Chapter Six

Types of Plant Tissue Culture

Prepared By:
Anon Chaulagain
Department of Biotechnology
HWIC, Putalisadak
Types of Plant Tissue Culture
• There are various types of plant tissue culture which can be
categorized:

A) On the Basis of Explant used

1. Seed Culture 7. Bud Culture
2. Shoot-tip/Apical meristem Culture 8. Anther and Pollen Culture
3. Root Culture 9. Ovary Culture
4. Leaf/Leaf primordia Culture 10. Endosperm Culture
5. Complete flower Culture 11. Hairy-root Culture
6. Embryo culture 12. Protoplast Culture

B) On the Basis of type of in vitro growth

1. Callus culture 2. Suspension Culture
1. Seed Culture
• Seeds that are difficult to germinate in vivo may be cultured in
vitro (in suitable nutrient medium and proper environment) after
surface sterilization to generate seedlings or plants; growth
regulators are usually not necessary for seed germination
• This technique is important in propagation of plants like orchids
• In nature, germination of orchid seedling is dependent on
symbiotic relationship with a fungus
• However, asymbiotic germination of orchid seedling is possible in
vitro
• Seed culture technique is applied to increase the efficiency of
germination of seed of various plants & to produce clean
seedling as explant for meristem culture
2. Meristem/Shoot-tip Culture
• A meristem is a tissue in plants that consists of undifferentiated
cells (meristematic) and actively dividing cells

• Shoots of all angiosperms and gymnosperms grow by virtue of

their apical meristem

• The apical meristem is usually a dome-shaped tissue located at

the extreme tip of shoot

• The meristem or shoot-tip can be used as explant to regenerate

the whole plant when cultured in a suitable nutrient media

• The meristem grows directly into a simple leafy shoot or multiple

shoots
Shoot apex
Protocol:

 The young twigs are removed from the healthy plant

 The tip portion of the twig is cut that should be of 1cm

 The shoot tip is subjected to the surface sterilization in the

sodium hypochlorite solution for 10 minutes

 The explants are thoroughly rinsed with the distilled water for
about 4 times

 Then, each explant is transferred to a sterilized petri-plate

 The outer leaves of the shoot tip are dissected

 After the dissection of outer leaves, the apex region will get
exposed which is separated by the help of a scalpel

 The apex or apical meristem is transferred to the MS (Murashige

and Skoog’s) nutrient medium; generally, supplemented with
high cytokinin (esp. BAP) and low auxin (esp. NAA)

 The culture is incubated under 16 hours light at 250C

 After the development of single or multiple shoots from callus, it

is transferred to the hormone-free medium for the root
development

 Then, the plants are transferred to the pots containing compost

and kept under greenhouse condition for hardening
• Meristem-tip culture have been such that it has become the most
efficient technique for obtaining completely virus-free plants
• Apical meristems in the infected plants are generally either free
or carry a low concentration of viruses; the reasons attributed to
the escape of the meristems by virus invasion are:
 Viruses readily move in a plant body through the vascular
system which is absent in the meristem and the method of
cell-to-cell movement of the virus through plasmodesmata is
rather too slow to keep pace with the actively growing tip
 The high metabolic activity in the actively dividing meristem
cells does not allow virus replication
 A high endogenous auxin level in shoot apices may inhibit
virus multiplication
• This technique can be applied for micropropagation i.e.
vegetative propagation of plants (production of large no. of
plants) using plant tissue culture techniques
• The produced plants will be genetically uniform (clones)
• Many plants produce seeds that are highly heterozygous in
nature
• Such seeds are not stored as genetic resources; hence, meristems
from such plants can be stored in vitro (i.e. germplasm
conservation)
• Apical meristem cultures have shown successful results in potato,
sugarcane, banana etc.
3. Root Culture
• Root culture can be defined as the culture of excised radical tips
of aseptically germinated seeds in a liquid medium where they
are induced to grow independently under controlled condi­tions

• Intact in vivo plants are not suitable for isolation of intact root
tips because the roots of the plant are buried deeply in the soil

• Also, root tips of young seedlings are sensitive to toxic sterilants

• So it is better to avoid the surface sterilization of young root tips

for the es­tablishment of root cultures

• Hence, root cultures can be successfully initiated from the

excised radicle tips of aseptically germinated seeds
• Root tip cultures are generally maintained in moving liq­uid
medium

• In culture, root tips are induced to grow like that of root system
of an intact plant

• A clone of excised roots can also be established from a single root

culture by repeatedly cutting and transferring of the main root
tips or of lat­eral tips into fresh medium in every subculture at the
interval of definite period

• Growth of ex­cised roots can be expressed in terms of fresh and

dry weight, increase in length of the main axis, number of
emergent laterals and total length of laterals per culture
Protocol:

 Seeds are surfaced sterilized by the conven­tional methods and

germinated on moist fil­ter paper or White’s basal medium at
25°C in the dark

 When the seedling roots are 20 to 40 mm in length, 10 mm apical

or radical tips (tip inoculum) are excised with a scalpel and each
transferred to the flasks containing 40 ml of liquid medium

 Flasks are incubated for 10 days at 25°C in the dark

 The root material (10-day-old tip culture) derived from a single

radi­cle tip could be multiplied and maintained in continuous
culture (moving liquid medium)
 Such genetically uniform root cultures are referred to as a clone
of iso­lated roots
 A 10-day-old established root cul­ture is transferred to a sterile
petri dish containing ster­ile medium
 Next, using flamed scissors, the main axis of root is cut into a
number of pieces; each piece is called sector inoculum or initial
with each bearing four or five young laterals
 The individual sector inoculum is transferred aseptically to a flask
liquid medium and in­cubated for 7 days at 25°C in the dark
 Such sector culture can be used to initiate further tip culture
using 10mm apical tips of laterals of a growing sector inoculums
or the growing sector is again cut into 4-5 sectors to initiate the
sector culture
Fig. Root culture technique & maintenance of isolated root in continuous culture
• Root cultures have increased our knowledge of carbohydrate
metabolism, role of mineral ions, vitamins etc. in root growth

