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  1. 1. Mutation Breeding
  2. 2. Introduction :  Mutation is a sudden heritable change in a characteristic of an organism .  Gene/Point mutation :- Mutation produced by changes in base sequence of genes Chromosomal mutation :- Mutation may be produced by changes in chromosome structure or even in chromosome number.  Other terms- cytoplasmic or plasmagene mutation  -bud or somatic mutation
  3. 3. Historical Account : Hugo de Vries: 1900. introduced the term “Mutation”. Mutants were observed before this: e.g., short legged sheep was discovered by an English Farmer in 18th century. This sheep was used to develop a breed-c/a Ancon. Muller(1927)discovered the action of X-rays on Drosophilla and Stadler discovered the effect of gamma-rays in barley(H.vulgare) and Maize(Z.mays). Muller was awarded nobel prize in 1946
  4. 4. Historical Account :  Auerbach and Robson-nitrogen mustards produced mutation in Drosophila.  subsequently a number of chemicals with mutagenic action was discovered  Mutation Breeding Programme was started in Sweeden, USSR and Germany in 1927 after the discovery by Muller.
  5. 5.  Swaminathan is known as "Indian Father of Green Revolution" for his leadership and success in introducing and further developing high-yielding varieties of wheat in India.  Swaminathan and his team produced a mutant breed of wheat by gamma irradiation of a Mexican variety (Sonora 64) resulting in Sharbati Sonora.  The mutant variety Sharbati Sonora was officialy approved in 1967. It was developed by irradiation of seeds with gamma rays (200 Gy). Main improved attributes of mutant variety are amber grain color, early maturity, bread and chapati making and high protein content (16,5%).
  6. 6. Type of mutation  Spontaneous (natural) mutation  Frequency of mutation is one in 10 lakhs  Different genes show different mutation rate  R-locus in maize mutate at a frequency 4.92x10-4, su at 2.4x10-6,Wx-highly stable.  Rate of spontaneous mutation is affected by genetic background and mutator gene
  7. 7.  Induced mutations- artificially induced by certain physical of chemical mutagens  Mutagens-agents used to induce mutation  Mutation breeding -utilization of mutations for crop improvement  Induced mutation
  8. 8. General characteristics  Generally recessive ,dominant also occur  Generally harmful to the organism -most cases-deleterious,some-beneficial  Random-occur in any gene,some show high rate  Recurrent-same mutation can occur again and again  Induced mutations show pleiotropy due to closely linked genes.
  9. 9. Effects of mutations on survival  Based on the effect on viability mutations are Lethal-kill each and every individual -dominant mutants do not survive -recessive survive only heterozygous condition Sublethal-kill more than 50% individual Subvital-kill less than 50% individual Vital-do not reduce the viability
  10. 10. Mutation  A heritable change in the base sequence of DNA  Base mutation- change in a single base position  Transitions –purine Purine, (A-G,T-C) pyramidine pyramidine (A-T or c,G-T or C)  Transversions-purine- pyramidine, pyramidine-purine Affect one codon-affect one aminoacid in the concerned protein Substitution that generate codon that donot code any aminoacid(nonsense codon-it acts as a terminator of polypeptide chain)
  11. 11. Consequences of Mutation  Silent Mutation---base change, no amino acid change  Neutral Mutation--- Base change resulting in a change that does not affect protein function – EX. Aspartic acid (D) Glutamic acid (E)  Missense mutation---altered codon, new aa with different chemical properties. Function affected.  Nonsense mutation---base pair substitution results in a stop codon (and shorter polypeptide)  Frameshift mutations —additions or deletions. Peptide may be longer or shorter.  Sense mutation?
  12. 12. Chromosomal mutation  Mutation associated with splitting and subsequent changes in the structure of the chromosomes  The end of the split chromosomes may fuse to form structure again, but the new chromosomes are not always exactly what the used to be  The microscopic structures of chromosomes may be characterized by deletion or deficiency (loss of a chromosomal segment), duplication (doubling of a chromosomal segment), inversion (rearrangement of a group of genes in a chromosomal segment in a such a way that their order is reversed; rearrangement of genetic material in a chromosome results from loss of segment, its rotation by 180°, and fusion of the separated ends) and translocation (change in a position of a chromosome or more often exchange of segments between different chromosomes)
  13. 13. Genome mutation Changes in sets of chromosomes Remarks: 1. Breeders are more interested in gene mutation, because chromosomal rearrangement usually produce negative results, such as lower fertility of the offspring 2. Mutant are aften of great value for breeding as sources of new, previously unknown useful characters 3. Mutagenesis may be instrumental in obviating the technical difficulties arising in the crossing of such a small flowered crops such as millet
  14. 14. Effects Of Mutation :  Lethal :- They kill each & every individual that carry them in appropriate genotype .  Dominant lethal : It can’t survive. Recessive lethal : kill in homozygous state.  Sub Lethal & Sub Vital :- Both mutation reduce viability but don’t kill all the individual carrying them in appropriate genotype.  Sub Lethal : Kill more than 50%.  Sub Vital : Kill less than 50%. Vital :- a) Don’t reduce the viability. b) Crop improvement can utilize only such mutations. 
