3. Plant tissue culture broadly refers to the in vitro cultivation of plant parts under
aseptic conditions.
Tissue culture is a collection of techniques used to maintain or grow plant cells,
tissues or organs under sterile condition on a nutrient culture medium
A. INTRODUCTION
Figure 1, Tissue Culture 3
5. Central concept of Plant tissue culture
Totipotency: The property of plant cell to express the total genetic potential of the
parent plant under correct stimuli.
Fig.-2,Totipotency
Plasticity: Plant cell exhibits high degree of plasticity (One type of tissue or organ to be
initiated from another type).
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6. Dedifferentiation- Formation of undifferentiated cells from differentiated cells.
Differentiation: Formation of specific cell types from undifferentiated mass of cells
Redifferentiation: The capacity of an already differentiated cell type like parenchyma or collenchyma to
produce another cell type
Transdifferentiation :If a fully differentiated living cell like a leaf mesophyll cell directly transforms itself,
without undergoing any change in size or shape, into a tracheary element or a sieve element by PCD.
Figure 3,Cytodifferentiation 6
7. C. BASIC TECHNIQUES
1. Preparation of suitable nutrient medium.
2. Selection of explants.
3. Sterilization of explants.
4. Inoculation of the explants.
5. Incubation & callus formation.
6. Regeneration.
7. Hardening.
8. Transfer to the field.
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8. • Inorganic nutrients: In addition to C , H & O , media provide the 12 essential elements for plant
growth i.e N , P , K , Ca , S & Mg {macronutrients}, Fe , Zn , Mn , Cu , B & Mo {micronutrients}
• Vitamins: Inositol, thiamine, pyridoxine, nicotinic acid, pantothenic acid, biotin, folic acid, p-amino
benzoic acid, riboflavin & Ascorbic acid
• Carbon & energy source: Sucrose, maltose, galactose, mannose & even starch.
• Growth regulators: Auxins- IAA, NAA, 2-4,D, IBA, NOA, 2,4,5-T, P-Cholorophenoxy acetic acid.
Cytokinin- Kinetin, 6-benzyl amino purine, zeatin, isopentenyladenine, BAP, 2-IP.
• Complex organic additives:
Yeast extract, coconut milk, casein hydrolysate, corn milk, malt extract & Tomato juice.
Gelling agents: Agar is used as gelling agent. 0.5-1% agar is used to form gel.
• PH: 5.0-6.0 is suitable for in vitro growth of explants.
1.PREPARATION OF SUITABLE NUTRIENT MEDIUM
The culture medium is selected depending upon the plant material to be cultured. A media of tissue
culture contains the following ingredients:
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9. Common culture medium used for tissue culture:
The commonly used tissue culture media are:
White’s medium- for root culture.
MS medium- organogenesis & regeneration.
B5 medium- for cell suspension & callus cultures.
N6 medium- for cereal anther culture.
During preparation: Media, containers
and small instruments are made
contamination free by suitable methods
such as Steam, Dry, Filter & UV sterilization
Table-1 9
10. 2. SELECTION OF EXPLANT
The excised plant part for culture is called explants.
The following portion may be used as explant materials:
i. Root
ii. Stem
iii. Leaf
iv. Meristem
v. Endosperm
vi. Nucellus
vii. Embryo
viii. Anther, Pollen
ix. Ovule
Figure.5 Explant for PTC
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11. 3. STERILIZATION
The process of killing microbial contamination on the surface of explant is known as
surface sterilization.
It is carried out using:
Sodium hypochlorite: (1-1.4%)
Calcium hypochlorite: (9-10%)
Hydrogen Peroxide: (10-12%)
Silver Nitrate: (1%)
Mercuric Chloride: (0.1-1%)
Bromine Water: (1-2%) 11
12. 4.INOCULATION OF EXPLANTS
The process of transfer explants to culture medium under aseptic conditions, is called
Inoculation.
