This is the slide presented to my class of plant tissue culture.

1. Sushil Nyaupane
Department of Natural Resources and Environmental Design
North Carolina A&T State University

2. What is tissue culture?
Tissue culture is the term used for “the process of growing
cells artificially in the laboratory”.
Tissue culture involves both plant and animal cells.
Tissue culture produces clones in which all product cells
have the same genotype.

3. Plant Tissue Culture
 Plant tissue culture refers to the techniques of
growing plant cells, tissues, organs, seeds or
other plant parts in a sterile environment on a
nutrient medium.

4. Who they are ?
Haberlandt T. Murashige F. Skoog

5. A glance in History
 In 1902, Haberlandt; father of plant tissue culture ( He proposed
that plant cells could be cultured.
 In 1930- White cultured tomato root tip and subculutred to fresh
medium containing salts, yeast extract and sucrose and vit B
 During this period, some plant growth regulators, additives and
vitamins was discovered for the plant micro- propagation.
 discovery of first PGR ---IndoleAcetic Acid , called IAA, in 1937
 In 1962, Murashige and Skoog published a recipe for MS media.
 In 1972, protoplast fusion has been done in toabacco.

7.  Micropropagation is rapid clonal in vitro
propagation of plants from cells, tissues or organs
cultured aseptically on defined media contained in
culture vessels maintained under controlled
conditions of light and temperature.
 Briefly, it is the art and science of multiplying plants
in- vitro.

8. Explants
Explant is an excised piece of tissue or organ taken
from the plant to initiate a culture.
They can be :
• shoot meristem, tip, bud
• leaf or stem (internode)
• root
• anther / microspore
• ovule
• embryo associated seed parts

9. Shoot Explants
Meristem Tip Shoot Tip

10. Dormant Bud

11. Somatic Embryo Culture

12. Leaf Explants

13. Seed explants

14. Pollen culture

15. Plant tissue culture types for micro-propagation

19. Yes, this is from lab.

20. Tissue cultured banana

21. Steps of micro-propagation
 Stage 0- Selection and preparation of the mother
plant(Sterilization of the explant tissues)
 Stage I- Initiation of culture ( Explant placed into growth
media)
 Stage II- Multiplication ( Explant transferred to shoot
media and shoot can be constantly divided)
 Stage III- Rooting ( Explant transferred to rooting media)
 Stage IV- Transfer to soil ( Hardening off)

23. Why do micro-propagation
 A single explant can be multiplied into several thousand
plants in less than a year.
 Once established, it can give a continuous supply of
young plants throughout the year i.e. irrespective of
season.
 Taking an explant does not usually destroy the mother
plant, so rare and endangered plants can be saved
somehow.
 Clones through micro-propagation are ‘true to type’ as
compared with seedlings, which show greater variability

24. Why do micro-propagation?
 This allows fast selection for crop improvement - explants
are chosen from superior plants, then cloned.
 Plant ‘tissue banks’ can be frozen, then regenerated
through micro-propagation.
 Disease and virus free plants can be produced through
this technique.
 Greater credibility in international market as plantlets are
produced through micro-propagation( Phyto-sanitary
perspective)

25. How possible is this ??
 Micro-propagation of almost all vegetables and fruit
crops is possible.
 Some examples,mustard, corn, soybean, azalea,
dwarfing sweet cherry, strawberry, mango, banana,
rose, orchid, nutraceutical plants, rhododendron,
citrus, potato, tomato,legumes etc.

26. Applications of micro-propagation
 Somaclonal Variation
 Germplasm Conservation
 Mutation Breeding
 Inducing mutation
 Embryo culture
 Haploid and Dihaploid production
 In Vitro Hybridization- protoplast fusion
 Production of Disease free plants
 Molecular farming
 Genetic engineering
 Production of secondary metabolites

27. Applications ..
Somaclonal variation
Somaclonal variation is a general phenomenon of all
plant regeneration systems that involve a callus phase.
There are two types of Somaclonal variation:
Heritable genetic variation
Non- Heritable genetic variation

28. Applications..
Advantages of somaclonal variation
 It helps in crop improvement
 Creates additional genetic variants
 Plants with resistant and tolerant to toxins, herbicides,
high salt and even mineral toxicity
 Suitable for breeding purposes
 Increased and improved production of secondary
metabolites

29. Applications..
Germplasm conservation
 Micropropgation is utilized in conserving genetic
resources.
 Depending upon the crop species and method of
preservation, tissue culture can help in the
preservation of genetic resources from 1 to 15 years.
 Cyopreservation – the preservation of germplasm in a
dormant state at ultra-low temperatures, usually in
liquid nitrogen (-196 °C) – is a type of tissue culture
which can be used to preserve seeds.

30. Applications..
Mutation Breeding
 1927: Muller produced mutations in fruit flies using x-
rays
 1928: Stadler produced mutations in barley
 Basically, there are three groups of breeders
1) Mutation breeding is useless, we can accomplish the
same thing with conventional methods
2) Mutation breeding will produce a breakthrough
given enough effort
3) Mutation breeding is a tool, useful to meet specific
objectives

31. Applications..
Types of mutation
Spontaneous (natural mutation)
 Some type of spontaneous mutation have played an
outstanding role in development of valuable crop
cultivars and hybrids.
 But limitation of this is that, it can not form the basis
of modern plant breeding
Induced mutation
 Any mutation found in nature can be induced by
mutation breeding.

