Polyploidy Hybrids from Wide Crosses Between Hordeum Vulgare and H

Mississippi State University Scholars Junction

Theses and Dissertations Theses and Dissertations

1-1-2018

Polyploidy Hybrids from Wide Crosses between Hordeum Vulgare and H. Bulbosum for Improving Salinity Tolerance Using Embryo Rescue

Abullah Hassn Mohammed

Follow this and additional works at: https://scholarsjunction.msstate.edu/td

Recommended Citation Mohammed, Abullah Hassn, "Polyploidy Hybrids from Wide Crosses between Hordeum Vulgare and H. Bulbosum for Improving Salinity Tolerance Using Embryo Rescue" (2018). Theses and Dissertations. 3560. https://scholarsjunction.msstate.edu/td/3560

This Dissertation - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Template B v3.0 (beta): Created by J. Nail 06/2015

Polyploidy hybrids from wide crosses between Hordeum vulgare and H. bulbosum for

improving salinity tolerance using embryo rescue.

By TITLE PAGE Abdullah Hassn Mohammed

A Dissertation Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Plant and Soil Sciences in the Department of Plant and Soil Sciences

Mississippi State, Mississippi

December 2018

2018

Polyploidy hybrids from wide crosses between Hordeum vulgare and H. bulbosum for

improving salinity tolerance using embryo rescue.

By APPROVAL PAGE Abdullah Hassn Mohammed

Approved:

______Brian S. Baldwin (Major Professor)

______W. Brien Henry (Committee Member)

______Shaun R. Broderick (Committee Member)

______J. Brett Rushing (Committee Member)

______Ebrahiem M. Babiker (Committee Member)

______Michael S. Cox (Graduate Coordinator)

______George M. Hopper Dean College of Agriculture and Life Sciences

Name: Abdullah Hassn Mohammed ABSTRACT Date of Degree: December 14, 2018

Institution: Mississippi State University

Major Field: Plant and Soil Sciences

Major Professor: Brian S. Baldwin

Title of Study: Polyploidy hybrids from wide crosses between Hordeum vulgare and H.

bulbosum for improving salinity tolerance using embryo rescue.

Pages in Study 348

Candidate for Degree of Doctor of Philosophy

Salinity is a critical challenge facing productivity of crops around the world, causing major reduction in growth, yield, and quality. It is necessary to produce varieties with the ability to tolerate salinity. However, the lack of genetic variation among H. vulgare genotypes prevents progress in developing salt tolerant varieties. H. bulbosum is a source of tolerance to stress conditions. Consequently, five accessions of domestic barley and six of wild barley were used in this study. Accessions were screened for salinity tolerance. Genotypes 7, 9, and 10 germinated at 2% NaCl. Lines of H. vulgare showed reduction of root and shoot length greater than H. bulbosum. Crosses were made between diploid and tetraploid H. vulgare ♀ and tetraploid H. bulbosum ♂. Immature embryos were rescued. Murashige and Skoog medium was found to be generally better for most crosses.

Number of successful crosses varied among families. Female 5, for diploid crosses, and female 2, for tetraploid crosses, have high GCA and compatibility with bulbosum males, and 17.9% and 17.6% of their progeny exceeding the mean grain yield,

respectively. Parent 9 had also high GCA and compatibility with vulgare parents (2x), and its progeny seem to exceed the mean in many cases with most families.

During germination screening, progeny of diploid females 1 and 2 were found to be highly desirable for saline tolerance. Among the tetraploid crosses, Family 1 had greatest percentage of superior progeny (18.8%), while Families 2 and 3 had greatest number of superior individuals (8 and 7, respectively).

For seedling growth, diploid Families 2 and 3 crosses had the greatest shoot dry weight and tolerant saline index (SSI<1). Families 2 and 4 of tetraploid crosses had 12 of

39 and eight of 26 crosses show greatest shoot dry weight and tolerance as measured by

SSI, respectively.

Final germination percentage (FG%) showed positive association with plant height, while associating negatively with tiller number, fertility, cSW, and grain yield. In diploid crosses, FG% associated positively with tiller number. Shoot dry weight showed negative association with plant height, while it associated positively with tiller number, fertility, cSW, and grain yield.

DEDICATION

This dissertation is dedicated to my mother and father, whose support, love, and well wishes have made this work possible. This is also dedicated to my wonderful and supportive family: my loving wife, Amel, and amazing sons, Abdulrahman, Mohammed,

Ammar, and Anmar. Furthermore, I dedicate this work to my brothers, sisters, and all other family members for the prayers and sacrifices they made for me.

اإلهداء

- إلى نبع الحنان الدافئ: )أمي(، والى من أضاء في نفسي شعاع ال ح ب االول للعلم والمعرفة: )أبي(.. برا ب ه ما.

- إلى من شد هللا تعالى بها ازري م خففا بها عنائي: زوجتي المخلصة )أمل( .. مودة و ح با .

- إلى غدي المشرق وفلذات كبدي: أوالدي )عبد الرحمن، محمد، عمار، أنمار(.

- إلى رفاق دربي في صفوتي وشقوتي: إخوتي وأخواتي.

- إلى كل من ارشدني، وكان عونا لي في اتمام هذا العمل حتى استوى على سوقه.. ب را ووفا ء ، واعترافا بالجميل.

- إلى كل من أحبني في هللا تعالى واحببته فيه ... إكراما .

أهدي ثمرة جهدي هذا...

ACKNOWLEDGEMENTS

My sincere gratitude to my major professor Dr. Brian S. Baldwin for his invaluable advice, invariable encouragement and guidance. His patience and kindness will never be forgotten. I would like to thank my dissertation committee members, Dr. W.

Brien Henry, Dr. Shaun R. Broderick, Dr. J. Brett Rushing, and Dr. Ebrahim M. Babiker for their advice and encouragement.

I am also grateful to GRIN, National Small Grains Collection/USDA-ARS for providing the seed for my study. I would like to express my thanks to Dr. Timothy A.

Rinehart and USDA-ARS- Thad Cochran Southern Horticultural Research Laboratory at

Poplarville, MS for using flow cytometry. A special thanks to the Department of Plant and Soil Sciences and the College of Agriculture and Life Sciences at Mississippi State

University for supporting and providing the facilities to conduct the research. I am grateful to the Iraqi Ministry of Higher Education and Scientific Research for financial support.

I greatly appreciated the time and help provided by Dr. Jesse Morrison and graduate students, Eric Billman, Michael Nattrass, John McLemore, Sarah Montgomery,

Salah Jumaa, Riyadh Mohsin, Firas Alsajri, Firas Jasim and Omar Ali. This gratitude is extended to all those who have helped me through every step of my education.

Finally, my greatest expression of gratitude goes to all my family and relatives who supported and encouraged me on the journey to my educational success.

iii

TABLE OF CONTENTS

DEDICATION ...... ii

ACKNOWLEDGEMENTS ...... iii

LIST OF TABLES ...... viii

LIST OF FIGURES ...... xxvii

CHAPTER

I. INTRODUCTION ...... 1

II. LITERATURE REVIEW ...... 4

Barley Breeding ...... 4 Flower Structure ...... 12 Commercial Cultivars ...... 14 Iraq ...... 14 IPA 99 ...... 14 IPA 265 ...... 15 Tuwaitha ...... 15 Shuaa ...... 15 United States ...... 16 Winter Barley ...... 17 Maja ...... 17 Charles ...... 18 Spring Barley ...... 18 Goldeneye ...... 18 Merit 57 ...... 19 Salt Tolerance ...... 19 Tissue Culture to Overcome Sexual Incompatibilities ...... 23 Mechanics of Chromosome Elimination ...... 27 REFERENCES ...... 28

III. PRELIMINARY SCREENING FOR SALT TOLERANCE ...... 39

Introduction ...... 39 Materials and Methods ...... 40 Salt tolerance at germination stage: ...... 41 iv

Salt tolerance at seedling growth stage: ...... 42 Results and Discussion ...... 43 Salt tolerance at germination stage: ...... 43 Salt tolerance at seedling growth: ...... 47 Conclusion ...... 52 REFERENCES ...... 53

IV. INTERSPECIFIC CROSSES AND EMBRYO RESCUE TO OVERCOME SEXUAL INCOMPATIBILITIES ...... 56

Introduction ...... 56 Materials and Methods ...... 58 Crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum: ...... 58 Interspecific crosses: Diploid H. vulgare x diploid and tetraploid H. bulbosum ...... 58 Embryo culture of resulting progeny ...... 60 Crosses between tetraploid H. vulgare and tetraploid H. bulbosum ...... 61 Results and Discussion ...... 62 Crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum: ...... 62 Crosses between tetraploid H. vulgare and tetraploid H. bulbosum ...... 70 Conclusion ...... 78 REFERENCES ...... 79

V. MORPHOLOGICAL CHARACTERISTICS OF BARLEY PROGENIES...... 82

Introduction ...... 82 Materials and Methods ...... 84 Progenies of interspecific crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum: ...... 85 Flow Cytometry: ...... 85 Colchicine Treatment: ...... 86 Progenies Growouts: ...... 87 Progenies of interspecific crosses between tetraploid H. vulgare and tetraploid H. bulbosum: ...... 88 Results and Discussion ...... 89 Genotypes of diploid H. vulgare crosses ...... 89 Conclusion ...... 114 Genotypes of tetraploid H. vulgare crosses ...... 115 Conclusion ...... 143 REFERENCES ...... 145

VI. EVALUATING SALINITY TOLERANCE IN PROGENY OF DOMESTIC (HORDEUM VULGARE) AND WILD BARLEY (H. BULBOSUM) AT GERMINATION STAGE ...... 150

Introduction ...... 150 Materials and Methods ...... 151 Results and Discussion ...... 154 Genotypes of diploid H. vulgare crosses ...... 154 Genotypes of tetraploid H. vulgare crosses ...... 166 Conclusion ...... 182 REFERENCES ...... 183

VII. EVALUATING SALINITY TOLERANCE IN PROGENY OF DOMESTIC (HORDEUM VULGARE) AND WILD BARLEY (H. BULBOSUM) AT SEEDLING GROWTH STAGE ...... 186

Introduction ...... 186 Materials and Methods ...... 188 Results and Discussion ...... 191 Genotypes of diploid H. vulgare crosses ...... 191 Genotypes of tetraploid H. vulgare crosses ...... 205 Conclusion ...... 222 REFERENCES ...... 224

VIII. SELECTION INDEX ...... 227

Introduction ...... 227 Materials and Methods ...... 227 Results and Discussion ...... 228 Genotypes of diploid H. vulgare crosses ...... 228 Genotypes of tetraploid H. vulgare crosses ...... 231 REFERENCES ...... 237

APPENDIX

A. CROSSING CAPACITY BETWEEN H. VULGARE AND H. BULBOSUM ...... 238

Crossing capacity of diploid H. vulgare crosses ...... 239 Percentage of germinated embryo at MS and Gamborg’s B-5 media of diploid H. vulgare crosses ...... 242 Crossing capacity of tetraploid H. vulgare crosses ...... 245 Percentage of germinated embryo at MS and Gamborg’s B-5 media of tetraploid H. vulgare crosses ...... 248

B. GENOME EVALUATION OF BARLEY GENOTYPES VIA FLOW CYTOMETRY ...... 251 vi

C. MEAN, STANDARD DEVIATION, AND COEFFICIENTS OF VARIATION OF BARLEY GENOTYPES FOR DIFFERENT QUANTITATIVE CHARACTERISTICS ...... 267

Mean, standard deviation, and coefficients of variation of barley genotypes (2x H. vulgare crosses) for different quantitative characteristics ...... 268 Mean, standard deviation, and coefficients of variation of barley genotypes (4x H. vulgare crosses) for different quantitative characteristics ...... 274

D. MEAN FINAL GERMINATION PERCENTAGE, CORRECTED GERMINATION PERCENTAGE, AND GERMINATION INDEX OF BARLEY GENOTYPE UNDER SALINITY STRESS ...... 283

Genotypes of diploid H. vulgare crosses ...... 284 Mean final germination percentage under salinity stress ...... 284 Mean corrected germination percentage under salinity stress ...... 289 Mean germination index under salinity stress ...... 295 Genotypes of tetraploid H. vulgare crosses ...... 301 Mean final germination percentage under salinity stress ...... 301 Mean corrected germination percentage under salinity stress ...... 310 Mean germination index under salinity stress ...... 319

E. MEAN SHOOT DRY WEIGHT, AND SALINITY SUSCEPTIBILITY INDEX OF BARLEY GENOTYPE UNDER SALINITY STRESS ...... 328

Genotypes of diploid H. vulgare crosses ...... 329 Mean shoot dry weight under salinity stress ...... 329 Mean salinity susceptibility index under salinity stress ...... 333 Genotypes of tetraploid H. vulgare crosses ...... 337 Mean shoot dry weight under salinity stress ...... 337 Mean salinity susceptibility index under salinity stress ...... 343

vii

LIST OF TABLES

2.1 Species in the genus Hordeum, distribution, chromosome numbers, and life form (Kant et al. 2016): ...... 5

3.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study ...... 40

3.2 Key for scoring of salt tolerance at germination for barley accessions Mano and Takeda 1998 ...... 42

3.3 Analysis of variance of the parental barley genotypes, salinity, and their interaction for root and shoot length of initial germination screening experiment ...... 43

3.4 Mean root length (mm) of parental barley genotypes after 10 days germination at 0% (0mM), 1% (171 mM), 1.5% (257 mM), and 2% (342 mM) NaCl solution of initial germination screening experiment...... 45

3.5 Mean shoot length (mm) of parental barley genotypes after 10 days germination at 0% (0mM), 1% (171 mM), 1.5% (257 mM), and 2% (342 mM) NaCl solution of initial germination screening experiment...... 46

3.6 Key for scoring salt tolerance at germination for parental barley genotypes derived from Mano and Takeda 1998 ...... 47

3.7 Analysis of variance for growth index of the parental barley genotypes at 500 mM NaCl solution of initial screening at seedling growth ...... 48

3.8 Mean growth index of parental barley genotypes at 500 mM NaCl solution of initial screening at seedling growth ...... 49

4.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study ...... 59

viii

4.2 Analysis of variance of the Family 1 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 63

4.3 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 1 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses)...... 63

4.4 Analysis of variance of the Family 2 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 65

4.5 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 2 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses)...... 65

4.6 Analysis of variance of the Family 3 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 66

4.7 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 3 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses)...... 67

4.8 Analysis of variance of the Family 4 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 68

4.9 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 4 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses)...... 68

4.10 Analysis of variance of the Family 5 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 69

4.11 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 5 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses)...... 69

4.12 Analysis of variance of the Family 1 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 70

4.13 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 1 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses)...... 71

4.14 Analysis of variance of the Family 2 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 72

4.15 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 2 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses)...... 72

4.16 Analysis of variance of the Family 3 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 74

4.17 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 3 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses)...... 74

4.18 Analysis of variance of the Family 4 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 75

4.19 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 4 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses)...... 76

4.20 Analysis of variance of the Family 5 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number...... 77

4.21 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 5 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses)...... 77

5.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study ...... 84

5.2 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 1 crosses (2x H. vulgare crosses)...... 91

5.3 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 92

5.4 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 2 crosses (2x H. vulgare crosses)...... 96

5.5 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 97

5.6 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 3 crosses (2x H. vulgare crosses)...... 101

5.7 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 102

5.8 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 4 crosses (2x H. vulgare crosses)...... 105

5.9 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 106

5.10 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 5 crosses (2x H. vulgare crosses)...... 109

5.11 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 110

5.12 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 1 crosses (4x H. vulgare crosses)...... 117

5.13 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 118

5.14 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 2 crosses (4x H. vulgare crosses)...... 122

5.15 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 123

xii

5.16 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 3 crosses (4x H. vulgare crosses)...... 128

5.17 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 129

5.18 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 4 crosses (4x H. vulgare crosses)...... 134

5.19 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 135

5.20 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 5 crosses (4x H. vulgare crosses)...... 139

5.21 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY)...... 140

6.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study ...... 152

6.2 Analysis of variance of barley genotypes (2x H. vulgare crosses), salinity, and their interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl...... 155

xiii

6.3 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 156

6.4 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 159

6.5 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 161

6.6 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 162

6.7 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 164

6.8 Analysis of variance of barley genotypes (4x H. vulgare crosses), salinity, and their interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl...... 167

6.9 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 168

6.10 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 171

xiv

6.11 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 174

6.12 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 177

6.13 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D)...... 179

7.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study ...... 188

7.2 Analysis of variance of barley genotypes (2x H. vulgare crosses), salinity, and their interaction for shoot dry weight (SDW) at 0, 100, 200, and 300 mM NaCl...... 192

7.3 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 13 genotypes of Family 1 (2x H. vulgare crosses)...... 193

7.4 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 194

7.5 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 13 genotypes of Family 2 (2x H. vulgare crosses)...... 196

7.6 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 197

7.7 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 6 genotypes of Family 3 (2x H. vulgare crosses)...... 198

7.8 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 199

7.9 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 3 genotypes of Family 4 (2x H. vulgare crosses)...... 200

7.10 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 201

xvi

7.11 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 18 genotypes of Family 5 (2x H. vulgare crosses)...... 202

7.12 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 203

7.13 Analysis of variance of barley genotypes (4x H. vulgare crosses), salinity, and their interaction for shoot dry weight (SDW) at 0, 100, 200, and 300 mM NaCl...... 205

7.14 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 20 genotypes of Family 1 (4x H. vulgare crosses)...... 206

7.15 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 208

7.16 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 45 genotypes of Family 2 (4x H. vulgare crosses)...... 209

7.17 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 211

xvii

7.18 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 27 genotypes of Family 3 (4x H. vulgare crosses)...... 213

7.19 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 214

7.20 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 32 genotypes of Family 4 (4x H. vulgare crosses)...... 216

7.21 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 217

7.22 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 22 genotypes of Family 5 (4x H. vulgare crosses)...... 219

7.23 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E)...... 220

8.1 Selection index of diploid H. vulgare crosses of all families using grain yield plant-1 (GY), plant height (PH), 100-seed weight (cSW), final germination percentage (FG%) and shoot dry weight (SDW) at 300 mM NaCl...... 229

xviii

8.2 Selection index of tetraploid H. vulgare crosses of all families using grain yield plant-1 (GY), plant height (PH), 100-seed weight (cSW), final germination percentage (FG%) and shoot dry weight (SDW) at 300 mM NaCl...... 231

A.1 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 1 crosses (2x H. vulgare crosses)...... 239

A.2 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 2 crosses (2x H. vulgare crosses)...... 239

A.3 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 3 crosses (2x H. vulgare crosses)...... 240

A.4 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 4 crosses (2x H. vulgare crosses)...... 240

A.5 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 5 crosses (2x H. vulgare crosses)...... 241

A.6 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 1 crosses (2x H. vulgare crosses)...... 242

A.7 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 2 crosses (2x H. vulgare crosses)...... 243

A.8 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 3 crosses (2x H. vulgare crosses)...... 243

A.9 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 4 crosses (2x H. vulgare crosses)...... 244

A.10 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 5 crosses (2x H. vulgare crosses)...... 244

xix

A.11 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 1 crosses (4x H. vulgare crosses)...... 245

A.12 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 2 crosses (4x H. vulgare crosses)...... 245

A.13 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 3 crosses (4x H. vulgare crosses)...... 246

A.14 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 4 crosses (4x H. vulgare crosses)...... 246

A.15 Mean percentage of seed set, inviable seed, abnormal, and normal germinated embryo of Family 5 crosses (4x H. vulgare crosses)...... 247

A.16 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 1 crosses (4x H. vulgare crosses)...... 248

A.17 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 2 crosses (4x H. vulgare crosses)...... 248

A.18 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 3 crosses (4x H. vulgare crosses)...... 249

A.19 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 4 crosses (4x H. vulgare crosses)...... 249

A.20 Mean percentage of ungerminated embryo, abnormal, and normal germinated embryo at MS and Gamborg’s B-5 media of Family 5 crosses (4x H. vulgare crosses)...... 250

B.1 Identification of diploid, triploid, tetraploid parameters of each barley genotypes (2x H. vulgare crosses)...... 252

B.2 Identification of ploidy level of parental barley genotypes (4x H. vulgare) after colchicine treatment...... 256

B.3 Identification of diploid, triploid, tetraploid parameters of each barley genotypes (4x H. vulgare crosses)...... 258

C.1 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 268

C.2 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 269

C.3 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 270

C.4 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 271

C.5 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 272

C.6 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 274

xxi

C.7 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 276

C.8 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 278

C.9 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 280

C.10 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) for flag leaf length (FLL), flag leaf width (FLW), chlorophyll content (CHC), awns length (AWL), spike number plant-1 (SPN/P), spike length (SL), spikelet number spike-1 (SPTN/S), and seed number spike-1 (SN/S)...... 282

