Plant tissue culture method and types of culture methods

1. PLANT TISSUE
CULTURE
Presented By:
Sushant Mangesh Gawali
(A701118421007)
1st Sem M.Tech Food Biotech.

2. CONTENTS
• Plant Tissue Culture
• General technique of Plant Tissue Culture
• Culture medium
• Types of Plant Tissue Culture
• Micropropagation
• Advantages and disadvantages
• Applications of Plant Tissue Culture
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3. PLANT TISSUE CULTURE
• Tissue culture is in-vitro cultivation of plant cell or tissue under
aseptic and controlled environment conditions, in liquid or on
semisolid well-defined nutrient medium for the production of
primary and secondary metabolite or to regenerate plant.
• Plant tissue culture relies on the fact that many plant cells have the
ability to regenerate a whole plant (totipotency).
• Gottlieb Haberlandt, a German botanist, in 1902 cultured fully
differentiated plant cells isolated from different plants. This was
the very first step for the beginning of plant cell and tissue culture.
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4. Cont…
• The mostly used MS media was invented early by T. Murashige and F.
Skoog in 1962 during the invention of plant growth regulator.
• In India, Shri S. C. Maheshwari and Sipra Guha made a remarkable
contribution in the development of anther culture in 1970, that
opened the new era of androgenesis.
• The main requirements of plant tissue culture are:
(1) Laboratory Organization
(2) Culture Media
(3) Aseptic Conditions
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5. Fig. 1: General Technique of Plant Tissue Culture
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6. Selection and Sterilization of Explant
Suitable explant is selected and is then excised from the
donor plant. Explant is then sterilized using disinfectants.
Preparation and Sterilization of Culture Medium
A suitable culture medium is prepared with special attention
towards the objectives of culture and type of explant to be
cultured. Prepared culture medium is transferred into sterilized
vessels and then sterilized in autoclave.
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Detail steps involved in PTC

7. Inoculation
Sterilized explant is inoculated
(transferred) on the culture medium
under aseptic conditions.
Incubation
Cultures are then incubated in the culture
room where appropriate conditions of light,
temperature and humidity are provided for
successful culturing.
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8. Sub culturing
Cultured cells are transferred to a fresh
nutrient medium to obtain the plantlets.
Transfer of Plantlets
After the hardening process (i.e.
acclimatization of plantlet to the
environment), the plantlets are transferred to
green house or in pots.
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Fig. 2: Flow chart of
plant tissue culture
technique

10. A Laminar air flow i.e. LAF hood/cabinet is
an enclosed workstation that is used to create
a contamination - free work environment
through filters to capture all the particles
entering the cabinet.
Laminar air flow or LAF
Fig. 3: LAF cabinet
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The formulation or the medium on which the explant is cultured is called culture
medium. It is composed of various nutrients required for proper culturing. A number of
media have been devised for specific tissues and organs. Some important of them are:
1. MS (Murashige and Skoog) Medium
2. LS (Linsmaier and Skoog) Medium
3. B5 (Gamborg’s) Medium
4. White’s Medium, etc.
CULTURE MEDIUM

12. Important constituents of a culture medium are:
(i) Organic supplements:
• Vitamins like thiamine (B1), Pyridoxin (B6), Nicotinic Acid (B3), etc.
• Antibiotics like Streptomycin, Kanamycin, etc.
• Amino Acids like Arginine, Asparagine, etc.
(ii) Inorganic Nutrients:
• Micronutrients as Iron (Fe), Manganese (Mn), Zinc (Zn), Molybdenum (Mo), Copper (Cu),
Boron (B).
• Macronutrients include six major elements as Nitrogen (N), Phosphorus (P), Potassium (K),
Sulphur (S), Calcium (Ca), Magnesium (Mg).
(iii) Carbon and Energy Source:
• Most preferred carbon source is Sucrose.
• Others include lactose, maltose, galactose, raffinose, cellobiose, etc.
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13. (iv) Growth Hormones:
• Auxins - mainly for inducing cell division.
• Cytokinins - mainly for modifying apical dominance and shoot differentiation.
• Abscisic Acid (ABA)
• Gibberellic acid (GA)
(v) Gelling Agents:
• Added to media to make them semisolid or solid.
• Agar, Gelatin, Alginate etc.
(vi) Other Organic Extracts:
• Sometimes culture media are supplemented with some organic extracts also like coconut
milk, orange juice, tomato juice, potato extract, etc.
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14. The culture room means the room for
keeping or incubating the culture under
controlled temperature, light and humidity.
The relative humidity of the culture room
is maintained above 50%. The optimum
temperature in growth room is about 20°C
to 30°C and the light intensity required is
about 1000-2000 lux.
Fig. 4: Culture growth room
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Cell or suspension culture
Shoot culture
Root culture
Protoplast culture
Embryo culture
Endosperm Culture (Triploid Production)
Anther and pollen culture (Haploid Production)
TYPES OF PLANT TISSUE CULTURE

16. Cell Culture:
• Cell culture is actually, the process of producing clones
of a single cell. First attempts for cell culture were
made by Haberlandt in 1902.
• It is important that the cell cultures require a suitably
enriched nutrient medium and it should be done in
dark because light may deteriorate the cell culture.
Suspension Culture:
• A culture which consists of cells or cell aggregates
initiated by placing callus tissues in an agitated liquid
medium is called as a suspension culture.
• The general technique of suspension culture involves
basically two types of cultures: batch culture and
continuous cultures.
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Fig. 5: Cell culture
Fig. 6: Suspension culture

