The document describes a seminar presentation on using plant tissue culture techniques to induce variation and improve crops. It provides background on tissue culture, discusses types of culture and variation that can occur. It also presents a case study on developing insect-resistant transgenic brinjal (eggplant) and studies on tissue culture-induced variation in tomato and torenia plants. The conclusion states that tissue culture has been valuable for plant biotechnology and improving food security.

2. SEMINAR
ON
Mr. Jadhao Gopichand Prakash
Regd. No. 014/042
Seminar In charge
Dr. D. V. Kusalkar
Professor of Botany
Tissue culture- Induce variation and crop
improvement
DEPARTMENT OF AGRICULTURAL BOTANY
POST GRADUATE INSTITUTE
MAHATMA PHULE KRISHI VIDYAPEETH.
RAHURI – 413 722

3. Presentation on
INDUCE VARIATION
AND
CROP IMPROVEMENT

4. Introduction
 The propagation of a plant by busing a plant parts or single cell
or group cell in test tube under controlled and hygienic
conditions called as “tissue culture”.
 Plant tissue culture is a collection of techniques used to
maintain or grow plant cells, tissues or organs under sterile
conditions on a nutrient culture medium of known
composition.
 Plant tissue culture is widely used to produce clones of a plant
in a method known as micropropagation.
 Different techniques in plant tissue culture may offer certain
advantages over traditional methods of propagation.

5.  The production of exact copies of plants that produce
particularly good flowers, fruits, or have other
desirable traits.
 To quickly produce mature plants.
 The production of multiples of plants in the absence
of seeds or necessary pollinators to produce seeds.
 The regeneration of whole plants from plant cells that
have been genetically modified.

6.  The production of plants in sterile containers that allows
them to be moved with greatly reduced chances of
transmitting diseases, pests, and pathogens.
 The production of plants from seeds that otherwise have
very low chances of germinating and growing, i.e.: orchids
andNepenthes.
 To clean particular plants of viral and other infections and
to quickly multiply these plants as 'cleaned stock' for
horticulture and agriculture.
 .

7. Somaclonal variation
 Somaclonal variation is a general phenomenon of all
plant regeneration systems that involve a callus phase.
 There are two general types of Somaclonal
Variation:Heritable, genetic changes (alter the DNA).
 Stable, but non-heritable changes (alter gene
expression

8. Basic Steps of PTC
Stage 0:
preparative
stage-involving
selection of
species.
Stage 1:
production of
axenic, viable
cultures.
Stage 2:
Multiplication
stage.
Stage 3: planlet
production
stage.
Stage 4:
Establishment
under in vivo
condition.

9. Types of In Vitro Culture
 Culture of intact plants
 Embryo culture (immature embryo culture)
 Organ culture
 Callus culture
 Cell suspension culture
 Protoplast culture
 Somatic Embryogenesis
 Micropropagation
 Somaclonal variation

10. Types of Plant Tissue Culture

11. Advantages of Tissue culture
 They produce exact copies of plants required that have
desirable traits.
 They produce mature plants quickly.
 Multiple plants are produced in the absence of seeds.
 Whole plants are produced regenerated from plant cell
that are genetically modified.
 Many plants that are clones of each othercan be
produced.
 Disease resistant plants are produced by
micropropagation.
 High rate of fecundity is obtained.

12. Disadvantages of Tissue culture
 Micropropagation is not method of multiplying
plants.
 It is labour intensive and expensive process.
 A monoculture that is produced after
micropropagation which leads to the lack of
disease resistance all the progeny plants are
vulnerable to the same infections.
 An infected sample plants produced infected
progenies.
 All plants cannot be successfully tissue cultured.

13. Basic Techniques
 Setting up of a tissue culture lab requires proper
planning.
 It is divided into 5 areas
 Media preparation room
 Aseptic transfer area
 Culture room
 Analytical room
 Acclimatization room

14. Media Preparation Room
 Refrigerator & freezer
 Water purification & storage system
 Glassware washing facility
 Continuous supply of single & double distilled
water
 Culture media, washing powder, disinfectants
 Cabinets or shelves

15. Aseptic Transfer Area
 Laminar air flow
 Dissecting microscopes
 Dissection instruments
 Gas outlet
 Vacuum facility
 Sterilizer

16. Culture Room
 Environmentally controlled
 Incubators with controlled temperature
 Rotary shakers
 Lux meter
 Space for cultures requiring complete darkness

18. Media
 No single medium supports growth of all tissues.
 Some basic factors
 Callus induction
 Organogenesis
 Murashige-Skoog medium, White’s medium, woody
plant medium

19. Media Components
 Inorganic salts
 Major Elements
○ Calcium
○ Iron
○ Magnesium
○ Nitrogen
○ Phosphorus
○ Potassium
○ Sulphur
 Minor Elements
○ Boron
○ Chlorine
○ Cobalt
○ Copper
○ Iodine
○ Manganese
○ Molybdenum
○ Zinc

20. Media Components
 Organic Chemicals
 Carbohydrates
○ Sucrose
○ D-Mannitol
○ D-Sorbitol
 Vitamins
○ Vit B complex
○ Adenine
○ Choline
○ Folic Acid
○ Riboflavin
○ Thiamine

21. Media Components
 Growth regulators
 Auxins
 Cytokinnins
 Gibberellins
 Abscisic acid
 Amino Acids
 L-alanine
 L-arginine
 Antibiotics
 Ampicillin
 Cefotaxime
 Gentamicin sulphate
 Polymyxin B

