MICROPROPOGATION:

SEXUAL SEED PROPOGATIOON ASEXUAL SEED PROPOGATION (VEGETATIVE PROPOGATION)

SEED: Seed is a result of pollination Grafting, Budding, Layering & Cutting
&fertilization between male and female Where as in asexual propagation, progeny is obtained by vegetative
granules. So variations in characters are propagation of a single plant or individual called as clone. So the plant
seenbecause of segregation. derived from clone will have the same genotype i.e. they are true to type.
Conventionally, vegetative reproduction is achived by cutting, budding, grafting etc, similarly. Tissue
culture also enables rapid clonal propagation of plants called "micropropagation"
Micropropagation is a method of propagating plants by cultivating very sma'l parts of plant -called
explants under aseptically controlled conditions on an artificial medium".
It derived from the miniature shorts/plantlets initially produced from this method ofplant propagation.
This technique provides a rapid and reliable system for production of large number of genetically uniform
disease free plants within a short period.
This is the only process of plant biotechnology, which is being utilized by industries in India for
commercial production of plantlets mainly ornamental plants and fruits ex: banana.
George Morelin 1960 was the first to show the potential of clonal propagation using in-vitro technique in orchid.
(Lymbidium)
Using the technique of aseptic micropropagation, it was possible to produce several million plantlets from,
a single shoot
tip explants in a single year.
1. Meristem Culture: Cultivation of
axillary or apical shoot meristems is known as meristem culture. The shoot
apical meristem is the upper most portion of the plant body or it is the portion distal to the youngest leaf
primordium. It is up to about 100 uM in diameter and 250 uM in length. The shoot apex/tip consists of the apical
meristem plus 1-3young leaf primordia and would measure
up to 500M.
For meristem culture a very small explants
(0.1-0.5mm) are taken from the apical or auxiliary bud, usually
with the help of very fine scalpels and under a stereo microscope.
The expalnt comprises of the apical dome and only one or tvo
pairs of the youngest leaf primordia. This involves
the development of an already existing shoot meristem into
shoot, thus regeneration of adventitious roots from the
developed shoots gives rise to complete plantlets.
Since the meristems will be free from the viruses, it is best nurtured to obtain
virus free plants.
2. SHOOT TIP CULTURE: in these method
relatively large explants (up to Icmin lengti) consisting of the shoot
apical meristem and a number of leaf primordial and small unexpanded leaves at different stages of
are needed. development
The dominance of the apical meristems removed and development of precocious auxiliary shoots is
stimulated by incorporation of growth regulators. (Usually cytokinin) in the growth medium. This results in a
highly branched shoot system
3. SINGLE NODE CULTURE/PROLIFERATIONOF AXILLARY
BUDS: An auxiliary meristem is
one located in the axil of a leaf, which
gives rise to an axillary bud. These do not develop into shoots under in-
vivo, but under in-vitro conditions with the addition of cytokinins the
hidden axillary bud proliferates into shoots.
apical dominance is suppressed at the
It is useful in plants, were bud break is difficult with
available cytokinins. Singe nodes of
mature plants have been used to initiate shoot
tip culture and further they can be used for mass seedlings or
4. SEED CULTURES: From seed culture, it is propagation.
possible to obtain multiple shoots instead of one
thus it is useful in many monocot and
dicotyledonous species.
seedling,
obtain 10 million shoots of almond from According to Hisajima
(1982) it is possible to
single seeding a year.
5. ADVENTIVE (DIRECT) ORGANOGENESIS: In this method, adventitious shoots
from the tissues of the explants without any callus formation. In this may arise directly
type, the precursor of the new organs is cells
 1 the
explant itself. The exogenously supplied organs can facilitate the process of organogenesis but are not
required for the organogenesis to occur.
ne induction of direct shoot regeneration depends upon the plant organ from which the explant is
derived and above alI, the plant species used.
in some species the adventitious shoots arise in-vitro on pieces of tissues derived from different organ
(example leafs on the stem, petal or roots); while in others they occur on limited range of tissues. The plants
produced will be true to type. It is useful in plants where auxiliary and apical meristem are difficult to disinfect of
establish in cultures.
It is widely followed in plants like
Begonia-from leaves.
Blackberry from shoots.
Bulbous crops from leaf
6. INDIRECT ORGANOGENESIS:
This type of organogenesis is not "adventitive" and involves a "de-differentiation" of the explant, elaboration of
callus tissue along cut edges of the explant and the induction of new organs from the newly formed callus tissues.
Exogenously supplied phytohormone not only, control the process but are required for organogenesis to
occur. This type of multiplication has a disadvantage that, the propagules may not be true to type and a callus
phase may result in genotypic and phenotypic abnormalities.
7.
SOMATIC EMBRYOGENESIS:-In somatic embryogenesis, somatic cells develop by division to form complete
embryos analogous to zygotic embryos. The bipolar structure of the somatic embryo contains both shoots and root
meristem. As the embryos develop, they progress through the distinct structural steps of an azygotic embryo and
another means of clonal propagation.
are

