Training managment training 14-18 feb,2021

1. Dr. Md. Abdul Malek
CSO
Plant Breeding Division
BINA
Advancement of Mutant Population

2. SELECTING PARENTS AND HANDLING M1TO
M3 GENERATIONS FOR MUTANT SELECTION
A successful mutation breeding programme starts with well-
defined objectives for improvement of a defined plant
phenotype/genotype. Common targets are:
1. To improve one or a few specific traits of a preferred variety or an
elite line;
2. To induce a morphological marker (colour, awns, bracts, hairiness,
etc.) in order to establish distinctness in a promising line to make it
easy to identify and meet the requirement for variety registration;
and/or
3. To induce male sterility or fertility restoration making a line useful
as a component for hybrid variety production.

3. LIFDCs
a. Isolation of mutants
Parent cultivar
seeds
(multicellular embryo)
Mutagenic treatment
Radiation
Chemical mutagenesis
Insertional mutagenesis
Mo
M1
(embryo) (M1 plant,
chimera)
(meiosis)
M2 seeds
(embryo)
Segregating M2 population
(Selection on single plant basis)
M3

4. LIFDCs
b. Handling of mutants
Segregating M2 population
M3 - Homozygosity test of
putative mutants selected in M2
M4 - Homozygosity test of selected
mutants, preliminary evaluation,
seed multiplication
Selection M5 - Mn
Multilocation trials
Direct release
Cross-breeding
Segregating M3
population
(Selection of mutants among lines
derived from individual M2 plants)
M4 - Homozygosity test of selected
mutants, preliminary evaluation,
seed multiplication
Cross-breeding

5. Planning for the M1 generation
A control (untreated) population should always be grown to
serve three purposes:
 Provide a comparison of the treatment effects on germination,
growth, survival, M1 injury and sterility;
 Assess the phenotypic variability of the parent genotype stock
used to produce M1 and;
 Provide a 're-purified' parent genotype as a back-up for initiating
a new M1 generation to be grown during the same season with the
M2 generated from the first M1 if needed.

6. Sowing of M1 seeds
Considering the detrimental effects of mutagens on seed
viability, the M1 must be handled with more care than untreated
controls. The M1 should therefore be grown in benign
conditions:
 Greenhouse conditions;
 Field conditions;
 Time of sowing M1;
 Condition of the treated M1 seeds;
 Density of sowing; and
 Weed control.

7. Isolation of M1 material
Generally, it can be assumed that some level of genetic
heterogeneity is always present even in populations of parent
material from self-pollinating plants. Therefore, several
disrupting hazards may be expected, especially in field
plantings, which may affect the certainty of the origin of the
variability observed in a mutation breeding programme as
presented below:
 Outcrossing;
 ‘Volunteer’ crop;
 Bird damage; and
 Soil borne toxicity, disease or in some cases, parasitic weeds etc.

8. Caring of M1 seeds
Care during cultivation and data recording:
 Emergence;
 Seedling survival;
 M1 chimera induction;
 Delayed development;
 Survival to maturity; and
 Sterility in M1

9. Figure: Example of reduced fertility (seed set) in M1 plants of
barley with increased gamma irradiation dose rate from 0 (control)
to 300 Gy. Courtesy of A. Mukhtar Ali Ghanim.
0 Gy 100 Gy 200 Gy 300 Gy

10. Harvest of M1 seeds
A consideration of the relation of these factors to the methods of
managing mutagen treated populations of different plant forms
is presented below:
 Tiller, branch or plant progeny methods;
 Single or multiple seeds bulk methods; and
 Mass bulk methods

