Plant tissue culture by s. kasthuri

MORPHOGENESIS
ORGAN
CULTURE TISSUE CULTURE
VEGETATIV
E ORGANS
Root tip
culture
Shoot tip
culture
Leaf tip
culture
REPRODUCTIVE ORGANS
Complete flower culture
a. Isolated ovary culture
b. Ovule culture
c. Anther culturepollen culture
d. Embryo culture
e. Seed and fruit culture
f. Seed and fruit culture

Tissue culture
Tissue culture involves the
aseptic culture of an
isolated homogenous mass
of cells.
1. Cell suspension culture
2. Single cell culture 1.Isolation of Protoplast
2.Protoplast culture
3.Somatic hybridization
or Parasexual hybridization
4.Somoclonal variation
5.Cryopreservation
6.Secondary metabolites
7.Meristem culture
8.Micro propagation
Or Somatic propagation
9.Somatic embryogenesis
10.Artificial seeds
11.Application of tissue
culture in Horticulture and forestry.

Morphology
Morphology is study of structure and form of
plant organs.
Morphogenesis
Formation of shape , Formation of body
organization and symmetry.

Differentiation (Division of Labour) Morphogenesis (Origin of Form)
Growth (Cell Multiplication)
Changes of form
and Function,
(organells, cells
Tissues and
organ)
Formation of shape,
Formation of body
organization and
symmetry
Quantitative increase
Cell Division and Cell
Enlargement
Development
(Cell Differentiation)

Trimming of Explants for Micro propagation

Stages in Banana Micropropagtion
Initiation

Multiple shoots Shoot Elongation

In vitro rooting Weaning of Agar

NEXT PREVIOUS END
Field view of Tissue culture banana plants

NEXT PREVIOUS END
Synchronous fruiting of banana plants raised through
tissue culture.

MICRO PROPAGATION
 The asexual or vegetative propagation of whole
plants using tissue culture technique.
Banana belonging to the genus Musa sps are
among the most important food crops and they are
the stable food for atleast 400 million people.
Banana ranks third in importance among the
fruits of the world.
In India it ranks second both in area and
production accounting for nearly 12% (4,3300ha)
of the total area under fruits crops and over
30%(10.46 million tonnes) of the total fruit
production.

• In Tamil Nadu the total area under
cultivation is 83,398 ha with an annual
production of 3.69 million tonnes.
• Its a major tropical food crop with annual
world production of around 40 million
tonnes.
• Bananas being parthenocarpic and seed
sterile in nature can be propagated only be
vegetative.

II Laboratory facilities: Propagation and
Sterilization media and glassware.
III Choice of explant
Cultivar Explant Rate of Propagation
Musa accuminata Rhizome tip Slow growth
Poovam Rhizome tip Slow growth
Monthan Rhizome tip Slow growth
Nendran Rhizome tip Fast growth
Rasathali Rhizome flower tip Fast, slow,
moderate.
Neypoovam Rhizome flower tip No growth

PROTOCOL FOR BANANA
Stage I Explanting shoot tip from sword sucker
(18cm diam and 35 cm long)
washed well in raining tap water to remove
soil particles
Surface sterilization (0.1% Hgcl2 ;30minutes)
Rinse 2-3 times in double distilled water
Stage II Culture of shoot tips in liquid medium followed by
transfer to semisold medium
Incubate all the cultures of 25±2c
Temperature
Stage III Multiple shoot culture production
Shoot culture transfer to rooting medium

Stage IV Rooted plants were seperated washed in tap water and
transplanted in 20X13cm Polythene bags filled with plotting
mixture (1:1:1) of river sand farmyard manure (Fym) and topsil.
The plants have were hundred in a mist chamber maintained at 70-
80% R.H for 3-4 weeks.
Transfer the plants in poly bags to the open for sale.

Micropropagation Of Eucalyptus
 Eucalyptus is a forest tree recently introduced into india.
 It is valuable for the fragrant oil, its leaves and for it wood.
 Plant propagated by seeds do not breed true to the parent.
 Vegetative propagation of Eucalyptus by rooting of cutting or by
grafting has been successful in a few species.
II Laboratory facilities:
Propagation and sterilization media and glassware.
III choice of explant : Eucalyptus mature tree
IV Protocol for Eucalyptus

Stage I Select and cut a twig from mature elite trees.
(60-90m cm ,19 -15cm wide)
Cut them into small pieces of about 5-8 cm Auxillary shoot buds.
transfer the buds to a sterile 250ml conical
flask.
Surface sterilization
Inoculate 2 pieces to each tube on medium
B5
Satge II Incubate at 15±2ºC and at 1500 lux light intensity for 72 hrs.
after 72hr to another incubator maintained
at 25±2ºC 1500 lux light intensity for 16 hrs
photoperiod
After 25 days the young buds start sprouting
Transferred to liquid medium D

Incubate the flask on a rotary shaker at 120rpm at 500 lux
light intensity
After 10-15 days formation of multiple shoots is observed.
transfer the multiple shoots from the flask to a sterile petridish
aseptically.
StageIII Seperate the shoots under sterile condition and cut them into pieces.
Incubate the culture at 1500 luc light intensity with 16hr
photoperiod at 25±2ºc
After 25 days and about 10 shoots are formed per explant
Seperate the shoots aseptically and culture them on medium
. Incubate these culture at 25-28c for 48 hr
remove the shoots from medium E and put them on medium for
aseptically .
Incubate these culture at 25±2ºc under 1500 lux light intensity with 16 hrs Photoperoid.
Stage IV Formation of roots is observed after 8-10 days.
Remove the plants after 15 days and transfer them to pots (sterile soil: Sand mixture (1:1)

Result :
In a year over 100,000 plants of
Eucalyptus from single bud of mature tree

Introduction:
Plant tissue culture:
Plant tissue culture is culturing of any part of the plant in a
specially defined growth media under aseptic laboratory condition
in petri dishes, test tubes or in any other suitable glass containers.

