cskhpkv palampur lpu.... shoolini university

1. PROJECT-SEMINAR
ON
PRODUCTION OF HAPLOID WHEAT PLANTS FOLLOWING WHEAT X MAIZE
AND WHEAT X IMPERATA CYLINDRICA METHODS
OF CHROMOSOME ELIMINATION TECHNIQUE
Under the guidance of
Dr. H.K. Chaudhary
Prof. & Incharge
Molecular Cytogenetic and Tissue Culture Lab
Department Of Plant Breeding & Genetics
CSK HPKV Palampur-176062
Himachal Pradesh
SAMPY DUGGAL
REG. NO.1040070064
BTECH. BIOTECH.

2. 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 upgradation of bread 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 programmes (Sunega et al, 1994). Amongst the available
techniques for the development of haploids and doubled haploids in bread wheat,
chromosome elimination approach following wheat x maize system is one of the most
efficient approach (Laurie and Bennett, 1988).
- Inter-generic hybridization of wheat with maize and imperata cylindrica leads to the
development of haploid wheat embryos, because the maize chromosomes in the hybrid zygote
are rapidly eliminated in the first few cycles of cell division due to the failure of their
centromeres to attach to the spindle of wheat origin during meiosis.

3. INTRODUCTION CONTINUE…
- Further, doubled haploidy breeding is the fastest way of combining and fixing the desirable
features of two diverse wheat genotypes into a common genetic background.
- Double haploid breeding serves as novel tool in wheat improvement by providing instant
homozygosity which leads to fixation of desirable characters.
- The production of wheat haploid plants through wide hybridization followed by chromosome
elimination was first used successfully by crossing wheat with Hordeum bulbosum, which is
commonly reffered to as the bulbosum technique (Barclay, 1975).
- The chromosome elimination approach following wheat x maize system has been
successfully utilized for the production of doubled haploids from the winter x spring hybrids
(Chaudhary et al., 2002 and Sharma et al., 2005).
- The chromosome elimination approach following wheat x imperata cylindrica system has
been successfully utilized for the production of haploid wheat plants (chaudhary et al ., 2005).

4. AIM
- Production Of Haploid Wheat Plants Following Wheat X Maize And Wheat
X Imperata Cylindrica Method Of Chromosome Elimination Technique.
OBJECTIVE
- To check the relative efficiency of the maize and imperata cylindrica
mediated system of haploid induction in bread wheat.
- To acquire technical know how about the protocol of wide hybridization in
wheat genotypes with maize and cogon grass (imperata cylindrica).

5. MATERIAL USED
Different Genotypes Of Wheat Crop :
1. Pure line of wheat : sonalika
2. F2s :
a) DH100 X DH40.
b) DH40 X DH 5.
c) DT123 X HPW249.
Material Used For Pollination With Wheat Varieties :
1. Maize (zea mays).
2. Cogon grass ( imperata cylindrica ).

6. METHODOLOGY
Wheat Wheat
Variety A Variety B
F1
F2
F3
1.CONVENTIOANL BREEDING ->
F4
F5
F6
Homozygous Population
4 year Testing
New variety

7. 2.WHEAT X IMPERATA CYLINDRICA : newly invented system -
parent 1 parent 2
Wheat or triticale X wheat
F1 hybrid X imperata cylindrica
Zygote
Genomic in situ hybridization
Chromosome elimination at 1st mitotic division
Wheat Haploid
100% Homozygisity

8. PROTOCOL USED :-
1. EMASCULATION OF THE WHEAT SPIKES :

9. COLLECTING POLLENS OF MAIZE AND IMPERATA CYLINDRICA FROM FIELDS AND POLYHOUSE

10. 2. POLLINATION OF THE EMASCULATED WHEAT SPIKES :

11. 3. Injecting 2,4-D in the uppermost internode of the pollinated wheat
Spikes for 3 days

12. 4. Spikes harvested after 18-20 days of pollination.
Before harvesting After harvesting

13. 5. Sorting of Psuedoseeds which have embryo carrying seeds.

14. 6. Embryo floating in Psuedoseed.

15. -> Preparation of MS media.

16. 7. Embryo separated after the dissection of pseudo seed .

17. 8. Embryo being rescued and cultured on MS medium .

