Training managment training 14-18 feb,2021

1. WHEAT 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 may
contain 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 few days
remaining to anthesis
(pollen shed).
The anthers
are green in
this spike.
Note. If the anthers near the centre of the
spike appear pale yellow, the spike is too
old to emasculate.

8. Take off the top spikelets and the
down spikelets, which are too
young.

9. Take out the middle florets
only keep the lateral florets

10. From bottom to top,
clip individual
florets just above
the anthers.

11. Clipping of the spike is complete when the awn
and the upper part of each floret is removed.

12. The female spike is emasculated by carefully removing the
three (3) anthers from each floret, not hurting the stigma.
Re-check the florets after
emasculation to make sure
no anthers have been
missed.

13. The label on the glassine bag indicates the
day the emasculation was made.
After emasculation of the female spike has been completed,
place it in a labeled glassine bag.

14. The female spike will be ready to
pollinate when the florets open,
and the stigma becomes featery
and protrudes, or shows a gap
between the lemma and palea.
Gapping of the florets will occur 2 to 7
days after emasculation, depending
upon temperature and the age of the
selected spike.

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

16. 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 floret.
Selection of the male parent for irradiation (Method 1).
Remove tiller which has a
spike ready for irradiation

17. Cover tiller with paper
bag to avoid pollen
contamination until
irradiation.
Remove tiller(s) from
water and place in a
paper bag and irradiate
(we used gamma
irradiation).
Put the base of the
cut tiller into water (to
maintain tiller and
pollen viability)

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

19. The anther can be
easily broken to shed
pollen grains.
Remove one anther from the male
floret for pollination.

20. 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.
Harvest the anthers and place in a
tube, the tubes may be place inside
a paper bag, irradiate immediately.
Irradiation of anthers
using gamma ray

21. FDA DAPI
Pollen vitality and fertility using fluorescence staining
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

22. Dosage (Gy) Pollen viability
0 F= 18.78%
V= 48.67%
100 F= 18.75%
V= 40%
200 F= 11.29%
V= 6.24%
250 F= 1.7%
V= 11.4%
300 F= 2.6%
V= 18.63%
500 F= 0.67%
V= 13.3%
Dose treatments of 100-300
Gy may be used as
mutagenic treatments. Pollen
is viable and can be used in
crossing to introduce
mutations via fertilisation.
Dose treatments of 500 Gy
and greater kill pollen and
these may be used in
pollinations to induce haploid
embryo development.
Effects of irradiation on pollen fertility (F) and vitality (V)

23. Place the irradiated anther(s)
above the open female flower
and break the anther.
and brush the stigma slightly. Each
anther can be used to pollinate 2, 3,
or florets.

24. 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.
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.

25. If the cross is successful, enlarging seeds
(caryopses) will be visible in 7 to 10 days.
This time, glassine bag can be removed.

26. Immature embryos may be rescued by in vitro culture at
21 days after pollination
Immature seed
(caryopses) at 21
days after pollination
(different size of
seeds)
Seed are surface sterilised by
soaking in 70% alcohol (5 min)
and 5% chlorox (10 min) before
embryo excision and culture.

27. Picture of embryo excision
Farzaneh?

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

29. 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)

30. 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

31. 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

32. 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

33. Acknowledgements
This protocol was developed by:
Heru Rusfiandi (PT. London Sumatra, Tbk, Indonesia),
Gilbert Seballos, Farzaneh Shirazi & Abdelbagi MA Ghanim
Edited and compiled by: Brian P. Forster & Abdelbagi MA
Ghanim
Plant Breeding and Genetics Laboratory, Joint FAO/IAEA
Division,Vienna, Austria.