• These cultures proved the dependence of roots on shoots for the

growth hormones

• Culture of isolated roots can be maintained continuously for

many years

• However, in some species e.g. Atropa, Convolvulus arvensis,

shoots can be induced to regenerate from cultured roots

• Root culture of leguminous plants help us to study the process of

nodule formation by nitrogen fixing bacterium i.e., Rhizobium sp.
• Root cultures also help us in locating the biosynthesis of alkaloids
by the roots and also to get increased production of such
compounds

• For e.g. Hyoscyamine is an alkaloid commercially produced from

the root cultures of Datura stramonium
4. Leaf/Leaf primordia Culture
• Leaf culture is the culture of excised young leaf primordia or
immature young leaf of the shoot apex in a chemically defined
medium whe­re they grow and follow the developmental
sequences under controlled conditions
• Leaf primordia or very young leaves are ex­cised, surface sterilized
and inoculated on an agar solidified medium where they will
remain in a healthy conditions for a long period
• Leaves can be taken from aseptically grown plants for culture
• The growth of leaves on the culture medium depends upon the
stage of the maturity of leaves during excision
• Explants from immature young leaves or leaf primordia grow
better than explants from older (mature) leaves
• Most of the work on leaf culture has been done with lower
plants, particularly fern (Osmunda)

• Higher plant species, such as tobacco and sunflower, have also

been used

• In cul­ture, the fern leaf primordia (1.2 mm), excised from

underground buds, develop into leaves hav­ing a normal
morphology

• However, they are much reduced in size than in vivo leaves due
to a reduced number of cells rather than a decrease in cell size

• Also, the growth of cultured leaf primor­dia is completed earlier

than intact leaf
• It has also been found that there is a corre­lation between leaf
primordia size and its mode of development in vitro

• In Osmunda cinnamomea, smallest leaf primordia (300 µm in

length) give rise to shoots instead of leaf in cul­ture

• However, with increasing size of primordia, there is an increased

tendency to form leaves

• This might indicate that some unidentified leaf forming

substances gradually accumulate as the primordia develop
Protocol:
 Vegetative bud or very young leaf from shoot apex is detached at
the vegetative phase of the plant
 The explants are washed thoroughly with running tap water
 The leaf buds or young leaves are immersed in 5% Teepol for 10
minutes
 The explants are washed to remove Teepol
 Leaf buds or young leaves are surface ster­ilized by immersion in
70% v/v Ethanol for 30 seconds
 This treatment is followed by 10-15 minutes incubation in sodium
hypo­chlorite solution with 0.8% available chlo­rine
 The explants are rinsed 3-4 times in sterile distilled water

 The leaf primordia is excised from the leaf bud with the help of
surgical scalpel

 The leaf primordia or young leaf is inoculated onto 20 ml of

solidified medium in a culture tube 

 The culture is incubated at 25° C under 16 hrs. light

 The regenerated plantlets are transferred to the pots containing

compost and kept under greenhouse condition for hardening
• Leaf culture is useful to study the effects of various nutrients,
growth regulators and environmental factors on leaf growth

• In Ferns, leaf culture helps us in studying the various

developmental stages of sporangia and the size at which a
primordium is des­tined to become a leaf

• Leaf culture of solanaceous species can be used as clonal micro-

propagation
5. Complete Flower Culture
• Flowers two days after pollination are excised, sterilized by
immersion in 5% calcium hypochlorite, repeatedly washed with
sterilized water and transferred to culture medium
• When cultured, such flowers produce fruits
• Larger fruits are obtained on medium supplemented with growth
hormones
• Flowers excised before pollination do not produce fruits; in some
cases, parthenocarpic fruit (the natural/artificially produced fruit
without fertilization of ovules making fruit seedless)
development can be observed, particularly in presence of auxins
• This culture system is useful in studying microclimates or
nutritional effects on the vegetative and reproductive processes
of the plant & fruit development
6. Embryo Culture
• Embryo culture is the technique of sterile isolation and in vitro
growth of a mature or an immature embryo into a viable plant
a) Mature embryo culture:
• When the embryos remain dormant for long periods or there is
low survival of embryos in vivo, mature embryo cultures are done
• Also, to avoid inhibition in the seed for germination or to convert
sterile seeds to viable seedlings, mature embryo cultures are
carried out
• Mature embryos are isolated from ripe seeds and cultured in
vitro
• Very small globular embryos require a delicate balance of the
hormones
• Embryo culture is relatively easy as they can be grown on a
simple inorganic medium supple­mented with energy source
(usually sucrose)
• This is possible since the mature embryos excised from the
developing seeds are autotrophic in nature
• Hence, mature embryo culture helps in overcoming seed
dormancy and seed sterility
b) Immature embryo culture (Embryo Rescue):
• Embryo rescue involves the culture of immature embryos to
rescue them from unripe or hybrid seeds which fail to germinate
• This approach is very useful to avoid embryo abortion and
produce a viable plant
• Wild hybridization might hinder normal development of zygote
and seed due to genetic barriers

• Consequently, hybrid endosperm fails to develop leading the

abortion of hybrid embryo

• The endosperm may also produce toxins that ultimately kill the
embryo

• Embryo abortion can be avoided by isolating and culturing the

hybrid embryos prior to abortion

• The most important application of embryo rescue is the

production of interspecific and inter-generic hybrids from wild
plant species
• The isolation of immature embryos often poses some difficulty
• The aseptically isolated embryos can be grown in a suitable
medium under optimal conditions
• In general, a complex nutrient medium is required for culture
methods involving embryo rescue
• For adequate nutritional support of immature embryos, embryo-
endosperm transplant is used because endosperm and maternal
tissues supply nutrient for developing embryo in its early phase
Embryo-endosperm transplant:
 The hybrid embryo from the ovule in which endosperm
development has failed is taken out by excision
 Another normally developed ovule with endosperm enclosing an
embryo is chosen
 This ovule is dissected and the normal embryo is removed which
leaves a normal endosperm with an exit hole
 Now, the hybrid embryo can be inserted into the normal
endosperm through exit hole
 This results in embryo-endosperm transplant which can be
cultured in a suitable medium
• By this technique, many interspecific and inter-generic plants
have been raised e.g. hybrid plants of legumes
Protocol:

