. The roots then release ABA which is translocated to the foliage through
the vascular system[6] and modulates the potassium and sodium uptake within
the guard cells, which then lose turgidity, closing the stomata.[7][8] ABA exists
in all parts of the plant and its concentration within any tissue seems to mediate
its effects and function as a hormone, its degradation or more properly
catabolism within the plant affects metabolic reactions and cellular growth and
production of other hormones.[9] Plants start life as a seed with high ABA levels,
just before the seed germinates ABA levels decrease; during germination and
early growth of the seedling, ABA levels decrease even more. As plants begin to
produce shoots with fully functional leaves - ABA levels begin to increase,
slowing down cellular growth in more "mature" areas of the plant. Stress from
water or predation affects ABA production and catabolism rates which mediate
another cascade of effects triggering specific responses from targeted cells.
Scientists are still piecing together the complex interactions and effects of this
and other phytohormones.
Auxins
Auxins are compounds that positively influence cell enlargement, bud
formation and root initiation. They also promote the production of other
hormones and in conjunction with cytokinins, they control the growth of
stems, roots, fruits and convert stems into flowers.[10] Auxins were the first
class of growth regulators discovered.[11] They affect cell elongation by
altering cell wall plasticity. Auxins decrease in light and increase where its
dark. They stimulate cambium cells to divide and in stems cause secondary
xylem to differentiate. Auxins act to inhibit the growth of buds lower down
the stems (apical dominance), and also to promote lateral and adventitious
root development and growth. Leaf abscission is initiated by the growing
point of a plant ceasing to produce auxins. Auxins in seeds regulate specific
protein synthesis,[12] as they develop within the flower after pollination,
causing the flower to develop a fruit to contain the developing seeds. Auxins
are toxic to plants in large concentrations; they are most toxic to dicots and
less so to monocots. Because of this property, synthetic auxin herbicides
including 2,4-D and 2,4,5-T have been developed and used for weed
control. Auxins, especially 1-Naphthaleneacetic acid (NAA) and Indole-3butyric acid (IBA), are also commonly applied to stimulate root growth when
taking cuttings of plants. The most common auxin found in plants is
indoleacetic acid or IAA.
Lack of the plant hormone auxin can
cause abnormal growth (right)
Cytokinins
Cytokinins or CKs are a group of chemicals that influence cell division and
shoot formation. They were called kinins in the past when the first cytokinins
were isolated from yeast cells. They also help delay senescence or the aging
of tissues, are responsible for mediating auxin transport throughout the plant,
and affect internodal length and leaf growth. They have a highly-synergistic
effect in concert with auxins and the ratios of these two groups of plant
hormones affect most major growth periods during a plant's lifetime.
Cytokinins counter the apical dominance induced by auxins; they in
conjunction with ethylene promote abscission of leaves, flower parts and
fruits.[13]
Cytokinins
Cytokinins (CK) are a class of plant growth substances (plant hormones)
that promote cell division. They are primarily involved in cell growth,
differentiation, and other physiological processes. Their effects were first
discovered through the use of coconut milk in the 1940s by a scientist at
the University of Wisconsin-Madison named Folke Skoog. [1]
There are two types of cytokinins: adenine-type cytokinins represented
by kinetin, zeatin and 6-benzylaminopurine, as well as phenylurea-type
cytokinins like or (TDZ). The adenine-type cytokinins are synthesised in
stems, leaves and roots, which is the major site.[citation needed]
Cambium and possibly other actively dividing tissues are also sites of
cytokinin biosynthesis.[2] There is no evidence that the phenylurea
cytokinins occur naturally in plant tissues.[3] Cytokinins are involved in
both local and long distance signalling, the latter of which involves the
same in planta transport mechanism as used for transport of purines and
nucleosides.[4]
The cytokinin zeatin , Zea,
in which it was first
discovered in immature
kernels.
Gibberellins
Gibberellins or GAs include a large range of chemicals that are produced
naturally within plants and by fungi. They were first discovered when
Japanese researchers, including Eiichi Kurosawa, noticed a chemical
produced by a fungus called Gibberella fujikuroi that produced abnormal
growth in rice plants.[15] Gibberellins are important in seed germination,
affecting enzyme production which mobilizes food production used for
growth of new cells. This is done by modulating chromosomal transcription.
In grain (rice, wheat, corn, etc.) seeds, a layer of cells called the aleurone
layer wraps around the endosperm tissue. Absoption of water by the seed
causes production of GA. The GA is transported to the aleurone layer, which
responds by producing enzymes that break down stored food reserves within
the endosperm, which are utilized by the growing seedling. GAs produce
bolting of rosette-forming plants, increasing internodal length. They promote
flowering, cellular division, and in seeds growth after germination.
Gibberellins also reverse the inhibition of shoot growth and dormancy
induced by ABA.[16]
What is Plant Tissue Culture?
•
The physiological state of the plant does have an influence on its
response to attempts to initiate tissue culture. The parent plant must be
healthy and free from obvious signs of disease or decay. The source,
termed explant, may be dictated by the reason for carrying out the
tissue culture. Younger tissue contains a higher proportion of actively
dividing cells and is more responsive to a callus initiation programme.
The plants themselves must be actively growing, and not about to enter
a period of dormancy.
•
The exact conditions required to initiate and sustain plant cells in
culture, or to regenerate intact plants from cultured cells, are different
for each plant species. Each variety of a species will often have a
particular set of cultural requirements. Despite all the knowledge that
has been obtained about plant tissue culture during the twentieth
century, these conditions have to be identified for each variety through
experimentation.
