innovative ideas of tissue culture in crop improvement

1. Application of tissue
culture in crop
improvement
Presented by,
M. Uma Maheshwari,
M. Phil., (Botany)

2. Introduction
 Plant tissue culture comprises a set of invitro techniques, methods,
strategies that are part of plant
biotechnology.
 Tissue culture had been exploited to create genetic variability, to
improve the health of planted material, increase the number of desired
germplasms.
 Tissue culture are combined with molecular techniques, successfully used
toincorporatespecifictraitsthroughgenetransfer.
 Thecultureofprotoplasts,anthers,microspores,ovulesandembryos used
tocreatenewgeneticvariation.
 Cell culture produced somaclonal and gametoclonal variants with crop
improvementpotential.
 The culture of single cells and meristems can be effectively used to
eradicatepathogenstherebyincreasingtheyieldofthecrops.

3. Application of tissue culture in crop
improvement
Newer molecular and cellular technologies have a broad significant
impactoncropimprovement
The various application of tissue culture approaches to crop
improvement are-
Breedingandbiotechnology
Widehybridization
Haploids
Somaclonalvariation
Micropropagation
Syntheticseeds
Pathogeneradication
Germplasmpreservation

4. Breeding and biotechnology
Plantbreedingseparatedintotwoactivities
 Manipulatinggene
 Plantevaluation
Controlled pollination of plants lead to specific crosses result in new generation
performedbetterintheyield
Tissue–culture techniques is having significant impact in manipulating genetic
diversity
More than 50 different species have already been genetically modified either by
vector dependent (Agrobacterium) or vector independent (Biolistic, micro-
injectionandliposome)methods
Tissue culture techniques have been played a major role in the development of
plantgeneticengineering,especiallyinefficientgenetransferandtransgenicplant
recovery

5. Wide hybridization
Acriticalrequirementforcropimprovementistheintroductionofnew
geneticmaterialintothecultivatedlinesofinterest,whetherviasingle
genes, throughgeneticengineeringormultiplegenesthroughconventional
hybridizationortissueculturetechniques
Theprocessoffertilizationcanbeblockedatanyofthestages,resultingin
thefunctionalbarriertothehybridizationandtheblockageofgenetransfer
betweenthetwoplants.
Twotypesof barriers
Pre-zygoticbarriers
Post-zygoticbarriers
Toovercometheseproblems,thefollowingproceduresarefollowed
invitrofertilization
Embryoculture
Protoplastfusion

6. in vitro fertilization
 IVF has been used to facilitate both interspecific and
intergeneric crosses, to overcome physiological
basedselfcompatibilityandtoproducehybrids
Awiderangeofplantshavebeenrecovered suchas
 Tobacco
 Clover
 Corn
 Rice
 Canola
 Cotton
 Poppy

7. Embryo culture
Itdealswiththesterileisolationandinvitrogrowthofamatureoranimmatureembryo
ofobtainingaviableplant
Seeddormancycanbesuccessfullybyepassed
Twotypesofembryoculture
Matureembryoculture
Immatureembryoculture/Embryorescue
Matureembryoculture
o Matureembryosareisolatedfromripeseedsandare culturedin
vitro
o Itiscarriedoutduringfollowingcondition
o Embryosremaindormantforlongtime
o Lowsurvivalofembryosinvivo
o Toavoidinhibitioninseedgermination
o Convertsterileseedsintoviableseedlings

8. Embryo culture

9. It has been successfully applied to a number of agricultural
crops including cotton, barley, banana, orchids, roses,
tomato, rice, jute and some brassicas
Application of embryo culture
o Prevention of embryo abortion
o Overcoming seed dormancy
o Shortening of breeding cycle
o Production of haploids
o Overcoming seed sterility
o Clonal propagation
Embryo rescue
o It involves the culture of immature embryos to rescue
them from unripe or hybrid seeds that fails to germinate

10. Protoplast fusion
It has been suggested as a means of developing unique hybrid
plantswhichcannotbeproducedbyconventionalhybridization
Protoplasts of two plants can be fused by chemical or physical
methods
Applicationofprotoplastfusion
Ithas opened new possibilities for thein vitro genetic manipulation
oftoimprovethecrops
 Diseaseresistance
 Environmentaltolerance
 Qualitycharacters
Cytoplasmicmalesterility

11. Protoplast fusion

12. Haploids
 Haploid plants are characterized by possessing only a single set of
chromosomes.
 Haploid plants are of great significance for the production of homozygous
lines
 At least 171 plant species have been used to produce haploid plants by
pollen,antherandmicrosporeculture
 Cereals(barley,maize,rice,rye,wheat)
 Fruits(grapeandstrawberry)
 Oil-seeds(rapeandcanola)
 Trees(apple,litchi,poplarandrubber)
 Plantationcrops(cotton,sugarcaneandtobacco)
 Vegetable crops (asparagus, Brussels sprouts, cabbage, carrot,
pepper,potato,sugarbeet,tomatoandwingbean)

