Mutation Breeding

1. Mutation Breeding Taryono Faculty of Agriculture Gadjah Mada University

2. Mutation - Mutant • Mutation Changes in genes and chromosomes • Mutated Altered genes • Mutant New organism with a mutated gene or rearranged chromosomes

3. Mutation Breeding • Advantages • Screen very high populations (cell based) • Can apply selection to single cells • Disadvantages • Many mutations are non-heritable • Requires dominant mutation (or double recessive mutation); most mutations are recessive • Can avoid this constraint by not applying selection pressure in culture, but you loose the advantage of high through-put screening – have to grow out all regenerated plants, produce seed, and evaluate the M2 • Alternative: perform on haploid cell lines

4. Mutation • May involve any trait • All kind of transition are encountered, from drastic morphological changes deviations in physiology so minute as to be almost indiscernible • Harmful or even lethal

5. Type of mutation • Spontaneous (natural) mutation • Some have played an outstanding role in development of valuable crop cultivars and hybrids • Unfortunately, it can not form the basis of modern plant breeding due to its low frequency and difficulties in detection • Induced mutation

6. Genetic structure changes • Gene (point mutation) • Chromosome • Genome Gene (point) mutation → a change in specific sequence of nucleotides in DNA molecules leading to the formation of a new type of protein or preventing that of the normally protein → take place at the molecular or sub-microscopic level → Such change may be accompanied by the emergence of a new trait inherited in accordance with Mendel’s Laws

7. 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)

8. Genome mutation • Changes in sets of chromosomes • Remarks: • Breeders are more interested in gene mutation, because chromosomal rearrangement usually produce negative results, such as lower fertility of the offspring • Mutant are aften of great value for breeding as sources of new, previously unknown useful characters • Mutagenesis may be instrumental in obviating the technical difficulties arising in the crossing of such a small flowered crops such as milled

9. Story of induced mutation • X rays can significantly promote mutation in fungi (1925) • X rays produced pronounced mutagenic effect on the fruit fly Drosophila (1927) • Artificial mutants can serve as good source material in plant breeding; X rays induce mutations in Maize and barley (1928) • Today there are three groups of breeders: • Mutation breeding is useless, we can accomplish the same thing with conventional methods • Mutation breeding will produce a breakthrough given enough effort • Mutation breeding is a tool, useful to meet specific objectives

10. Technique for inducing mutation • Physical mutagens • Chemical mutagens Physical mutagens • Various sources of ionizing radiations are explored, most often X and gamma rays, UV radiation, fast and slow neutron, alpha ray, beta ray • Radioactive isotopes P-32 and S-35 are not convenient for use due to the storage and application difficulties • The usual sources of gamma rays in laboratories are radioactive cobalt (Co-60) and Cesium (Cs-137) placed in cobalt bomb

11. Physical mutagens 4. 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)

12. Chemical mutagens • Mutagenic substances belonging to different classes of chemical compounds, such as ethylene imine, diethyl sulfate, dimethyl sulfate, N-nitrosoethyl urea, N-nitrosomethyl urea, methal 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

13. Chemical mutagens • 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

14. Use of mutations in sexually reproduced crops • More valuable in self than cross pollinated. The probability of producing desirable mutations and genetic variability is theoretically higher • Seeds • Very young seedling

15. 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

16. 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

17. 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)

18. 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)

19. Requirements for Mutation Breeding • Effective screening procedure • Most mutations are deleterious • With fruit fly, the ratio is ~800:1 deleterious to beneficial • Most mutations are recessive • Must screen M2 or later generations • Consider using heterozygous plants? • But some say you should use homozygous plants to be sure effect is mutation and not natural variation • Haploid plants seem a reasonable alternative if possible • Very large populations are required to identify desired mutation: • Can you afford to identify marginal traits with replicates & statistics? Estimate: ~10,000 plants for single gene mutant • Clear Objective • Can’t expect to just plant things out and see what happens; relates to having an effective screen • This may be why so many early experiments failed

20. Mutation detection • Detection, isolation and testing 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 recession of the mutation) and crops must be taken into account

21. Trend in plant breeding based on mutation • Mutagens are used to induce mutations within a broad range and at a high frequency to obtain ample source of material for selection • Mutant with a specific changes in certain characters are created in order to correct some defects in crop varieties. It is important that the other economic characters remain unaltered • Mutagens can be used to solve special problems in plant breeding for instance by increasing the number of genetic recombinations and breaks of undesirable linkages, transferring chromosomal fragment from one plant species into chromosomes of another during hybridization, obtaining homozygous mutant through irradiation of haploids with subsequent doubling of chromosome number

