Types techniques nutritional requirements

1. Prepared By
Shivanee Vyas
M Pharmacy
Lakshmi Narain College of Pharmacy, Indore (MP)

2.  Tissue culture is anTissue culture is an
experimental processexperimental process
through which a massthrough which a mass
of cells(callus) isof cells(callus) is
produced from anproduced from an
explant tissue and theexplant tissue and the
callus produced in thiscallus produced in this
way can be usedway can be used
directly either todirectly either to
regenerate plantlets orregenerate plantlets or
to extract someto extract some
primary or secondaryprimary or secondary
metabolites.metabolites.

3. CALLUSCALLUS
→→
New plantletsNew plantlets
↓↓

4.  The production of exact copies of plantsThe production of exact copies of plants
that produce particularly good flowers,that produce particularly good flowers,
fruits, or have other desirable traits.fruits, or have other desirable traits.
 To quickly produce mature plants.To quickly produce mature plants.
 The production of multiples of plants inThe production of multiples of plants in
the absence of seeds or necessarythe absence of seeds or necessary
pollinators to produce seeds.pollinators to produce seeds.
 The production of plants in sterileThe production of plants in sterile
containers that allows them to be movedcontainers that allows them to be moved
with greatly reduced chances ofwith greatly reduced chances of
transmitting diseases, pests, andtransmitting diseases, pests, and

5.  The tissue obtained from the plant toThe tissue obtained from the plant to
culture is called an explantculture is called an explant
 Risk of microbial contamination increasesRisk of microbial contamination increases
with inappropriate explants.with inappropriate explants.
 The most commonly used tissue explantsThe most commonly used tissue explants
are the meristematic ends of the plants likeare the meristematic ends of the plants like
the stem tip, auxiliary bud tip and root tip.the stem tip, auxiliary bud tip and root tip.
 The soil microflora can form tightThe soil microflora can form tight
associations with the root systems whichassociations with the root systems which
overgrow the tissue cultureovergrow the tissue culture

6.  Surface sterilizing agents: SodiumSurface sterilizing agents: Sodium
hypochlorite, bromine water, hydrogenhypochlorite, bromine water, hydrogen
peroxide, mercuric chloride, silverperoxide, mercuric chloride, silver
nitrate.nitrate.
 The seeds are treated with 70%ethanolThe seeds are treated with 70%ethanol
and washed with sterile distilled water,and washed with sterile distilled water,
treated with surface sterilizing agents.treated with surface sterilizing agents.
 The aerial portion of plants are sterilzedThe aerial portion of plants are sterilzed
by submerging for 2-3 minutes inby submerging for 2-3 minutes in
70%ethanol followed by 2-3 rinses in70%ethanol followed by 2-3 rinses in

7.  General OrganizationGeneral Organization
 GlasswareGlassware
 High purity waterHigh purity water
 AutoclaveAutoclave
 Laminar air flow cabinetsLaminar air flow cabinets
 Oven for dry sterilizationOven for dry sterilization
 ShakersShakers
 pH meterpH meter
 Plant materialPlant material

8.  GautheretGautheret
 White’ mediumWhite’ medium
 Nitsch and NitschNitsch and Nitsch
 Murashige and skoogMurashige and skoog
 ErikssonEriksson
 B5 MediumB5 Medium
 N6 mediaN6 media

9.  Macro elementsMacro elements
 MicroelementsMicroelements
 Organic supplementsOrganic supplements
 Carbon sourceCarbon source
 Gelling agentsGelling agents
 Plant growth regulatorsPlant growth regulators

10.  These are present in large amounts forThese are present in large amounts for
plant growth and development.plant growth and development.
 Nitrogen, phosphorus, potassium,Nitrogen, phosphorus, potassium,
magnesium, calcium and sulphur ,carbon,magnesium, calcium and sulphur ,carbon,
are usually regarded as macro elements.are usually regarded as macro elements.
 These elements usually comprise at leastThese elements usually comprise at least
0.1% of the dry weight of plants.0.1% of the dry weight of plants.
 Nitrogen is most commonly supplied as aNitrogen is most commonly supplied as a
mixture of nitrate ions and ammoniummixture of nitrate ions and ammonium
ions.ions.
 Phosphorus is usually supplied as thePhosphorus is usually supplied as the
phosphate ion of ammonium, sodium orphosphate ion of ammonium, sodium or
potassium salts.

