Plant tissue culture techniques

1. Welcome

2. Plant Tissue Culture
PG and Research Department of Botany
Government Arts College (Autonomous)
Coimbatore 641 018
Dr. K. KALIMUTHU, M.Sc., M.Phil., Ph.D.
Assistant Professor

3. •Plant Tissue Culture refers to the technique of growing
plant cells, tissues, organs, seeds or other plant parts in
a sterile environment on a nutrient medium.
•It is mainly based on the totipotency of the cell. That is
every living cell, of a multicellular organism is capable of
independent development, when provided with suitable
conditions (white 1963). Morgan (1901) coined the term
totipotency.

4. Differentiation (De-)
The physiological and
morphological changes that occur in
a cell, tissue, or organ during
development.
Organogenesis
The development of tissues and/or organs from
individual cells not from pre-existing meristems.

5. Characteristic of Plant Tissue Culture Techniques
Environmental condition optimized
nutrition,
light,
temperature.
Ability to give rise to callus,
embryos,
adventitious roots and shoots.
Ability to grow as single cells
(protoplasts, microspores,
suspension cultures.

6. HISTORY
 German botanist Gottlieb Haberlandt (1902),
regarded as the father of plant tissue culture, first
developed the concept of in vitro cell culture. He was
the first to culture isolated and fully differentiated plant
cells in a nutrient medium.
 Gautheret, White and Nobecourt largely contributed to the
developments made in plant tissue culture (1934-1940).
 Steward and Reinert (1959) first discovered somatic embryo production
in vitro. Maheswari and Guha (1964) from India were the first to develop
anther culture and pollen culture for the production of haploid plants.

7. Applications of Plant Tissue Cultures
•Micropropagation
•Somatic embryogenesis and artificial seeds production.
•Recovery of pathogen free stocks.
•Germplasm conservation and transfer.
•Embryo rescue and culture.
•Haploid and triploid production.
•Secondary metabolite production.
•Protoplast isolation, culture and fusion.
•Vector mediated gene transfer.

8. Advantages
In terms of product development and
enhancement
•It is the only method of rapid, high volume and less space
occupying plant multiplication system (e.g.) Foliage
ornamentals.
•Plantlets produced are uniform, true to type or with
improved phenotype (e.g.) Syngonium
•Plantlets are disease free (e.g.) Banana, Chrysanthemum
•Only way through which genetically engineered products
of the future can be brought into market place.

9. In terms of product format and marketability
•A variety or choice is offered as rooted, clumps or
hardened plants specifically for exports.
•Movement of plantlets easy due to relatively less
cumbersome quarantine.
•No seasonal production in artificial environment 24
hours a day can be contemplated.
•New plant species can be introduced quickly and in
large quantities to the market.
•In general, a balance between the plants for both local
and export markets and reasonable pricing can pave the
way for a sustained, viable Plant Tissue Culture
Industry.

10. THE FACILITY

14. THE EQUIPMENTS AND MATERIALS
•Autoclave
•Laminar air flow cabinets with glass bead sterilizer
•Culture racks with lighting arrangement
•Water distillation unit/Millipore unit (for larger operations)
•Electronic weighing balance
•pH meter & pH stick
•Magnetic stirrer/hot plate
•Low power stereo dissection microscope

15. • Filter units and Vacuum pump for filter sterilizing
• Microwave oven/electric or gas stove
• Media dispenser (for larger operations)
• Bottle trays
• Hot air oven
• Culture bottles of appropriate size
• Automatic dish washing machine (for larger operations)
• Refrigerator
• Trolleys
• Illuminated Gyratory /Platform shakers (for R & D and suspension
culture work)
• Lux meter
• Thermo hygrometers

16. CULTURE RACKS

19. Horizontal
– Maintains sterile working
area
– Air blows directly at the
Investigator
– Enough to protect your
cultures but not you

