Download Basis for Plant Tissue Culture

PLANT
BIOTECHNOLOGY
PLANT BIOTECHNOLOGY
 Manipulating plants for the benefit of mankind
 A process to produce a genetically modified plant
by removing genetic information from an
organism, manipulating it in the laboratory and
then transferring it into a plant to change certain
of its characteristics
Improved food crops
Higher yields
Improved nutrition
Environmental tolerances
Improved production of valuable molecules
Production of novel molecules
 It chiefly involves the introduction of foreign
genes into
economically important plant species, resulting in
crop improvement and the production of novel
products in plants.
 Today, biotechnology is being used
 as a tool to give plants new traits that benefit
agricultural production, the environment, and
human nutrition and health.
 The goal of plant breeding is to combine desirable
traits from different varieties of plants to produce
plants of superior quality.
 This approach to improving crop production has
been very successful over the years.
 Plant Biotechnology is a rapidly expanding field
within Biotechnology.
 Today plant biotechnology encompasses two major
areas, plant tissues culture and plant genetic
engineering (Transformation).
Transgenic Plants:
 A transgenic crop plant contains a gene or genes
which have been artificially inserted instead of the
plant acquiring them through pollination.
 The inserted gene sequence (known as the
transgene) may come from another unrelated
plant, or from a completely different species
Tissue culture
Tissue culture is the term
used for “the process of
growing cells artificially in
the laboratory”.
It involves both plant and animal
cells.
Tissue culture produces clones
from the explant. (In clones all
the produced plants have the
same genotype).
Tissue culture of every living cell of
every plant is possible.
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pathogen-derived genes
Exogenous genes
(non-plant genes)
bacterial genes
any other organism
Pathogen resistance
Applications:
Herbicide resistance
 transgenic
bioreactors
Delivery systems
Gottlieb Haberlandt (1902) - Who grew palisade cells from
leaves of various plants. (Father of Tissue Culture)
1932 - White , Developed tissue culture medium for in vitro
culturing of meristematic cells of tomato [medium containing
salts, yeast extract and sucrose and 3 vit B (pyridoxine,
thiamine, nicotinic acid)].
1957 Miller and Skoog, proposed that root-shoot differentiation
is regulated by auxin – cytokinin ratio.
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• 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.
A more recent advance is the use of plant and animal
tissue culture along with genetic modification using viral
and bacterial vectors and gene guns to create
genetically engineered organisms.
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Totipotency:
It is the ability of a single plant cell, (with protoplasm) to divide and to
produce all the differentiated cells in an organism.
Medium:
The basic nutritive material on which explant is placed in laboratories for
callus or organ formation is called medium . It can be in liquid or solid.
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Explant:
•Cell, tissue or organ of a plant
that is used to start in vitro
cultures.( in vitro = in + glass)
•Any part of the plant can be
used as explants for micro
propagation, but axillary buds
and meristems are most
commonly used.
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Callus:
A. Is a natural response of the plant tissue to wounding.
B. A unorganised mass of actively dividing undifferentiated cells
produced by plant tissue explant.
Usually a explant with morphologically uniform cells produces a
uniform type of callus and explant with variety of cell types
produces a mixed callus.
Callus cultures are transferred to fresh medium at 4 to 6 weeks
interval.
Failure of transfer to fresh medium leads to death of callus due
to exhaustion of nutrient.
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Organoganesis: ( organ + genesis- birth)
The process of initiation and development of root,
stem & leaf (but not embryo) in plant tissue culture.
Starts due to presence of chemicals in the medium
especially growth hormones.
Embryogenesis: (embryo + genesis-birth)
The process of initiation and development of embryo
or embryo like structure from somatic cells in tissue
culture.( somatic embryogenesis).
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Stem cells:
undifferentiated cells that are able to differentiate into
more than one cell type.
Stem cells can divide to produce differentiated type of
cells and also retain the ability to divide to maintain
the stem cell population.
Pluripotent:
The most versatile stem cells that have the ability to give
rise to all tissues of the body are also called
pluripotent stem cells.
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Micropropagation:
(Murishige 1974) Also known as clonal propagation
in vitro culture
It is nothing but multiplication of genetically
identical copies of plant by asexual or vegetative
method.
As minute sized propagules are used in culture so is
named as micropropagation.
It is beneficial as it produces large number of plants
in short period.
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1. Tissue culture laboratory:
requires all the nutrient and physicochemical factor mention in all
laboratory.
Good laboratory must have:
 Nutrient medium preparation, sterilization, cleaning and storage
of supply
 Aseptic condition for working the living material
 A control environmental condition for the growth and
development of culture
 Observation and evaluation of the culture as hope.
 Recording the observation during the experiment.
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2. Nutrient Media Composition and Preparation
 Vital activity of a cell as the absorption of nutrient through cell
membrane and rapid proliferation into innumerable cell.
 Nutrient medium contains inorganic salts, sugar, vitamins,
growth hormones and a few amino acids.
a. INORGANIC:
 It included N, P, K, Ca, Mg, S.