22. Mutation useful for crop improvement • Useful mutation Any mutational change in a character which can be put to practical use • Improve nutrition value of crop product • Short stem • High lodging resistance • Disease resistance

23. Successes of Mutation Breeding Herbicide Resistance and Tolerance • Resistance: able to break-down or metabolize the herbicide – introduce a new enzyme to metabolize the herbicide • Tolerance: able to grow in the presence of the herbicide – either ↑ the target enzyme or altered form of enzyme • Most successful application of somaclonal breeding have been herbicide tolerance • Glyphosate resistant tomato, tobacco, soybean (GOX enzyme) • Glyphosate tolerant petunia, carrot, tobacco and tomato (elevated EPSP (enolpyruvyl shikimate phosphate synthase)) • But not as effective as altered EPSP enzyme (bacterial sources) • Imazaquin (Sceptor) tolerant maize • Theoretically possible for any enzyme-targeted herbicide – it’s relatively easy to change a single enzyme by changing a single gene

24. D Backcrossing x x M3 plant mut-1/mut-1 M3 plant mut-1/mut-1 Wild type +/+ M3 plant mut-2/mut-2 Legend Case 1: Case 2: x Mutant phenotype BC1 plant mut-1/+ Wild type +/+ mut-1/+ mut-2/+ No allelism Two genes mut-1/mut-2 Allelism Single gene E Careful phenotypic study Multiple backcrosses to remove background mutations Wild-type phenotype C Allelism Tests BC2 plant mut-1/+ “How many genes are involved?” F Mapping Outcross Screen M2 pools (1, 2, etc.) for mutant phenotypes +/+ mut-1/mut-1 x “Where is the gene located?” M2 seedlings Wild type +/+ Strain B mut-1/mut-1 Strain A Mutant mut-1 G Gene Cloning B Screening mut-1/mut-1 Propagate mutant from mut1/mut-1 or from its mut-1/+ heterozygous siblings Option 1: Backcross mut-1/+ Strain A/Strain B “How does the gene function?” x Re-screening mut-1 Strain A M3 seedlings x Establish segregation ratio Option 2: Selfing - Recessive or dominant? - Monogenic or polygenic? - Penetrance? Examine co-segregation of mutant phenotype versus strain-specific (visible or molecular) traits Initiate mutant characterization Mapping population Mapping population Mutagenesis - Overview Wild type Mutagen A Mutagenesis (chemical, radiation, T-DNA,…) M1 plants Harvested In pools Pool 1 Pool 2 Pool 3, etc. Strain A/A Pools of M2 seeds “What exactly is wrong?” Strain A/B Strain B “Recessive phenotypes appear here”

25. A Mutagenesis Wild type Mutagen Legend (chemical, radiation, T-DNA,…) M1 plants Harvested In pools Mutant phenotype Pool 1 Pool 2 Pool 3, etc. Pools of M2 seeds Wild-type phenotype

26. B Screening Screen M2 pools (1, 2, etc.) for mutant phenotypes Legend M2 seedlings Mutant mut-1 Propagate mutant from mut1/mut-1 or from its mut-1/+ heterozygous siblings Mutant phenotype Re-screening M3 seedlings Establish segregation ratio - Recessive or dominant? - Monogenic or polygenic? - Penetrance? Wild-type phenotype Initiate mutant characterization

27. x C Allelism Tests “How many genes are involved?” M3 plant mut-2/mut-2 M3 plant mut-1/mut-1 Legend Case 1: Case 2: Mutant phenotype mut-1/+ mut-2/+ No allelism Two genes mut-1/mut-2 Allelism Single gene Wild-type phenotype

28. D Backcrossing x M3 plant mut-1/mut-1 x/x Wild type +/+ X/X x BC1 plant mut-1/+ x/X Wild type +/+ X/X Multiple backcrosses to remove background mutations BC2 plant mut-1/+ X/X BC2 plant mut-1/mut-1 E Careful phenotypic study

29. F Mapping Outcross “Where is the gene located?” +/+ mut-1/mut-1 x Wild type +/+ Strain B mut-1/mut-1 Strain A G Gene Cloning mut-1/mut-1 Option 1: Backcross mut-1/+ Strain A/Strain B “How does the gene function?” x mut-1 Strain A x Option 2: Selfing Strain A/A Strain A/B Examine co-segregation of mutant phenotype versus strain-specific (visible or molecular) traits Strain B Mutant Heterozygote Mapping population Mapping population Wild type