Procedure for Production of Haploid embryos

1. Name- Patil Kulbhushan Savindra
PID.- 12020442
Department of Genetics and plant
Breeding
Topic- Wheat X Maize Wide Hybridization For Obtaining
Haploid Embryos
Assignment - I
Dr. Sanjeet Singh
Lovely Professional University

2. 1. Introduction
• Wheat is the staple food of millions of people. It is also an important part of the daily
diet of many millions more. There has been a tremendous increase in wheat
production in our country since the times of the Green Revolution.
• However, at present, the wheat production has almost reached a plateau and another
breakthrough is required in order to meet the ever-increasing food demand of the
nation.
• The genetic up gradation of wheat through conventional breeding approaches require
longer time there is a need to assist these methods following certain biotechnological
tools so as to shorten the breeding cycle and Doubled Haploidy (DH) breeding is one
such tool which has been widely used in breeding programs (Sunega et al, 1994).
Amongst the available techniques for the development of haploids and doubled
haploids in wheat, chromosome elimination approach following wheat x maize
system is one of the most efficient approach (Laurie and Bennett, 1988).

3. • The use of doubled haploid (DH) plants has revolutionized modern plant breeding
and genetic mapping studies in many important cereals and fruit crops.
• DH technology fixes rare alleles and may play an important role in evaluation of
genetic diversity. Thus, doubled haploid technology is useful in plant improvement
for gene transfer and production of breeding lines.
• Doubled haploids are homozygous plants developed by androgenesis (microspore
and anther culture), gynogenesis (ovary and ovule culture), and wide hybridization.
• In wheat, the wide hybridization method is the most effective for producing
doubled haploids(Niu et al, 2014).
• The method of crossing between species of the same or different genera is called
wide hybridization.
• Wide hybridization has been becoming popular in wheat since late 1970s which
has been described in several literatures.

4. • Haploid production through wide hybridization is considered gynogenesis because
the haploid developed from this procedure contain the maternal haploid genome.
• The haploid production through wide hybridization was first discovered in crosses
between barely and bulbous barely.
• In 1986 Laurie and Bennett established the wide hybridization system between
wheat and maize. He reported that wheat ovules were fertilized by maize pollen
and the haploid wheat embryos were produced through the elimination of maize
chromosomes.
• The efficiency of haploid induction in common wheat is usually higher than durum
wheat using wide hybridization with maize.
• High ploidy level and the D genome of common wheat may play an important
role in DH production.

5. 2. Objectives
1. To speed the process of breeding and production of breeding lines.
2. To identify the population development in crop improvement, genetic
manipulation, plant genome and gene mapping.
3. Factors affecting efficiency of DH production in wide
hybridization
1. The position of spikelet in the flower and time for pollination.
2. Temperature, light intensity and photoperiod during the plant growth period.
3. The type and concentration of plant growth regulators applied after pollination.
4. The biochemical elements added to the rescue media.
5. The concentration of colchicine used for chromosome doubling and the plant
growth stage, duration and temperature when the colchicine is applied.

6. 4. Material required for obtaining haploid embryos / DH laboratory-
1. Wheat seeds ( F1, F2 or other generations) and maize genotypes.
2. Glassine bags, surgical scissors, forceps, scalpels, and stapler.
3. Laboratory glassware's.
4. Refrigerator, laminar flow cabinet, distilled water unit, autoclave, pH meter, digital
balance with four-digit precision, stirrer with hot plate and aluminum foil.
5. Hydrochloric acid (1 N HCL) and sodium hydroxide (1 and 10 N NaOH) solutions.
6. Sodium hypochlorite (bleach) and cheesecloth.
7. Alcohol burner or germinator™ 500.
8. 2,4-D solution for spray.
9. MS media.
10. Colchicine stock solution (5%)

7. 5. Procedure for obtaining haploid embryos-
Maize
Planting and
Management
Wheat
vernalization
, planting
and
management
Emasculatio
n and
Pollination
Hormone
Spray
Embryo
Rescue
Regeneration
of embryos
Vernalization
of Haploid
Plantlets for
Winter
Wheat
Colchicine
Treatment

8. 5.1 Maize Planting and Management-
1. Maize was planted 3 weeks before transplanting vernalized wheat seedlings and
is then planted every week to ensure a continuous supply of pollen. At every
planting time, seven to ten pots were planted with three seeds in each pot.
2. Pots were watered as needed and were fertilized weekly with a fertilizer solution.
3. Maize plants should be healthy in order to get viable pollen and should be
monitored from time to time for diseases and pests.

