Presented by :-Presented by :- Anuj Kumar Sharma Anuj Kumar Sharma M.Sc. Biotechnology 2 M.Sc. Biotechnology 2ndnd sem. sem.

Contents:-Contents:-

1.1. introduction to Somaclonal Variationintroduction to Somaclonal Variation2.2. Schemes for obtaining somaclonal variationSchemes for obtaining somaclonal variation3.3. Detection and Isolation of Somaclonal VariantsDetection and Isolation of Somaclonal Variants4.4. Factor affecting somaclonal variationFactor affecting somaclonal variation5.5. Mechanism of Somaclonal VariationsMechanism of Somaclonal Variations6.6. Advantages of Somaclonal VariationsAdvantages of Somaclonal Variations7.7. Disadvantages:Disadvantages:8.8. Application of somaclonal variation in crop improvement Application of somaclonal variation in crop improvement 9.9. Control of Somaclonal VariationControl of Somaclonal Variation10.10. To Increase Somaclonal VariationTo Increase Somaclonal Variation11.11. Targets for Somaclonal VariationTargets for Somaclonal Variation12.12. ReferencesReferences

It is the term used to describe the It is the term used to describe the variation seen in plants that have been variation seen in plants that have been produced by plant tissue culture.produced by plant tissue culture.

Chromosomal rearrangements are an Chromosomal rearrangements are an important source of this variationimportant source of this variation

Kohleria „Orange Glow“, eine durch mutageneBehandlung von Gewebekulturen erhaltene Mutante(Oben) im Vergleich zur Ausgangsform (links).

Schemes for obtaining Schemes for obtaining somaclonal variationsomaclonal variation

Two schemes Two schemes

1.Without in vitro selection 1.Without in vitro selection

2.With in vitro selection 2.With in vitro selection

1.Without in vitro selection 1.Without in vitro selection

A flow diagram for generation of somaclonal variation A flow diagram for generation of somaclonal variation without in vitro selection without in vitro selection

2. With in vitro selection :-2. With in vitro selection :-

Continue………….Continue………….

Continue……….Continue……….

Detection and Isolation of Detection and Isolation of Somaclonal VariantsSomaclonal Variants

1.1. Analysis of morphological charactersAnalysis of morphological charactersQualitative characters: Plant height, maturity date, Qualitative characters: Plant height, maturity date, flowering date and leaf sizeflowering date and leaf sizeQuantitative characters: yield of flower, seeds and wax Quantitative characters: yield of flower, seeds and wax contents in different plant parts contents in different plant parts

2.2. Variant detection by cytological StudiesVariant detection by cytological StudiesStaining of meristematic tissues like root tip, leaf tip Staining of meristematic tissues like root tip, leaf tip with feulgen and acetocarmine provide the number and with feulgen and acetocarmine provide the number and morphology of chromosomes. morphology of chromosomes.

3.3. Variant detection by DNA contentsVariant detection by DNA contentsCytophotometer detection of feulgen stained nuclei can Cytophotometer detection of feulgen stained nuclei can be used to measure the DNA contentsbe used to measure the DNA contents

4.4. Variant detection by gel electrophoresis Variant detection by gel electrophoresisChange in concentration of enzymes, proteins and hemical Change in concentration of enzymes, proteins and hemical products like pigments, alkaloids and amino acids can be products like pigments, alkaloids and amino acids can be detected by their electrophoretic patterndetected by their electrophoretic pattern

5.5. Detection of disease resistance variant Detection of disease resistance variantPathogen or toxin responsible for disease resistance can Pathogen or toxin responsible for disease resistance can be used as selection agent during culture.be used as selection agent during culture.

6.6. Detection of herbicide resistance variant Detection of herbicide resistance variantPlantlets generated by the addition of herbicide to the cell Plantlets generated by the addition of herbicide to the cell culture system can be used as herbicide resistance plant.culture system can be used as herbicide resistance plant.

Detection and Isolation of Detection and Isolation of Somaclonal VariantsSomaclonal Variants

7.7. Detection of environmental stress tolerant Detection of environmental stress tolerant variantvariant

Selection of high salt tolerant cell lines in tobaccoSelection of high salt tolerant cell lines in tobacco

Selection of water-logging and drought resistance cell lines Selection of water-logging and drought resistance cell lines in tomatoin tomato

Selection of temperature stress tolerant in cell lines in pear.Selection of temperature stress tolerant in cell lines in pear.

