The ability of an explant to regenerate into a whole plant under in vitro asceptic conditions by providing a proper artificial nutrient medium is called as Plant Tissue Culture.

1. Presented by,
Dr. Manoj R. Kakpure
Head & Assistant Professor,
Late R. Bharti Arts, Comm. & Smt. S. R. Bharti Science College
Arni, Dist-Yavatmal.

2. Plant tissue culture is a collection of techniques used to
maintain or grow plant cells, tissues or organs under in vitro
asceptic conditions on a nutrient culture medium of known
composition to develop a whole plant.
 Plant tissue culture relies on the fact that many plant cells
have the ability to regenerate a whole plant (totipotency).
 Single cells, plant cells without cell walls (protoplasts),
pieces of leaves, stems or roots can often be used to
generate a new plant on culture media given the required
nutrients and plant hormones.

3. Plant Tissue culture origins at the
beginning of the 20th century with
the work of German Scientist
Gottleib Haberlandt (1902). His
original ideas was called
totipotentiality “theoretically all
plant cells are able to give rise to a
new plant”.
History

4. Advantages
• True-to-type clones.
• A single explants can be multiplied into several thousand.
• To produce disease free plants.
• Long-term germplasm storage with tissue banks.
• Plant cultures easier to export than are soil-grown plants.
• To conserve rare or endangered plant species.
• To cross distantly related species by protoplast fusion and
regeneration of the novel hybrid.
• Production of haploid.
• Production of secondary metabolites.

5. Basis for Plant Tissue Culture
• Two Hormones Affect Plant Differentiation:
– Auxin: Stimulates Root Development (IAA, IBA, NAA, 2,4-D)
– Cytokinin: Stimulates Shoot Development (Kn, BAP, Za)
• Generally, the ratio of these two hormones can determine plant
development:
–  Auxin ↓Cytokinin = Root Development
–  Cytokinin ↓Auxin = Shoot Development
– Auxin = Cytokinin = Callus Development

6. How can plant cell or tissue develop?
 Totipotency
The potential or inherent capacity of a plant cell to develop into an entire
plant if suitably stimulated. It implies that all the information necessary for
growth and reproduction of the organism is contained in the cell.
 Dedifferentiation
Capacity of mature cells to return to meristematic condition and
development of a new growing point, follow by redifferentiation which is
the ability to reorganize into new organ.
 Competency
The endogenous potential of a given cells or tissue to develop in a
particular way.

7. PTC Lab Design
• Washing & Storage Room
• Media Preparation Room
• Instrumentation
• Asceptic Transfer Area
/ Inoculation Room
• Growth Culture Room
Washing Room
Media Preparation Room
Instrumentation Room
Inoculation / Asceptic
transfer Room
Growth Room

8. Instrumentation
PH meter
PH should maintained 5.8
PH can be adjusted (lower /
increase) with 1N HCl & 1N
NaOH.

9. Autoclave
Autoclaves use pressurized steam
as their sterilization agent. The
basic concept of an autoclave is to
have each item sterilized whether it
is a liquid, plastic wares or
glassware come in direct contact
with steam at a specific
temperature and pressure for a
specific amount of time.
Temperature: 121°C (250 °F)
for 15–20 minutes
Pressure : 15 lbs

10. Laminar Air Flow
Laminar airflow is defined
as air moving at the same
speed and in the same
direction, with no or minimal
cross-over of air streams (or
“lamina”).
It is used to separate volumes
of air or prevent airborne
contaminants from entering an
area.
A laminar flow hood consists of a filter pad, a fan and a HEPA (High Efficiency
Particulates Air) filter. The fan sucks the air through the filter pad where dust is
trapped. After that the prefiltered air has to pass the HEPA filter where contaminating
fungi, bacteria, dust etc are removed.

11. Growth Culture Racks

12. Sterilization Techniques
The materials, i.e. explants, instruments, medium and other
requirements used in culture work must be free from microbes. This
can be done by one of the following approach –
1. Dry heat sterilization
2. Flame sterilization
3. Autoclaving
4. Wiping with 70 % ethanol
5. Surface sterilization

13. MS- Media
Murashige and Skoog Medium (MS) was originally formulated in 1962.
VITAMINS
myo-Inositol
Nicotinic acid (free acid)
Pyridoxine HCl
Thiamine hydrochloride
AMINO ACID
Glycine Total(gms/litre) 4.4
Stock solution of PGR’s
Others
Cacl2 440 mg
Sucrose 30 gm
Agar-agar 8 gm
PH should maintained 5.8
Composition
Ingredients mg/L
MACROELEMENTS
Ammonium nitrate 1650.000
Calcium chloride 332.200
Magnesium sulphate 180.690
Potassium nitrate 1900.000
Potassium phosphate 170.000
MICROELEMENTS
Boric acid 6.200
Copper sulphate 0.025
EDTA disodium salt 37.300
Ferrous sulphate 27.800
Manganese sulphate 16.900
Potassium Iodide 07.023
Zinc sulphate 14.365

14. Cellular totipotency
Steward et al., (1962)

15. Importance of Totipotency
1. In protoplast, cell and tissue culture is reconstruction of plants from
totipotent cell.
2. It provides lot of information during differentiation in culture.
3. The potentiality of somatic cell has been exploited in vegetative
propagation of many economical, medicinal as well as agricultural
important plant species.
4. It helps in the production of homozygous diploid plants through
haploid cell culture, somatic hybridization and mutation.
5. It can be utilized successfully for germplasm conservation of
endangered plant species.