32. Applications..
 Inducing Mutations
 Physical mutagens (Irradiation)
These mutagens helps in the chromosome aberrations
and point mutations.
Neutrons, Alpha rays
Gamma, Beta, X-rays
 Chemical mutagens
By using different carcinogenic chemicals which are
highly toxic in nature and eventually result in point
mutations.

33. Applications..
Embryo Culture
 Embryo culture is usually done from the need to
rescue embryo from wide crosses where fertilization
occurred, but not the embryo development.
 Objectives:
Rescue F1 Hybrid from wide crosses
Overcome seed dormancy by addition of hormone to
media. For ex. GA
To overcome immaturity in seed
To rescue valuable genotype from dead or dying plant
To speed up the generations in a breeding program

34. Applications..
Embryo culture as a source of genetic variation
 Hybridization
Can introduce new genetic combinations through
inter-specific crosses
Can transfer mutant alleles between species
 Polyploidy
It combines embryo culture with chromosome
doubling to create new polyploid species

35. Application…
Haploid and dihaploid production
 Plants produced through the anther culture are the
haploids.
 Doubling the chromosomes without going into series
of backcrossing produce homozygous plants.
 This technique shortens the time of breeding by half.

36. Application…
In Vitro Hybridization( Protoplast fusion)
 Created by degrading the cell wall using enzymes.
 Very fragile in nature.
 Protoplasts can be induced to fuse with one another.
Methods:
 Electrofusion
 Poly Ethylene Glycol(PEG)
 Addition of calcium ions at high PH Values

37. Applications..
Production of Disease free plant
Heat treatment
Plants grow faster than viruses at higher temperature.
Meristemming
Viruses are transported from cell to cell through
plasmodesmata and vascular tissue.
Apical meristem are virus free in nature. So, micro-
propagation of these cells gives virus free plantlets.
Even, not all the cells in the plant are infected. For
example, adventitious shoots formed from single cells
can give virus free shoots.

38. Applications..
Molecular farming
 Where plants are treated as bioreactors for the production
of specific compounds.
 Range from simple peptides to a thermoplastic.
Two types of products:
1)High value compounds with small scale production
requirements such as pharmaceutical products.
For ex. Malaria epitope.
2)Compounds needed on a bulk scale with low production
costs (plant biotechnology has greatest potential in this
area)
For ex. α-Amylase (food + detergent industries)-starch
manipulation

39. Applications…
Genetic Engineering
 Genetic engineering would not be possible without the
development of plant tissue culture.
 Genetic engineering requires the regeneration of
whole plants from single cells.
 Efficient regeneration systems are required for
commercial success of genetically engineered
products.

40. Application..
 Protoplast fusion between male sterile cabbage and
normal cabbage was done and cybrids were selected
that contained the radish mitochondria and cabbage
chloroplast.

41. Applications..
Production of secondary metabolites
 Secondary metabolites are those cell constituents
which are not essential for survival.
 For example, alkaloids, glycosides,
terpenoids,latex,tannins etc.
 In vitro production of secondary metabolites is much
higher from differentiated tissues compared to non-
differentiated tissues.
 Azadirachtin from Azadirachta indica Insecticidal
 Berberine from Coptis japonica Antibacterial, anti
inflammatory
 Capsaicin from Capsicum annum Cures Rheumatic
pain

42. Problems of micro-propagation
 Expensive laboratory equipment and service
 No possibility of using mechanization
 Plants are not autotrophic
 Poor Acclimatization to the field is a common
problem (hyperhydricity)
 Risk of genetic changes if 'de novo' regeneration is
used
 Mass propagation cannot be done with all crops to
date. In cereals much less success is achieved
 Regeneration is often not possible, especially with
adult woody plant material.

43. Do you have any idea about this orchid?

44. Micro-propagation a tool for commercialization (Taiwan story)
According to Taiwan Today, Jan 1, 2016
 The nation’s orchids are exported to 36 countries in
Northern America, Northern Europe and South Africa.
 This country began the export with US $ 23 million in
2004 but in 2015 they have exported with worth US $
130 million.
 They are selling a Phalaenopsis or moth orchid in
Dubai at the price of US $ 1,000.
 Won the bid to host the 23rd World Orchid Conference
in 2020.

45. What may be the reason for this achievement ?
 According to John Feng, CEO of SOGO TEAM CO
LTD., they are exploiting micro-propagation as a
technique in mass production as well as in variety
improvement.
 In I-Hsin Biotechnology Corp’s Tissue Culture Lab
only, they have conducted 12,000 breeding
experiments which have yielded 2,300 Phalaenopsis
varieties.
 This all is being possible through tissue culture.

46. ALBIFLORA ORCHID OF NEPAL
In Nepal a total of 377 species of orchids belonging to 100
genera reported.

47. Future of micro-propagation
 ……………………………………………………………………………………
 Perhaps some of the greatest discoveries of plant tissue
culture are yet to come.
 SO BE READY !

48. THANK YOU !