Enabling techniques to enhance efficiency

of mutation breeding

OFID training course 4 Oct 2016

Abdelbagi MA Ghanim
PBGL Seibersdorf

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Generating and identifying mutants
Mutagenic
treatment

Mo seed M1 plants M2, M3 ! ..

Hin Studies, Fluorescence

900
850

800 Mutation

750
700
650 Hin 96
Hin 72
600
Hin 48
550 Hin 32
500 Hin 24
Hin 16

Intensity (mV)
450 Hin 10

Seed resource of
400 Hin 8
Hin 6
350 Hin 4
300 Hin 2
Hin WT
250
Hin Hom
200
150

identified mutants 100

50
0

5.0 5.5 6.0 6.5 7.0 7.5 8.0

Genotyping Time (min)

Phenotyping

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Mutant line Identified mutant

development
Check for potential alleles Rounds of selfing

Produce segregating population

Varietal
development

Bulk segregant analysis

Adaptive backcross to elite line

Fine Map

Others
Varietal
development

International Atomic Energy Agency

Food and Agriculture Organization of the United Nations
Time scales in mutation breeding

Mutagenesis: seconds/minutes

Mutation detection: months/years

Mutant variety development: 10-12 years

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
How to speed the course for mutant delivery

- Rapid generation cycling

- Doubled haploidy
- Markers

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Accelerated breeding by rapid cycling
Grow plants in small pots
Grow plants under continuous lighting
Culture embryos 12-14 days after
flowering
Doubled haploidy
Screening for mutant trait
(genotyping/phenotyping)
Upto M5-M6 can be advanced in one
year
REPEAT

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Established techniques to accelerate mutants development

Repeated
cycles to
advance
mutant
generations

Wheat Sorghum Barley

Shortening the crop cycle by management

techniques such as planting in small pots,
light watering and continuous light Rescue of immature
embryos to gain time
 Normal and accelerated generations time and the respective
number of generations per year for wheat, barley and sorghum
Plant Species Normal Generations Accelerated Generations/
generation time /year 1) generation time 2) year

Wheat (Spring) 4-5 months 1-2 45 days 7

Barley 4-5 months 1-2 40 days 8

Sorghum 5-6 months 1-2 60 days 6

In very few environments two crop generations can be grown per year
These are averages for 5-10 varieties from each crop species with the aid of embryo rescue

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
 Doubled haploid technologies

Haploid
a plant with gametic (haploid = n) number of chromosomes
in somatic tissues
Doubled haploid
a plant obtained after chromosome doubling in a haploid cell,
tissue, or individual

Dihaploid Polyhaploid
a haploidplant in a a haploid plant in a
tetraploid species (n=2X) polyploid species (e.g. n=3X)
 maturation

bi-cellular mature pollen

pollen male
gametophyte

stress
uni-cellular
microspore:
cell with embryogenesis
restricted
developmental
potential embryogenic microspore embryo, sporophyte

totipotent
cell
 Stages of microspore development in barley

a b c dd

e f g h

a) tetrads; b) early uninucleate; c) early-mid uninucleate; d) mid

uninucleate; e) mid-late uninucleate; f) late uninucleate; g) anaphase of the
first microspore division; h) bi-nucleate.
 Value of Haploids in Breeding

Haploids are very valuable in plant breeding for several

reasons
Since they carry only one allele of each gene,
mutations and recessive characteristics are expressed
in the plant.
Plants with lethal genes are eliminated from the gene
pool.
Can produce homozygous diploid or polyploid plants -
valuable in breeding
Shorten the time for inbreeding for production of
superior hybrids genotypes.
Widely used methods of Doubled Haploid
:

Anther Culture
Microspore culture
Ovule culture
Irradiated pollen
Wide hybridization (cereals)

Food and Agriculture Organization of the United Nations International Atomic Energy Agency
Doubled Haploid for efficient and rapid mutants development
Donor plant (2n)
Meiosis Mutagenic
treatment
In vitro culture of haploid
cells (n)
In vitro Sporothytic development
selection Haploid embryos/calli (n)
Regeneration

Haploid plants (n) In vivo

Chromosome doubling
selection

Doubled haploid mutants (2n)

Seed increase
Lab test & field
evaluation DH mutant lines (2n)
 Accelerated mutation breeding by DH
Mutant induction

Recurrent Donor
parent mutant
F1

BC1
Doubled Haploidy
for efficient & fast
BC2 mutant selection
Doubled
Haploidy BC3

BCn Selfing
Homozygous
mutant lines
FAST SLOW

Backcross inbred lines

 Factors influencing success of DH production
in wheat by anther/microspre culture
a) Genotype and the cytoplasm
b) Culture conditions of donor plants
c) Optimum stage for spike collection
d) Pretreatment of spikes Response of wheat genotypes

60

e) Culture media 50

% mean
40
%Green plants
30
%Albinos
20
10
0

DHS1

DHS2

DHS3

DHS7

DHS9

DHS10

DHS11

DHS12

DHS14

DHS15
Genotypes
 Anther Culture for Wheat Doubled
Haploid Production

Pretreatment of the Anthers in liquid Developing of embryos in liquid

donor plants at 4C induction medium induction medium

Regeneration on solid Haploid plants at Haploid plants in DH plants in the

regeneration medium acclimatization stage plastic house field
 Wide Hybridization with Maize, Zea
mays and Anther Culture