The plant nutrient media consists of macro and micro salts, vitamins
and desired levels of plant growth hormones.
Depending upon the plant species, genetic nature and with help of
above supportive media, various forms of callus / embryos / shoots /
roots or direct plantlets can be induced.
Obtaining plants through the above techniques is generally known as
plant tissue culture.

Banana is a globally important fruit crop with 97.5 million tones
of production.
In India, it supports livelihood of millions of people.
Banana occupies 20% area among the total area under crop in
India and contributes 37% of the total fruit production and ranks
second in importance next to mango with a total annual
production of 16.91 million tons from 490.70 thousand ha. With
national average of 33.5 T/ha.

Maharashtra ranks second in area and first in
productivity and production with 60 T/ha.
As per an estimate, India occupies the third place in
annual banana production.
However, in spite of large potentialities, there is no
appreciable presence in the export trade.

This is due to several factors; chief among them is poor yield due
to biotic losses.
One of the major impediments to extending the area under
cultivation of banana is non-availability of disease diagnosed
planting material.
In India banana is grown under diverse conditions and
production systems.

Selection of varieties therefore is based on a large number of
varieties catering to various kinds of needs and situations. Around
20 cultivars viz.
Dwarf Cavendish, Robusta, Monthan, Poovan, Nendran, Red
banana, Nyali, Safed Velchi, Basarai, Ardhapuri, Rasthali,
Karpuravalli, Kathali and Grand naine etc., were cultivated in
different parts of India.

Edible bananas do not produce seeds.
The main method of vegetative propagation in banana is by means
of daughter suckers formed at the base of the pseudo stem suckers
(5 to 10 in number depending on the variety).
Traditionally, sword suckers with narrow leaves, weighing
approximately 500-1000 gm are the preferred planting material for
vegetative propagation.

The major constraint for conventionally propagating banana is the
lack of ready availability of large quantities of sword suckers at any
given time.
The problem is felt more acutely in non-availability of sword suckers
consistently.
Besides, suckers generally may be infected with some pathogens
and nematodes.

Similarly due to the variation in age and size of sucker, the crop is
not uniform, harvesting is prolonged and management becomes
difficult.
Therefore, in vitro clonal propagation i.e. tissue culture plants
(properly hardened secondary seedlings) are recommended for
planting as they are healthy, disease free, uniform and authentic.

The sterile operational nature of tissue culture procedures excludes
fungal, bacteria, and pests from the production system, which
means that sigatoka, Panama disease, weevils, and nematodes
cannot be transmitted through the TC micro-propagation process.
However, viruses, such as the banana bunch top and the episomal
form of banana streak virus, are not eliminated by tissue culturing
unless measures are taken to prevent the transmissions from
happening (e.g., virus indexing).

Banana plants produced from tissue culture are free from diseases at
the time of supply and they give high yields since they are made from
selected high yielding mother plants.
If proper care is taken, as per instructions, they grow into strong healthy
plants and give high yields of good quality fruits.
Since they are produced under controlled laboratory conditions using
selected nutrients, they usually give yields one or two month earlier
than conventionally propagated plants.

Advantages of Tissue Culture micro propagation :
1. Initiation and establishment of rapidly multiplying aseptic shoot
cultures can eliminate the problem of low sucker multiplication rates
effectively and economically.
2. Large number of uniform propagules can be generated in a relatively
short period of time.
3. Variability encountered in size and propagules density especially in
clones suckering erratically can be minimized.
4. It could allow for rapid bulking of novel clones when used in concert
with breeding programs.

It would facilitate transcontinental exchange of disease diagnosed
planting material.
With refinement in preservation techniques, in vitro culture of
bananas can be of immense value in germplasm conservation
True to the type of mother plant under well management.
Pest and disease free seedlings.
Uniform growth, increases yield.
Early maturity of crop - maximum land use is possible in low land
holding country like India.

Round the year planting possible as seedlings are made available
throughout the year.
Two successive ratoons are possible in a short duration which
minimizes cost of cultivation.
No staggered harvesting.
95% - 98% plants bear bunches.
New varieties can be introduced and multiplied in a short
duration.

Establishing the tissue culture work, ideally a plant tissue culture
facility must consist of separate rooms for media preparation, aseptic
transfer, culture incubation and illuminated rack systems.
The process of tissue culture consists of five important steps:
Initiation, Multiplication, Shooting & rooting, Primary Hardening in
green houses and Secondary Hardening in shade houses.

Strict adherence to aseptic standards and micro-climatic
conditions and care during the hardening process alone can
ensure success.
The tissue culture process involves the micro-propagation of a
sucker growing point under sterile conditions.

A sucker is detached from the nursery parent plant and brought
to a laboratory where the outside tissue is pared away until only
the growing point remains inside a plug of 10 mm³.
This is placed in a jar on agar containing a nutrient solution in a
sterile environment and under controlled conditions of
temperature and light.
The growing point subdivides into several shoots, which are
subdivided and re-established on fresh agar. This process, called
sub-culturing.

The sub culturing, continues about five or eight times (one month
per sub-culture) until approximately 1000 plants are produced
from one original growing point.
These plants are then transferred to a rooting medium and when
fully rooted, they are transferred from in vitro conditions (sterile
under glass) to in vivo conditions (seedling trays in a greenhouse
environment).