Somatic embryogenesis (like organogenesis) can be initiated in two different

ways
I n some cultures, embryogenesis occurs directly in the absence of callus
any formation from cells of the explant
tissue. Such cells are called "pre-embryonic determined cells" that are
programmed for embryonic development.
.The second type of development requires dedifferentiation of the cells of the
explant leading to callus formation.
The S.E. originates from "induced embryogenic cells" within the callus.
Plants grown from S. E. remain genetically stable.

ADVANTAGES OF MICROPROPAGATION: (IN-VITRO

1.
PROPOGATION)
Rapid mass propagation to produce genetically uniform plants.
2. Making clones of species which are very slow growing & difficult. Propagate by other methods.
3. Plants can be maintained in vitro in a pathogen free state
4. Propagation can be carried out throughout the year i.e. it is independent of seasons.
5. Long term storage of germ plasm is possible within a small space.
6. Large -scale propagation through somatic embryogenesis is another
7. Artificial seed production using S.Es has led to the
advantage of micropropagation.
development of mass propagation and delivery
number of crop plants. system for a

8. Micropropagation is one of the finest ways of plant multiplication by in vitro

DISADVANTAGES: techniques.
1. It requires skilled workers.
2. Laboratory investment and maintenance cost is very high.
3. Loss of micro propagated plant is electively high during hardening and
4. Bacterial contamination. transferring to the field.
5. Morphological and somaclonal variation.
 STAGES OF MICROPROPAGATION:
in micropropagation of
Murashige (1974) and Debergh and Maene (1981) suggested following stages
plants viz.
STAGE-0: - MOTHER PLANT SELECTION AND PROPAGATION.
reflects the overall quantity of the stock plant that is used as a source
The behavior of the cultured Explant
of the explant. Stock plants must be healthy and free from visual disease symptoms.
treatments.(if
They must quality light, temp, appropriate fertilizers and growth regulators
receive
mother plant selection.
required).influence of seasonal variation must be kept in mind during
STAGE-I: -
INITIAL ESTABLISHMENT OF ASEPTIC CULTURE:
or
microbes.Removalof external micro-organisms
Plants will be usually cohabited with different
decontamination or surface sterilization of explants is a pre-requisite
for aseptic culture. The surface sterilization
or raised in a glass
and kind of Explant i.e. whether the material is field grown
process depends on the nature
house.
sterilized following plants may carry a
Explants obtained from glass house grown plants may be surface
contamination load or entophytic contamination (bacteria /fungi inside
the Explant), so they should be
high Bavistine.
Trimithoprim. Ex; trimithoprim. & fungicide ex:
treated with the suitable antibiotics
house and theseplants should be
Therefore, cuttings from field grown plants should be grown in glass
a

used as explant source.

STAGE-II:-MULTIPLICATION STAGE: (In-vitro Shoot Proliferation and Multiplication).
from each culture.
This step consists of production of few to several shoots /somatic embryos
at which plantlets are produced. Most of the
Multiplication stage is the most crucial stage as it determines the rate
failures in micro-propagation occur during this stage.
Multiplication can be achieved by the following three approaches:
1. Enhaced proliferation of auxiliary shoot buds.
2. Induction of adventitious buds, bulbs. Proto corms

3. Somatic embryogenesis.
PROLIFERATION OF AXILLARY BUDS:
After 2-3 weeks of cultured initials the cultures are transferred to a shoot multiplication medium designed
to promote auxiliary branching. The multiplication media generally contain a cytokinin (usually 1-2 mg/1, but up
to 30 mg/l has been either alone or in combination with an auxin (0.1-1 mg/l) depending on plant species.
BAP is the most commonly used cytokinin but in some species like garlic, blue berry and rhododendrons
etc.2-ip is much it promotes callusing. The cytokinin stimulates pre-existing shoot buds present in the Explant
nodal develop into shoots. After 4-6 weeks the
(apical meristem in shoot tips and auxiliary buds in explant) to

auxiliary brunching in a culture reaches a maximum.