20. TECHNIQUES & TOOLS

21. SOME OF THE IMPORTANT BASAL MEDIA USED IN
PLANT TISSUE CULTURE
1. Murashige and Skoog medium (MS)
2. Linsmaer and Skoog medium (LS)
3. Gamborg’s medium (B5)
4. Woody plant medium (WPM)
5. Vacin and Went medium (VW)
6. Knudson ‘C’
7. White’s medium

23. S.No Item Chemical Formula 1 lit 5 lit 10 lit 25 lit 50 lit
Dissolve
in
1 Major
Ammonium
nitrate
1.65g 8.25g 16.5g 41.25g
25lit/5
00
ml
25lit
500ml
20ml/litPotassium
nitrate
KNO3 1.9g 9.5g 19g 47.5g
Potasssium di
hydrogen
orthophosp
hate
KH2PO4 0.17g 0.85g 1.7g 4.25g
Calcium
chloride
CaCl2 2H2O 0.44g 2.2g 4.4g 11g
Magnesium
sulphate
MgSO4.7H2O 0.37g 1.85g 3.7g 9.25g
2 Minor
Manganese
sulphate
MnSO4.4H2O 16.8m
g
84mg 168m
g
420mg 840mg 50 lit
100
ml
2ml/lit
Zink sulphate ZnSO4.6H2O 8.6 mg 43 mg 86 mg 125 mg 430 mg
Boric acid H3BO3 6.2 mg 31 mg 62 mg 155 mg 310 mg
MS MEDIUM - STOCK COMPOSITION

24. 3 Micro
Sodium
molybdate
Na2MoO4.2H2
O
0.25
mg
1.25
mg
2.5 mg 6.25
mg
12.5
mg
50 lit
100ml
2ml/litColbatous
chloride
CoCl. 6H2O 0.025
mg
0.125
mg
0.25
mg
0.625
mg
1.25
mg
Copper sulphate
CuSO4.5H2O 0.025
mg
0.125
mg
0.25
mg
0.625
mg
1.25
mg
4 KI Potassium iodide
KI 0.83
mg
4.15
mg
8.3 mg 20.75
mg
41.5
mg
50 lit
100ml
2ml/lit
5 Iron Ferrous sulphate
FeSO4.7H2O
Na2
EDTA2H2O
27.86
mg
37.3m
g
139.3
mg
278.6
mg
696.5
mg
932.5
mg
25 lit
250ml
10ml/li
t

25. 6
Amino
acid
Glycine
2 mg 10 mg 20 mg 50 mg 100
mg
50 lit
100ml
2ml/lit
7 Vitamins
Nicotinic acid
0.5 mg 2.5 mg 5 mg 12.5
mg
25 mg 50 lit
100ml
2ml/lit
Thiamine HCL 1 mg 5 mg 10 mg 25 mg 50 mg
Pyridoxine
HCL
0.5 mg 2.5 mg 5 mg 12.5
mg
25 mg
8
Meso Inosital 100 mg
Sucrose C12H22O11 30gm
Agar Agar 8gm

26. ONE LITRE MEDIUM PREPARATION
• Add stocks as follows
900 ml of distilled water in 1L flask
Major stock - 20 ml
Add CaCl2. 2H2O - 440 mg
Minor stock - 2 ml
Micro stock - 2 ml
Vitamins stock - 2 ml
Amino acid - 2 ml
KI stock - 2 ml
Iron EDTA stock - 10 ml
Myo- inositol - 100 mg
Add Sucrose - 30 g
• Adjust pH to 5.8 to 6 with 0.5N HCL or 0.2N NaOH and make up to
1000 ml.

27. • After add required amount of growth regulators.
• Keep the medium over a micro wave/ heater and dissolve the Agar
with constant stirring.
• Dispense the medium in culture bottles.
• Cover tightly with non-absorbent cotton wool/ autoclavable caps
and cling wrap the bottles.
• Store the medium in a sterile environment.

