SOMACLONAL VARIATION

BY : KALPESH GHEVARIYA Guided by: Dr. Monisha kottayi

SOMACLONAL VARIATION (LARKIN AND SCOWCRAFT IN 1981)

The GENETIC VARIATION found in the in vitro cultured cells are collectively referred as SOMACLONAL VARIATIONS. The plants derived from such cells are referred to SOMACLONES.

Some uses calliclones(culture obtained from callus) and protoclones(culture obtained from protoplast).

TERMS
Gametoclonal variation:- proposed for the variability present among pollen derived cells/plants. S.C.V. are obtained in homozygous condition in plants regenerated from cells cultured in vitro(Ro generation), but most variants are recovered in the selfed progeny of tissue culture-regenerated plants(R1 generation)

STEPS IN INDUCTION
Callus Tissue

Organogenesis

Somaclonal Variants

Regenerated plants

Hardening and Selfing

BASIS OF SOMACLONAL VARIATION

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

Causes of Somaclonal Variations

Physiological Cause

Biochemical Cause

Genetic Cause

Physiological Cause Exposure of culture to plant growth regulators Culture conditions

Genetic Cause
1. 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

2. Change in chromosome structure

Deletion Inversion Duplication Translocation

Gene Mutation
Tansition (purine to purine & ) Transversion (purine to pyrimidine & ) Insertion Deletion

Transposable element activation Silent mutator activation Plasmagene mutation

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.

Biochemical Cause
Lack of photosynthetic ability due to alteration in carbon metabolism Biosynthesis of starch via carotenoid pathway Nitrogen metabolism Antibiotic resistance

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

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

Detection of disease resistance variant

Pathogen or toxin responsible for disease resistance can be used as selection agent during culture.

Detection of herbicide resistance variant

Plantlets generated by the addition of herbicide to the cell culture system can be used as herbicide resistance plant

SCREENING
In general, R1 progeny (progeny of regenerated, Ro plant) are used for the identification of variants and R2 progeny are used as confirmation. Computer based cell sorting devices are also used..

SELECTION METHODS
Positive selection Negative selection

Selection
Positive selection- allow only mutant cells to survive or divide 1.Direct selection 2.Rescue method 3.Stepwise selection 4.Double selection

 Direct selection -Cells resistant to selective agent survive and divide to form colonies Used for isolation of cells resistant to toxins, herbicides antibiotics etc Rescue method- wild type cells killed by the agent & variants survive but do not divide due to unfavourable conditions Removal of agent facilitates recovery of variant cells

 Stepwise selection: selection gradually increased from low to high levels Resistant clones of each step can be subjected to higher selection pressure Double selection: selection of one parameter on one hand and other parameter on the other

 Negative selection: wild type cells divide normally & are killed by selection agent: mutant cells cannot divide so they escape the agent Variant cells can be rescued by removal of counter selection agent Can be used for recovery of low temperature and aluminium resistant mutants

APPLICATION
DISEASE RESISTANCE: I. SPECIFIC TOXIN: EX. Helminthosporium victoriae produce toxin oat Helminthosporium maydis -maize II. NONSPECIFIC TOXIN: EX. Alternaria sp. ,many bacteria etc

 STRESS RESISTANT AND OTHER MUTANTS: 1. Plants resistant to 4-5 times the normally toxic salt concentration have been isolated 2. Saline condition tolerant 3. Cell lines resistant to chilling have been isolated in some cases., chillies, Nicotiana sylvestris etc.

 MUTANTS FOR AGRONOMIC CHARACTERS: 1. In vitro regenerated plants often show variation in quntitative Characters. Ex. Tobacco, maize, barley, tomato, sorghum, wheat, carrot, brassica, celery, sugarcane, potato etc..

ACHIVEMENTS
Total 20+ varieties are realeased ono sugarcane fiji disease resistant of susceptible cultivar pindar also have yield advantage. Sweet potato cultivar scarlet by shoot tip culture disease resistant and yield improvement but have dark and more stable color.

Fiji disease in sugarcane

CHARLESTON SCARLET SWEETPOTATO

D. Michael Jackson, Janice R. Bohac, Judy A.Thies, and Howard F. Harrison in USDA-ARS. IN 2010

MARKET TYPE ATTRACTIVE, SCARLET COLORED SKIN HIGHLY RESISTANT TO INSECTS AND NEMATODES MODERATELY RESISTANT TO Fusarium WILT. EXCELLENT COOKING CHARACTERISTICS

 A gerenium variety called velvet rose is a somaclone of robbers lemon rose; -variety has twice the chromosome number. DETECTION OF SOMACLONAL VARIATION IN POTATO CALLUS INDUCED BY UV- RADIATION USING RAPD-PCR A. Ehsanpour, S. Madani, M. Hoseini, isfahan
university, iran (STRESS AND DROUGHT RESISTANT)

MERA BHARAT MAHAN

Variant of citronella java realeased as bio-13 for commercial cultivation by CIMAP,LUCKNOW. YIELD: 37% more oil and 39% more citronellol than control one. Variant of B.juncea variety varuna has been realeased for commercial cultivation as pusa jai kishan. It has bolder seeds and some yield advantages.

 Variant of khesari(Lathyrus sativus) has low neurotoxin content; it has been realeased as ratan for commercial cultivation.

YOU ALSO CAN REALEASE A NEW VARIETY

BEST VARIETY Ro R1 IMPROVED SOMACLONES VERIFY STABILITY BREEDING VARIETY REALEASED.BY YOU NAMED LIKE G-KALLU001(20__)

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 High frequency of variants than conventional mutagenesis.

Disadvantages
Clonal uniformity not obtained which is important in horticulture and forestry where elite genotypes are propagated using tissue culture 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.

Only used for those species regenerate complete plant.

which

REFERENCES
Biotechnology- expanding horizons by B.D.SINGH Biotechnology- by U.SATYANARAYANA Plant cell and tissue culture- by BHOJWANI AND RAZDAN www.ddr.nal.usda.gov/bitstream www.scribd.com/General and applied plant physiology(2007)

QUIZ
pre-existing variation in somatic cell called_______, variation generated during tissue culture is called________???? A. heritable variation, genetic variation B. Inheritable variation, epigametic variation C. Genetic variation, inheritable variation D. Epigenetic variation, heritable variation

 Variation caused by phenotypic changes is called????? A. Genetic B. Epigenetic C. Heritable D. Inheritable

 Due to alteration in carbon metabolism there is a lack of________ in plants.????? A. Photosynthesis B. Leaf color C. Root size D. Stress resistance

 In one DNA sequence, the GC is replaced by CG, then its called????? A. Inversion B. Translocation C. Transition D. Transversion

 Computer based cell sorting devices used in somaclonal variation technique for___????? A. Selection of variants B. Screening of variants C. Screening of alkaloids D. Selection of plant

 In cytophotometer, to measure DNA content, nuclei is stained by..????? A. Acetic acid B. Carmine fixative C. Aceto carmine D. Fuelgen

 In rescue method, the mutant cells can______ in favourable condition????????? A. Survives then killed B. Survives but not divides C. Divides but not survive D. Survives and divides

 Toxins produced by many pathogens are toxic to plant species and varieties in general called______?????? A. Specific toxins B. LD 50 toxins C. Non-specific toxins D. Killer toxins

 Somaclonal variation is used for the plants which can not regenerate whole plant by tissue culture.-statement is TRUE/FALSE

 Who used the term somaclonal variation first???? A. Salman khan B. Shahrukh khan C. Katrina kaif D. Larkin and Scowcraft