Training managment training 14-18 feb,2021

1. Dr. M A SAMAD, Principal Scientific Officer and Head,
Plant Breeding Division
Bangladesh Institute of Nuclear Agriculture(BINA)
BAU Campus, Mymensingh-2200, Bangladesh
Advancement of Mutant Poulation

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.

8. Molecular Biology of Induced
Mutations in Plants
•Plant DNA damage and repair
•Plant mutagenesis and induced mutation
•Molecular basis of Induced mutations
•Overall strategies on use of molecular
markers for mutant characterization

9. DNA Replication

10. a master blueprint subject to
constant insults
1. Definition: modifications of molecular
structure
2. Classification: spontaneous and
environmental
DNA Damage

11. 1. Mismatchs
2. Tautmoeric shifts ( T in enol form
matchs G in keto form)
3. Deamination bases (C to U)
4. Incorporation of Uracil into DNA
5. Loss of Bases: depurination and
depyrimidination
6. Oxidative damage to DNA
Types of DNA Damage
Spontaneous

12. 1. Irradiation: base damage;
sugar and strand breaks;
cyclobutane pyrimidine
dimers, DNA cross links,
etc.)
2. Chemical: cross-linking;
base analog,
3. Damage to chromatin
structure
Types of DNA Damage
Environmental

13. The consequence
of DNA damages
may cause
mutations that
produce a
variety of direct
molecular effects
Mutagenesis, mutation and mutants

14. Key Definitions
Term Definition
Mutation A heritable change in the sequence of an organism’s
genome (the full complement of an organism’s genetic
information)
Mutant An organism that carries one or more mutations in he
genome
Genotype The genetic information that an organism encodes in its
genome
Phenotype The ensemble of observable characteristics of an
organism
Mutagen An agent that leads to an increase in the frequency of
occurrence of mutations
Mutagenesis The process by which mutations are produced
After Friedberg et al. 1995

15. DNA Repair Systems

16. Mismatch Repair
DNA repair during replication through proof reading
DNA polymerase editting function

17. Mismatch Repair
Blue: parent
strand
Red: mutant
strand
Green: repaired
strand

18. Mismatch Repair: Genes
TABLE 1 Eukaryotic MutS and MutL homologs
E. coli Plants Function
MutS MSH1 Mutation avoidance in mitochondria
MSH2 Forms heterodimers with MSH3 and MSH6 to:
Repair replication errors
Repair mismatches in recombination
intermediates
Remove nonhomologous tails (MSH2-MSH3
only)
Inhibit recombination between nonidentical
sequences

19. Mismatch Repair: Proteins
TABLE 1 Eukaryotic MutS and MutL homologs
E. coli Plants Function
MutS MSH3 Forms heterodimers with MSH2
MSH4 Forms heterodimers with MSH5 to promote
crossing-over in meiosis
MSH6 Forms heterodimers with MSH2
MutL PMS2 Forms heterodimers with MLH1 to:
Repair replication errors
Repair mismatches in recombination intermediates
Inhibit recombination between nonidentical
sequences
Respond to DNA damage (mammals)

20. Mismatch Repair: Proteins
TABLE 1 Eukaryotic MutS and MutL homologs
E. coli Plants Function
MutS MLH1 Forms heterodimers with PMS1, MLH2 and MLH3
MLH3 Forms heterodimers with MLH1 to:
Repair replication errors
Promote crossing-over in meiosis
HARFE & JINKS-ROBERTSON

21. Mismatch Repair: Mutation Avoidance
•The MMR system plays a key role in the elimination of
Mutational intermediates generated during DNA
synthesis and thus helps to insure that DNA replication
is a high-fidelity process.
•Although generally considered to be detrimental,
elevated mutation rates in microorganisms can be
advantageous when environmental conditions demand
rapid adaptation.

22. Mismatch Repair: Mutation Avoidance

23. Mismatch Repair: Mutation Avoidance

24. Mismatch Repair: Mutation Avoidance

25. Double Strand Break Repair
Errol C. Friedberg 2003 Nature 436-440

26. Double Strand Break Repair in Plants

27. Double Strand Break Repair in Plants
Homologous sequences at different
positions in the genome can be used
as matrix for the repair of a DSB
Schematic diagram of the experimental set-up that
was used to demonstrate that a DSB could be repaired
by a combination of homologous recombination (HR)
and non-homologous end-joining (NHEJ). By
expression of I-SceI a DSB is induced in the target
locus. The break can be repaired by restoration of the
kanamycin gene (KANA) by homologous
recombination with an incoming T-DNA. Two different
T-DNAs were used: one with homologies to both ends
of the break and the other with homology to only one
end of the break. Both kinds of constructs could be
targeted to the genomic locus. As only the SDSA
model of recombination is able to predict both kinds
of events, this mechanism may be the most
appropriate to account for gene conversion events in
plants.

28. Double Strand Break Repair in Plants
Allelic recombination

29. Double Strand Break Repair in Plants
Detection of HR and NHEJ Repair
NHEJ might lead, in
a certain fraction of
cases, to genomic
changes such as
deletions, insertions
or various kinds of
genomic
rearrangements

30. DSB HR System in Plants

31. DSB HR System in Plants
Genes invovled in DSB HR:
ATM: Double-strand break (DSB) signaling,
cell-cycle checkpoint
ATR: DSB signaling, cell-cycle checkpoint
BRCA2c: Strand invasion
BRU: Chromatin state, silencing
Centrin2: Nucleotide excision repair (NER)
DMC1: Meiotic interstrand invasion
ERCC1: NER
Total 26

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34. Thanks for your patience hearing