D.1 Mean final germination percentage (FG%) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 284

D.2 Mean final germination percentage (FG%) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 285

D.3 Mean final germination percentage (FG%) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 286

D.4 Mean final germination percentage (FG%) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 286

D.5 Mean final germination percentage (FG%) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 287 xxii

D.6 Mean corrected germination percentage (CG%) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 289

D.7 Mean corrected germination percentage (CG%) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 290

D.8 Mean corrected germination percentage (CG%) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 291

D.9 Mean corrected germination percentage (CG%) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 292

D.10 Mean corrected germination percentage (CG%) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 293

D.11 Mean germination index (GI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 295

D.12 Mean germination index (GI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 296

D.13 Mean germination index (GI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 297

D.14 Mean germination index (GI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 298

D.15 Mean germination index (GI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 299

D.16 Mean final germination percentage (FG%) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 301

D.17 Mean final germination percentage (FG%) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 303

D.18 Mean final germination percentage (FG%) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 305

xxiii

D.19 Mean final germination percentage (FG%) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 307

D.20 Mean final germination percentage (FG%) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 309

D.21 Mean corrected germination percentage (CG%) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 310

D.22 Mean corrected germination percentage (CG%) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 312

D.23 Mean corrected germination percentage (CG%) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 314

D.24 Mean corrected germination percentage (CG%) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 316

D.25 Mean corrected germination percentage (CG%) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 318

D.26 Mean germination index (GI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 319

D.27 Mean germination index (GI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 321

D.28 Mean germination index (GI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 323

D.29 Mean germination index (GI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 325

D.30 Mean germination index (GI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 327

E.1 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 329

xxiv

E.2 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 330

E.3 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 331

E.4 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 331

E.5 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 332

E.6 Mean salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 333

E.7 Mean salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 334

E.8 Mean salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 335

E.9 Mean salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 335

E.10 Mean salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 100 and 200 mM NaCl...... 336

E.11 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 337

E.12 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 338

E.13 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 340 xxv

E.14 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 341

E.15 Mean shoot dry weight (SDW), g plant-1 of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 0, 100, and 200 mM NaCl...... 342

E.16 Mean salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 343

E.17 Mean Salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 344

E.18 Mean Salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 346

E.19 Mean Salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 347

E.20 Mean Salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 100 and 200 mM NaCl...... 348

xxvi

LIST OF FIGURES

3.1 Relationship between growth index and root length under saline conditions (1, 1.5, and 2% NaCl) of parental barley genotypes...... 50

3.2 Relationship between growth index and shoot length under saline conditions (1, 1.5, and 2% NaCl) of parental barley genotypes...... 51

5.1 Mean grain yield (GY), g plant-1 of parental genotypes and Family 1 crosses (2x H. vulgare crosses)...... 93

5.2 Mean grain yield (GY), g plant-1 of parental genotypes and Family 2 crosses (2x H. vulgare crosses)...... 98

5.3 Mean grain yield (GY), g plant-1 of parental genotypes and Family 3 crosses (2x H. vulgare crosses)...... 103

5.4 Mean grain yield (GY), g plant-1 of parental genotypes and Family 4 crosses (2x H. vulgare crosses)...... 107

5.5 Mean grain yield (GY), g plant-1 of parental genotypes and Family 5 crosses (2x H. vulgare crosses)...... 112

5.6 Mean grain yield (GY), g plant-1 of parental genotypes and Family 1 crosses (4x H. vulgare crosses)...... 119

5.7 Mean grain yield (GY), g plant-1 of parental genotypes and Family 2 crosses (4x H. vulgare crosses)...... 125

5.8 Mean grain yield (GY), g plant-1 of parental genotypes and Family 3 crosses (4x H. vulgare crosses)...... 131

5.9 Mean grain yield (GY), g plant-1 of parental genotypes and Family 4 crosses (4x H. vulgare crosses)...... 136

5.10 Mean grain yield (GY), g plant-1 of parental genotypes and Family 5 crosses (4x H. vulgare crosses)...... 141

6.1 Mean final germination percentage (FG%) of barley diploid crosses (2x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl...... 166 xxvii

6.2 Mean final germination percentage (FG%) of barley tetraploid crosses (4x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl...... 181

7.1 Mean shoot dry weight (SDW), g plant-1 of barley diploid crosses (2x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl...... 204

7.2 Mean shoot dry weight (SDW), g plant-1 of barley tetraploid crosses (4x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl...... 221

xxviii

CHAPTER I

INTRODUCTION

Domestic barley (Hordeum vulgare L.) is one of the oldest, most domestic field crops. It belongs to the tribe Triticeae of Poaceae family. Barley is considered one of the most widely cultivated cereal crops in the world (FAO 2004) and is the fourth major cereal crop in terms of production and area after wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea mays) (Goedeke et al. 2007, Blake et al. 2011). In the past, barley was essential food in the baking industry and soup and porridge dishes. However, barley has become a multipurpose crop. The grain is used as a feed for animals and in alcoholic beverages. The plant is used in pastures, as a cover crop to prevent erosion/ improve the soil, and curb weeds (Poehlman 1985). Starch extracted from barley is used for either food or for the chemical industry. Sugars and starches derived from barley are used in malting, milling and flaking, and production of alcoholic and non-alcoholic beverages

(OECD 2004). In Iraq, wheat and barley are the most important crop covering 80% of cultivated land. Greater than 80% of barley is used as fodder (FAO 2008).

Barley was domesticated in the Middle East over 10,000 years ago. It grows well in a wide range of environments, which extend from the equator to the 65° N latitude to

50° S latitude (Dev 2004). Also, barley can be found growing at different elevations, from 330 meters below sea level in the Middle East to 4200 meters in Bolivia (Akar and

Dusunceli 2004). Consequently, its widespread cultivation makes it one of the most

widely adapted cereal crops in modern agriculture (Goedeke et al. 2007). In 1492, barley introduced into North America by Columbus, and again by immigrants from Europe

(Tiwari 2010). Total harvested land area in 2018 was 48.12 million hectares, and the annual production was 144.27 million Mg worldwide (USDA 2018).

Salinity and drought are the most important problems facing the productivity of crops over the world. More than 6% of the world’s total land area (900 million hectares) is impacted by salt, accounting for more than 20% of the total agricultural land. In addition, the global economic losses due to saline soils amount to $27.3 billion annually

(FAO 2013). Human population growth and lack of fresh water available for agriculture are also problematic. Climate change and population growth have exacerbated desertification, salinity, drought, and corresponding water shortages. The FAO (2009) estimated 76% of cereals would need to be imported to Iraq. In 2008, Iraqi wheat farmers experienced a 55% decrease in production because severe drought and fixed irrigation water (FAO 2012). War has only exacerbated productivity decline. Thus, there is a need to use more saline water to irrigate crops, and a corresponding need to breed barley varieties with high water use efficiency with the ability to tolerate salinity.

The continuous cultivation of barley has led to a narrowing of its genetic base, as well as the loss some its traits still present in wild species. Consequently, the wild species are an important genetic resource for inclusion in breeding programs. The two species used in this study are related, but very different. Domestic barley (H. vulgare subsp. vulgare) is an annual crop with some salinity tolerance, while bulbous (H. bulbosum) is perennial with strong tolerance to salinity (Mano and Takeda 1998).

The objectives of this study are to:

* Screen H. bulbosum germplasm for saline tolerance and transfer that tolerance to H.

vulgare through interspecific hybridization, embryo rescue, and diploidization.

* Comparison between the hybrid progeny and their parents.

* Verification of their salinity tolerance at germination and seedling growth.

CHAPTER II

LITERATURE REVIEW

Barley Breeding

Barley belongs to the genus Hordeum, which contains both annual species, (H. vulgare and H. marinum) and perennial species, H. bulbosum. Hordeum genus belongs to the tribe Triticeae of the family Poaceae. This genus includes 32 species (Table 2.1). All

Hordeum species have a basic chromosome number of x=7. Consequently, cultivated barley, H. vulgare L. ssp. vulgare, and its wild progenitor [H. vulgare L. ssp. spontaneum

(C. Koch.) Thell.] are diploid (2n = 2x= 14). Other wild species are diploid, tetraploid

(2n=4x=28) or hexaploid (2n=6x=42) (Komatsuda et al. 1999; Kant et al. 2016). This great genetic diversity as well as the geographic diversity suggests that barley can tolerate a wide variety of environmental conditions, including cold and salinity (Von Bothmer

1992; Von Bothmer et al. 2003).

Barley is one of the most important crops in the world, but the genetic diversity for this crop as a level in many breeding programs is limited, although there are ongoing research and breeding programs to improve performance of the crop. Barley is a self- pollinated plant. Furthermore, it has been intensively bred for improved performance and quality, resulting in a reduced genetic reservoir in the elite cultivars. Genetic variation is essential in breeding programs for improvement and development of any crop (Matus and

Hayes 2002).

Table 2.1 Species in the genus Hordeum, distribution, chromosome numbers, and life form (Kant et al. 2016):

No. Species Subspecies 2n Growth Distribution Habit 1 H. Vulgare L. -vulgare L. 14 Annual E Mediterranean -spontaneum (C. Koch.) Thell 2 H. bulbosum L. - 14, 28 Perennial Mediterranean 3 H. murinum L. -murinum 14, 28, Annual Europe, -glaucum 42 Mediterranean to -leporinum Afghanistan 4 H. pusillum Nutt. - 14 Annual USA, N Mexico, S Canada 5 H. intercedens - 14 Annual SW California Nevski and N Mexico 6 H. euclaston - 14 Annual C Argentina, Steud. Uruguay, S Brazil 7 H. flexuosum - 14 Annual/Per Argentina and Steud. . Uruguay 8 H. muticum Presl - 14 Perennial W South America 9 H. chilense - 14 Perennial C Chile and W Roem. & Schult Argentina 10 H. cordobense - 14 Perennial Argentina Bothm. Et al. 11 H. stenostachys - 14 Perennial Argentina, Godr. Uruguay, S Brazil 12 H. pubiflorum - pubiflorum 14 Perennial W Argentina, Hook. F. -breviaristatum Chile, Bolivia, Peru 13 H. halophilum - 14 Perennial Argentina, Chile, Grisebach Bolivia, Peru 14 H. comosum - 14 Perennial Chile and W Presl Argentina 15 H. jubatum L. - 28 Perennial W North America to E Russia 16 H. arizonicum - 42 Annual/Per S USA and N Covas . Mexico 17 H. procerum - 42 Perennial C Argentina Nevski 18 H. lechleri - 42 Perennial Chile and (Steud.) Schenk Argentina 19 H. marinum -marinum 14, 28 Annual Mediterranean to Huds. -gussoneanum Afghanistan

Table 2.1 (Continued)

20 H. secalinum - 28 Perennial W Europe and N Schreb. Africa 21 H. capense - 28 Perennial South Africa and Thunb. Lesotho 22 H. bogdanii Wil. - 14 Perennial C Asia 23 H. roshevitzii - 14 Perennial S Siberia, Bowd. Mongolia, N China 24 H. - brevisubulatum 14, 28, Perennial Asia brevisubulatum -violaceum 42 (Trin.) Link -turkestanicum -nevskianum -iranicum 25 H. -brachyantherum 14, 28, Perennial W N America to brachyantherum -californicum 42 Kamchatka Nevski 26 H. depressum - 28 Annual W USA (Scribn. & Sm.) Rydb. 27 H. guatemalense - 28 Perennial N Guatemala Bothm. et al. 28 H. erectifolum - 28 Perennial C Argentina Bothm. et al. 29 H. tetraploidum - 28 Perennial S Argentina Covas 30 H. fuegianum - 28 Perennial S Argentina and S Bothm. et al. Chile 31 H. parodii Covas - 42 Perennial S Argentina and S Chile 32 H. patagonicum -patagonicum 14 Perennial S Argentina and S (Haum.) Covas -mustersii Chile -santacrucense -setifolium -magellanicum

Plant breeding is the art of genetic improvement of crops and genetic variation of quantitative traits which is the key to this genetic improvement (Lakew 2009). Breeding programs are a cyclical process that are aimed to produce new cultivars. Each cycle in breeding program consists of three major steps. The first step is generating genetic

variability (making crosses, inducing mutation, introducing exotic germplasm, or using genetic engineering techniques). Second, selection and testing to identify superior recombinants. Finally, release, distribution, and adoption of new cultivars (Ceccarelli

2015).

The breeding methods commonly used in genetic improvement of barley are based on selecting desirable genotypes from genetically heterogeneous landraces (pure- line method) or from the segregating progeny of crosses between lines with superior traits

(pedigree method). Another method that can be used is the bulk-population method, where the segregating progeny of multiple crosses between superior lines are harvested and propagated in bulk. Depending on the characteristic and location of selection, between seven and 10 cycles are required to obtain a new variety by using these methods

(Jensen 1988; Ibrahim and Barrett 2001; Gomez-Pando et al. 2009).

In 1929, Harlan and Martini proposed the composite cross method for breeding barley. This method is essentially similar to the bulk-population method except that the hybridization stage involves crossing a large number of varieties of diverse origin, and genetic make-up, in order to create a heterogeneous population of recombinant genotypes. This population would then be grown under normal agricultural conditions in the areas where locally adapted varieties were needed. Natural selection acts on the available variation during the successive generation, leading to an increase in frequency of locally adapted genotypes without creating a genetically uniform population. Mak and

Harvey (1982) state that this method not only produces new genetic variation during the hybridization and segregation stages but also preserves the variation in an exploitable form. Composite cross populations can provide dynamic gene pools, which in turn

provide a means of conserving germplasm resources. However, the major drawback of this method is the length of time required to produce new varieties. Successful barley varieties obtained from this method have all been selected from well-advanced generations, all beyond F15 (S14) (Ibrahim and Barrett 2001; Phillips and Wolfe 2005).

In addition, the continuous cultivation of barley led to a narrowing of its genetic base as well as loss of some its characters which are present in the wild species. The lack of genetic variation among vulgare genotypes prevents the production of varieties with high productivity in self-pollinated crops (Agdew et al. 2014). Therefore, in order to improve this crop must expand its genetic base (Matus et al. 2003). Thus, the wild species are an important source of genetic differences for inclusion in breeding programs to generate varieties tolerant to stress conditions (Ellis et al. 2000).

Haploid methods are also used to produce homozygous diploid plants in conjunction with colchicine in a single generation. These homozygtes are fertile, uniform, and do not segregate. They can be used as inbred lines. In conventional plant breeding methods, this goal requires six to seven generations after the initial cross. Consequently, haploid methods reduce the time of a breeding cycle, save space, and money (Jha and

Biswajit 2005).

Two species in the Hordeum genus; H. vulgare and H. bulbosum, are considered to share a common genome (Von Bothmer 1992). Domestication and continued breeding practices have led to a narrowing of vulgare’s genetic diversity, so wild relatives act as a source of genetic diversity in breeding programs (Hajjar and Hodgkin 2007). The species, bulbosum represents the secondary gene pool of the genus. This species is used for

introgression into improve elite barley germplasm; making bulbosum the most important resource for barley improvement (Wendler et al. 2014).

The Hordeum genus of barley has three types of wild relatives, which may serve as the potential gene pools for stress tolerant alleles in barley breeding (von Bothmer et al. 1995). These divisions are based on different criteria; such as ease of interspecific hybridizations, cytogenetic analyses, and molecular homology (Zhang et al. 2001). The first gene pool contains domesticated barley (H. vulgare L. ssp. vulgare) and wild common barley (H. vulgare L. ssp. spontaneum). The species spontaneum is often used to improve the species vulgare elite cultivars such as resistance to disease and abiotic stresses (Kalladan et al. 2013). Also, there are no obstacles in crossing programs with cultivated barley. Hordeum bulbosum composes the only member for secondary gene pool of barley. Crosses between the two are used in the production of homozygous diploids from haploids. The third gene poll for barley contains of 30 species of genus

Hordeum, but there are sexual barriers to hybridization between these species and cultivated barley (Pickering and Johnston 2005).

Crossing barriers between bulbosum and vulgare can be overcome by modifying environmental conditions, use of specific genotypes, and biotechnological tools, such as embryo rescue (Pickering 1984). Embryo rescue is considered one of the techniques that provides an opportunity for the exploitation of wild relatives of barley as a source of genetic variation in barley breeding programs. Chromosome elimination of one of the genomes in F1 through crosses between species or genus is a way to produce haploid that can be doubled for plant breeding. Kasha and Kao (1970) were the first researchers to interpret this phenomenon correctly between H. vulgare and H. bulbosum. After which

the use of this techniquic spread among intergeneric and interspecies crosses of other species (Jones and Thomas 2009). Haploid production in barley is best obtained by hybridization between cultivated species as females (H. vulgare) and (H. bulbosum) as male (Murovecand and Bohanec 2011).

Interspecific crosses between H. vulgare (2x) as a female and H. bulbosum (2x) as a male produce haploids. Zygote induction is fairly high and the chromosomes of H. bulbosum are rapidly eliminated from the developing embryos. Developing endosperm also aborts after about 2 - 5 days of growth which necessitates the rescuing of embryos in order to complete their development. As a result, immature embryos are cultured by using nutritionally rich medium and results in complete haploid plants of H. vulgare.

Haploid plants have to be treated with colchicine to double the chromosome number and to restore their fertility (Chahal and Gosal 2006). In crosses between H. vulgare genotypes and H. bulbosum, seed setting can be very low due to germplasm specific incompatibility issues (Pickering and Johnston 2005). Also, in the absence of gibberellic acid (GA3) application following pollination the developing seed degenerate before embryo rescue, so GA3 and 2,4-dichlorophenoxyacetic acid (2,4- D) are applied as a spray to florets 1-2 days after pollination to enhance seed development and embryo size

(Devaux 2003; Houben et al. 2011).

Haploid formation from diploid cross combination is known to depend on genetic factors and temperature after fertilization (Pickering and Morgan 1985). Pickering

(1984) determined that temperatures above 18° C during the early stages of embryo growth can promote chromosome elimination. However, temperature less than 17.5° C during the first few days after pollination is more suitable for obtaining hybrids from

diploid cross combinations (Pickering 1985). These hybrids are completely sterile, but occasionally set seed when they are pollinated by H. vulgare. The progeny usually resemble barley. Also, fertility of these hybrids can be restored with colchicine treatment to obtain fertile tetraploid hybrids (Thomas and Pickering 1983; Pickering 1991; Zhang

2001). Kasha and Kao (1970) proposed that the genome balance between the parental species is important in chromosome stability. When the genome ratio V (H. vulgare): B

(H. bulbosum) is 1 or more, chromosome elimination occurs, whereas 1V: 2B results in chromosome retention and hybrid production.

Triploid hybrids from interspecific cross between H. vulgare and H. bulbosum have been produced for introgressing desirable genes from wild species into H. vulgare.

Successful introgression of useful genes from wild species (H. bulbosum) into cultivated barley was achieved recently through backcrossing partially fertile triploid hybrids

(VBB) to H. vulgare (Pickering et al. 2000; Zhang et al. 2001; Pickering and Johnston

2005). Triploid hybrid (VBB) can be originated from a cross between H. vulgare (2x) and

H. bulbosum (4x) and then culture embryo on defined media. These hybrids are completely sterile. However, triploid hybrids from this combination have been obtained with some fertility by pollinating H. vulgare (2x) with tetraploid H. bulbosum (4x) derived from colchicine treated diploid plants (Pickering, 1988). Fertility was also observed in triploid hybrids (BBV) by using H. bulbosum as female (Lange and

Jochemsen 1976).

Seed set on crosses between H. vulgare and H. bulbosum are very low because of incompatibility. Pickering (1991) tried to produce large number of triploid hybrids by crossing two cultivars of H. vulgare (4x) and four genotypes of H. bulbosum (2x). Seed

set was ranged from 1.2 - 43.7%, and hybrid plant generation ranged from 0 - 13.2%. In reciprocal crosses, these percentages were 2.3 - 7.2% for seed set and 0% for hybrid plants. Also, Bjornstad (1986) found that not all seed formed from this interspecific cross had an embryo. There were 22.9 - 80% of seed had embryos. In addition, Hung et al.

(1984) reported that not all embryos can regenerate plants. They found about 21.7 -

30.1% of cultured embryos grew into plants.

