Somoclonal variation and its applications

somoclonal variation in plants,introduction, basic features, nomenclature, mechanism, factors influencing, applications, causes, detection and isolation of somoclonal variants, advantages & disadvantages, Gametoclonal Variations-features,
production of Gametoclones, Source etc

1. SOMOCLONAL VARIATION AND ITS
APPLICATIONS
Dr. SHILPY SINGH
ASSISTANT PROFESSOR
(MIMT, GR. NOIDA)

2. Geneticvariationsinplantsthathavebeenproducedbyplanttissue
cultureandcanbedetectedasgeneticorphenotypictraits
Somoclonalvariation-Thevariabilitygeneratedbytheuseofatissue
culturecycle
“Soma”-somaticcellsand“clones”-generations
ThetermSomoclonalvariationwascoinedbyLarkinandScowcroft
(1981)

3. Variations for Karyotype, isozymecharacteristics and morphology
in somaclonesmay also observed.
•Calliclone-clones of callus
•Mericlone-clones of meristem
•Protoclone-clones of Protoplast
Variation occurs in both qualitative and quantitative traits.
Generally heritable mutation and persist in plant population even
after plantation into the field.
Basic Features of Somaclonal Variations

4. Nomenclature
Chaleff(1981) has labelled the plants regenerated from tissue culture
as R or R0 plants and the self fertilized progeny of R0 plants as R1.
Subsequent generations produced by self-fertilization are termed as
R2, R3, R4 etc.
Larkin and Scowcroft (1981) have referred regenerated plants as
SC1(=R0) and subsequent self fertilized generations as SC2, SC3, SC4
etc.

5. Mechanism of Somaclonal Variations
• Pre-existing variations in the somatic cells of explant
• Caused by mutations and other DNA changes
• Occur at high frequency
• Variations generated during tissue culture
• Caused by temporary phenotypic changes
• Occur at low frequency
Genetic (Heritable Variations)
Epigenetic (Non-heritable Variations)

6. SCHEMES FOR OBTAINING SOMOCLONAL VARIATION
Two schemes, with and without in vitro selection have been generally followed for
getting somoclonalvariation in crop plants.
Without in vitro selection
Anexplantisculturedonasuitablemediume.g.smallshootsegments(1-2cm)of
sugarcane,cotyledons,hypocotyls,protoplasts,leaves,embryosetc.
Thebasalmediumissupplementedwithgrowthregulatorswhichsupport
dedifferentiationstage,i.e.callus.Normallytheseculturearesubculturedand
thentransferredtoshootinductionmediumforplantregeneration.
Theregeneratedplantsaretransferredtopots,growntomaturityandanalysed
forvariants.
Disadvantage
Thisapproachistimeconsumingduetothefertilizationstepandrequires
screeningofmanyplants.

7. Without in vitro Selection
Explant
Shoot regeneration
Plant
Explant derived callus
Transfer to the field
Screening for desirable traits
Agronomic traits

8. With in vitro selection
Thededifferentiatedculturei.e.callusissubjectedtoselectionagainstinhibitorslike
antibiotics,aminoacidanalogs,pathotoxinsetc.
Differentselectioncyclesareperformedtogettolerantcells/callusculturesthatare
subsequentlyregeneratedintoplants.Theseplantsaretheninvivoscreenedagainst
theinhibitor.
Iftheplantsareresistanttotheinhibitor,thenstabletransmissionofthatcharacteris
analyzedinsubsequentgenerations.
Inthisapproach,variantsforaparticularcharacterareselectedratherthanthegeneral
variationobtainedinfirstcasewhereselectionisdoneattheplantlevel.

9. Explant
Explant derived callus
Multiplication of callus
Purified culture filtrate
Pathogen
Toxin isolation
Determination of lethal concentration of toxin for
tissue
Tolerant calli
Regeneration
In vivotesting against toxin/pathogen (Putative resistant plants R0
or SC1
Progeny rows from each plant
Test for disease resistance
Small pieces of calli on toxic medium
Selection cycles
Multiplication of callus
Disease resistant plants (R1 or SC2)
Agronomic trials
Self pollination
Vegetative
propagation
With in vitro Selection

10. Factors influencing Somoclonal Variation
Genotype
Explant source
Duration of cell culture
Culture conditions
Selection propagule(cells, protoplasts or calli)
Selective agent (Inhibitors used can be an amino acid analog, herbicide, a
synthetic toxin isolated from fungal liquid culture) Selection technique
Regeneration of plants
In vivo testing
Agronomic analysis
Resistance stability

