Micronutrient deficiencies, especially those arising from zinc and iron, pose serious human health problems. Wheat is a major source of dietary energy and protein for the growing population of the world. Its potential to assist in reducing micronutrient-related malnutrition can be enhanced via integration of agronomic fertilization practices and delivery of genetically manipulated micronutrient rich wheat varieties. Biofortified wheat emerges as a promising approach to address food security and malnutrition problems. To evaluate the performance of twenty (including two checks; Gautam and Zinc Gahun 1) biofortified bread wheat genotypes, a field experiment was conducted at the Directorate of Agricultural Research (DOAR), Parwanipur, Nepal from November 2020 to March 2021. The grain iron and zinc were analyzed by energy-dispersive X-ray fluorescence spectrometry (EDXRF) instrument, whereas grain protein was analyzed by Kjeldhal method.

1. EVALUATION OF BIOFORTIFIED BREAD WHEAT GENOTYPES FOR
YIELD AND QUALITY RELATED TRAITS
Presenter
Prabesh Koirala
Roll No – PLB-06M-2019
Department of Genetics and Plant Breeding
Agriculture and Forestry University, Rampur, Chitwan
1

2. ADVISORY COMMITTEE
Chairman – Krishna Hari Dhakal, PhD, Assistant Professor, Department of
Genetics and Plant Breeding, AFU
Member – Mr. Surya Dhungana, Assistant Professor, Department of Genetics and
Plant Breeding, AFU
Member – Mr. Rajendra Prasad Yadav, Technical Officer, National Wheat
Research Programme/NARC, Bhairahawa
2

3. Introduction
Literature Review
Materials and Methods
Results and Discussion
Conclusion
Recommendation for further Research
Acknowledgement
Outlines of Presentation
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4. • Wheat is a staple source of food for 40% of the world's population providing 20% of the daily
protein and food calories (Giraldo et al., 2019)
• Production status of wheat in Nepal
• Wheat grain contains
• Carbohydrate: 75-78%
• Protein: 12-14%
• Fat and minerals: 2%
 Zinc : 20-115 ppm
 Iron : 23-88 ppm
Introduction
4
Total cultivable land of cereals Total production of cereals Productivity
20.4% 18.8% 2.99 mt/ha
(MOALD, 2022)
(Velu et al., 2015)

5. • Developing more nutrient-dense food crops could help to reduce malnutrition and hidden
hunger problems by exploring natural genetic variation (Calderini & Ortiz-Monasterio,
2003; Pfeiffer et al., 2007)
• Only five biofortified bread wheat varieties (Zinc Gahun 1, Zinc Gahun 2 for terai and
Khumal Shakti, Himganga and Bheriganga for hill) are released, which are 20-40% higher
in Zn and Fe than commercial varieties (CIMMYT, 2020)
Introduction
5
Age groups Iron deficient Zinc deficient
Children (6-59 months) 43% (25% mildly, 18% moderately,
and >1% severely)
21%
Women (15-49 years) 34% (18% mildly, 15% moderately,
and 1% severely)
24.3%
(MoHP, 2022)
Table 1. Prevalence of Iron and Zinc deficiency in children and women in Nepal.

6. General objective
• Assess the extent of genetic variability and diversity in biofortified bread wheat genotypes
Specific objectives
• Estimate the genetic parameters and character association among the yield and quality
related traits of biofortified bread wheat genotypes
• Evaluate and identify high yielding, zinc and iron rich biofortified bread wheat genotypes
Objectives
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7. • Production status of wheat in Nepal
• Nutritional value of wheat
• Hidden hunger and its solutions
• Biofortification
• Current status of biofortified crop
• History of wheat biofortification in the world and Nepal
• Genetic parameters and their associations
Genetic variability
Correlation analysis
• Multivariate analysis
Principal component analysis (PCA)
Literature Review
7

8. • Experiment was conducted from November 2020 to March 2021 at the Directorate of
Agricultural Research (DoAR), Parwanipur, Bara, Madhesh Province
• Location: 27° 2' North Latitude, 84° 53' East Longitude
• Altitude: 115 meter above sea level
• Subtropical region
Materials and Methods
Experimental site
8
Figure 1. Location map of experimental site (DoAR, Parwanipur)
DOAR

9. Properties Content Category
PH 4.85 Acidic
Available nitrogen (%) 0.088 Low
Available phosphorus (kg/ha) 21.92 Low
Available potassium (kg/ha) 98.4 Low
Organic matter (%) 1.73 Low
Soil texture Loam
Table 2. Physical and chemical properties of soil of experimental site at Parwanipur, Bara (2020)
Materials and Methods
Physical and chemical properties of soil
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Soil and Fertilizer Testing Laboratory, Makwanpur

