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

Contents:- 1.introduction to Somaclonal Variation 2.Schemes for obtaining somaclonal variation 3.Detection and Isolation of Somaclonal Variants 4.Factor affecting somaclonal variation 5.Mechanism of Somaclonal Variations 6.Advantages of Somaclonal Variations 7.Disadvantages: 8.Application of somaclonal variation in crop improvement 9.Control of Somaclonal Variation 10.To Increase Somaclonal Variation 11.Targets for Somaclonal Variation 12.References

It is the term used to describe the variation seen in plants that have been produced by plant tissue culture. Chromosomal rearrangements are an important source of this variation

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

Schemes for obtaining somaclonal variation Two schemes 1.Without in vitro selection 2.With in vitro selection

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

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

Continue……….

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 meristematic tissues like root tip, leaf tip with feulgen and acetocarmine provide the number and morphology of chromosomes. 3.Variant detection by DNA contents Cytophotometer detection of feulgen stained nuclei can be used to measure the DNA contents

4. Variant detection by gel electrophoresis Change in concentration of enzymes, proteins and hemical 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. Detection and Isolation of Somaclonal Variants

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) Detection and Isolation of Somaclonal Variants

Factor affecting somaclonal variation 1.Genotype 2.Explant source 3.Duration of cell culture 4.Culture condition

Mechanism of Somaclonal Variations 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: 1.changes in chromosome 1.changes in chromosome 2.chromosome structure 2.chromosome structure 3.DNA sequence 3.DNA sequence

1.changes in chromosome numbers ; Euploidy- changes in whole chromosome sets Aneuploidy- changes in parts of chromosome sets Ploidy: number of basic chromosome sets (a diploid has 2 sets; a hexaploid has 6 sets) Euploid: organism containing multiples of the basic chromosome set Monoploid: organisms with one chromosome set (in essentially diploid taxa) Polyploid: organism containing more than two chromosome sets

2.Structural Changes in Chromosomes DeletionInversion DuplicationTranslocation

3.DNA sequence (base mutations). Change at DNA level Detected using restriction enzyme analysis (altered fragment size) Detected using restriction enzyme analysis (altered fragment size) Changes at protein levels Loss or gain of protein band Loss or gain of protein band Altered levels of specific proteins Altered levels of specific proteins DNA methylation Methylation of a gene inactivates its transcription Methylation of a gene inactivates its transcription

B] Epigenetic change Due to gene expression not gene alteration Often temporary Some examples Morphological change - leaf shape Morphological change - leaf shape Earlier flowering Earlier flowering Improved adventitious rooting Improved adventitious rooting Increased vigor Increased vigor Plageotropic growth Plageotropic growth

Advantages of Somaclonal Variations Benefit of somaclonal variation is in plant improvement Creation of additional genetic varitions 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 Characteristics for which somaclonal mutants can be enriched during in vitro culture include resistance to disease pathotoxins, herbicides and tolerance to environmental or chemical stress.

Disadvantages: No control - may change in undesirable direction Variations are random in nature May not be genetically stable Require extensive field testing May not occur for complex agronomic traits

Application of somaclonal variation in crop improvement :- Improve by 1.selecting novel varients 2.Disease resistant 3.Abiotic stress resistance 4.Salt tolerance 5.Aluminium tolerance 6.Herbicide resistance 7.Insect resistance 8.Seed quality

Disease Resistant Success using Somaclonal Variation *Shown to be heritable through sexual propagation **Shown to be stable through vegetative propagation CropPathogenToxin Alfalfa Colletotrichum sp. Culture filtrate Banana Fusarium sp. Fusaric acid Coffee Colletotrichum sp Partially purified culture filtrate Maize Helminthosporium maydis T-toxin Oat* Helminthosporium victoriae Victorin Sugarcane** Helminthosporium sp. Culture filtrate Potato** Phytophthora infestans Culture filtrate Rice Xanthomonas oryzae Culture filtrate Tobacco* Alternaria alternata Partially purified toxin

Somaclonal variation in trees: Somatic variation may be a better mans for tree improvement Direct gene insertion or agrobacteruim – mediated transformation have many technical difficulties that must be overcome with this methods. It have the potential to significantly change tree breeding programs by reducing the time required for selecting and improving desired traits.

Applying soma clonal technology to forest trees offers an even greater advantage because of the long generation times of trees and the opportunity to introduce desired traits not possible through traditional breeding. Somaclonal technology is simpler to exploit than other types of genetic engineering.

Control of Somaclonal Variation: Regular reinitiation of clones from new explants might reduce variability over time. Avoid 2,4-D in the culture medium, as this hormone is known to introduce variation. The duration of the culture cycle may influence the variability.

To Increase Somaclonal Variation: Callus and suspension cultures for several cycles Regeneration of large number of plants from long-term cultures Testing of selected somaclones for genetic stability Multiplication of genetically stable somaclones for developing new cultivars

Targets for Somaclonal Variation Specific amino acid accumulators Specific amino acid accumulators – Screen for specific amino acid production – Screen for specific amino acid production – e.g. Lysine in cereals – e.g. Lysine in cereals Abiotic stress tolerance Abiotic stress tolerance – Add or subject cultures to selection agent – Add or subject cultures to selection agent – e.g.: salt tolerance, temperature stresses, etc… – e.g.: salt tolerance, temperature stresses, etc… Disease resistance Disease resistance – Add toxin or culture filtrate to growth media – Add toxin or culture filtrate to growth media – Examples shown on next slide → – Examples shown on next slide →

References: Chawla.H.S.(2008),introduction to plant biotechnology, somaclonal variation, Oxford & IBH publishing co.( ) Rajdan.M.K.(2007),plant tissue culture, somaclonal variation.( ) Collen.H.A. and Edwards.S(2009),plant cell culture,selection of somaclonal variants,bioes scientific publisher (91-101) Improvement-Biotechnology genomics/tissue-culture-micropropagation-somaclonal- variation