21. Mutation Breeding in crop improvement

Dr. NAVEENKUMAR K.L
Assistant Professor
Dept. of GPB

1. Mutation Breeding in crop improvement Dr. NAVEENKUMAR K.L Assistant Professor Dept. of GPB

2. I. Application in crop improvement 2 1. General Methods 2 . Special Methods 3. Population Improvement Basis of classification methods Plant introduction, Pure line selection, Mass selection, progeny selection, pedigree method, bulk method, back cross method, SSD, clonal selection, heterosis breeding, synthetics and composites. Mutation breeding, Polyploidy breeding, transgenic breeding, molecular breeding Recurrent selection, disruptive selection, diallel selective approaches mating system, biparental mating.

3. ‘Mutation ' refers to all the heritable changes in the genome, excluding those resulting from incorporation of genetic material from other organisms. Sudden heritable change in genetic material or character of an organism is known as mutation A mutation is an abrupt qualitative or quantitative change in the genetic material of an organism. Mutations may be intragenic or intergenic . Intergenic mutations, of which chromosomal changes in structure are examples, involve long regions of DNA (deletion or addition). The unit of gene mutation is the muton . Mutation

4. History 1791 -English farmer Seth Wright recorded mutation first time in male lamb with unusual short legs in Ancon breed of sheep. 1900- the term mutation is coined by Hugo de Vries in by his observation in Oenothera . 1910-Systematic study of mutation was started in when T.H Morgan genetically analyzed white eye mutant of Drosophila. 1927- H. J. Muller induced mutation in Drosophila by using X- rays. he was awarded with Nobel prize in 1946 for Physiology and Medicine . 1929- Stadler described the mutagenic effects of X- rays in Barley. (First plant breeding Programme designed to exploit induced mutation breeding, Sweden, Germany &USSR). 1946- Auerbach & Robson, discovered mutagenic effects of mustard gas and other chemical compounds.

5. Characteristics of Mutation The mutant alleles are recessive to their wild type or normal alleles. But some mutations are dominant, Ex: Epiloia and achondroplastic dwarfness in man, Notch wing in Drosophila etc. Mutations are random events in terms of the time of their occurrence and the gene in which they occur. Mutations occur at very low frequencies in nature without any known cause such mutations are called Spontaneous mutations. The rate of spontaneous mutations for most of the genes is very low ranging between 10 -7 and 10 -4 . The rate of spontaneous mutations varies considerably from one gene to another . Some genes e.g. waxy locus of maize, are highly stable, while some others, yellow body locus of Drosophila, show a relatively high mutation rate 6. Some genes show exceptionally high rates of spontaneous mutations they are known as mutable genes . 7. Most of the mutations have harmful effects, but some mutations are beneficial. 8. Mutations are recurrent.

6. Cont …. Some genes increase the spontaneous mutation rates of some other genes of the genome; such genes are called mutator genes . Some still other genes suppress the mutation of some other genes of the genome they are termed as antimutator genes . Mutations producing the same phenotypic effect may be located at different sites within the same gene. Some sites within genes show very high rates of mutation, while some other sites show little or no mutation. The highly mutable sites within a gene are commonly known as hot spots . Many agents, both physical and chemical, increase the frequency of mutations by several folds they are known as mutagens . The rate of induced mutations varies considerably from one gene to the other. Mutations can occur in any tissue/ cell of an organism. Mutational events can occur during any developmental stage of organisms. Mutations occur in both forward and reverse directions. The rate of forward mutations are higher than those for reverse mutations.

7. How Does a Mutation Act? The genes act by controlling the rate of production of specific proteins (enzymes). The scheme of protein synthesis in most organisms

8. Forward mutation When mutation occurs from the normal/wild type allele to mutant allele are known as forward mutation A + a forward recessive a + A forward Dominance 2 . Reverse mutation When mutation occurs in reverse direction that is from mutant allele to the normal/wild type allele are known as reverse mutation Classification of mutation I. Based on direction of mutation

9. II. Based on causes of mutation 1 . Spontaneous mutation Spontaneous mutation occurs naturally without any cause. The rate of spontaneous mutation is very slow. Eg - Methylation followed by deamination of cytosine. Rate of spontaneous mutation is very low. Eukaryotes (10 -4 - 10 -6 /gene/ generation) Prokaryotes ( 10 -5 - 10 -7 /gene/ generation) . The possible sources of origin of spontaneous mutations are Errors during DNA replication, Mutagenic effects of the natural environment of organisms Transposons and insertion sequences, and Methylation , followed by spontaneous deamination of DNA bases, especially cytosine.

