12. meristem culture

MERISTEM CULUTRE
PTC course for Horticulture students. By: Dr. Rafail S. Toma
1

What is meristem?
A meristemis the tissue in all plants
consisting of undifferentiated cells
(meristematic cells), found in zones of the
plant where growth can take place.
2

What is meristem?
Groups of cells that are the source of new cells form
tissue called meristem.
Meristemcells aren't specialized, but when they
divide, some of the new cells specialize into tissues.
Areas of growth that lengthen the tips of roots and
stems are called apical meristems.
Lateral meristems, found all along woody roots and
stems, increase the thickness of these plant parts.
3

Meristem Culture History
Though meristemculture technique is known since
1933 it was made successful only in 1965 by Morel.
In the early period (1949) of this adventure,
Wetmore and Morel regenerated plantlets fromthe
meristemof ferns on simple defined medium.
But this did not workwell with the angiosperms
since they required complex mediumfortheirin
vitro development.
4

Meristem Culture History
In 1946, Ball found the initiation of root
primordiumformation and the plantlets
regeneration fromthe young meristems of
Trpaeolummajus and Lupinus albus.
Following this, Morel established the technique of
meristemculture with orchids.
Of late meristemculture technique is being
increasingly applied in micropropagation as an
alternative means forsexual propagation of
economically important crop plants.
5

Explants
The explant of meristemculture
may eitherbe the apical dome
(apical meristem) ormore
frequently, the apical dome plus a
few subjacent leaf primordia (the
sub apical meristematic region).
The apical meristemis located at
the extreme tip of a shoot and
measure 0.1 mmin diameterand
0.25 to 0.30 in length.
6

The culture of meristeminvolves three stages:
1. culture establishment,
2. multiplication of the propagules and
3. root regeneration
7
MeristemCulture Technique

Culture can be established from meristem, shoot tips or
axillary buds.
Forshoot regeneration from meristem, young development
stage of meristemhas been found to be optimum.
Therefore, it is desirable to excise terminal explants for
culture.
Axillary buds are preferred since there would be only one
terminal bud pershoot.
Further, the explants should be largerenough forgetting
successful results.
So largerexplants like shoot tips and buds have to be chosen
instead of minute meristems.
8
Culture Establishment

Afterexplant excision, they are inoculated into culture
medium.
Generally there is no necessity forthe addition of
exogenous hormones in the mediumsince sufficient
quantity of endogenous hormone is present in the shoot
apices.
However, there are cases in which exogenous auxin is
applied to get betterresults.
Among the auxins, NAA is the auxin routinely used for
meristemtip and bud cultures.
9
Culture Establishment

The propagules multiplication in meristem/shoot
tip/axillary bud culture can be accomplished by the
three methods as given below:
Explant - callus - meristemoids -shoot/roots plantlets
Explant - callus - embryoids/embryos- plantlets
Explant – axillary buds - multiple shoots-roots plantlets
10
Multiplication of propagules

Among the three methods, axillary shoot
proliferation is considered as the best because of
the lowerriskof genetic instability than the other
two systems of multiplication and is easily
achievable in most plant species.
In this system, the concentration of cytokinin used
is comparatively higherand is done to overcome the
apical dominance.
11

The incorporation of cytokinin enhances the branching of
lateral buds from leaf axils.
Too high a concentration of auxin may not only inhibit
axillary bud branching but also induces callus formation,
especially when 2,4-Dis used.
12

The purpose of this stage, in
the meristemculture is to
induce regeneration of roots
fromthe shoot multiplied in
the previous stage.
Adventitious root formation
can be induced quite readily in
many species, but it can be
very much recalcitrant in most
woody species.
13
Roots Regeneration

The induction of rooting does not
always have to be carried out invitro.
Good rooting can be obtained in
greenhouse by placing shoots into
pasteurized sand underintermittent
mist.
Forbetterrooting, the proliferated
shoots may be dipped in auxin
solution orcommercial rooting
powderbefore planting into rooting
medium.
14
Roots Regeneration

Size of the explant
The size of the explant determines the survival of the
culture.
In general, the largerthe explant, the betterthe chance of
survival.
Meristemof the smallest size within the regenerable range
should be used forvirus elimination.
When very small explants are used, the presence of leaf
primordia appears to determine the capability of an
explant to develop.
15
Factors influencing meristem culture

Physiological state of the explant
Explants taken fromthe tip of a shoot are in a younger
stage of development than explants taken fromthe base.
Young developmental stage has often been found to be
optimumforbettershoot regeneration.
16

Culture media
White's medium(1943) was the most widely used medium
during the early days of meristemculture.
There is no general purpose mediumyet available for
meristem, shoot tip and bud culture.
Murashige and Skoog (1962) mediumwith some
modification is the one used more frequently and with
great success.
17

Growth regulators
The requirement of growth regulators varies form species
to species, fromone stage of culture development to
another.
Presence of cytokinin at higherlevel during proliferation
stage is felt to overcome the apical dominance.
Similarly, presence of auxin is headed forgood rooting.
18

A. Invitro micropropagation,
B. Production of pathogen free plants, and
C. Cryopreservation of germplasm.
19
Applications of meristem culture

A. Invitro micropropagation
The micropropagation technique through meristem
orshoot tip culture favors production of thousand
and thousands of plants froma single explant
within a short period.
Moreover, once a stockof multiple shoot culture is
established, it can continuously serve as the source
material instead of having to restart fromfresh
explant cultures periodically.
20

The greatest success using this technique has been
achieved in most of the herbaceous horticultural
species.
Compared to herbaceous plants, the
micropropagation of woody species has lagged far
behind.
21

The majorproblems encountered with the
propagation of woody species are:
1. Most of the forest species are recalcitrant to
culture condition because of the presence of large
quantity of polyphenolic compounds in the tissues
and
2. The otherdifficulty experienced is rooting of in
vitro cultures.
22

B. Production of pathogen free plants
The most important application of
micropropagation technique viameristemculture
is the production of pathogen free plants,
especially viruses as they are absent in apical
meristem.
23

Generally, viruses infect plant species systemically
making the plants to die.
But the evidences fordecrease in virus particles
toward apical meristemmade Morel and Martin
(1952) to postulate the concept of culturing apical
meristemof systemically infected plant invitro in
orderto obtain virus free plants, genetically
identical to the "motherplant”
24

Forexample potato virus Xinfection could not be totally
eradicated since these viruses maintain theirreplication in
actively growing meristem.
In some cases of meristemtip culture the heat therapy has
necessarily to be followed to eliminate the viruses.
Forexample, in the carnation, heat therapy of plants at
38°C fortwo months followed by meristem culture
eradicated all the viruses.
25

The proposed reasons formeristem’s virus freeness:
1. The absence of vascularconnections
2. The high metabolic activity of the meristematic cells
which prevent virus multiplication
3. The high activity of the affective virus abolishing group
in meristems
4. The high auxin levels in apical meristems inhibits virus
multiplication
26

C. Cryopreservation of germplasm
The conventional systemof seed storage has the following
disadvantages:
1) the loss of viability of seeds,
2) destruction by pathogen and pest attacks,
3) problems in clonally propagated crops,
4) high cost of maintenance and transport and
5) material loss due to environmental hazards.
27

Considering the above disadvantages, the feasibility
of invitro storage was extensively studied.
The potential advantages of this method are:
1) relatively little space is needed,
2) the plants are maintained free frompest and
pathogens,
28

3) maintenance of vegetatively propagated species
is easier,
4) the materials can be multiplied as and when
needed and
5) the pest pathogen free nature favors easy and
quickinternational germplasmexchanges.
29

12. meristem culture

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