Then individual shoots are excised and sub-cultured on to fresh medium to initiate a new cycle of
multiplication by maxillary branching.
In most plant S.M each explant would produce 5-6 shoots in 4-5 weeks, this would give up to 5 10-6 12
plants in one year from a single initial explant. Provided all the shoots produced could be cultured and these were
100% survival.

INDUCTION OF ADVENTITIOUS BUDS /BULBS/PRODUCTION:

In many plant S.M. rapid clonal prorogation can be archived by inducing ad Bunch/ bulbs/ protocorms:
: In orchid, excised shoot tips (<Imm) can produce round bodies similar to the pratocorms forms by their
embryos during seed germination. These develop from the sub epidermal cells of the shoot tip leaf primordia.
 OMATIC EMBRYOGENESIS:
In some plant S.M. the explants having embryogenic potential form somatic embryos directly from the
explant. ex: alfalta, carrot yellow-popular etc.
Now protocols are available for large scale production of S.E in these crops.

STAGE-III IN-VITRO ROOTING OF PROPAGULES:

Shoot produced at stage-Il are small in size (miniature or micro). At stage -Illstem are taken to elongate
and proliferate the micro shoots and then root induction.
in general, the rooting medium has low salt (ex. % or salts of M.S., medium and reduced sugar levels

(usually 1g) for rooting. But in moss a species o.1 -I mg/l of NAA or IBA is required for rooting
Root of sho0ots is a very important part of plant tissue culture. Micro shoots (2.5cm long) are excised and
rooted. Roots may be induced in-vitro i.e. aseptically within the micro shoots are taken to the green house for
rooting directly in vermiculture /potting mix.
In-vitro rooting involves transfer of individual shoots of 2 cm on to the rooting medium in a culture vessel.
Plantlets with o.5 to lem roots are usually transplanted into pots since longer roots tend to get damaged during the
transfer.
Ex -vitro rooting/ in vivo rooting: In these methods, the cut ends of shoots are treated with a suitable
auxin solution or rooting power mix, transplanted in pots and kept under high relative humidity and low light
intensity. This saves cost as rooting and soil transplant stages are combined and the rooting medium is eliminated.
Rooting taken about 10-15 days, depending mainly species.

EX-VITRO ROOTING OFFERS SEVERAL ADVANTAGES OVER IN-VITRO ROADING:

. Structural and functionally better roots.
2. Better rooting ofrootingof difficult to root species.
3. No riek of labor & media reagent required for in-vitro rooting and hardening steps.
Further, it may be pointed out that roots produced in vitro are often thick, lack root hairs and have poor
vasculator connections as a result; they often die during transfer to soil.

STAGE -Iv.EX-VITRO ESATBLISHMENT/ HARDENING

This stage involves transfer of n-vitro plants to the soil after acclimatization. Acclimatization is the
process that must undergo in order to make the transition from the culture environment (RH-98-99%, low light
intensity, complete aseptic environment using sucrose as the carbon source) to the green house of field.
Thus to make the micropropagation plants survive on transfer to the field, they are gradually hardened to
withstand the exposure to the stress of lower. Rh (20%-60%), higher light intensities exposure to pathogens and
autotrophic growth.

The Procedure For Ex-vitro Hardening (Outside Culture Vessels)Involves The Following Steps:
1. Rooted shoots are removed from the medium, agar sticking to their roots is washed with tap water and they are
transferred to plastic cups/portrays containing potting mix.
2. Plants are kept for 10-15 days in high (90% or more) humidity and initially low light intensities (high humidity
can be attained by producing fog, water drop 10mm or less)
3. Potting mixture should not be too wet and water drops should not form on the plantlet.
4. The high humidity is gradually reduces to the ambient level over a period of 15-30
days and the light intensity is
also gradually increased.
5. The plants are finally exposed to the G.H. conditions.
 OMATIC EMBRYOGENESIS:
In some plant s.M. the explants having embryogenic potential form somatic embryos directly from the

explant. ex: altalfa, carrot yellow-popular etc.