28. A SELECTED LIST OF ELEMENTS AND THEIR FUNCTIONS IN PLANTS
ELEMENT FUNCTION (S)
Nitrogen
Essential component of proteins, nucleic acids and some coenzymes.
(Required in most abundant quantity)
Calcium Synthesis of cell wall, membrane function, cell signaling.
Magnesium Component of chlorophyll, cofactor for some enzymes.
Potassium Major inorganic cation, regulates osmotic potential.
Phosphorus
Component of nucleic acids and various intermediates in respiration and
photosynthesis, involved in energy transfer.
Sulfur
Component of certain amino acids (methionine, cysteine and cystine, and
some cofactors).
Manganese Cofactor for certain enzymes.
Iron Component of cytochromes, involved in electron transfer.
Chlorine Participates in photosynthesis.
Copper Involved in electron transfer reactions, cofactor for some enzymes.
Cobalt Component of vitamin B12.
Molybdenum
Component of certain enzymes (e.g., nitrate reductase), cofactor for some
enzymes.
Zinc Required for chlorophyll biosynthesis, cofactor for certain enzymes.

29. EFFECT OF AUXIN AND CYTOKININ

30. PLANT HORMONES IN PLANT TISSUE CULTURE
Auxins
Main effects in tissue culture
systems
Modulators of metabolism, action or
transport
indole-3-acetic acid (IAA)
indole-3-butyric acid (IBA)
1-naphthaleneacetic acid (NAA)
phenyl acetic acid (PAA)
2,4-dichlorophenoxyacetic acid
(2,4-D)
2,4,5-trichlorophenoxyacetic acid
(2,4,5-T)
picloram
dicamba
p-chlorophenoxyacetic acid (CPA)
1.Adventitious root formation
(at high conc.).
2. Adventitious shoot formation
(at low conc.).
3. Induction of somatic embryos
(in part. 2,4-D).
4. Cell division.
5. Callus formation and growth.
6. Inhibition of outgrowth of
axillary buds
7. Inhibition of root growth.
1. 2,3,4-Triiodobenzoic acid (TIBA) and 1-
N-naphthylphthalamic acid (NPA) inhibit
polar auxin transport.
2. p-Chlorophenoxyisobutyric acid (PCIB)
inhibits auxin action as a genuine antiauxin
by binding to the auxin receptor.
3.Phenolic compounds (e.g. ferulic acid or
phloroglucinol) inhibit auxin oxidation.
4. riboflavin strongly promotes
photooxidation of IBA and IAA.
Cytokinins
Main effects in tissue culture
systems
Modulators of metabolism, action or
transport
zeatin (Z)
zeatinriboside (ZR)
isopentenyladenine (iP)
isopenenyladenosine (iPA)
6-bezylaminopurine (BAP)
kinetin
thidiazuron (TDZ)
N-(2-chloro-4-pyridyl)-N’-
phenylurea (CPPU)
1.Adventitious shoot formation
(at high conc.).
2. Adventitious root formation.
3. Cell division.
4. Callus formation and growth.
5. Stimulation of outgrowth of
axillary buds.
6. Inhibition of shoot elongation.
7. Inhibition of leaf senescence.
Compounds have been described that inhibit
cytokinin synthesis (lovastatin), degradation
and action. The various effects are,
however, not yet well studied or ambiguous.

31. Gibberellins
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
gibberellic acid (GA3)
gibberllin 1 (GA1)
gibberellin 4 (GA4)
gibberellin 7 (GA 7)
1.Shoot elongation.
2. Release from dormancy in
seeds, somatic embryos, apical
buds and bulbs.
3. Inhibition of adventitious root
formation.
4. Synthesis-inhibitors promote
root formation.
5. Synthesis-inhibitors promote
tuber, corm and bulb formation.
6. Synthesis-inhibitors facilitate
acclimatization.
Paclobutrazol and ancymidol inhibit
gibberellin sysnthesis and thereby result in
short shoots.
Ethylene Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
1.Senescence of leaves.
2. Ripening of fruits.
3. Promotion or inhibition of
adventitious regeneration
(depending on the time of
application or on the genotype?).
1.1-aminocyclopropane-1-carboxylic acid
(ACC) is a precursor of ethylene and is
metabolized by plant tissues to ethylene.
2. Aminoethoxyvinylglycine (AVG)
inhibits ethylene synthesis. Co2+, ?-
aminooxy-acetic acid and ?-
aminoisobutyric acid also inhibit ethylene
synthesis but have a lower efficiency.
3. Silver inhibits ethylene action. Silver is
applied preferably as silverthiosulphate
(STS).

