MICROPROPAGATION (source from link below)
http://www.biologydiscussion.com/biotechnology/clonal-propagation/micro-propagation-technique-factors-applications-and-disadvantages/10732
http://www.biologydiscussion.com/biotechnology/clonal-propagation/micro-propagation-technique-factors-applications-and-disadvantages/10732
MICRO
PROPAGATION:
Plants
can be propagated by sexual (through generation of seeds) or asexual (through
multiplication of vegetative parts) means.
Clonal
propagation refers to the process of asexual reproduction by multiplication of
genetically identical copies of individual plants. The term clone is used to
represent a plant population derived from a single individual by asexual
reproduction.
Asexual
reproduction through multiplication of vegetative parts is the only method for
the in vivo propagation of certain plants, as they do not produce viable seeds
e.g. banana, grape, fig, and chrysanthemum. Clonal propagation has been
successfully applied for the propagation of apple, potato, tuberous and several
ornamental plants.
Advantages
of Vegetative Propagation:
Asexual
(vegetative) propagation of plants has certain advantages over sexual
propagation.
i. Faster
multiplication — large number of plants can be produced from a single
individual in a short period.
ii. Possible to
produce genetically identical plants.
iii. Sexually —
derived sterile hybrids can be propagated.
iv. Seed — raised
plants pass through an undesirable juvenile phase which is avoided in asexual
propagation.
v. Gene banks can be
more easily established by clonally propagated plants.
In
Vitro Clonal Propagation:
The
in vivo clonal propagation of plants is tedious, expensive and frequently
unsuccessful. In vitro clonal propagation through tissue culture is referred to
as micro propagation. Use of tissue culture technique for micro propagation was
first started by Morel (1960) for propagation of orchids, and is now applied to
several plants. Micro propagation is a handy technique for rapid multiplication
of plants.
Technique
of Micro propagation:
Micro
propagation is a complicated process and mainly involves 3 stages (I, II and
III). Some authors add two more stages (stage 0 and IV) for more comprehensive
representation of micro- propagation. All these stages are represented in Fig.
47.1, and briefly described hereunder.
Stage
0: This is the initial step in micro-
propagation, and involves the selection and growth of stock plants for about 3
months under controlled conditions.
Stage
I: In this stage, the initiation and
establishment of culture in a suitable medium is achieved. Selection of appropriate
explants is important. The most commonly used explants are organs, shoot tips
and axillary buds. The chosen explant is surface sterilized and washed before
use.
Stage
II: It is in this
stage, the major activity of micro propagation occurs in a defined culture
medium. Stage II mainly involves multiplication of shoots or rapid embryo
formation from the explant.
Stage
III: This stage
involves the transfer of shoots to a medium for rapid development into shoots.
Sometimes, the shoots are directly planted in soil to develop roots. In vitro
rooting of shoots is preferred while simultaneously handling a large number of
species.
Stage
IV: This stage
involves the establishment of plantlets in soil. This is done by transferring
the plantlets of stage III from the laboratory to the environment of
greenhouse. For some plant species, stage III is skipped, and un-rooted stage
II shoots are planted in pots or in suitable compost mixture.
The different stages described above for micro propagation
are particularly useful for comparison between two or more plant systems,
besides better understanding. It may however, be noted that not all plant
species need to be propagated in vitro through all the five stages referred
above.
Micro propagation mostly
involves in vitro clonal propagation by two approaches:
1.
Multiplication by axillary buds/apical shoots.
2.
Multiplication by adventitious shoots.
Besides
the above two approaches, the plant regeneration processes namely organogenesis
and somatic embryogenesis may also be treated as micro propagation.
3.
Organogenesis: The formation of individual organs such as shoots, roots,
directly from an explant (lacking preformed meristem) or from the callus and
cell culture induced from the explant.
4.
Somatic embryogenesis: The regeneration of embryos from somatic cells, tissues
or organs.
1. Multiplication by Axillary Buds and Apical Shoots:
Quiescent
or actively dividing meristems are present at the axillary and apical shoots
(shoot tips). The axillary buds located in the axils of leaves are capable of
developing into shoots. In the in vivo state, however only a limited number of
axillary meristems can form shoots. By means of induced in vitro multiplication
in micro propagation, it is possible to develop plants from meristem and shoot
tip cultures and from bud cultures.