Now protocols are available for large scale production of S.E in these crops.

STAGE -II IN-VITRO ROOTING OF PR0PAGULES:

Shoot produced at stage -II are small in size (miniature or micro). At stage -IlI stem are taken to elongate

and proliferate the micro shoots and then root induction.

levels
In general, the rooting medium has low salt (ex. h or h salts of M.S., medium and reduced sugar
or IBA is required for rooting.
(usually 1g/l) for rooting. But in moss aspecies o.1 -I mg/l of NAA and
Micro shoots (2.5cm long) are excised
Root of shoots is
very a important part of plant tissue culture. house for
rooted. Roots may be induced in-vitro i.e. aseptically within the
micro shoots are taken to the green

rooting directly in vermiculture /potting mix. medium in a culture vessel.

involves transfer of individual shoots of2 cm on to the rooting
In-vitrorooting the
roots tend to get damaged during
Plantlets with o.5 to l cm roots are usually transplanted into pots since longer
transfer.
are treated with a
suitable
Ex -Vitro rooting/ in vivo In these methods, the cut ends of shoots
rooting: and low light
power mix, transplanted in pots
and kept under high relative humidity
auxin solution or rooting
the rooting medium is eliminated.
intensity. This saves cost as rooting and soil
transplant stages are combined and
Rooting taken about 10-15 days, depending mainly species.
OVER IN-VITRO ROADING:
EX-VITRO ROOTING OFFERS SEVERAL ADVANTAGES
I. Structural and functionally better roots.
2. Better rooting of rooting of difficult to root species.
3. No rick of labor & media reagent required for in-vitro rooting and hardening steps.
in vitro are often thick, lack root hairs and have poor
Further, it may be pointed out that roots produced
vasculator connections as a result; they often die during transfer to soil.

STAGE-IV.EX-VITRO ESATBLISHMENT/HARDENING;
after acclimatization. Acclimatization is the
This stage involves transfer of In-vitro plants to the soil
process that must undergo in
order to make the transition from the culture environment (RH-98-99%, low light
sucrose as the carbon source) to the green house of field.
intensity, complete aseptic environment using
to the field, they are gradually hardened to
Thus to make the micropropagation plants survive on transfer
withstand the exposure to the stress of lower. Rh (20%-60%), higher light intensities exposure to pathogens and

autotrophic growth.

The Procedure For Ex-vitro Hardening (Outside Culture Vessels)Involves The Following Steps:
1. Rooted shoots are removed from the medium, agar sticking to their roots is washed with tap water and they are
transferred to plastic cups/portrays containing potting mix.
2. Plants are kept for 10-15 days in high (90% or more) humidity and initially low light intensities (high humidity
can be attained by producing fog, water drop 10mm or less)
3. Potting mixture should not be too wet and water drops should not form on the plantlet.
4. The high humidity is gradually reduces to the ambient level over a period of 15-30 days and the light intensitvy is
also gradually increased.
5. The plants are finally exposed to the G.H. conditions.
 6

Micro propagation
sexual. Sexually
of propagation may be either asexual or
In native, the methods plant are not true
-

to
of heterogeneity since their seed progeny
propagated plants demonstrate a high amount
on the other hand, gives
have been derived from inbred lines. Asexual reproduction,
-type unless they perpetuation of the
which genetically identical to the parent plant and this permits
rise to plants are

the cultivars. Asexually plants can be perpetuated

through in vivo and invitro
unique characters of and even
of certain plants is often difficult, time consuming
techniques. The in vive clonal propagation
alternative means of vegetative
unsuccesstul the invitro or tissue culture techniques offer an
to as "micro
The clonal propagation through tissue culture is popularly referred
propagation. to
can be achieved in a short
time and space. Thus, it is possible
propagation". Micro propagation
from a single individual. Use of
TC for micro propagation was
in
produce plants large numbers starting
for micro propagation.
initiatedby G. Morel in 1960 who found this as the only commercially approach