32. Abscisic acid
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
1.Maturation of somatic
embryos.
2. Facilitation of
acclimatization.
3. Bulb and tuber formation.
4. Promotion of the
development of dormancy.
Fluridone inhibits ABA synthesis. As it
acts by inhibiting an early step in
carotenoid synthesis, plants are unable to
synthesize chlorophyll. However,
fluridone does not seem to be toxic.
Paclobutrazol also inhibits ABA
synthesis.
Polyamines
Main effects in tissue
culture systems
Modulators of metabolism,
action or transport
putrescine
spermidine
spermine
1. Promotion of adventitious
root formation.
2. Promotion of shoot
formation.
3. Promotion of somatic
embryogenesis.
1. DL-a-difluoromethylarginine
(DFMA) and a –
difluoromethylornithine (DFMO)
block the synthesis of putrescine.
2. Methylglyoxal-bis-guanylhydrazone
(MGBG) and cyclohexylamine (CHA)
block the synthesis of spermidine and
spermine.
3. Amino-guanidine (AG) blocks the
degradation of putrescine.

33. Support Systems
 Agar (from seaweed)
 Agarose
Phytagel
 Mixtures (Phytagar)
 Mechanical (bridges, rafts)
 Sand

34. Natural substances in tissue culture media
 Coconut water
 Yeast extract
 Malt extract
 Potato extract
 Banana homogenate

35. Charcoal
• Activated charcoal is used as a detoxifying agent.
Detoxifies wastes from plant tissues, impurities
– Impurities and absorption quality vary
– Concentration normally used is 0.3 % or lower
• Charcoal for tissue culture
– acid washed and neutralized
– never reuse

36. MEDIA STERILIZATION
Media is sterilized at 121ºC (1.12kg/cm2) for 20 minutes
Filter sterilization
Compound such as certain amino acid, vitamins and hormones are
usually destroyed during autoclave (eg) calcium pantothenate, IAA (40%
loss), IBA (20% loss), zeatin, kinetin and thiamine HCl (high above [pH
5.5).
Such thermolabile compounds are sterilized by ultrafilteration
through size or Millipore filtration unit with 0.2um or .45 um pore size
filters. Media prepared for polypropylene containers/liquid media are filter
sterilized using this technique (or) alternatively by using UV or electrostatic
filters
STORAGE
Autoclaved medium is stored at room temperature to cool in sterile
environment.

37. Functions of medium
 Provide water
 Provide mineral nutritional needs
 Provide vitamins
 Provide growth regulators
 Access to atmosphere for gas exchange
 Removal of plant metabolite waste

38. TYPES OF EXPLANTS
• Seeds (eg.) Orchids, Cacti, Pterocarpus, Jatropha etc.
• Tender explants like meristems, leaf bits, shoot tips buds of
herbaceous plants. (eg.) Chrysanthemum, Strawberry,
Crossandra, Asparagus etc.
• Moderately hard explants like shoot tips, nodal sections of
shrubs etc. (eg.) Rose, Sugarcane, Dieffenbachia, Bamboo,
Phillodendron.
• Hardwood explants like nodal / shoot tip sections of trees
like Teak, Eucalyptus Pterocarpus, Jatropha etc.
• Explants from plants with compressed internodes length
(or) Gerbera, Timonium, Anthurium etc.
• Suckers, Corms, tubers, rhizome and bulb explant (eg,)
Banana, Caladium, Potato, Ginger, Turmeric, Calla lily,
Gladioli etc.