Flower Structure

The floral structure of the barley plant is the spike. A spikelet is the flowering unit. It is attached directly to the rachis (central axis). Rachis is the extension of the stem that supports the spike. At each node, there are three spikelets called triplets. Spikelets alternate on opposite sides of the spike. Each spikelet is made up of two glumes and one floret that includes the lemma, the palea, and the enclosed reproductive components. The floret is perfect meaning that it contains both male (stamen) and female (pistil) floral components. Depending on the variety, each lemma is an extended as awn, and more rarely a hood. In hulled or husked varieties, the palea and lemma adhere to the grain. In hull-less or naked varieties, the palea and lemma are not attached and separate from the grain on threshing.

In six-row barley, all of the spikelets in a triplet are fertile and able to develop into grain, leading to six vertical rows of seed on the spike. However, in two-row barley, only the central spikelets are fertile. Therefore, the lateral spikelets are sterile, and only a single seed is produced at each node of the spike, leading to two rows of seed on the spike. Anthesis begins in florets near the center of the spike and progresses to the top and bottom. Anthesis usually begins while the spike is still partially to totally enclosed in the 12

flag leaf sheath (boot). As the fertile florets consist of both male and female reproductive structures, and fertilization occurs as the spikes emerge from the boot, barley is thus predominantly self-pollinated (OGTR. 2008). Consequently, emasculation of barley plant is more difficult than wheat.

As two-rowed phenotype found in wild barley, resulting that the two-rowed spike is the ancestral form, which was changed to a six-rowed spike in cultivated barley by spontaneous mutation during domestication (Von Bothmer and Komastuda 2011). The six-rowed phenotype is controlled by five independent loci. Six-rowed spike 1 (vrs1) is a recessive gene located on chromosome 2HL. This gene is observed in all six-rowed cultivars. Wild barley has dominant alleles for Vrs1, while cultivated barley has a dominant Vrs1 (two-rowed) allele or a recessive vrs1 (six-rowed) allele depending on phenotype. Cultivated barley with recessive allele at vrs1 are completely six-rowed. This gene (vrs1) is expressed only in lateral spiklets at the immature stage, so it only affects the development of lateral spikelets. Six-rowed spike 2 (vrs2), six-rowed spike 3 (vrs3) and six-rowed spike 4 (vrs4) are recessive genes located on chromosome 5HL, 1HL and

3HL, respectively. Alleles at these loci enhance the development of lateral spikelets to various degrees depending on their position in the spike. Six-rowed spike 5 (vrs5 or int-c) is a recessive gene located on chromosome 4HS (Lundqvist et al. 1997). Alleles at the int-c locus modify the degree of fertility in lateral spikelets and produce an intermediate spike type. Two alleles of this locus were observed in cultivars. Intermedium spike c.b

(Int-c.b) allele (dominant state) prevents anther development in lateral spikelets, and Int- c.h allele (recessive state) allows the development of anthers and promotes occasional seed set in lateral spikelets. Among those five, six-rowed spike loci only the natural

mutation on vrs1 and vrs5/int-c are observed in barley cultivars (Pourkheirandish and

Komatsuda 2007; Von Bothmer and Komastuda 2011). In addition to six-row and two- row types of barley, there are spring and winter barley. Winter barley is planted in late fall and requires vernalization to initiate floral development. Spring barley is planted in the spring and does not require vernalization.

Commercial Cultivars

Iraq

In Iraq, the breeding program of barley was divided into two subprograms. The first program is used for irrigated areas around Baghdad. The second is used for rain-fed areas in the North. The first subprogram was for improving six-row types that are used for forage and grain yields. This program was dependent on the segregating populations received from International Center for Agricultural Research in the Dry Areas

(ICARDA), and released some of successful cultivars such as IPA 7, IPA 9, IPA 265. In the North, although farmers preferred the two-row black-seed barley, the second subprogram attempted to introduce six-row types. This program achieved many successes, including the release of the cultivar Rihane-03. This cultivar was cultivated on

250,000 ha in the late 1990s. Currently, Iraq is conducting a limited number of variety trials using new materials from ICARDA (Ceccarelli et al. 2011).

IPA 99

IPA 99 was released by State Board of Seeds Testing and Certification/ Ministry of Agriculture (SBSTC). It was produced from OAP-4AP-7L, Sel/ ICARDA. It is a six- rowed winter barley (Al-Hadeithi, 2016). IPA 99 can be used as dual-purpose (grazing

and grain). Plant height ranges between 120 - 130 cm. It has good tolerance for lodging and seed shattering and is resistant to leaf rust disease (Puccinia hordei). IPA 99 is suitable for the central and southern irrigated areas of Iraq (Ghani and Salman 2011).

IPA 265

IPA 265 was released by SBSTC. It was produced from a cross of Brigs and

9cr.279 (OAP- 2AP-4AP-03355_79/ICARDA). IPA 265 a six-rowed winter barley (Al-

Hadeithi, 2016). It is one of semi-prostrate varieties with mean plant height of 116 cm.

This cultivar highly resistant to lodging and seed shattering. It is resistant to leaf rust and powdery mildew disease (Blumeria graminis f. sp. hordei). IPA 265 is suitable for the central and southern irrigated areas of Iraq (Ghani and Salman 2011).

Tuwaitha

Tuwaitha was released by SBSTC. It was produced from irradiated local black x

Arevat-IRAQ. It is a two-rowed winter barley (Al-Hadeithi, 2016). Tuwaitha requires

125 days from germination to maturity under irrigated conditions in the central area of

Iraq and 165 days under normal rainfall conditions of the northern areas. It is resistant to lodging and powdery mildew. This cultivar is suitable for both irrigated and normal rainfall areas where consistent rainfall occurs (Ghani and Salman 2011).

Shuaa

Shuaa was released by SBSTC. It was produced from irradiation Arevat seed-

IRAQ. It is a six-rowed winter barley (Al-Hadeithi, 2016). Shuaa is an early maturing cultivar that requires 120 days from germination to maturity in the central region of Iraq.

It has excellent lodging resistance with an average height of 80 - 100 cm. This cultivar is

suitable for irrigated and normal rainfall areas that provide consistent rainfall (Ghani and

Salman 2011).

United States

The area of barley production in the United States can be subdivided into four regions: The East, Upper Midwest, West, and Southwest. The Midwest and West regions account for over 80% of the area sown to barley. North Dakota is the largest barley- producing state in the United States followed by Idaho and Montana (USDA-NASS).

Over 90% of the barley sown in the Upper Midwest region of Minnesota, North Dakota, and South Dakota are six-row malting barley cultivars. Production in this region is typically conducted under dryland conditions, and the preponderance of six-row barley goes back to barley improvement efforts in the early 1900s (Friedt et al. 2011).

Barley breeding in the United States is largely conducted by public institutions located in the northern tier of states. Institutions with breeders working solely on barley include: Montana State University, North Dakota State University (NDSU), Oregon State

University, the University of California-Davis, the University of Minnesota, Washington

State University, and the United States Department of Agriculture Agricultural Research

Service (USDA-ARS) at Aberdeen, Idaho. Public programs with breeders working on barley and other small grain crops are located at the University of Georgia, University of

Maryland, University of Nebraska, Utah State University, and Virginia Polytechnic

Institute and State University (VPI). The programs in California, Minnesota, Montana,

North Dakota, and Washington work primarily on developing spring barley cultivars; the programs in Georgia, Maryland, Nebraska, Oregon, and Virginia work almost exclusively in developing winter types; and the USDA-ARS program in Idaho works on developing 16

both spring and winter barley cultivars. In addition, Minnesota, North Dakota, and

USDA-ARS work primarily on developing malting barley cultivars. The breeding program at the University of Minnesota focuses on the development of six-row barley cultivars, whereas the NDSU and USDA-ARS programs work on the development of both two-row and six-row cultivars. The public programs in California, Georgia,

Maryland, Nebraska, Utah, and Virginia develop primarily non-malting barley cultivars, and the Montana, Oregon, and Washington programs work on barley for malting and non-malting uses. The VPI, USDA-ARS, Oregon, and Washington breeding programs also work on hulless barley for food, feed, and industrial uses. The program at VPI is particularly working on hulless barley for ethanol production in collaboration with the

USDA-ARS Eastern Regional Research Center at Wyndmoor, PA (Friedt et al. 2011).

Winter Barley

Maja

Maja is a six-rowed winter feed and malt barley. It was released in 2009 by

Oregon AES as a winter malt variety. It is a doubled haploid developed from the F1 of the cross of Strider/88 Ab536. Maja is an adaptive cultivar, which is similar to its 88Ab536 parent. This trait can lead to early maturity under some environmental conditions, flexibility in the planting date, irrigation savings, and drought avoidance. The mean plant height is 105 cm. It is resistant to stripe rust (Puccinia striiformis) and susceptible to scald (Rhynchosporium secalis). It has had a very high-test weight with mean protein and a very low lodging rating. (Jackson 2011; Marshal et al. 2016).

Charles

Charles (Reg. no. CV-321, PI 637845) is a two-rowed winter malting barley. It was released in 2005 by the USDA-ARS, and the University of Idaho AES. Charles was released as the first two-rowed winter malting barley that putatively meets malting and brewing industry standards. It was selected from a cross between Bearpaw'/81Ab1702.

Charles was selected for maturity, height, lodging resistance, resistance to shattering.

Charles’ yields and test weights are lower than the winter feed variety average. It is short, early maturing and has a tendency to lodge. Charles has excellent yields in the Twin

Falls, ID area even when severe winter conditions reduce the stand. Charles can suffer significant stand losses under cold winter conditions (Obert et al. 2006; Marshal et al.

2016).

Spring Barley

Goldeneye

Goldeneye is a six-rowed spring feed barley. It was released in 2005 by the Utah

AES. It was selected from the cross ID633019/Woodvale//Steptoe//OR3. It is erect- growing and early heading. Plant height is mid-tall. Goldeneye has very high yields under irrigated conditions, above average yields under dryland production, and an above average test weight. When cut at soft dough, Goldeneye has proven to be a high-yielding and high quality forage variety. Goldeneye also has high plumps and protein. At the time of release, it was resistant to loose smut (Ustilago nuda) and covered smut (Ustilago hordei) but susceptible to stripe rust (Roche et al. 2005, Jackson 2011, Marshal et al.

2016).

Merit 57

Merit 57 is a two-rowed spring malting barley. It was released in 2009 by Busch

Agricultural Resources. It was selected from the backcross Merit//Merit/2B94-5744 made in 1996 in Fort Collins, Colorado. It has high levels of enzymes, like α-amylase, and is good for diastatic power. Merit 57 is late maturing, with high yield potential. It has an intermediate plant height and straw strength and average lodging. It has good resistance to shattering and fair to good tolerance of straw breakage and drought. At the time of release, Merit 57 was moderately susceptible to scald, net blotch (Pyrenophora teres), race MCC of stem rust (Puccinia graminis), and spot blotch (Cochliobolus sativus). It is also susceptible to barley yellow dwarf virus (BYDV) (Jackson 2011, Marshal et al.

2016).

Salt Tolerance

Salinity and drought are the most important challenges facing the productivity of crops around the world. Salinity causes a major reduction in cultivated land area, productivity, and crop quality (Zhou et al. 2012). More than 20% of the world’s total cultivated land and 33% of irrigated land are impacted by salt (Machado and Serralheiro

2017). Pimentel et al. (2004) estimated that 10 million ha of the world’s agricultural land is destroyed by salt each year. These challenges are exacerbated by population growth and lack of fresh water available for agriculture. Thus, there is a need for the judicious use of slightly saline water for crop irrigation. Therefore, there is a need to develop varieties with efficient water use, and the ability to tolerate salinity. In order to improve

the productivity and quality of barley, it is essential to develop new varieties able to withstand various stress conditions.

Breeding programs of barley aim to improve agricultural traits, and the quality characteristics of grain and malt. The wild species H. bulbosum and H. murinum are important because they are a source of quality and disease resistance genes (Knezevic et al. 2004). The strategies of barley saline tolerance include: selecting saline tolerant lines, analyzing inheritance, and pyramiding the genes that influence salt tolerance (Mano et al,

1996). In the Heilongjiang Region of China, Takeda et al. (1995) selected wheat and barley varieties with highly salt tolerance from more than 10,000 lines.

According to Matsumoto (1989), in soils with a high water table, salinity is more severe at the soil surface compared to lower water tables, so varieties tolerant to salinity at germination is the most important. Mano et al. (1996) tested the salt tolerance at germination of 6,712 barley accessions from around globe. They found that Chinese six- rowed barley were highly salt tolerant while two-rowed, black lemma varieties from

West Asia were highly salt sensitive. Mano and Takeda (1997) reported that the development of salt tolerant varieties is the best way to overcome the problems of crop productivity in saline areas. The first stages of plant growth affect all subsequent growth and hence final yield of plant, so testing salt tolerance of varieties at germination and the seedling stage are of primary importance. Their study also indicated salt tolerance at germination and during the seedling stage was controlled by multiple genes located at different loci. This was confirmed by Maddur (1977). El Madidi et al. (2014) reported that seed germination percentage had a greater reduction than the other parameters of early plant growth under salt stress.

Mano and Takeda (1998) reported that most wild Hordeum species had higher

NaCl tolerance than cultivated barley at the seedling stage. They evaluated 340 accessions of Hordeum for salt tolerance; however, at germination salt tolerance was higher in cultivated barley than wild Hordeum species. Garthwaite et al. (2005) tested salt tolerance in eight wild Hordeum species (H. jubatum, H. lechleri, H. secalinum, H. intercedens, H. patagonicum, H. bogdanii, H. marinum, and H. murinum) and one cultivated barley (Hordeum vulgare L. ssp. vulgare cv. ‘Golf’) using concentrations of

0.2 (control), 150, 300, or 450 mM NaCl. They found all wild species (except H. murinum) had better Na+ and Cl- exclusion and maintained higher leaf K+ than H. vulgare.

Kook et al. (2009) compared between two varieties of domestic barley, G41

(Japanese six-rowed naked barley) a salt-susceptible variety and T76 (Tunisian six-rowed covered barley) as salt-tolerant variety by testing them at 0, 150, 300, or 450 mM NaCl solutions. These varieties showed extreme differences in the growth of shoot and root at germination stage. Germination of T76 and G41 was 95% and 51% at 150 mM NaCl, respectively. At 450 mM NaCl germination rate was 69% and 16% in T76 and G41, respectively. Likewise, at 150 mM NaCl the rate of shoot growth was 93% in T76 while

45% in G41. Shoot growth rate was 29% in T76 and 9% in G41 at 450 mM. They reported that high salt tolerance of T76 cultivar was due to exclusion of Na+ . Sodium ion content was about three times higher in G41 (susceptible) than in T76 in the shoot of the seedlings treated for 5 days at 200 mM NaCl.

Adgel et al. (2013) tested twelve genotypes of barley (Hordeum vulgare L.) at four salt stress levels (0, 50, 100, 150 mM) during germination and seedling growth

stages. They found as salinity increased from none to 150 mM NaCl treatment, the germination percentage decreased from 86.0 to 50.9%; the speed of germination slowed from 16.2 to 8.3% day-1; the coleoptile length decreased from 2.5 to 1.5 cm; the root length decreased from 35.4 to 8.3 cm; and the number of roots decreased from 5.1 to 3.1 roots. The average shoot fresh weight decreased from 718.2 mg to 520.0 mg seedling-1 as salinity increased from 0 to 150 mM NaCl treatment. The average roots fresh weight decreased from 642.5 mg to 210.78 mg/seedling as salinity increased from 0 to 150 mM

NaCl treatments. They indicated that the reductions in traits associated with increased

[Na+], decreased [K+] and K+:Na+. Fricke et al. (2006) reported a 68% and 64% biomass reduction in Clipper and Arivat barley cultivars, respectively under 250 mM vs. control.

Seed germination under salinity is reduced due to osmotic stress. High levels of

NaCl reduce the bioavailability of water (caused by Na+) resulting in what is effectively a water deficit (Mano et al. 1996; Parida and Das 2005). Additionally, Na+ shows toxic effects on the germinating embryo as it displaces essential K+ (Mazher et al. 2007). This interferes with enzyme functions in the seed, which decreases activity of polyphenol- oxidases and amylases (Khemiri et al. 2004). These findings were confirmed by Othman et al. (2006); they found that increasing salt concentration significantly reduced K+ concentration in barley seed after 24 hr of imbibition.

Salinity affects plant growth through water stress, changes nutrient uptake and translocation, and ion toxicities of NaCl which can deactivate cellular functions and physiological processes (Marschner 1995; Mano et al. 1996; Bagci et al. 2003; Kook et al. 2009). Furthermore, osmotic stress reduces cell division and cell expansion rates in root tips and young leaves resulting in reduction of the photosynthetic area, and causes

stomatal closure, reducing photosynthesis (Munns 2002; Munns and Tester 2008).

Salinity inhibits plant growth through displacement of Ca+2 by Na+ from critical cell wall binding sites, which disrupts cell wall synthesis, modifies the metabolic activities, and limits the cell wall elasticity (Xue et al. 2004; Khan et al. 2008).

Flowers (2004) and Foolad (2004) reported that salinity tolerance is not constant over growth stages. For example, Munns (2002) found that barley has been tolerant to salinity at the germination stage, but then becomes sensitive at the seedling and early vegetative growth stages, then again tolerant at maturity stage. As a result, wild Hordeum species are good sources of genetic materials for salt tolerance breeding. Levels of salinity tolerance vary depending on the genotype, stage of plant development, and geographical origin of the germplasm.

Tissue Culture to Overcome Sexual Incompatibilities

Tissue culture plays an important role with breeding programs to aid in the propagation of improved varieties for resistance to disease and stress conditions, such as salinity and drought. It is important in genetic transformation and the production of secondary metabolites (Hussain et al. 2012). Tissue culture itself is considered a source of genetic variation, where culture conditions lead to genetic somaclonal variations.

Environmental conditions, medium composition, ploidy level, donor genotype, and explants source influence the generation and isolation of somaclones (Veilleux and

Johnson 1998; Bednarek et al. 2007).

Tissue culture techniques use trans-species embryos, microspores, ovules and anthers to generate haploid plantlets, as well as the exploitation of somaclonal, protoplasts and gametoclonal variations. Regenerated plants from tissue culture may 23

exhibit somaclonal variation. This occurs throw gene activation or silencing, nucleotide mutations, and chromosome abnormalities (Geelen 2012). Tissue culture propagation produces large numbers of improved genotypes for commercial use as well as germplasm conservation. Moreover, tissue culture used in conjunction with other technology such as genetic engineering, and molecular techniques, has been successful in integrating desired characteristics into domestic crops (Brown and Thorpe 1995). Plant breeders have improved some important traits in barley by using tissue culture e.g., malting quality, disease resistance and stress tolerance (Mohan et al. 1988).

Anther (pollen) culture of barley is widely used in haploid breeding (doubled haploid) (Castillo et al. 2000; Lazaridou et al. 2005; Wei et al. 2013). Barley is a diploid species and haploids can be produced from male or female gametophytes using gynogenesis (ovule culture), androgenesis (culture of anthers or isolated microspores), and chromosome elimination following interspecific crosses with H. bulbosum (Devaux

1995). However, the high regeneration of lutescent plantlets and low regeneration of green plants limits use of anther culture (Caredda and Cl´ement 1999). Problems with the production of double haploids from the anther culture presists (Grauda et al. 2008).

Results vary based on the genotype used (Zamani et al. 2003). Anther culture is becoming more common within the commercial sector of barley breeding (Guasmi et al.

2013). Conversely, immature embryos can be used without the associated microspore problems (Khush and Virmanim1996; Serhantova et al. 2004). Barley is highly problematic in tissue culture. Most regeneration techniques rely on immature embryos as an explant excised from donor plants grown under controlled conditions, making the process time consuming (Rostami et al. 2013).

In plant breeding programs, plant breeders can produce haploid plants through crossing of distantly related species, leading to the elimination of chromosomes from one parent (usually the male) after fertilization. However, some recombination does occur between the chromosomes of the two species. This process provides a haploid plantlet which can be made into a diploid by use of colchicine. This is important because doubled haploids are homozygous at all loci. This process accelerates breeding programs by eliminating the need for several generations of inbreeding. Centromere inactivity arising from loss of a centromere specific histone (CENH3) plays an important role in uniparental chromosome elimination in trans-species hybrid embryos of barley (Devaux and Pickering 2005; Sanei et al. 2011).

Haploid embryos can be produce from ovules without the male gamete, termed parthenogenesis, or from gametophytic cell other than the ovule, termed apogamy. San

Noeun (1976) was the first to describe the development of haploid from ovule culture in barley. However, this method does not apply to barley because the production of haploids by this method remains very inefficient. By using this method, only 0.2 - 1.4% of cultured ovules produce haploid plants (Forster & Powell, 1997). Also, there were many unsuccessful attempts to induce haploid development by culturing unfertilized ovules

(Mukhambetzhanov 1997; Germana 2006). Consequently, techniques other than ovule culture have been developed. However, this technique is successful in many plant species such as wheat (Sibi et al. 2001), sweet potato (Ruth et al. 1993) and maize (Tang et al.