After 6 to 8 weeks, the 5 cm plants are relocated from the
greenhouse trays to nursery bags in a netted shade house.
After another 6 to 8 weeks, the 20 cm plants are ready for planting
out in the field.
The entire process from excavating the original sucker to planting
out 200 mm plants in the field takes about 10 months.

Media details:
1. Initiation and multiple shoot induction: MS+ BAP 5 mg/L
2. Shoot Elongation: MS+ BAP 2 mg/L + IAA 0.5 mg/L
3. Rooting: ½ MS + IBA 0.5 mg /L + NAA 0.5 mg /L + 0.05%
activated charcoal
4. Hardening: Ex-agar plants in mist chamber in coco peat and
then in shade house for secondary hardening with sand: Red
soil: FYM 1:2:1 ratio for 15-45 days.

STAGES:
EXPLANT PREPARATION AND DISINFECTION:
Sword suckers are carefully removed from field grown fruiting
banana.
plants and traces of soil particles adhering over are removed by
repeated washing thoroughly in tap water and a solution of the
diluted detergent teepol.
Teepol are removed by repeated washing and the extraneous
rhizome tissues are carefully chopped with a stainless steel knife.

Trimmed suckers are now soaked in a solution of Bavistin (0.5%) –a
fungicide and streptocycline antibiotic for six to eight hours.
To prevent the oxidation of phenolic compounds, the trimmed buds
are stored in antioxidant solution (100 mg Ascorbic acid + 150 mg
Citric acid per litre of sterile water.) till the buds are taken to
laminar flow chamber for inoculation.

Shoot tips containing rhizome tissue and measuring 2.5 to 3.5 cm
in length are isolated, surface sterilized using 70% ethanol for 1
min and then with mercuric chloride.
Two different concentrations of mercuric chloride were used
First the sucker was sterilized using 0.12% mercuric chloride for
2 min. After that, the mercuric chloride was removed and the
sucker was washed using sterile distilled water.

At first, the sterile distilled water was added and the bottle was
shaken for 1min., then the water was removed and fresh sterile
distilled water was added, shaken for another one min and then
the water was removed with the following timings 1 min, 2 min, 3
min, 5 min and 12 min.
After the first sterilization, a layer of the sucker is removed
carefully.

The suckers are again sterilized with 0.1% mercuric chloride for 5
min. After that they were washed with sterile distilled water
following the timings 1min,1min, 2 min,3 min,5 min and 12 mi
Finishing the above process, another layer of the sucker was
removed. The sterilized shoot tip explants are handled using
sterilized stainless steel scalpels.

Cut surfaces of the rhizomatous tissue and leaf bases are further
trimmed so that shoot tips finally contain at least six to eight
overlapping leaf bases enclosing auxiliary buds.
A vertical cut is given (to arrest the apical dominance) and the
buds are inoculated in the semi-solid prepared for multiple shoot
induction.

The explants are now ready for inoculation and measures 1 to 2
cm. The optimum size of the explants depends on the purpose.
For rapid multiplication, relatively larger explants (3-10 mm) are
desirable despite its higher susceptibility to blackening and
contamination.
When virus or bacteria elimination is needed, meristem tip culture
is the preferred option.

The explants are further reduced in size (0.5-1 mm length) leaving
a meristematic dome with one or two leaf initials.
Meristem cultures have the disadvantage that they may have a
higher mortality rate and poor initial establishment.
Cultures should be incubated in the basal nutrient media
supplemented with plant growth regulators.
Thereupon the healthy, contamination free explants should be
taken for next multiplication stage.

For banana micro propagation, MS based media are widely
adopted. Generally they are supplemented with sucrose as a
carbon source at a concentration of 30-40 g/L.
Media are poured in a glass bottle where suckers are propagated.
In most banana micro propagation systems, semisolid media are
used.

. As a gelling agent, agar (5-8g/L) is frequently added to the
culture medium.
Banana shoot tip cultures are incubated at an optimal
temperature of 26±2⁰C in a light cycle of 12-16 h with a
photosynthetic photon flux(PPF) of 60µE/m2s
After 2 weeks, the suckers will become greenish in color and the
multiple shoots will arise from the base of the suckers

The shoots are cut at the base, separated and placed in a fresh
medium. In each bottle, three-five shoots were inoculated.
After 2-3 weeks, multiple shoots arise from the inoculated shoot.
Again they are separated and placed in afresh medium.
The sub culturing is done until they require amount of plants are
needed.

The shoots are every day checked for contamination and the
contaminated shoots are transferred to a fresh medium.
Meanwhile a set of well grown healthy shoots are taken for rooting.
MASS MULTIPLICATION
Contamination free explants are further cultured on multiplication
media supplemented with plant growth hormones which help in
proliferation of auxiliary buds (cytokinins) into multiple shoots.

These shoots are divided and multiplied to bulk up the multi
culture stock.The multiplication cycles are restricted to 8 because
beyond that banana is genetically highly unstable.
SHOOTING
Multi cultures are further divided and transferred to shooting
media which is composed of auxins (PGR) to get the elongation.
In this stage, leaves will develop and the whole plant will grow up
to 4 to 5 cm.

ROOTING:
Plantlets from shooting media are separated and single plantlets
are transferred to media containing charcoal and auxins or
medium without any growth regulators.
It will take 2-3 weeks for rooting and fresh roots arise at the base
of the shoot.
In this stage, roots will develop and plants will be ready for
dispatch from laboratory.

