micropropagation.ppt

Micropropagation

1. Dr Nidhi Mahendru
Asst. Professor & Head
Dept of Biotechnology
Guru Nanak Khalsa College

2. Thecultureofplantseeds,organs,tissues,
cells,orprotoplastsonnutrientmediaunder
sterileconditions.

3. TwoHormonesAffectPlantDifferentiation:
◦Auxin:StimulatesRootDevelopment
◦Cytokinin:StimulatesShootDevelopment
Generally,theratioofthesetwohormonescan
determineplantdevelopment:
◦Auxin↓Cytokinin=RootDevelopment
◦Cytokinin↓Auxin=ShootDevelopment
◦Auxin=Cytokinin=CallusDevelopment

4. Micropropagation
Germplasm Preservation
Somaclonal variation
Embryo Culture
Haploid Production
In vitro Hybridization-Protoplast fusion

5. InvitroClonalPropagation.
Micropropagationisthepracticeofrapidly
multiplyingstockplantmaterialtoproducea
largenumberofprogenyplants,usingmodern
planttissueculturemethods.

6. Clone is a plant population derived from a
single individual by asexual reproduction.
Clonal Propagation is the multiplication of
genetically identical individuals by asexual
reproduction.

7. Clonal reproduction
Multiplication stage can be recycled many
times to produce an unlimited number of
clones
Easy to manipulate production cycles
Disease-free plants can be produced

8. 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

9. Commercialization of Micropropagation 1970s & 1980s
Murashige (1974)
Broad commercial application

10. Tip
bud
Leaf
Axillary
bud
Internode
Root
Starting material for
micropropagation

11. Part of plant
Genotype
Physiological condition
Season
Position on plant
Size of explant

12. Minerals
Sugar
Organic ‘growth factors’
Growth regulators
Gelling agent
Other additives

13. Temperature
Moisture
Light

14. 1. Selection of plant material
2. Establish aseptic culture
3. Multiplication
4. Shoot elongation
5. Root induction / formation
6. Acclimatization

15. Stage I –Establishment
◦Selection of the explant plant
◦Sterilization of the plant tissue takes place
◦Establishment to growth medium
Stage II -Proliferation
◦Transfer to proliferation media
◦Shoots can be constantly divided
Stage III –Rooting & Hardening
◦explant transferred to root media
◦explant returned to soil

16. Organogenesis
◦Organogenesis via callus formation
◦Direct adventitious organ formation
Embryogenesis
◦Direct embryogenesis
◦Indirect embryogenesis
Microcutting
◦Meristem culture (Mericloning)
◦Bud culture

17. PGRsareprob.themostimportantfactoraffecting
organogenesis
◦cytokininstendtostimulateformationofshoots
◦auxinstendtostimulateformationofroots
Thecentraldogmaoforganogenesis:
◦ahighcytokinin:auxinratiopromotesshootsand
inhibitsroots
◦ahighauxin:cytokininratiopromotesrootsand/or
callusformationwhileinhibitingshootformation

18. Theprocessofinitiationanddevelopmentofa
structurethatshowsnaturalorganformand
function.
Theabilityofnon-meristematicplanttissuesto
formvariousorgansdenovo.
Theproductionofroots,shootsorleaves.
Theseorgansmayariseoutofpre-existing
meristemsoroutofdifferentiatedcells.
This,likeembryogenesis,mayinvolveacallus
intermediatebutoftenoccurswithoutcallus.

19. Tissue culture maintains the genetic of the cell
or tissue used as an explant.
Tissue culture conditions can be modified to
cause to somatic cells to reprogram into a
bipolar structure.
These bipolar structures behave like a true
embryo -called somatic embryos.

20. AnEmbryoismadeupofactivelygrowing
cellsandthetermisnormallyusedtodescribe
theearlyformationoftissueinthefirststages
ofgrowth.

21. Theprocessofinitiationanddevelopmentof
embryosorembryo-likestructuresfrom
somaticcells
Theproductionofembryosfromsomaticor
“non-germ”cells.
Usuallyinvolvesacallusintermediatestage
whichcanresultinvariationamongseedlings

22. Somaticembryogenesisisauseful
regenerationpathwayformanymonocotsand
dicots,butisespeciallyusefulforthegrasses
Typesofembryogenesis
◦zygoticembryogenesis–theresultofnormal
pollinationandfertilization
◦somaticembryogenesis–embryosfrom
(cultured)sporophyticcells,thatisembryos
ariseindirectly

23. Thecompositionoftheculturemedium
controlstheprocess-
◦auxin(usually2,4-D)addedcauses
induction,theformationofembrygogenic
clumpsorproembryogenicmasses(PEMs)
(inductionmedium)
◦auxinisdeletedandtheclumpsbecome
matureembryos(maturationmedium)

24. Stagesofdevelopment
◦earlycelldivisiondoesn'tfollowafixed
pattern,unlikewithzygoticembryogenesis
◦laterstagesareverysimilartozygotic
embryos(dicotpattern)
globularstage(multicellular)
heart-shapedstage(bilateralsymmetry)–
bipolarity
torpedo-shapedstage–consistsofinitial
cellsfortheshoot/rootmeristem

26. SomaticEmbryogenesis
Stimulationofcallusorsuspensioncellstoundergoa
developmentalpathwaythatmimicsthedevelopmentofthe
zygoticembryo.

27. •From one to many propagules rapidly.
•Multiplication in controlled lab conditions.
•Continuous propagation year round.
•Potential for disease-free propagules.
•Inexpensive per plant once established.

28. •Specializedequipment/facilitiesrequired.
•Moretechnicalexpertiserequired.
•Protocolsnotoptimizedforallspecies.
•Plantsproducedmaynotfitindustry
standards.
•Relativelyexpensivetosetup.

29. •Equipment/facility intensive operation
•Technical expertise in management positions
•Protocols not optimized for all species
•Liners may not fit industry standard
•Propagules may be too expensive

30. •Rapidincreaseofstockofnewvarieties.
•Eliminationofdiseases.
•Cloningofplanttypesnoteasilypropagated
byconventionalmethods.
•Propaguleshaveenhancedgrowthfeatures
(multibranchedcharacter;Ficus,Syngonium)