Transcript and cultured.

Type of in vitro
culture
 Culture of intact plants (Seed orchid culture)
 Embryo culture (embryo rescue)
 Organ culture
1.
2.
3.
4.
shoot tip culture
Root culture
Leaf culture
anther culture
 Callus culture
 Cell suspension and single cell culture
 Protoplast culture
Seed culture
 Increasing efficiency of germination of seeds that are
difficult to germinate in vivo
 Precocious germination by application of plant growth
regulators
 Production of clean seedlings for explants or meristem
culture
Embryo rescue
 This is useful technique
in plant tissue culture
where embryo abortion is
reported at early stage
and seed setting is failed.
 Here immature embryo,
ovary or ovule is rescued
( separated ) and cultured.
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Endosperm culture
 As endosperm is triploid so are the
plantlets formed from it.
 This technique may be exploited as
an alternative to crossing tetraploid
and diploid parents for raising
triploids during plant improvement
programme.
 The triploid plants are self-sterile and
usually seedless.
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Organ culture
 Any plant organ can serve as an explant to initiate
cultures
Shoot apical meristem culture
 Culturing of apical meristem (the region of shoot apex laying
distal to leaf primordium)
 Also known as meristem culture, meristemming & mericlones.
 Extensively used in horticulture, agriculture and forestry.
 Production of virus free germplasm
 Mass production of desirable genotypes
 Facilitation of exchange between locations (production of
clean material)
 Cryopreservation (cold storage) or in vitro conservation of
germplasm
Root tip culture
 By using meristematic cells such as those that operate
in the root tip or bud.
 Conceived by Kotte (germany) and Robbins (USA) in
1922.
new leaf
tunica
apical
meristem
corpus
leaf trace
axillary
meristem
procambium
cortex
pith
Meristematic tissues ...
 Shoot ... apical,
… axillary
Meristematic tissues ...
 Shoot ... apical,
… axillary
Leaf
Root
Meristematic tissues ...
 Shoot ... apical,
… axillary
Leaf
Root
Adventitious
Cambial tissues
Anther and pollen grain culture
Haploid plants are derived from microspores
(pollen) cultured individually or in anthers
First report of Haploid plant from anther and pollen
culture is by eminent Indian embryologists Guha &
Maheshwari from the plant Datura stramonium.
Wheat, Rice, Maize, Rye, Tobacco, Potato, Brassicas has
been used in pollen & anther culture to release the
cultivars having superior chts. , high yield & disease
resistance.
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Ovary or ovule culture
 Production of haploid plants
 A common explant for the initiation of somatic
embryogenic cultures
 Overcoming abortion of embryos of wide hybrids at very
early stages of development due to incompatibility
barriers
 In vitro fertilization for the production of distant hybrids
avoiding style and stigmatic incompatibility that inhibits
pollen germination and pollen tube growth
Bajaj, Y.P.S. 1983. In D.A. Evans, W.R. Sharp, P.V. Ammirato, and Y. Yamada (eds.), Handbook of Plant Cell Culture. Volume 1.
Techniques for Propagation and Breeding. MacMillan, New York. p. 228-287.
Callus Cultures
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Callus is formed via 3 developmental stages:
1. Induction
2. Cell division
3. Differentiation
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Callus formation is governed by
The source of Explants
Nutritional composition of medium
And environmental factors
 The unique feature of callus is that the abnormal growth has
biological potential to develop normal root, shoot, and
embryo ultimately forming plant.
Need for Callus Cultures
 In some instances it is necessary to go through a
callus phase prior to regeneration via somatic
embryogenesis or organogenesis
 For generation of useful somaclonal variants
 As a source of protoplasts and suspension cultures
 For production of metabolites
 Used in in vitro selection
Cell suspension culture
 The technique of isolation and culturing a single
selected cell
 When callus pieces are agitated in a liquid medium,
they tend to break up.
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Uses:
Induction of somatic embryos and shoots
In vitro mutagenesis and selection of mutants
Genetic transformation studies
Production of secondary metabolites etc.
Applications of plant cell culture
Suspension culture
Embryogenesis
Protoplast
Secondary
metabolites
Mutagenesis
Cell
modification
Artificial seeds
Secondary
products
Mutant
Gene transfer
Cell fusion
Protoplast culture
Cell without cell wall is called protoplast.
For tissue culture application protoplast can be isolated
from almost any part of the plant but leaves are
preferentially used for this purpose as these are easy to
handle.
Technique involves following steps;
A. Sterilization of the explant by 2% sodium hypochloride
for about 30 minutes.
B. Peeling of the epidermal cells.
C. Digestion of cell wall by enzymes, macerozymes,
cellulase, hemicellulase and pectinase.
D. Washing of the released protoplasts.
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Protoplast fusion
The common methods employed for the fusion of the
released protoplasts is as:
A. Spontaneous fusion of protoplast.
B. Mechanical induction fusion.
C.NaNO3 induced fusion.
D. High p H and Ca++ induced fusion.
E. Poly Ethylene Glycol induced fusion (PEG).
F. Electro fusion.
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The most popular method of protoplast fusion is PEG
induced fusion.
 In this freshly isolated protoplasts from desired parents
are mixed in appropriate mixture.
 The mixture is treated with 28-50% PEG for 15-30
minutes to facilitate fusion.
 Protoplasts are subject to gradual wash with culture
medium.
 Fusion frequency can be increased by 50% if high Ca++
concentration is used at p H 9-10.
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Somatic embryogenesis
 The process of a single cell or group of cells initiating
the development pathways that leads to reproducible
regeneration of non zygotic embryo capable of
germination to form complete plant.
OR
 a process embryo initiation and development from
somatic cell (non zygotic cell)
Haploid - Gametic number of chromosomes, n which
may not be equivalent to x (x is one genomic complement)
Monoploid - haploid derived from a diploid, x
(one genomic
complement)
Polyhaploid - haploid from a polyploid (nx), prefix
indicates genome complement number, e.g. tobacco is a
dihaploid
Agricultural applications for haploids Rapid generation of homozygous
genotypes after chromosome doubling
Reduce time for variety development, e.g. 10 to 6
years or less
Homozygous recombinant line can be developed in
one generation instead of after numerous
backcross generations
Selection for recessive traits in recombinant lines is
more efficient since these are not masked by the
effects of dominant alleles
Processes Leading to Production of
Haploid Plants
Androgenesis – haploid plant derived from male gamete,
most common method in vitro
Parthenogenesis - from unfertilized egg
Apogamy - from other cells of the mega-gametophyte,
example
Chromosome elimination - chromosome elimination in
somatic cells, most common method used with plant
breeding