39. EXPLANT STERILIZATION
S.
NO.
CHEMICAL CONCENTRATION EXPOSU
RE TIME
(min)
Surface Sterilant
1.
Ethyl alcohol (enables removal of waxy epidermal coating and better
surface sterilant contact.)
70 0.5-5
2. Sodium hypochlorite (bleaches the explant and needs correct conc./time) 0.5-5 5-30
3. Mercuric chloride (Carcinogenic and needs careful handling) 0.1-2 1-15
4. Commercial bleach (bleaches the explant and needs correct conc./time) 10-20 5-30
Detergent
5. Teepol (for large, hardy explants) 1-2 2-10
6.
Tween 20 (mild, safe wetting agent – reducing surface tension, cleans dust / dirt
and facilitates better surface contact.)
One or few
drops
5-15
Fungicide
7. Bavistin (carbendazin) (for large explants like corms, suckers etc) 0.1-0.2 5-15
Antibiotics
(rarely employed as it promotes contamination on withdrawal (or) with a lag phase
in latter stages)
8. Rifampicin (for explant treatment) 5-10 ml /100 ml 5-30
9. Streptomycin (for explant treatment) 25–50 mg/100 ml 5-30
10. Ampicillin (for explant treatment) 25–50 mg/100 ml 5-30

40. STAGES OF MICROPROPAGATION
• Stage O – Mother Plant selection and preparation
• Stage I – Initiation
• Stage II – Multiplication
• Stage III – Shooting
• Stage IV – Rooting
• Stage V – Hardening

41. Micropropagation
Direct Indirect
Node and Shoot tip Leaf, Petiole, Stem and root
Surface sterilization Surface sterilization
Inoculation
( MS + Growth regulators
Observation and
subculturing

42. (Node, Shoot tip, internode,
leaf, petiole and root)
(Node, Shoot tip, internode,
leaf, petiole and root)
in vivo in vitro
Explant
Inoculation
(MS + Growth regulators)
Subculturing & Observation
Surface
sterilization
Steps Involved in Micropropagation

43. THE PRINCIPLE METHODS OF MICROPROPAGATION

44. Types of Culture
 Seed culture
 Embryo culture
 Ovary / ovule culture
 Anthers / microspore culture
 Shoot tip / meristem culture
 Somatic embryo culture
 Callus culture
 Protoplast culture

45. SEED CULTURE
Seed culture is the type of tissue culture that is primarily used for
plants.
Advantages
1. The production of exact copies of plants that produce
particularly good flowers, fruits or have other desirable
traits.
2. The production of plants in sterile conditions with greatly
reduced chances of transmitting diseases, pests and
pathogens.
3. The production of plants from seeds that otherwise have low
chances of germinating and growing.
4. Mass propagate plants for commercial use.
5. Disease-free plants

46. Tobacco Seed germination

47. Tobacco Seed germination and callus initiation

48. Callus initiation from seed

49. EMBRYO CULTURE
Mature embryos are isolated from ripe seeds and cultured in
artificial medium.
Advantage
 Recovery of distant hybrids.
 Recovery of haploid plants from interspecific crosses
 Propagation of orchids
 Shortening the breeding cycle
 Overcoming dormancy
 Ovule and ovary can also be cultured

54. Orchid multiple shoot formation, rooting and hardening

57. CALLUS AND NODAL CULTURE

58. GF
A B
Ceropegia pusilla - Habit observation
A- Ebbanad hills. B and C- Plants with tubers. D and E -Plants with flowers.
F -Plants with follicles G -Seed dispersal. H - Plant with flower. I - Seed.
I
SEED
ED
C

