Plant tissue culture tech Unit-III Pcog -I

Detail information about plant tissue culture techniques

1. UNIT-III
PLANT TISSUE CULTURE

2. Introduction
•What is plant tissue culture?
–Plant tissue culture is a technique of
growing plant cells, tissues, organs, seeds or
other plant parts in a sterile environment
on a nutrient medium.
–The growth or regeneration of plant cells,
tissues, organs or whole plants in artificial
medium under aseptic conditions.

3. Introduction
•There are numerous methods to propagate
plants in tissue culture.
•But the one principle that is constant is
totipotency – all plants and plant parts have
this potential.

4. Why does tissue culture work?
•Totipotency:
–The ability of a cell to differentiate and develop
into a whole plant when given the correct
conditions. This is because every cell has the
genetic potential of the parent plant.

5. Totipotency
Plant cell
Tissue Callus
Plant organ Embryo
New plant

6. EXPLANT
•Explant
–Living tissue transferred
from a plant to an
artificial medium for
culture.
–It can be any portion of
the shoot, leaves, roots,
flower or cells from a
plant.

7. •Living plant materials from the environment
are naturally contaminated on their surfaces
(and sometimes interiors) with
microorganisms, so surface sterilization of
starting material (explants) in chemical
solutions (usually alcohol and sodium or
calcium hypochlorite is required).

8. •Explants are then usually placed on the
surface of a solid culture medium, but are
sometimes placed directly into a liquid
medium, when cell suspension cultures are
desired.
• Culture /Nutrient media are generally
composed of inorganic salts plus a few organic
nutrients, vitamins and plant hormones.

13. Nutrient Medium
•When an explant is isolated, it is no longer
able to receive nutrients or hormones from
the plant, and these must be provided to
allow growth in vitro.
•The composition of the nutrient medium is
mostly similar, although the exact
components and quantities will vary for
different species and purpose of culture.
• Types and amounts of hormones vary greatly.

14. •A nutrient medium is defined by its mineral
salt composition, carbon source, vitamins,
plant growth regulators and other organic
supplements
•The composition of plant tissue culture
medium can vary depending upon the type of
plant tissues or cell that is used for culture.
•The most widely used nutrient medium is MS
medium
(developed by Murashige and Skoog).

15. A typical nutrient medium consists of:
•Inorganic salts (both micro and macro ele
­
ments),
•A carbon source (usually sucrose),
•Vitamins, amino acids,
•Growth regulators (e.g. auxins and cytokinins
and gibberellins).

16. •Generally, a gelling agent agar is added to the
liquid medium for its solidification.
• An optimum pH (usually 5-7) is also very
important for the proper growth of explant.

22. Hormones used in Plant Tissue Culture
•Auxins
•Cytokinins
•Gibberellins
•Abscisic Acid
•Polyamines
•Plant hormones play an important role in the
growth and differentiation of cultured cells
and tissues.

23. 23
 Cell growth expansion
 Cell wall acidification
 Initiation of cell division
 Initiation of callus
 Initiation of roots).
Initiation of flower
Sex determination
Fruit development
Functions of Auxins

24. Functions of Cytokinins
24
Promotes cell division.
Morphogenesis.
Lateral bud development.
Delay of senescence.
Stomatal opening.
Rapid transport in xylem stream.

25. 25
General cell elongation.
Breaking of dormancy.
Promotion of flowering.
Functions of Gibberelins

26. Basic Methodology/technique of Plant Tissue
Culture
•1.Preparation of suitable nutrient medium: As
per the selection of plant ,medium is autoclaved.
•2. Selection of explant: Any excised part of
healthy plant to be used e.g. Bud,leaf, root, seed
etc.
•3. Sterilisation of explants: By sodium
hypochlorite, mercuric chloride etc. and washed
asceptically for 6-10 times with sterilised water.

27. •4. Inoculation (Transfer): The sterile explant is
inoculated on solidified nutrient medium
under asceptic condition.
•5. Incubation: Growing the culture in the
growth chamber.
 After the defined period of
incubation, an unorganized and
undifferentiated (no root and shoot) mass of
cells called
 
callus 
is obtained from each
explant. Cultures are incubated at of 25±2°C
and at a relative humidity upto 50-70% for 16
hrs of photo period.