  15. 15. Mutation breeding   The genetic improvement of crop plants for various economic traits through the use of induced mutations is referred to as mutation breeding.  Used in S.P. and clonally propagated crops.  Eg:Wheat, Sugarcane 
  16. 16. Mutation breeding is successful when…… :  Desired variability exhausts in cultivated species and germplasm.  There is tight linkage between desirable and undesirable traits.  Only one or two characters are to be improved in a fruit crop without changing its taste.  Crop does not have sexuality, thus lacks variability.  The generation cycle is very long, such as plantation crops, fruit trees….there mutation breeding is the shortcut way for genetic improvement. In ornamental plants Mutant Dog
  17. 17. Mutation breeding is successful when…… :  A particular reaction is to be blocked e.g., in opium the synthesis of morphine takes place in stepwise manner. If the reaction is stopped at bane level, it will block the synthesis of morphine without affecting the conversion process of the bane into useful pharmaceutical products. A B C D E H I J K Enzyme1 Enzyme 2 Enzyme 4 Enzyme 5 Enzyme6 Enzyme 7 Enzyme 8 Enzyme 3 
  18. 18. Induced Mutations  Caused by exposure to a mutagen  Causes – Exposure to base analogs – Chemical mutagens – Intercalating agents – Uv- radiation – Transposable elements – Mutator genes
  19. 19. Procedure for mutation breeding :  Procedure for mutation breeding Mutagenesis :- Treating a biological material with a mutagen in order to induce mutation. Irradiation :- Exposure of biological material to one of the radiation(X-rays, gamma rays etc.).  Mutation Breeding: muations are induced for crop improvement ,the entire operation of the induction and isolation etc. of mutants
  20. 20. Selection of the variety for mutagen treatment :  Selection of the variety for mutagen treatment Generally the variety selected for mutagenesis should be the best variety available in the crop.  Note : In certain situations, it may be desirable to isolate variants in varieties other than the best one,  e.g., Dwarf & semi dwarf mutants would have to be isolated from tall varieties in cereal crops (wheat, rice etc.)
  21. 21. Part of the plant to be treated :  Seeds, pollen grains, buds/cuttings or complete plant can be used for mutagenesis.  It depends on whether the crop is sexually or asexually propagated & type of mutagen to be used.  Sexually propagated crops : Seeds are commonly used because seed can tolerate extreme environmental conditions. 
  22. 22. Part of the plant to be treated Clonal crops : Buds/cuttings are used for mutagenesis. Pollen grains are also infrequently used because :-They are difficult to collect in large quantities . Hand pollination(with treated pollens) is rather difficult. Survival percentage of pollens is relatively low. Note:- Pollen grains are the only plant part which can be treated with ultra violet rays.
  23. 23. Dose and duration of the mutagen :  The usefulness of a mutagen and its efficiency depends on the mutagenic agent employed as well as on specific characteristics of the biological system to be treated.  An optimum dose is the one which produce the maximum frequency of mutation and causes minimum killing.  LD50 dose should be optimum  LD5o-dose of the mutagen which kill 50% of the treated individuals-varies from crop to crop.  Vicia faba-46 krad Brassica napus oleifera-120- 140
  24. 24. Giving mutagen treatment  selected plant is exposed to the desired dose  Plants are immediately planted to raise the M1  In the case of chemical mutagens,seeds are usually pre-soaked with for few hours to initiate metabolic activities,exposed to desired mutagen and washed in running water for saurface dried with blotting paper about to four hours to remove the mutagen present in them
  25. 25. Procedure :  Treated seed/buds/cuttings Pollination with treated pollen are M1 Plants M1 generation- Large no. of plants are grown in wider spacing. Dominant mutations are recorded if any (generally mutations are recessive and do not express in M1) Chlorophyll sectors and fertility is recorded. M1 plants are selfed and their seed is harvested separately. Procedure M2-----Seed obtained from M1 is sown in wide spacing Selected mutants are selfed. Oligogenic mutations are detected in M2 and are harvested separately M3-----M3 progeny is raised from selected M2 and evaluated for homozygosity. Selected homozygous M3 progenies are bulked together to conduct yield trials in M4. M4-----M4 progeny are raised in replicated trials using local check for comparison M5-M9----Selected lines are tested in multiplication coordinated trials
  26. 26. Handling of mutagen treated population  Number of mutations depends on the no. of treated seeds-6000 seeds for 10 desired mutation  M2 size should be -1000-1500  Monocots-primary tillers are good-arise from shoot bud primordia present in the seed at the time of mutagen treatment  Dicots seeds produced in the main shoot are show high frequency of mutations.
