The green revolution dwarfing genes Rht1 &Rht2 are found in indian wheat cultivars.The dwarfing genes lead to a no. of benefits.

1. Molecular & Physiological Evidence
of Rht Genes Expression in Few
Indian Wheat Cultivars

2. WHEAT( Genus :Triticum )
• Staple crop
• Wheat production must increase to meet the increasing demand
• Species of Triticum are grouped into three ploidy
classes:
1. Diploid (14)
2. Tetraploid (28)
3.Hexaploid (42)
• Common wheat (T. aestivum) is an allohexaploid : AABBDD

3. One strategy is to increase wheat productivity by optimizing
plant architecture:
• Determined significantly by stem elongation
•Decrease in stem stature increases yield:
Reduces risk of lodging
More fertile florets per spikelet
Increase in partitioning of assimilates to the grain
Increase in harvest index
Increased grain numbers
•Decrease in stem stature is Achieved by:
Introduction of the "Dwarfing genes "

4. Dwarfing Genes Produce More
Active DELLA Proteins

5. GA‐DELLA Signalling Mechanism
Dwarfing gene
present
Normal wheat

6. DELLA Protein
Rht genes cause changes in this
domain

7. Dwarfing gene Mechanism
Rht‐B1b ; Rht‐D1b
Single nucleotide substitutions that introduce
premature stop codons in the N‐terminal coding
region
Rht‐B1c
Cause an intragenic insertion, in‐frame 90‐bp
insertion in the transcript and a predicted 30‐
amino acid insertion within the highly conserved
amino‐terminal DELLA domain
Rht‐B1d ;Rht‐B1e
Introduce premature stop codons within the
amino‐terminal coding region
Rht‐D1c
Overexpression of the semidwarfing Rht‐D1b
allele

8. MATERIALS & METHODOLOGY

9. Plant young leaves samples were taken from wheat genotypes :
1. WH1105
2. HD3086
3. C323
4. C591
5. CB313
6. CB314
7. CB315
8.CB318
9. CB320
10.CB322
11.CB324
12.CB273
13. PBW321
14. DBW88
15. 5218
16. 5219.

10. DNA Extraction BY CTAB Method

11. Small pieces of young leave samples + 800µl CTAB buffer in pestle
and mortar
↓
Crush and then put the green soup so obtained in eppendorf
↓
Put on water bath at temp. 65 ̊Cfor 1 hour
↓
Add chloroform: isoamyl alcohol (24:1) (800 µl)
↓
Place on revolver for 1 hour
↓
Centrifuge at 10,000 rpm for 10 minutes
↓
Transfer the supernatant to clean sterile eppendorf & discard the
pellet
↓

12. Add isopropanol (800 µl) & incubate in refrigerator for 1 hour
↓
Centrifuge at 10,000 rpm for 10 minutes
↓
Discard supernatant & add 300 µl of 70% ethyl alcohol to pellet
↓
Centifuge at 10,000 rpm for 10 minutes
↓
Discard supernatant & allow pellet to dry
↓
Add 200µl of TE buffer & leave at room temperature for few
hours to allow DNA to dissolve. Tap with fingertips
↓
Store at 4 ̊C

13. QUANTIFICATION OF DNA
1.BY GEL ELECTROPHORESIS
2.BY NANODROP

14. Agarose Gel Electrophoresis
Agarose was dissolved in TBE electrophoresis buffer
The mixture was heated till the agarose dissolved completely ( became
transparent)
It was cooled down to 55-60 ̊C with constant stirring & 5 µl of EtBr
The solution was then poured into already prepared gel mould and was
left for 40-50 min for solidification; then combs are removed
In the meanwhile, DNA samples were prepared for loading by adding
loading dye, Bromophenol blue to the DNA
After the gel solidified, the DNA samples were loaded into the wells
The gel was run & then visualized under UV transilluminator

15. 2. BY NANODROP
The quantification of DNA was also conducted by using Nanodrop
spectrophotometer
1.BY GEL ELECTROPHORESIS
•For quantification of DNA 0.8%gel was used
•For quantification 2 microlitre DNA sample + 8 microlitre
bromophenol dye is loaded.
• Run at 80V/cm after loading

16. Sr.no Genotypes Quantity (ng)
1 WH1105 1626.61
2 HD3086 50
3 C323 891.48
4 C591 75.71
5 CB324 501.82
6 CB313 378.66
7 DBW88 923.18
8 CB315 262.53
9 CB320 1448.59
10 PBW321 151.30
11 CB318 128.14
12 CB314 375.23
13 CB322 324.08
14 5218 2257.90
15 5219 1000.74
16 C273 905.68