Meristem
and Shoot Tip Cultures:
Apical
meristem is a dome of tissue located at the extreme tip of a shoot. The apical
meristem along with the young leaf primordia constitutes the shoot apex. For
the development of disease-free plants, meristem tips should be cultured.
Meristem
or shoot tip is isolated from a stem by a V-shaped cut. The size (frequently
0.2 to 0.5 mm) of the tip is critical for culture. In general, the larger the
explant (shoot tip), the better are the chances for culture survival. For good
results of micro propagation, explants should be taken from the actively
growing shoot tips, and the ideal timing is at the end of the plants dormancy
period.
The
most widely used media for meristem culture are MS medium and White’s medium. A
diagrammatic representation of shoot tip (or meristem) culture in micro
propagation is given in Fig 47.2, and briefly described hereunder.
In
stage I, the
culture of meristem is established. Addition of growth regulators namely
cytokinins (kinetin, BA) and auxins (NAA or IBA) will support the growth and
development.
In
stage II, shoot
development along with axillary shoot proliferation occurs. High levels of
cytokinins are required for this purpose.
Stage
III is associated
with rooting of shoots and further growth of plantlet. The root formation is
facilitated by low cytokinin and high auxin concentration. This is opposite to
shoot formation since high level of cytokinins is required (in stage II).
Consequently, stage II medium and stage III medium should be different in
composition. The optimal temperature for culture is in the range of 20-28°C
(for majority 24-26°C). Lower light intensity is more appropriate for good
micro propagation.
Bud
Cultures:
The
plant buds possess quiescent or active meristems depending on the physiological
state of the plant. Two types of bud cultures are used— single node culture and
axillary bud culture.
Single
node culture:
This
is a natural method for vegetative propagation of plants both in vivo and in
vitro conditions. The bud found in the axil of leaf is comparable to the stem
tip, for its ability in micro propagation. A bud along with a piece of stem is
isolated and cultured to develop into a plantlet. Closed buds are used to reduce
the chances of infections.
A
diagrammatic representation of single node culture is depicted in Fig 47.3. In
single node culture, no cytokinin is added.
Axillary
bud culture:
In
this method, a shoot tip along with axillary bud is isolated. The cultures are
carried out with high cytokinin concentration. As a result of this, apical
dominance stops and axillary buds develop. A schematic representation of
axillary bud culture for a rosette plant and an elongate plant is given in Fig
47.4.
For a good axillary bud culture, the cytokinin/ auxin ratio
is around 10: 1. This is however, variable and depends on the nature of the
plant species and the developmental stage of the explant used. In general,
juvenile explants require less cytokinin compared to adult explants. Sometimes,
the presence of apical meristem may interfere with axillary shoot development.
In such a case, it has to be removed.
2. Multiplication by Adventitious Shoots:
The stem and leaf structures that are naturally formed on
plant tissues located in sites other than the normal leaf axil regions are
regarded as adventitious shoots. There are many adventitious shoots which
include stems, bulbs, tubers and rhizomes. The adventitious shoots are useful
for in vivo and in vitro clonal propagation. The meristematic regions of
adventitious shoots can be induced in a suitable medium to regenerate to
plants.
3. Organogenesis:
Organogenesis is the process of morphogenesis involving the
formation of plant organs i.e. shoots, roots, flowers, buds from explant or
cultured plant tissues. It is of two types — direct organogenesis and indirect
organogenesis.
Direct
Organogenesis:
Tissues from leaves, stems, roots and inflorescences can be
directly cultured to produce plant organs. In direct organogenesis, the tissue
undergoes morphogenesis without going through a callus or suspension cell
culture stage. The term direct adventitious organ formation is also used for
direct organogenesis.
Induction of adventitious shoot formation directly on roots,
leaves and various other organs of intact plants is a widely used method for
plant propagation. This approach is particularly useful for herbaceous species.
For appropriate organogenesis in culture system, exogenous addition of growth
regulators—auxin and cytokinin is required. The concentration of the growth
promoting substance depends on the age and nature of the explant, besides the
growth conditions.