Techniques (1978)
Micro propagation is complicated process requiring many steps or stages. Murashige
a
of clones commercially
proposed 4 distinct stages that can be adopted for overall production technology
where as stage IV is accomplished in a greenhouse
stages 1-l are followed under in vitro conditions,
environment. Debergh and Maene (1981) suggested an additional stage o for various micro propagating
and product
systems. The adoption of all these stages not only simplifies the daily operation accounting
cost. but also allows for greater ease in coommunication with other laboratories. Thus, a particular plant
can be marketed or requested by specifying its stage.
The different stages of micro propagation are as follows.
Stage 0: seiection and maintenance of stock plants for culture initiation.
Stage I: Initiation and establishment of aseptic culture (Main steps : explant isolation, surface
sterilization. washing and establishment on appropriate culture medium).
Stage II: Multiplication of shoots or rapid somatic embryo formation using a defined culture medium.
Stage II: Germination of somatic embryos and /or rooting of regenerated shoots in vitro.
Stage IV: Transfer of plant lets to sterilized soil for hardening under green house environment (in a few
cases this stage may also include the in vivo rooting of regenerated shoots by shipping stage II).

Stage 0: This is an initial stop of micro propagation in which stock plants used for culture initiation are
grown for at least 3 months under carefully monitored conditions. Stock plants are grown at a relatively
low humidity and watered either with irrigation tubes or by capillary sand beds or mats. This stage also
includes measures to be adopted for reduction of surface and systemic microbial contaminants.
Stage I: The main steps involved are preparation of the explants followed by its establishment on a
suitable culture medium. Cultures are initiated from explants of several
organs but shoot tips and
axillary buds are most often used for commercial micro propagation. Stage I lasts for few
months to 2
years and requires at least few passage of the sub culture.
Stage Il: This stage takes up the bulk of micro
propagation activity using a defined culture medium that
stimulates maximum profanation of regenerated shoots. Various
(i) Multiplication through the growth and
approaches are:
shoots of the parent plant.
proliferations of meristems excised
from apical and
axillary
 Gi) Induction and multiplication of adventitious met stems through processes of organogenesis
somatic embryogenesis directly on explants.
(ii) Multiplication of call derived from organs, tissues, cells, or protoplasts and their subsequent
expression of either organogenesis or somatic embryogenesis in serial sub cultures.

passage or harvest cycle generally requires 4 weeks. Shoots are harvested from the multiplying
culture to either be sold as a stage -II product or carried on to stage -III. Generally stage -II lasts 10-36
months with a large number of subcultures of similar age.
Stage M: Shoots proliferated during stage II are transferred to a rooting (storage) medium. Sometimes
shoots are directly established in soil as micro cuttings to develop roots. Since such a possibility
depends on the particular species and at present, a large no of species cannot be handled in this manner,
the shoots are generally rooted in vitro.
Stage IV: Here the plantlets of stage Ill form the aseptic environment of the laboratory are transferred
to the greenhouse. Uprooted stage II shoots are also acclimatized in suitable compost mixture or soil in
pots under controlled conditions of light, temp, and humidity inside the greenhouse. In such cases stage
s shipped. Supplying bottom heat and maintenance of a dense fine-particle fog system within the
green house enhances the rooting process. Complete plants cannot be established in the artificial
growing media such as soil less mixes, or even sponges. It takes 4-16 wecks for the finished product to
be ready for sale or shipment.

Modes of invitro multiplication: In vitro multiplication can be done by any of the three ro
1)Multiplication by axillary and apical shoots: Axillary and apical shoots contain quiesc
active men stems depending on the physiological state of the plant. Vascular plant with an indeterminate
mode of growth have in their leaf axils subsidiary men stems with the potential or growing in to a
shoo. However, only a limited number of axillary meristems have the capacity to develop in vivo if the
type of branching of a particular spp displays apical dominance. When the shoots of such spp are grown
on culture media containing cytokinin, axillary shoots develop precociously which prolife rats to form
clusters of secondary and tertiary shoots. These clusters can be further subdivided when sub cultured on
a fresh medium. About 5-10 multiplication rates can be achieved with in 4-8 weck micro propagation
cycle, which may ultimately lead to extremely rapid clonal propagation. In general, the technique of
proliferation by axillary shoots is applicable to any plant that produces regular axillary shoots and
responds to cytokinin such as BAP, 2-1P and zeatin. Many forest and orchid spp are good examples for
micro propagation using axillary shoots. Apical shoots are normally cultured on media containing
mixtures of auxin (0.01-0.1 mg ) and cytokinin (0.05-0.5mge") The level of cytokinin in raised in
subsequent subcultures to induce an acceptable rate of proliferation without yellowing or distortion of
shoots.
2) Multiplication by adventitious shoots: Adventitious shoots are stem and leaf structures that
arise naturally on plant tissues located in sites other than at the normal leaf axils. These include stems,
bulbs, corms, tubers and rhizomes. Almost every one of these organs can be used as a cutting in
conventional clonal propagation. A similar type of adventitial shoot development can be induced in
cultures by using a suitable explants from preconditioned plant material and appropriate levels of growth