9. 5.2 Wheat vernalization, planting and management-
1. F1 plants obtained from crossing between desired parents are used as female
parents for the wide hybridization. The plants should be healthy without
incidence of diseases or pests.
2. Winter wheat needs 8 weeks of vernalization whereas spring wheat seeds may be
planted directly in pots.
3. As soon as vernalization is over, wheat seedlings are transplanted two seedlings
per one gallon pot.
4. Watering is done on alternate days or if the soil appears dry.
5. Staggered planting of wheat seedlings by 7–10 days may be an option to spread
the workload and reduce the need for large amounts of maize pollen on any
single day.

10. 5.3 (a) Emasculation
1. Selection of the correct developmental stage of the wheat floret is very important.
2. A floret approximately 2/3 of the way up the spike should be checked to determine if
the spike is ready for emasculation.
3. Each wheat floret contains three anthers along with a pistil and the process of
removing three anthers form each floret is called emasculation.
4. If the anthers are starting to change from a dark green color to a light green color and
the stigma is fluffy, the spike can be emasculated.
5. Starting at the bottom of the spike, emasculation is done by removal of the central
florets of each spikelet leaving only the two lateral florets.
6. Anthers are then removed using a forceps from the lateral florets in a systematic
pattern from the bottom to the top of the spike.
7. Once emasculation is complete, the spike is bagged with a glassine bag that is
properly labeled with the name of the cross and the day of emasculation.

11. Male Reproductive
parts
Wheat spike
ready to be
emasculated
Clipping of spikelet's prior to anther removal
during the emasculation process

12. 5.3 (b) Pollination
1. Anthers are collected from maize plants when the anthers are starting to emerge,
being very careful not to disturb the other anthers as the pollen will be lost.
2. Anthers from multiple maize plants are collected at the same time and placed in
a petri dish.
3. The glassine bags are removed from the emasculated wheat spikes 1 day after
emasculation, or when the stigma has a feathery appearance.
4. The pollen is then brushed onto each stigma on the emasculated spikes using a
small paint brush; the pollen should flow easily from the anther to the
emasculated florets. Once the entire spike is pollinated, the date of pollination is
recorded on the glassine bag and the bag is replaced over the spike.

13. Emasculated wheat flower
Fresh pollen collected in a
petri dish

14. 5.4 Hormone Spray
1. The day after pollination the spikes should be sprayed with 2,4-D (Sigma)
solution (0.5 mg/L ).
2. Wedzony and Van Lammeren in 1996 demonstrated that 2,4-D increased the
number of the pollen tubes that reached the micropyle and multiplied the number
of sperm cells in the pollen tube, thus increasing successful intergenric
fertilization.
3. The hormone treatment is applied by spraying the solution on the spike using a
small aerosol sprayer bottle. The hormone treatment should be done within 24–
48 hrs. after pollination.
4. After hormone treatment, the glassine bags are replaced and secured with a
stapler.

15. 5.5 Embryo Rescue
1. From 16 to 19 days after pollination, spikes are cut off leaving approximately 20 cm
of stem with the spike; spikes with stems are placed in a container of double
distilled water.
2. The caryopses (hybrid seeds) are removed carefully with forceps.
3. The culture of the embryos should be done under sterile conditions in a laminar
hood.
4. Embryos should be rescued within 7 days after collecting the spikes. The spikes are
kept at 4 C until embryos are rescued.
5. The caryopses are surface sterilized with 70% alcohol for 1 min. The ethanol is
drained and the seeds are covered with a 60% sodium hypochlorite (bleach) solution
for 10 min with 2 or 3 drops of Tween 20.

16. 6. The bleach solution is poured off and seeds are rinsed five times with sterilized double
distilled water. Seeds are transferred into sterile petri dishes and then dissected
carefully.
7. Dissection can be achieved in one of the following two ways-
a. From the embryo end, hold the caryopsis with the forceps, and cut the brush
end of the caryopsis using a scalpel. Tilt the open end of the caryopsis toward the
microscope with the forceps. The embryo is a small white or translucent structure within
the caryopsis. Since there is no solid endosperm present, it should be fairly easy to see if
there is an embryo present.
b. To open the caryopsis is to steady the caryopsis with forceps or scalpel and use
another set of forceps to grab the seed coat 2/3 of the way from the embryo end and pull
back exposing the embryo.

17. Harvesting pollinated and hormone
treated wheat heads after 16 days
Embryo rescue under stereo microscope
Removal of hybrid seeds from wheat-
maize hybrid heads
Sterilization of wheat- maize hybrid seeds

18. 5.6 Regeneration of embryos
1. After excision, the embryos are placed in ½ strength Murashige–Skoog (MS) or
B5 basal medium.
2. The concentration of sucrose supplemented in the media is the major element
affecting the germination of the rescued embryos.
3. The embryos are placed in a dark incubator at 23 C for 1–2 weeks.
4. Once good roots and shoots form, the small plantlets are transferred to a lighted
incubator at 23 C . The lighted incubator is maintained using a 16 h day and 8 h
night photoperiod.
5. Sometimes very small embryos are rescued and these embryos do not grow in
normal hormone-free media. Those small embryos are placed in hormone media
fortified with kinetin and phenyl acetic acid (PAA).