Plant tissue culture facility
Clean bench
Shelving unit
Autoclave
petridish
SI-1 Media
SI-1 Media
SI-2 Media
SE-1 Media
selfing
1 : inbreed
2 : to pollinate with pollen from the same
flower or plant
Somaclonal variation
• It is the term used to describe the variation seen in plants
that have been produced by plant tissue culture.
Chromosomal rearrangements are an important source of
this variation.
• Somaclonal variation is not restricted to, but is particularly
common in plants regenerated from callus. The variations
can be genotypic or phenotypic, which in the later case
can be either genetic or epigenetic in origin. Typical
genetic alterations are: changes in chromosome numbers
(polyploidy and aneuploidy), chromosome structure
(translocations, deletions, insertions and duplications) and
DNA sequence (base mutations). Typical epigenetic
related events are: gene amplification and gene
methylation.
Predigestion
digestion
Isolation of protoplast
A protoplast is a plant, bacterial or fungal cell that had its cell wall completely
or partially removed using either mechanical or enzymatic means.
Protoplasts - Have their cell wall entirely removed
Agrobacterium
• Agrobacterium is a genus of Gram-negative
bacteria that uses horizontal gene transfer to
cause tumors in plants. Agrobacterium
tumefaciens is the most commonly studied
species in this genus. Agrobacterium is well
known for its ability to transfer DNA between
itself and plants, and for this reason it has
become an important tool for plant
improvement by genetic engineering.
• The ability of Agrobacterium to transfer genes to plants and fungi is
used in biotechnology, in particular, genetic engineering for plant
improvement. A modified Ti or Ri plasmid can be used. The plasmid
is 'disarmed' by deletion of the tumor inducing genes; the only
essential parts of the T-DNA are its two small (25 base pair) border
repeats, at least one of which is needed for plant transformation.
Marc Van Montagu and Jozef Schell at the University of Ghent
(Belgium) discovered the gene transfer mechanism between
Agrobacterium and plants, which resulted in the development of
methods to alter Agrobacterium into an efficient delivery system for
gene engineering in plants[5][6]. A team of researchers led by Dr
Mary-Dell Chilton were the first to demonstrate that the virulence
genes could be removed without adversely affecting the ability of
Agrobacterium to insert its own DNA into the plant genome (1983).
• The genes to be introduced into the plant are cloned into a plant
transformation vector that contains the T-DNA region of the
disarmed plasmid, together with a selectable marker (such as
antibiotic resistance) to enable selection for plants that have been
successfully transformed. Plants are grown on media containing
antibiotic following transformation, and those that do not have the
T-DNA integrated into their genome will die.
• Plant (S. chacoense) transformed using Agrobacterium. Transformed
cells start forming calluses on the side the leaf pieces
• Transformation with Agrobacterium can be achieved in two ways.
Protoplasts, or leaf-discs can be incubated with the Agrobacterium
and whole plants regenerated using plant tissue culture. A common
transformation protocol for Arabidopsis is the floral-dip method: the
flowers are dipped in an Agrobacterium culture, and the bacterium
transforms the germline cells that make the female gametes. The
seeds can then be screened for antibiotic resistance (or another
marker of interest), and plants that have not integrated the plasmid
DNA will die.
• Agrobacterium does not infect all plant species, but there are several
other effective techniques for plant transformation including the
gene gun.
• Agrobacterium is listed as being the original source of genetic
material that was transferred to these USA GMO foods [7]:
–
–
–
–
–
–
–
–
Soybean
Cotton
Corn
Sugar Beet
Alfalfa
Wheat
Rapeseed Oil (Canola)
Creeping bentgrass (for animal feed)
Agrobacterium-mediated gene transfer
Agrobacteria
Plant cell
http://www.ppws.vt.edu/~sforza/prokaryote.html
A. tumefaciens exhibits polar
attachment to the plant cell.
The production of cellulose fibrils
serve to anchor the bacteria to
the plant
as well as trap other
bacteria. Once the
concentration of lignin
precursors
reaches approximately 10-5 M,
the virulence genes of the Ti
plasmid are induced
and the T-DNA is processed
The actual mechanism of T-DNA
transfer is difficult to animate.
The model at left
lacks the double stranded
intermediate of the T-DNA,
but shows in an approximate
manner how the T-DNA
moves into the plant cell and is
passed
through the nuclear pores.
http://www.ppws.vt.edu/~sforza/prokaryote.html
Gene gun PDS1000
Gene gun accessory
The gene gun or the Biolistic Particle Delivery
System, originally designed for plant
transformation, is a device for injecting cells with
genetic information . The payload is an elemental
particle of a heavy metal coated with plasmid DNA.
This technique is often simply referred to as
biolistics. Another instrument that uses biolistics
technology is the PSD-1000/He particle delivery
system This device is able to transform almost any
type of cell, including plants, and is not limited to
genetic material of the nucleus: it can also
transform organelles, including plastids.
The Helios Gene Gun by BioRad
The Helios Gene Gun is a new way for in vivo transformation of cells
or organisms (i.e. gene therapy and genetic immunization (DNA
vaccination)). This gun uses Biolistic ® particle bombardment where
DNA- or RNA-coated gold particles are loaded into the gun and you
pull the trigger. A low pressure helium pulse delivers the coated gold
particles into virtually any target cell or tissue. The particles carry the
DNA so that you do not have to remove cells from tissue in order to
transform the cells. (Go to BioRad and search for "Helios" to learn
more.)
Gene gun (유전자총I) PDS1000
휴대용 유전자총 II (Helios Gene Gun by BioRad)
7 가지 크기의 금가루 사용: 0.4, 0.6, 0.8, 1.0, 1.2 and 6.0 micron
1 micron= 1/1,000,000 M
금가루를 이용하여 세포에 유전자 총 발사