regulators in the medium. Clonal propagation by embryo formation is another useful

adventitious
can arise directly from a group of cells
approach followed for many plant spp. Adventitious embryos
Orchids produce a large number of embryoids at
within the original explants or from primary embryoids.
 p of leaver in vivo, while cultivars of Citrus and mango develop polyembryos from the nucellar
uSsuc. Similarly adventitious embryos obtained by inducing embryogenesis on explants, are goou
materials for micro
propagation. For example, leaf pieces of coffee form embryos directly when culture
on a basal MS medium
containing high levels of cytokinin.
3 Multiplication through cultured culture: Differentiation of plants from cultured cells via shoot
root formation or somatic embryogenesis, where applicable, can be the fastest method of shoot

most series drawback in the use of callus

multiplication and cloning of plant species. However, the
cultures for shoot multiplication is the genetic mist ability of their cclls due to which the initial plant
regeneration capacity of the tissue may decline with the passage of time.

Factors affecting shoot multiplication:

1) Physiological statures of plant material: Explants isolated from the more recently produced
of
parts of a plant are more regenerative than those from older regions. The regenerative potential
tissue culture diminishes with each year of maturation, particularly in perennial woody species. The
season can also be linked to regenerative capacity of tissue cultures. Papaya tissue cultures can be
established in hot summer months, whereas flower-stem explants of tulips give rise to shoots only
hen excised during dry storage (document) phase. Once elongation of stems has commenced

following dormancy the regenerative potential of will be lost. Nodal explants ot

tulip explants
Dioscorea alata yams produce axillary shoot growth only when excised from donor plants growing
under a 16-hr photoperiod. Exposure to shorter photoperiods given rise to explants which either do not

grow or show prolific callus development.

Familiarity with the donor plants natural propagation mechanism, especially with reference to season
and the growth stage is very much helpful in determining the more suitable explant source.
2) Culture media: The standard TC media are more suitable for achieving stages I & Il of micro
propagation. Only stage II requires some modifications. The relative proportion of mineral
components and two types of growth regulating substances in the culture medium largely govern the
overall micro propagation performance of a culture system. Among minerals, the relative
concentrations of NH, and K' in the medium influence the number and size of shoots produced from
explants. These mineral components also affect the induction of somatic embryogenesis in cultures.
To induce adventitious root formation, cytokinin is usually omitted and auxin added. Depending on
the spp. Root formation may also medium without hormones. Gibberellins and ABA in the
occur on a
medium are reported to inhibit root formation. Activated charcoal may also induce the
formation of
adventitious roots in some species. Its pres3ence in the medium reduces the
light supply to invitro
regenerated shoots and helps in removing inhibition by the adsorption of all such compounds released
in cultures.
3) Culture environment: Although green, the in vitro
regenerated shoots grow as heterotrophs
since they derive all of their nourishment
(organic well as inorganic) from the culture medium.
as
Studies have indicated that light, apparently absorbed
by photosynthetic pigments in cultured
plays an imp role in inducing the morphogenesis of these tissues. The tissues,
shoot multiplication in most of the species IS T000 lux. optimal light intensity found for
The quality of light also
organogenic differentiation and growth of shoots in cultures. For eg. Blue controls the
formation in tobacco shoots and doubled the number of light (467 mm) induced bud
lettuce shoots. The red
induction of flower buds and far red light stimulates root light stimulates
production.
 notoperiodic effects are dependent on the
relative sensitivity of the individual species. A diurn
lumination of 16 hr day and 8 hr
might is generally found satisfactory for multipiicann and
proliferation of shoots
although there are exceptions such as cauliflower which require 9 hr day light
regimes. Icmperature also forms an important part of the culture environment, in most micro
propagating cultures, it is maintained at 25+2°c. Some cultures may
require initial low temperature
(18C: Begonia x cheimantha
hybrid tissues) for morphogenic response. Another factor that affects the
periormance of cultures during
Culture vessels. And micropropagation is the constitution of the gas phase with in the
ethylene, oxygen, carbon dioxide, ethanol and
active gases with
possible effects on morphgenesis and may
acetaldehyde are metabolically
of all these gases is complex and requires detailed studies tounorganized
interaction promote growth of cells. 1he
gas phase on the micro ascertain the real effects of the
4)
propagation systems.
Genotype: Screening and selection of
fruitful approach in the genotypes among segregating populations could be a
improvement micro propagation capabilities of plant species which are
of
recalcitrant in tissue culture. The different
cultivars of a species may differ with
regenerating capacity. Hence protocol developed for one cultivar regard to in intro
of a species may have to be
altered while extending it for
another cultivar. slightly