11. Application of Somoclonal Variation
Asaresultofsomaclonalvariations,severalnovelvariantsofexistingcropshavebeen
developede.g.AnimprovedscentedGeraniumvarietynamed‘VelvetRose’,purethorn-less
blackberriesetc.
Alistofsomoclonalvariantsobtainedfromdifferent
cropspecieswiththeirmorphologicalcharacters
Productionofagronomicallyusefulplants(NovelVariants)

12. Abiotic/Biotic stress resistance
Lazaretal.,1988developedsomoclonalvariantsforfreezingtoleranceinNorstarwinterwheat.A
significantpositivecorrelationbetweenprolinelevelandfrosttolerancehasbeenfoundinabroad
spectrumofgenotypes.
Invitroselectionandregenerationofhydroxyprolineresistantlinesofwinterwheatwithincreased
frosttoleranceandincreasedprolinecontenthasbeenreported(Dorfflingetal.,1997).Theresults
showedstrongcorrelationofincreasedfrosttolerancewithincreasedprolinecontent.
Planttissueculturetechniqueshavebeensuccessfullyusedtoobtainsalttolerantcelllinesorvariants
inseveralplantspecies,viz.tobacco,alfalfa,rice,maize,Brassicajuncea,Solanumnigrum,sorghum
etc.
Mandaletal.,1999developedasalttolerantsomacloneBTS24fromindigenousricecultivarPokkali.
In vitro selection and regeneration of salt tolerant plants
Cold tolerance
Salt tolerance

13. Inrecentyears,considerableresearchhasbeenfocusedontheunderstandingofphysiological,genetic
andmolecularprocessesthatleadtoaluminiumtolerance.
Despitetheproblemsencounteredinadaptingculturemediaforinvitroselectionforaluminium
resistance,celllineshavebeenisolatedinseveralspecieseg.Alfalfa,carrot,sorghum,tomato,tobacco.
Janetal.,1997elicitedaluminiumtoxicityduringinvitroselectioninricebymakingseveralmodifications
inthemediaviz.lowpH,lowphosphateandcalciumconcentrations,andunchelatedironandaluminium
alongwithaluminiumsulphate.
Wangetal.,1997reportedasorghumsomoclonalvariantline(R111)resistanttodroughtstress.
Droughttolerantricelineswereobtainedbyinvitroselectionofseedinducedcallusonamedia
containingpolyethyleneglycolasaselectiveagentwhichsimulatedtheeffectofdroughtintissueculture
conditions(Adkinsetal.,1995).
Zemetraetal.,1993usedinvitroselectiontechniqueforgenerationofsomoclonalvariantsforRussian
wheataphid(Diuraphisnoxia)inwheat.
Croughanetal.,1994reportedvariantofBermudagrass(Cynodondactylon)calledBrazosR-3with
increasedresistancetofallarmyworm.
Aluminium tolerance
Drought tolerance
Insect Resistance

14. Disease resistant crop plants obtained without in
vitro selection
Disease Resistance
Thegreatestcontributionofsomoclonalvariationtowardsplantimprovementisinthe
developmentofdiseaseresistantgenotypeinvariouscropspecies.Carlson(1973)firsttested
thepossibilityofinvitroselectionfordiseaseresistanceforwildfirediseaseoftobacco
causedbyPseudomonassyringaepvtabaci.
Disease resistant crop plants obtained by in vitro
selection

15. Herbicide resistance
Through in vitro selection several cell lines resistant to herbicides have been isolated and few have
been regenerated into complete plants.
Important achievements are tobacco, soyabean, wheat, maize etcresistant to various herbicide
such as glyphosate, sulfonylurea, imidazolinonesetc.
Seed Quality
Recently,avarietyBioL212ofLathyrussativahasbeenidentifiedforcultivationincentralIndia
whichhasbeendevelopedthroughsomoclonalvariationandhaslowODAP(β-N-oxalyl-2-α,β
diaminopropionicacid),aneurotoxin(MehtaandSantha,1996),indicationthepotentialof
somoclonalvariationsforthedevelopmentofvarietieswithimprovedseedquality.
Enhancingaliengeneintrogressionintocultivatedspecies
Importantinenhancinginter-specificcrosseswherethegenomesshownohomologySothe
chromosomalexchangeresultsInnewcombinationsandtransferofalienchromosomesegment.
Some examples of somoclonalvariants for herbicide resistance