18. 9. Regeneration of embryo in dark.

19. 10. Shifting of embryo to the light.

20. 11. Regenerating embryo after shifting intoo light.

21. 12. Regenerating haploid plantlet.

22. 13. Cytology of the root of haploid pantlet.

23. 14. Slide of root of haploid pantlets containing 21 chromosomes.

24. RESULTS -
1. Emasculation and pollination of the different genotypes of wheat spikes
• 25 spikes of cross DH100 X DH40 , 10 spikes of pure line of wheat : sonalika,15 spikes of DT123
X HPW249 and 8 spikes of DH40 X DH5 are raised in the experimental farm of the Department of
Plant Breeding and genetics were emasculated during march and April, 2011. These spikes were
pollinated on the following day with freshly collected pollen of maize genotype growing in the
polyhouse.
• 23 spikes of DH100 X DH40 , 9 spikes of pure line of wheat:sonalika ,17 spikes of DT123 X
HPW249 and 10 spikes of DH40 X DH5 were emasculated and pollinated with the freshly collected
pollen of cogon grass (imperata cylindrica) nearby the fields of wheat.
2. Hormonal treatment
To enhance the seed and embryo formation, the spikes were injected with 100 ppm 2,4-D at the
uppermost internode after 24 hrs of pollination. The injections of 2,4-D were repeated after 48 and
72 hrs of pollination.
3. Harvesting of the spikes and sorting of the seeds
The spikes were harvested 18-20 days after pollination. In the lab the seeds were detached from the
spikes and were counted. In 1015 pollinated florets 686 seeds formed i.e. there was 67.5 per cent
seed formation in case of wheat x maize,and in case of wheat x imperata cylindrica : in 1004
pollinated florets 734 seeds formed i.e. there was 73.1 per cent seed formation . These pseudo seeds
were then observed under light to separate the embryo carrying seeds. Out of 686 seeds formed 177
embryo carrying seeds in case of wheat x maize system were separated i.e. 26 per cent because of
different genotypes of wheat and in case of wheat x imperata cylindrica out of 734 seeds formed
305 embryo carrying seeds were separated i.e. 41.5 per cent embryo carrying seeds.

25. RESULTS CONTINUE….
4. Embryo rescue and culturing
The embryo carrying seeds were washed with autoclaved distilled water and then surface
sterilized by giving 0.1% Hgcl2 treatment for two minutes. After Hgcl2 treatment, seeds were
again washed with water 2-3 times to remove Hgcl2 solution. The embryo was excised out
from the seeds with the help of sterilized forceps in the aseptic conditions and was cultured on
the medium.
5. Shifting to the growth Room
The cultured embryos were then given cold treatment at 40C for 24 hrs and then transferred to
the dark room in the growth chamber for initiation of roots and shoot. They were observed
after one week and the regenerated embryos were shifted to light for further growth. Out of
177 embryos, 80 embryos get regenrated i.e. 46 per cent embryos regenerated in case of
wheat x maize and out of 305 embryos, 191 get regenerated i.e. 62.6 per cent regenrated in
case of wheat x imperata cylindrica.

26. . Relative efficiency of maize and imperata cylindrica mediated system of doubled haploidy breeding in
bread wheat :
s.no. genotypes Florets pollinated Florets pollinated Psuedo- Pseudo-seeds Embryo Embryo Regener-ation Regener-ation in
with with imperata seeds Formed in case of Formed Formed in case of Case of
maize cylindrica Formed in case imperata in case of in case of Maize (%) Imperata
of maize cylindrica maize imperata Cylindrica
(%) (%) (%) Cylindrica (%)
(%)
1. DH100 470 465 329 349 102 157 45 94
X (70%) (75%) (31%) (45%) (44%) (60%)
DH40
(F2)
2. SONALIKA 210 190 132 142 36 58 18 40
(PURE (63%) (74.7%) (27%) (41%) (51%) (69.3%)
-LINE
OF
WHEAT )
3. DT123 225 237 150 163 14 51 6 32
X (66.7%) (69%) (9%) (31.2%) (40.1%) (62.7%)
HPW249
(F2)
4. DH40 110 112 75 80 25 39 11 25 (63.7%)
X (68.3%) (71.3%) (34%) (49%) (42%)
DH5
(F2)
TOTAL 1015 1004 686 734 177 305 80 191
(67.5%) (73.1%) (26%) (41.5%) (46%) (62.6%)