AGAR WEANING OF PLANTS
Well developed single plantlets need to be removed from the culture
incubation room and exposed to ambient conditions in the culture
vessel for four to five days.
The plantlets are then carefully removed and the roots washed in
running tap water.

Depending on the parameters such as location/the site of
planting, soil quality and the climatic conditions defined by the
customer, the ex-agar plant for sale could be in vitro rooted plants
or only the shoots.
When the tissue culture plants are sold at this stage, the plants are
washed in sterilized water to remove the agar medium.

The plants after being removed from nutrient media should
preferably be transplanted within 72 hours.
Polybags is separated from the plant without disturbing the root
ball of the plant and then plants are planted in the pits keeping
the pseudo-stem 2 cm below the ground level.Soil around the
plant is gently pressed. Deep planting should be avoided.

PRIMARY HARDENING
A quick dip in 0.5% Bavistin solution follows and finally in-
vitro plants are transferred to trays containing sterilized
coco peat.
These trays are kept under tunnels made of transparent PP
Plastic sheets to maintain the humidity above 80%.
These tunnels should be under 50% to 75% shade nets.

Primary hardening will take at least 4 weeks depending upon the
climatic conditions. In final week, these trays are gradually exposed
to 50% shade by removing plastic sheets.
These plantlets are sprayed with fungicides, bactericide, and water
soluble fertilizers as per schedule.
SECONDARY HARDENING
Primary hardened plants after 4 to 5 weeks are transferred to Poly
bags (Nursery Bags) of suitable size.

Soil mixture is prepared by mixing sand, soil and farm yard
manual into 1:2:1 ratio.
The plants are kept in these Poly bags for 6 to 8 weeks under 50%
shades.
Humidity is maintained around 60% to 70% and regular foliar
sprays of plant protection chemicals and water soluble fertilizers
are given regularly.

Any possible variation if observed is discarded at this stage
The plant ready for sale will be having 5 to 6 opened leaves and
almost 1 feet in height.
The plantlets after acclimatization should be transported to the
required place.
Normal transportation is done where the plants are placed and
grown in plastic bags.

Well grown plants are removed to provide space in green house for the
next cycle of plants and also to lower the cost of storage.
Problem of Banana micro propagation
Banana tissues often suffer from excessive blackening caused by
oxidation of ployphenolic compounds released from wounded tissues.

Therefore, during first 4-6 weeks, fresh shoot tips are transferred
to new medium every 1-2 weeks.
Alternatively, freshly initiated cultures can be kept in complete
darkness for one week.
Anti oxidants such as ascorbic acid or citric acid in concentrations
ranging from 10-150 mg/L, are added to the growth medium to
reduce blackening or the explants are dipped in anti oxidant
solution (Cysteine 50 mg/L) prior to their transfer to culture
medium.

Cryopreservation
Or
Cryobiology
Or
Germ plasm preservation
Or
Longterm invitro Storage

(Cryopreservation extreme cold derived from
latin word kruos=frost)
Preservation in the frozen state.
Definition :
The biological materials were generated
stored under a low temperature. This branch
of science that deals with the long term
storage of plant and animal materials under a
low temperature is named Cryobiology.

The storage of living specimen is called
Cryopreservation this is also named longterm
invitro storage. This cryobiology of plants was
focused on the preservation of fruits vegetables
and various products.

Storage of Germplasm
Insitu
Biosphere reserves
National park
Gene sanctuaries
Exsitu
1. Botanical garden
2. Nurseries
3. Seed Bank:
At room temperature
At cold temperature
4. Invitro:
 Slow growth technology
 Cryopreservation
5. Freezing method

Differentiate: Invitro - preservation &
Insitu
• Larger amount of material can be
preserved in small area.
• It provides large amount of plant material
for culturing, It overcomes the destruction
due to environmental hazards.

APPLICATION OF
CRYOPRESERVATION
1. Conservation of genetic uniformity.
2. Preservation of rare genomes.
3. Freeze storage of cell cultures and cell
lines.’
4. Maintenance of disease free material
5. Cold acclimasation and frost resistance.
6. Retention of Morphogeneic potential
inlong term cultures.

ADVANTAGES
1. Germplasm can be stored within small area.
2. Rare species can also be stored.
3. Maintenance plant material easy.
4. Disease free clones can be maintained.

Achievements made through
Cryopreservation
a) Cryopreservation of cell lines:
ex; cell suspension, somatic, hybrid, protoplasm.
(soybean, tobacco,carrot,etc)
b) Cryopreservation of pollen and pollen embryos: Ex; fruit
crops, trees, mustard,carrot,etc.
c) Cryopreservation of exised meristem Ex; sugar cane ,
potato, chick pea,etc.
d) Cryopreservation of germplasm of vegetatively
propagated crops ex; potato, sugar cane.
e) Cryopreservation of recalcitrant seeds and embryos.large
sized seeds that are short lived and abortive, such as oil
palm, coconut, walnut, mango,etc.

Plant cells bank / germ plasm / cell
Cryobank
• Cryopreservation of genetic stock i.e.
germplasm is a novel approach for their
conservation in liquid nitrogen on a long
term basis ( vegetative propagated crops,
rare plant species, horticultural, medicinal
plants, VAM fungi.

GENE BANK (OR) GERM PLASM BANK (SOME
OF THESE ORGANISATION
• ICAR- Indian council of agricultural
research: new Delhi
• IBPGR – International Bureau of plant
Genetic Resources ;UK
• NBPGR – National Bureau of Plant Genetic
resources ; New Delhi.
• CSIR - Council of Scientific and industrial
research: New Delhi.