Indirect
Organogenesis:
When the organogenesis occurs through callus or suspension
cell culture formation, it is regarded as indirect organogenesis (Fig 47.5 B
and C). Callus growth can be established from many explants (leaves, roots,
cotyledons, stems, flower petals etc.) for subsequent organogenesis.
Direct
Somatic Embryogenesis:
When the somatic embryos develop directly on the excised
plant (explant) without undergoing callus formation, it is referred to as
direct somatic embryogenesis (Fig 47.6A). This is possible due to the presence
of pre-embryonic determined cells (PEDQ found in certain tissues of plants. The
characteristic features of direct somatic embryogenesis is avoiding the
possibility of introducing somaclonal variations in the propagated plants.
Indirect
Somatic Embryogenesis:
In indirect embryogenesis, the cells from explant (excised
plant tissues) are made to proliferate and form callus, from which cell
suspension cultures can be raised. Certain cells referred to as induced embryo
genic determined cells (IEDC) from the cell suspension can form somatic
embryos. Embryogenesis is made possible by the presence of growth regulators
(in appropriate concentration) and under suitable environmental conditions.
Somatic
embryogenesis (direct or indirect) can be carried on a wide range of media
(e.g. MS, White’s). The addition of the amino acid L-glutamine promotes embryogenesis.
The presence of auxin such as 2, 4-dichlorophenoxy acetic acid is essential for
embryo initiation. On a low auxin or no auxin medium, the embryo genic clumps
develop into mature embryos.
Indirect
somatic embryogenesis is commercially very attractive since a large number of
embryos can be generated in a small volume of culture medium. The somatic
embryos so formed are synchronous and with good regeneration capability.
Artificial seeds can be made by encapsulation of somatic
embryos. The embryos, coated with sodium alginate and nutrient solution, are
dipped in calcium chloride solution. The calcium ions induce rapid
cross-linking of sodium alginate to produce small gel beads, each containing an
encapsulated embryo. These artificial seeds (encapsulated embryos) can be
maintained in a viable state till they are planted.
Factors Affecting Micro propagation:
For a successful in vitro clonal propagation (micro
propagation), optimization of several factors is needed.
Some
of these factors are briefly described:
1.
Genotype of the plant:
Selection of the right genotype of the plant species (by
screening) is necessary for improved micro propagation. In general, plants with
vigorous germination and branching capacity are more suitable for micro-
propagation.
2.
Physiological status of the explants:
Explants (plant materials) from more recently produced parts
of plants are more effective than those from older regions. Good knowledge of
donor plants’ natural propagation process with special reference to growth
stage and seasonal influence will be useful in selecting explants.
3.
Culture media:
The standard plant tissue culture media are suitable for
micro propagation during stage I and stage II. However, for stage III, certain
modifications are required. Addition of growth regulators (auxins and
cytokinins) and alterations in mineral composition are required. This is
largely dependent on the type of culture (meristem, bud etc.).
4.
Culture environment:
Light:
Photosynthetic pigment in cultured tissues does absorb light
and thus influence micro- propagation. The quality of light is also known to
influence in vitro growth of shoots, e.g blue light induced bud formation in
tobacco shoots. Variations in diurnal illumination also influence micro
propagation. In general, an illumination of 16 hours day and 8 hours night is
satisfactory for shoot proliferation.
Temperature:
Majority of the culture for micro propagation requires an
optimal temperature around 25°C. There are however, some exceptions e.g.
Begonia X Cheimantha hybrid tissue grows at a low temperature (around 18°C).
Composition
of gas phase:
The constitution of the gas phase in the culture vessels
also influences micro propagation. Unorganized growth of cells is generally promoted
by ethylene, O2, CO2 ethanol and acetaldehyde.
Factors Affecting in Vitro Rooting:
A general description of the factors affecting micro
propagation, particularly in relation to shoot multiplication is given above.
For efficient in vitro rooting during micro- propagation, low concentration of
salts (reduction to half to one quarter from the original) is advantageous.
Induction of roots is also promoted by the presence of suitable auxin (NAA or
IBA).
