Plant Tissue Culture

TOPIC 5 (PART 2)
BY NALLAMMAI SINGARAM
Learning outcomes
1. To identify and exemplify the techniques of plant
tissue culture.
2. To describe different types of tissue culture
propagation and somaclonal variation.
Different types of Cultures
 Organ culture
 Shoots
 Roots
 Callus
 Cell suspension
 Protoplast
 Embryogenic culture and
 others
Shoot Tip and Meristem Culture
 Shoot tips (which contain the shoot apical
meristem) can be cultured in vitro, producing
clumps of shoots from either axillary or
adventitious buds.
 Useful for clonal propagation.
 Shoot meristem cultures are less genotype
dependent and more efficient (seedlings can be
used as donor material) – young, active cells are
very efficient for culture.
Shoot apical meristem culture
 Production of virus free material
 Mass production of desirable
genotypes
 Facilitate material exchange
between locations (production of
clean material)
 Cryopreservation (cold storage) or in
vitro conservation of germplasm
Meristem Culture
 Cultures can also be established directly from the
rapidly dividing cells of meristematic tissues or
embryos, or from haploid cells.
 Roots and shoots contain meristematic tissue,
which is the source of all dividing cells in the
elongating roots and stem.
 Root and shoot tips can be excised and cultured
directly on solid medium, and will give rise to new
organs that can be clonally propagated.
Root Culture
 Can be established in vitro from explants of the
root tip of either primary or lateral roots.
 Can be cultured on fairly simple media.
 Growth of roots in vitro is potentially unlimited, as
roots are indeterminate organs.
 But root cells are not widely used in plant
transformation studies. Why?
Root Culture
 Production of important
economical secondary metabolites
eg. medicinal compounds…etc.

 Study the physiology and

metabolism of roots, and primary
root determinate growth patterns
Root culture

Ref: http://www.rombio.eu/rbl1vol17/21%20Sobri.pdf
Callus Culture
 Conditions that maintain cells in an undifferentiated
state.
 Explants, on suitable culture media grow into an
undifferentiated mass known as a callus.
 Can be propagated indefinitely by subdivision.
 Usually maintained in the dark because light can induce
differentiation of callus cells.
 correct balance of plant hormones are required
(phytohormones) to maintain the cells in an
undifferentiated state.
 Calli derived from mature embryo of barley

Fig. 1A-F Shape of different culture stages. (A):3 week-old calli, (B):1 day-old initiated
shoots, (C): 3 day-old initiated shoots, (D): 6 day-old initiated shoots, (E): 10 day-old
initiated shoots, and (F): plantlet

Ref: http://www.cropscience.org.au/icsc2004/poster/3/2/1/554_seong.htm
 Callus Types
Depending on the species and culture conditions, callus tissue can become
 Friable callus  Compact callus
• soft and easily breakable
• “hard and compact”
• loosely associated with each
other • difficult to form cell
• can be transferred into liquid suspension
medium, acts as inoculum and
form cell suspension, • Easy to subculture
• the cell mass breaks up to give • Necrosis might take place in
isolated cells, small clusters of the middle
cells, and larger aggregates
based on agitation .
Cell-Suspension Cultures
 Formed using friable callus in liquid medium
 Cells divide and the biomass of the culture
increases until nutrients in the medium are
exhausted and/or toxic byproducts build up to
inhibitory levels – called the stationary phase.
 Cells should be transferred as they enter the
stationary phase, otherwise, they will die.
Figure 2. Details of cocoa cell suspension culture for BIOA variety on liquid DKW
medium supplemented with vitamins, zeatine, and antioxidants. A. Cocoa cells in
exponential phase. B. Cell suspension cultures of four weeks after establishment.
Brown friable callus is obtained approximately at sixth months.
Ref: http://www.scielo.org.co/scielo.php?script=sci_arttext&pid=S0304-
35842008000200001
Protoplasts Culture
 are plant cells with the cell wall
removed, naked cells
 can be produced from suspension
cultures, callus tissue, or intact tissues,
o e.g. leaf mesophyll cells, by mechanical
disruption or, preferably, by treatment
with cellulolytic and pectinolytic enzymes.
 Two general approaches to removing
the cell wall: mechanical or enzymatic
isolation.
Enzymatic isolation of protoplasts
 Carried out in a simple salt solution with a high osmoticum, plus
the cell-wall-degrading enzymes (mix of both cellulase and
pectinase).
 Pectinase is necessary to break up cell aggregates into individual
cells and the cellulase digests away the cell wall.
 After enzyme treatment, protoplast suspensions are collected by
centrifugation, washed in medium without the enzyme, and
separated from intact cells and cell debris by flotation on a
cushion of sucrose.
 When plated onto nutrient medium, protoplasts will synthesize
new cell walls within 5-10 days and then initiate cell division.
Embryo Culture
 Embryos contain rapidly proliferating cells that
can be used as explants to generate callus
cultures or somatic embryos.
 Both immature and mature embryos can be
used as explants.
 The most widely applied strategy to
regenerate cereals and other
monocotyledonous plants is to use immature,
embryo-derived embryogenic callus.
Embryo culture
 Growing embryo aseptically on artificial nutrient
media.
 Developed from the need to rescue embryos (embryo
rescue) from wide crosses where fertilization occurred,
but embryo development did not occur.
 Aslo developed for the production of plants from
embryos developed by non-sexual methods (haploid
production).
 Overcoming embryo abortion due to incompatibility
barriers.
 Overcoming seed dormancy and self-sterility of seeds.
 Shortening of breeding cycle.
Anther and Microspore Culture
 culture source is the male gametophyte, or
microspores found in pollen grains.
 Haploid tissue can be cultured in vitro by using
pollen or anthers as an explant.
 Both callus and embryos can be produced from
pollen.
 Two main approaches can be taken to produce
cultures in vitro from haploid tissue.
Anther and Microspore Culture
METHOD 1 METHOD 2