22. MS media
 Macronutrients
 Ammonium nitrate (NH4NO3): 1,650 mg/l
 Boric acid (H3BO3): 6.2 mg/l
 Calcium chloride (CaCl2 · 2H2O): 440 mg/l
 Cobalt chloride (CoCl2 · 6H2O): 0.025 mg/l
 Magnesium sulfate (MgSO4 · 7H2O): 370 mg/l
 Cupric sulfate (CuSO4 · 5H2O): 0.025 mg/l
 Potassium phosphate (KH2PO4): 170 mg/l
 Ferrous sulfate (FeSO4 · 7H2O): 27.8 mg/l
 Potassium nitrate (KNO3): 1,900 mg/l
 Manganese sulfate (MnSO4 · 4H2O): 22.3 mg/l
 Potassium iodide (KI): 0.83 mg/l
 Sodium molybdate (Na2MoO4 · 2H2O):
0.25 mg/l
 Zinc sulfate (ZnSO4·7H2O): 8.6 mg/l
 Na2EDTA · 2H2O: 37.2 mg/l
•Common organic additives
•i-Inositol: 100 mg/l
•Niacin: 0.5 mg/l
•Pyridoxine · HCl: 0.5 mg/l
•Thiamine · HCl: 0.1 mg/l
•IAA: 1–30 mg/l
•Kinetin: 0.04–10 mg/l
•Glycine (recrystallized):
2.0 g/l
•Edamine (ethane-1,2-
diamine): 1.0 g/l
•Sucrose: 20 g/l
•Agar: 10 g/l

23. CASE STUDY

24. Insect-resistant transgenic brinjal plants. (Source-
Kumar et al.1998)
 Develop insect resistant transgenic brinjal plants. A synthetic
cry1Ab gene coding for an insecticidal crystal protein(ICP) of
Bacillus thuriengenesis(Bt) was transferred to brinjal by
cocultivating cotyledonary explants with Agrobacterium
tumefaciens. Transformed plants resistance to kanamycin were
regenerated. Hybridization experiments demonstrate gene
integration and mRNA expression. Double antibody sandwich
ELISA analysis revealed Bt toxin protein expression in transgenic
plants.It also resulted in significant insecticidal activity of
transgenic brinjal fruitbs against larvae of fruit borer.

25. A simple and efficient Agrobacterium-mediated procedure for transformation of tomato.
(Source- Sharma et al. 2009)
 Highly efficient and reproducible Agrobacteria-
mediated transformation protocol applicable to several
varieties of tomato.Optimizing various conditions of
co- cultivation period,bacterial concentration, BAP
concentration,zeatin and IAA tranformation was
confirmed by Southern blot analysis and B-
glucuronidase (GUS)assay.

26. Genetic transformation of agronomical important plant Solanum
melongena L. through hypocotyl explant. (Source- Ugandhar et al.
2011)
 Standardization of plant regeneration in S.melongena
cv.Pusa kranthi from hypocotyl explant using MS.media
supplemented with 0.5 mg/L IAA+3.0 mg/L BAP+100mg /L
kanamysin+200mg/L cefotaxime.Kanamycin sensitivity of
hypocotyl explants was assesed.Progeny analysis of
independent transgenic plant demonstrated that gus gene
was transmitted in a Mendelian pattern in 3 lines,indicating a
single copied gene was incorporated into the genome.

27. Tissue culture-induced somaclonal variation of decreased pollen
viability in torenia (Torenia fournieri Lind.) (SOURCE- Shun et al.)
 They studied the phenotypic variation in pollen viability of regenerants of torenia after
subculturing for one to nine generations. They found that pollen viability of regenerants
continuously decreased with increasing subculture time. High concentrations of plant
growth regulators applied to the Murashige and Skoog (MS) medium also resulted in
diminished pollen viability. Furthermore, antibiotic application during gene
transformation also decreased pollen viability of the transformants. However, the process
of long-term culture did not significantly change pollen viability. The mean methylation
level of regenerants showed a 0.28% to 3.95% decrease in seedlings subcultured in vitro
for nine generations. Moreover, when the ninth subcultured regenerants with reduced
pollen vibility were recovered in soil to get seeds, the pollen viability of seed-derive plants
was similar to that of the wild type.

28. CONCLUSION
 The ability to regenerate entire plants from cells or tissues has
been invaluable to plant biotechnology due to the totipotent
nature of plant cells.
 From the sole objective of demonstrating the totipotency of
differentiated plant cells, the technique now finds application in
both basic and applied researches in number of fields of enquiry.

 Plant tissue culture has generated intense interest among
molecular biologists, plant breeders and commercial
horticulturists.
 This technique should therefore be encouraged and supported
by African countries as it could aid in improving food security in
Africa.

29. References
 Kumar,P. A., Mandaokar, A., Sreenivasu, K., Chakrabarti, S. K.,
Bisaria, S., Sharma, S. R., Kaur, S. and Sharma, R. P. (1998).
Insect-resistant transgenic brinjal plants. Molecular Breeding
4:33-37
 Sharma, M. K., Solanke, A. U., Jani, D., Singh, U. and Sharma, A.
k. (2009). A simple and efficient Agrobacterium-mediated
procedure for transformation of tomato. J.Bioscience. 34(3):423-
433.
 Shun et al Tissue culture-induced somaclonal variation of
decreased pollen viability in torenia (Torenia fournieri Lind.)
Botanical studies 2013 54:36
 Ugandhar, T., Venkateshwarlu, M., Imran, M. A., Shekhar,G. P.
V. and Reddy, K. J. (2011). Genetic transformation of agronomical
important plant Solanum melongena L. through hypocotyl
explant. Plant Science Feed 1(7):142-146.