 The excised mature or immature embryo is placed on the filter-

paper bridge which is made on the tubes containing simple &
suitable media for growth (complex media may be required for
immature embryos)

 The culture is incubated at 24 - 26°C under darkness which are

later transferred to light for germination

 During the culture conditions, the embryos develop plumule

(shoot of the embryo)

 Further it is developed into plantlet, which is then transferred to

sterile soil for full-pledged growth to maturity
• Embryo culture has also been successfully used for the
production of haploid (or monoploid) plants e.g. barley

• Embryos are ideally suited for in vitro clonal propagation

• This is due to the fact that embryos are juvenile in nature with
high regenerative potential

• Further, it is possible to induce organogenesis and somatic

embryogenesis from embryonic tissues
7. Bud culture
• Axillary bud possess quiescent or active meristems depending on
the physiological state of the plant

• The axillary bud found in the axil of leaf

• Each bud has the potential to form shoots, either vegetative shoots
or reproductive shoots

• In axillary bud culture, a bud along with a piece of stem which is

known as a node is isolated and cultured in a suitable nutrient
medium under controlled conditions to develop into a plantlet

• Closed buds are used to reduce the chances of infections

• Axillary bud culture is used in micropropagation and production of

virus free plants
8. Anther and Pollen Culture
• The anther is a part of a flower which is bi-lobed and sac-like in
structure
• In flower, anther is the part of stamen which is a male
reproductive part
• Stamen comprises a long, narrow or stalk-like filament which
carries the anther at its tip
• An anther consists of microsporangia which produce pollen
grains by the meiotic cell division
• Anther culture is a type of tissue culture technique, where a part
of a plant, i.e. anther gives rise to an androgenic haploid plant
when cultured in a nutrient medium under optimal conditions
that are required for the growth of the cell
Fig. Structure of flower
• The process of anther culture comes under the process of
“Androgenesis” which merely refers to the induction of haploid
plants (plant possessing a single set of chromosomes i.e. n)

• In about 250 species, such as family of Gramineae, Solanaceae,

Ranunculaceae, Cruciferae, etc., this technique has been
implemented

• Anther culture can be done by two methods either through a

direct or indirect method

• The direct method of an anther culture involves “Embryogenesis”

• In this method, the anther behaves as a “Zygote” and forms

embryoid which gives rise to haploid plantlet
• The indirect method of an anther culture involves “Organogenesis”

• In this method, the anther undergoes cell division repeatedly to form

the callus tissue which later gives rise to the haploid plantlets

Protocol:

 First, unopened buds of size about 17-22mm in length are selected

The buds that are beginning to open or already open are rejected

While selecting buds, the size of the sepal is equal to the size of a
petal is ideal for the anther culture
 The buds are surface sterilized by 70% ethanol 1 minute and then
5% sodium hypochlorite for 10 minutes

 Then buds are washed in sterile water and transferred into a

sterile petridish

 With the help of sharp scalpel and using forceps the buds are
split open and anther lobes are taken out

 One of the anther lobes of each bud is checked by crushing into

acetocarmine stain under microscope for the proper stage of
microspore development

 The filament portions are removed from the selected anther

lobes
 The damaged anther lobes should be discarded and intact anther
lobes are placed into proper media (MS or White or Nitsch and
Nitsch medium)

 Then the culture is incubate it for 3-4 weeks at 24-28°C in the dark
for 12-18 hours in light and for 6-12 hours in the dark

 Then either by embryogenesis or organogenesis, the haploid

embryos or plantlets develop, come out by bursting the anther
lobes

 Individually these embryos or plantlets are removed and sub-

cultured on suitable media to develop further and root
development

 The developed plantlets are later transferred to pots for hardening

• The main disadvantage of anther culture is that the plants or
embryos or calli not only originate from microspore or pollen but
may also originate from various other parts of anther lobes
(anther wall, nucellus, tapetum) which are diploid tissues

• Furthermore, anther wall or other tissue may affect the way of

androgenesis by inhibiting the penetrance of media or
stimulating effect of hormones supplied

• This difficulty can be avoided by culturing the isolated

microspores or pollen

• Pollen or microspore culture is an in vitro technique by which

microspore or the immature pollen can be used as the explant to
get the haploid plants directly
Protocol:

 The flower buds are collected, surface sterilized and the anther
lobes are dissected out from the flower buds as before

 Then the anther lobes are squeezed with the help of a scalpel within
a tube or small beaker to col­lect the microspore or pollen in
nutrient media

 Then the anther tissue debris is removed by filtering the suspension

through a nylon sieve with a diameter slightly larger than the pollen
size (40µ-100µ) allowing the microspore only to pass through it

 Then the microspore-suspension washed and concentrated to a

plating density
 The microspores obtained are then mixed with an appropriate
culture medium at a density of 103-104 microspore/ml, and plated
in small petriplate

 To ensure good aeration, the layer of liquid in the dish should be

as thin as possible, and sealed with ‘parafilm’ to avoid
dehydration

 The responsive pollen will divide and form embryos or calli which
directly or indirectly will form the haploid plantlet

 By following the method of sub-culturing the whole plant

suitable for soil transfer can be obtained
• There are several advantages of pollen culture over anther
culture:

 The explants i.e., microspores or pollens are all haploid cells

which gives rise to purely haploid plant

 The sequence of androgenesis can be observed starting from

a single cell

 The microspores are ideal for uptake, transformation and

mutagenic studies, and the microspores are evenly exposed
to chemicals and physical mutagens

 Higher yields of plants/anther could be obtained

• There are various importance of anther and pollen culture

• Haploid plants derived from anther or pollen culture and useful in

cytogenetic studies

• Haploid plants may grow up to a flowering stage, but viable gametes

cannot be formed due to lack of one set of homologous
chromosomes

• Haploids can be diploidized (by duplication of chromosomes) to

produce homozygous diploid plants by colchicine treatment; it acts as
an inhibitor of spindle formation during mitosis and induces
chromosome duplication