10. Materials and Methods
Experimental materials
S.N Genotypes S.N Genotypes
1 NL 1461 11 NL 1526
2 NL 1462 12 NL 1527
3 NL 1463 13 NL 1528
4 NL 1464 14 NL 1529
5 NL 1467 15 NL 1530
6 NL 1469 16 NL 1531
7 NL 1470 17 NL 1532
8 NL 1523 18 NL 1533
9 NL 1524 19 Gautam (Check)
10 NL 1525 20 Zinc Gahun 1 (Check)
Table 3. List of twenty biofortified bread wheat genotypes used in the experiment (2020/2021)
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Experimental plots

11. Materials and Methods
Experimental Design Alpha lattice
Number of genotypes 20
Replications 2
No. of blocks per replication 5
No. of genotypes per block 4
Individual plot size 4m*2.5m = 10 m2
Seed rate 120 gm/plot
Distance between replications 1m
Distance between two plots 0.50 m
Net area of cultivation 400 m2
Area of field trail 573.5 m2
Experimental details
Figure 3. Layout of experimental field
B1 B2 B3 B4 B5
REP
2
36 37 38 39 40
37
m
35 0.5m 34 33 32 31
26 27 28 29 30
25 24 23 22 21
1.0m
REP
1
16 17 18 19 20
15 14 13 12 11
6 7 8 9 10
5 4 3 2 1
15.5 m
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12. Materials and Methods
Agronomic practices
13
Tillage Twice
Seeding method Hand drilling
Spacing Continuous at 25 cm row spacing
Fertilizer dose 100:50:50 NPK kg/ha
Fertilizer application Half dose N and full dose P, K was applied as basal
dose, and half dose of N applied in two split doses
Irrigations Two (crown root initiation and panicle initiation stage)
Weeding Herbicide Kross (sulfo sulfuron) after first irrigation
Harvesting Manually
Seed sowing

13. Morphological and yield-related observations
Quality observations
1 Emergence count 9 Productive tillers
2 Tiller count 10 Grains per spike
3 Plant height 11 Grain weight per spike
4 Spike length 12 Thousand grain weight
5 Peduncle length 13 Grain yield
6 Flag leaf area 14 Biological yield
7 Days to heading 15 Harvest index
8 Days to maturity
16 Crude protein percentage
17 Grain zinc concentration
18 Grain iron concentration
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Materials and Methods
Morphological and yield related observations

14. Materials and Methods
Micronutrient and protein analysis
• Grain Zn and Fe content analysis: Wheat Quality Lab, Banaras Hindu University,
India with a bench-top, non-destructive, energy-dispersive X-ray fluorescence
spectrometry (EDXRF) instrument (Paltridge et al., 2012)
• Grain protein analysis: Animal Nutrition Lab, Agriculture and Forestry University,
Rampur with the Kjeldahl method (Bradstreet, 1954)
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15. Materials and Methods
Statistical analysis
• Variance analysis, correlation analysis and LSD using package “agricolae” (Felipe
de Mendiburu, 2021)
• PCA analysis using the built-in functions prcomp() was done through R-Studio
version 4.3.0
• Tables and graphs were extracted from Excel (2016) and R-Studio version (4.3.0)
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16. Results and Discussion
• Analysis of variance of quantitative traits
• Estimation of PCV and GCV, broad sense heritability (H2) and genetic advance (GA)
• Correlation Analysis
• Multivariate Analysis:
 Principal Component Analysis
17