Pollen Bank
• The storage of pollen grains in liquid nitrogen
and establishment of pollen bank have also
been suggested to retain their viability for
various length of time.
• Growth at different places.
• Reducing the dissemination of disease by
pollination vectors.
• Maintenance of germplasm and enhancement
• Hybridization between plants with flowers at
different times.

Method of cryopreservation
• Establishment of cell, tissue and organ
cultures(selection of materials)
• Addition of cryoprotectants
Freezing
Storage of frozen cultures in liquid nitrogen
at 196°C

Plant Regeneration of plants
Thawing of retrieved cultures
Removal of cryoprotectants (or) Cryogens
Determination of viability
Reculture of retrieved material
Regeneration of plants

• Some examples
Species Organ Storage
period
Survival
percentage
Daucus
carorota
Somatic
embryo
60 days 100
Manihot
esculentu
m
Shoot tip Not known 21
Arachius
hypogea
Shoot tip 3 weeks 23-31
Solanum
tuberosam
Shoot tip 5 min 42-76

Hairy root culture for
secondary metobolites

Hairy Root Culture: History, Formulation and
Application
History of Hairy Root Culture:
The term “hairy root” was first coined by
Steward et al. (1900). In 1930, Ricker et al., first
named the hairy root causing organism Phytomonas
rhizogenes, which was later renamed A. rhizogenes.
The first transformation of higher plants using A.
rhizogenes was done by Ackermann in 1973.

• Formation of Hairy Root Culture:
The t-DNA of the agropine-type Ri-plasmid
consists of two separate t-DNA region tl-DNA
and tr- DNA. The genes encoding auxin
synthesis (tmsl and tms2) and agropine
synthesis (ags) have been localized on the tr-
DNA of the agropine type of Ri-plasmid.

• Gene Transfer Mechanism from Agro-
bacterium Rhizogenes to Plant Genome:
The vir gene expression, generation of t-
DNA copy, formation of T strand protein
complex, movement of the T-complex through
the bacterial membranes, targeting of the T
complex into and within the plant cell, targeting
of the T complex into the cell nucleus, it’s
stabilization, and finally integration of T strand
into cell DNA are seven successive steps of
transfer of DNA from Agrobacterium to plant
cell.

• Step 1:
• Bacterial colonization on the wounding site of
plant tissue is prerequisite for transformation. The
production of phenolic compounds at the
wounding site is sensed by one of the Vir A gene
product which initiates induction of expression of
remaining Vir loci.
Step 2:
• The product of Vir C and Vir D play pivotal role in
this step. Two Vir D specific product Vir D1 and
Vir D2 are essentially required for synthesis of t-
DNA strand. The Vir C locus decodes for two
polypeptides Vir Q and Vir C2 that are shown to
enhance t-DNA border nicking.

• Step 3:
• The t-DNA strand is likely to exist as a DNA
protein complex. The Vir E, specially Vir
E2 protein is the most abundant protein synthe-
sized in Vir induced Agrobacterium cells. The
Vir D2 bounds to the leading end of the T-
complex. Thus T-complex is compressed of the
t-DNA strand, Vir D2 and Vir E2.
• Step 4:
• The product of Vir B locus produces trans
membrane channel outside the bacterial cell
wall because of its 11 open reading frame
known as Vir B1 to Vir B11 the last one helps to
pump the T complex out of the bacterial cell.

• Step 5:
• The uptake of T-complex into the plant cell though
yet not understood clearly but assume this
mechanism somewhat analogues to bacterial
conjugation.
• Step 6:
• The T-complex (T- DNA strand, Vir D2 and Vir E2)
in this step enters within plant cell – nucleus. The
N terminal of Vir D2 has role to nick the T-DNA
border while C terminus helps in the nuclear
uptake of the T strand. The Vir E2 help to Vir D2 to
target the T complex to the nuclear pore in a polar
direction which facilitates it’s linear uptake.

• Step 7:
• Generally t-DNA insertions can occur in any
chromosome of the plant genome or it may
occur randomly.
• As hairy root formation involves the transfer of
DNA from the bacterium to the plant nucleus
and the response of plant cell to the root
inducing plant hormone-auxin. It was noticed
that t| DNA of Ri plasmid appear to sensitize the
transformed cell to auxin, which determined the
root growth and typical characteristics of hairy
roots.

• Hairy Root Induction and Establishment of
Hairy Root Culture:
• To succeed in establishing a hairy root culture
system for a certain species, several essential
conditions should be taken into consideration.
These conditions include the bacterial strain of
A. rhizogenes, an appropriate explants, a proper
antibiotic to eliminate redundant bacteria after
infection, and a suitable culture medium.

• The confirmatory test for hairy root culture:
• (a) Morphological Characteristics:
• Pal-geotropism is common phenomenon in the
roots transformed with A. rhizogenes, have an
alter phenotype such as profuse lateral
branching, as a result due to increase bio- mass
and consequent increase in the number of
elongating tips.

• (b) Biochemical Markers:
• The
• opines are effective biochemical marker for
identification of transformed roots has been
done through paper electrophoresis. Due to
instability of opine genes within transformed
roots this process is not popularly used.

• (c) Genetic Marker:
• t-DNA identification of the host plant
genome acts as a reliable genetic
marker to confirm transformation. The
most widely used procedure is Southern
blot hybridization. Other procedures
include DNA “dot blotting“, localization
of t-DNA in plant chromosome by “in
situ hybridization” and Polymerase
chain reaction.

