Meristem and shoot tip culture in horticultural crops

1. Media preparation
2. Explant selection
3. Establishment of explant in media
4. Callus development
5. Plantlet development
6. Hardening or acclimatization
7. Open field planting
Lecture 9: Meristem and shoot tip culture in horticultural crops
Course Code : HRT 552
Course Title : BIOTECHNOLOGY OF
HORTICULTURAL CROPs

Introduction to Tissue Culture
⚫Tissue Culture (also known as Micropropagation or In vitro culture) is:
⚫The growing of plant cells, tissues, organs, seeds or other plant parts in
a sterile environment on a nutrient medium.

Callus
Dedifferentiation
Callus growth &
cell division Redifferentiation
Plantlet
development

2) Explant:-
Totipotent somatic cell are used.
Immature inflorescence and Scutellar tissue of
immature seeds are used for the research. Ex:-
Triticum aestivum .
Epidermis, Procambial tissue are also produced
somatic embryo.

 EXPLANT PREPARATION
EXPLANT : It is defined as a portion of plant body, which has been
taken from the plant to establish a culture
•Explant may be taken from any part of the plant like
root,stem,leaf,or meristematic tissue like cambium, floral parts like
anthers, stamens etc..
•Age of the explant.
• Homozygous plants are preferred.
5

 TYPES OF CULTURE
 Callus culture
 Suspension culture
 Root tip culture
 Leaf or leaf primordial culture
 Shoot tip culture
 Complete flower culture
 Anther & pollen culture
 Ovule & embryo culture
 Protoplast culture 31

Callus culture Suspension
culture
Pollen culture
Ovule culture Root tip culture Shoot tip culture
32
Protoplast culture Leaf primordial culture Flower culture

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

STAGES OF MERISTEM
CULTURE
Murashige reported that there are three stages of culture:
Stage 1 is the culture establishment stage when explant may develop into
single shoot or multiple shoots.
At this stage explant are supplements with cytokinin like BA, kinetin and
2iP.

In stage 2 the objective is to multiply the propagule and for this axillary
shoot proliferation is followed as it maintains higher genetic stability.
In axillary shoot proliferation, high levels of cytokinin are utilized to
overcome the apical dominance.

The stage 3 purpose is regeneration of adventitious roots from the shoots
obtain in stage 2
Numerous studies have indicated that NAA is followed by IBA,IAA, 2,4-D
and other auxins are used for induction of root generation.

Components of medium
⚫ Inorganic nutrients (N2,P,Ca,Mg,S)
⚫ Carbon source (sugar)
⚫ Organic supplements including
Vitamins (Thiamine, nicotinic acid, panthonic acid, pyridoxine)
Amino acids (L-glutamine, L-asparagine, L-cysteine, L-glycine)
Complex organics (casein hydrolysate, coconut milk, yeast extract,
orange juice, tomato juice)
Plant growth hormones
Auxins (root)
Cytokinins (shoot)
Gibbrellins (internode elongation, meristem growth)
Abscissic acid (for culturing woody species)
Solidifying agent (agarose)
pH (optimum is 5.8) lower than 4.5 or higher than 7.5 greatly inhibit the
growth

PROTOC
OL
Remove the young twigs from a healthy plant. Cut the tip (1 cm)
portion of the twig
Surface sterilize the shoot apices by incubation in a sodium hypochlorite
solution (1% available chlorine) for 10 minutes. The explants are
thoroughly rinsed 4 times in distilled water
Transfer each explants to a sterilized petri dish.

Remove the outer leaves from each shoot
After the removal of all outer leaves, the apex is exposed.
Cut off the ultimate apex with the help of scalpel and transfer only those less
than 1 mm in length
Incubate the culture under 16hrs light at 25°C
As soon as the growing single leafy shoot or multiple shoots obtained from single
shoot tip or meristem, transfer them to hormone free medium to develop roots.
The plants are later transferred to pots containing compost and kept under
green house condition for hardening.

Application of Shoot-tip or Meristem
Culture
1. Virus Elimination
• Plants are often infected with more
than one type of virus, including
some even not known.
• A general term virus- free is used
by commercial horticulturist by this
method.

2. Micro Propagation
• Asexual or vegetative propagation
(vegetative part) of whole plants using
tissue culture techniques referred to as
micro propagation.
3. Storage of Genetic Resources
• Many plants produce seeds that are
highly heterozygous in nature or that is
recalcitrant. Such seeds are not
accepted for storing genetic resources.
So , the meristem from such plants can
be stored in vitro.

4. Use in Plant Breeding:
•In many plant breeding experiments the hybrid plants produce abortive
seeds or non viable seeds. As a result, it makes a barrier to crossibility in
plants where non-viable seeds are unable to develop into mature plants.
Shoot-tip or meristem from such hybrid plant can be cultured to speed up
breeding programme.
5. Quarantine
• Plantlets derived from shoot-tip or meristem
cultures are easily accepted by the quarantine
authority for international exchange without any
checking.
• Therefore, using this technique , crop plants can
be easily exchanged in crop improvement
programmes that are based on materials from
different parts of the world.