11.  These elements are required in traceThese elements are required in trace
amounts for plant growth andamounts for plant growth and
developmentdevelopment
 Manganese, iodine, copper, cobalt, boron,Manganese, iodine, copper, cobalt, boron,
molybdenum, iron and zinc usuallymolybdenum, iron and zinc usually
comprise the microelementscomprise the microelements
 Iron is usually added as iron sulphate,Iron is usually added as iron sulphate,
although iron citrate can also be used.although iron citrate can also be used.
Ethylenediaminetetraacetic acid (EDTA) isEthylenediaminetetraacetic acid (EDTA) is
usually used in conjunction with the ironusually used in conjunction with the iron
sulphate.sulphate.

12.  The two vitamins, thiamine (vitamin B1)The two vitamins, thiamine (vitamin B1)
and myoinositol (considered a B vitamin)and myoinositol (considered a B vitamin)
 Amino acids are also commonly includedAmino acids are also commonly included
in the organic supplement. The mostin the organic supplement. The most
frequently used is glycine (arginine,frequently used is glycine (arginine,
asparagine, aspartic acid, alanine,asparagine, aspartic acid, alanine,
glutamic acid, glutamine and proline areglutamic acid, glutamine and proline are
also used)also used)
 Amino acids provide a source of reducedAmino acids provide a source of reduced
nitrogen and, like ammonium ions, uptakenitrogen and, like ammonium ions, uptake
causes acidification of the medium.causes acidification of the medium.

13.  SucroseSucrose
 GlucoseGlucose
 MaltoseMaltose
 GalactoseGalactose
 SorbitolSorbitol

14.  AuxinsAuxins
 CytokininsCytokinins
 GibberellinsGibberellins
 Abscisic acidAbscisic acid
 EthyleneEthylene

15.  MS-Micronutrient stock solutionMS-Micronutrient stock solution
 VitaminsVitamins
 Calcium chlorideCalcium chloride
 Potassium iodidePotassium iodide
 2,4-D2,4-D
 NAANAA
 KinetinKinetin
 PH is around 5.5PH is around 5.5
 Agar (0.6 to 1% w/v)Agar (0.6 to 1% w/v)

16. Selection of the
plant tissue
(explant) from a
healthy vigorous
‘mother plant’ -
this is often the
apical bud, but can
be other tissue
This tissue must be
sterilized to
remove microbial
contaminants

17.  EstablishmentEstablishment of theof the
explant in a cultureexplant in a culture
medium. The mediummedium. The medium
sustains the plantsustains the plant
cells and encouragescells and encourages
cell division. It can becell division. It can be
solid or liquidsolid or liquid
 Each plant speciesEach plant species
(and sometimes the(and sometimes the
variety within avariety within a
species) hasspecies) has
particular mediumparticular medium
requirements thatrequirements that
must be establishedmust be established
by trial and errorby trial and error

18. Dividing shoots
Warmth and good light
are essential
Multiplication- The
explant gives rise to a
callus (a mass of
loosely arranged cells)
which is manipulated
by varying sugar
concentrations and
the auxin (low):
cytokinin (high) ratios
to form multiple shoots
The callus may be
subdivided a number of
times

19. Root formation - The
shoots are transferred
to a growth medium
with relatively higher
auxin: cytokinin ratios
The pottles on these racks are young banana
plants and are
growing roots

20. Tissue culture plants sold to
a nursery & then potted up
The rooted shoots
are potted up
(deflasked) and
‘hardened off’ by
gradually decreasing
the humidity
This is necessary as
many young tissue
culture plants have
no waxy cuticle to
prevent water loss

22.  Callus cultureCallus culture
 Cell-suspension culturesCell-suspension cultures
 ProtoplastsProtoplasts
 Shoot tip and meristem cultureShoot tip and meristem culture
 Microspore cultureMicrospore culture
 Root culturesRoot cultures
 Embryo cultureEmbryo culture
 Mass cell cultureMass cell culture
 Anther and Pollen CultureAnther and Pollen Culture
 Bergmann’s cell plating technique (cultureBergmann’s cell plating technique (culture
of single cells)of single cells)

23.  Explants, when cultured on the appropriateExplants, when cultured on the appropriate
medium, usually with both an auxin and amedium, usually with both an auxin and a
cytokinin, can give rise to an unorganised,cytokinin, can give rise to an unorganised,
growing and dividing mass of cells.growing and dividing mass of cells.
 . During callus formation there is some. During callus formation there is some
degree of Dedifferentiation both indegree of Dedifferentiation both in
morphology and metabolism as a resultmorphology and metabolism as a result
they lose their ability to photosynthesize.they lose their ability to photosynthesize.
 Callus culture is often performed in the darkCallus culture is often performed in the dark
(the lack of photosynthetic capability being(the lack of photosynthetic capability being
no drawback) as light can encourageno drawback) as light can encourage
differentiation of the callusdifferentiation of the callus
 Callus cultures are extremely important inCallus cultures are extremely important in
plant biotechnologyplant biotechnology