17. IN VITRO Amplification Through
Polymerase Chain Reaction (PCR)

18. Sr.
no
.
Primers
used
Sequence Tm
1. Rht-B1b F
5'-CAC TAC TAC TCC ACC
ATG TTC GAT TCT CTG-3'
59.5°C
2. Rht-B1b R
5'- GCG GCA GGA GCA
GCA GCC -3'
65.4°C
3. Rht-D1b F
5'- CCA CGA GAC GCT
GGG-3'
59.5°C
4. Rht-D1b R
5'- CCT TCC TTC TCC TCC
ACC TTG TAG-3'
58.4°C
Primers Used

19. Concentration of Various Components In PCR Reaction
Component Stock
concentration
Quantity Final
concentration
Sterile water - 7.6 µl -
PCR buffer 10X 2.0 µl 1X
MgCl2 25 mM 1.2 µl 1.5 mM
dNTP mix 1 mM 3.0 µl 0.15 mM
Forward Primer 5 µM 1.5 µl 0.375 µM
Reverse Primer 5 µM 1.5 µl 0.375 µM
Taq polymerase 5U/µl 0.2 µl 1U
Template DNA 20 ng/µl 3.0 µl 60 ng
Total 20 µl -
P
C
R
C
O
C
K
T
A
I
L

20. Steps Temperature (°C) Time (min)
Initial denaturation 95 5 min
Denaturation 94 20sec
Primer annealing 63 30 sec 42 times
Elongation 72 10 sec
Extension 72 2 min
Hold 4 ∞
Thermo-cycling Parameters for Rht B1-b

21. Steps Temperature (°C) Time (min)
Initial denaturation 95 5 min
Denaturation 94 20sec
Primer annealing 58 30 sec
Elongation 72 10 sec 42 times
Extension 72 2 min
Hold 4 ∞
Thermo-cycling Parameters for Rht- D1b

22. Visualization of PCR Products
•For visualization of PCR products 2.5% agarose gel was used.
•The PCR products were resolved by running gel at 120-150V/cm
for 3-5 hours
• Visualization in UV light

23. Effect of Rht Genes on Coleoptile Length &
Root Length In Wheat Genotypes

24. Cigar method
The germination paper was taken and a line drawn in middle of it
The seeds were placed on the line ,after wetting the paper little
bit
The paper was folded, rolled cigars were taped
The rolled cigars were wetted and laid in horizontal postion in a
tray for 3 days
After 3 days ,the cigars were placed in vertical position in a jar
The coleoptile length was measured with ruler after germination on 10th
and 15th
day for all the genotypes

25. EFFECT OF EXOGENOUS APPLICATION OF
GA3 ON COLEOPTILE LENGTH & ROOT
LENGTH IN WHEAT GENOTYPES

26. •The cigar method in this case was performed as replicate
for each variety
•But for a given variety one cigar was wetted with water
while the other with the the gibberalic acid solution (GA3)
(0.03 gm GA3 was dissolved in 1 litre of water)
• After germination in a growth chamber wetted with
water or GA3 solution ;The coleoptile length was measured
with ruler on the 10th and 15th day
•Data comparison

27. Effect of Rht Genes on Plant Height
The plant height of the genotypes were measured by a
ruler in the field and the heights were noted down.

28. Results and Discussions

29. a) Rht -B1b
b) Rht -D1b
Results of Molecular Analysis
1. WH1105 2. HD3086 3. C323 4.C591 5.CB324 6.CB313 7.DBW88
8. CB315 9.CB320 10. PBW321 11.CB318 12.CB314 13.CB322
14.CB273 15.5218 16.5219

30. Results of CIGAR Method

31. Genotypes on the 10th
day of Cigar Method

32. GENOTYPE COLEOPTILE LENGTH
(cm)
ROOT LENGHT
(cm)
HD 3086 1 7.5 13
2 10 13
3 8.5 15
4 7 12
5 8 12
MEAN 8.2 13
C323 1 12 12
2 12.5 12
3 12 10
4 14.5 9
5 10 9
MEAN 12.2 10.5
Measurements of the Coleoptile Length
and Root Length on 10th Day

33. WH1105 1 10 8.5
2 6 8
3 6.5 8.5
4 11.5 10
5 10.5 10
MEAN 8.9 9
C591 1 12 9
2 17 9.5
3 17 9.5
4 15 8.5
5 18 8
MEAN 12.9 8.9

34. Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)
1. HD3086 8.2 13
2. C323 12.2 10.5
3. WH1105 8.9 9
4. C591 64.5 8.9
Mean Coleoptile Length and Root Length
on 10th Day