11.  Initial parental germplasm: highly homogenous seeds
before treatment.
 1st mutant generation, treated seeds grown with narrow
spacing to maintain few tillers/branches.
 Ensure protection from out crossing.
 Harvest each plant separately or as appropriate bulking.
 2nd mutant generation, apply the screening method:
plant in head to row, and select individual mutant plant
separately (in case of qualitative traits).
 Control of untreated seeds should be planted
simultaneously to verify the identity of the mutant and
exclude contaminations.
 For cross-pollinated plant self M2
as appropriate to
produce M3.
 3rd mutant generation, seeds of selected mutants are
planted for further selection and verification.
 Selection for quantitative trait on row bases may start at
M3.
 Ensure homogeneity of the mutant seeds by appropriate
measures of isolation.
 Start selection in cross-pollinated plants in M3.
M0
M1
M2
M3
Figure: Scheme for mutant population development, identification, selection and
advancement of mutants from M0 to M3 generation

12. In practical mutation breeding, the nature of the trait sought,
the availability of space in the field, greenhouse or laboratory,
the labour needed, the possibility of mechanization, and other
resources will have an important bearing on the harvest
methods to be chosen and the precision and selection efficiency.
Selection of mutant traits is usually practiced for qualitative traits, in
self-pollinated crop plants, in the M2 generation as most of the
mutants are by then recessive, the mutant phenotype can thus, only
be seen in the M2 generation at the earliest. However, in cross-
pollinated plants mutant genes are likely to be heterozygous in M2
where further selfing should be practiced for producing M3
progenies in which homozygous individuals for the mutant genes
will segregate and selection can be applied. However, a useful
strategy on outcrossing species is to knock out the dominant allele at
heterozygous loci to unveil the recessive phenotype.
Management of M2 population

13. Fig.: Segregation of M2 tomato plants for chlorotic mutant
seedlings (yellow arrows) in tomato after seed (M0) treatment with
gamma irradiation at 300 Gy. Courtesy of A. Mukhtar Ali Ghanim.

14. Systems of handling M2 populations
All methods for the isolation of mutant genotypes in sexually
reproduced plants are based on the pedigree method, modified
to account for the chimeric structure of the M1 plants:
 M1 population bulk:
M1 population bulk to M2 single seed bulk to M3 ear to row
progenies; and
M1 population bulk to M2 ear to row progenies.
 M1 ear to row bulk:
This method, based on randomly harvested M1 ears, is
similar to method 5 below, but differs in that the relation of
the ears, branches, fruit, etc., to one another is not
maintained, permitting a type of bulk processing comparable
to that obtained with method 4 but requiring smaller M2
progenies (perhaps 25 – 30) and adaptable to semi-bulk
harvesting of units from M1 plants.

15. Systems of handling M2 populations
M1 single-seed or multiple-seeds bulk:
This method involves selecting a single seed at random from
each M1 spike (or fruit, branch etc.) of M1 plant to constitute
an M2 population of single plants from the resultant bulk. M2
single plants can be selected for mutant phenotypes that can
be further progeny-tested in the M3.
M1 plant to row:
In this method, all seeds or a sample of the seeds produced
from a given M1 plant are grown to produce the M2
generation, which is then screened for mutant phenotypes.
The success of its use will depend to a large extent on how
well the secondary tilling or branching has been controlled
because the secondary tillers tend to dilute the yield of M1
mutants.
Contd.

16. Systems of handling M2 populations
M1 ear, branch, pod, and fruit (within plant) to row :
Here, each ear taken from the M1 is processed as a separate
entity and sown out as an ear-row progeny, which is then
screened for mutant phenotypes.
Size of M2 population:
The M2 population size will, to some extent, be a function of
the available space and the screening methods to be used.
The size of the population may be assessed by either
sampling a few seeds from many M1 plants or more seeds
from fewer M1 plants. If the number of M1 plants is low, but
with relative high fertility then 20–25 seeds may be
harvested from 2–3 spikes per M1 plant.
Contd.

17. Screening methods and mutant selection techniques
 Visual methods of selection for identifying mutant phenotypes are
common and can be very efficient;
 Mechanical or physical methods of selection can also be used
very efficiently in screening for seed size, shape, weight, density.
etc., using appropriate sieving machinery as they are readily
adaptable for processing of large quantities of seeds.
 Other selection methods, such as chemical, biochemical,
physiological, physio-chemical, and various specific methods
may be needed for selecting certain types of mutants.
 Screening for abiotic stresses such as drought, salinity, heat etc.,
requires setting up of the selection pressure and maintaining a
uniform stress over the M2 and M3 populations.