Selection of mineral toxicities tolerant in sorghum plant Selection of mineral toxicities tolerant in sorghum plant (mainly for aluminium toxicity)(mainly for aluminium toxicity)

Detection and Isolation of Detection and Isolation of Somaclonal VariantsSomaclonal Variants

Factor affecting somaclonal Factor affecting somaclonal variation variation

1.Genotype 1.Genotype

2.Explant source2.Explant source

3.Duration of cell culture 3.Duration of cell culture

4.Culture condition 4.Culture condition

Mechanism of Somaclonal Mechanism of Somaclonal VariationsVariations

1. Genetic (Heritable Variations)• Pre-existing variations in the somatic cells of

explant• Caused by mutations and other DNA changes• Occur at high frequency

2. Epigenetic (Non-heritable Variations)• Variations generated during tissue culture• Caused by temporary phenotypic changes• Occur at low frequency

A] Genetic variations are: A] Genetic variations are:

1.changes in chromosome 1.changes in chromosome

2.chromosome structure 2.chromosome structure

3.DNA sequence 3.DNA sequence

1.changes in chromosome numbers1.changes in chromosome numbers; ;

Euploidy- changes in Euploidy- changes in wholewhole chromosome sets chromosome sets

Aneuploidy- changes in Aneuploidy- changes in partsparts of chromosome sets of chromosome sets

PloidyPloidy: number of basic chromosome sets (a diploid has : number of basic chromosome sets (a diploid has 2 sets; a hexaploid has 6 sets)2 sets; a hexaploid has 6 sets)

Euploid: Euploid: organism containing multiples of the basic organism containing multiples of the basic chromosome setchromosome set

Monoploid: Monoploid: organisms with one chromosome set (in organisms with one chromosome set (in essentially diploid taxa)essentially diploid taxa)

Polyploid: Polyploid: organism containing more than two organism containing more than two chromosome setschromosome sets

2.Structural Changes in 2.Structural Changes in ChromosomesChromosomes

Deletion Inversion

Duplication Translocation

3.DNA sequence (base mutations). 3.DNA sequence (base mutations).

Change at DNA level Change at DNA level Detected using restriction enzyme Detected using restriction enzyme

analysis (altered fragment size)analysis (altered fragment size)Changes at protein levels Changes at protein levels Loss or gain of protein bandLoss or gain of protein band Altered levels of specific proteinsAltered levels of specific proteinsDNA methylationDNA methylation Methylation of a gene inactivates its Methylation of a gene inactivates its

transcriptiontranscription

B] Epigenetic changeB] Epigenetic change

Due to gene expression not gene Due to gene expression not gene alterationalteration

Often temporaryOften temporary

Some examplesSome examples Morphological change - leaf shapeMorphological change - leaf shape Earlier floweringEarlier flowering Improved adventitious rootingImproved adventitious rooting Increased vigorIncreased vigor Plageotropic growthPlageotropic growth

Advantages of Somaclonal Advantages of Somaclonal VariationsVariations

Benefit of somaclonal variation is in plant improvementBenefit of somaclonal variation is in plant improvement

Creation of additional genetic varitionsCreation of additional genetic varitions

Increased and improved production of secondary Increased and improved production of secondary metabolitesmetabolites

Selection of plants resistant to various toxins, herbicides, Selection of plants resistant to various toxins, herbicides, high salt concentration and mineral toxicity high salt concentration and mineral toxicity

Suitable for breeding of tree speciesSuitable for breeding of tree species

Characteristics for which somaclonal mutants can be Characteristics for which somaclonal mutants can be enriched during enriched during in vitroin vitro culture include resistance to culture include resistance to disease pathotoxins, herbicides and tolerance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress.environmental or chemical stress.

Disadvantages:Disadvantages:

No control - may change in undesirable directionNo control - may change in undesirable direction

Variations are random in natureVariations are random in nature

May not be genetically stableMay not be genetically stable

Require extensive field testingRequire extensive field testing

May not occur for complex agronomic traitsMay not occur for complex agronomic traits

Application of somaclonal variation in Application of somaclonal variation in crop improvement :-crop improvement :-

Improve by Improve by 1.selecting novel varients 1.selecting novel varients 2.Disease resistant 2.Disease resistant 3.Abiotic stress resistance 3.Abiotic stress resistance 4.Salt tolerance 4.Salt tolerance 5.Aluminium tolerance 5.Aluminium tolerance 6.Herbicide resistance 6.Herbicide resistance 7.Insect resistance 7.Insect resistance 8.Seed quality 8.Seed quality

Disease Resistant Success using Somaclonal VariationDisease Resistant Success using Somaclonal Variation

*Shown to be heritable through sexual propagation*Shown to be heritable through sexual propagation**Shown to be stable through vegetative propagation **Shown to be stable through vegetative propagation

CropCrop PathogenPathogen ToxinToxin

AlfalfaAlfalfa Colletotrichum sp.Colletotrichum sp. Culture filtrateCulture filtrate

