Tissue culture techniques can be used for genetic improvement of plants. Some key applications include: 1. Meristem culture which allows production of virus-free plants by cultivating shoot meristems. This eliminates viruses that move through the vascular system. 2. Somaclonal variation which induces genetic variability that can be used for crop improvement traits like disease resistance. Selected variants require extensive field trials due to genetic instability. 3. Mutagenesis uses physical or chemical mutagens to induce desirable mutations and improve traits like disease resistance. However, frequency of desired mutations is low and screening large populations is required. 4. Embryo rescue promotes development of immature embryos, allowing interspecific hybridizations and

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Manoj C A
PALB 4210
Genetics & Plant Breeding
Tissue culture application for
genetic improvement of plants

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• What it is...
• Background of it
• Why do that only
• Different types of it
• Advantages
• Application in Plant Breeding
• Different techniques with case studies
• Disadvantages
• Conclusion

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6. Tissue culture
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It is the in vitro aseptic culture of cells,
tissues, organs or whole plant under
controlled nutritional and environmental
conditions.
It produces clones,resultant clones are true-to type
of the selected genotype (unless affected by
mutation during culture)

7. • Tissue culture had its
origins at the beginning
of the 20th century with
the work of Gottleib
Haberlandt
• 1902- proposed the
concept of in vitro cell
culture
• Pallisade cells, pith
cells, stamen hairs and
stomatal guard cells 7

8. Contd...,
• 1926 Went-first plant growth hormone - IAA
• 1941 Overbeek was first to add coconut milk
for cell division in Datura
• 1955 Skoog and Miller discovered kinetin as
cell division hormone.
• 1957 Skoog and Miller gave concept of
hormonal control (auxin: cytokinin) of
organ formation
• 1960 Kanta and Maheshwari developed test
tube fertilization technique
• 1962 Murashige and Skoog developed MS
medium with higher salt concentration.
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9. Contd...,
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• 1964 Guha and Maheshwari produced first
haploid plants from pollen grains of Datura
• 1972 Carlson produced first interspecific hybrid
of Nicotiana tabacum by protoplast fusion
• 1978 Melchers et al. carried out somatic
hybridization- Pomato.
• 1987 Klien et al. developed biolistic gene
transfer method for plant transformation.

10. Why do Plant Tissue Culture?
• Fast commercial propagation of new cultivars.
• Do not usually destroy mother plant.
• Continuous supply of young plants throughout the year.
• Virus free plants.
• Easier to export
• Fast selection for crop improvement.
• True to type
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General steps followed in plant tissue culture

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SEED CULTURE
Somatic embryogenesis

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PTC Advantages
• large number of clones from a single explant, in a
short time.
• Overcomes seasonal restrictions for seed
germination.
• It takes shortened time, no need to wait for the
whole life cycle of seed development.
• It helps to eliminate plant diseases through careful
stock selection and sterile techniques.
• In vitro cultures can be stored for long time through
cryopreservation.
• Breeding cycle can be shortened

14. Applications of tissue culture to plant breeding
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1. Meristem culture
Cultivation of axillary or apical shoot
meristems, particularly of shoot apical
meristem
Morel and Martin (1952)
were the first to use
meristem culture to
eradicate viruses from
potatoes

16. Why no virus in meristems
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• Viruses move through vascular system which in
meristems is absent
• A high metabolite activity in the actively dividing
meristematic cells does not allow virus
replication
• High endogenous auxin level - inhibit virus
multiplication.

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Case study
Objectives:
1.Virus free plantlet production by meristem culture
2.Evaluate BBTV and BMV resistance in the banana plants under
green house and field conditions

18. Materials and Methods
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• Planting material - Amritasagar
• Culture media- MS media
• shoot proliferation –Four levels of BAP (0, 3, 4 and 5
mg/l) and 5 levels of NAA (0.0, 1.0, 1.5, 2.0 and 2.5
mg/l)
• Root formation- four levels of IBA (0, 1, 2 and 3 mg/l)
and 4 levels of NAA (0, 2, 3 and 4 mg/l)
• ELISA for confirmation of virus

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Results
Conclusion to meristem culture:

20. 2. Somaclonal variations
• Genetic variations in plants that have been produced
by plant tissue culture and can be detected as
genetic or phenotypic traits.
• 1981- term- Larkin and Scowcroft
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21. • Creates additional genetic
variability
• Production of secondary
metabolites
• Selection of plants resistant
to various toxins, herbicides,
high salt concentration and
mineral toxicity
• Suitable for breeding of tree
species
• Sometime leads to
undesirable results
• Selected variants are
random and genetically
unstable
• Require extensive and
extended field trials
• May develop variants with
pleiotropic effects which
are not true.
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SV

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Objective:
To get regenerants that are having agronomically superior traits
Case study

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• 3 genotypes : Pavon, Glennson, PAK-16171
• Callus culture- mature embryos
• CIM- Linsmaier & skoog’s media
• Subculture- fifth passage-on to PEG-4000(0,10,15,20 & 25%
w/v)
• Subculture- 90 days with 30 day interval
• Transferred- L&S Media, 2,4-D replaced with IAA & BA for
plant regeneration
Materials and methods

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Pavon
PAK-16171

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Conclusion to SV:

26. 3. Mutagenesis
• Any fluctuation of the genome of the
organism by physical or chemical mutagen
• To gain one or two of the traits of greatest
interest without excessively changing
important agronomic traits.
• Physical & chemical mutagens.
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27. • Induction of desirable
mutant alleles, which
may not be present in the
germplasm
• Improves specific
characteristics of a well
adapted high Yielding
variety.
• Mainly useful to clonal
and ornamental crops
• Frequency is very low
• Breeder has to screen
large populations to
select desirable
mutations.
• Undesirable side effects
• Detection of recessive
mutations is almost
impossible in clonal crops
and is difficult in
polyploidy species
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Objectives:
 To develop new lines resistant to broom rape
 To generate new breeding material
Case study

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Materials and methods
• Fertility restorer line- 147R
• Embryo culture- Immature zygotic embryos(11-13 days
old)
• Treated- ultrasound- dose of 25.5W/cm2
for 5, 7, 9, 11 &
13 min before plating on MS media.
• Long term selfing & individual selection.(R5 generation).
• Evaluation – 0.2mg of broomrape seeds+ mixture of soil
& sand(2:1)
• Observation- forty five days after planting.