17. Table 4. Mean performance of biofortified bread wheat genotypes for morphological traits
Genotypes
Emergence
count per m2
Tillers per
plant
Flag leaf area
(cm2)
Days to
Heading
Days to
maturity
Effective tillers
per m2
Plant
height (cm)
Spike
length (cm)
Peduncle
length (cm)
NL 1461 199 6 35.29 82 122 332 89.0 9.9 17.9
NL 1462 204 6 33.37 87 125 340 90.2 10.1 15.8
NL 1463 226 5 37.31 82 124 349 88.4 9.7 18.7
NL 1464 210 3 35.89 82 123 338 90.9 10.1 18.6
NL 1467 273 6 30.20 80 122 308 95.4 10.3 17.2
NL 1469 241 6 36.67 85 126 333 95.1 10.3 17.4
NL 1470 235 5 43.46 83 124 365 91.5 10.7 18.1
NL 1523 173 5 41.31 85 124 357 89.2 11.0 18.1
NL 1524 225 5 47.05 85 123 459 89.2 11.2 14.2
NL 1525 167 6 42.59 85 126 417 90.1 11.3 18.2
NL 1526 184 6 38.74 86 124 371 87.8 10.5 16.6
NL 1527 155 6 43.52 84 127 407 87.7 10.4 15.1
NL 1528 188 5 37.25 83 123 363 90.7 11.2 16.7
NL 1529 174 4 31.52 84 124 372 89.0 10.4 16.2
NL 1530 162 5 36.72 83 124 340 93.1 10.3 20.5
NL 1531 255 4 38.45 85 127 342 84.6 10.5 15.1
NL 1532 172 5 35.14 84 125 314 89.8 9.8 18.3
NL 1533 158 6 38.67 85 124 398 89.1 11.0 16.3
Gautam (Check) 212 5 36.24 82 125 313 94.4 11.9 21.9
Zinc Gahun 1 (Check) 182 5 42.87 82 124 371 100.8 10.8 18.0
CV 20.35 20.99 7.18 2.2 0.69 14.47 2.09 3.77 3.75
LSD 100.18 2.53 5.89 4 1.85 111.15 4.59 0.88 1.39
F-value 1.4094 1.0808 4.4979 1.7623 4.5728 1.1632 6.9743 3.8706 11.38
F-Test * * ** * ** * ** ** **
* and ** denotes significant at 5% and 1% significance level respectively 18

18. Table 5. Mean performance of biofortified bread wheat genotypes for grain yield and quality-related traits
Genotypes
Grain per
spike
Grain weight
per spike (gm)
Thousand grain
weight (gm)
Biological
yield (t/ha)
Grain yield
(t/ha)
Harvest
index
Crude
protein %
Grain Iron
content (ppm)
Grain Zinc
content (ppm)
NL 1461 41.2 1.575 35.29 7.57 3.05 0.404 12.5 45.25 39.95
NL 1462 44.6 1.538 31.42 6.61 2.66 0.411 12.3 45.95 37.15
NL 1463 45.8 1.438 27.47 7.55 2.70 0.357 11.8 44.25 37.20
NL 1464 46.1 1.663 34.43 7.77 3.13 0.403 10.8 47.40 45.10
NL 1467 47.6 1.180 24.35 6.26 2.39 0.380 12.1 40.80 37.00
NL 1469 40.9 1.412 33.89 7.57 2.72 0.359 11.8 51.05 44.30
NL 1470 50.9 1.675 30.41 7.37 2.69 0.364 10.9 58.00 35.70
NL 1523 48.0 1.160 28.49 6.50 2.27 0.349 11.5 56.00 46.90
NL 1524 45.0 1.261 29.74 6.47 2.48 0.385 11.9 44.65 42.00
NL 1525 41.0 1.350 36.17 7.13 2.75 0.385 12.7 54.15 48.50
NL 1526 44.2 1.365 30.13 7.40 2.67 0.361 12.9 53.80 32.30
NL 1527 40.7 1.368 36.96 6.91 2.79 0.407 12.6 49.15 48.20
NL 1528 44.3 1.249 27.65 6.97 2.62 0.376 12.0 43.20 36.00
NL 1529 42.7 1.255 30.08 7.07 2.59 0.367 11.3 43.65 39.90
NL 1530 42.7 1.805 31.21 7.24 2.98 0.413 11.1 37.40 37.25
NL 1531 51.0 1.443 27.88 7.11 2.57 0.362 11.9 46.70 33.60
NL 1532 46.6 1.719 36.90 7.42 2.87 0.389 11.4 55.55 39.85
NL 1533 43.9 1.344 33.87 6.44 2.56 0.398 12.0 51.35 40.55
Gautam (Check) 40.9 1.491 34.05 6.84 2.62 0.382 11.6 46.10 36.50
Zinc Gahun 1 (Check) 39.4 1.680 38.94 8.03 2.95 0.367 12.3 46.70 42.40
CV 8.49 9.75 8.91 5.64 4.17 5.6 6.82 14.22 10.12
LSD 8.38 0.32 6.16 0.87 0.24 0.04 2.3 16.08 8.67
F-value 1.5578 3.5881 3.3002 2.2852 5.1638 1.732 1.371 1.2751 2.6302
F-Test * * * * ** * ns * *
*, **, and ns denotes significant at 5% , 1% significance level and non significant respectively 19