BananaBanana Fusarium sp.Fusarium sp. Fusaric acidFusaric acid

CoffeeCoffee Colletotrichum spColletotrichum sp Partially purified culture Partially purified culture filtratefiltrate

MaizeMaize Helminthosporium maydisHelminthosporium maydis T-toxinT-toxin

Oat*Oat* Helminthosporium victoriaeHelminthosporium victoriae VictorinVictorin

Sugarcane**Sugarcane** Helminthosporium sp.Helminthosporium sp. Culture filtrateCulture filtrate

Potato**Potato** Phytophthora infestansPhytophthora infestans Culture filtrateCulture filtrate

RiceRice Xanthomonas oryzaeXanthomonas oryzae Culture filtrateCulture filtrate

Tobacco*Tobacco* Alternaria alternataAlternaria alternata Partially purified toxinPartially purified toxin

Somaclonal variation in trees:Somaclonal variation in trees:

Somatic variation may be a better mans for tree Somatic variation may be a better mans for tree improvementimprovement

Direct gene insertion or agrobacteruim –Direct gene insertion or agrobacteruim –mediated mediated transformation have many technical transformation have many technical difficulties that must be overcome with this difficulties that must be overcome with this methods.methods.

It have the It have the potential to significantly change tree potential to significantly change tree breeding programs by reducing the time breeding programs by reducing the time required for selecting and improving desired required for selecting and improving desired traits .traits .

Applying soma clonal technology to forest Applying soma clonal technology to forest trees offers an even greater advantagetrees offers an even greater advantage

because of the long generation times of because of the long generation times of trees and the opportunity to introduce trees and the opportunity to introduce desired traits not possible through desired traits not possible through traditional breeding.traditional breeding.

Somaclonal technology is simpler to Somaclonal technology is simpler to exploit than other types of genetic exploit than other types of genetic engineering.engineering.

Control of Somaclonal Variation:Control of Somaclonal Variation:

Regular reinitiation of clones from new Regular reinitiation of clones from new explants might reduce variability over time.explants might reduce variability over time.

Avoid 2,4-D in the culture medium, as this Avoid 2,4-D in the culture medium, as this hormone is known to introduce variation.hormone is known to introduce variation.

The duration of the culture cycle may The duration of the culture cycle may influence the variability.influence the variability.

To Increase Somaclonal VariationTo Increase Somaclonal Variation::Callus and suspension cultures for several Callus and suspension cultures for several cyclescyclesRegeneration of large number of plants Regeneration of large number of plants from long-term culturesfrom long-term culturesTesting of selected somaclones for genetic Testing of selected somaclones for genetic stabilitystabilityMultiplication of genetically stable Multiplication of genetically stable somaclones for developing new cultivarssomaclones for developing new cultivars

Targets for Somaclonal VariationTargets for Somaclonal Variation

• • Specific amino acid accumulatorsSpecific amino acid accumulators – – Screen for specific amino acid productionScreen for specific amino acid production – – e.g. e.g. Lysine in cerealsLysine in cereals

• • Abiotic stress toleranceAbiotic stress tolerance – – Add or subject cultures to selection agentAdd or subject cultures to selection agent – – e.g.e.g.: salt tolerance, temperature stresses, etc…: salt tolerance, temperature stresses, etc…

• • Disease resistanceDisease resistance – – Add toxin or culture filtrate to growth mediaAdd toxin or culture filtrate to growth media – – Examples shown on next slide →Examples shown on next slide →

References:References:Chawla.H.S.(Chawla.H.S.(2008),introduction to plant biotechnology , 2008),introduction to plant biotechnology , somaclonal variation , Oxford & IBH publishing co.(110-somaclonal variation , Oxford & IBH publishing co.(110-123)123)

Rajdan.M.KRajdan.M.K.(2007),plant tissue culture , somaclonal .(2007),plant tissue culture , somaclonal variation .(125-135) variation .(125-135)

Collen.H.A.Collen.H.A. and Edwards.S(2009),plant cell and Edwards.S(2009),plant cell culture ,selection of somaclonal variants,bioes scientific culture ,selection of somaclonal variants,bioes scientific publisher (91-101)publisher (91-101)

www.amazon.co.uk/Somaclonal-Variation-Crop-www.amazon.co.uk/Somaclonal-Variation-Crop-Improvement-BiotechnologyImprovement-Biotechnology

http://www.studentsguide.in/plant-biotechnology-http://www.studentsguide.in/plant-biotechnology-genomics/tissue-culture-micropropagation-somaclonal-genomics/tissue-culture-micropropagation-somaclonal-variationvariation