35. Genotypes on the 15th
day of Cigar Method

37. GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT
(cm)
HD 3086 1 10.5 14
2 12.5 14
3 11.0 14
4 8 11
5 10 9
MEAN 10.4 12.4
C323 1 14 15
2 23 15.5
3 18 16
4 20 15.2
5 22 15
MEAN 19.4 15.34
Measurements of the Coleoptile Length and Root Length on 15th day

38. WH1105 1 15 15
2 9 11
3 14 13
4 15.5 14
5 15 12
MEAN 13.7 11
C591 1 28 15
2 21 14
3 28 14
4 30 15
5 27 15
MEAN 26.8 14.6

39. Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)
1. HD3086 10.4 12.4
2. C323 19.4 15.34
3. WH1105 13.7 11
4. C591 26.8 14.6
Mean Coleoptile Length and Root Length on 15th
Day

40. Effect of Exogenous Application of GA3 on Coleoptile
Length & Root Length in Wheat Genotypes

41. Genotypes on the 10th day of Cigar Method
(GA3 treated cigars)

42. GENOTYPE COLEOPTILE
LENGTH (cm)
ROOT
LENGHT
(cm)
HD 3086 1 7.6 14
2 7.5 13
3 7 13
4 6.8 12
5 6 10
MEAN 6.98 12.4
C323 1 21 19
2 21.5 30
3 20.6 25
4 22 29
5 19.6 15
MEAN 20.94 23.5
Measurements of the Coleoptile Length and Root Length on 10th
Day
(GA3 Treated)

43. WH1105 1 9.1 19
2 11.2 15
3 10 14
4 10.5 13
5 9 15
MEAN 9.96 15.2
C591 1 24.6 30
2 25.2 29
3 26 32
4 24.5 17
5 25.6 21
MEAN 25.18 20

44. Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)
1. HD3086 6.98 12.4
2. C323 20.94 23.5
3. WH1105 9.96 15.2
4. C591 25.18 20
Mean Coleoptile length and Root Length on
10th Day
(GA3treated)

45. Genotypes on the 15th day of Cigar Method
(GA3 treated )

46. GENOTYPE COLEOPTILE LENGTH
(cm)
ROOT LENGHT
(cm)
HD 3086 1 10.2 13.2
2 9.8 12.2
3 9 13.5
4 10 12.5
5 10.5 13
MEAN 9.9 12.8
C323 1 20 20
2 25 32
3 26.5 29
4 25.5 32
5 22.5 19
MEAN 23.9 26.4
Measurements of the Coleoptile Length and Root Length on 15th Day
(GA3treated)

47. WH1105 1 12 23.2
2 9 19
3 12 18
4 14 15.5
5 16 20
MEAN 12.6 19.14
C591 1 29 32
2 29.5 31
3 28.5 32.5
4 27.5 19
5 26.5 25
MEAN 28.2 22.9

48. Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)
1. HD3086 9.9 12.8
2. C323 23.9 26.4
3. WH1105 12.6 19.14
4. C591 28.2 22.9
Mean Coleoptile Length and Root Length on 15th
day(GA3treated)

49. Sr.No GENOTYPE COLEOPTILE LENGTH (cm) ROOT LENGHT (cm)
Water GA3 treated Water GA3 treated
1. HD3086 10.4 9.9 12.4 12.8
2. C323 19.4 23.9 15.34 26.4
3. WH1105 13.7 12.6 11 19.14
4. C591 26.8 28.2 14.6 22.9
Comparison of the Data of Water & Gibberalic Acid
Treated Cigars

50. Effect of Rht Genes on Plant Height
Sr. no. Genotype Plant height(cm)
1. C323 125
2. C591 128
3. HD3086 103
4. WH1105 100
Plant Height Data For Genotypes C323,C591,HD3086,WH1105

51. Conclusion

52. 1.Dwarfing genes PRESENT in all genotypes taken
except C323, C591
•Amplification of their DNA on PCR using dwarfing gene specific primers
• In Cigar method these genotypes showed shorter coleoptile length
• Thus, these varities are dwarf (high grain yields; less lodging)
2.Dwarfing genes ABSENT in C323, C591
•No amplification of their DNA on PCR using dwarfing gene specific primers
•In Cigar method these genotypes showed longer coleoptile length
•Plant height data from field that C323, C591 have longer plant heights
• Thus, these are not dwarf (less grain yields; more prone to lodging)

53. 3. Rht -B1b & Rht -D1b genes are the GIBBERLIN
INSENSITIVE dwarfing genes
•As No increase in coleoptile length of dwarf varities in
response to GA3application.
4. Rht -B1b & Rht -D1b genes have NO effect
on the root length
• As by cigar method No Effect of Rht -B1b & Rht -D1b
genes was found on the root length

54. THANK YOU