Training managment training 14-18 feb,2021

1. BARLEY CROSSING
PROTOCOL
USING
IRRADIATED POLLEN

2. Plants for crossing can be grown in the field, or in glasshouses.
Indoor grown plants are
generally easier to work with
and crossing success is often
better.

3. Pots are labeled and arranged
on glasshouse benches.
The label for each pot
contains information on the
nursery name, plant
identification number and
line name or code.

4. When working with plants,
it is often better to remove
the pots from the bench.
Select the pot and place it
in a position so that it is
convenient to sit while
doing the crossing.

5. Tools used for crossing.

6. Special
magnifying
glasses may be
used to see
spikelets and
their anthers.

7. Choose the female parent,
a spike that has several
days of growth before
anthesis (pollen shed).
Note. If the anthers near the centre of the
spike appear pale yellow, the spike is too
old to emasculate.
The anthers
are green in
this spike.

8. Clip off the spike near the tip of the
last spikelet; remove the flag leaf and
the upper part of the awns.
Expose the female spike
by unrolling the flag leaf
sheath.

9. Clip individual spikelets just
above the anthers.
The anthers are generally
visible inside the spikelet.

10. Clipping of the spike is
complete when the awn and
the upper half of each
spikelet are removed.
Use a fine-pointed tweezers to
remove the anthers (3) from
each spikelet.

11. The female spike is emasculated by removing the three (3)
anthers from each spikelet.
Re-check the spikelets after
emasculation to make sure
no anthers have been
missed.

12. After emasculation of the female spike has been completed,
place it in a numbered glassine bag.
The number on the glassine bag
indicates the day the
emasculation was made.
The female spike will be ready to
pollinate when the spikelets
open ----- or show a gap
between the lemma and palea.
Gapping of the spikelets will
occur 2 to 7 days after
emasculation, depending upon
temperatures.

13. Female spikes ready for pollination
Before pollination,
check to determine if
any of spikelets have
already set seed,
these should be
removed.
A closed spikelet
may indicate seed
development.

14. Selection of the male parent for irradiation (Method 1).
Chose the plant to be used as an irradiated male. Find a spike
that has not shed pollen. The anthers should be pale yellow
and still at the base of each spikelet.
Remove tiller which has a
spike ready for irradiation
Put the base of the cut tiller
into water (to maintain tiller
and pollen viability)
Remove tiller(s) from water
and place in a paper bag and
irradiate (here we use gamma
rays).

15. After irradiation, clip the spikelets above the anthers.
Male spike
prepared
for anther
harvest.
One to five
minutes after
clipping the
spikelet of the
male, the anthers
start to enlarge
(puff up).
When the anthers
start to enlarge,
they will shed
pollen and can
be used for
crossing.

16. Remove one anther from the
irradiated male spikelet for
pollination.
The anther can be
easily broken to shed
pollen grains.

17. Selection of the male parent for irradiation (Method 2).
Chose the plant to be used as an irradiated male. Find a spike
with pollen that has not shed, but close to shedding. The
anthers should be pale yellow and at the base of each spikelet.
Clip the spikelet
above the anthers.
Irradiation of anthers
using gamma ray
Harvest the anthers and
place into a tube, tubes can
be placed in a paper bag
and irradiated immediately.

18. Observations of pollen vitality and fertility using
fluorescence staining
FDA DAPI
Bright fluorescence with FDA indicates fertility
Dull fluorescence with FDA indicates vitality
No fluorescence with FDA indicates dead pollen
With DAPI staining the three nuclei of mature fertile pollen can be seen,
non-staining (empty) indicates non-viable pollen

19. Effects of irradiation on pollen fertility (F) and vitality (V)
Dosage (Gy) Pollen viability
0 F= 11.15%,
V= 33.86%
100 F= 5.3%,
V= 47.45%
200 F= 1.33%,
V= 22.66%
250 Abnormal
300 Abnormal
500 Mostly dead
Dose treatments of 100-200
Gy may be used as
mutagenic treatments. Pollen
is viable and can be used in
crossing to introduce
mutations via fertilisation.
Dose treatments of 250-500
Gy kill pollen and these may
be used in pollinations to
induce haploid embryo
development.

20. Place the anther(s) above the
open female spikelet (flower)
and break the anther.
Each anther can be used to
pollinate 2, 3, or more female
flowers.

21. When pollination is completed, replace the glassine bag over
the female spike. Fix the glassine bag with a paperclip.
Place the
tag near
the bottom
of the
glassine
bag.
Write a label for the cross with
the female parent listed first,
then the male parent. Also,
write the date when the cross
was made.

22. If the cross is successful, enlarging
seeds (caryopses) will be visible in 7
to 10 days.

23. Immature embryos may be rescued and cultured 21 days
after pollination
Surface sterilized of
seed by soaking in
70% alcohol (5 min)
and 5% chlorox (10
min) before embryos
are excised and
cultured.

24. Slide on embyro excision

25. Germination of Immature
embryos 1 week after culture.
In vitro cultured of immature embryos on solid MS media
Immature embryo cultured on
media.
In vitro seedling ready to
be sampled for ploidy
analysis

26. Sample preparation for flow cytometry
Leaf or root samples of
the embryo culture,
samples can be put in
petri dish or tube. Put
the label on it
Add CyStain® UV Ploidy solution
Note: all of this work should be conducted in the dark
(dark room)

27. Sample preparation for flow cytometry
Note: all of this work should be conducted in the dark
(dark room)
Chopping samples, should be
on ice
Samples are ready for
incubation

28. Sample preparation for flow cytometry
Incubate samples at 50C for
about 1 hour in the dark
Sample filtration and ready for
ploidy analysis using flow
cytometry

29. Ploidy analysis using flow cytometry
Flow cytometer for ploidy
analysis
Mixoploid (n-2n)
Diploid (2n)
Flow cytometry result showing
peak which determined ploidy
Put the sample tube to the flow
sample port

30. Acknowledgements
This protocol was developed by:
Heru Rusfiandi (PT. London Sumatra, Tbk, Indonesia),
Gilbert Seballos, Farzaneh Shirazi
Jerry Franckowiak, Julie McKavanagh & Simon Hamlet
Queensland Department of Plant Industry and Food,
Queensland, Australia.
and
William Thomas
James Hutton Institute, Dundee, UK.
Edited and compiled by Brian P. Forster & Abdelbagi MA
Ghanim, Plant Breeding and Genetics Laboratory, Joint
FAO/IAEA Division,Vienna, Austria.