13. Haploids

14. Androgenesis
 Haploidproductionoccurs
throughantherorpollen
culture
 Intheplantbiotechnologyprogramme,haploidproductionisachievedby
twomethods
 Androgenesis
 Gynogenesis
Gynogenesis
 Haploidproductionoccurs
throughovaryorovule
culture
Pollenculturepreferablethanantherculturebecause
• Androgenesis,startingfromasinglecellcanberegulated
• Isolated micro spores are ideal for several genetic manipulations
(transformation,mutagenesis)
• Theyieldof haploidplantsisrelativelyhigher
• Undesirableeffectofantherwallandassociatedtissuescanbeavoided

15. Somaclonal variation
 The genetic variations found in the in vitro cultured cells are collectively
calledsomaclonalvariations.Theplantsarecalledsomaclones
 Itoccursduetogeneticheterogeneityinplanttissuecultures
 Expressionofgeneticdisorders
 Spontaneousmutationduetocultureconditions
The geneticchangesinclude
 Polyploidy
 Aneuploidy
 Chromosomal
breakage
 Deletion
 Translocations
 Geneamplification
 Severalmutations

16.  Many of the changes observed in plants are
regenerated in vitro have potential
agriculturalandhorticulturalsignificance
 Thealterationsinclude
 Plantpigmentation
 Seedyield
 PlantvigourandSize
 Essentialoils
 Leafandflowermorphology
 Fruitsolids
 Diseasetoleranceorresistance
Such variations are observed in wheat, maize, tomato,
sugarcane,oats,oilseedrape,celery,potato

17. Somaclonal variation

18. Micro propagation
invitroclonalpropagationthroughtissuecultureis
calledmicropropagation
Themicropropagationtechniqueispreferredto
conventionalasexualpropagation
Smallamountoftissueisneeded
Possiblealternativefordevelopingresistance
Meansforinternationalexchangeofplant
materials
invitrostockcanbeeasilyproliferated
Micropropagationinvolvesinvitroclonalpropagationbytwo
approaches
 Multiplicationbyaxillarybuds/apicalbuds
Multiplicationbyadventitiousroots

20. • Selection of mother plant and its
maintenanceStage 0
• Initiation and establishment of
cultureStage 1
• Multiplication of shoots or rapid
somatic embryo formationStage 2
• in vitro germination of somatic
embryos and or rooting of shootsStage 3
• Transfer of plantlets to sterilized
soil for hardening under green
house condition
Stage 4
Major stages involved in Micro propagation

21. Micro propagation
Applications
 High rate of plant
propagation
 Production of disease-free
plants
 Production of seeds in
some crops
 Cost-effective process
 Automated micro
propagation
Disadvantages
x Contamination of cultures
x Brewing of medium
x Genetic variability
x Vitrification
x Cost factor

22. Synthetic seeds
Usually, Sodium or Calcium alginate is selected for encapsulation
becauseitislesstoxictoembryosandeasytohandle
The artificial seeds can be maintained in a viable state still they are
planted
Asynthetic or artificial seed has been defined as a somatic embryo
encapsulatedinsideacoatingandisconsideredtobeanalogoustoa
zygoticseed
Differenttypesofsyntheticseedsare
Somaticembryosencapsulatedinawatergel
Dried andcoatedsomaticembryos
Driedanduncoatedsomaticembryos
Somaticembryosuspendedinafluidcarrier
Shootbudsencapsulatedinawatergel

23. Pathogen eradication
Plant species are infected with pathogens-viruses, bacteria, fungi, mycoplasma and
nematodesthatcausesdiseases
Tissue culture techniques employing meristem-tips are successfully used for the
productionofdisease-freeplants
List of plants with virus-elimination by meristem-culture
Plant species Virus eliminated
Solanum tuberosum Leaf roll potato virus-A, X, Y, S
Allium sativum Mosaic virus
Petunia spp. Tobacco mosaic virus
Musa spp. Cucumber mosaic virus
Brassica oleracea Cauliflower / turnip mosaic virus
Armoracia
rusticena
Turnip mosaic virus

24. Germplasm preservation
Germplasm broadly refers to the hereditary material
transmittedtotheoffspringthroughgermcells
The main objective of germplasm conservation is to
preserve the genetic diversity of a particular plant or
geneticstockforitsuseatanytimeinfuture
in vitro methods employing shoots, meristems and
embryosareideallysuitedforgermplasmconservation
Threemainapproaches
 Cryopreservation
 Coldstorage
 Low-pressure and Low-oxygen
storage

25. Advantages
 Largequantitiescanbestoredinasmallarea
Maintained in an environment free from
pathogens
Protectedagainstnature’shazards
From germplasm stock, large number of
plantscanbeobtainedwheneveritisneeded
Obstacles for their transport through national
and international borders are minimal (since
theyaremaintainedunderasepticcondition)