Microelements:
 B, Mo, Cu, Zn, Mn, Fe, Cl
b. Growth Hormones:
 It will stimulate the biological activities in culture materials.
c. Organic Constituents:
2. Nutrient Media Composition and Preparation
e. Amino acid:
Serve as a source of nitrogen. Most commonly use aminoacid are
L-aspartic acid, L-Aspragin, L-glutanic acid, L-glutamine, Largine.
f. Solidifying agent:
Most commonly ager
Glidium amansii.
(0btain from seaweed) i.e. red alge,
It do not react with constituent of media and not digested by
enzyme. Generally from 0.5 to 1%.
g. pH Effect:
Affect the uptake of ions, optimum 5-6 is required for
development of culture tissue. pH should be mention before
sterilization of media.
3. Maintenance of Aseptic Environment
a. Sterilization of glass waves (160-180C, 2-4 hours)
b. Sterilization of Instruments:
The metallic instruments will be claim sterilize (dipping
them in 75% ethanol followed by flaming and cooling. It
is called incineration. Sterilization of culture room and
transfer area.
c. Sterilization of Nutrient media (15 psi) 121C for 20
minutes.
 Vitamins, plant extract, amino acid and hormone are
denatured by auto calving, they should be sterilized by
0.2 um filter paper.
The basic step required for regeneration of a whole plant
from plant cell tissue or organ culture:
 Preparation suitable nutrient medium (per objective of
the culture and transferred into a suitable container and
then sterilized)
 Selection of ex-plant: Always young and healthy part of
the plant are selected as an ex-plant.
 Sterilization of ex-plant
 Inoculation (under aseptic condition)
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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
Factors Affecting Plant Tissue Culture
 Growth Media
 Minerals, Growth factors, Carbon source
 Environmental Factors
 Light, Temperature, Photoperiod
 Explant Source
 Types
Usually, the younger, less differentiated the explant, the better for tissue
culture
 Genetics
1. Different species show differences in amenability to tissue culture
2. In many cases, different genotypes within a species will have variable
responses to tissue culture; response to somatic embryogenesis has
been transferred between melon cultivars through sexual
hybridization
Choice of explant
Desirable properties of
an explant
 Easily sterilisable
 Juvenile
 Responsive to culture
 Shoot tips
 Axillary buds
 Seeds
 Hypocotyl (from germinated
seed)
 Leaves
Medium Constituents
 Inorganic salt formulations
 Source of carbohydrate
 Vitamins
 Water
 Plant hormones - auxins, cytokinins, GA’s
 Solidifying agents
 Undefined supplements
Carbohydrates
 Plants in culture usually cannot meet their needs
for fixed carbon. Usually added as sucrose at 2-3%
w/v.
 Glucose or a mixture of glucose and fructose is
occasionally used.
 For large scale cultures, cheaper sources of sugars
(corn syrup) may be used.
Inorganic Salt
Formulations
 Contain a wide range of Macro-elements (>mg/l)
and microelements (<mg/l).
 A wide range of media are readily available as
spray-dried powders.
 Murashige and Skoog Medium (1965) is the most
popular for shoot cultures.
 Gamborgs B5 medium is widely used for cell
suspension cultures (no ammonium).
Vitamins
 A wide range of vitamins are available and may be
used.
 Generally, the smaller the explant, the more exacting
the vitamin requirement.
 A vitamin cocktail is often used (Nicotinic acid,
glycine, Thiamine, pyridoxine).
 Inositol usually has to be supplied at much higher
concentration (100mg/l)
Plant hormones (Growth regulators)
 Auxins
 Cytokinins
 Gibberellic acids
 Ethylene
 Abscisic Acid
 “Plant Growth Regulator-like compounds”
Auxins
Absolutely essential.
Only one compound, Indole-3-acetic acid. Many
synthetic analogues (NAA, IBA, 2,4-D, 2,4,5-T,
Pichloram) - cheaper & more stable
Generally growth stimulatory. Promote rooting.
Produced in meristems, especially shoot meristem
and transported through the plant in special cells
in vascular bundles.
Cytokinins
 Absolutely essential.
 Single natural compound, Zeatin.
 Synthetic analogues Benyzladenine (BA), Kinetin.
 Stimulate cell division (with auxins).
 Promotes formation of adventitious shoots.
 Produced in the root meristem and transported throughout
the plant as the Zeatin-riboside in the phloem.
Gibberellins (GA’s)
 A family of over 70 related compounds, all forms of
Gibberellic acid.
 Commercially, GA3 and GA4+9 available.
 Stimulate etiolation of stems.
 Help break bud and seed dormancy.
 Produced in young leaves.
Abscisic Acid (ABA)
 Promotes leaf abscission and seed dormancy.
 Plays a dominant role in closing stomata in response
to water stress.
 Has an important role in embryogenesis in preparing
embryos for desiccation.
 Helps ensure ‘normal’ embryos.
‘Plant Growth Regulator-like
substances’
 Polyamines - have a vital role in embryo
development.
 Jasmonic acid - involved in plant wound responses.
 Salicylic acid.
 Not universally acclaimed as plant hormones since
they are usually needed at high concentrations.
Undefined Supplements
 Sources of hormones, vitamins and polyamines.
 e.g. Coconut water, sweetcorn extracts
 Not reproducible