16. Differentiation & Morphogenesis
 In nature, totipotency of somatic cells observed in many taxa
where stem, root & leaf are able to differentiated into root & shoot.
 In vitro studies are (Skoog & Miller, 1957) indicated that
totipotency is not restricted to few species, most species if
provided with appropriate conditions would show differentiation.
 Plant production through organogenesis can be achieved by –
a) Shoot bud differentiation
i) organogenesis by callus formation (indirect regeneration)
ii) direct regeneration
b) Through somatic embryogenesis

17. Differentiation & Morphogenesis

20. Callus Culture
 Callus is an unorganised proliferative mass of parenchyma cells
produced from explants when grown asceptically on a artificial
nutrient medium under controlled condition.
 In vitro culture of callus tissue was carried out by Gautheret R.J.
(1937) and Steward (1948).
 For successful initiation of callus culture, three important criteria
should be accomplished –
i) asceptic preparation of plant materials (explants).
ii) selection of suitable nutrient medium supplemented with
appropriate ratio of auxin & cytokinin.
iii) incubation of culture under controlled conditions.

21.  A fresh tap root of carrot is taken and washed thoroughly under running tap
water to remove all surface detritus.
 The tap root is then dipped into 5% ‘Teepol’ for 10 minutes and then the root is
washed.
 The carrot root, sterilized forceps, scalpels, other instruments, autoclaved
nutrient medium petridishes are then transferred to laminar air flow in
inoculation chamber.
 Throughout the manipulation sequence forceps, scalpels must be kept in 95%
ethanol and flamed thoroughly before use.
 The tap root is surface sterilized by immersing in 70% ethanol for 60
seconds, followed by 20-25 minutes in sodium hypochlorite
 The root is washed three times with sterile distilled water to remove completely
the hypochlorite.
 The carrot is then transferred to a sterilized petridish containing a filter paper. A
series of transverse slice 1 mm in thickness is cut from the tap root using a sharp
scalpel.
Protocol

22.  Each piece is transferred to another sterile petridish. Each piece contains a
whitish circular ring of cambium around the pith. An area of 4mm2 across the
cambium is cut from each piece so that each small piece contains part of the
phloem, cambium and xylem. Size and thickness of the explants should be uniform.
 The closure (cotton plug) from a culture tube is removed and flamed the uppermost
20 mm of the open end. While holding the tube at an angle of 45°, an explants is
transferred using forceps onto the surface of the agarified nutrient medium. Nutrient
medium is Gamborg’s B5 or MS medium supplemented with 0.5 mg/L 2, 4-D.
 The closure is immediately placed on the open mouth of each tube. The forceps are
always flamed before and after use. Date, medium and name of the plant are
written on the culture tube by a glass marker pen.
 Culture tubes after inoculation are taken to the culture room where they are placed
in the racks. Cultures are incubated in dark at 25+2°C.
 Usually after 4 weeks of culture, the explants incubated on medium with 2, 4-D will
form a substantial callus. The whole callus mass is taken out aseptically on a sterile
petridish and should be divided into two or three pieces.
 Each piece of callus tissue is transferred to a tube containing fresh same medium.

24.  The whole plant can be regenerated in large number from callus tissue
in the culture medium. This phenomenon is known as plant
regeneration or organogenesis or morphogenesis.
 Cluster of embryos can be achieved directly from the somatic cells of
callus tissue. These embryos are called somatic embryos. This
phenomenon is known as somatic embryogenesis.
 Callus tissue is good source of genetic or karyotype variability, so it
may be possible to regenerate a plant from genetically variable cells of
the callus tissue.
 Callus culture is very useful to obtain commercially important
secondary metabolites. So, this alternative technique helps the
conservation of medicinal plants in nature.
 Several biochemical assays can be performed from callus culture.
Significance

25. Micropropagation
 Micropropagation is the artificial process of producing plants
vegetatively from a single explant.
 In this artificial process of propagation, plants are produced in vitro by
asexual means of reproduction or by vegetative propagation.
 Clonal propagation refers to the process of asexual reproduction by
multiplication of genetically identical copies of individual plants, where
the term clone is used to represent a plant population derived from a
single individual by asexual reproduction.
 In vitro clonal propagation through tissue culture is referred to as micro-
-propagation.
 Micropropagation is the practice of rapidly multiplying stock plant
material to produce a large number of progeny plants.
 Early discovery about this technique were made by G. Haberlandt
(1902), White (1932) and later by Skoog and Miller (1963), steward
(1964) Maheswari (1966).

26. Stages Methods Involved
Stage 0 Preparative stage: Selection of an explants
Stage I Initiation stage and establishment
Stage II Shooting & its multiplication (sub-culture)
Stage III Rooting of the shoots
Stage IV
Hardening: Transfer of plantlets in the greenhouse
environment
Stages of Micropropagation

29. Inoculated explant Initiation (Growing shoot) Multiple shoot
Ready to subculture Multiple shooting Rooting

30. Primary Hardening
Secondary Hardening

31. Advantages of Micropropagation
 This is an alternative method for vegetative propagation with enhanced
multiplication rate.
 Large quantities of identical plants can be obtained from a single plant tissue
within a very short time period.
 The germplasm stocks can be maintained for several years using this technique.
 It helps in the production and maintenance of pathogen-free plant varieties.
 In a dioecious plant, the seed progeny yield is 50% male and 50% female. This
method helps in obtaining the desired sex of the plant.
 Millions of plantlets can be maintained in the cultural vials.
 Genetic uniformity of the propagules can be maintained through this technique.
 It is a cost-effective process.
 New varieties of species can be propagated & is independent of season and can
be carried out anytime.