2006).

Haploids in barley can be produced by crossing H. vulgare and H. bulbosum.

Chromosomes in H. bulbosum are eliminated several days after fertilization and is

independent of the crossing direction (Kasha and Kao 1970). Trans-species hybrids often include both sets of parental chromosomes. Chromosome elimination depends on genetic factors (Humphreys 1978) and temperature after fertilization (Pickering 1985). Embryos will develop into true hybrid plants that contain both parental sets of chromosomes and are unstable for di-haploid production when temperatures are less than 18 ̊ C during fertilization process (Houben, 2011). Hybrids between Emir variety (H. vulgare) and Cb

2929/1 (H. bulbosum) produced a high proportion (51%) of hybrid embryos at temperatures less than 15 ̊ C, which retained the chromosomes of H. bulbosum. The proportion of hybrids were 4.4% at temperature 21.5 ̊ C (Pickering 1983). This method

(bulbosum method) was the first haploid induction method to produce large numbers of haploids across most genotypes and quickly entered into breeding programs (Murovec and Bohanec 2011).

Haploid generation is also important for genetic transformation. Chauhan et al.

(2011) transformed amphi-haploid bread wheat with HVA1 gene to obtain drought tolerance in the hexaploid. Another approach is for haploid cells themselves, especially microspores, to be targets of transformation. For instance, Chugh et al. (2009) transformed triticale (x Triticosecale) microspores using cell-penetrating peptides with plasmid DNA and regenerated poly-haploid transformed plants. More recently, the approach for transformation of haploid induction focused on the centromeric location

(Ravi and Chan 2010). The centromere is a key chromosomal component for sister chromatid coherence, and important for kinetochore assemblage and spindle fiber attachment, key for chromatid separation during anaphase of mitosis (Yi et al. 2013).

Mechanics of Chromosome Elimination

Sani et al. (2011) studied the role of the centromere for specific histone H3 variant in chromosome elimination through hybridization between the two species of barley which are H. vulgare and H. bulbosum. They found centromere inactivity of bulbosum chromosomes triggers the mitosis-dependent process of uniparental chromosome elimination; centromeric loss of CENH3 protein rather than uniparental silencing of CENH3 genes causes centromere inactivity; in stable species combinations, trans-species incorporation of CENH3 occurs despite centromere-sequence differences.

Not all CENH3 variants get incorporated into centromeres if multiple CENH3s are present in trans-species combinations. Finally, they found wild diploid barley species encode two CENH3 variants, the proteins of which are intermingled within centromeres throughout mitosis and meiosis. Interspecific hybrids between wild barley species (H. marinum and H. bulbosum) were generated and tested for chromosomal stability and chromatin properties. Contrary to the hybridization between H. vulgare x H. bulbosum, there was no effect of temperature on uniparental chromosome elimination or chromosome retention during hybrid seed development. Seven chromosomes from each parent were detected in the offspring. The centromere-specific histone H3 gene (CENH3) of both parental genomes was active in hybrid progeny (Sanei et al. 2010).

REFERENCES

Adjel, F., H. Bouzerzour, and A. Benmahammed. 2013. Salt stress effects on seed germination and seedling growth of barley (Hordeum vulgare L.) genotypes. J. Agric. Sustain 3:223-237.

Agdew, B., S. Verma, and R. Saharan. 2014. Comparison of variability generated through biparental mating and selfing in barley (Hordeum vulgare L.). For. Res. 40:98- 105.

Akar, T., M. Avci, and F. Dusunceli. 2004. Barley: Post-harvest operations. Cent. Res. Inst. Field. p. 1-64. Crophttp://www.fao.org/fileadmin/user_upload/inpho/docs/Post_Harvest_Compe ndium_-_BARLEY (accessed 25 May 2018).

Al-Hadeithi, Z.S. 2016. Detection of genetic polymorphism in Iraqi barley using SSR- PCR analysis. Iraqi J. Sci. 57:1158-1164.

Anonymous. 2008. The biology of barley, Hordeum vulgare L. Office Gene Technol. Regulator, Dep. Health Ageing, Gov. Australia. p. 1-41. www.ogtr.gov.au/internet/ogtr/ publishing.nsf. (accessed 25 May 2018).

Bagci, S.A., H. Ekiz, and A. Yilmaz. 2003. Determination of the salt tolerance of some barley genotypes and the characteristics affecting tolerance. Turk. J. Agric. For. 27:253-260.

Bednarek, P.T., R. Orłowska, R.M. Koebner, and J. Zimny. 2007. Quantification of the tissue-culture induced variation in barley (Hordeum vulgare L.). BMC Plant Biol. 7(1):10.

Bjornstad, A. 1986. Partial incompatibility between Scandinavian six‐rowed barleys (Hordeum vulgare L.) and H. bulbosum L., and its genetical basis. Hereditas. 104(2):171-191.

Blake, T., V. Blake, J. Bowmanand, and H. Abdel-Haleem. 2011. Barley feed uses and quality improvement. In: S.E. Ullrich, editor, Barley production, improvement and uses. Wiley-Blackwell, Southern Gate, Chichester, West Sussex, UK. p.522- 531.

Bohanec, B. 2003. Ploidy determination using flow cytometry. In: M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht. p. 397-403.

Brown, D.C.W., and T.A. Thorpe. 1995. Crop improvement through tissue culture. World J. Microbio. Biotech. 11(4):409-415.

Burun, B., and E.C. Poyrazoglu. 2002. Embryo culture in Barley (Hordeum vulgare L.). Turk. J. Biol. 26:175-180.

Caredda, S., and C. Clement. 1999. Androgenesis and albinism in Poaceae: Influence of genotype and carbohydrates. In: C. Clement, E. Pacini, and J.C. Audran, editors, Anther and pollen from biology to biotechnology. Springer-Verlag, Berlin. p. 211-228.

Castillo, A.M., M.P Valles, and L. Cistue. 2000. Comparison of anther and isolated microspore cultures in barley, effects of culture density and regeneration medium. Euphytica. 113(1):1-8.

Ceccarelli, S. 2015. Efficiency of plant breeding. Crop Sci. 55(1):87-97.

Ceccarelli, S., S. Grando, and F. Capettini. 2011. Barley breeding history, progress, objectives, and technology, Near East, North and East Africa and Latin America. In: S.E. Ullrich, editor, Barley: Production, improvement and uses. Wiley- Blackwell, Southern Gate, Chichester, West Sussex, UK. p. 210-220.

Ceccarelli, S., S. Grando, V. Shevstov, H. Vivar, A. Yayaoui, M. El-Bhoussini, and M. Baum. 1999. The ICARDA strategy for global barley improvement. Rachis. 18(2):3-12.

Chahal, G.S., and Gosal, S.S. 2006. Principles and procedures of plant breeding. Biotechnological, and conventional approaches. Alpha Sci. Int. Ltd. Harrow, UK. p.429-456.

Chauhan, H., and P. Khurana. 2011. Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics. Plant Biotech. J. 9(3):408-417.

Chugh, A., E. Amundsen, and F. Eudes. 2009. Translocation of cell-penetrating peptides and delivery of their cargoes in triticale microspores. Plant Cell Rep. 28(5):801- 810.

Collins, H.M., R.A. Burton, D.L. Topping, M. Liao, A. Bacic, and G.B. Fincher. 2010. Review: Variability in fine structures of noncellulosic cell wall polysaccharides from cereal grains: Potential importance in human health and nutrition. Cereal Chem. 87(4):272-282.

Dahleen, L.S., M. Manoharan. 2007. Recent advances in barley transformation. In vitro Cell Dev. Biol. Plant. 43(6):493-506.

Dev, O.E. 2004. Consensus document on compositional considerations for new varieties of barley (Hordeum vulgare L.): key food and feed nutrients and anti-nutrients. Doc ENV/JM/MONO 20, Ser Saf Nov Foods Feeds 12. Organ Econ Co-op Dev, Paris. 29

Devaux P, and R. Pickering. 2005. Haploids in the improvement of poaceae. In: Palmer, C.E., W.A. Keller, K.J. Kasha, editors. Haploids in crop improvement II, Biotechnology in agriculture and forestry series. Berlin: Spr. p. 215-242.

Devaux, P. 1995. Production and use of doubled haploids for breeding barley. In: Proceedings of the 7th Australian barley technical symposium, Perth, Australia. 195-199.

Devaux, P. 2003. The Hordeum bulbosum (L.) method. In: M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht. p.15-19.

El Madidi, S., B. El Baroudi, and F.B. Aameur. 2004. Effects of salinity on germination and early growth of barley (Hordeum vulgare L.) cultivars. Int. J. Agric. Biol. 6(5):767-770.

Ellis, R.P., B.P. Forster, D. Robinson, L.L. Handley, D.C. Gordon, J.R. Russell, and W. Powell. 2000. Wild barley: a source of genes for crop improvement in the 21st century? J. Exp. Bot. 51(342):9-17.

FAO. 2004. BARLEY: Post-Harvest Operations, 1 -64. http://www.fao.org/3/a-au997e. (accessed 19 July 2018).

FAO. 2009. Iraq food prices analysis, 1-10. ttp://www.fao.org/GIEWS/english/index.htm (accessed 21 July 2018).

FAO. 2012. Iraq- Agriculture Sectore Note, 1-72. http://www.fao.org/investment/en/ (accessed 21 July 2018).

FAO-AQUASTAT. 2013. Area equipped for irrigation and percentage of cultivated land. Available at http://www.fao.org/nr/water/aquastat/globalmaps/index.stm (accessed 19 July 2018).

Flowers, T.J. 2004. Improving crop salt tolerance. J. Exp. Bot. 55: 307-319.

Foolad, M.R. 2004. Recent advances in genetics of salt tolerance in tomato. Plant Cell Tiss. Org. 76:101-119.

Forster, B.P., and W. Powell. 1997. Haploidy in barley. In: Jain, S.M., S.K. Sopory, and R.E. Veilleux, editors, Vitro haploid production in higher plants. Kluwer Academic Publishers, Netherlands. p. 99-115.

Fricke, W., G. Akhiyarova, W. Wei, E. Alexandersson, et al. 2006. The short-term growth response to salt of the developing barley leaf. J. Exp. Bot. 57(5):1079- 1095.

Friedt, W., R.D. Horsley, B.L. Harvey, D.M. Poulsen, R. Lance, S. Ceccarelli, and F. Capettini. 2011. Barley breeding history, progress, objectives, and technology. In: S.E. Ullrich, editor, Barley: Production, improvement and uses. Wiley-Blackwell, Southern Gate, Chichester, West Sussex, UK. p. 160-220.

Garthwaite, A.J., R. Von Bothmer, and T.D. Colmer. 2005. Salt tolerance in wild Hordeum species is associated with restricted entry of Na+ and Cl− into the shoots. J. Exp. Bot. 56(419):2365-2378.

Gent, J., C. Topp, R. Dawe, C. Rodriguez, K. Schneider, and G. Presting. 2011. Distinct influences of tandem repeats and retrotransposons on CENH3 nucleosome positioning. Epigenetics and Chromatin. 4(1):3, doi:10.1186/1756-8935-4-3.

Germana, M. 2006. Doubled haploid production in fruit crops. Plant Cell Tissue Organ Cult. 86:131-141

Ghani, A.J., and K.A. Salman. 2011. Barley from planting to harvesting. (In Arabic.). Pub. Author. Agri. Res. Abu Graib Res. Station. http://agriculturresearch.blogspot.com/2013/05/blog-post.html (accessed 5 July 2018).

Goedeke, S., G. Hensel, E. Kapusi, M. Gahrtz, and J. Kumlehn. 2007. Transgenic barley in fundamental research and biotechnology. Transgenic Plant J. 1:104-117.

Gomez-Pando, L., J. Jimenez-Davalos, A. Eguiluz-De La Barra, E. Aguilar-Castellanos, J. Falconi-Palomino, M. Ibanez-Tremolada, and J. Lorenzo. 2009. Estimated economic benefit of double-haploid technique for Peruvian barley growers and breeders. Cereal Res. Commun.37(2):287-293.

Grauda, D., L. Lapina, A. Mikelsone, I. Kokina, and I. Rashal. 2008. Obtaining of double haploid lines for Latvian breeding programs: barley, wheat and triticale anther culture optimization. In: J. Prohens, and M.L. Badenes, editors, Modern variety breeding for present and future needs. Valencia, Valencia, Spain, p.131-132

Guasmi, F., W. Elfalleh, H. Hannachi, K. Feres, L. Touil, N. Marzougui and A. Ferchichi. 2013. Influence of various physical parameters on anther culture of barley. J. Plant Nutrition. 36(5):836-847.

Hajjar, R., and T. Hodgkin. 2007. The use of wild relatives in crop improvement: a survey of developments over the last 20 years. Euphytica, 156:1-13.

Horsley, R.D., and B.L. Harvey. 2011. Barley breeding history, progress, objectives, and technology, North America. In: S.E. Ullrich, editor, Barley: Production, improvement and uses. Wiley-Blackwell, Southern Gate, Chichester, West Sussex, UK. p. 171-186.

Houben, A., M. Sanei, and R. Pickering. 2011. Barley doubled-haploid production by uniparental chromosome elimination. Plant Cell Tissue Organ Cult. 104(3):321- 327.

Hua, W., Y. Shang, Q. Jia, J. Wang, J. Zhu, F. Lin, and J. Yang. 2013. Rapid culture method for barley anther and effect of genotype difference in anther culture response. Acta Agr. Zhejiangensis. 25(3):425-430.

Huang, B., J.M. Dunwell, W. Powell, A.M. Hayter, and W. Wood. 1984. The relative efficiency of microspore culture and chromosome elimination as methods of haploid production in Hordeum vulgare L. J. Plant Breed. 92(1), 22-29.

Humphreys, M.W. 1978. Chromosome instability in Hordeum vulgare x H. bulbosum hybrids. Chromosoma. 65:301-307.

Hussain, A., H. Nazir, I. Ullah, and I.A. Qarshi. 2012. Plant tissue culture: current status and opportunities. In Leva, A., and L. Rinaldi, editors, Recent advances in plant in vitro culture, In Tech. Open Access Publisher. p. 1-28. doi: 10.5772/50568.

Ibrahim, K.M., and J.A. Barrett. 2001. Evolutionary changes in Cambridge composite cross five of barley. Broadening the genetic base of crop production. International Plant Genetic Resources Institute, Food and Agriculture Organization of the United Nations and CABI Publishing. New York, NY, USA. p. 271-282.

Jackson, L. 2011. Barley Cultivars for California. P. 37. Dep. Plant Sci.Univ. California, Davis. p. 1-37. http://smallgrains.ucdavis.edu/cereal_files/BarCvDescLJ11. (accessed 29 June 2018).

Jain, S.M., R.J. Newton, and N.A. Tuleen. 1988. Tissue culture and gene transfer in barley. Current Sci. 57:59-70.

Jensen, N. 1988. Breeding and selection methods; double haploid method. Plant breeding methodology. Wiley, New York. p.15-62.

Jha, T.B., and G. Biswajit. 2005. Plant tissue culture: basic and applied. Univ. Press India. p. 96-103.

Jones, R.N., and H. Thomas. 2009. Genome restructuring in hybrids: marriages of inconvenience. Genetyka i genomika w doskonaleniu roślin uprawnych. 11-29. http://bluetreeband.org/pdf/paperpdfs/127.pdf. (accessed 15 June 2018).

Kalladan, R., S. Worch, H. Rolletschek, V.T. Harshavardhan, L. Kuntze, C. Seiler, N. Sreenivasulu, and M.S. Roder. 2013. Identification of quantitative trait loci contributing to yield and seed quality parameters under terminal drought in barley advanced backcross lines. Mol. Breed. 32:71-90.

Kant, L., S. Amrapali, and K.B. Banisetti. 2016. Barley. In: M. Singh, and H.D. Upadhyaya, editors, Genetic and genomic resources for grain cereals improvement. Academic Press, Waltham, p 125-157.

Kasha, K.J., and K.N. Kao. 1970. High frequency haploid production in barley (Hordeum vulgare L.). Nature. 225:874-876.

Khemiri, H., H. Belguith, T. Jridi, M. Ben El Arbi, and J. Ben Hamida. 2004. Biochemical characterization of an active amylase during germination process of colza seed (Brassica napus L.). Enzymology and metabolism. Congres Int. Biochimie. Marrakech. p.146-149.

Knezevic, D., N. Przulj, V. Zecevic, N. Dukic, V. Momcilovic, D. Maksimovic, and B. Dimitrijevic. 2004. Breeding strategies for Barley quality improvement and wide adaptation. Kragujevac J. Sci. 26:75-84.

Komatsuda, T., K. Tanno, B. Salomon, T. Bryngelsson, and R. Von Bothmer. 1999. Phylogeny in the genus Hordeum based on nucleotide sequences closely linked to the vrs1 locus (row number of spikelets). Genome. 42:973-981.

Kook, H.S., T. Park, A. Khatoon, S. Rehman, and S.J. Yun. 2009. Avoidance of sodium accumulation in the shoot confers tolerance to salt stress in cultivated barley. Pak. J. Bot. 41:1751-1758.

Lakew, B. 2009. The genetic potential of wild barley (Hordeum spontaneum C. Koch) to improve adaptation to low rainfall environments. Ph.D. diss., Southern Cross Univ.

Lange, W., G. and Jochemsen. 1976. The offspring of diploid, triploid and tetraploid hybrids between Hordeum vulgare and H. bulbosum. Proc. Int. Barley Genet. Symp. 3rd. p.252-259.

Lazaridou, T.B., A.S. Lithourgidis, S.T. Kotzamanidis, and D.G. Roupakias. 2005. Anther culture response of barley genotypes to cold pretreatments and culture media. Russian J. Plant Physiol. 52(5):696-699.

Lundqvist, U., J.D. Franckowiak, and T. Konishi. 1997. New and revised descriptions of barley genes. Barley Genet. Newsletter. 26:22-516.

Machado, R.M.A., and R.P. Serralheiro. 2017. Soil salinity: Effect on vegetable crop growth, management practices to prevent and mitigate soil salinization. Hortic. 3(2):1-30. Mak, C. and B.L. Harvey. 1982. Exploitable genetic variation in a composite bulk population of barley. Euphytica. 31:85-92.

Mano, Y., and K. Takeda. 1998. Genetic resources of salt tolerance in wild Hordeum species. Euphytica. 103(1):137-141.

Mano, Y., H. Nakazumi, and K. Takeda. 1996. Varietal variation in and effects of some major genes on salt tolerance at the germination stage in barley. Breed. Sci. 46:227-233.

Marschner, H. 1995. Mineral nutrition of higher plants. 2nd ed. Academic Press, San Diego, CA.

Marshal, J., C. Jackson, T. Shelman, L. Jones, and K. O’Brien. 2016. 2015 Small Grains Report for Southcentral and Southeastern Idaho. University of Idaho. p. 133. https://www.cals.uidaho.edu/edcomm/pdf/RES/RES188.pdf. (accessed 14 June 2018).

Matus, I., A. Corey, T. Filichkin, P.M. Hayes, M.I. Vales, J. Kling, and R. Waugh. 2003. Development and characterization of recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a Hordeum vulgare subsp. vulgare background. Genome. 46(6):1010-1023.

Matus, I.A., and P.M. Hayes. 2002. Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats. Genome. 45:1095-1106.

Mazher, A.A.M., F.E.M. EL-Quensi, and M.M. Farahat. 2007. Responses of ornamental plants and woody trees to salinity. World J. Agric. Sci. 3:386-395.

Mukhambetzhanov, S. K. 1997. Culture of nonfertilized female gametophytes in vitro. Plant Cell Tissue Organ Cult. 48:111-119.

Munns, R., and M. Tester. 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59:651-681. http://dx.doi.org/10.1146/annurev.arplant.59.032607.092911 (accessed 28 July 2018).

Munns, R. 2002. Salinity, growth and phytohormones. In: A. Lauchli and U. Luttge, editors, Salinity: Environment-plants-molecules. DordrechtKluwer Academic Publishers. p. 271-290.

Newton, A.C., A.J. Flavell, T.S. George, P. Leat, B. Mullholland, L. Ramsay, and I.J. Bingham. 2011. Crops that feed the world 4. Barley: a resilient crop? Strengths and weaknesses in the context of food security. Food Security. 3(2):41-178.

Obert, D.E., D.M. Wesenberg, D.E. Burrup, B.L. Jones, and C.A. Erickson. 2006. Registration of 'Charles' barley. Crop sci. 46(1):468-470.