18. Figure: Screening for salinity tolerance, in hydroponic nutrient solution, of rice
mutants at four levels of salt (NaCl2) (0, 5, 10 and 15 dS/m) showing variation in
shoot and root growth after 14 days of applied stress (The Joint FAO/IAEA Plant
Breeding and Genetics (PBGL), Seibersdorf, protocols 2014). Top row depicting
show and lower row for roots. Courtesy of A. Mukhtar Ali Ghanim.
0 dS/m 5 dS/m 10 dS/m 15 dS/m

19. Figure: Screening lentil mutants, in hydroponic solution, for drought tolerance using
PEG6000 at four levels of concentrations (A,B,C and D), respectively: 0, 10, 15 and
20%. Photos were taken 6 weeks applying stress pressure protocol optimization
experiment at PBGL, Seibersdorf, Austria in 2014. Courtesy of A. Mukhtar Ali
Ghanim.

20. Management of the M3 generation
 Progeny tests are essential for the identification of all mutant
lines useful for plant improvement and re-selection from M3;
 This is done to establish that the trait is heritable. Further progeny
tests, may be necessary to stabilize a potentially useful variant.
 Furthermore, it is not uncommon that a mutant may be
homozygous for the desired character but segregate for other
undesirable ones, they still can be chosen when their selection
might be useful for improving the genetic background of the
desirable mutant.
 In several situations, M3 progeny tests may be essential for the
detection of mutants particularly of those not easily discernible
from single (M2) plants. This may be particularly true for traits
that are influenced by environment, e.g. pigmentation and some
biochemical or physiological mechanisms.

21. Contamination in self-pollinated mutant crops
 Sometime, authenticating the genetic origin of the variation in
mutagen-treated plant material may be of interest and concern
not only to geneticists but also to breeders;
 Contamination is a common problem in any plant-breeding
programme and causes even greater problems in mutation
breeding;
 This is potentially a bigger problem for a non-breeding
organization that must purchase seeds of a commercially
available cultivar rather than a breeding company utilizing its
own pure stock seeds

22. Figure: Outcrossing in the pale green mutant rice fields. The dark tall green plants
are likely a result from outcrossing of the wild type with the mutant.
Outcrossing rates are often higher than mutation rates. Courtesy of Q. Shu.

23. Genotypic and phenotypic analyses
 In general, for practical purposes, the criteria described below are
the most critical, even though they can seldom be performed for
large numbers of variants. However, tests of large numbers of
variants are rarely necessary as, in most instances; the breeder
finally uses relatively few of the variants isolated. The following
analyses will usually permit a conclusion relative to the origin of
any specific genetic variant:
1. Genotypic
2. Phenotypic

24. Genotypic analyses
 Progeny testing:
Selected variants should breed true in progeny grown from
selected M3, but might not do so in M2 progeny tests. If
segregation does occur in progenies from M2 single plants, the
variation should be limited to the selected trait although it may
sometimes occur for a few (1 or 2) others.
 Backcross and other crossings:
Selected variants should show a relatively simple genetic
segregation in backcrosses to the parental genotype and to some
other strains (1 or at most 2 gene segregations).

25. Phenotypic analyses
 Morphological characteristics :
In general mutants should show a measure of similarity to the
parent genotype except for modifications related to the mutation
involved. Often a whole complex of changes in phenotype could
be caused by a simply inherited mutant, but a similarity in many
other features should still remain.
 Physiological, phyto-pathological and biochemical characteristics :
The variant should show similarity to the parent genotype in a
large number of measured traits, especially in traits governed by
several different genes or complexes, such as quality
characteristics, disease and pest resistance, and biochemical traits.

26. Thanks for your patience hearing
Mutation Result!!!!