Applications of Micro propagation:
Micro propagation has become a suitable alternative to
conventional methods of vegetative propagation of plants. There are several
advantages of micro propagation.
High Rate of Plant Propagation:
` Through micro propagation, a large
number of plants can be grown from a piece of plant tissue within a short
period. Another advantage is that micro propagation can be carried out
throughout the year, irrespective of the seasonal variations. Further, for many
plants that are highly resistant to conventional propagation, micro propagation
is the suitable alternative. The small sized propagules obtained in micro
propagation can be easily stored for many years (germplasm storage), and
transported across international boundaries.
Production of Disease-free Plants:
It is possible to produce disease-free plants through micro
propagation. Meristem tip cultures are generally employed to develop
pathogen-free plants .In fact, micro propagation is successfully used for the
production of virus-free plants of sweet potato (Ipomea batatus), cassava
(Manihot esculenta) and yam (Discorea rotundata).
Production of Seeds in Some Crops:
Micro propagation, through axillary bud proliferation
method, is suitable for seed production in some plants. This is required in
certain plants where the limitation for’ seed production is high degree of
genetic conservation e.g. cauliflower, onion.
Cost-effective Process:
Micro propagation requires minimum growing space. Thus,
millions of plant species can be maintained inside culture vials in a small
room in a nursery. The production cost is relatively low particularly in
developing countries (like India) where the manpower and labour charges are
low.
Automated Micro propagation:
It has now become possible to automate micro propagation at
various stages. In fact, bio- reactors have been set up for large scale multiplication
of shoots and bulbs. Some workers employ robots (in place of labourers) for
micro- propagation, and this further reduces production cost of plants.
Disadvantages of Micro propagation:
Contamination of Cultures:
During the course of micro propagation, several slow-growing
microorganisms (e.g. Eswinia sp, Bacillus sp) contaminate and grow in cultures.
The microbial infection can be controlled by addition of antibiotics or
fungicides. However, this will adversely influence propagation of plants.
Brewing of Medium:
Micro propagation of certain plants (e.g. woody perennials)
is often associated with accumulation of growth inhibitory substances in the
medium. Chemically, these substances are phenolic compounds, which can turn the
medium into dark colour. Phenolic compounds are toxic and can inhibit the
growth of tissues. Brewing of the medium can be prevented by the addition of
ascorbic acid or citric acid or polyvinyl pyrrolidone to the medium.
Genetic Variability:
When micro propagation is carried out through shoot tip
cultures, genetic variability is very low. However, use of adventitious shoots
is often associated with pronounced genetic variability.
Vitrification:
During the course of repeated in vitro shoot multiplication,
the cultures exhibit water soaked or almost translucent leaves. Such shoots
cannot grow and even may die. This phenomenon is referred to as vitrification.
Vitrification may be prevented by increasing the agar concentration (from 0.6 to
1%) in the medium. However, increased agar concentration reduces the growth
rate of tissues.
Cost Factor:
For some micro propagation techniques, expensive equipment,
sophisticated facilities and trained manpower are needed. This limits its use.
Production of Disease-Free Plants:
Many plant species are infected with pathogens — viruses,
bacteria, fungi, mycoplasma and nematodes that cause systemic diseases.
Although these diseases do not always result in the death of plants, they
reduce the quality and yield of plants. The plants infected with bacteria and
fungi frequently respond to chemical treatment by bactericides and fungicides.
However,
it is very difficult to cure the virus-infected plants. Further, viral disease
are easily transferred in seed- propagated as well as vegetatively propagated
plant species. Plant breeders are always interested to develop disease-free
plants, particularly viral disease-free plants. This have become a reality
through tissue cultures.
Apical Meristems with Low Concentration of Viruses:
In
general, the apical meristems of the pathogen infected and disease harbouring
plants are either free or carry a low concentration of viruses, for the
following reasons:
i.
Absence of vascular tissue in the meristems through which viruses readily move
in the plant body.
ii.
Rapidly dividing meristematic cells with high metabolic activity do not allow
viruses to multiply.
iii.
Virus replication is inhibited by a high concentration of endogenous auxin in
shoot apices. Tissue culture techniques employing meristem-tips are
successfully used for the production of disease-free plants, caused by several
pathogens — viruses, bacteria, fungi, mycoplasmas.