16. Causes of Somoclonal
Variation
Genetic Cause
Physiological
Cause
Biochemical
Cause

17. Exposure of culture to plant growth regulators
Culture conditions
a)Change in chromosome number
Euploidy: Changes chromosome Sets
Aneuploidy: Changes in parts of chromosome Sets
Polyploidy: Organisms with more than two chromosome sets
Monoploidy: Organism with one chromosomes set
Causes of Somaclonal Variations
Physiological Cause
Genetic Cause

18. b) Change in chromosome structure
Deletion
Inversion
Duplication
Translocation
c) Gene Mutation
Tansition
Transversion
Insertion
Deletion
d) PlasmageneMutation
e) Transposable elementactivation

19. f) DNA sequence
Change in DNA
•Detection of altered fragment size by using Restriction enzyme
Change in Protein
•Loss or gain in protein band
•Alteration in level of specific protein
Methylation of DNA
•Methylation inactivates transcription process
Lack of photosynthetic ability due to alteration in carbon metabolism
Biosynthesis of starch via Carotenoid pathway
Nitrogen metabolism
Antibiotic resistance
Biochemical Cause

20. Detection and Isolation of Somaclonal Variants
1. Analysis of morphological characters
Qualitative characters: Plant height, maturity date, flowering date and leaf size
Quantitative characters: yield of flower, seeds and wax contents in different
plant parts
2. Variant detection by cytological Studies
Staining of meristematictissues like root tip, leaf tip with feulgenand
acetocarmineprovide the number and morphology of chromosomes
3. Variant detection by DNA contents
Cytophotometerdetection of feulgenstained nuclei can be used to measure the
DNA contents

21. 4. Variant detection by gel electrophoresis
Change in concentration of enzymes, proteins and chemical products like
pigments, alkaloids and amino acids can be detected by their electrophoretic
pattern.
5. Detection of disease resistance variant
Pathogen or toxin responsible for disease resistance can be used as selection
agent during culture.
6. Detection of herbicide resistance variant
Plantlets generated by the addition of herbicide to the cell culture system can
be used as herbicide resistance plant.
7. Detection of environmental stress tolerant variant
Selection of high salt tolerant cell lines in tobacco
Selection of water-logging and drought resistance cell lines in tomato
Selection of temperature stress tolerant in cell lines in pear
Selection of mineral toxicities tolerant in sorghum plant (mainly for aluminium
toxicity)

22. Advantages
Help in crop improvement
Creation of additional genetic variations
Increased and improved production of secondary metabolites
Selection of plants resistant to various toxins, herbicides, high salt concentration and
mineral toxicity
Suitable for breeding of tree species
Disadvantages
A serious disadvantage occurs in operations which require clonal uniformity, as in the
horticulture and forestry industries where tissue culture is employed for rapid
propagation of elite genotypes
Sometime leads to undesirable results
Selected variants are random and genetically unstable
Require extensive and extended field trials
Not suitable for complex agronomic traits like yield, quality etc.
May develop variants with pleiotropic effects which are not true.
Advantages & Disadvantages of Somaclonal Variations

23. Gametoclonal Variations
Thevariationobservedamongplantsregeneratedfromculturedgameticcellsis
termedgametoclonalvariationascomparedtosomoclonalvariation,derived
fromsomaticcells.
Thetermgametoclones(inplaceofsomaclones)isusedfortheproductsof
gametoclonalvariations.
Asthesomaticcellsdividebymitosis,thegeneticmaterialisequallydistributed
tothedaughtercells.Incontrast,thegametes,beingtheproductsofmeiosis,
possessonlyhalfoftheparentcellgeneticmaterial.

24. The gametoclonalvariations differ from somaclonalvariations by three distinct
features:
1.Mutants obtained from gametoclonalvariations give rise to haploid plants since a single
set of chromosomes are present.
2.Meiotic crossing over is the recombination process observed in gametoclonalvariations.
3.The gametoclonescan be stabilized by doubling the chromosome number.

25. Gametoclonescanbedevelopedbyculturingmaleorfemalegameticcells.The
culturesofanthersorisolatedmicrosporesarewidelyused.Improvementshave
beenmadeinseveralplantspeciesthroughdevelopmentofgametoclonese.g.,
rice,wheat,andtobacco.
Production of Gametoclones
Variation among plants regenerated from gametophytictissue culture

26. Cellculturetechniquemayinducegeneticvariations.
Variationsmaybeinducedwhiledoublingthehaploid
chromosomes.
Geneticvariationsmayoccurduetoheterozygosityof
thediploids.
Source of Gametoclonal Variations

27. Reference
Introduction to Plant Biotechnology book by H. S. Chawla