 using the anther as the explant.  anthers can also be cultured in

liquid medium, and
 During embryogenesis, the
haploid tissue may undergo  pollen released from the anthers
spontaneous or induced can be induced to form embryos,
chromosome doubling, resulting although the efficiency of plant
in so-called di-haploid plants regeneration is often very low.
containing two copies of the same
haploid genome.  Immature pollen can also be
extracted from developing anthers
 Pollen-derived embryos are and cultured directly, although
subsequently produced via this is a very time-consuming
dehiscence of the mature anthers process.
at the correct stage.
Anther and microspore culture
 Production of haploid plants
 Production of homozygous
diploid lines through chromosome
doubling, thus reducing the time
required to produce inbred lines
 Uncovering mutations or recessive
phenotypes
Anther and Microspore Culture
 Some of the embryos produced from anther culture
may originate from the somatic anther tissue rather
than the haploid microspore cells.
 Using isolated pollen prevents mixed-embryo
formation, but the efficiency is low and the process
is time-consuming.
 Haploid tissue cultures can also be initiated from
the female gametophyte (the ovule) but rarely
studied.
Organogenesis and Somatic Embryogenesis
 Regeneration of fertile plants can occur through
organogenesis or somatic embryogenesis.
 The developmental plasticity of plant cells means
that whole fertile plants can often be regenerated
from tissue explants, callus, cell suspensions, or
protoplasts by placing them on appropriate media.
Organogenesis
 Organogenesis relies on the production of
organs, either directly from an explant or from
a callus culture.
 Somatic embryogenesis relies on plant
regeneration through a process analogous to
zygotic embryo germination.
 Organogenesis relies on the inherent plasticity
of plant tissues, and is regulated by altering
the components of the medium.
Organogenesis
 Formation of roots and shoots on callus tissue is known
as organogenesis.
 Culture conditions required to achieve organogenesis
vary from species to species, and will have to be
determined for different types of callus.
 Adventitious organogenesis of shoots and roots can
also occur directly from organized plant tissues such as
stem segments, without first passing through a callus
stage.
Organogenesis
 There are three methods to regenerate a plant via
organogenesis:
 The first two methods depend on adventitious
organs arising either from a callus culture or
directly from an explant.
 The third method is by axillary bud formation and
growth, which can also be used to regenerate
whole plants from some types of tissue culture.
Organogenesis
 It is the auxin to cytokinin ratio of the medium that
determines which developmental pathway the
regenerating tissue will take.
 It is usual to induce shoot formation by increasing
the cytokinin to auxin ratio of the culture medium.
 These shoots can then be rooted relatively simply.
Organogenesis
 Only cytokinin is required for shoot culture and only
auxin for root culture, therefore increasing the level of
cytokinins available to the callus induces shoot
formation and increasing the auxin level promotes root
formation.
 Ultimately plantlets arise through the development of
adventitious roots on shoot buds, or through the
development of shoot buds from tissues formed by
proliferation at the base of rootlets.
Organogenesis
Somatic Embryogenesis
 Cells or callus can be induced to undergo a
development process known as somatic
embryogenesis.
 In this process, the cells undergo a pattern of
differentiation similar to that seen in zygotes after
fertilization, to produce embryoids.
 Embryoids are embryo-like but are produced from
somatic cells (not from the fusion of two germ cells);
develop into plants without the need to induce root
and shoot formation.
Somatic Embryogenesis
 Somatic embryos can be produced either directly or
indirectly.
 Direct somatic embryogenesis - embryo is formed
directly from cells without the production of an
intervening callus.
 Commonly from usually reproductive tissues such as
the nucellus, styles, or pollen, direct somatic
embryogenesis is generally rare in comparison with
indirect somatic embryogenesis.
Somatic embryogenesis
usually proceeds in two
distinct stages:

(i) initial stage of embryo

initiation, and

(ii) second stage of embryo

production.
Somatic Embryogenesis
Somatic Embryogenesis

In indirect somatic embryogenesis, callus is first produced from the explant,

then embryos can be generated from the callus tissue or cell suspension.

