Mutation Breeding

By- SHRIKANT YANKANCHI

2

Introduction

Micro- and Macro-mutations

Procedures for micro mutations

breeding/polygenic

Handling of segregating population

Screening/selection

Breeding for A Biotic and Biotic stress

Methods for Validation of mutants

Achievements of mutation breeding

Advantages and limitations of breeding

Contents......

3

IntroductionThe term mutation breeding (‘Mutationszüchtung’) was first coined by Freisleben and Lein (1944) He referred mutation breeding as the deliberate induction and development of mutant lines for crop improvementMilestones in mutation breeding300BC- The ancient Chinese book “Lulan” provides the first documentation of mutant selection in plant breeding: maturity and other trait in cereals in China (Huang and Liang, 1980)1590 - The first verifiable (spontaneous) plant mutant described, ‘incisa’ mutant of greater celandine

4

1667 -The first known description of a graft-chimera; Bizarria-orange, Florence, Italy1901-1904 - de Vries suggests and promotes radiation to induce mutations in plants and animals1907-Cramer publishes extensive examples of spontaneous mutants in crop plants1927 - First proof of induced mutations in plants; radium ray treatment of Datura stramonium (Gager and Blakeslee)1927 - Muller working with Drosphila provides proof of mutation induction by X-rays Muller champions induced mutation for animal and plant breeding and opens a new era in genetics and breeding1928 Stadler publishes the first results of mutation induction in crop plants, barley, maize, wheat and oat, but is sceptical about the use of induced mutation for crop improvement

5

1936 The first induced mutant variety is released, tobacco var. ‘Chlorina’ using X-rays in Indonesia (then the Dutch East Indes)1942 First report of induced disease resistance in a crop plant; X-ray induced mildew resistance in barley (Freisleben and Lein, 1942)1944/46 First reports of chemical induced mutation (Auerbach and Robson, 1944)1949 First plant mutation experiments using 60Co gamma ray installations1954 The first release of a mutant variety in a vegetatively propagated crop: tulip var. Faraday with an improved flower colour and pattern (see van Harten and Broertjes, 1989)2000-2009 Development of high-throughput genotyping and phenotyping using automated, robotic and computerised systems2000 onwards Development of TILLING populations

6

Mutations called micro- and macro-mutations

depending on the magnitude of phenotypic effect

produced by them

Macromutation

produces a large phenotypic effect

easily recognizable on individual plant basis

Oligogenic in nature

Can be easily selected in M2 generation

Micro- and Macro-mutations

Micromutations

Produces Small phenotypic effect

Cannot be recognizable on individual plant basis

Detected only in group of plants and need treatment of

statistical data

Polygenic nature and selection delays till M3

Procedures for micro mutations

breeding/polygenic

9

M1

M2

M3

M4

M5

M6-M8

M9

i. Mutagen -treated seeds space plantedii. Seeds from individual plants harvested separately

i. Individual plant progenies are grown ii. Fertile, vigorous, normal looking plants harvested separately

i. Individual plant progenies from selected plants grown ii. Superior plants selected from superior progenies showing segregationiii. Homogeneous mutant progenies may be harvested in bulki. Individual plant progenies from selected plants grown ii. Superior homogeneous lines harvested in bulk

i. Preliminary yield trial with a suitable check ii. Superior lines selected

i. Multi-location yield trialii. Outstanding line released as new variety

i. Seed multiplication for distribution

10

1.Selection of the variety for Mutagen treatment

It should be the

best variety available in

crop

Seed shold be pure

Oladosu et al, 2016

2. Part of the plant to be treated

Seeds

Pollen grains

Vegetative propagules

Corns

bulbs

complete plants

12

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

13

Optimum mutagen dose is one, which produces maximum

frequency of mutations and causes the minimum killing

Close to LD50 dose is optimum

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

14

Optimum dose rate of physical and chemical mutagens for seed treatment of cereals

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 reducedR. Roychowdhury and J. Tah., 2013

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

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

Screening/selection

Mainly three types screening/selection techniques in

M2 and subsequent generation

i) Visual

ii) Mechanical/Physical

iii) Other methods

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.

ii) Mechanical/Physical-Very efficient for seed size, shape, weight, density,

etc., using appropriate sieving machinery

Iii) Other methodschemical, 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

21

Methods for Validation of mutants

Genome-wide chips

Difference screening

Microarray

PCR screening

TILLING and ECO-TILLING

Mutation breeding for Biotic and Abiotic stress

Methods for generating mutant varieties

mutagenesis

Forward genetics-chemicals-radiation

Reverse geneticsInsertional mutagenesis - Agrobacterium mediated

transformation- Virus induced gene silencing- RNA mediated interference- transposon tagging

TILLINGNext generation sequencing

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 virusChen et al, 2014

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 acidsThrough 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

Achievements of mutation breeding

Distribution of mutant crop varieties by continents

IAEA mutant database, http://mvgs.iaea.org (2015)

28

Number of mutant varieties released in the world

https://mvd.iaea.org/#!Search?page=1&size=15&sortby=Name&sort=ASC&Criteria[0][field]=Country&Criteria[0][val]=136

Officially released mutant varieties in the FAO/IAEA Mutant Varieties Database, July

2015

INDIA Mutant cultivars

popular mutant cultivars of chickpea developed in India

Variety Release year Main improved attributes

Pusa-408 (Ajay) 1985, Resistant to Ascochyta blight, high yield, profuse branching, semi erect, maturity 140-155 d

Pusa-413 (Atul) 1985, Resistant to Fusarium wilt, stunt virus & foot rot, high yield,profuse branching, semi erect, maturity 130-140 days

Pusa-417 (Girnar) 1985, 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)

Leading rice varieties obtained by mutation breeding

Mutant rice varieties released in India for cultivation

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

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

AdvantagesPossible to achieve instant progress in elite materialSingle trait improvements can be made to an established variety preferred by producers, processors and/or consumersLimited breeding effort requiredNovel variation can be producedSingle gene mutants with no negative pleiotropic effects are possibleFor 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 handleSpecific genes/traits can be targetedPossible to calculate chances of success (mutation frequency)

Limitations...

The process is generally random and unpredictable Useful mutants 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

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

References....Text books

Plant breeding – B. D. Singh Plant Mutation Breeding and Biotechnology

- Edited by Q.Y. Shu, B. P. Forster, H. Nakagawa

Review papers Principle and application of plant

mutagenesis in crop improvement: a review – By Oladosu et al., 2015 Mutagenesis - By Rajnikant Mishra (2012)

Thank

you