27. GRAPH FOR THE PSUEDOSEED FORMED IN MAIZE AND IMPERATA CYLINDRICA
(%)

28. GRAPH FOR THE EMBRYO FORMED IN MAIZE AND IMPERATA
CYLINDRICA (%)

29. GRAPH FOR THE REGENERATION IN MAIZE AND IMPERATA CYLINDRICA(%)

30. Composition of Murashige and Skoog medium (1962) stock solution
S.No. Strength Salts Quantity Use Amount in culture (mg/l)
ml/l
1.(2L) X 20 Ammonium nitrate 66.0g 50 1650
Potassium nitrate 76.0g 1900
Potassium dihydrogen phosphate 6.8g 170
Boric acid 0.248g 6.2
Manganese sulphate 0.892g 22.3
Zinc sulphate 0.344g 8.6
Potassium iodide 0.033g 0.825
Copper sulphate* 1.0ml 0.025
Cobalt chloride* 1.0ml 0.250
Sodium molybdate* 1.0ml 0.025
2.(1/2L) X 50 Calcium chloride 11 20 440
3.(1/2L) X 50 Magnesium sulphate 9.25g 20 370
4.(1L) X 100 Ferrous sulphate 2.78g 10 27.8
Disodium EDTA 3.728 37.28
5.(1L) X 100 Thiamine HCl 0.010g 10 0.1
Nicotinic acid 0.050g 0.5
Pyridoxine HCl 0.050g 0.5
Glycine 0.200g 2
6. Myoinositol 100
7. Sucrose 30000
8. Agar 8000
9. Glutamine 150
10. Kinetin** 0.5
*Dissolve 100mg of copper sulphate and cobalt chloride and 1 g of sodium molybdate in 100 ml of water
separately.
Than take 1ml each and add to stock number one.
**Dissolve 0.1g in 1N solution of NaOH (5 ml) & Water (95 ml)

31. Composition of MS medium (1 litre - working solid medium)
S.No. Constituents Quantity
1. Sucrose 30.00 gm
2. Agar- agar 8.00 gm
3. Myo- inositol 0.10 gm
4. Glutamine 0.15 gm
5. Kinetin 0.50 ml
6. Stock solution 1 50.00 ml
7. Stock solution 2 20.00 ml
8. Stock solution 3 20.00 ml
9. Stock solution 4 10.00 ml
10. Stock solution 5 10.00 ml

32. Composition of MS medium (1 litre - working liquid medium)
S.No. Constituents Quantity
1. Glutamine 0.15 gm
2. Myo-inositol 0.10 gm
3. IBA* 2.00 ml
4. NAA* 2.00 ml
5. Stock solution 1 50.00 ml
6. Stock solution 2 20.00 ml
7. Stock solution 3 20.00 ml
8. Stock solution 4 10.00 ml
9. Stock solution 5 10.00 ml
*Dissolve 100 mg in 5 ml 1/2N NaOH (heat slightly in test tube
till complete dissolution)

33. DISCUSSION
• Instant development of the homozygous lines.
• Shortening the breeding cycle.
• Development of the various varieties in the shortest period of time.
• Quick and precise method for the gene mapping populations
• Easy in vitro selection for the complex traits like drought and cold
tolerance.
• Easy to trigger the alien gene which develope transgenics.
• Smaller populations are required for selection.

34. CONCLUSIONS
• Imperata cylindrica is better than that of maize because :
a) High frequency of haploid embryos recovered.
b) No work raise for polyhouse for offseason maize.
c) We get the more percentage of embryo regeneration in case imperata
than that of maize , which is 62.6% (imperata cylindrica) and in case of
maize is 46%.
d) We can easily get the cogon grass (imperata cylindrica) around the
fields in maximum numbers.

35. REFERENCES
• Barclay, I.R. 1975. High frequencies of haploid production in wheat (Triticum aestivum)
by chromosome elimination. Nature (London) 256: 410-411.
• 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.
• 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). Efficienthaploid
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.

36. THANK YOU !!