OECD. 2004. Consensus document on compositional considerations for new varieties of barley (Hordeum vulgare L.): Key food and feed nutrients and anti-nutrients. Report No. 12, Environment Directorate, OECD, Paris. 34

OGTR. 2008. The Biology of Hordeum vulgare L. (barley). Office of the gene technology regulator, Australian Dep. of Health and Ageing. Canberra, Australia. p.41.

Othman, Y., G. Al-Karaki, A.R. Al-Tawaha, and A. Al-Horani. 2006. Variation in germination and ion uptake in barley genotypes under salinity conditions. World J. Agric. Sci. 2(1):11-15.

Parida, A.K., and A.B. Das. 2005. Salt tolerance and salinity effects on plants: A review. Ecotox. Environ. Safe. 60:324-349. http://dx.doi.org/10.1016/j.ecoenv.2004.06.010 (accessed 29 June 2018).

Phillips, S.L., and M.S. Wolfe. 2005. Evolutionary plant breeding for low input systems. J. Agri. Sci. 143(04):245-254.

Pickering, R., and P.A. Johnston. 2005. Recent progress in barley improvement using wild species of Hordeum. Cytogenet. Genome Res. 109(1-3):344-349.

Pickering, R.A. 1984. The influence of genotype and environment on chromosome elimination in crosses between Hordeum vulgare L.× Hordeum bulbosum L. Plant Sci. Letters. 34(1-2):153-164.

Pickering, R.A. 1985. Partial control of chromosome elimination by temperature in immature embryos of Hordeum vulgare L. x H. bulbosum. Euphytica. 14:869– 874.

Pickering, R.A. 1988. The production of fertile triploid hybrids between H vulgare L. (2n= 2 x= 14) and H. bulbosum L. (2n= 4 x= 28). Barley genet. Newsl. 18:25-29.

Pickering, R.A. 1991. Comparison of crossover frequencies in barley (Hordeum vulgare) and H. vulgare × H. bulbosum hybrids using a paracentric inversion. Genome. 34(4):666-673.

Pickering, R.A., S. Malyshev, G. Kunzel, P.A. Johnston, V. Korzun, M. Menke, I. Schubert. 2000. Locating introgressions of Hordeum bulbosum chromatin within the H. vulgare genome. Theor. Appl. Genet. 100:27-31.

Pimentel, D., B. Berger, D. Filiberto, M. Newton, B. Wolfe, E. Karabinakis, S. Clark, E. Poon, E. Abbett, and S. Nandaopal. 2004. Water resources: Agricultural and environmental issues. BioScience. 54:909-918.

Poehlman, J.M. 1985. Adaptation and distribution. In: Barley (Ed., D.C. Rasmussen) pp. 1-18. American Society of Agronomy, Madison, WI. FAO (2008) FAO crop estimate data http://faostat.fao.org/site/565/defact.aspx. (accessed 16 July 2018).

Pourkheirandish, M., and T. Komatsuda. 2007. The importance of barley genetics and domestication in a global perspective. Annals Bot. 100(5):999-1008. 35

Ravi, M., and S. W.Chan. 2010. Haploid plants produced by centromere-mediated genome elimination. Nature. 464(7288):615-618.

Roche, D., D.J. Hole, R.S. Albrechtsen, J.W. Clawson, and S.A. Young. 2005. Registration of ‘Goldeneye’ Spring Barley. Crop sci. 45(2005):2658.

Rostami, H., A. Giri, A.S. M. Nejad, and A. Moslem. 2013. Optimization of multiple shoot induction and plant regeneration in Indian barley (Hordeum vulgare) cultivars using mature embryos. Saudi J. Bio. Sci. 20(3):251-255.

Ruth, S.K., L.S. Stephen, and C.B. John. 1993. Ovule culture of sweet potato (Ipomoea batatas) and closely related species. Plant Cell Tissue Organ Cult. 32:77-82.

San Noeum, L.H. 1976. Haploides Hordeum vulgare L. par culture in vitro d'ovaires non fecondes. Ann Amelior Plant. 26:751–754

Sanei, M., R. Pickering, J. Fuchs, A. Banaei Moghaddam, A. Houben, and A. Dziurlikowska. 2010. Interspecific hybrids of Hordeum marinum ssp. marinum × H. bulbosum are mitotically stable and reveal no gross alterations in chromatin properties. Cytogenet. and Genome Res. 129(1-3):110-116.

Sanei, M., R. Pickering, K. Kumke, S. Nasuda, and A. Houben. 2011. Loss of centromeric histone H3 (CENH3) from centromeres precedes uniparental chromosome elimination in interspecific barley hybrids. Proc. Natl. Acad. Sci. U.S.A 108(33): E498-E505.

Serhantova, V., J. Ehrenbergerova, and L. Ohnoutkova. 2004. Callus induction and regeneration efficiency of spring barley cultivars registered in the Czech Republic. Plant Soil Environ. 50:456-462.

Sharma, L., M. Dalal, R.K. Verma, S.V. Kumar, S.K. Yadav, S. Pushkar, and V. Chinnusamy. 2018. Auxin protects spikelet fertility and grain yield under drought and heat stresses in rice. Environ. Exp. Bot. 150:9-24.

Sibi, M.L., A. Kobbaissi, and A. Shekafandeh .2001. Green haploid plants from unpollinated ovary culture in tetraploid wheat (Triticum durum Defs.). Euphytica. 122:351-359

Tang, F., Y. Tao, T. Zhao, G. Wang. 2006. In vitro production of haploid and double haploid plants from pollinated ovaries of maize (Zea mays). Plant Cell Tissue Organ Cult. 84:233-237

Temel, A., and N. Gozukirmizi. 2012. Retrotransposition events in barley callus cultures. In VII international symposium on in vitro culture and horticultural breeding. 961:175-179.

Temel, A., G. Kartal, and N. Gozukirmizi. 2008. Genetic and epigenetic variations in barley calli cultures. Biotechnol. Equip. 22 (4):911-914.

Thomas, H.M., and R.A. Pickering. 1983. Chromosome elimination in Hordeum vulgare x H. bulbosum hybrids. Theor. Appl. Genet. 66(2):135-140.

Tiwari, V. 2010. Growth and production of Barley. In: I. Aufl , editor, Soils, plant growth and crop production. Eolss Publishers Company Limited, Encyclopedia of Life Support Systems (EOLSS), Developed under the Auspices of the UNESCO, Eolss Publishers, Paris, France. http://www.eolss.net/sample-chapters/c10/E1-05A-11- 00.pdf (accessed 6 July 2018).

USDA-FAS. 2018. World agricultural production. Circular series WAP 9-18. https://apps.fas.usda.gov/psdonline/circulars/production.pdf (accessed 25 Sept. 2018).

Veilleux R.E., and A.A.T. Johnson. 1998. Plant Breed. Reviews. 16:229-268.

Von Bothmer R., K. Sato, T. Komatsuda, S. Yasuda, and G. Fischbeck. 2003. The domestication of cultivated barley. In: R. von Bothmer, T. Van Hintum, H. Knupffer, K. Sato, editors, Diversity in barley (Hordeum vulgare). Elsevier Science B.V., Amsterdam. p. 9-27.

Von Bothmer, R. 1992. The wild species of Hordeum: Relationships and potential use for improvement of cultivated barley. In: P.R. Shewry, editor, Barley: Genetics, biochemistry, molecular biology and biotechnology. C.A.B Int. Wallingford, Oxon. p. 3-18.

Von Bothmer, R., and T. Komastuda. 2011. Barley origin and related species. In: S.E. Ullrich, editor, Barley: Production, improvement and uses. Wiley-Blackwell, Southern Gate, Chichester, West Sussex, UK. p. 14-62.

Von Bothmer, R., N. Jacobsen, C. Baden, R.B Jorgensen and I. Linde-Laursen. 1995. An ecogeographical study of the genus Hordeum. 2nd ed. Systematic and ecogeographic studies on crop genepools 7 Rome: Int. Plant Genet. Resources Ins. Rome, Italy. p. 129.

Von Bothmer, R., N. Jacobsen, C. Baden, R.B Jorgensen and I. Linde-Laursen. 1995. An ecogeographical study of the genus Hordeum. Rome: International Plant Genetic Resources Institute (IPGRI).

Wendler, N., M. Mascher, C. Noh, A. Himmelbach, U. Scholz, B. Ruge‐Wehling, and N. Stein. 2014. Unlocking the secondary gene‐pool of barley with next‐generation sequencing. Plant biotech. J. 12(8): 1122-1131.

Xue Z., D. Zhi, G. Xue, H. Zhang, Y. Zhao and G. Xia. 2004. Enhanced salt tolerance of transgenic wheat (Triticum aestivum L.) expressing a vacuolar Na+/H+ antiporter gene with improved grain yields in saline soils in the filed a reduced level of leaf Na+. Plant Sci.167(4):849-859.

Yi, C., X. Dai, X. Li, Z. Gong, Y. Zhou, G. Liang, and W. Zhang. 2013. Identification and diversity of functional centromere satellites in the wild rice species Oryza brachyantha. Chromosome Res. 21(8):725-737. doi:10.1007/s10577-013-9374-8

Zamani, I.N., E. Gouli-Vavdinoudi, G. Kovacs, I. Xynias, D. Roupakias, and B. Barnabas. 2003. Effect of parental genotypes and colchicine treatment on the androgenic response of wheat f1 hybrids. Plant Breed. 122:314-317.

Zhang, L., R.A. Pickering, and B.G. Murray. 2001. Hordeum vulgare x H. bulbosum tetraploid hybrid provides useful agronomic introgression lines for breeders. New Zealand J. Crop Hortic. Sci. 29(4):239-246.

Zhou, G., P. Johnson, P.R. Ryan, E. Delhaize, and M. Zhou. 2012. Quantitative trait loci for salinity tolerance in barley (Hordeum vulgare L.). Mol. Breed. 29(2):427-436.

CHAPTER III

PRELIMINARY SCREENING FOR SALT TOLERANCE

Introduction

Salinity is one of the most important problems facing crop productivity in Iraq, as well as around the world. More than 6% of the world’s total land area (900 million hectares) is impacted by salt, accounting for more than 20% of the total agricultural land.

In addition, the global economic losses due to saline soils amount to US$27.3 billion annually (FAO 2013). Because of increased desertification, salinity, drought, and corresponding water shortages, the FAO (2009) estimated 76% of cereals would need to be imported to Iraq. In 2008, Iraqi wheat farmers experienced a 55% decrease in production because of severe drought and fixed irrigation water (FAO 2012). Thus, there is a need to produce plant varieties with high water use efficiency and the ability to tolerate salinity. In plant breeding programs, there is a need to screen genotypes for salt tolerance at germination and seedling growth stages to identify genotypes with salt stress tolerance (Mano and Takeda 1998). Also, genotypes can be screened for salt tolerance by using different phenotypic parameters such as germination percentage, radicle length, coleoptile length, relative water content, fresh and dry weight of roots and shoots (Tavili and Biniaz 2009; Adjel et al. 2013; Abu-El-lail et al. 2014; El Goumi et al. 2014; Sbei et al. 2014; Abdi et al. 2016; Chikha et al. 2016; Shahraki and Fakheri 2016; Angessa et al.

2017). The continuous cultivation of barley has led to a narrowing of its genetic base, as

well as the loss some of its traits still present in wild species. Consequently, the wild species are a valuable genetic resource for inclusion in breeding programs (Knezevic et al. 2004).

Materials and Methods

Two barley species were used in this study. Domestic barley (Hordeum vulgare) is an annual crop with some salinity tolerance, while bulbous barley (H. bulbosum) is perennial with strong tolerance to salinity. Seed of the two species used in this study were requested and received from GRIN, USDA-ARS/National Small Grains Collection. Five

H. vulgare germplasm lines were used to cross with six H. bulbosum. This material originated from several locations in Iraq as well as Jordan, Syria, Iran, Turkey and Israel

(Table 3.1). For convenience, the five H. vulgare species were labelled 1-5; the six H. bulbosum species were labelled 6-11 (Table 3.1). The experiments were conducted in greenhouses and on a gravel pad of the Plant and Soil Sciences Department at Mississippi

State University, Mississippi, USA (33.452928, -88.794104).

Table 3.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study

Domestic barley Bulbous barley H. vulgare subsp. spontaneum (2x = 14) H. bulbosum (2x = 14, 4x = 28) No. Plant Identity Origin No. Plant Identity Origin 1† PI 219796 HO08ID Iraq, Arbil 6 PI 219869 HO07ID Iraq, Arbil 2 PI 254894 HO05ID Iraq, As 7 PI 220054 HO15ID Iraq, As Sulaymaniyah Sulaymaniyah 3 PI 268243 HO08ID Iran, Ilam 8 PI 220055 HO14ID Iraq, As Sulaymaniyah 4 PI 296843 HO05ID Israel, Northern 9 PI 227242 HO11ID Iran, Fars 5 PI 560558 HO99ID Turkey, Siirt 10 PI 420909 HO93ID Jordan 11 PI 487248 HO00ID Syria, Halab † All genotypes were identified as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, respectively. These numbers were used as the identifier in all experiments.

Initial salt screenings were performed to assess salinity tolerance of germplasm lines used in this study. Germplasm was screened for salinity tolerance at two stages of growth: at germination and seedling growth stage according to the methods outlined by

Mano and Takeda (1998).

Salt tolerance at germination stage:

This experiment was conducted in a growth chamber (Percival Scientific, Mod.

GR-37L, Boone, Iowa) at the Department of Plant and Soil Sciences at Mississippi State

University (MSU), on October 2015. Seed were cleaned by hand to remove chaff and empty seed. Because of limited seed availability, one seed of each accession was placed on one layer of WhatmanTM filter paper (GE Healthcare, 85cm, Buckinghamshire, UK) moistened with 0, 171, 257, and 342 mM of NaCl solution which corresponds to 0, 1.0,

1.5, and 2.0% NaCl solution, respectively. Germination screening was conducted in plastic Petri dishes (Fisher Scientific, 90 x 15 mm, Suwanee, GA) with three replications of each treatment. The experimental design was completely randomized (CRD).

Germination tests were carried out at 20° C under a 12/12 h (light/dark) photoperiod in a growth chamber. After 10 days, root and shoot length were recorded. Salt tolerance was scored on a rating scale of 1 to 6; 1 being least growth and 6 showing greatest growth according to key descriptors by Mano and Takeda (1998) (Table 3.2). Due to limited seed availability and to obtain a sufficient amount of seed to conduct additional experiments, seedlings of the control treatment were transplanted into a field plot (180 cm x 180 cm) with plant density 30 cm x 30 cm at the H. H. Leveck Research Farm at MSU.

Table 3.2 Key for scoring of salt tolerance at germination for barley accessions Mano and Takeda 1998

NaCl Percentage Score 1 1.5 2 (171 mM) (252 mM) (342 mM) 1 x x x 2 O x x 3 ʘ O x 4 ʘ ʘ Δ 5 ʘ ʘ O 6 ʘ ʘ ʘ x: Only root elongated, Δ: Shoot elongated less than 10 mm, O: Shoot elongated 10 - 25 mm, ʘ: Shoot elongated over 25 mm.

The data were statistically analyzed as a CRD using PROC MEANS and PROC

GLM in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Significant mean separation among genotypes was determined using Fisher’s least significant difference (LSD) at α =

0.05 and 0.01.

Salt tolerance at seedling growth stage:

The germinated seed which were evaluated for salt tolerance at germination were also tested for salt tolerance at the seedling stage. After ten days, germinated seedlings were planted in pots (5 L) filled with a mixture (1:2 V/V) of fine sand and Sunshine® Mix

1 (Planet Natural, Bozeman, MT). The plant density was 3.0 cm x 2.5 cm. Pots were placed in a greenhouse under ambient November photoperiod at 20° C. When the seedlings were at the three-leaf stage, they were irrigated every third-fourth day with 500 mM NaCl solution for three weeks. The experiment was arranged as a randomized complete block design (RCBD) with three replications (pots) with one plant for each replication. After 21 days of salt treatment (5 - 6 weeks old), growth index was calculated to identify salinity 42

tolerance of seedlings of each accession. The equation for growth index (GI) was:

GI = No. of leaves x height of the tallest leaf (3.1)

The data were statistically analyzed as a RCBD using PROC MEANS and PROC

GLM in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Significant mean separation among genotypes was determined using Fisher’s least significant difference (LSD) at α =

0.05 and 0.01. The regression analyses were estimated using SigmaPlot version 13

(Systat Software Inc., San Jose, CA).

Results and Discussion

Salt tolerance at germination stage:

Analysis of variance revealed highly significant (P ≤ 0.001) differences among the parental genotypes, salinity, and parental genotypes x salinity combination for root and shoot length (Table 3.3). The significant variations among the genotypes and genotypes x salinity under four salinity levels indicate that there was a high genetic variation among the screened genotypes. Previous studies have also reported highly significant differences among genotypes and genotypes x salinity for different traits such as germination, root, and shoot length (Tavili and Biniaz 2009; El Goumi et al. 2014;

Abdi et al. 2016; Angessa et al. 2017).

Table 3.3 Analysis of variance of the parental barley genotypes, salinity, and their interaction for root and shoot length of initial germination screening experiment

Source of variance Root length Shoot length

Genotypes (G) *** *** Salinity (S) *** *** G x S *** *** *** Significant at the 0.001 probability level 43

Generally, mean root length was decreased as the level of NaCl increased for all genotypes. Mean barley root length of control plants were 10x the length of those at 2%

NaCl. The decrease was expressed with the increase of salinity. This condition was more intense in the H. vulgare species than in the H. bulbosum species (Tavili and Biniaz

2009). Genotypes showed a wide variation for root length under control (0%), 1%, and

1.5% NaCl conditions, which varied from 33 to 112.7 mm, 3 to 26 mm, and 0 to 10 mm, respectively (Table 3.4). Genotype 2 had the greatest root length compared to all other genotypes of both species. At the highest salinity level (2% NaCl), all genotypes of H. vulgare species failed to germinate. In contrast, three of six genotypes of H. bulbosum,

(genotypes 7, 9, and 10) germinated at the 2% NaCl level of salinity with mean root lengths of 2, 2, and 1 mm, respectively. These genotypes would have an advantage for improving barley tolerance for salinity at the germination stage. Varieties tolerant to salinity at the germination stage is most important because seed germination is the first stage of plant growth and takes place near the surface soil where a high level of salt accumulates, especially soil with high levels of groundwater (Matsumoto 1989). Previous studies have also reported that there were significant variations in salt tolerance among barley genotypes at germination stage (Tajbakhsh et al., 2006; Kook et al. 2009). Seed germination under salinity is reduced due to osmotic stress, and high levels of NaCl reduce the bioavailability of water caused by Na+, resulting in what is effectively a water deficit (Mano et al. 1996; Parida and Das 2005; Abdi et al. 2016). Additionally, Na+ displayed toxic effects on the germinating embryos (Mazher et al. 2007). Furthermore, salinity may act to interfere with enzyme functions in the seed, which decreased activity of polyphenol-oxidases and amylases (Khemiri et al., 2004).

Table 3.4 Mean root length (mm) of parental barley genotypes after 10 days germination at 0% (0mM), 1% (171 mM), 1.5% (257 mM), and 2% (342 mM) NaCl solution of initial germination screening experiment

Root length (mm) Salinity NaCl Percentage 0 1 1.5 2 Genotypes (0 mM) (171 mM) (252 mM) (342 mM) H. vulgare 1 84.0† 22.0 3.0 0.0 2 112.7 26.0 10.0 0.0 3 53.5 3.0 0.0 0.0 4 78.3 18.0 2.0 0.0 5 64.3 14.3 1.7 0.0 H. bulbosum 6 33.0 5.5 2.0 0.0 7 47.0 6.5 4.0 2.0 8 44.5 4.0 3.0 0.0 9 47.7 8.0 4.3 2.0 10 33.5 4.7 3.0 1.0 11 39.3 5.0 3.5 0.0

Mean (x̅ ) 58.0 10.6 3.3 0.5 Range 33.0 - 112.7 3.0 – 26.0 0.0 – 10.0 0.0 – 2.0 LSD (0.05) 13.308 1.988 1.524 0.000 LSD (0.01) 18.088 2.702 2.072 0.000 † Each value represents the mean of 3 replications with one seed for each replicate.

Barley genotypes in this study showed wide variation of shoot length under control conditions. The 10-day shoot length varied from 29.3 mm in genotype 3 to 81 mm in genotype 2 (Table 3.5). Most genotypes of H. vulgare had greater shoot length than H. bulbosum under the control conditions (Mano and Takeda 1998; Tavili and

Biniaz 2009).