24.  The growth rate of the suspension culturedThe growth rate of the suspension cultured
cells is generally higher than that of thecells is generally higher than that of the
solid culture. The former is more desirablesolid culture. The former is more desirable
particularly in production of usefulparticularly in production of useful
metabolites in a large-scale.metabolites in a large-scale.
 A piece of the callus is transferred to aA piece of the callus is transferred to a
liquid medium in a vessel such as anliquid medium in a vessel such as an
Erlenmeyer flask and the vessel placed onErlenmeyer flask and the vessel placed on
a rotary or reciprocal shaker.a rotary or reciprocal shaker.
 The cells are cultivated at 100 rpm,on aThe cells are cultivated at 100 rpm,on a
rotary shaker at 25° C by subculturing forrotary shaker at 25° C by subculturing for
several generations, a fine cell suspensionseveral generations, a fine cell suspension
culture containing small cell aggregatesculture containing small cell aggregates
and single cells is establishedand single cells is established

25.  Batch cultureBatch culture
 Continuous cultureContinuous culture
 Immobilized cultureImmobilized culture

26.  Protoplasts are plant cells with the cell wallProtoplasts are plant cells with the cell wall
removedremoved
 Protoplasts are most commonly isolated fromProtoplasts are most commonly isolated from
either leaf mesophyll cells or cell suspensions.either leaf mesophyll cells or cell suspensions.
 Two general approaches to remove the cellTwo general approaches to remove the cell
wall-mechanical or enzymatic isolation.wall-mechanical or enzymatic isolation.
 Mechanical isolation:Mechanical isolation: It results in lowIt results in low
yields, poor quality and poor performance inyields, poor quality and poor performance in
culture due to substances released fromculture due to substances released from
damaged cells.damaged cells.
 Enzymatic isolationEnzymatic isolation :It is usually carried:It is usually carried
out in a simple salt solution with a highout in a simple salt solution with a high
osmoticum, plus the cell wall degradingosmoticum, plus the cell wall degrading
enzymes(cellulase and pectinase ) whichenzymes(cellulase and pectinase ) which
must be of high quality and purity.must be of high quality and purity.

27.  Root cultures can be established in vitroRoot cultures can be established in vitro
from explants of the root tip of eitherfrom explants of the root tip of either
primary or lateral roots and can beprimary or lateral roots and can be
cultured on fairly simple media.cultured on fairly simple media.
Shoot tip and meristem cultureShoot tip and meristem culture
 The tips of shoots (which contain the shootThe tips of shoots (which contain the shoot
apical meristem) can be cultured in vitro,apical meristem) can be cultured in vitro,
producing clumps of shoots from eitherproducing clumps of shoots from either
auxillary or adventitious budsauxillary or adventitious buds

28.  Besides, roots, shoots, and pollen, embryosBesides, roots, shoots, and pollen, embryos
can also be cultured to produce haploidcan also be cultured to produce haploid
plants. The embryo culture is very useful inplants. The embryo culture is very useful in
conditions where embryo fails to develop dueconditions where embryo fails to develop due
to degeneration of embryonic tissues.to degeneration of embryonic tissues.
 It has been used as a routine technique inIt has been used as a routine technique in
orchid propagation, in breeding of speciesorchid propagation, in breeding of species
showing dormancyshowing dormancy
 EMBRYO RESCUE:EMBRYO RESCUE: Technique of growingTechnique of growing
immature embryo is termed as ‘embryoimmature embryo is termed as ‘embryo
rescue’.rescue’.

29.  The anthers with their filaments are removedThe anthers with their filaments are removed
from the flower buds after surface sterilization.from the flower buds after surface sterilization.
 Under aseptic conditions, the anthers areUnder aseptic conditions, the anthers are
excised and crushed in 1% acetocarmine toexcised and crushed in 1% acetocarmine to
test the stage of pollen development.test the stage of pollen development.
 The anthers in correct stage of developmentThe anthers in correct stage of development
are separated and inoculated on a nutrientare separated and inoculated on a nutrient
medium.medium.
 The anther cultures are maintained at 280CThe anther cultures are maintained at 280C
and alternating photo periods of light (12-18and alternating photo periods of light (12-18
hrs) and darkness (6-12 hrs).hrs) and darkness (6-12 hrs).
 The anthers proliferate and produce callusThe anthers proliferate and produce callus
which forms an embryo and the embryowhich forms an embryo and the embryo
subsequently develops into a haploid plant.subsequently develops into a haploid plant.