List of the plants from which viruses have been eliminated by
meristem cultures

ADVANTAGES:
Lack of vascular tissue.
High auxin concentration.
Production of virus free plants
Facilitation of exchange between locations
Cryopreservation or in-vitro conservation of germplasm
• DISADVANTAGES:
Isolation is difficult
 Low survival rate & regeneration time for explants may be long(abo
months for potato explant)

CONCLUSION
It is very effective method of cloning of plant material and to
develop disease free clean plant stock. Shoot Tip Culture is a
part of plant tissue culture which is a sun-rise technology and
working as a catalyst of agricultural and industrial
development

What is meristem?
A meristem is the tissue in all plants consisting of undifferentiated cells
(meristematic cells), found in zones of the plant where growth can take place.
2
6

What is meristem?
Groups of cells that are the source of new cells form
tissue called meristem.
Meristem cells aren't specialized, but when they
divide, some of the new cells specialize into tisues.
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.
2
7

Meristem Culture History
2
8
• Though meristem culture 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 from the
meristem of ferns on simple defined medium.
• But this did not work well with the angiosperms
since they required complex medium for their in
vitro development.

Meristem Culture History
2
9
1. In 1946, Ball found the initiation of root primordium formation and the
plantlets regeneration from the young meristems of Trpaeolummajus and
Lupinus albus.
2. Following this, Morel established the technique of meristem culture with orchids.
3. Of late meristem culture technique is being increasingly applied in
micropropagation as an alternative means for sexual propagation of economically
important crop plants.

Explants
The explant of meristem culture may either be
the apical dome (apical meristem) or more
frequently, the apical dome plus a few
subjacent leaf primordia (the sub apical
meristematic region).
The apical meristem is located at the extreme
tip of a shoot and measure 0.1 mm in
diameter and
0.25 to 0.30 in length.
6

The culture of meristem involves three stages:
1. culture establishment,
2. multiplication of the propagules and
3. ro t regeneration
Meristem Culture Technique
3
1

• Culture can be established from meristem, shoot tips or axillary buds.
• For shoot regeneration from meristem, young development stage of meristem has 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 per shoot.
• Further, the explants should be larger enough for getting successful results.
• So larger explants like shoot tips and buds have to be chosen instead of minute meristems.
3
2
Culture Establishment

After explant excision, they are inoculated into culture medium.
Generally there is no necessity for the addition of exogenous hormones in
the medium since sufficient quantity of endogenous hormone is present in
the shoot apices.
However, there are cases in which exogenous auxin is applied to get better
results.
Among the auxins, NAA is the auxin routinely used for meristem tip and bud
cultures.
3
3
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
3
4
Multiplication of propagules

Among the three methods, axil ary shoot proliferation is
considered as the best because of the lower risk of 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.
3
5

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.
3
6

The purpose of this stage, in
the meristem culture is to
induce regeneration of roots
from the 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.
Roots Regeneration
3
7

The induction of rooting does not
always have to be carried out in
vitro.
Good rooting can be obtained in
greenhouse by placing shoots into
pasteurized sand underintermittent
mist.
For better rooting, the proliferated
shoots may be dipped in auxin
solution or commercial rooting
powder before planting into rooting
medium.
Roots Regeneration
3
8

Size of the explant
The size of the explant determines the survival of the
culture.
In general, the larger the explant, the better the chance of
survival.
Meristem of the smal est size within the regenerable range
should be used for virus elimination.
When very smal explants are used, the presence of leaf
primordia appears to determine the capability of an
explant to develop.
3
9
Factors influencing meristem culture

Physiological state of the explant
Explants taken from the tip of a shoot are in a younger
stage of development than explants taken from the base.
Young developmental stage has often been found to be
optimumfor better shoot regeneration.
4
0

Culture media
White's medium(1943) was the most widely used medium
during the early days of meristem culture.
There is no general purpose medium yet available for
meristem, shoot tip and bud culture.
Murashige and Skoog (1962) medium with some
modification is the one used more frequently and with
great success.
4
1

Growth regulators
The requirement of growth regulators varies form species
to species, from one stage of culture development to
another.
Presence of cytokinin at higher level during proliferation
stage is felt to overcome the apical dominance.
Similarly, presence of auxin is headed for good rooting.
4
2

A. In vitro micropropagation,
B. Production of pathogen free plants, and
C. Cryopreservation of germplasm.
Applications of meristem culture
4
3

A. In vitro micropropagation
The micropropagation technique through meristem
or shoot tip culture favors production of thousand
and thousands of plants from a single explant
within a short period.
Moreover, once a stock of multiple shoot culture is
established, it can continuously serve as the source
material instead of having to restart from fresh
explant cultures periodical y.
4
4

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.
4
5

The major problems 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 other difficulty experienced is rooting of in vitro cultures.
4
6

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

General y, viruses infect plant species systemical y
making the plants to die.
But the evidences for decrease in virus particles
toward apical meristem made Morel and Martin
(1952) to postulate the concept of culturing apical
meristem of systemical y infected plant in vitro in
order to obtain virus free plants, genetical y
identical to the "mother plant”
4
8

For example potato virus X infection could not be total y
eradicated since these viruses maintaintheir replication in
actively growing meristem.
In some cases of meristem tip culture the heat therapy has
necessarily to be fol owed to eliminate the viruses.
For example, in the carnation, heat therapy of plants at
38°C for two months fol owed by meristem culture
eradicated al the viruses.
4
9

The proposed reasons for meristem’s virus freeness:
1. The absence of vascular connections
2.The high metabolic activity of the meristematic cel s
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
5
0

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

C. Cryopreservation of germplasm Considering the above
disadvantages, the feasibility of in vitro storage was extensively
studied.
The potential advantages of this method are:
1) relatively little space is needed,
2)the plants are maintained free from pest and pathogens,
5
2

C. Cryopreservation of germplasm
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 quick
international germplasm exchanges.
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3

Meristem and shoot tip culture in horticultural crops

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