Table 3.5 Mean shoot length (mm) of parental barley genotypes after 10 days germination at 0% (0mM), 1% (171 mM), 1.5% (257 mM), and 2% (342 mM) NaCl solution of initial germination screening experiment

Shoot length (mm) Salinity NaCl Percentage 0 1 1.5 2 Genotypes (0mM) (171 mM) (252 mM) (342 mM) H. vulgare 1 77.3† 0.0 0.0 0.0 2 81.0 5.3 0.0 0.0 3 29.3 0.0 0.0 0.0 4 52.0 0.0 0.0 0.0 5 58.3 1.0 0.0 0.0 H. bulbosum 6 31.7 0.0 0.0 0.0 7 31.0 5.3 0.0 0.0 8 37.0 0.0 0.0 0.0 9 32.7 6.3 0.0 0.0 10 29.5 4.3 0.0 0.0 11 37.3 1.3 0.0 0.0

Mean (x̅ ) 45.2 2.1 0.0 0.0 Range 29.3 – 81.0 0.0 - 6.3 0.0 0.0 LSD (0.05) 22.628 3.598 0.000 0.000 LSD (0.01) 30.755 4.8904 0.000 0.000 † Each value represents the mean of 3 replications with one seed for each replicate.

Mean shoot length declined sharply at 1% NaCl for all genotypes compared to control; which decreased from 45.2 mm at 0% NaCl to 2.1 mm at 1% NaCl and 0 mm at both 1.5 and 2% NaCl. Bagci et al. (2003) reported that increasing [NaCl] treatments showed a significant decrease in shoot length with significant differences between genotypes. As [NaCl] increased, shoot length of H. vulgare genotypes was highly reduced compared to H. bulbosum genotypes at 1% NaCl. Genotypes 2 (Hv), 7 (Hb), 9

(Hb) and 10 (Hb) had greatest shoot length 5.3, 5.3, 6.3 and 4.3 mm, respectively, at 1%

NaCl. All genotypes failed to produce shoots at 1.5 and 2% NaCl. These results indicate that shoot length was more affected by salt stress than root length for all genotypes. Also, 46

the genotypes of H. vulgare were more susceptible to salinity than H. bulbosum. Abdi et al. (2016) reported that coleoptile’s emergence of barley cultivars showed greater sensitivity to salinity than radicle’s emergence.

According to keys described by Mano and Takeda (1998) (Table 3.2), salt tolerance of genotypes at germination was scored on a binary rating scale of 1 to 2 (Table

3.6). All H. vulgare genotypes were scored a value of one, which indicates they were more sensitive to salinity at germination. Three genotypes from H. bulbosum were scored a value of 2, which meant they were more tolerant to salinity than H. vulgare genotypes.

Table 3.6 Key for scoring salt tolerance at germination for parental barley genotypes derived from Mano and Takeda 1998

NaCl Percentage Genotypes 1 1.5 2 Score (171 mM) (252 mM) (342 mM) 1 x x - 1 2 Δ x - 1 H. vulgare 3 x - - 1 4 x x - 1 5 Δ x - 1 6 x x - 1 7 Δ x x 2 H. bulbosum 8 x x - 1 9 Δ x x 2 10 Δ x x 2 11 Δ x - 1 x: Only root elongated, Δ: Shoot elongated less than 10 mm.

Salt tolerance at seedling growth:

The analysis of variance indicated highly significant (P ≤ 0.001) differences among the genotypes at the seedling growth stage under saline conditions for growth index (Table 3.7). This significant phenotypic variation corresponds to a high degree of genetic variation among the genotypes. 47

Table 3.7 Analysis of variance for growth index of the parental barley genotypes at 500 mM NaCl solution of initial screening at seedling growth

Source of variance Growth Index Block NS† Genotypes (G) *** *** Significant at the 0.001 probability level. NS†, significance level P > 0.05.

Growth index reflects the ability of plants to grow under stressful conditions, which can be estimated by leaf number and plant height. Growth index varied widely among the genotypes in response to salinity treatment. Growth index ranged from 36.23 in genotype 8 to 108.20 in genotype 3 (Table 3.8). Genotype 3 (H. vulgare) had the greatest growth index (108.20) compared to all other genotypes of both species.

Genotype 11 from H. bulbosum species had the greatest growth index (70.67) compared to all genotypes of the same species. The differences of growth indexes between the two species was not due to salinity but was due to growth habit. H. bulbosum is a perennial crop (grows slowly and develops a crown), and H. vulgare is an annual crop (rapid growth) (von Bothmer et al. 1995; Bara et al. 2006). As a result, the mean growth index for H. vulgare species was greater but showed more leaf injury than H. bulbosum. Tavili and Biniaz (2009) also reported that the stem length of H. vulgare genotypes was greater than H. bulbosum in their experiment at 60, 120, 180, 240, and 300 mM NaCl.

Table 3.8 Mean growth index of parental barley genotypes at 500 mM NaCl solution of initial screening at seedling growth

Growth Index Genotypes 500 mM NaCl H. vulgare 1 85.80† 2 72.87 3 108.20 4 55.00 5 76.80 H. bulbosum 6 64.00 7 40.50 8 36.23 9 70.67 10 48.07 11 56.00

Mean (x̅ ) 64.92 Range 36.23-108.2 LSD (0.05) 10.251 LSD (0.01) 13.982 † Each value represents the mean of 3 replications with one seedling for each replicate.

Overall plant growth of barley was reduced under saline conditions due to water stress, changes in nutrient uptake and translocation, and ion toxicities of NaCl which can deactivate cellular functions and physiological processes (Marschner 1995; Mano et al.

1996; Bagci et al. 2003). Kook et al. (2009) reported that high salt tolerance of T76 cultivar was due to exclusion of Na+ accumulation in the shoot under high salt conditions.

Regression analysis of growth index against root length under saline conditions

(1, 1.5, and 2% NaCl) of parental barley genotypes was not significant (P ˃ 0.05), r2 =

0.07, 0.04, 0.1, respectively (Figure 3.1); showing that there was no relationship between growth index and root length under saline conditions.

160 1.0% [NaCl], Y = 57.89 + 0.66x; r² = 0.07 140 1.5% [NaCl], Y = 70.15 - 1.58x; r² = 0.04 2.0% [NaCl], Y = 68.58 - 8.06x; r² = 0.10

120

100

Seedling test Seedling index) (Growth

20 0 5 10 15 20 25 30 Germination test (Root length, mm)

Figure 3.1 Relationship between growth index and root length under saline conditions (1, 1.5, and 2% NaCl) of parental barley genotypes.

Regression analysis of growth index against shoot length under saline conditions

(1, 1.5, and 2% NaCl) of parental barley genotypes was also not significant (P ˃ 0.05), r2

= 0.05, 0, 0, respectively (Figure 3.2), indicating that there was no correlation between growth index and shoot length under saline conditions.

180 1.0% [NaCl], Y = 68.86 - 1.84x; r² = 0.05 † 160 1.5% [NaCl], Y = 0.00 + 0.00x; r² = 0.00 2.0% [NaCl], Y = 0.00 + 0.00x; r² = 0.00† 140

120

100

Seedling test Seedling index) (Growth 80

20 0 1 2 3 4 5 6 7 Germination test (Root length, mm)

† No shoots were present

Figure 3.2 Relationship between growth index and shoot length under saline conditions (1, 1.5, and 2% NaCl) of parental barley genotypes.

The results of regression analysis indicated that there was no relationship between salinity tolerance at germination and tolerance at the seedling growth stage, confirmed by

Munns 2002; El Madidi et al. 2004; Foolad 2004; Kook et al. 2009; and Adjel et al. 2013.

Consequentially, salt tolerance of barley during germination and early growth is controlled by different genetic mechanisms (Mano et al. 1996; Mano and Takeda 1998;

Bagci et al. 2003; Qiu et al., 2011). Therefore, in a breeding program, it is important to screen for salt tolerance at different growth stages. Munns and Tester ( 2008) pointed out

that germination and seedling growth stage were the most salt sensitive stages of plant growth but given the lack of correlation between these two stages, screening for reliable phenotyping must be a two-step process.

Conclusion

Root length and shoot length decreased as the level of NaCl increased for all genotypes. As expected this decrease was greater with H. vulgare species than H. bulbosum. Genotype 2 had the greatest root length at 0, 1, 1.5% NaCl, but it did not germinate at 2% NaCl. Only genotypes 7, 9, and 10 germinated at the greatest level of salinity. Genotypes 2, 7, 9, and 10 had greatest shoot length at 1% NaCl. No genotypes tested produced shoots at 1.5 and 2% NaCl. Seedling screening showed genotype 3 had the greatest growth index compared to the other genotypes. Genotypes 9 had greatest growth index among H. bulbosum. These data indicate there was no relationship between salinity tolerance at germination and tolerance at the seedling growth stage. Therefore, in a breeding program, it is important to screen for salt tolerance at different growth stages.

Based on this study, wild Hordeum species may be a source of resilience of salinity in a breeding program. Levels of salinity tolerance vary by genotype, stage of plant development, and geographical origin; yet success is still dependent upon yield at maturity.

REFERENCES

Abdi, N., S. Wasti, M.B. Salem, M. El Faleh, and E. Mallek-Maalej. 2016. Study on germination of seven barley cultivars (Hordeum vulgare L.) under salt stress. J. Agric. Sci. 8:88-97.

Abu-El-lail, F.F.B., K.A. Hamam, K.A. Kheiralla, and M.Z. El-Hifny. 2014. Salinity tolerance in 280 genotypes of two-rows barley. Egypt. J. Plant Breed. 18:331-345.

Adjel, F., H. Bouzerzour, and A. Benmahammed. 2013. Salt stress effects on seed germination and seedling growth of barley (Hordeum vulgare L.) genotypes. J. Agric. Sustain 3:223-237.

Angessa, T.T., X.Q. Zhang, G. Zhou, S. Broughton, W. Zhang, and C. Li. 2017. Early growth stages salinity stress tolerance in CM72 x Gairdner doubled haploid barley population. Plos One 12:1-16.

Bagci, S.A., H. Ekiz, and A. Yilmaz. 2003. Determination of the salt tolerance of some barley genotypes and the characteristics affecting tolerance. Turk. J. Agric. For. 27:253-260.

Bara, I., G. Capraru, and E. Maxim. 2006. Hordeum vulgare L. cytotaxonomical aspects. Analele stiintifice ale universitatii "Alexandru Ioan Cuza" din Iasi Sec. II a. Genetic Biol. Mol. 7:227-234.

Chikha, M.B., K. Hessini, , R.N. Ourteni, A. Ghorbel, and N. Zoghlami. 2016. Identification of barley landrace genotypes with contrasting salinity tolerance at vegetative growth stage. Plant Biotechnol. 33:287-295.

El Goumi, Y., M. Fakiri, O. Lamsaouri, and M. Benchekroun. 2014. Salt stress effect on seed germination and some physiological traits in three Moroccan barley (Hordeum vulgare L.) cultivars. J. Mater. Environ. Sci. 5:625-632.

El Madidi, S., B. El Baroudi, and F.B. Aameur. 2004. Effects of salinity on germination and early growth of barley (Hordeum vulgare L.) cultivars. Int. J. Agric. Biol. 6(5):767-770.

FAO. 2009. Iraq food prices analysis, 1-10. ttp://www.fao.org/GIEWS/english/index.htm (accessed 21 July 2018).

FAO. 2012. Iraq- Agriculture Sectore Note, 1-72. http://www.fao.org/investment/en/ (accessed 21 July 2018).

FAO-AQUASTAT. 2013. Area equipped for irrigation and percentage of cultivated land. Available at http://www.fao.org/nr/water/aquastat/globalmaps/index.stm (accessed 19 July 2018).

Foolad, M.R. 2004. Recent advances in genetics of salt tolerance in tomato. Plant Cell Tiss. Org. 76:101-119.

Knezevic, D., N. Przulj, V. Zecevic, N. Dukic, V. Momcilovic, D. Maksimovic, and B. Dimitrijevic. 2004. Breeding strategies for barley quality improvement and wide adaptation. Kragujevac J. Sci. 26:75-84.

Kook, H.S., T. Park, A. Khatoon, S. Rehman, and S.J. Yun. 2009. Avoidance of sodium accumulation in the shoot confers tolerance to salt stress in cultivated barley. Pak. J. Bot. 41:1751-1758.

Mano, Y., and K. Takeda. 1998. Genetic resources of salt tolerance in wild Hordeum species. Euphytica. 103:137-141.

Mano, Y., H. Nakazumi, and K. Takeda. 1996. Varietal variation in and effects of some major genes on salt tolerance at the germination stage in barley. Breed. Sci. 46:227-233.

Marschner, H. 1995. Mineral nutrition of higher plants. 2nd ed. Academic Press, San Diego, CA.

Matsumoto, S. 1989. Controversial points from soil diversity for arid land developments. Sand Dune Res. 36:1-10.

Mazher, A.A.M., F.E.M. EL-Quensi, and M.M. Farahat. 2007. Responses of ornamental plants and woody trees to salinity. World J. Agric. Sci. 3:386-395.

Munns, R. 2002. Comparative physiology of salt and water stress. Plant Cell Environ. 25: 239-250.

Munns, R., and M. Tester. 2008. Mechanisms of salinity tolerance. Annu. Rev. Plant Biol. 59:651-681. http://dx.doi.org/10.1146/annurev.arplant.59.032607.092911 (accessed 28 July 2018).

Parida, A.K., and A.B. Das. 2005. Salt tolerance and salinity effects on plants: A review. Ecotox. Environ. Safe. 60:324-349. http://dx.doi.org/10.1016/j.ecoenv.2004.06.010 (accessed 29 July 2018).

Qiu, L., D.Z. Wu, S. Ali, S.G. Cai, F. Dai, X.L. Jin, … and G. Zhang. 2011. Evaluation of salinity tolerance and analysis of allelic function of HvHKT1 and HvHKT2 in Tibetan wild barley. Theor. Appl. Genet. 122:695-703.

SAS Institute. 2011. SAS guide to macro processing. Vol. 11. SAS Inst., Cary, NC.

Sbei, H., K. Sato, T. Shehzad, M. Harrabi, and K. Okuno. 2014. Detection of QTLs for salt tolerance in Asian barley (Hordeum vulgare L.) by association analysis with SNP markers. Breed. Sci. 64:378-388.

Shahraki, H., and B.A. Fakheri. 2016. QTLs Mapping of morpho-physiological traits of flag leaf in Steptoe × Morex doubled haploid lines of barley in normal and salinity stress conditions. Int. J. Farm. Alli. Sci. 5:356-362.

Tajbakhsh, M., M. Zhou, Z. Chen, and N.J. Mendham. 2006. Physiological and cytological response of salt-tolerant and non-tolerant barley to salinity during germination and early growth. Aust. J. Exp. Agri. 46:555-562.

Tavili, A., and M. Biniaz. 2009. Different salts effects on the germination of Hordeum vulgare and H. bulbosum. Pak. J. Nutr. 8:63-68.

Von Bothmer, R., N. Jacobsen, C. Baden, R.B Jorgensen, and I. Linde-Laursen. 1995. An ecogeographical study of the genus Hordeum. 2nd ed. Systematic and ecogeographic studies on crop genepools 7 Rome: Int. Plant Genet. Resources Ins. Rome, Italy. p. 129.

CHAPTER IV

INTERSPECIFIC CROSSES AND EMBRYO RESCUE TO OVERCOME SEXUAL

INCOMPATIBILITIES

Introduction

Domestic barley (Hordeum vulgare) is one of the most important crops in the world. Genetic variation is essential in breeding programs for improvement and development of any crop (Matus and Hayes 2002). However, barley has been intensively bred for improved performance and grain quality, resulting in a reduced genetic reservoir in the elite cultivars as well as loss of some characteristics which are present in its wild progenitors. Consequently, the lack of genetic variation among H. vulgare genotypes prevents the development of varieties with high productivity expected with a self- pollinated crop (Agdew et al. 2014). Therefore, in order to improve this crop, we must expand its genetic base (Matus et al. 2003). Thus, wild species act as a source of genetic diversity for inclusion in breeding programs to obtain tolerance to stress conditions (Ellis et al. 2000).

Two species in the Hordeum genus, H. vulgare and H. bulbosum, are considered to share a common genome (Von Bothmer 1992). The wild species H. bulbosum represents the secondary gene pool of the genus. This species is used for introgression to improve elite H. vulgare germplasm; making H. bulbosum the most important wild resource for barley improvement (Wendler et al. 2014). However, there are sexual

barriers to hybridization between these species, which can be overcome by modifying environmental conditions, the use of specific genotypes, and biotechnological tools, such as embryo rescue (Pickering 1984). Consequently, tissue culture plays an important role in breeding programs to overcome sexual incompatibilities and aid in the propagation of improved varieties for resistance to disease, such as stripe rust (Puccinia striiformis) and scald (Rhynchosporium secalis), and stress conditions, such as salinity and drought

(Hussain et al. 2012). For example, seed set can be very low due to incompatibility

(Pickering and Johnston 2005), causing the developing seed to degenerate before embryo maturity. Plant growth regulators, such as GA3 and 2,4-dichlorophenoxyacetic acid (2,4-

D) are applied to florets 1 - 2 days after pollination to enhance seed development, embryo number, embryo size, survival, and reduce seed shriveling (Devaux 2003; Houben et al.

2011).

In addition, the developing endosperm aborts after 2 - 5 days of growth, which necessitates rescuing the embryo in order for them to complete their development. As a result, immature embryos need to be cultured on a nutritionally rich medium in order to recover a plant from these sexually incompatible crosses (Pickering and Johnston 2005;

Chahal and Gosal 2006). Immature embryos require a more complex culture medium than a mature embryo. Also, an embryo may fail to grow in an insufficient culture medium (Burun and Poyrazoglu 2002). Therefore, it is important to identify efficient culture medium for immature embryos that can provide sustainable growth and development of the embryo.

Materials and Methods

Crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum:

Crosses were made between two species of barley to transfer salinity tolerance from H. bulbosum germplasm to H. vulgare. Dihaploid genotypes were produced through interspecific hybridization followed by embryo rescue. Diploidization (chromosome doubling) was conducted with the use of colchicine in an effort to restore fertility and resulted in many polyploids.

Interspecific crosses: Diploid H. vulgare x diploid and tetraploid H. bulbosum

All accessions (Table 4.1) were germinated during the winter of 2015/2016 and grown in 1 L pots filled with a mixture (1:2 V/V) of fine sand and Sunshine® mix 1

(Planet Natural, Bozeman, MT). Seedlings were placed in a growth chamber (Conviron,

Mod. E15, Controlled Environments. Winnipeg, MB, Canada) at 15/5° C (light/dark) with a 12 h photoperiod and a light intensity of 97 μmol m−2 s−1. Seed of each of the 11 accessions were planted in six sequential dates, one week apart from Oct. 13 to Nov. 17,

2015, to ensure nicking at flowering. Seedlings were fertilized once a week with 150 ppm

N of Peters® Professional 20:20:20 fertilizer (Everris NA Inc., Dublin, OH). After six weeks, seedlings were transferred to the greenhouse for a seven day acclimation, then transplanted to 6 L pots (same soil mix) and placed outside on a gravel pad. The plants grew and jointed, producing inflorescence. Crosses between each H. vulgare (♀) and H. bulbosum (♂) were made by the method described by Devaux (2003) and Houben et al.

(2011). Hordeum vulgare spikes were observed to be ready for emasculation when the anthers were light green and beginning to turn yellow, but before pollen shed. Tips of the lemma and palea were removed just distal to the anthers; the three anthers were excised 58

using fine forceps. Magnifying OptiVISOR® (7X) glasses (Donegan Optical Co., Lenexa,

KS) was used to visualize florets and their anthers. Emasculated spikes were covered with trimmed pearl millet pollination bags Canvasback® #G27 (Seedburo Equipment Co.,

Des Plaines, IL) and labeled. Bamboo sticks were used to support bagged, emasculated spikes. Pollination was made with each male accession 1 - 2 days later. In order to increase the percentage of seed set and embryo size, pollinated spikes were sprayed with a mixture of GA3 (Sigma-Aldrich Co., G-7645, St. Louis, MO), 2,4- dicholrophenoxyacetic (Sigma-Aldrich Co., D-7299, St. Louis, MO), and dicamba

(Sigma-Aldrich Co., D-5417, St. Louis, MO) (75 mg L-1, 2 mg L-1, and 1 mg L-1, respectively), plus 12 drops L-1 of Tween 20 (Sigma-Aldrich Co., P-2287, St. Louis, MO) once and then again 2 days after the pollination (Houben et al. 2011). This solution was made fresh every 2 - 3 days and stored at 5° C in darkness.