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Shoot Culture:
• The practical application of this method was proposed by
Morel and Martin (1952) after they successfully recovered the
complete Dahalia plant from shoot-tips cultures.
• In this technique, the shoot apical meristem is cultured on a
suitable nutrient medium. This is also referred to as Meristem
Culture.
Fig. 7: Shoot culture
Root Culture:
• Pioneering attempts for root culture were made by Robbins and
Kotte during 1920s. Root culturing of a number of plant
species of angiosperms as well as gymnosperms has been done
successfully.
• Root cultures are usually not helpful for giving rise to complete
plants but they have importance’s of their own.
Fig. 8: Root culture

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Protoplast Culture:
• A protoplast is described as a plasma membrane bound vesicle
which consists of a naked cell formed as a result of removal
of cell wall.
• At present, there are available a number of enzymes which
have enabled the isolation of protoplasts from almost every
plant tissue.
• Production of hybrid plants through the fusion of protoplast
of two different plant species or varieties is called somatic
hybridization and the hybrid plant obtained is known as
somatic hybrid
• It not only serves for genetic manipulations in plants but also
for biochemical and metabolic studies in plants.
• In-vitro culturing of protoplasts has immense applications in
the field of plant biotechnology.
Fig. 9: Protoplast culture

19. Endosperm Culture (Triploid Production):
• Endosperm tissue is triploid therefore the plantlets originating by the culture of endosperm are
also triploid. Endosperm is formed after the double fertilization of one male nucleus with two
polar nuclei.
• The triploid plants are usually seedless therefore this technique is most beneficial for increasing
the commercial value of fruits like apple, mango, grapes, watermelon, etc.
• Endosperm culture is helpful for studying biosynthesis and metabolism of certain natural
products also.
Embryo Culture:
• The technique of embryo culture involves the isolation and
growth of an embryo under in-vitro conditions to obtain a
complete viable plant.
• First success for embryo culture was made by Hannig in
1904.
• Embryo culture is advantageous for in-vitro micro
propagation of plants, overcoming seed dormancy and for
production of beneficial haploid plants. Fig. 10: Embryo culture

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Anther and Pollen Culture (Haploid Production):
• Haploid plants are those which contain half the number of
chromosomes (denoted by n).
• There are two approaches for in-vitro haploid production and these are:
(a) Androgenesis: The technique of production of haploids through anther
or microspore culture is termed as androgenesis. It is achieved either by
another culture or by microspore (pollen) culture. Androgenesis is
preferred over gynogenesis.
(b) Gynogenesis: It is an alternative source of in-vitro haploid production. It
refers to the production of haploid plant from ovary culture or ovule
culture.
Fig. 11: Anther
culture

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• Tissue culture helps in the rapid propagation of plants by the technique of micro-
propagation or clonal propagation in-vitro. The asexually produced progeny of a cell or
individual is called as clone and the clones have an identical genotype.
• Micropropagation is the technique of in-vitro production of the clones of plants i.e., it
produces the progeny plants which have an identical genotype as their parents, by cell,
tissue or organ culture.
• It is nothing but the use of plant tissue culture at large scale. It serves as an alternate
method to conventional vegetative propagation methods.
• Micro propagation may be achieved by shoot tips, axillary buds, adventitious buds, bulbs
or somatic embryos.
MICRO-PROPAGATION

22. The stages in general Micro-propagation technique
Stage I - Initiation
This stage also involves the preparatory process for achieving better establishment of aseptic
cultures of explant. Suitable explant is selected from the mother plant. Then, the explant is
sterilized and transferred to the nutrient medium for culture.
State II - Multiplication
This is the most important stage of micro propagation. In this stage, there occurs the
proliferation or multiplication of shoots (or embryoids) from the explant on medium. It
occurs either by the formation of an intermediary callus or by induction of adventitious buds
directly from the explant.
Stage III - Sub-culturing
The shoots are transferred to rooting medium (sub-cultured) to form roots. As a result,
complete plantlets are obtained.
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23. Stage IV - Transplantation
In this stage, the regenerated plantlets are transferred out of culture. These are grown in pots
followed by field trials.
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Fig 12:
Micropropagation
in Banana

24. • The plantlets are obtained in a very short time with a small amount of plant
tissue.
• The new plants produced are disease-free.
• The plants can be grown throughout the year, irrespective of the season.
• A large space is not required to grow plants by tissue culture technique.
• The production of new varieties in the market place speeds up.
• This technique is being used for the production of ornamental plants such as
dahlia, chrysanthemum, orchids, etc.
ADVANTAGES
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25. • Complex and time consuming
• Dependent to plant genotype
• Require efficient in-vitro plant regeneration protocol
• Requires complete sterile conditions throughout the culturing
• Labor intensive
• High risk of contamination, etc.
DISADVANTAGES
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1. Large scale and fast multiplication
2. Clonal propagation
3. Germplasm conservation in form of cryopreservation
4. Genetic transformation
5. Synthetic seeds
6. Somatic hybrids and cybrids
7. Early flowering
8. Seedless fruits and vegetables
9. Breaking dormancy and overcoming male sterility
10. Secondary metabolite production, etc.
APPLICATIONS OF PLANT TISSUE CULTURE