59. Ceropegia callus initiation and multiple shoot initiation

60. Ceropegia callus initiation and multiple shoot initiation

61. C. pusilla nodal culture and multiple shoot formation

62. C. pusilla nodal culture and tuber formation

63. C. pusilla nodal culture and in vitro flower formation

64. Cadaba nodal culture and multiple shoot formation

65. Cadaba multiple shoot formation

66. C. juncea nodal culture and multiple shoot formation

67. C. juncea nodal culture and multiple shoot formation

68. Caralluma habitat

69. Caralluma multiple follicles formation and flower

70. Caralluma nodal culture and multiple shoot formation

71. Caralluma nodal culture and multiple shoot formation

72. Caralluma multiple shoot formation, rooting and hardening

73. Caralluma diffusa multiple shoot formation, rooting and hardening

75. Plate 1– Habit
BA
D
Entire Plant Leaf Enlarged Flower
Fruit Seeds
E
B

76. Brinjal nodal culture and multiple shoot formation

77. Vernonia different stages of callus formation

78. Turnera ulmifolia - Initiation of shoots from callus

80. Tylophora multiple shoot formation, rooting and hardening

81. Tylophora multiple shoot formation, rooting and hardening

83. Syngonium multiple shoot formation and rooting

84. SOMATIC EMBRYO CULTURE
The developmental pathway of numerous well-organized,
embryoids resembling to zygotic embryo from the embryogenetic
potential somatic plant cells of the callus, tissue or cell suspension
culture is known as somatic embryogenesis.
History
J. Reinert (1958-59): Reported his first observation of in vitro
somatic embryogenesis in Daucus carota (Carrot).
N. S. Rangaswami (1961): Studied in detail the somatic
embryogenesis in Citrus sp.
Advantages
1. Production of artificial seed
2. Production of adventitious embryo
3. Mutagenic studies
4. Free of viral and other pathogenic infection

85. Somatic embryogenesis

86. Somatic embryogenesis

87. Synthetic seeds

88. Gloriosa

89. OVARY / OVULE CULTURE
Ovule culture is an elegant experimental system by which are aseptically isolated from the ovary
and are grown aseptically on chemically defined nutrient medium under controlled conditions.
Advantages
1. Produce haploid plants and embryology study
2. Study of genetic recombination in higher plants
3. Mutation study
4. Heritability studies are simplified (recessive mutation are easily
identified.

90. ANTHERS / MICROSPORE CULTURE
Anthers culture
 Culturing of anther obtained from unopened flower bud in the
nutrient medium under aseptic condition.
 Callus tissue or embryoids that give rise to haploid plantlets
either through organogenesis or embryogenesis.
Microspore culture
 Pollen or microspore culture is in vitro technique by which the
pollen grains preferably at uninucleated stage, are squeezed
out aseptically from the intact anther and then culture on
nutrient medium.
 The micropores develop into haploid embryoids or callus tissue
that give rise to haploid plantlets by embryogenesis or
organogenesis.

91. History of anther culture
 1964, 1966 Datura innoxia (Guha and maheshwari) the Indian Scientist
 1967 (Bourign and Nitsch): 1st haploid plants from isolated
anthers Nicotiana.
Advantage of anther culture
1. Simple
2. Less time consuming
3. Responsive
4. Utility of anther and pollen
culture for basic research
5. Cytogenetic studies
6. Genetic recombination in
higher plants
7. Controlling pollen
embryogenesis of higher plant

92. SHOOT TIP / MERISTEM CULTURE
Shoot tip culture may be described as the culture of
terminal portion of a shoot comprising the meristem together
with primordial and developing leaves and adjacent stem tissue.
Advantages
 Virus elimination
 Storage genetic recourses
 Use in pant breeding
 Quarantine

94. Aloe vera

96. Vannila multiple shoot formation, rooting and hardening

98. Banana multiple shoot formation, rooting and hardening

100. Cell suspension culture
When callus pieces are agitated in a liquid medium, they tend to
break up.
Suspensions are much easier to bulk up than callus since there is
no manual transfer or solid support.

102. HAIRY ROOT CULTURE Normal roots from leaf segments
Hairy roots from callus culture
Typical hairy root development

103. Hairy root culturewith
Bioreactors

104. Typical hairy root formation