Figure 6- Inoculation
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13. 5.INCUBATION & CALLUS FORMATION
The explant inoculated flask are kept in a constant Temperature room or Incubator for a
considerable time.
Temperature adjusted to 25+-2 Oc with 50-60% relative humidity & 16 hrs. of
photoperiod. After define period callus develops on the medium from explants.
Figure 7-Callus induction 13
14. Subculture: As callus grows, depletion of nutrients occur in the medium. So subculture
required.
The maintenance of fragmented callus in fresh medium is called Subculture.
Generally Subculture Practice performed in the interval of 4 weeks.
Figure-8, Subculture
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15. 6.REGENERATION
The development of young plants looking like Seedlings, is called Plant regeneration.
It can be achieved by two ways:
(a).Organogenesis
(b). Somatic-embryogenesis
a. Organogenesis: refers to development of organs:
adventive roots and shoots.
i. Direct organogenesis
ii. Indirect organogenesis
Organogenesis from callus is determined by the
concentration of Auxin and Cytokinin in the medium.
Explant Callus meristemoid organ primordia
Explant meristemoid organ primordia Figure 8,Regeneration 15
18. b. Somatic embryogenesis:
The process of development of embryos from somatic cells, tissues or organs is regarded
as somatic embryogenesis.
Somatic embryos are structurally similar to zygotic embryos. They can be excised from
the parent tissue & induced to germinate in tissue culture media
Globular Heart Torpedo
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Figure 11, Somatic embryogenesis
19. There are 2 types Somatic embryogenesis :
(i) Direct somatic embryogenesis
(ii) Indirect somatic embryogenesis
i. Direct somatic embryogenesis:
When the somatic embryos develop
directly from the explant due of presence
of pre-embryonic determined cells
(PEDC).
Explant form callus, from which
Somatic embryo develops due to
activities of induced embryogenic
determined cells (IEDC)
ii. Indirect somatic embryogenesis:
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Figure 12, Somatic embryogenesis
20. 7.HARDENING
The process of acclimatization of the in vitro grown plants to the normal environment is called hardening.
First stage
The regenerated plantlets are transferred to large bottles contain nutrient medium and kept in constant
temperature room with illumination . It helps in growth of root and shoot within a week.
Second Stage
Grown up plantlets are planted in pots/polythene bags contain soil, nutrients. Plantlets are maintained in green
house for 15-30 days.
Figure 13, A. Primary hardening B) Secondary hardening
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22. Types of Culture: On the basis of plant material used culture is following types
Cell culture
Tissue culture
Embryo Culture
Shoot Apical meristem culture
Endosperm Culture
Anther culture
Pollen Culture
Ovule culture etc
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23. D.APPLICATIONS
Micropropagation /clonal propagation
Production of virus free plants
Rejuvenation plant materials
Somaclonal variation
Production of somatic hybrids and cybrids
Production of secondary metabolites
Embryo rescue
Production of Haploid plants
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24. E. CONCLUSION
Plant tissue culture is one of the most rapidly growing areas of Biotechnology because of its
high potential for rapid multiplication of plants, in addition it is associated with,
production of secondary metabolites, germplasm conservation, development of improved
crops varieties & ornamental plants, Hybrids etc.
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25. REFERENCES
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Otoni, W. C. (2018). Light quality in plant tissue culture: does it matter?. In Vitro Cellular &
Developmental Biology-Plant, 54, 195-215.
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changes. Plant Cell, Tissue and Organ Culture (PCTOC), 140(2), 245-257.
3. Brar, D. S., & Khush, G. S. (2021). Cell and tissue culture for plant improvement. In Mechanisms of Plant
Growth and Improved Productivity Modern Approaches (pp. 229-278). CRC Press.
4. Dias, M. I., Sousa, M. J., Alves, R. C., & Ferreira, I. C. (2016). Exploring plant tissue culture to improve
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for production of biological active compounds. Planta, 248, 1-18.
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perspectives. Plant cell culture protocols, 3-13.
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