10. 2. Induced Mutation Mutations produced due to treatment with either a chemical or physical agent are called induced mutation. The agents capable of inducing such mutations are known as mutagen. The process of inducing mutation through treatment with a mutagen is known as mutagenesis. The exploitation of induced mutation for crop improvement program is known as mutation breeding . Eg . X- rays causing mutation in cereals Induced mutations are useful in two different ways In genetic and biochemical studies In crop improvement

11. III. Based on dominance Relationship Dominance mutation Recessive mutation Co-dominance mutation Incomplete dominance mutation

12. Lethal mutation: when mutation causes death of all individuals undergoing mutation are known as lethal Sub lethal mutation: causes death of 90% individuals Sub vital mutation: such mutation kills less than 90% individuals Vital mutation: when mutation don’t affect the survival of an individual are known as vital Supervital mutation: This kind of mutation enhances the survival of individual IV. Based on the survival of an individual

13. V . Based on tissue of origin Somatic mutation A mutation occurring in somatic cell is called somatic mutation. In asexually reproducing species somatic mutations transmits from one progeny to the next progeny 2 . Germinal Mutation: When mutation occur in gametic cells or reproductive cells are known as germinal mutation . In sexually reproductive species only germinal mutation are transmitted to the next generation

14. VI. Type of trait affected Visible mutation : Those mutation which affects on phenotypic character and can be detected by normal observation are known as visible mutation . 2. Biochemical mutation : mutation which affect the production of biochemical and which does not show any phenotypic character are known as biochemical mutation

15. VII. Quantum of Morphological Effect Produced Macro mutations: produces large enough changes in the phenotype that can be detected without any confusion ex: short legged mutation in sheep. 2. Micro mutations: mutations in quantitative characters generally produce small morphological effects, which are not easily detected.

16. VIII . Cytological basis Chromosomal : number or structure Nuclear Gene: base sequence of nuclear gene Cytoplasmic : plastids and mitochondria

17. IX. Chromosomal / Molecular Basis Deletion Duplication Inversion Translocation Nondisjunction Point mutation: mutation in genes due to a changes in single base pair.

18. X . Types of Amino Acid Replacement Missense : cause the replacement of a single amino acid of the respective polypeptides by another amino acid. They are produces base substitutions. 2. Nonsense : produces nonsense codon. Such codon does not code for any amino acid Terminate the growth of polypeptide chains. 3. Frame shift: mutation produced by deletion or addition of bases that are not in multiple of three, as result reading frame changes downstream of the site of addition or deletion. Tautomeric shifts : hydrogen atoms move from one position in a purine or pyrimidine to another position.

19. Induced Mutation Mutagens: The agents capable of inducing such mutations are known as mutagen. Mutagen is a natural or human-made agent (physical or chemical) which can alter the structure or sequence of DNA. The different mutagenic agents may be classified into the following two broad groups: Physical mutagens: the different types of radiations having mutagenic properties. Chemical mutagens:

20. Mutagens I. Physical mutagens II. Chemical mutagens Present in solar radiation or mercury- vapour lamps or tubes Wavelength ranges 10-390 nm The maximum absorption of UV rays by DNA 254nm Direct effect: pyrimidine dimmers & pyrimidine hydrates Ionizing Radiations 1A . Particulate Radiations : Beta ( 3 H, 32 P, 35 S ), Alpha rays, fast neutrons & thermal neutrons. 1B . Non Particulate Radiations: X rays: X-ray tubes Gamma rays: 14 C, 60 Co), 2. Non- Ionizing Radiations: UV rays

21. Physical mutagens contd.., Various sources of ionizing radiations are explored, most often X and gamma rays, UV radiation, fast and slow neutron, alpha ray, beta ray. 2. Radioactive isotopes P-32 and S-35 are not convenient for use due to the storage and difficulties in application . 3. The usual sources of gamma rays in laboratories are radioactive cobalt (Co-60) and Cesium (Cs-137) placed in cobalt bomb. may cause DNA breakage and other damages .

22. Physical mutagens contd.. The object can be irradiated in two ways : With an aid of a powerful source of a short-duration gamma rays for short duration radiation. Need special units for irradiating living object. A much weaker radiation but operating continuously (gamma field). The dosage must be varied depending not only on the plant species whose seeds/organs are irradiated, but also on many other factors. Plant must be irradiated heavily enough to ensure as many inherited changes as possible but without seriously affecting the germination, growth and fertility of plant directly emerging from the irradiated seeds or vegetative organs ( critical radiation dose : dosage which strong enough to assure many mutation not yet so strong as to kill plants )

23. Mutagens

24. Chemical mutagens contd., Mutagenic substances belonging to different classes of chemical compounds, such as ethylene imine , diethyl sulfate, dimethyl sulfate, N- nitrosoethyl urea, N- nitrosomethyl urea, metal sulfonate , diepoxy butane, ethyleneoxide . Most are highly toxic , usually result in point mutations. Use in solution in the concentration ranging from tenth – hundredths even thousandths of percent. Many chemical mutagens are much more effective than physical one. If irradiation of crops produces 10 – 15% of viable inherited changes, chemical mutants do the same at a rate of 30 to 60 %. They often exert more specific and finely tuned action on the cell.