28. •6. Transfer of callus and Regeneration.
Plantlets regenerated after transferring a
portion of callus into another medium and
induction of roots and shoots takes place.
•7. Hardening: Is the gradual exposure of
plantlets for acclimatization to environment
condition.
•8. Plantlet transfer: Plantlet are transferred to
green house or field conditions.

30. Preparation of an explant
Inoculation
After incubation

31. Various stages of plant growth

37. Laminar Air Flow

38. Types of tissue culture
1.On the basis of Explant used- Organized culture:
The culture of whole or parts of a plant.
–Bud culture
–Seed culture
–Meristem/shoot tip culture
–Embryo culture
–Ovary culture
–Leaf culture
–Root tip culture
–Protoplast culture
–Hairy root culture

39. Types of tissue culture
2.Unorganized culture:
•Callus culture
•Cell suspension culture
•Organogenesis
•Somatic embryogenesis

40. 1. On the basis of Explant used- Organized
culture:
1. Seed culture:
•Seeds may be cultured in-vitro to generate
seedlings or plants. It is the best method for
raising the sterile seedling.
•Germination of seeds in in-vitro conditions
involves the proper inoculation of seed in the
medium.
• It is better to inoculate the seeds without seed
coats as it reduce the germination time and
increases the germination potential.

42. Advantages
•Increased efficiency seed germination.
•Preparation of clean seedlings for explant/
meristem culture.

43. 2. Embryo culture
•Embryo culture is the isolation and growth of
mature or immature embryo in-vitro with the
aim of obtaining a viable plant.
•In some plants, seed dormancy may be due to
chemical inhibitors or mechanical resistance
etc. Excision of embryos and culturing them in
nutrient media help in developing viable
seedlings.

45. Both monocot and dicot embryos have a
plumule that forms the leaves, a hypocotyl that
forms the stem, and a radicle that forms the root.

47. 3. Meristem Culture
•A Meristem is a localized group of cells, which
are actively dividing and undifferentiated &
give rise to permanent tissue.
•The apical meristem of shoots is cultured to
get the disease-free plants even if the plant is
infected.
•Advantages-
•Disease free plants
•Germplasm conservation

50. 4. Anther Culture:
•Anthers of some plants are cultured on a
suitable medium to produce haploid plants, it
is called anther culture. Sometimes, pollen
grains are separated from anthers and
cultured on a
 suitable medium.
•This technique was first used in
 
India 
to
produce haploids of Datura(n).

51. 5.Root Tip culture
•Root culture can be defined as the culture of excised
radicle tips of germinated seeds in aseptical conditions
in a liquid medium where they are induced to grow
independently under controlled conditions.
•Root Explant also be used for Root culture.
•Advantages-
•The root material derived from a single radicle tip could
be multiplied and maintained in continuous culture.
• Such genetically uniform root cultures are referred to
as a clone of iso
­lated roots. Initiation of root clones is a
very simple technique.
•Production of virus free plants

53. 6.Leaf Culture
•Leaf culture is the culture of excised young
leaf primordia or immature young leaf of the
shoot apex in a chemically defined medium
where they grow and follow the
developmental sequences under controlled
conditions.
•Leaf primordia or very young leaves are
excised, surface sterilized and inoculated on
an agar solidified medium.

54. •• In culture, leaf remains in healthy condition
for a long period.
•• Leaves can be taken from aseptically grown
plants for culture.
•• Leaf primordia or very young leaf have more
growth potential than nearly mature leaves.

56. Advantages
•Culture of excised leaf primordia is valuable to
study the effects of various nutrients, growth
factors and changing environmental
conditions on leaf development

57. 7.Bud Culture
•In this, axillary bud as an explant is cultured to
produce the new plantlet.
•Actively dividing meristems are present at the
axillary and apical shoots (shoot tips).
•The axillary buds located in the axils of leaves
are capable of developing into shoots.

59. Unorganized culture:
1.Cell Suspension culture:
•Cell culture is the culture of isolated individual
cells, obtained from an explant tissue or
callus. These cultures are carried out in
dispersion medium and are referred to as cell
suspension cultures.
•Cell or suspension culture can be obtained
either directly from explant or from callus.

60. •Pieces of undifferentiated calli are transferred
to liquid medium which is continuously
agitated to obtain a suspension culture.
•Tissue and cells cultured in a liquid medium
produces a suspension of single cells and cell
clumps of few to many cells which is called
suspension culture.