• By comparing the heterozygous diploid with haploid homozygous

diploid population recessive phenotypic characters can be identified
very easily
• Critical genetic analysis of haploid population derived from
individual microspores of pollen tetrad is very useful for the
study of genetic recombination in higher plants

• The series of cell divisions and mode of differentiation

(embryogenesis or organo­genesis), starting from single cell
(microspore) and ending in whole organism can be studied under
microscope

• Double haploid, that are homozygous and fertile are readily

obtained, ending the selection of desirable gene combination

• Culture of isolated pollen provides a novel experimental system

for the study of factor controlling pollen embryogenesis of
higher plants
• Study of meiotic behaviour of haploids provides valuable clues to
measure chromosome duplication within a species and for
understanding a phylogenetic relationship among species

• It also provides information for the interpretation of

chromosome homology

• Genetic analysis could be performed on haploid population to

establish inheritance patterns

• The use of haploids in the production of monosomics,

nullisomics and other aneuploids
9. Ovary Culture
• An ovary is a part of a flower which is the female reproductive part
and also refers as “Gynoecium”

• Within the ovary, there is an ovule which also refers to as

“Megasporangium” as it carries megaspore or egg-cell; the egg
cell undergoes fertilization by which a zygote is produced that later
develops into a mature embryo

• An ovary can regenerate into a fully differentiated plant

• In the ovary culture, the flowers are excised from the plant either
in pollinated or non-pollinated stage, and from that pistil
containing ovary is removed

• Ovaries in an aseptic condition are cultured in an appropriate

nutrient medium and under optimal conditions
• Ovary culture gives rise to haploid plantlets by the fertilization of
egg-cell or megaspore either through embryogenesis or
regeneration from callus
Protocol:
 First, the open flowers are collected from a healthy plant, in a
sterilized zip-lock bag
 The flowers are washed with the distilled water
 Then, the flowers are dipped into the 5% of Teepol solution for 10
minutes
 Again, the flowers are washed with the distilled water
 Then the flowers are surface-sterilized by immersing in a 5% of
sodium hypochlorite solution for 5-7minutes
 And, then flowers are washed with the distilled water

 To a sterile Petri-plate, the surface sterilized flower are

transferred and by using sharp scalpel the calyx, petal, anther
filaments etc. are dissected out to separate the ovary

 Then, the ovary can be cultured either through induction or

regeneration process

 In induction, the ovaries float over the liquid medium

 And, in regeneration culture the ovaries on the solid nutrient

medium

 The cultures are incubated for 16 hours at 25°C

 For the regeneration of ovary, the culture plates are kept in a
daylight regime by using a fluorescent lamp

For the induction process the culture tubes are placed in a dark

 After 2 weeks, haploid plantlets grow either through

embryogenesis or through plant regeneration from callus

• Haploids can be produced by ovary culture and used in the

various studies

• Dormancy period of seeds can be reduced

• Ovary culture helps in the study of the early stages of embryo

development
• Gynogenesis also helps in the study of fruit development and its
physiology including maturation

• From the culture of the un-pollinated ovary, the effect of

phytohormones on the parthenocarpic fruit can be studied

• Role of floral organs can be studied which plays a significant role

in fruit development

• Ovary culture also explains the in-vitro pollination and seed

formation method

• Ovary culture also helps to understand the hybridization process

to obtain hybrid varieties of seedlings by crossing over of
interspecific and intergenic species
Limitations:

However, there are some limitations of ovary culture:

• The frequency of haploid production is very low in ovary culture

as only 1-5% of the responding ovaries are there

• This technique requires high technical skills and management

• In some case, besides a haploid plant, some different traits may

develop

• Gynogenesis can be employed only in limited species, for

example, wheat, rice, maize, sugar beet etc.
10. Endosperm Culture
• Endosperm is a unique tissue

• In the majority of flowering plants (over 81% of families) it

originates from the fusion product of three haploid nuclei (one
from the male gametophyte and two from the female
gametophyte) and is, therefore, triploid

• The endosperm is the main nutritive tissue for the embryo and,
also, a dynamic centre of developmental influences on the
embryo
• Triploid cells of endosperm are totipotent thus endosperm tissue
being triploid, the plantlets formed from it are also triploid

• This technique of endosperm culture may be profitably exploited

as an alternative to crossing tetraploids and diploids for raising
triploids plants (seed sterile) in plant improvement programmes

• The cereal endosperm proliferates only if excised during a proper

period of development; e.g. 8 - 11 DAP in Zea mays

• Initial association of the embryo was essential for inducing

proliferation of the endosperm from fully mature and dried seeds

• It is speculated that embryo is essential for the activation of

mature and dried endosperm by secretion of GA3
• Shortly after the endosperm has started callusing, the embryo
can be removed without affecting the growth of the former

• The callus phase is followed by shoot bud differentiation or

embryogenesis

• Endosperm grows well in MS medium supplemented with 2,4-D

or IAA, Kinetin, yeast extract or tomato extract or casein
hydrolysate depending on the plant species

Protocol:

 The immature seed is dissected under aseptic conditions and

endosperm along with embryo is excised, sometimes mature
seed can also be used
 The excised endosperm is cultured on a suitable medium
supplemented with growth regulators and embryo is removed
after initial growth

 The initial callus phase is followed by shoot bud differentiation or

embryogenesis

 The shoots and roots may subsequently develop and complete

triploid plant is formed

• Some of the economically important plants whose triploids are

already in commercial use include several varieties of apple,
banana, mulberry, sugar beet, tea, and watermelon

• Seedless fruits can be produced from endosperm culture

• Some species of plant produces larger and tastier fruits

• Some have more desirable pulpwood characteristics than its

diploid

• Endosperm culture provides an excellent experimental system to

study biosynthesis and metabolism of these natural products

Fig. Seedless watermelon

11. Hairy-root Culture
• Certain soil bacteria of the genus Agrobacterium (gm–ve)
bacteria) infects a wide range of plant species and causes the
infection in plant termed as “Hairy root” disease