Table 4.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study

Embryo culture of resulting progeny

In interspecific crosses, the developing endosperm aborts after 2-5 days of growth, which necessitates embryo rescue in order for them to complete their development. As a result, immature embryos need to be cultured using nutritionally rich medium. Consequently, spikes from the crosses (interspecific crosses expt.) were harvested 12 - 14 days after pollination when embryos were approximately 1.5 mm in length. Hybrid embryos within their glumes were excised and prepared for media culture.

Spikes were cut from the plant and immersed in a beaker of tap water in the dark at 4° C until dissection (Devaux 2003). Individual spiklets containing embryos were disinfested in a bleach solution (0.825% NaOCl) for 6 mins and rinsed three times with sterile distilled water. Embryos were dissected from their caryopses and placed in 100 ml jars containing either Murashige and Skoog (Sigma-Aldrich Co., M-5519, St. Louis, MO) or

Gamborg B-5 (Sigma-Aldrich Co., G-5893, St. Louis, MO) media (no PGRs) and labeled. Jars were wrapped with Parafilm M® PM-996 (Bemis Company Inc., Neenah,

WI) to reduce contamination. This process was carried out under a laminar flow hood

(ENVIRCO, Environmental Air Control, Inc., Hatfield, PA). Comparisons of embryo growth were made between the two media types. Murashige and Skoog medium was supplemented with 3% sucrose (Fisher Scientific, S5-500, Fair Lawn, NJ) and 0.8% agar

(Sigma-Aldrich Co., A-1296, St. Louis, MO); pH was adjusted to 5.7 by using 0.1 N

NaOH. Gamborg’s B-5 medium was supplemented with 2% sucrose and 0.8 % agar, and pH adjusted to 5.5 (Burun and Poyrazoglu 2002).

Cultured embryos were incubated in darkness to mature for 1 - 4 weeks in a growth chamber (Percival, Mod. 135LLVL, Controlled Environments. Boone, IA).

Germinated embryos reaching 1 - 2 cm were transferred to a second growth chamber at

25/20° C (light/dark) with a 16 h/8 h photoperiod (light/dark) and a light intensity of

92 μmol m−2 s−1. Seedlings were allowed to develop for 1 - 3 additional weeks. When shoots reached 4 - 5 cm (had at least two leaves and a visible root system), they were vernalized in a third growth chamber at 4° C and 8 h photoperiod for 6-8 weeks.

Seedlings with intact root systems were removed from the media, identified by cross, and transferred to 0.5 L pots containing Sunshine® Mix 1 (Planet Natural, Bozeman, MT).

For acclimation, seedlings were placed at 10/8° C (light/dark) with photoperiod 10 h/14 h in a growth chamber for two weeks; though during the first two days, seedlings were covered with a glass beaker to maintain humidity. After two weeks, the photoperiod and temperature were changed to 12 h/12 h at 15/10° C (light/dark) for one week to acclimate the seedlings and enhance tillering.

Crosses between tetraploid H. vulgare and tetraploid H. bulbosum

During 2016/2017 winter season, all accessions were germinated in a growth chamber following the same method mentioned prior (2x H. vulgare crosses). Seedlings of diploid H. vulgare were treated with colchicine to double chromosomes number (2x →

4x). Flow cytometry was used to identify the ploidy level of the H. vulgare seedlings and verify chromosome doubling. At the flowering stage, each line of tetraploid H. vulgare

(4x) was crossed with five lines of tetraploid H. bulbosum (4x) following the same method mentioned prior (2x H. vulgare crosses).

The term, “2x H. vulgare crosses” is used to identify crosses between diploid (2x)

H. vulgare lines served as females and diploid or tetraploid (2x, 4x) H. bulbosum lines

served as males. The term, “4x H. vulgare crosses” is used to identify crosses between tetraploid (4x) H. vulgare lines and tetraploid (4x) H. bulbosum. Family name depends on

H. vulgare female number given in Table 4.1. For example, “Family 1” means H. vulgare

(♀) parent 1 crossed with all other H. bulbosum lines (♂).

To determine crossing capacity between H. vulgare and H. bulbosum the number of pollinated florets, seed collected, inviable seed, abnormally germinated embryos, and germinated embryos were recorded (Appendix A). Comparisons were made between MS and Gamborg B-5 media for the number of days to germinate (1-2 cm length of coleoptile), leaf and root number, shoot and root length.

A Chi square (χ2) test was applied to crossing capacity parameters to determine differences. The two types of media were analyzed for growth parameters as a complete randomized design (CRD) using PROC MEANS and PROC GLM in SAS (Version 9.4,

SAS Institute, 2011, Cary, NC). Mean separation among traits was determined using

Fisher’s least significant difference (LSD) at α = 0.05 and 0.01.

Results and Discussion

Crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum:

Analysis of variance of Family 1 (Table 4.2) showed significant effects for the genotype x medium interaction, superseding other factors for days to germinate and root length. Significant effects were observed for root number due to both genotype and medium. Significant (P ≤ 0.05) effects for the genotype and highly significant (P ≤ 0.01) effects between media for shoot length and leaf number were observed. Previous studies

have reported significant differences among genotypes and the interaction with media

(Chung 1994; Mihailescu and Giura 1996; Burun and Poyrazoglu 2002, Han et al. 2011).

Table 4.2 Analysis of variance of the Family 1 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) ** * NS * * Media (M) NS† * ** ** ** G x M ** NS ** NS NS * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. † NS, significance level P > 0.05.

Table 4.3 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 1 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 1 x 6‡ 12.0b 9.0 c 5.0ab 4.7ab 4.5b 3.8b 6.2bc 5.9bc 1.7b 1.3b 1 x 9 10.0bc 8.0c 4.3b 3.0c 4.9b 4.2b 7.3b 5.3c 2.3ab 1.3b 1 x 10 10.0bc 15.0a 5.7a 4.5ab 8.3a 3.0b 8.8a 6.4bc 3.3a 1.5b

Mean -† - 5.0* 4.1* - - 7.4** 5.9** 2.4** 1.4** Means within a column followed by the same letter are not significantly different (P > 0.05). * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. † Significant interaction precludes the calculation of means. ‡ Other crosses (1x7, 1x8, 1x11) failed to germinate (Appendix 4.A).

Among the crosses of Family 1, cross 1x10 had longest time to germinate (15 days) on Gamborg’s B-5 medium compared to all other crosses (Table 4.3). Cross 1x6 performed better on B-5 medium than MS (9 days vs 12 days, respectively) for germination. These data confirm individual genotypes responded differently to the media 63

types. Chen et al. (2011) reported similar difficulty drawing conclusions about the most suitable medium composition for different species. Type of medium and genotype had significant effect on root number. Root number ranged from 3 (1x9) in B-5 to 5.7 (1x10) in MS. Cross 1x9 performed better in MS medium than in B-5 (4.3 vs 3 roots, respectively) for root number. Though the practicality of the difference may be limited, these results indicate MS medium is better (5 roots) than B-5 (4.1 roots) for mean root number. Shoot length was affected by both media and genotype. Cross 1x10 had greatest shoot length (8.8 cm) in MS medium compared to all other crosses. Cross 1x9 performed better in MS medium (7.3 cm) than B-5 (5.3 cm). Even though there was a genotype effect, MS medium stimulated shoot length better than B-5. Mean leaf number ranged from 1.3 (1x6, 1x9) in B-5 to 3.3 (1x10) in MS. Cross 1x10 produced more leaves in MS medium (3.3 leaves) than B-5 (1.5 leaf). MS medium produced more leaves (2.4) compared to B-5 (1.4). Burun and Poyrazoglu 2002 listed the media used in their study from best to worst as follows: Randolph and Cox (RC), MS, 1/2 MS and B-5 based on barley plant development from embryos. They suggested that MS medium is better for barley embryo performance than B-5 medium. MS medium contains more nitrate and ammonium salts compared to B-5, providing enough nutrients to allow the embryo to grow into a seedling. In addition, MS medium contains glycine, an amino acid that is essential in purine synthesis, an important part in the porphyrin ring structure of chlorophyll (Beyl 2011). B-5 medium lacks glycine. Mihailescu and Giura (1996) reported B-5 medium to be better than MS based on barley embryo germination; however, we did not observe that with our results.

Analysis of variance of Family 2 (Table 4.4) showed highly significant (P ≤

0.001) interactions for all of the characteristics measured, indicating each genotype responded to each medium type independently.

Table 4.4 Analysis of variance of the Family 2 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) ** * *** *** * Media (M) NS† ** *** NS NS G x M *** *** *** *** ** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Table 4.5 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 2 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 2 x 7‡ 0.0d 10.5ab 0.0d 5.0a 0.0d 2.9b 0.0d 4.7bc 0.0c 2.0ab 2 x 8 13.3a 8.0bc 4.0ab 4.0ab 4.9a 3.2b 5.3b 3.8c 3.0a 1.3b 2 x 9 8.0bc 6.7c 3.3bc 3.7abc 5.2a 1.7c 7.2a 6.6a 2.3ab 2.0ab 2 x 11 9.0bc 9.3bc 3.3bc 2.3c 5.3a 1.3c 4.6bc 3.9c 3.0a 2.0ab

Mean -† ------Means within a column followed by the same letter are not significantly different (P > 0.05). † Significant interaction precludes the calculation of means. ‡ Other crosses (2x6, 2x10) failed to germinate (Appendix 4.A).

Among the crosses of Family 2, cross 2x7 did not respond to MS medium for all characteristics measured (Table 4.5). No other discernable trends among genotype for

day to germinate were observed. In general, because the genotype x medium interaction was significant for all characteristics, not single type of media will be able to produce optimal results for all genotypes. As a result, both media have been used by many researchers for barley embryo culture (Pickering 1989; Chen and Hayes 1991; Burun and

Poyrazoglu 2002; Hayes, et al. 2003; Houben et al. 2011; and Kahani et al. 2012).

Analysis of variance of Family 3 (Table 4.6) revealed highly significant (P ≤

0.001) effects for the interactions among all characteristics measured, except leaf number.

These interactions preclude the identification of either medium as superior to the other with respect to behavior of individual crosses. However, media was highly significant (P

≤ 0.001) for leaf number among the genotypes tested. MS medium clearly enabled greater leaf number on these barley crosses than B-5 for this family of crosses.

Table 4.6 Analysis of variance of the Family 3 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** *** *** *** NS† Media (M) *** *** *** *** *** G x M *** *** *** *** NS *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Table 4.7 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 3 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 3 x 6‡ 14.0a 0.0e 6.0b 0.0d 5.1c 0.0f 8.5a 0.0d 2.7a 0.0c 3 x 8 16.0a 11.0c 6.7ab 3.0c 10.0a 2.0e 6.9b 3.4c 3.0a 1.3b 3 x 9 10.0c 8.3d 3.7c 2.7c 3.2d 2.0e 6.1b 2.9c 2.0ab 1.3b 3 x 10 11.3c 7.0d 8.0a 4.0c 6.1b 3.5d 8.7a 7.0b 2.5a 1.0bc

Mean -† ------2.5*** 0.9*** Means within a column followed by the same letter are not significantly different (P > 0.05). *** Significant at the 0.001 probability level. † Significant interaction precludes the calculation of means. ‡ Other crosses (3x7, 3x11) failed to germinate (Appendix 4.A).

Among the crosses of Family 3, cross 3x6 did not respond to B-5 medium for all characteristics measured (Table 4.7). However, the same cross did germinate in 14 days on MS medium. Leaf number was affected by media type. MS medium produced more leaves compared to B-5 (2.5 vs 0.9 leaves), indicating MS medium stimulated leaf number better than B-5. This result reflected the high concentration of nitrate, potassium and ammonia in MS compared to B-5 (Misawa 1994). Han et al. 2011 found that frequency of germinating embryos in B-5 media was slightly higher (10%) compared to

MS media, indicating that embryo germination may be reasonable at low concentrations of sucrose. However, the highly significant interactions of genotype x medium preclude verification of Misawa’s and Han et al.’s results with our own.

Analysis of variance of Family 4 showed highly significant effects for the genotype x medium interaction superseding other factors for number of days to germinate and root length (Table 4.8).

Table 4.8 Analysis of variance of the Family 4 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) NS† NS *** NS NS Media (M) NS NS *** NS NS G x M ** NS *** NS NS ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

No significant differences were observed for the other characteristics measured

(Table 4.9).

Table 4.9 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 4 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 4 x 6‡ 13.7b 16.0ab 5.0a 4.0a 4.0b 10.0a 6.5ab 7.0a 3.0a 3.3a 4 x 8 17.0a 14.0b 7.3a 5.7a 3.0b 3.1b 6.5ab 5.0b 2.7a 2.0a

Mean -† - 6.2ns 4.8ns - - 6.5ns 6.0ns 2.8ns 2.7ns Means within a column followed by the same letter are not significantly different (P > 0.05). † Significant interaction precludes the calculation of means. ‡ Other crosses (4x7, 4x9, 4x10, 4x11) failed to germinate (Appendix 4.A).

Analysis of variance of Family 5 revealed highly significant (P ≤ 0.001) effects for the interactions among all characteristics measured (Table 4.10). These interactions preclude the identification of either medium as superior to the other with respect to behavior of individual crosses. This data was confirmed by previous studies (Pickering

1989; Arabi et al. 1991; Burun and Poyrazoglu 2002; Hayes et al. 2003).

Table 4.10 Analysis of variance of the Family 5 genotypes (2x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** *** *** *** *** Media (M) *** *** *** *** *** G x M *** *** *** *** *** *** Significant at the 0.001 probability level.

Previous studies reported MS medium better than B-5 for root number, root and shoot length (Burun and Poyrazoglu 2002; Han et al. 2011). Mihailescu and Giura (1996) found B-5 medium to be more efficient by giving greater values for barley embryo germination compared to MS. These results confirmed individual genotypes responded differently to the media types, so it is difficult to draw broad conclusions about the most suitable medium composition for a broad spectrum of genotypes (Chen et al. 2011).

Table 4.11 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 5 genotypes in MS and Gamborg’s B-5 media (2x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 5 x 6 14.0a 10.0bc 6.0ab 5.7ab 5.2ab 4.9abc 7.1bcd 6.3ed 2.7abc 2.3bcd 5 x 7 13.3ab 8.0c 4.3b 2.3c 4.2abc 2.6d 8.9a 4.0f 2.3bcd 1.7d 5 x 8 13.0ab 15.0a 4.3b 4.7ab 4.8abc 3.7bcd 7.6b 5.9e 2.0cd 1.7d 5 x 9 8.0c 0.0d 6.5a 0.0d 5.5a 0.0e 7.3bc 0.0g 2.5abcd 0.0e 5 x 10 9.3c 0.0d 5.7ab 0.0d 4.2abc 0.0e 7.4bc 0.0g 3.3a 0.0e 5 x 11 13.0ab 14.3a 5.7ab 6.0ab 4.7abc 3.5cd 6.6cde 5.9e 3.0ab 2.7abc

Mean -† ------Means within a column followed by the same letter are not significantly different (P > 0.05). † Significant interaction precludes the calculation of means.

Crosses between tetraploid H. vulgare and tetraploid H. bulbosum

Analysis of variance of Family 1 (Table 4.12) showed highly significant (P ≤

0.001) effects for the genotype x medium interaction superseding other factors of number of days to germinate. Highly significant (P ≤ 0.001) effects were observed for root number and shoot length due to media. Highly significant (P ≤ 0.01) effects were observed for root length due to both genotype and medium. Previous studies have also reported significant differences among genotypes and interactions with media

(Mihailescu and Giura 1996; Burun and Poyrazoglu 2002, Han et al. 2011).

Table 4.12 Analysis of variance of the Family 1 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** NS† ** NS NS Media (M) *** *** ** *** NS G x M *** NS NS NS NS ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Type of medium had significant effect on root number (Table 4.13). Crosses 1x7 and 1x8 performed better on MS medium (4.5 and 4.3 roots, respectively) than on B-5

(2.8 and 1.8 roots, respectively). Because genotype was not significant, these results indicate MS medium is better (4 roots) than B-5 (2.6 roots) for root number. Root length was affected by both media and genotype. Among the crosses, cross 1x8 performed better in MS medium (3.9 cm) than B-5 (0.9 cm) for root length. Even though there was a genotype effect for root length, MS medium stimulated root length more than B-5. Shoot length was also affected by media type. Cross 1x8 performed better in MS medium (7.1 70

cm) than B-5 (2.9) for shoot length. These data indicate MS medium better than B-5 for root number, root length, and shoot length for Family 1 crosses similar to Gu et al. 1990,

Burun and Poyrazoglu 2002, and Russowski et al. 2006 who found similar results. The levels of inorganic nutrients in the MS medium are higher than in B-5 medium, because

MS contains more nutrients, this allows growth and development of embryo (Russowski et al. 2006; Beyl 2011). However, some studies indicated that B-5 medium resulted very positive results (Mihailescu and Giura 1996; Han et al. 2011), but because genotypic performance varied based on media, no single recommendation could be made.

Table 4.13 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 1 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 1 x 6 7.3c 5.0c 3.0bcd 2.8cd 4.4ab 2.9bcd 6.4ab 4.2bc 1.3ab 1.0b 1 x 7 11.0b 23.0a 4.5a 2.8cd 4.5ab 3.3abcd 5.8abc 4.4bc 1.5ab 1.5ab 1 x 8 5.8c 7.0c 4.3ab 1.8d 3.9abc 0.9e 7.1ab 2.9c 1.0b 1.0b 1 x 10 14.0b 20.5a 3.5abc 2.5cd 2.5cde 1.9de 5.6abc 3.2c 1.3ab 1.0b 1 x 11 5.8c 11.8b 4.5a 3.3abc 4.2abc 4.9a 8.7a 5.6abc 1.3ab 2.0a

Mean -† - 4.0*** 2.6*** 3.9** 2.8** 6.7*** 4.0*** 1.3ns‡ 1.3ns Means within a column followed by the same letter are not significantly different (P > 0.05). ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level. † Significant interaction precludes the calculation of means. ‡ NS, significance level P > 0.05.

Analysis of variance of Family 2 (Table 4.14) revealed no interactions and highly significant (P ≤ 0.01) effects for the genotype for number of days to germinate and shoot length affected by media, significant (P ≤ 0.05) effects for root number, root length, and

shoot length based on genotype, and highly significant (P ≤ 0.001) effects between media for root number.

Table 4.14 Analysis of variance of the Family 2 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) ** * * * NS Media (M) NS† *** NS ** NS G x M NS NS NS NS NS * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Table 4.15 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 2 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 2 x 6 9.8bc 12.8abc 7.0a 3.0bcd 5.0ab 3.1bcd 7.4ab 5.6ab 2.0ab 1.5ab 2 x 7 17.0a 14.0ab 3.8bc 3.0bcd 3.6bcd 4.5abc 6.7ab 5.5abc 1.8ab 1.5ab 2 x 8 8.5bc 6.8c 4.8b 2.3cd 5.0ab 3.9bcd 7.6a 5.5abc 2.3a 1.5ab 2 x 10 15.0ab 11.8abc 3.8bc 3.0bcd 6.8a 3.7bcd 7.5ab 5.3abc 1.5ab 1.3ab 2 x 11 6.5c 9.5bc 3.5bc 1.5d 2.3cd 1.8d 4.8bc 2.8c 1.0b 1.3ab

Mean 11.4ns† 11.0ns 4.6*** 2.6*** 4.5ns 3.4ns 6.8** 4.9** 1.7ns 1.4ns Means within a column followed by the same letter are not significantly different (P > 0.05). ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level. † NS, significance level P > 0.05.

Mean number of days to germinate ranged from 6.5 days for cross 2x8 in MS medium to 17 days for cross 2x7 in the same medium, which confirmed the variation

among genotype (Table 4.15). Number of days to germinate was not affected by media type. Each genotype ranked the same across both media types. In contrast, root number was affected by both media and genotype. Among the crosses of Family 2, cross 2x6 had greatest root number (7 roots) in MS medium compared to all other crosses. Also, crosses

2x8 and 2x11 performed better in MS medium (4.8 and 3.5 roots, respectively) than B-5

(2.3 and 1.5 roots, respectively) for root number. Even though there was a genotype effect, MS medium stimulated root number better than B-5. Mean root length varied among the crosses and ranged from 1.8 (2x11) in B-5 medium to 6.8 (2x10) in MS confirmed the variation among the genotype. Cross 2x10 performed better in MS medium than B-5 (6.8 vs 3.7 cm, respectively); however, root length was not affected by media.

Mean shoot length was affected by both media and genotype. Mean shoot length was better in MS medium (6.8 cm) than B-5 (4.9 cm). These data indicate that MS medium is better than B-5 for root number and shoot length for Family 2 crosses.