Somatic Embryogenesis
Question for discussion
What is somatic Embryogenesis
In Plant tissue culture, the developmental pathway
of numerous well-organised, small embryoids
resembling the zygotic embryos from the
embryonic potential somatic plant cell of the callus
tissue or cells of suspension culture is known as
somatic embryogenesis.
What is embryogenic potential?
The capability of the somatic plant cell of a
culture to produce embryoids is known as
embryogenic potential.

What is Embryoid?
Embryoid is a small, well-organised structure
comparable to the sexual embryo, which is
produced in tissue culture of dividing embryo-
genic potential somatic cells.
Brief Historical Background
Reinert (1958-59)
Reported his first observations of invitro somatic
embryogenesis in Daucus carota.
N.S. Rangaswamy (1961)
Studied in detail the somatic embryogenesis in
Citrus sp.

TYPES OF SOMATIC EMBRYOGENESIS
DIRECT
EMBRYOGENEISIS
(When explants
without production of
Callus)
INDIRECT
METHOD
EMBRYOGENES
IS
(When explants
produce callus
forms embryos)

Microscopic observation of suspension culture derived somatic
embryos of coriander showing different stages of development
a. Globular stage (X 20)
b. Heart stage (X 20)
c. Torpedo stage (X 20)
d. Cotyledonary stage (X 20)
e. Bipolar stage (X 10)
f. Bipolar stage. Note the first
leaf emergence (X 10)

INTRODUCTION
• Coriandrum sativum L. Commonly called coriander, is one of the
earliest spices.
• Coriander has been extensively cultivated in india and other Asian
countries.
MEDICINAL IMPORTANCE:
• Seeds and leaves
• Treatment of various oilments
• Indigestion, diuretic, body cooling, soothing, etc.,
Hence, we have depending on plant tissue culture
techniques and genetic transformation for improving the crop.

Leaf and stem explants
showing organogenesis
a,b: Shoot induction and
multiplication from leaf
explants
c,d: Shoot induction and
multiplication from stem
explants
e: Established plants in soil

Somatic embryogenesis from leaf and stem explants of B.
monnieri
a: Small protuberance emerge from cut ends of leaf explants.
b-c: Globular somatic embryos developed all over the surface of
the explants.
d: Maturation of embryos acquiring different shapes.
e: Germination of embryos.
f- Scattered somatic embryos.
g- Small protuberances emerge from cut ends of stem explants.
h- developing embryos.
i- Embryos maturation and germination.
j: Cross section of an embryo.
k-l: Multiplication of plantlets.

IN VITRO FLOWERING AND SEED FORMATION
 Seeds of coriander developing from the allogamous flowers arre
genetically variable in nature.
 Initiation of flowering and complete seed formation in vitro may
become a valuable research tool for plant breeders.
 To ensure seed purity.
Materials and Methods
• Explants : shoot tip, nodal segments
• Media : SH (Schenk and Hildebrandt, 1972)
• Growth regulators : GA3+IAA, GA3+IBA, GA3+NAA,GA3+2,4-
D and BAP

In vitro flowering and seed setting from shoot tip derived
explants of coriander.
a.Induction of in vitro flowers from
shoot tip explant.
b.Initiation of in vitro flowers.
c. In vitro seed setting.
The highest frequency of response -
number of roots and leaves were
obtained on BAP with combination of
GA3 and IAA (0.44µM +0.28µM +
(0.28µM). Among the other
combination (IBA, NAA, 2,4 -D)
induced the lowest response.

In vitro flowering and seed setting from nodal explants of coriander
a. Induction of in vitro flowers
from nodal explants.
b. Initiation of flowers.
c. Initiation of flowers showing
clusters of umbels.
d. In vitro seed formation showing
cluster of seeds.
e. A single node showing the well
developed in vitro seeds.
The nodal explants also responded to
a maximum 90% for regeneration
with combination of GA3, IAA and
BAP at concentration of 0.28 + 0.28
+ 0.44 µM respectively among the
other hormone concentration.

Ex situ germination of in vitro
derived coriander seeds
a. Germination of in vitro developed
coriander seeds and successful
initiation of flowering.
b. Seed formation from coriander
seedlings growing ex situ.

Organogenesis
Explants : Hypocotyl, cotyledon, cotyledonary
node, immature leaflet.
Media : Murashige and Skoog, 1962
Growth regulators : IAA, NAA, IBA, 2,4-D + BAP, 2ip + IAA,
NAA, IBA, 2,4-D

• a. Callus induction
• b. Multiple shoot induction
• c. Single shoot for elongation
• d. Shoot elongation
• Rooted shoot
• f. Well developed plantlets
established in a plastic cup.
The highest frequency of callus
induction (87.1%) was observed
on MS basal medium
combination with IBA and BAP
(9.84 µM + 2.22 µM ).
The maximum frequency of shoot bud
differentiation (88.7%) on basal
medium supplemented with 2iP +
IBA (14.76 µM + 2.46 µM)
Regeneration from cotyledonary explants of
coriander

 a. Callus formation
 b. Shoot bud development from
hypocotyl explant.
 c. Multiple shoot bud production from
hypocotyl derived callus cultures.
 d. Rooting of Regenerated shoot.
 e. Well developed plantlet established
in plastic cups.
The highest frequency of callus induction
(74.1%) was observed on MS basal
medium combination with IBA and
BAP (9.84 µM + 2.22 µM ).
differentiation (88.4%) on basal
medium supplemented with 2iP + NAA
(14.76 µM + 2.69 µM).
The highest frequency of root induction
medium combination with IBA and
KIN (9.84 µM + 2.32 µM ).
Regeneration from hypocotyl explants of coriander

Regeneration from cotyledonary nodal explant of coriander
a. Callus induction from cotyledonary
nodal explant.
b. Shoot bud formation.
c. Multiple shoot induction from
cotyledonary node derived callus.
d. Shoot elongation.
e. Rooted plantlet.
f. Regenerated plant growing in a
plastic cub showing successful
flowering in ex situ condition.
The combination of IBA and BAP (9.84
µM + 2.22 µM ) was found to be high
frequency of callus initiation (75.4%)
followed by 2,4 –D, IAA, NAA with
combination of BAP.
The maximum number of multiple shoots
on MS medium containing BAP or 2iP +
NAA (13.32 µM + 24.60 µM + 5.37 µM).