• One of such bacteria is Agrobacterium rhizogenes which contains

Ri plasmid (Hairy root inducing plasmid)

• When A. rhizogenes infects the plant, the segment of their Ri

plasmid gets incorporated into the genome of the plant cells

• The produced copious root (cloned roots) by inoculating A.

rhizogenes with explant grown in a hormone-free medium are
referred to as 'transformed roots' or 'hairy roots‘

• These are fast growing, highly branched adventitious roots at the

site of infection
• They are genetically stable and affect a wide range of
dicotyledonous plants and have same metabolic features

Establishment/Methodology of Hairy root culture:

 The explant material is inoculated with a suspension of A.

rhizogenes generated by growing bacteria in YMB medium for
two days at 25°C with gyratory (round circle) shaking, pelleting by
centrifugation (5×10 rpm; 20min) and suspending the bacteria in
YMB medium to form a thick suspension

 Transformation may be induced as aseptic plants grown from

seeds, of on detached leaves, leaf discs, petioles of stem
segments from green house plants following sterilization of
excised tissue with 10% (v/v) domestos for 20 minutes
 Scratching the midrib of a leaf or the stem of a plantlet, with the
needle of a hypodermic syringe containing the thick bacterial
suspension allows inoculation with small (about 5-10μl) droplets
containing A. rhizogenes

 In some species a profusion of roots may appear directly at the

site of inoculation, but in other a callus will form initially and
roots emerge subsequently from it

 In either case hairy rot normally appear with in 1-4 weeks

although the susceptibility of species to infection is variable

 From any of the process, the hairy roots are selected and
cultured in liquid medium for scaling up using bioreactors or
transformed plants can be regenerated
Advantages of Hairy Root Culture:

• Hairy root culture shows genetic and growth kinetic stability over
prolonged period of growth in vitro

• It is simple technique to culture in vitro without using phytohormones

• Many plant cell culture systems, which did not produce adequate
amount of desired compounds is being reinvestigated using hairy root
culture methods

• A diversified range of plant species has been transformed using

various bacterial strains

• Plant regeneration, plant improvement and genetic manipulation in

plant can be done
• Capability to synthesize (novel) secondary metabolites specific to
that plant species from which they have developed in equal or even
higher amount compared to field grown plants

• More accumulation of secondary metabolites can be achieved with

hairy root culture as compared to normal root culture

• E.g. Artopine (anticholinergics, antidote) from Atropa belladonna

Taxol (anticancer) from Taxus sps.

• Significantly higher amount of secondary metabolites can be

obtained with hairy root culture as compared to callus or suspension
cultures

• E.g. steroidal alkaloid Solasodine (diuretic, anticancer, etc.) from

Solanum aviculare
12. Protoplast Culture
• Protoplasts are cells without a cell wall, but bounded by a cell
membrane or plasma membrane

• Plant protoplast are isolated by the removal of cell wall by either

enzymatically or mechanical means from the artificially
plasmolysed plant cells

• The isolated protoplast is only a naked plant cell surrounded by

plasma membra­ne which has potential of cell wall regeneration,
cell division, growth and plant regen­eration in culture

• Protoplast culture is the aseptic isolation of large number of

intact living protoplasts and culturing them on a suitable nutrient
medium under controlled environmental conditions for their
requisite growth and development into plantlets
• Protoplast can be isolated from varieties of plant tissues

• Convenient and suitable materials are leaf mesophyll and cells

from liquid suspension culture

• Protoplast yield and viabil­ity are greatly influenced by the

growing condi­tion of the plant as well as the cells

• The essential step of the isolation of proto­plast is the removal of

the cell wall without dam­aging the cell or protoplasts

• The cell wall exerts the inward pressure upon the enclosed
protoplasts

• Like­wise, the protoplast also puts equal and oppo­site pressure

upon the cell wall; thus, both the pressures are balanced
• Now if the cell wall is re­moved, the balanced pressures will be
disturbed

• As a result, the outward pressure of protoplast will be greater

• And at the same time in absence of cell wall, irresistible

expansion of protoplast takes place due to huge inflow of water
from the external medium

• Greater outward pressure and the expansion of protoplast cause

it to burst

• So, the isolated protoplast is an osmotically frag­ile structure at its

nascent stage
• Therefore, if the cell wall is to be removed to isolate proto­plast,
the cell or tissue must be placed in a hy­pertonic solution of a
metabolically inert sugar such as mannitol at higher
concentration (13%) to plasmolysis the cell away from the cell
wall

• Mannitol, an alcoholic sugar, is easily transported across the

plasmodesmata, provides a stable osmotic environment

• This prevents the usual expansion and bursting of protoplast

even after loss of cell wall

• Thus, this hypertonic solution is known as os­motic stabilizer or

plasmolyticum or osmolyticum
• Once the cells are stabilized in such a man­ner by plasmolysis the
protoplasts are released from the containing cell wall either
mechanically or enzymatically

• Mechanical isolation involves breaking open each cell

compartment to liberate the protoplast

• This operation can be done carefully on small pieces of tissue

under a microscope using a micro-scalpel

• But very few protoplasts are obtained for a lot of time and effort

• Also, the protoplast can be damaged with micro-scalpel if not

handled with care
• A considerably more efficient way of liberating the protoplasts is
by enzymatic method

• In this method, the cell walls around them are digested, using cell
wall de­grading enzymes such as cellulase, hemicellulase,
pectinase or macerozyme etc.