30.  The pollens are extracted by pressing andThe pollens are extracted by pressing and
squeezing the anthers with a glass rodsqueezing the anthers with a glass rod
against the sides of the beaker.against the sides of the beaker.
 The anther tissue debris is removed byThe anther tissue debris is removed by
filtering the pollen suspension and large andfiltering the pollen suspension and large and
healthy pollen are washed and collected.healthy pollen are washed and collected.
 These pollen are cultured on a solid orThese pollen are cultured on a solid or
liquid medium and the callus or the embryoliquid medium and the callus or the embryo
formed is transferred to a suitable mediumformed is transferred to a suitable medium
to produce a haploid plant.to produce a haploid plant.

31.  In this technique, free cells areIn this technique, free cells are
suspended in a liquid medium.suspended in a liquid medium.
 Equal volumes of liquid and agar mediaEqual volumes of liquid and agar media
are mixed and rapidly spread in petriare mixed and rapidly spread in petri
dish, which makes the cells evenlydish, which makes the cells evenly
distributed in a thin layer afterdistributed in a thin layer after
solidification.solidification.
 After sealing the Petri dishes withAfter sealing the Petri dishes with
parafilm, they are examined under theparafilm, they are examined under the
inverted microscope to mark the singleinverted microscope to mark the single
cells.cells.

32.  LAG PHASELAG PHASE: In this phase the cell regais: In this phase the cell regais
the ability to divide following subculture.the ability to divide following subculture.
 EXPONENTIAL PHASEEXPONENTIAL PHASE:Rapis cell:Rapis cell
division occurs and the duration of thisdivision occurs and the duration of this
phase varies according to the cell and itsphase varies according to the cell and its
nutrient management.nutrient management.
 LOG PHASELOG PHASE:The growth in this phase:The growth in this phase
foolows a linear pattern with respect tofoolows a linear pattern with respect to
time.time.
 STEADY STATE PHASESTEADY STATE PHASE:The rate of:The rate of
production of cells will be equal to theproduction of cells will be equal to the
rate of their death.rate of their death.

33. Growth profile and measurements of
plant Tissue Culture
0
0.1
0.2
0.3
0.4
0.5
0.6
1 2 3 4 5 6 7 8
number of days
numberofcells

34.  LAG PHASELAG PHASE: next to inoculation the cell regains: next to inoculation the cell regains
the ability of division and the tissue shows slowthe ability of division and the tissue shows slow
growth.growth.
 EXPONENTIAL PHASEEXPONENTIAL PHASE:This phase shows rapid:This phase shows rapid
growth due to the rapid cell division .The level ofgrowth due to the rapid cell division .The level of
nutrients comes down due to the uptake ofnutrients comes down due to the uptake of
nutrients by tissue.nutrients by tissue.
 DECLINE PHASEDECLINE PHASE:Decline in the growth of callus:Decline in the growth of callus
tissue because of the minimal amount oftissue because of the minimal amount of
nutrients in the medium.nutrients in the medium.
 STATIONARY PHASESTATIONARY PHASE:Growth does not occurs:Growth does not occurs
and for growth subculturing is necessary.and for growth subculturing is necessary.

35.  FRESH AND DRY WEIGHTFRESH AND DRY WEIGHT
MEASUREMENTSMEASUREMENTS
 CELL NUMBERCELL NUMBER
 PACKED CELL VOLUMEPACKED CELL VOLUME
 CELL VIABILITYCELL VIABILITY
 NUTRIENT UTILISATIONNUTRIENT UTILISATION

36.  Text book of Pharmacognosy andText book of Pharmacognosy and
Phytochemistry by EDWINPhytochemistry by EDWIN
JARALD,SHEEJA EDWIN JARALDJARALD,SHEEJA EDWIN JARALD
 Plant cell culture edited by RADIXONPlant cell culture edited by RADIXON
AND RAGONZALESAND RAGONZALES
 An introduction to Plant TissueAn introduction to Plant Tissue
Culture by MK RAZDANCulture by MK RAZDAN

37. THANK YoUTHANK YoU