25. Some substances ( supermutagen ) are capable of causing inherited changes in plants at a rate up 100 %. Chemical mutagens aim at the most vulnerable spot of a living organism (DNA) to induce changes in nucleotides and alter the genetic information (Sometimes causes specific mutation ) It provides a powerful tool to induce desire changes in a trait

26. 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.

27. Mutation Breeding Definition: 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. Ex: Wheat ,Sugarcane etc

28. Mutation breeding is successful when…… :  Desired variability exhausts in cultivated species and germplasm. When a desirable variety has an oligogenic genetic defect. There is tight linkage between desirable and undesirable traits. Only one or two characters are to be improved Ex : 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

29. 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)

30. Part of the plant to be treated Part of the plant to be treated are 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 .

31. Selection of the variety or plant parts for mutagen treatment in sexually propagated crops 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.)

32. More valuable in self than cross pollinated. The probability of producing desirable mutations and genetic variability is theoretically higher Seeds Very young seedling Pollen grains

33. Use of mutations in asexually produced crops It has been much easier and quicker to obtain variant plant types Specific location of the mutation event (segmental chimera) becomes important. The mutant must be in meristematic tissue that will produce faithfully through cutting or other vegetative means Bud Scion Cutting Tuber bulbs

34. Mutation detection Detection, isolation and testing of mutants are extremely difficult. Due to the sporadic nature of viable useful mutations, it is advisable to have larger plant population. When mutagens are used in breeding, the biological nature of the trait (dominance or recessive of the mutation ) and crops must be taken into account.

35. Detection of mutations in plants : Number of plants having the recessive form of the traits governed by the gene Frequency of mutant gene (%) = --------------------------------------------------------------X 100 total number of plants in the progeny

36. Procedure for Vegetative propagated crops In apical buds, auxiliary buds and adventitious buds, there are two functional layers, outer layer and inner layer. 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. Procedure for Vegetative propagated crops

37. Chimera? Vegetative mutations are expressed in the form of chimeras. Periclinial chimera (whole of inner and outer layer) Sectorial chimera(part of inner/outer layer) Seed propagated crop-inner layer important…seed formation Chimera – a plant or plant part composed of genetically different layers

38. M1 M2 M3 M4 M5 M6 - M8 M9 Mutagen -treated seeds space planted Seeds from individual plants harvested separately Individual plant progenies are grown Fertile, vigorous, normal looking plants harvested separately Individual plant progenies from selected plants grown Superior plants selected from superior progenies showing segregation Homogeneous mutant progenies may be harvested in bulk Individual plant progenies from selected plants grown Superior homogeneous lines harvested in bulk Preliminary yield trial with a suitable check Superior lines selected Multi-location yield trial Outstanding line released as new variety i. Seed multiplication for distribution 9

39. 1.Selection of the variety for Mutagen treatment It should be the best variety available in crop Seed sho u ld be pure Oladosu et al , 2016 10

40. 2 . Part of t he plant to be t r e a t e d Seeds Pollen grains Vegetative propagules Corns bulbs complete plants

41. Mutagen Treatments reduces germination growth rate vigour & fertility Mutagens generally induce a high frequency of chromosomal changes and meiotic and mitotic irregularities 3. Dose of mutagen 12

42. Optimum mutagen dose is one, which produces maximum frequency of mutations and causes the minimum killing Close to LD 50 dose is optimum LD50: the dose of a mutagen , which will kill 50 per cent of the treated individual. Varies with crops eg:- 46 krad for Vicia faba , 120-140 krad for Brassica napus varies with mutagens eg: EMS – 0.3-1.5 %, for 2-6 hours 13

43. Handling of segregating population M1 generation Seeds treated with chemical mutagens should be washed thoroughly and be planted as soon as possible Large M1 generation is raised from treated seeds (Wider spacing) E.g :- 25,000 plants are to be grown to obtain a useful mutation in M1 generation Mutagens with high mutation frequency - M1 generation size can be reduced R. Roychowdhury and J. Tah., 2013

44. Continued...... The M1 plants should not be allowed to cross pollinate M1 population should be planted 75-100 m apart from the parental or other genotypes of the same crop species Mechanical isolation M1 generation Dominant mutations are selected each plant selfed and harvested separately for M2

45. M2 generation Two methods of sowing M2 generation can be followed M1 plant to row where all seeds produced from a single plant are grown in row M1 spike or branch to row , Oligogenic mutants with distinct features are identified and selected

46. Screening/selection Mainly three types screening/selection techniques in M2 and subsequent generation Visual Mechanical/Physical Other methods

47. i) Visual screening -most effective and efficient method for identifying mutant phenotypes -Visual selection often is the prime basis for selecting for disease resistance , earliness, plant height, colour changes, ion-shattering, adaptation to soil, climate, growing period etc.