Factors affecting invitro rooting: Media

for having low concentration of auxin have proven satisfactory
rooting of shoots micropropagated at stage II. For
species in which induction of shoot multiplication
requires a full -strength MS medium, reduction of the salt
found satisfactory for
concentration to half or one-quarter has been
rooting. Roots are mostly induced in the presence of a suitable auxin (NAA or
IBA 0.1- 1 mgl') in the medium
although the shoots of some plants (gladiolus, Narcissus and
strawberry) may readily root on a hormone-free medium. Riboflavin is
of root system in reported to improve the quality
Eucalyptus similarly Phloroglucinol has been found to promote
rosaceous fruit trees. rooting in a number of
Hardening /acclimatization
Micropropagation on a large scale can be successful only when plants after transfer from culture
to the soil show high survival rates
and the cost involved in the
process is low. TC plants generally show
some structural and
physiological abnormalities like a) abnormal leaf
-

morphology and anatomy, b)

poor photosynthetic efficiency c) marked decrease in
epicuticular was and d) mal functioning of
stomata. These characteristics as well as a
heterotrophic mode of nutrition and a poor mechanism of
water-loss control further render micropropagated
plants vulnerable to transplantation shocks.
Therefore, the transfer of individual plantlets to a potting mix and their acclimatization under
greenhouse conditions require the application of various methods to harden the plants for
transplantation.
Plants are transferred to the soil
after in invitro rooting stage. However, the induction of
usually
invivo rooting of cultured shoots may be more economical besides producing
good quality roots it is
essential that the lowest parts of micro shoots be washed thoroughly before their transfer to the
potting
mix (peat, vermiculite, soil, sand or their mixtures in difierent proportions). Transplanted plantlets are
immediately irrigated with an inorgamic nutrient solution and maintained under high humidity for initial
4-5 weeks. This is recued because plantlets during culture are adapted to almost 90-100% humidity.
High humidity can be built up around transplanted plants by covering them with clean
transparent plastic bags having a small hole for air cireulation. The size of the hole can be enlarged after
to 4 weeks order to reduce the humidity. This enables ramets to adapt well to green nouse
in

conditions and to establish functional roots. Partial defoliation of ramets and applicatiorn or
antitranspirants (1/ Acropol v/u) in the initial stages improved the survival frequency due to reauction
waterloss by plantlets. Most commercial labs have computerized hardening rooms or mist chambers

with controlled conditions of light, temp and humidity.

Direct transplantation of cultured plants (rice, tobacco) to the field has also been tried.
and grease or
Transplants survived with high frequencies provided a thin film comprising 50% glycerol
brush betore
paraffin in an equal amount of diethyl either is applied on the surface of leaves with
a

transplantation.
Attempts have been made to harden the shoot system by inducing autotrophism and development
induced either
of surface way on invitro formed leaves. Increase in epicuticular way deposition could be
Dy exposing cultures (cabbage) to CaClh These treatments also helped
in reducing humidity. However,
RH can also be reduced
CaClh may prove detrimental to overall growth and development of plants. The
This approach resulted in
by opening the culture tube inside a desicecator with CaSOg as the desiccant.
hardening of TC of carnation in vitro
plants of way after of days. These plants then
by development
acclimatized casily and had a higher survival rate (96%).
and can be
Storage organs induced in the cultured shoots of several spp do not require hardening
of
directly transplanted to additional step of rooting of shoots is altogether eliminated. Transplantation
show high germination (75-80%) under
micropropagated plants can circumvent this problem as they
field conditions.