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Control genotypes
147 R
MUTANT LINES- 116RM, 117RM, 118RM
Conclusion to mutagenesis:

31. 4. Embryo rescue
• Oldest and most successful in vitro procedures
• To promote the development of an immature or
weak embryo into a viable plant.
• Used for producing plants from interspecific
hybridizations.
• Artificial nutrient medium serves as a substitute
for the endosperm.
• First achieved in 1904 by Hannig in crucifers
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32. Applications of Embryo rescue
• Breeding incompatible interspecific and intergeneric species
• Overcoming seed dormancy & seed sterility
• Recovery of maternal haploids that develop as a result of
chromosome elimination following interspecific hybridization
• Used in studies on the physiology of seed germination and
development.
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Objectives:
• To transfer TMV resistant genes from C. chinensis to C. annuum
• To find out optimum timing for embryo rescue
Case study

34. Materials & methods
• Three species including two varieties
• Embryos from Young immature fruits: 27-36 DAP
• MS media
• Highest % of embryo growth- Casein hydrolysate+ yeast
extract
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C. annuum – Kashi Anmol, Pusa jwala
C. chinense (COO-304)
C. frutescence (COO904)

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Conclusion to embryo rescue:

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5. Double haploid lines
• Method to accelerate the production of homozygous
lines.
• Populations of lines in perfect homozygosity can be
produced in just one generation.
• Savings in time and costs.
• First report of the haploid plant was published by
Blakeslee et al. (1922) in Datura stramonium
• Optimum concentration of colchicine- 0.1 to 0.5%

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A – Selfings; B – haplodiploidization of microspores
AAAAbb

39. Applications OF DH
• It is a rapid approach to homozygosity that shortens the
time required for development of new cultivar
• Haploids- valuable to detect and fix desirable recessive
traits.
• It accelerates the breeding cycle and allow better
discrimination between genotype.
• F1 production through DH would facilitate the use of
interspecific crosses.
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Conclusion to DH:

40. 6. Synthetic seeds: A novel concept
• It is living seed-like structure
derived from somatic embryoids
in vitro culture after
encapsulation by a hydrogel.
• Encapsulated by protective gel
like calcium alginate against
microbes and desiccation.
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41. Advantages of Synthetic seeds
 Direct delivery of tissue cultured plants to the field
 Propagation of desirable genotypes with genetic uniformity
 Reduction in cost of vegetative propagated elite plants
 Preservation of germplasm and convenience in germplasm
exchange
 Reduction in dormancy period
 Large production identical embryos in
short time
 It can be produced through out the year 41

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Development of synthetic seeds involving androgenic and
pro-embryos in elite indica rice
Bidhan Roy and Asit B Mandal, 2008
Objective:
• Encapsulation of embryos, pro-embryos and embryo like
structures of androgenic origin
• Assessment of their germination invitro & invivo conditions
Case study

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Materials and methods
• Indica rice- IR 72
• Antherculture- mid uninucleate anthers
• CIM- N6 Media
• Regeneration media- MS+ Different combinations of
BAP, Kn, NAA
• Encapsulation- sodium alginate(4%w/v)

44. Results
• Anther to callus response-60%
• Regeneration of green plants from calli-22.2%
• High rate of multiplication of embryos, pro-embryos and
embryo like structure recorded on MS With 4-6mg/l of BAP.
• Germination of synthetic seeds- high germination % with BAP in
combination with lower concentration of NAA(87.5%)
• Addition of Kn in MS reduced the germination percentage(20%)
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Conclusion to synthetic seeds:

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• Conservation of endangered genotypes.
• Helps to keep the genetic background of a crop
• Helps to avoid the loss of the conserved patrimony
due to natural disasters, whether biotic or abiotic
stress.
• Germplasm exchange
• Easy transport & safe exchange of genotypes
7. Germplasm conservation &
Exchange

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Contd..,
• Sterile plants or ‘recalcitrant’ seeds can
successfully be preserved.
• Cryopreservation - longterm in vitro conservation
method
• It involves the storage of in vitro cells or tissues in
liquid nitrogen.
• Cryobionomics - new approach to study genetic
stability in the cryopreserved plant materials.

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• provides the mean of transfer of genes with desirable trait into
host plants and recovery of transgenic plants
• promising role for the introduction of agronomically important
traits such as increased yield, better quality and enhanced
resistance to pests and diseases
• It can be achieved by either vector-mediated (indirect gene
transfer) or vector less (direct gene transfer) method.
8. Genetic transformation

48. Contd..,
• Among vector dependant, Agrobacterium-mediated genetic
transformation is most widely used .
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49% or 181.5 million hectares were biotech
Conclusion:

50. Tissue culture disadvantages
 Specialized equipment/facilities required
 More technical expertise required
 No possibility of using mechanization
 Protocols not optimized for all species
 Plants produced may not fit industry standards
 Relatively expensive to set up
 Equally vulnerable to environmental factors,
infections and pests because of same genetic material.
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Tissue culture- Nothing but Sophisticated
PLANT BREEDING...,

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