• These enzymes are isolated from fungi and available

commercially e.g. Aspergillus niger

• Period of treatment and concentration of enzymes are the critical

factors and both factors should be standardized for particular
plant tis­sue
• There are two methods of enzymatic isolation of protoplast

• In the sequential enzyme treatment (two step method),

• intact tissue can be incubated with a pectinase or macerozyme

solution which will dissolve the middle lamella between the cells
& so sep­arate them

• Subsequent treatment with cellu­lase will digest away the

cellulosic layer of the cell wall

• In mixed enzyme treatment (one step method), both cellulase &

pectinase or macerozyme are mixed so that the entire wall is
broken down in a single operation
• The isolated protoplasts can be cultured ei­ther static liquid or
agarified medium using techniques either Hanging-droplet
method of culture, Co-culture or Plating of protoplasts

• The pro­toplast media consist of mineral salts, vitamins, carbon

sources & plant growth hormones as well as osmotic stabilizers &
possibly organic nitrogen sources, coconut milk & organic acids

• In culture, protoplast can reform a new cell wall around them

• Once the wall is formed, the proto­plast becomes a cell

• The cells from protoplasts subsequently enter cell division which

is followed by the formation of callus and cell cultures

• Such callus also retain the capacity for morphogenesis and plant
regeneration
• A brief list of plant regeneration from plant protoplast culture is
given below
Fig. Protoplast culture (using Enzymatic isolation & Plating of Protoplast Technique)
• Protoplast culture has been widely used to develop Novel hybrid
(somatic hybrids, cybrids, etc.) plant through protoplast fusion

• Single cell cloning can be easily performed with protoplasts

• In single cell derived colony, isolation of mutants through

mutagens is easier

• Regeneration of entire plant or plant improvement through

protoplast culture can be achieved

• Genetic transformation through DNA uptake can be achieved

• Reproducible protoplast to plant systems are now available for

many plants of agronomic value
Protoplast Fusion:

• Protoplast fusion is the technique in which two or more

protoplast are fused into a single cell protoplast

• The protoplast fusion allows us to bring an desirable plant traits

in combination that are not possible by sexual means

• This fusion may occur between same (interspecific) or different

(intergeneric) plant

• Mutinucleate protoplasm obtained after fusion

• There are three main phases of protoplast fusion: Agglutination,

Formation of cytoplasmic bridge and Rounding off
1. Agglutination: Plasma membrane of two or more protoplast are
brought into close proximity i.e. (A and B)

2. Membranes of protoplasts agglutinated by fusogen get fused at

the point of adhesion

This results in the formation of cytoplasmic bridge between

the protoplast

3. Rounding off of the fused protoplast due to the expansion of

cytoplasmic bridge forming a spherical homokaryons (A-A or B-
B) hetrokaryon (A-B) i.e. Binulceate heterokaryons

• The fusion of the nuclei results in a tetraploid hybrid cell; also

cybrid cell is formed with a selective chromosome loss
Methods of Protoplast Fusion:

• Protoplast fusion could be spontaneous during isolation of

protoplast or it can be induced by mechanical, chemical and
physical means

• During spontaneous process, the adjacent protoplasts fuse via

plasmodesmata together as a result of enzymatic degradation of
cell walls forming homokaryons or homokaryocytes, each with
two to several nuclei

• The occurrence of multinucleate fusion bodies is more frequent

when the protoplasts are prepared from actively dividing callus
cells or suspension cultures
• Since the somatic hybridization or cybridization require fusion of
protoplasts of different origin, the spontaneous fusion has no
value

• Freely isolated protoplast from different sources are fused with

the help of fusion inducing agents

• Fusion needs an inducing agent that actually reduces

electronegativity and allows the isolated protoplast to fuse
Chemical Fusion:

• Types of fusogens are used like PEG, NaNo3, Ca2+ ions, Polyvinyl
alcohol (PVA) etc.

• The cell membrane posses negative charge and after treatment

with such chemical agents when cell membrane are brought into
close physical contact they fuse

Mechanical Fusion:

• It is not dependent upon the presence of fusion inducing agent

• Physical fusion of protoplasts is done under microscope by using

micromanipulator and perfusion micropipette
Electrofusion:

• This process involves a two-step process

• First, the protoplasts are introduced into a small fusion chamber

containing parallel wires or plates which serve as electrodes

• Second, a low-voltage and rapidly oscillating AC field is applied,

which causes protoplasts to become aligned into chains of cells
between electrodes

• This creates complete cell-to-cell contact within a few minutes

• Once alignment is complete, the fusion is induced by application

of a brief spell of high-voltage DC pulses (0.125-1 kVcm-1)
• A high voltage DC pulses induces a reversible breakdown of the
plasma membrane at the site of cell contact, leading to fusion
and consequent membrane reorganization

• The entire process can be completed within 15 min

Somatic Hybridization & Cybridization:

• The technique of hybrid production through the fusion of isolated

somatic protoplast under in-vitro conditions and subsequent
development of their product to a hybrid plant is called somatic
hybridization

• Various stages of somatic hybridization are as follows:

1. Isolation of protoplasts
2. Fusion of isolated protoplasts of desired species/varieties
3. Identification and Selection of somatic hybrid cells
4. Culture of the hybrid cells
5. Regeneration of hybrid plants
1. Isolation of protoplast:

• At first, cells are stabilized using osmolyticum (mannitol) and

protoplast is isolated by mechanical methods (using micro-
scalpel) or by enzymatic methods (cellulase, hemicellulase,
pectinase or macerozyme)

2. Fusion of isolated protoplasts of desired species/varieties:

• Isolated protoplasts of desired species/varieties are fused using

chemical agents or fusogens such as PEG, NaNo3, Ca2+ ions,
Polyvinyl alcohol (PVA) etc. or mechanical fusion such as
micromanipulator and perfusion micropipette or electrofusion
technique
3. Identification and Selection of somatic hybrid cells:

• The protoplasts of two parents may be labeled by different

fluorescent compounds, which will then enable for the
identification and selection of hybrids

• Identification is based on difference between the parental cells

and hybrid cell with respect to Pigmentation, Cytoplasmic
markers, Presence of chloroplast, Nuclear staining etc.