Regeneration from immature leaf explants of coriander
a. Callus initiation.
b. Shoot bud initiation from
immature leaf explants.
c. Shoot elongation.
d. Rooted shoot.
e. Regenerated plants
established in a plastic cup note
the flowering.
The highest frequency of callus initiation
medium combination with IBA and BAP
(9.84 µM + 2.22 µM ).
differentiation (88.4%) on basal medium
supplemented with 2iP + IBA (14.76 µM +
2.46 µM).

Somatic embryogenesis
Explants : cotyledon, hypocotyl, immature leaflet
Media : SH, Whites
Direct somatic embryogenesis
Growth regulators
Induction : NAA + BAP, NAA + KIN, NOA + Zeatin,
NOA + KIN
Germination : ABA + 2iP, GA3 + 2iP
Indirect somatic embryogenesis
Induction : 2,4-D, 2,4-D + Zeatin, NAA, NAA + Zeatin
Germination : 2iP + Zeatin, 2iP + ABA, 2iP + GA3

Direct somatic embryogenesis from cotyledonary explant of
coriander a. Induction of direct somatic
embryogenesis.
B. Initiation of somatic embryos.
c. Maturation of somatic embryos.
d. Maturation of somatic embryos
shows simultaneous shoot and
root development.
e. Somatic embryo derived plantlet
growing in a pot.
The NOA (0.98 µM) + Zeatin (0.04 µM)
combination was found to be the maximum
frequency of embryogenic response in
cotyledon (61.9%).
The NAA (0.55 µM) + BAP (0.44 µM) was
found to be the maximum percentage of
somatic embryos in Cotyledon (53.3%)
The embryo maturation and germination
was found on the combination of ABA (3.02
µM) + 2iP (4.91 µM) nearly (70.5%).

Direct somatic embryogenesis from hypocotyl explants of
coriander a.Induction of direct somatic embryogenesis (X
2.1)
b.Magnified view of direct somatic
embryogenesis (X 10)
c.Maturation of somatic embryos (X 1.6)
d.Cluster of germinated somatic embryos (X
1.5)
e.A single somatic embryo showing
simultaneous shoot and root formation (X 1.4)
f.Somatic embryo derived plantlet was
successfully transformed and established in a
plastic cups (X 0.6)
The NOA (0.98 µM) + Zeatin (0.04 µM) combination
was found to be the maximum frequency of
embryogenic response in hypocotyl (69.1%).
The embryo maturation and germination was found on
the combination of ABA (3.02 µM) + 2iP (4.91 µM)
nearly (75.3%) respectively within 35 days culture.

Direct somatic embryogenesis from immature leaflet explant of coriander
a. Induction of direct somatic
embryogenesis (X 1.7)
b. Magnified view of direct somatic embryos
showing shoot and root pole (arrows ) (X
2.2)
c. Cluster of matured somatic embryos
from immature leaflet explant (X 1.8)
d. Somatic embryo derived plantlet growing
in a plastic cup (X 0.6)
The NOA (0.98 µM) + Zeatin (0.04 µM) combination
was found to be the maximum frequency of
embryogenic response in immature leaf (65.1%).
The embryo maturation and germination was found on
the combination of ABA (3.02 µM) + 2iP (4.91 µM)
nearly (70.4%).

Indirect somatic embryogenesis from cotyledonary explants of
coriander.
a.Callus induction (X 2.2)
b.Induction of indirect somatic
embryogenesis (X 2.2)
a.Maturation of somatic embryos (X 1.4)
b.Cluster of somatic embryos germinated
with simultaneous shoot and root
formation (X 2.2)
c.A single somatic embryo derived plantlet
growing in a test tube showing well
developed root and shoot (X 1.8)
d.Well developed somatic embryo derived
plantlet growing in a plastic cup (X 0.7)
The embryogenic mass induction was high on
SH medium with 2,4 –D (0.9 µM) + Zeatin (0.9
µM) and casein hydrolysate (600mg/L)
combination produced the highest frequency of
somatic embryogenesis (78.2%).
The highest number of somatic embryos (367.5
embryo/callus) from cotyledon explants.

Indirect somatic embryogenesis from hypocotyl derived
explants of coriander
a. Embryogenic callus induction from hypocotyl
explant b. Induction of somatic embryogenesis.
c. Maturation of somatic embryos.
d. A single somatic embryo derived plantlet
growing in a test tube showing well developed
root and shoot.
e. A successful transfer of from somatic
embryo derived plantlet in ex situ condition.
The highest number of somatic embryos
(400.4 embryo/callus) from hypocotyl explants.

Indirect somatic embryogenesis from immature leaf explants
of coriander
a.Embryogenic callus induction.
b.Maturation of somatic embryos showing
simultaneous shoot and root formation.
c.A single somatic embryo derived plantlet
growing in a test tube showing well
developed shoot and root.
d.Somatic embryo derived plantlet
growing in a plastic cup.
The highest number of somatic embryos (368.4
embryo/callus) from immature explants.