Efficiency of somatic embryogenesis can be further improved by supplying a

source of reduced nitrogen, such as specific amino acids or casein hydrolysate.
Somaclonal Variation
 Cell suspensions, callus, plantlets can be maintained
indefinitely by subculture.
 However, some of these products will experience
genetic instability, that will add genetic heterogeneity.
 Long-term culture of plants generally results in the
accumulation of mutations, this is known as somaclonal
variation.
 This may adversely affect the vitality and fertility of
regenerated plants.
Normal and mutated Nicotiana spp. plant occurred in
plant tissue culture.
(A) Normal leaf of N. debneyii (B) Mutated N. debneyii:
tubular shoots (leaves looks fleshy with cyndrical
extension) (C) Mutated N. benthamiana: bushy shoot (D)
Mutated MATF: fleshy and tubular shoot
Detection of somaclonal variation using Methyl-Sensitive Transposon Display (MSTD)
Tnt1 in Nicotiana benthamiana
Left plant: Normal regenerated Musa acuminata. The leaves were smooth and soft.
Right plant: Mutated regenerated plant. The plant grew likes ‘cabbage’ where there was
no proper shoot and the leaves were hard and crunchy.
Detection of somaclonal variation in banana mutant using Methyl-Sensitive Transposon
Display (MSTD) : Copia33-Mad I element insertions
In vitro propagation of Anthurium andreanum (a) Initiation of shoot bud (b) Multiple shoot
regeneration (c) Root induction and elongation (d) Ex vitro acclimation of micropropagated
plantlets (e) Micropropagated plants at bloom stage and (f) Gel electrophoresis using ISSR
marker [(GT)8A] showing the monomorphic bands ensures clonal fidelity (Lane M- 50 bp
ladder, C1 to C7- in vitro regenerated clones and P- mother plant).
Ref: http://scialert.net/fulltext/?doi=ijb.2010.207.219
Plant Tissue Culture Applications
 A single explant can be multiplied into several
thousand plants in less time - fast commercial
propagation of new cultivars.
 Plant tissue culture can continuously supply young
plants throughout the year.
 Allows faster selection for crop improvement -
explants are chosen from superior plants for cloning.
 Plant ‘tissue banks’ can be frozen, then regenerated
through tissue culture.
 Plant Tissue Culture Applications
 Tissue culture clones are ‘true to type’ as
compared with seedlings, which show genetic
variation [but beware of somaclonal variation].
 Conservation - rare and endangered plants can be
cloned safely.
 To facilitate trade - plant cultures in sterile media
are easier to export than soil-grown plants, as
they are pathogen free and take up little space).
Plant Tissue Culture Applications
 To produce virus-free plants from meristem
cultures.
 It is also possible to maintain transformed plant
cell lines or tissues (e.g. root cultures) producing
recombinant proteins or metabolites, indefinitely
using plant tissue culture.
Plant Tissue Culture Applications
 Foreign DNA can be introduced into most types of
plant material - protoplasts, cell suspensions,
callus, tissue explants, gametes, seeds, zygotes,
embryos, organs, and whole plants.
 The ability to recover fertile plants from such
material is often the limiting step in plant genetic
engineering rather than the DNA transfer process
itself.
Summary
 Some crops may be amenable to a variety of
regeneration and transformation strategies, and
others may currently only be amenable to one
particular protocol.
 Some protocols are clearly more efficient than
others for particular plant species such as for
monocots/dicots.
 Not all regeneration protocols are compatible with
all plant transformation techniques.
Plant Tissue Culture
R E F E RENCES:
[ I ] CHA PT ER 2 : P L A N T T I S SUE CU LT U R E. OX FOR D
UN I V ERSITY P R ESS.
WWW.OUP.COM/UK/ORC/BI N/9780199282616/C H02. PDF
[ I I ] P R I M ROSE S B & T W YM A N R M . 2 0 0 6. P R I N CIPL ES OF
G E N E M A N I PU LATI ON A N D G E N OMICS. 7 T H E D. , BL ACKW E L L
P UBL I S HI NG.
Web-based study resources
http://aggie-horticulture.tamu.edu/tisscult/tcintro.html
http://hort201.tamu.edu/YouthAdventureProgram/TisueCulture/TissueCulture.
html
www.oup.com/uk/orc/bin/9780199282616/ch02.pdf
www.accessexcellence.org/LC/ST/st2bgplant.html
http://www.liv.ac.uk/~sd21/tisscult/what.htm
http://www.phytocultures.com/
http://www.youtube.com/watch?v=kje0YczE0Do
http://www.youtube.com/watch?v=5ZveqLwGecs&feature=related (pineapple
tissue culture Part 1)
http://www.youtube.com/watch?v=Hy9tA4G8uDk&feature=relmfu (pineapple
tissue culture Part 2)
http://www.youtube.com/watch?v=D7Glb3l9mbY