Maize plants Emasculated Spikes after pollination Seeds formation

spikes with maize pollen grains

Embryo rescue chromosome doubling DH plants at the field after colchicine

and haploid with colchicine (0.2%) doubling and seed multiplication
plant
Inter-specific crosses with maize for doubled wheat haploid production,
while H. bulbosum is used for doubled haploid production in barley
Stages in producing wheat doubled haploids through
ultra-wide crosses
a) Wheat spikes at ear emergence
(left), emasculation (center) and
seed setting (right).
b) Germination of maize pollen on
wheat stigma. Bar 0.1 mm.
c) c) Injection of 2,4-D into wheat
spike using a syringe.
d) d) Pearl millet pollen at
collection (upper) and after being
dried for two hours (lower). Bar
0.1 mm.
e) e) Detached-tiller culture of
wheat at emasculation (left),
pollination (center) and seed
setting (right).
f) f) Wheat seeds obtained from
self-pollination, and from crosses
with maize, pearl millet and
sorghum (from left to right). Bar
2 mm.
 Isolated Microspore
Culture

Grinding spikes at 18.000 rpm

a) Cut florets in blender cup ready to be blended;
b) b) Blender cup assembled to the blender for blending
to release microspores
DH population

Seed increase

Evaluation
 In vitro mutagenesis using isolated
microspore culture

Application of
mutagenic agent

n or 2n

In vitro selection
of mutants
 Critical dose evaluation tests
Survival of microspores of B.napus after gamma irradiation

100
90
80
70
60
50
40
30
20
10
0
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

Gamma-rays (Gy)

Polsoni et al., 1988

 High quantities of embryos can be
produced (1,000-2000 per ml)

Density can be
enriched by sieving
High uniformity
 Mutagenic treatments applied to
isolated microspore cultures
Species Mutagen Dose
Brassica napus ENU 20 mM
MNU 0.2-1.5 mM/3h
gamma rays 5-40 Gy
X-rays 10-40 Gy
UV 10-60 s, 33 erg mm-2
s-1
Brassica carinata EMS 0.1-.5%/30 min
Hordeum vulgare NaN3 10-4 M/1h, 10-5 M/1h
EMS - ethyl methanesulfonate; ENU - N-ethyl-N-nitroso urea; MNU - N-methyl-N-
nitroso urea; NaN3 - sodium azide; UV ultra violet light

Szarejko, 2003
In vitro mutagenesis of haploid explants or plant organs
that contain gametophytes - selected examples
Species Organ/Explant Mutagen Dose
treated
Brassica napus flower buds gamma rays 10-50 Gy
anthers fast neutrons 10-16 Gy
secondary embroids gamma rays 80-240 Gy

Hordeum vulgare spikes before culture gamma rays 1-10 Gy

anthers NaN3 10-3 M/ 6 h, 10-4 M/ 6 h

Oryza sativa panicles before culture gamma rays 1-20 Gy

anthers EI 0.5-1 ml/L/ 20 h

EMS 0.5%/6h
MNNG 25-100 mg/L/ 15 h
microspore-derived calli gamma rays 10-50 Gy
Nicotiana haploid protoplasts UV 32 erg mm-2 s-1
sylvestris
Solanum inflorescences gamma rays 2-30 Gy
tuberosum
X-rays 10 Gy
Tritium aestivum spikes before culture gamma rays 1-3 Gy

EI ethylenimine; MNNG - N-methyl-N'-nitro-N-nitrosoguanidine; MNU - N-methyl-N-

nitroso urea; NaN3 - sodium azide; UV ultra violet light Szarejko, 2003
 In vitro selection applied in haploid systems
Species Selection objective Selection factor Concentration
Brassica napus Tolerance to imidazolinone Herbicide Pursuit 40 g/L
herbicides
Improved resistance to Oxalic acid (OA) 3 mM
Sclerotinia sclerotiorum

Salt tolerance NaCl 0.6 and 0.7%

Cold tolerance Salycilic acid (SA) 0.1-0.2 mM*

Reduced levels of saturated Heat 35oC during embryo -

fatty acids maturation,
HPLC analysis of embryo
fatty acid composition
Brassica Improved resistance to culture filtrates (OD680 ~ 1.8- 30-40% (w/v)
campestris Erwinia carotovora 2.0)
Nicotiana Salt tolerance NaCl and KCl 200 mM
plumbaginifolia
Water stress tolerance Polyethylene glycol (PEG) 25%

Resistance to amino acid L-valine 5 or 10 mM

valine

Nicotiana Resistance to amino acid L-valine 8 mM

tabacum valine
Szarejko, 2010
 DH system in mutation techniques

Advantages
- rapid fixing of mutated genotypes
- avoiding chimerism
- screening for recessive mutants in the first
generation after mutagenic treatment
- shortening the production of pure mutant/
recombinant lines
- increased selection efficiency of desired mutants
- application of the selection factor at the haploid or
DH cell/embryo/plant level
DH mutants in barley
(DH1 generation)
 Development of salt-tolerant mutants in wheat
Explants: anthers or young spikes Radiation treatment with 100-150Gy

Callus induction under salt-stressed condition

Differentiation under salt-stressed condition Regenerated plantlets

Seed propagation for 1-2 generations without salt-stress

Selection and identification for multi-generations in

natural saline soil
Physiological and biochemical
identification in the lab

Obtained stable and inheritable salt-

tolerant mutants
Main species for which DH technology is available

Gynogenesis

Ovary/ovule culture: Pollination with irradiated

pollen (250-2500 Gy)
sugar beet, onion,
sunflower, gerbera
barley, rice, wheat, maize, tobacco In vitro culture
(ovules, ovaries, immature embryos)

Induced parthenogenesis:
apple, pear, cucumber,
melon, rosa
Inoculated onion flowers after two days of culture
Microspore culture in ornamentals:
Zantedeschia

C
Tulip microspore culture
Brassica microspore culture
 Verification of haploids

1. Seed setting/pollen fertility

2. Cytology
3. Histology
4. Vigour
5. Flowcytometery
6. Marker.

Fertile DH

wheat
Wheat DH Protocol at PBGL