Microscopic observation of somatic embryogenesis
a.Magnified view of single
piece of embryogenic calli note
the proembryos (arrows X 20)
b.Cluster of somatic embryos
(note globular (arrow) and
heart stage somatic embryos
(arrows) (X 20)
c.Cluster of torpedo stage
somatic embryos (arrow X 20)
d.Torpedo (arrow) and
cotyledonary stage somatic
embryos ( arrow X 20)

Somatic embryogenesis from suspension cultures and production
of “Synthetic seeds”
Explants : Cotyledon, Hypocotyl
Media : SH, Whites
Growth regulators
Induction : 2,4-D, 2,4-D+Zeatin, 2,4-D+NAA
Maturation : Whites, GA3+BAP, GA3+ABA, ABA+BAP
Germination :Whites, 2iP, 2iP+ABA, 2iP+ABA+NOA
Synthetic seeds
Explant : Somatic embryos, apical buds
Media : Modified MS, Sodium alginate
Storage : viability in months (0,2,4)

Somatic embryogenesis from suspension cultures of
coriander
a.Induction of somatic
embryos.
b.Maturation of somatic
embryos.
The maximum percentage of somatic
embryos in cotyledon (92.2%) and
hypocotyl (77.2%) with in the combination
of 2,4-D, (0.04 µM), Zeatin (0.09 µM) and
CM (15%) + glutamine (10 mg/L).
The highest number of somatic embryos
were noticed in hypocotyl (998.9
embryos/culture ) and cotyledon (925.0).

Germination of somatic embryos from suspension
cultures of coriander
a.Germination of somatic
embryos
b.Magnified view of somatic
embryos showing
simultaneous root and shoot
development
The maximum percentage of germination
(70.6%) showed in combination with the
optimum concentration 2iP (1.96 µM), NOA
(0.24 µM) and ABA (5.67 µM) .

Microscopic observation of suspension culture derived
somatic embryos of coriander showing different stages of
development
a. Globular stage (X 20)
b. Heart stage (X 20)
c. Torpedo stage (X 20)
d. Cotyledonary stage (X 20)
e. Bipolar stage (X 10)
f. Bipolar stage. Note the first leaf
emergence (X 10)

Storage and regrowth viability of synseeds derived
from somatic embryos a.A mechanical apparatus designed
for preparation of synseeds (X 0.7)
b.Sodium alginate encapsulated
somatic embryos (X 0.8)
c. One month old viable encapsulated
somatic embryos of treatment 2 (X
2.3)
d.One month old viable encapsulated
somatic embryos of treatment 3 (X
2.3)
e.One month viable encapsulated
somatic embryos of treatment 4 (X 2)
f.One month viable encapsulated
somatic embryos showing shoot and
root development (X 1.8)
g.Ex situ germination of synseeds
derived from somatic embryos (X 1.1)

Storage and regrowth viability of synseed derived from
apical buds of coriander
a.Sodium alginate encapsulated
apical bud synseeds (X 0.8)
b.Germinability of encapsulated
apical bud of treatment 2 (X 1.6)
c.Germinability of encapsulated
apical bud of treatment 3 (X 1.6)
d.Germinability of encapsulated
apical bud of treatment 4 showing
shoot development (X 1.6)
e.Ex situ germination of apical bud
derived synseeds (1.2x)

Anther/pollen culture
• Method to produce haploid plants
• Spontaneous occurrence in low frequency
• Induction by physical and/or chemical
treatment (nitrous oxide)
• Chromosome elimination following
interspecific hybridization

Haploid/anther culture
• Anther culture – 1966 – pollen grains of
Datura.
• Typically haploids can only be produced in
polyploid plants – wheat, tobacco, clover.
• Used in over 200 species

Haploid culture advantages
• Technique is fairly simple
A large proportion of the anthers may
respond
• Haploids can be produced in large numbers
very quickly

Haploid culture disadvantages
• The majority of plants produced may not be
haploid
• May be albino or chimeric
• Tedious

Haploids are useful because:
• They carry only one allele of each gene.
Thus any recessive mutation or
characteristic is apparent.
• Plants with lethal genes are eliminated from
the gene pool.
• One can produce homozygous diploid or
polyploid plants – inbreeding.

Normal pollen development
• Pollen mother cells are in anther
primordia.
• First phase – meiosis – pollen mother cell
• A tetrad forms from each PMC
• Second phase – microspores released
from tetrads

• Third phase – microspores mature into
pollen grains – first pollen mitosis
• Second pollen mitosis, maybe after
germination
• Generative and vegetative cells formed.

Nutritional requirements
• Sucrose essential
• Mineral salts – iron
• Complex organics – coconut milk
• Activated charcoal

Hormones
• Species requiring no hormones – tobacco,
petunia – direct embryogenesis
• Species requiring hormones – callus phase -
monocots

Haploid production by the bulbosum method
in barley
Pollen is collected from plants of
Hordeum bulbosum, a wild relative
of cultivated barley (H. vulgare).

The H. bulbosum pollen is brushed
onto emasculated barley florets.

A hybrid zygote forms, but during the first
few cell divisions the H. bulbosum
chromosomes are eliminated.
The seeds that develop contain haploid
embryos with one set of H. vulgare
chromosomes.

The haploid embryos must be germinated
in vitro.

Uses of haploids and doubled
haploids
• Completely homozygous plants
• Inbred lines
• Mutation studies
• Breeding (equal ploidy levels)
• Mapping

Plant tissue culture by s. kasthuri

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