19. Parameters
Genotypic coefficient
of variation
Phenotypic coefficient
of variation
Broad sense
heritability
Genetic advance as
percentage of mean
Emergence per square meter 8.63 22.56 0.14 6.80
Tillers per plant 4.22 21.41 0.03 1.71
Flag leaf area (cm2) 8.78 13.30 0.43 11.94
Days to heading 1.35 2.58 0.27 1.47
Days to maturity 0.79 1.26 0.39 1.02
Effective tillers per square meter 2.57 14.70 0.03 0.92
Plant height (cm) 3.60 4.16 0.74 6.42
Spike length (cm) 4.22 6.26 0.45 5.85
Peduncle length (cm) 9.81 11.05 0.78 17.95
Grains/spike 4.27 9.69 0.19 3.88
Grains weight per spike (gm) 11.08 14.77 0.56 17.14
Thousand grain weight (gm) 10.04 13.88 0.52 14.96
Biomass yield (ton/ha) 3.04 9.20 0.10 2.08
Grain yield (ton/ha) 5.73 9.69 0.34 6.97
Harvest index 3.71 6.95 0.28 4.10
Crude protein percentage 4.71 4.95 0.82 10.21
Table 6. Estimation of genotypic coefficient of variation, phenotypic coefficient of variation, broad sense
heritability, and genetic advance as percentage of mean
20

20. Discussion
• Medium PCV and GCV were observed in grain weight per spike, thousand-grain
weight, grain zinc content and grain iron content, which indicated the existence of
considerable genetic base among the genotypes and possibility of genetic improvement
through direct selection (Ghawat & Sakhare, 2010; Gupta et al., 2009)
• Plant height, crude protein content, and peduncle length have high transmissibility from
generation to generation, since they have high heritability (Ashfaq et al., 2014; Mohsin
et al., 2009)
• Maximum genetic gain can be achieved by using traits peduncle length, crude protein,
grain weight per spike, and thousand-grain weight under selection, since they have high
genetic advance (Gupta et al., 2009)
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21. EPMS TPP FLA DH DM ET PH SL PL GPS GWPS TGW BY GY HI CP% GFe GZn
EPMS 1 -0.05 -0.234 -0.106 -0.163 -.314* 0.062 -0.164 0.003 0.281 -0.036 -0.24 -0.033 -0.09 -0.109 -0.112 0.079 -0.226
TPP 1 .317* 0.188 0.071 0.166 0.213 0.08 -0.049 -0.12 -0.053 0.044 0.138 0.176 0.096 .384* 0.00 -0.143
FLA 1 0.202 .439** .625** 0.064 0.212 -0.14 0.016 0.125 0.192 .320* 0.228 -0.186 0.112 0.221 0.037
DH 1 0.227 0.248 -.413** 0.074 -.334* 0.073 -0.195 0.066 0.028 -0.075 -0.147 0.219 0.277 -0.047
DM 1 0.237 -0.109 -0.108 -0.048 -0.026 0.171 0.27 0.248 0.15 -0.175 0.108 0.126 0.007
ET 1 -0.042 0.118 -0.295 0.049 -0.031 0.077 0.086 0.069 -0.015 0.207 0.13 0.088
PH 1 0.105 .406** -.321* 0.263 0.23 0.298 0.308 -0.006 -0.091 -0.138 0.041
SL 1 0.001 -0.14 -.317* -0.146 -.412** -.439** -0.059 0.078 0.108 0.173
PL 1 -0.172 .385* 0.221 0.257 .315* 0.071 -.372* -0.038 -0.125
GPS 1 0.118 -.391* -0.053 -0.154 -0.199 -.333* 0.255 -0.197
GWPS 1 .453** .411** .568** 0.273 -0.312 0.195 -0.049
TGW 1 .431** .550** 0.212 0.139 0.239 .337*
BY 1 .870** -0.297 -0.063 -0.015 -0.21
GY 1 0.208 -0.061 -0.046 -0.106
HI 1 0.039 -0.065 0.177
CP% 1 0.028 0.046
GFe 1 .348*
GZn 1
Table 7. Pearson’s correlation coefficient among yield and quality related traits of biofortified bread wheat genotypes
EPMS = Emergence count per meter square, TPP = Tillers per plant, FLA = Flag leaf area, DH = Days to heading, DM = Days to maturity, ET = Effective tillers
per square meter, PH= plant height (cm), SL = Spike length (cm), PL = Peduncle length (cm), GPS = Grain/spike (No.), GWPS = Grain weight/spike, TGW =
Thousand grain weight, BY = Biological yield, HI = Harvest index, GY = Grain yield (ton/ha), CP% = Crude protein percentage (%), GFe = Grain iron
concentration (ppm), GZn = Grain zinc concentration (ppm)
22