Methods to Eliminate Viruses in Plants:
In general, plants are infected with many viruses; the
nature of some of them may be unknown. The usage virus-free plant implies that
the given plant is free from all the viruses, although this may not be always
true. The commonly used methods for virus elimination in plants are listed
below, and briefly described next.
i.
Heat treatment of plant
ii.
Meristem-tip culture
iii.
Chemical treatment of media
iv.
Other in vitro methods
Heat Treatment (Thermotherapy) of Plants:
In the early days, before the advent of meristem cultures,
in vivo eradication of viruses from plants was achieved by heat treatment of
whole plants. The underlying principle is that many viruses in plant tissues
are either partially or completely inactivated at higher temperatures with
minimal injury to the host plant. Thermotherapy (at temperatures 35-40°C) was
carried out by using hot water or hot air for elimination viruses from growing
shoots and buds.
There
are two limitations of viral elimination by heat treatment:
1.
Most of the viruses are not sensitive to heat treatment.
2.
Many plant species do not survive after thermotherapy.
With
the above disadvantages, heat treatment has not become popular for virus
elimination.
Meristem-Tip Culture:
A general description of the methodology adopted for
meristem and shoot tip cultures has been described (see Fig 47.2). For viral
elimination, the size of the meristem used in cultures is very critical. This
is due to the fact that most of the viruses exist by establishing a gradient in
plant tissues.
In general, the regeneration of virus-free plants through
cultures is inversely proportional to the size of the meristem used. The
meristem-tip explant used for viral elimination cultures is too small. A
stereoscopic microscope is usually employed for this purpose.
Meristem-tip
cultures are influenced by the following factors:
i.
Physiological condition of the explant — actively growing buds are more
effective.
ii.
Thermotherapy prior to meristem-tip culture — for certain plants (possessing
viruses in the meristematic regions), heat treatment is first given and then
the meristem-tips are isolated and cultured.
iii.
Culture medium —MS medium with low concentrations of auxins and cytokinins is
ideal.
A
selected list of the plants from which viruses have been eliminated by meristem
cultures is given in Table 47.1.
Chemical Treatment of Media:
Some workers have attempted to eradicate viruses from
infected plants by chemical treatment of the tissue culture media. The commonly
used chemicals are growth substances (e.g. cytokinins) and antimetabolites (e.g
thiouracil, acetyl salicylic acid).
There
are however, conflicting reports on the elimination viruses by chemical
treatment of the media. For instance, addition of cytokinin suppressed the
multiplication of certain viruses while for some other viruses, it actually
stimulated.
Other in Vitro Methods:
Besides meristem-tip culture, other in vitro methods are
also used for raising virus-free plants. In this regard callus cultures have
been successful to some extent. The callus derived from the infected tissue
does not carry the pathogens throughout the cells. In fact, the uneven
distribution of tobacco mosaic virus in tobacco leaves was exploited to develop
virus-free plants of tobacco. Somatic cell hybridization, gene transformation
and somaclonal variations also useful to raise disease-free plants.
Elimination of Pathogens Other than Viruses:
Besides the elimination of viruses, meristem-tip cultures
and callus cultures are also useful for eradication bacteria, fungi and
mycoplasmas. Some examples are given
i.
The fungus Fusarium roseum has been successfully eliminated through meristem
cultures from carnation plants.
ii.
Certain bacteria (Pseudomonas carophylli, Pectobacterium parthenii) are
eradicated from carnation plants by using meristem cultures.
Merits and Demerits of Disease-Free Plant Production:
Among the culture techniques, meristem-tip culture is the
most reliable method for virus and other pathogen elimination. This, however,
requires good knowledge of plant pathology and tissue culture.
Virus-free plants exhibit increased growth and vigour of
plants, higher yield (e.g. potato), increased flower size (e.g. Chrysanthemum),
and improved rooting of stem cuttings (e.g. Pelargonium).Virus-free plants are
more susceptible to the same virus when exposed again. This is the major
limitation. Reinfection of disease-free plants can be minimized with good
knowledge of greenhouse maintenance.