48. ii) Mechanical/Physical -Very efficient for seed size, shape, weight, density, etc., using appropriate sieving machinery Iii) Other methods chemical, biochemical, physiological etc. E.g.- Low alkaloid content mutants can be selected using colorimetric tests -chromatographic or electrophoresis techniques may be used to select isolate protein variants

49. Mutation breeding for Biotic and Abiotic stress

50. Breeding for disease resistance Numerous mutants have been developed through mutation induction, showing enhanced resistance to various diseases (virus, bacterial, and to some extent fungi ) E.g.: locus (ml-o) - located on the short arm of chromosome 4H in barley Induced mutations at the locus confers resistance to powdery mildew and barley yellow mosaic virus

51. Quality, nutrition and functionality starch, protein, fatty acid, vitamins, etc . Elimination of undesired substances such as anti-nutritional factors Raising or lowering the concentration of specific substances such as fatty acids Through mutation is by inducing knock-outs in genes involved in the metabolic pathways Eg : high quality edible oil of canola was achieved by lowering the levels of glucosinolates and the erucic acid by gene knock outs induced by gamma ray irradiation

52. Application in crop improvement Development of improved varieties Induction of male sterility Production of haploids Creation of genetic variability Overcoming self Incompatibility Improvement in Adaptation

53. Distribution of mutant crop varieties by continents IAEA mutant database, http://mvgs.iaea.org (2015)

54. Number of mutant varieties released in the world https://mvd.iaea.org/#!Search?page=1&size=15&sortby=Name&sort=ASC&Criteria[0][f ield]=Country&Criteria[0][val]=136 28

55. P opular mutant cultivars of chickpea developed in India Variety Release year Main improved attributes P u sa - 4 8 ( Aj a y ) 1 9 8 5 , Resistant to Ascochyta blight, high yield, profuse branching, semi erect, maturity 140-155 d Pusa-413 ( Atul ) 1 9 8 5 , Resistant to Fusarium wilt, stunt virus & foot rot, high yield, profuse branching, semi erect, maturity 130-140 days Pusa-417 ( Girnar ) 1 9 8 5, High resistance to Fusarium wilt & moderate resistance to Ascochyta blight, stunt virus, high yield, profuse branching, maturity 110-130 days Kharkwal M. C . et al (1985)

56. Leading rice varieties obtained by mutation breeding

57. Mutant rice varieties released in India for cultivation

58. Gamma radiation-induced rice mutants were released in India as high-yielding varieties under the series ‘PNR’. Two early ripening and aromatic mutation- derived rice varieties ‘PNR381’ and ‘PNR102 ’ currently popular in Haryana and UP Mutant Varieties database https://mvd.iaea.org/#!Home

59. The primary research centres and institutes in India that participated in the development and release of various mutants Indian Agricultural Research Institute (IARI)- New delhi Bhabha Atomic Research Centre- Mumbai Tamil Nadu Agricultural University –TN and National Botanical Research Institute – Lucknow, UP

60. Advantages Possible to achieve instant progress in elite material Single trait improvements can be made to an established variety preferred by producers, processors and/or consumers Limited breeding effort required Novel variation can be produced Single gene mutants with no negative pleiotropic effects are possible For some mutagenic treatments such as gamma and X-ray, there is neither residual radiation nor chemical contamination of the treated material. The treated material is safe to handle Specific genes/traits can be targeted Possible to calculate chances of success (mutation frequency)

61. Limitations... The process is generally random and unpredictable Useful mutants (very low frequency at 0.1 %) are rare and predominantly recessive Large population sizes and effective mass screening methods are required to select rare mutants Mutants can have strong negative pleiotropic effects on other traits Most of the mutations are deleterious and undesirable.

62. Continued ..... Health risks: handling, chemical mutagens ; radiations, fast neutrons treatments Most mutants are of no use to breeding even if a large number of mutants can be produced Field trialling and germplasm storage can be expensive and require a lot of space and careful management if large mutant populations are handled