22. Discussion
• Peduncle length, grain weight per spike, thousands-grain weight, biological yield, and
spike length are crucial for increasing yield, since they have significant correlation with
grain yield (Joshi et al., 2008; Kashif & Khaliq, 2004)
• Thousand grain weight is an important trait for selection, since it has significant
positive correlation with grain yield as well as grain zinc content (Velu et al., 2015)
• Grain zinc and iron concentration can be simultaneously improved through selection,
since they have significant positive correlation with each other (Velu et al., 2012;
Pfeiffer & McClafferty, 2007)
• The non-significant negative relation between grain iron content and grain zinc content
with grain yield suggests that breeding for high zinc and iron does not have significant
negative effect on grain yield potential (Velu et al., 2012) 23

23. Quantitative Traits PC1 PC2 PC3 PC4 PC5 PC6
Emergence per square meter -0.060 -0.765 -0.243 0.025 0.025 -0.247
Tillers per plant -0.241 0.049 0.144 0.105 0.815 -0.053
Flag leaf area (cm2) 0.058 -0.059 0.304 0.009 0.055 0.839
Days to heading -0.213 0.138 0.554 -0.580 0.133 0.242
Days to maturity 0.066 0.146 0.704 -0.158 0.050 0.046
Effective tillers per square meter -0.176 0.170 0.008 -0.289 0.121 0.882
Plant height (cm) 0.193 -0.047 -0.203 0.818 0.133 0.002
Spike length (cm) -0.551 0.077 0.149 0.465 -0.022 0.474
Peduncle length (cm) 0.189 0.136 0.096 0.757 -0.264 -0.375
Grains/spike -0.222 -0.569 0.133 -0.388 -0.471 -0.219
Grains weight per spike (gm) 0.776 0.200 0.084 0.161 -0.240 -0.258
Thousand grain weight (gm) 0.570 0.571 0.324 0.253 0.191 0.233
Biomass yield (ton/ha) 0.928 -0.142 0.083 0.160 -0.085 0.045
Grain yield (ton/ha) 0.890 0.367 -0.153 0.075 -0.055 -0.068
Harvest index 0.154 0.784 -0.333 -0.146 0.072 -0.171
Crude protein percentage -0.060 0.069 0.012 -0.181 0.893 0.172
Grain iron content 0.045 -0.135 0.848 -0.030 0.048 0.192
Grain zinc content 0.047 0.443 0.202 0.147 0.045 0.498
Eigen Value 4.274 3.72 2.113 1.772 1.337 1.089
Table 8. Principal component analysis for yield and quality related traits of biofortified bread wheat genotypes
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24. S.N PC1 PC2 PC3 PC4 PC5 PC6
1
Biomass yield
(ton/ha)
Harvest index Grain iron
content
Plant height
(cm)
Crude protein
percentage
Effective tillers
per square meter
2
Grain yield
(ton/ha)
Thousand grain
weight (gm)
Days to
maturity
Peduncle
length (cm)
Tillers per
plant
Flag leaf area
(cm2)
3
Grain weight per
spike (gm)
Days to
heading
Grain zinc
content
4
Thousand grain
weight (gm)
Table 9. Principal component analysis scores for the traits having values >0.5 in each principal component
Results
• Yield contributing traits were having the highest variation in PC1 followed by PC2. And, PC3,
PC5, and PC6 should be considered for the quality-related variations
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25. Conclusion
• The present study found significant variability for yield and quality related
traits, which suggests wide genetic background and can be used for future
biofortified bread wheat improvement programs
• Biofortified bread wheat genotypes i.e. NL 1461, NL 1464 and NL 1527 are
promising genotypes, which could be used as parents for future biofortified
bread wheat breeding program
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26. Recommendation for Further Research
• Further multi-environment evaluation and stability analysis could be performed to
generate strong proof for their superiority
• Additionally, research could be conducted on how these genotypes respond to various
biotic and abiotic stresses, as well as evaluation of flouring and baking quality
27

27. Acknowledgement
Advisory Committee
Department of Genetics and Plant Breeding (Entire Team)
Agriculture and Forestry University, Rampur, Chitwan
Dean, Faculty of Agriculture, and Entire Team
Directorate of Research and Extension
Directorate of Agricultural Research (DOAR), Parwanipur, Bara.
National Wheat Research Programme (NWRP), Bhairahawa
Seniors, colleagues and juniors.
28

28. Thank You!
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