19. Regeneration of rescued embryos in dark
incubator
Regenerated plants with well- developed
roots and shoots in a lighted incubator

20. 5.7 Vernalization of haploid plantlets for winter wheat
1. Once the regenerated plants show good root and shoot development in the tubes
we place them in a refrigerated chamber at 4 C for 8 weeks.
2. We transfer haploid plants to potting soil in flat trays after 8 weeks and treat them
with colchicine solution when plants become healthy.
3. Vernalized plants are planted into a soil medium with good nutrition in a flat
having 48–96 cells.
4. Once plants reach the 5–6 leaf stage, we remove them from the soil and wash the
roots to remove the soil. The roots are then trimmed to about 1 cm in length prior
to dipping into the colchicine solution.

21. 5.8 Colchicine Treatment
1. We treat the plants with colchicine after vernalization for winter wheat.
2. Colchicine is an alkaloid obtained from Colchicum species.
3. Colchicine disrupts mitosis by inhibiting formation of spindle fibers and
disturbing normal polar chromosomal migration, resulting in chromosome
doubling (Jensen 1974).
4. Treatment is done at the 5–6 leaf stage. Colchicine treatment is done under a
fume hood and plants are given overnight treatment (14–15 h) under dark.
5. The optimal colchicine treatment should have a high rate of embryo germination
and a high plant survival rate, with a high rate of chromosome doubling.
6. Roots of rinsed plants are kept in tap water for 2–3 h before planting them into
pots or soil beds.

22. Planting chromosome- doubled plants
in a soil bed in the greenhouse
Wheat spike with seeds in chromosome
doubled plants

23. 6. Chromosome elimination????
• Chromosome elimination is a powerful tool in the production of haploid plants. It
is achieved by conducting wide interspecific crosses.
• Ho and Kasha who first used this technology and discovered that certain
chromosomes carry genetic elements that account for genetic stability in
interspecific crosses.
• Various theories have been proposed to explain the mechanism of Chromosome
elimination could be caused by the difference in timing of mitotic processes
(Gupta 1969), the genomic balance (Kasha and Kao 1970) and the failure of the
chromosome to initiate or to complete either congregation at metaphase or
migration to the poles at anaphase (Bennett et al., 1976).

24. • Ho and Kasha developed chromosome elimination by emasculating Hordeum
vulgare and pollinated it with Hordeum bulbosum.
• After pollination, the formation of embryos occurred with approximately 68.5%
being haploid and a few diploid hybrids and aneuploids.
• In hybrid embryos between wheat and maize, the maize chromosome to be
eliminated were peripherally located on the metaphase plates and lagged behind
the what chromosomes at anaphase (Laurie and Bennett 1989).

25. 7.References
• Barclay, I.R. 1975. High frequencies of haploid production in wheat (Triticum
aestivum) by chromosome elimination. Nature (London) 256: 410-411.
• Bennet, M. D., Barclay, I. R. and Finch, R. A. 1976. The time rate and
mechanism of chromosome elimination in Hordeum hybrids. Chromosoma 54,
175-200.
• Blakslee, A. F., Belling, J. F. M. and Berger, A. D. 1922. A haploid mutant in the
jimson weed Datura stramonium. Science 55: 646.
• Chaudhary, H. K., Singh, S. and Sethi, G. S. 2002. Interactive influence of wheat
and maize genotypes on haploid induction in winter x spring wheat hybrids.
Journal of Genetics & Breeding 56: 259-266.
• Matzk, F and Mahn, A. 1994. Improved techniques for haploid production in
wheat using chromosome elimination. Plant Breeding 113:125-129.

26. • Suenaga, K. 1994. Doubled haploid system using the intergeneric crosses
between wheat (Triticum aestivum) and maize (Zea mays). Bulletin of National
Institute of Agrobiological resources 9: 83-139.
• Laurie, D.A. and Bennet, M.D. 1988. The production of haploid wheat plants
from wheat x maize crosses. Theoretical and Applied Genetics 76: 393-397.
• Chaudhary H.K., Sethi G.S., Singh S., Pratap A. & Sharma S. (2005). Efficient
haploid induction in wheat by using pollen of Imperata cylindrica. Plant Breeding
124(1): 96-98.
• Yamamoto, M. & Mukai, Y. (1989).Application of fluorescence in situ
hybridization to molecular cytogenetics of wheat. Wheat Inf. Serv. 69:30-32.

27. Thank You.