• In the mixture of both fused and unfused protoplast Selection of

hybrid is done on the basis of several procedures like Genetic
complementation, antibiotic-resistant cell lines, isoenzyme
analysis, Phytotoxins, Specific amino acid, Auxin autotrophy,
Auxotrophic and metabolic mutants, Chromosomal analysis by
hybrid cell, Herbicides, etc.
4. Culture of the hybrid cells

• Hybrid cells are cultured on suitable medium provided with the

appropriate culture conditions

5. Regeneration of hybrid plants

• Plants are induced to regenerate from hybrid cells and via

Callogenesis and Organogenesis somatic hybrid plants are
produced

• These hybrid plants must be at least partially fertile, in addition

to having some useful property, to be of any use in breeding
schemes
Fig. Somatic Hybridization
Process
Cybridization:

• Sexual hybridization involves fusion of the nuclear genes of both

the parents but somatic hybrids involves even cytoplasm from
both the parental species in hybrid obtained by protoplast fusion

• However, in another case somatic hybrids containing nuclear

genome of one parent but cytoplasm from both the parents, are
termed as cybrids

• The approach is time consuming and require several years of

crossing plants provides an opportunity to study inter-parental
mitochondrial, chloroplast fusion giving rise to plants with novel
genomes
Methods to produce cybrids:

• They are produced in variable frequencies in normal protoplast

fusion experiments due to one of the following methods:

1. Fusion of normal protoplast with an enucleated protoplast; the

enucleated protoplast can be produced by high speed
centrifugation (20,000-40,000xg) for 60 min with 5-50% percoll

2. Fusion between a normal protoplast and another protoplast

with a non-viable nucleus or suppressed nucleus

3. Elimination of one of the nuclei after heterokaryons formation

4. Selective elimination of chromosomes at a later stage

5. Irradiating (with X-rays or gamma rays) the protoplasts of one
species prior to fusion in order to inactivate their nuclei

6. By preparing enucleate protoplasts (cytoplasts) of one species

and fusing them with normal protoplasts of the other species

Cybrids provide the following unique opportunities:

 Transfer of plasmogenes of one species into the nuclear

background of another species in a single generation even in
sexually incompatible combinations

 Recovery of recombinants between the parental mitochondrial

or chloroplast DNAs (genomes) & production of a wide variety of
combinations of the parental and recombinant chloroplasts with
the parental or recombinant mitochondria
Advantages of somatic hybridization:

• This is a novel approach for introducing or increasing genetic

variability at nuclear or extra nuclear organelle genome level, in
higher plants

• Novel interspecific and intergenic hybrid can be produced e.g.

Pomato (Hybrid of potato and tomato)

• Somatic hybridization is significant in improvement of plants

such as banana, potato, sugarcane, and yam

• Production of fertile diploids and polypoids from sexually sterile

haploids, triploids and aneuploids can be achieved
• Transfer of gene for disease resistance, abiotic stress resistance,
herbicide resistance and many other quality characters can be
achieved

• Heterozygous lines in the single species which cannot be

propagated by vegetative means can be produced

• Studying cytoplasmic genes may be helpful to carry out plant

breeding

• Unique hybrids of nucleus and cytoplasm can be produced

Limitations of somatic hybridization:

• Application of protoplast methodology requires efficient plant

regeneration system from isolated protoplasts; protoplasts from
two species can be fused, however, production of somatic
hybrids is not easy

• Lack of a proper selection method for fused products (hybrids)

poses a problem

• The end product of somatic hybridization are often unbalanced

(sterile, misformed and unstable)

• Somatic hybridization of two diploids leads to formation of

amphidiploids which is unfavorable
• It is not sure for a character to completely express after somatic
hybridization

• The regeneration products of somatic hybridization are often

variable due to somaclonal variation, chromosome elimination,
organelle segregation

Somaclonal variation is defined as genetic variation observed

among progeny of plants regenerated from somatic cells cultured
in vitro

• All diverse intergeneric somatic hybrids are sterile and, therefore,

have limited chances of development of new varieties

• To transfer useful genes from wild species to cultivated crop, it is

necessary to achieve intergeneric recombination or chromosome
substitution between parental genomes
Types of Plant Tissue Culture
• There are various types of plant tissue culture which can be
categorized:

A) On the Basis of Explant used

1. Seed Culture 7. Bud Culture
2. Shoot-tip/Apical meristem Culture 8. Anther and Pollen Culture
3. Root Culture 9. Ovary Culture
4. Leaf/Leaf primordia Culture 10. Endosperm Culture
5. Complete flower Culture 11. Hairy-root Culture
6. Embryo culture 12. Protoplast Culture

B) On the Basis of type of in vitro growth

1. Callus culture 2. Suspension Culture
1. Callus Culture
• Callus is undifferentiated mass of actively dividing parenchyma
cells

• Callus culture is the production & maintenance of callus on a

solidified nutrient medium under aseptic & controlled condition

• Via callus plant can be regenerated and secondary metabolites

production can be done

• For production of secondary metabolites, callus is cultured in the

liquid nutrient medium

• Loosely arranged thin-walled cells (callus) is shaken in the liquid

medium which is easily broken into individual cells or cell
aggregates
• Such cell aggregates when cultured in a nutrient medium
produce secondary metabolites of therapeutic interest which is
extracted from the nutrient medium

Steps involved in callus culture:

• Selection and collection of explant material:

Leaf, stem, root, etc. can be used as explant for callus culture

• Selection and preparation of culture medium:

According to the nature (nutritional requirement) of explant

material, culture media is prepared; sterilized MS media is
generally preferred for callus culture
• Surface sterilization of explant material:

Microbial contaminant is removed using chemical; if leaf is used

as explant material, it is first washed with distilled water, and if
waxy substance is present, then it is again washed with detergent
(Tween 80) followed by dipping in ethanol, then after dipped in
chemical sterilizing agent (mercuric chloride or sodium
hypochlorite) and again washed with distilled water

• Preparation of explant from surface sterilized explant material:

The surface sterilized explant material is cut into small pieces;

the small pieces of plant material or tissue is called as explant;
this explant is used for production of callus
• Inoculation (transfer) of explant:

The sterilized explant is transferred to the sterilized semi- solid

nutrient medium using forceps; this action is performed in
laminar air hood

• Incubation of culture:

The culture flask containing explant is placed under the proper

controlled environment (temperature, light, etc.); under these
conditions, after few weeks, callus is formed from the explant
• Subculture of callus:

With the formation, multiplication & growth of callus, nutrient

along with water goes on depleting; also due to many metabolic
reactions inside culture flask, toxic materials might get
accumulated; these condition effects the callus growth; so to
maintain the growth of callus, it is then transferred in the fresh
nutrient medium, the process known as subculture; for this the
callus is cut into small pieces and cultured in the fresh medium

• Regeneration of plant from callus:

Plant is regenerated from the developed callus via either of the

process i.e. Organogenesis & Embryogenesis
In organogenesis, root and shoot are developed; for this
developed callus is transferred in plant regeneration media
containing proper concentration of auxin and cytokinin; for e.g.
in leaf culture, developed callus is transferred in shooting media
(containing high concentration of cytokinin as compared to
auxin) where shoot is developed; the developed shoot with small
portion of the callus is separated and transferred in the fresh
nutrient medium containing the high concentration of auxin as
compared to cytokinin (rooting media); root formation starts and
eventually develops into the young plantlets

In embryogenesis, the callus is developed in the proper nutrient

medium, where embryoid is formed from the callus; from this
embryo plantlet is developed
Fig. Callus culture
2. Suspension Culture
• Single cell or small aggregates of cells multiply while suspended
in agitated liquid medium, the process termed as suspension
culture

• Here, uniform suspension of separate cells in the liquid medium

occurs

Preparation of suspension culture:

• Isolation of single cell:

There are two methods for isolation of single cell i.e. callus
culture (from which callus is developed from explant and single
cell is isolated) & plant organ (plant part is taken and single cell
is isolated)
Plant organ:

First, the plant part is taken, surface sterilized and cut into small
pieces; cell is isolated from explant by either of the process i.e.
mechanical (grinding) or enzymatic (macerozyme)

Mechanical method: explant is surface sterilized and ground in

mortar and pestle, cleaned, filtered and filtrate is centrifuged to
isolate single cell; single cell is transferred in specific nutrient
medium for establishment of suspension culture

Enzymatic method: explant is surface sterilized and dipped in

enzymatic solution using macerozyme which isolates single cell;
enzymatic solution is replaced with specific nutrient medium for
establishment of suspension culture
 Callus culture:

In callus culture, first, the plant part is taken, surface sterilized

and cut into small pieces (explant); explant is inoculated in semi-
solid nutrient medium and incubated in suitable growth
conditions; callus is developed after few weeks

Callus is cut into small pieces and transferred aseptically into

liquid medium containing flask; this flask is placed in shaker;
since callus is loosely massed cells because of agitation breaks
into individual cell or cell aggregate

Nutrient media is filtered; large cell aggregates are removed and

filtrate is centrifuged or resuspended in to fresh medium; this
process is known as sub-culture
 After subculture, it is incubated at 250C in dark or low intensity
light in BOD incubator with shaker so as to prevent larger cell
aggregates and maintain separated cells in suspended form

As compared to callus culture, subculture of suspension culture is

generally performed at the interval of 7-21 days because cells in
the suspension culture grow at higher rate; so to maintain the
cells in the suspension culture periodic subculturing is done

• Plating of cell suspension:

Small volume of suspension culture containing cells is taken and

mixed with same volume of agar medium

The mixture is transferred to petri-plate where cells are distributed

in thin layer after the solidification of agar
 Plate is incubated where single cells divide to form the group of
cells known as cell colonies

• Selection of more productive clones:

More productive cell colonies are selected with the help of

various techniques such as clone pigmentation, HPLC, etc.

• Extraction of secondary metabolite or Plant regeneration:

The clones of cells are then transferred to bioreactor for

extraction of secondary metabolites or plant can be regenerated
via formation of embryoid
Importance of Suspension culture:

• Since single cells are used in suspension culture, cell physiology,

biochemistry, etc. can be studied

• In suspension culture, we can perform in vitro mutagenesis by

adding mutagens in liquid medium and mutants are selected and
plant is regenerated (crop improvement program)

• Biotransformation and Genetic engineering is possible with

suspension culture:

• Biotransformation is the process whereby a substance is

changed from one chemical to another (transformed) by a
chemical reaction within the body
• For biotransformation, substrate is added in the suspension
culture; cells in the suspension culture utilize the substrate and
convert into desired product

• For genetic engineering, desired gene is inserted in the genetic

material of plant cell in the suspension culture; this transformed
cell is further cultured and developed into transgenic plant with
desired characteristics
Growth profile of suspension culture:

• Under appropriate light, temperature, aer­ation and nutrient

medium the growth of sus­pension culture follows a predictable
pattern or growth curve

• To maintain the cells in the suspension culture, sub-culturing is

done

• The time for sub-culturing and to maintain the growth in the

suspension culture, the growth of cells in the suspension culture
is monitored

• For this the growth curve is constructed and growth curve is used
for monitoring
• Growth in suspension culture is monitored by counting the no. of
cells present per unit volume of the suspension culture with
respect to the time (day of the culture)

• In particular time interval (with in 1-2 days), specific volume of

liquid from suspension culture is taken and cells are counted and
growth curve of suspension culture is constructed

• Following this growth curve, growth of suspension culture is

maintained
• The growth curve for a typical higher plant suspension culture
consists of lag phase, loga­rithmic phase or exponential phase,
linear phase and stationary phase

• The lag phase is the period where the cells adjust themselves to the
nutrient medium & undertake all necessary synthesis prior to
division

• The logarithmic phase is the period where cells divide, multiply and
increase in number

• A further period of rapid cell division results in a linear increase in

number and the phase is called linear phase

• As nutrients are depleted and some of the cells of the culture begin
to show senescent charac­teristics, the rate of cell division within the
cul­ture declines & passes through station­ary phase; sub-culturing is
done just before reaching this phase or just after reaching this phase
Maintenance of Suspension culture:

1. Batch Suspension Culture: More amount of nutrient medium is

added to the original culture or cells are transferred to the fresh
medium in the closed system

2. Semi-continuous Suspension Culture: Periodic removal of

culture and addition of fresh medium

3. Continuous Suspension Culture: Fresh medium and culture are

added and withdrawn respectively which means the volume of
the culture remain constant