  27. 27. Procedure for Vegetative propagated crops :  In Veg. Prop. Crops mutations are expressed in the form of chimeras.  The chimera refers to genetically different tissue in an individual.  The individual has one type of tissue in one part and another type of tissue in another part. 
  28. 28. Procedure for Vegetative propagated crops :  In apical buds, axillary buds and adventitious buds, there are three functional layers, outer layer , inner layer and middle layer  L1-epidermis  L2-part of leaf mesophyll and gametes.  L3-rest of the plant body  When the changes occur in entire inner or outer layer, it is also known as Periclinal Chimera and when only a part of inner or outer layer is altered, it is called Sectorial Chimera. 
  29. 29. Chimera ? : Chimera - a plant or plant part composed of genetically different layers The most common example is a "variegated" plant where different regions of the leaf are yellow or white due to the lack of chlorophyll synthesis, i.e. these are chlorophyll mutants. However, there are many kinds of chimeras. Thornless blackberries are chimeras where the L-I epidermis lacks the ability to produce thorns. Some fruits have sweet and sour regions of flesh, which may be a chimera. 
  30. 30. Chimera ? : Mericlinal Chimera A mutation occurs in one layer and along the side of the apex. Due to its position, the cell division products of those mutated cells occur as a layer on only one side of the plant. In other words, only a section of one of the layers is mutated. Mericlinal chimeras are not stable.
  31. 31. Chimera ? : Periclinal Chimera A mutation occurs in one (or more) layer at the top of the apex. Due to its position, the cell division products of the mutated cells spread and cover the entire layer of the apex. In other words, the entire layer is mutated. Periclinal chimeras are stable to very stable, and comprise the most common type chimeras in horticulture.
  32. 32. Chimera ? : Sectorial Chimera A mutation occurs in multiple layers at the top of the apex. In other words, an entire section of the plant is mutated. Sectorial chimeras are stable to very stable, and comprise the most common type chimeras in horticulture. Chimera ?
  33. 33. Sectorial chimera
  34. 34. Traditional Mutation Breeding Procedures  Treat seed with mutagen (irradiation or chemical)  Target: 50% kill  Grow-out M1 plants (some call this M0) – Evaluation for dominant mutations possible, but most are recessive  Grow-out M2 plants – Evaluate for recessive mutations – Expect segregation  Progeny test selected, putative mutants – Prove mutation is stable, heritable
  35. 35. Mutation breeding scheme for seed propagated crop  Mutagenic application  Growing the plants (M1 generation)  Identification of induced mutation, seed harvest from mutated plants (M2)  Continue the identification and selection of induced mutation (M3)  First agronomic evaluation. Propagation of promising lines (M4)  Multilocation trials of stable mutant and recombinant lines (M5 – M8)  Official testing and releasing of mutant (M9)
  36. 36. Mutation breeding for oligogenic traits
  37. 37. Mutation breeding for oligogenic traits
  38. 38. Mutation breeding scheme for vegetative propagated crop  Mutagenic application  Cutting back the M1V1 shoot, bud grafting, or in vitro propagation via axillary buds  Isolation of induced somatic mutation, establishment of clones, cutting back of non-mutant shoots from chimeric plants (M1V2)  Further isolation of somatic mutations, vegetative propagation of mutant plant (in vivo or in vitro), preliminary evaluation of mutants (M1V3)  Evaluation of mutant clone performance, assesing segregation from mutant crosses and reselection of desired recombinants. Released of improved mutant (M2V4)
  39. 39. Application of mutation breeding :   Induction of desirable mutant alleles which may not be present in the germplasm available to the breeder.  In improving specific characteristics of well adapted high yielding variety .  Mutagenesis has been successfully used to improve various quantitative characters including yield.
  40. 40. Limitations :  Limitations The frequency of desirable mutants is very low.  Desirable mutations are commonly associated with undesirable side effects.  There may be problems in the registrations of a mutant variety.  Mutations in quantitative traits are usually in the direction away from the selection history of the parent variety.  Most of the mutations are recessive