61. 2. Protoplast culture
•Protoplast is a biological term refers to the
cell in which the cell wall has been removed.
•In this, isolated protoplasts are cultured on a
suitable medium under the aseptic condition.
•The protoplast culture is aimed to develop
genetically transformed plant.
•Somatic hybridization of two plant species
through proto
­plast fusion is done by
protoplast culture.

62. 3. Callus culture
•Callus is the unorganised and undifferentiated
dividing mass of cells.
•Callus culture involves the growth of callus
from explant by providing appropriate
conditions. Darkness and solid medium gelled
by agar stimulate callus formation.
•Darkness
 is required as light can encourage
differentiation of the callus.

65. Stages of Callus Culture
5 Days 10 Days
15 Days
20 Days 25 Days
30 Days
65

66. Plant Tissue Culture Applications
•The commercial production of plants.
•To
 conserve rare or endangered plant species.
•To screen cells rather than plants for advantageous
characters, e.g.
 herbicide resistance/tolerance.
•Large-scale growth of plant cells in liquid culture
in
 bioreactors for production of valuable compounds,
like plant-derived secondary metabolites
and
 recombinant proteins used as biopharmaceuticals.

67. Plant Tissue Culture Applications
•To cross distantly related
 species by protoplast
fusion
 and regeneration of the novel hybrid.
•To produce clean plant material from
 stock
infected by viruses or other pathogens.
•Production of identical sterile hybrid species
can be obtained

68. •A large number of plants having identical
features to the parents can be produced by
this method.
•To conserve rare or endangered plant species.
•Many plantlets can be produced without
seeds.

69. •Healthy and disease free plants can be
propagated by this technique.
•Large-scale production of artificial seeds
through somatic embryogenesis.
•Genetic variation present among plant cells of
a culture is called soma clonal variation. This
variation has been used to develop several
useful varieties.

70. Advantages
•Mass production of various plant cultivars
–6 million plants per year from one explant.
–Much higher production rate than other asexual
propagation methods.
•Especially beneficial for:
–Plants in high demand or valuable plants.
–Plants that are slow or difficult to propagate.
–Endangered species.

71. Advantages
•Production of pathogen-free plants
–Maintaining disease-free plants by micropropagation.
•Germplasm preservation
–Germplasm: the DNA of a species
–In the past: seeds
–limited shelf-life
–don’t preserve uniform characteristic (variability)

72. Advantages
•Continuous year round production
– Unaffected by climate
•Propagated in controlled lab conditions
–The ability to change specific conditions to meet
the needs of a particular plant species.
–Mainly, nutrient, light and temperature
requirements.

73. Disadvantages
•Specialized equipment required
–Laminar flow cabinets
–Autoclave
–Water purification systems
–Glassware etc…
•High labor cost is the most limiting factor
–Skilled labor required

74. Disadvantages
•Contamination risks
–Maintenance of aseptic (sterile) environment difficult.
–Rapid spread of contaminants = widespread loss.
•Risk of mutation arising
–Artificial environment induces mutations.
•Responses to tissue culture conditions varies
–Trial and error to determine optimum media or conditions。

75. Applications of Plant Tissue
Culture in Pharmacognosy

76. What are secondary
metabolites/Phytoconstituents?
• Secondary metabolites are generally defined
as small organic molecules produced by an
organism that are not essential for their
growth, development and reproduction.
• They may include pharmaceuticals, flavours,
fragrance, food additives, feedstock etc.

77. Type of secondary metabolites
•Alkaloids - Caffeine, Codeine, Quinine
•Saponin glycoside -Diosgenin Progesterone
•Flavonoids - Quercetin, Procyanidins
•Tannins and Phenolic compounds- Gallic acid
•Carotenoids - α-carotene, β-carotene and
lycopene
•Terpenoids – Menthol, Camphene
•Steroids – Glycyrhetinic acid

78. Why in vitro production?
•According to WHO survey approximately 70-80% of
world’s total population depends on herbal drugs.
• Some compounds are difficult to synthesize
chemically due to their structural complexity.
•Some novel compounds produced in cell cultures
are not produced in intact plants. At least 85 novel
compounds including 23 alkaloids, 19 terpenoids,
30 quinones and 11 phenyl compounds have been
isolated from some 30 different plant culture
systems.

79. •Yield of some secondary metabolites can be
improved by cell cultures compared to natural
growth. Ex Vincristine and Vinblastine.
•No need to wait for season of collection of
crude drug for production of
phytoconstituents.