Technical problems encountered in micropropagation:

1) Contamination of cultures: During large scale micropropagation of some plants certaint types of
slow growing (bacterial) contaminants persist even after initial surface sterilization of explants. Such
contaminants (Pseudomonas sp, Erwinia sp and Bacilturs sp) may persist for may generations
without being noticed and cause reduction in vigour or chlorosis of propagated plants. Addition of
antibiotics or fungicides to the culture medium may control the infection by microbial contaminants.
2) Browning of the medium: A problem more frequently associated with micropropagation of woody
perennials is the accumulation of in habiting substances in the growth medium during initiation of
cultures. Explants from adult tissues of these species often produce excessive amounts of Phenolic
substances which up on oxidation turn the medium dark brown such a medium is toxic to the tissues
and inhibits their growth. Quick transfer of explants to a fresh medium at short intervals could avoid
this problem in orchid bud cultures. Browning of the medium may also be prevented by dissecting
explant tissues under the surface of liquids or by incorporating ascorbic acid or citric acid in culture
media.

FACTORS INFLUENCING IN VITRO GROWTH AND MORPHOGENESIS.

A) The effects of genotype:

The growth of cultures tissues or organs and invitromorphogenesis appears to be profoundly
influenced by gem type than by any other factor. 'Thus the media
and the cultural
need to be varied from one genus or
species of plant to another, and even
environment often
plants can differ in their cultural
requirements. 'The results obtained in closely related varieties of
any particular test depend on
 the medium, growth regulators protocols are obviously those which produce. Reproducible resui
among the widest range of genetic material.
Many investigators have encountered differences among cultivars in the degree of and pattern of shoot
bud proliferation in vitro. These difference have been observed not only among, hut also within the

different genomic groups of musa section of bananas. The varying degrees of in vitro bud proliferation
suggest that the levels of endogenous growth regulators different between genotypes (George and
Sherrington, 1984).
B) The effects of the environment:
i)Temperature: The average temperature, maintained in culture rooms may be higher or lower than
those which could be experienced by plants of the same species in invo. Most tissus cultures are
maintained at the same divrnal temperature, The average
temperature employed in a large sample
Cxperimental reports to be 25°C (with a range between 17'C to 32°c). Tropical and subtropical plants
tend to respond letter when cultured at stightly higher temperatures (av. 2707 C range 24.0°c 27.0°C).
ii) Humidity:
The relative humidity in growth chambers or rooms used for cultures is frequently given as 70 % but
that inside culture vessels needs to be
greater. Jones (1974) found that carrot embryodis were best kept
at a relative humidity of 80 to 90
per cent. They all died if it was less than 60 per cent. By contrast,
Ziv et of (1983) found high humidity (98%
RH) inside culture vessels appeared to assist in the
formation of vitrescent shoots of sorghum become necrotie and died off it
they were covered by water
droplets formed due to condensation.
ii) Light:
Light is an important factor in growth and development of plants, since both photosynthesis and
photomorphogenesis are dependent on it. The three qualities of light which most clearly influence the
morphogenesis in vitro are wavelength, density and plotoperiod. The effects of light on photosynthesis
in vitro be of less
seems to importance due to the heterotrophic nature of the cultures. The initial cell
division of explants and the
growth of the callus tissues are sometimes prevented by light.
iv) Containers/ Closures:
The nature of the vessel used for tissue culture studies affects the
growth of explants. Glass tubes or jars
have an advantage can be washed and reused
though relatively expensive because they are made of
borosilicate glass.
Vessel size seems to affect initiation,
proliferation and morphogenesis of explants in vitro.
C) Tissue dependent factors:
The type of explant, its size and the manner in which
it is cultured, can all affect the initiation of
cultures and further morphogenetic
response. However in choosing a suitable explant, the
should consider the following points: investigator
The explant age
Explant size and source
Polarity of regeneration
Type of in vitro technique tor which the
explant is being used.