Analysis of variance of Family 3 (Table 4.16) showed highly significant (P ≤

0.001) effects for the genotype x medium interaction superseding other factors for number of days to germinate, root number, and shoot length. Highly significant (P ≤

0.001) effects were observed for root length due to media.

Table 4.16 Analysis of variance of the Family 3 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** ** NS ** NS Media (M) NS† *** *** *** NS G x M *** ** NS ** NS ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Table 4.17 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 3 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 3 x 6 10.3b 8.0b 4.5a 3.8ab 5.7a 4.4abc 7.0abc 7.7ab 2.0a 1.5ab 3 x 7 13.3a 8.8b 4.5a 1.3d 4.7abc 1.7d 6.5bc 3.7f 1.8ab 1.5ab 3 x 8 7.8b 5.0c 4.0ab 3.0bc 5.1ab 3.2bcd 6.4bcd 5.9bcde 1.8ab 1.0b 3 x 10 5.0c 15.5a 4.3a 2.5c 6.5a 2.6cd 8.9a 4.4def 1.3ab 1.3ab 3 x 11 8.5b 8.3b 3.0bc 2.5c 3.1bcd 2.4cd 5.3cdef 4.0ef 1.0b 1.3ab

Mean -† - - - 5.0*** 2.8*** - - 1.6ns‡ 1.3ns Means within a column followed by the same letter are not significantly different (P > 0.05). *** Significant at the 0.001 probability level. † Significant interaction precludes the calculation of means. ‡ NS, significance level P > 0.05.

Root length was affected by type of media. Crosses 3x7 and 3x10 performed better on MS medium than B-5 (4.7 and 6.5 cm vs 1.7 and 2.6 cm, respectively) for root length. MS medium stimulated root length better (5 cm) than B-5 (2.8 cm). Both media have been used by many researchers for barley culture embryo (Chen and Hayes 1991;

Burun and Poyrazoglu 2002; Hayes, et al 2003; Houben et al 2011). Previous studies 74

claimed difficulty to drawing conclusions about the most suitable medium composition for different species (Chen et al. 2011).

Analysis of variance of Family 4 showed significant interactions among all characteristics measured, except leaf number (Table 4.18). These interactions preclude the identification of either medium as superior to the other with respect to behavior of individual crosses. However, media was highly significant (P ≤ 0.01) for leaf number among the genotypes tested. MS medium clearly enabled greater leaf number on these barley crosses than B-5.

Table 4.18 Analysis of variance of the Family 4 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** NS† *** ** NS Media (M) *** *** *** *** ** G x M *** ** ** ** NS ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Leaf number was affected by media type. MS medium produced more leaves compared to B-5, making MS medium stimulated leaf number better than B-5. This may have been caused by the high concentration of nitrate, potassium and ammonia in MS medium compared to B-5 (Misawa 1994). Han et al. 2011 found that frequency of germinating embryos in B-5 medium was slightly higher (10%) compared to MS, indicating that embryo germination may be reasonable at low concentrations of sucrose.

However, the highly significant interactions of genotype x medium preclude verification of Misawa’s and Han et al.’s results.

Table 4.19 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 4 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 4 x 6 7.0e 8.3ed 4.8a 2.0ed 7.8a 3.9edf 7.1ab 3.5e 1.8a 1.0b 4 x 7 18.0a 5.3e 3.8ab 2.3cde 4.1ed 2.6fg 6.2bc 4.9cde 1.8a 1.3ab 4 x 8 5.0e 5.0e 3.3bc 3.0bcd 6.3b 6.1bc 8.3a 5.7bcd 1.0b 1.0b 4 x 10 12.0bc 10.8cd 4.5a 1.5e 4.9cd 1.9g 7.4ab 3.2e 1.5ab 1.0b 4 x 11 14.5ab 11.0bcd 3.0bcd 3.0bcd 3.3efg 2.7fg 4.6cde 4.5de 1.3ab 1.0b

† Mean ------1.45** 1.05** Means within a column followed by the same letter are not significantly different (P > 0.05). † Significant interaction precludes the calculation of means. ** Significant at the 0.01 probability level.

Analysis of variance of Family 5 (Table 4.20) revealed significant effects for the genotype x medium interaction for number of days to germinate, root length, and shoot length. These interactions preclude the identification of either medium as superior to the other with respect to behavior of individual crosses. Previous studies have also reported significant differences among genotypes and interactions with media (Burun and

Poyrazoglu 2002, Han et al. 2011). No significant differences due to genotype or media were observed for root number or leaf number.

Table 4.20 Analysis of variance of the Family 5 genotypes (4x H. vulgare crosses), media treatments (MS and B-5), and their interaction for days to germinate, root number, root length, shoot length, and leaf number.

Days to Root Root Shoot Leaf Source of variance germinate number length length number Genotypes (G) *** NS† *** *** NS Media (M) *** NS *** *** NS G x M *** NS *** ** NS ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level † NS, significance level P > 0.05.

Table 4.21 Mean number of days to germinate, root number, root length, shoot length, and leaf number of Family 5 genotypes in MS and Gamborg’s B-5 media (4x H. vulgare crosses).

Days to Root number Root length Shoot length Leaf number Cross germinate (cm) (cm) MS B-5 MS B-5 MS B-5 MS B-5 MS B-5 5 x 6 10.0b 6.8d 2.5ab 2.0b 2.8gf 3.0ef 3.7g 4.0fg 1.3a 1.3a 5 x 7 13.0a 6.5d 2.5ab 2.3b 4.6cd 4.9bc 4.9ef 5.3de 1.3a 1.0a 5 x 8 5.0d 6.3d 3.5a 2.5ab 4.3cd 2.0f 8.2a 5.7de 1.5a 1.3a 5 x 10 9.0bc 5.0d 3.0ab 3.0ab 5.6b 3.3ef 6.9bc 5.6de 1.5a 1.0a 5 x 11 5.5d 7.0cd 3.5a 3.0ab 7.2a 3.9ed 7.9ab 6.1cd 1.5a 1.3a

Mean -† - 3.0ns‡ 2.6ns - - - - 1.4ns 1.2ns Means within a column followed by the same letter are not significantly different (P > 0.05). † Significant interaction precludes the calculation of means. ‡NS, significance level P > 0.05.

Previous studies reported MS medium is better for root and shoot length (Burun and Poyrazoglu 2002; Han et al. 2011) because MS medium contains more nitrate and ammonium salts providing enough nutrients to allow the embryo to grow into a seedling

(Beyl 2011). Other studies reported that B-5 medium better than MS for germination

(Mihailescu and Giura 1996). Chen et al. (2011) claimed source of organic nitrogen and carbohydrates essential components to modify culture media. However, the highly

significant interactions of genotype x medium for germination time, root length, and shoot length, indicate that each genotype responded to each medium type independently for these characteristics.

Conclusion

Most studied traits were significantly and individually affected by genotypes and culture media. Also, genotypes were affected differently in each culture medium as well as both media, which indicate that immature embryos in culture are affected by medium components and genotype. Some crosses failed to germinate on MS or B-5 culture media.

MS medium was found to be the better medium for most crosses compared to B-5. Our results confirmed that definitive conclusion are hard to draw.

REFERENCES

Agdew, B., S. Verma, and R. Saharan. 2014. Comparison of variability generated through biparental mating and selfing in barley (Hordeum vulgare L.). For. Res. 40:98- 105.

Arabi, M.I., A. Sarrafi, J. Barrault, and L. Albertini. 1991. The influence of parental genotype and period of pollination on haploid barley production in Hordeum vulgare L.× H. bulbosum L. crosses. Cereal Res. Commun. 19:405-412.

Beyl, C.A. 2011. Getting started with tissue culture media preparation, sterile technique, and laboratory equipment. In R.N. Trigiano and D.J. Gray, editors, Plant tissue culture, development, and biotechnology. CRS Press, Florida, USA. p. 11-26.

Burun, B., and E.C. Poyrazoglu. 2002. Embryo culture in Barley (Hordeum vulgare L.). Turk. J. Biol. 26:175-180.

Chahal, G.S., and S.S. Gosal. 2006. Principles and procedures of plant breeding: Biotechnological and conventional approaches. Alpha Sci. Int. Ltd. Harrow, UK. p.429-456.

Chen, J.F., L. Cui, A.A. Malik, and K.G. Mbira. 2011. In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tissue Organ Cult. 104(3):311-319.

Chen, F., and P. Hayes. 1991. Effect of exogenous plant growth regulators on in vitro seed growth, embryo development, and haploid production in a cross between H. vulgare (L.) and H. bulbosum (L.). Plant Cell Tissue Organ Cult. 26:179-184.

Chung, D.H., K.S. Min, and J.U. Chun. 1994. Improvement of plant generation rate in in vitro cultured haploidy embryos from Hordeum vulgare pollinated with H. bulbosum. (In Korean, with English abstract.) Korean J. Crop Sci. 39:193-197.

Devaux, P. 2003. The Hordeum bulbosum (L.) method. In: M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht. p.15-19.

Ellis, R.P., B.P. Forster, D. Robinson, L.L. Handley, D.C. Gordon, J.R. Russell, and W. Powell. 2000. Wild barley: A source of genes for crop improvement in the 21st century? J. Exp. Bot. 51:9-17.

Gu, D.F., X.K. Wu, Y.Z. Zhang, and W.Q. Zhang. 1990. Factors affecting callus induction and plantlet regeneration in in vitro culture of immature barley embryos. J. Jilin Agric. Univ. 12:1-107.

Han, Y., X.L. Jin, F.B. Wu, and G.P. Zhang. 2011. Genotypic differences in callus induction and plant regeneration from mature embryos of barley (Hordeum vulgare L.). J Zhejiang Univ Sci B. 12:399-407.

Hayes, P., A. Corey, and J. DeNoma. 2003. Doubled haploid production in barley using the Hordeum bulbosum (L.) technique. In: Maluszynski M., K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht, p 5-14.

Houben, A., M. Sanei, and R. Pickering. 2011. Barley doubled-haploid production by uniparental chromosome elimination. Plant Cell Tissue Organ Cult. 104(3):321- 327.

Hussain, A., H. Nazir, I. Ullah, and I.A. Qarshi. 2012. Plant tissue culture: Current status and opportunities. In Leva, A., and L. Rinaldi, editors, Recent advances in plant in vitro culture, In Tech. Open Access Publisher. p. 1-28. doi: 10.5772/50568.

Kahani, F., F. Bakhtiar, R. Bozorgipour, S. Hittalmani, H.N. Khah, and K. Zargari. 2012. Production and evaluation of doubled haploid lines of barley via detached-tiller culture method. Afr. J. Biotechnol. 11:6075-6082.

Matus, I., A. Corey, T. Filichkin, P.M. Hayes, M.I. Vales, J. Kling, and R. Waugh. 2003. Development and characterization of recombinant chromosome substitution lines (RCSLs) using Hordeum vulgare subsp. spontaneum as a source of donor alleles in a H. vulgare subsp. vulgare background. Genome. 46:1010-1023.

Matus, I.A., and P.M. Hayes. 2002. Genetic diversity in three groups of barley germplasm assessed by simple sequence repeats. Genome. 45:1095–1106.

Mihailescu, A., and A. Giura. 1996. Production of winter barley haploids by bulbosum system. 2. Influence of barley genotype on in vitro haploid regeneration. Romanian Agric. Res. 6:5-20.

Pickering, R.A. 1989. Plant regeneration and variants from calli derived from immature embryos of diploid barley (Hordeum vulgare L.) and H. vulgare L. x H. bulbosum L. crosses. Theor. Appl. Genet. 78:105-112.

Pickering, R.A. 1984. The influence of genotype and environment on chromosome elimination in crosses between Hordeum vulgare L.× Hordeum bulbosum L. Plant Sci. Lett. 34:153-164.

Pickering, R., and P.A. Johnston. 2005. Recent progress in barley improvement using wild species of Hordeum. Cytogenet. Genome Res. 109(1-3):344-349.

Russowski, D., N. Maurmann, S.B. Rech, and A.G. Fett-Neto. 2006. Role of light and medium composition on growth and valepotriate contents in Valeriana glechomifolia whole plant liquid cultures. Plant Cell Tissue Organ Cult. 86:211- 218.

SAS Institute. 2011. SAS guide to macro processing. Vol. 11. SAS Inst., Cary, NC.

CHAPTER V

MORPHOLOGICAL CHARACTERISTICS OF BARLEY PROGENIES

Introduction

The main objective of plant breeding programs in barley is to produce new lines with high-yields and better quality, and then release them as cultivars. The breeding methods commonly used in genetic improvement of barley are based on selecting desirable genotypes from genetically heterogeneous landraces (pure-line method) or from the segregating progeny of crosses between lines with superior traits (pedigree method).

Another method that can be used is the bulk-population method, with segregating progeny of multiple crosses between superior lines harvested and propagated in bulk.

Depending on the characteristic and geographic location of selection, between seven and

10 cycles are required to obtain a new homozygous variety by using these methods

(Jensen 1988; Ibrahim and Barrett 2001; Gomez-Pando et al. 2009).

Haploid methods (pollen culture) are used to produce homozygous diploid plants in conjunction with colchicine in a single generation. These homozygtes are fertile, uniform, and do not segregate. Consequently, doubling of haploids reduces the time of a breeding cycle, saving space, and money (Jha and Biswajit 2005; Jauhar et al. 2009).

Haploid methods can be used in the final stages of breeding program to achieve genetic uniformity which is a requirement for release new cultivars (Devaux and Kasha 2009).

Also, it is useful in restoring fertility to interspecific hybrids (dihaploids), which fixes

traits rapidly in desirable combinations in a genotype. Dihaploid lines can be evaluated quickly with more confidence and accuracy due to their uniformity (Wedzony et al.

2009). Additional research indicates that doubled haploid methods are a useful tool for hastening plant breeding programs, genome mapping, induced mutagenesis, transferring and fixing alien gene insertion, and unraveling genetic control of chromosome pairing

(Devaux 2003; Jauhar et al. 2009; Wedzony et al. 2009; Chen et al. 2011; Kindiger

2012).

Haploid production in barley can be obtained by hybridization between cultivated species as females (Hordeum vulgare) and (H. bulbosum) as males (Pickering and

Johnston 2005; Murovecand and Bohanec 2011). Haploid plants have to be treated with colchicine to double the chromosome number to restore fertility (Chahal and Gosal

2006). Hordeum bulbosum also serves as a source of desirable traits that are useful to transfer to H. vulgare such as pest and disease resistance, abiotic stress tolerance, and seed quality (Pickering and Johnston 2005). Many modern commercial cultivars of barley originated from dihaploid lines (Thomas et al. 2003).

Materials and Methods

Crosses were made between two species of barley H. vulgare and H. bulbosum

(Table 5.1) through interspecific hybridization followed by embryo rescue. Seedlings of rescued progenies were vernalized in a growth chamber (Percival, Mod. 135LLVL,

Controlled Environments. Boone, IA) at 4° C with an 8 h photoperiod for 6 - 8 weeks.

Seedlings with intact root systems were removed from the media, identified by cross, and transferred to 0.5 L pots containing Sunshine® Mix 1 (Planet Natural, Bozeman, MT).

For acclimation, seedlings were placed at 10/8° C (light/dark) with a photoperiod of 10 h/14 h in a growth chamber for two weeks. During the first two days, seedlings were covered with a glass beaker to maintain high humidity. After two weeks, the photoperiod and temperature were changed to 12 h/12 h at 15/10° C (light/dark) for one week to acclimate the seedlings and enhance tillering. Seedlings were moved to a greenhouse under a long day conditions using supplemental lighting.

Table 5.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study

The term “2x H. vulgare crosses” indicates crosses between diploid (2x) H. vulgare lines serving as females and diploid or tetraploid (2x, 4x) H. bulbosum lines serving as males. The term “4x H. vulgare crosses” refers to crosses between tetraploid

(4x) H. vulgare lines as females and tetraploid (4x) H. bulbosum as males. Family name depends on H. vulgare female identifying number given in Table 5.1. For example,

“Family 1” means H. vulgare (♀) parent 1 crossed with all other H. bulbosum lines (♂).

Progenies of interspecific crosses between diploid H. vulgare and diploid and tetraploid H. bulbosum:

Flow Cytometry:

Flow cytometry was used to determine ploidy level of individual crosses and the cytotype of their parents. The day of the analysis, a healthy leaf was cut from each plant.

Each sample was wrapped by a piece of a damp paper towel, labeled, and put in a plastic bag and then placed in a cooler. Samples transported to the USDA-ARS lab in

Poplarville, MS. Using standard protocols, refined by Dr. Timothy Rinehart (Research

Plant Molecular Geneticist, USDA ARS Thad Cochran Southern Horticultural Research

Laboratory, Poplarville, MS), samples were screened for ploidy level on a BD Accuri C6 cytometer (BD Biosciences: Becton, Dickinson and Company, Franklin Lakes, NJ).

CyStain® PI Absolute P supplied by Sysmex (Sysmex Partec, Görlitz, Germany) was used during preparation of the sample for analysis.

Approximately 0.5 cm2 of leaf tissue was excised from each plant sample placed in a 90 mm round polystyrene Petri dish along with 250 μL of nuclei extraction buffer

(proprietary, Sysmex). A double-sided razor blade was used to chop leaf material into a

fine, even consistency. Chopping events lasted no longer than 60 seconds. Razors were replaced after four samples (two samples per side). Dish contents were washed with 1 ml of staining solution. Staining solution was made daily for every 20 samples by mixing

120 μL of propidium iodide (PI) stock solution as a fluorescent stain with 60 μL RNAase

A stock solution to avoid RNA staining. It was then diluted with 20 ml of staining buffer

(proprietary, Sysmex). Staining solution was stored at 2 - 8° C in darkness.

The nuclei suspension was filtered through a 50 μm mesh filter cap into a 3.5 ml

(12 x 75 mm) test tube. Plates and filter caps were discarded after each use. Filtered samples were labeled and placed in the dark at 3° C for 30 minutes before analysis.

Garden pea (Pisum sativum L.) was used as an external standard. Barley progeny were compared to their parents. Every screening was allowed to progress for at least 500 events within a predetermined gate-width (Appendix B).

Colchicine Treatment:

Haploid (1x) seedlings resulting from chromosome elimination between diploid

H. vulgare (2x) and diploid H. bulbosum (2x) crosses were treated with colchicine to generate dihaploids (1x → 2x) following methods of Devaux (2003) and Houben et al.

(2011). When seedlings developed at least two tillers, they were removed from the soilless media and the roots were washed and cut back to 1 - 2 cm. Leaves were also cut to make the final plant size about 20 cm in length. A single incision was made into each tiller base. Rinsed, trimmed, and cut seedlings were immersed in an aqueous solution of

0.05% colchicine (Sigma-Aldrich Co., C-3915, St. Louis, MO) and 2% dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., D-4540, St. Louis, MO) for 5 h at 25 - 30° C using water bath, flow hood, artificial light, and aerator. After colchicine treatment, 86

seedlings were rinsed in tap water, and transferred into pots (1 L) filled with a mixture

(1:2 V/V) of fine sand and Sunshine® Mix 1 (Planet Natural, Bozeman, MT) and placed in growth chamber with a photoperiod 12 h/12 h at 15/8° C (light/darkness) for two weeks. Seedlings were transferred to 6 L pots containing the same soilless mix as described earlier and placed in a 20° C greenhouse under ambient September light conditions.

Progenies Growouts:

During September 2016, parents and progenies were planted in 6 L pots filled with a mixture (1:2 V/V) of fine sand and Sunshine® Mix 1 (Planet Natural, Bozeman,

MT) and placed in greenhouse at 25 ± 4/17° C under a long day conditions using supplemental lighting to stimulate flowering. Plants were spaced at 30 cm and fertilized once a week with 150 ppm N of Peters® Professional 20:20:20 fertilizer (Everris NA Inc.,

Dublin, OH). At flowering stage, triploid hybrids (♀) identified by flowcytometry were backcrossed with their H. vulgare parents (♂). However, there were no seed set. Spikes of the other ploidy progenies were covered with trimmed pearl millet pollination bags

(Canvasback® #G27, Seedburo Equipment Co., Des Plaines, IL) to ensure self- pollination. Morphological characteristics were measured, and seed was collected to determine morphological differences due to the interspecific crossing.

The characteristics measured were; chlorophyll content of the flag leaf (SPAD-

502 chlorophyll photometer, Minolta, Ramsey NJ) at the flowering stage, flag leaf length

(cm; measured from the base of ligule to the tip), width (cm; measured at the widest part), leaf area (cm2; measured with a Li-Cor Portable Area Meter (LI-3000, Lincoln, NE); plant height (cm; measured from the ground to the tip of the spike, excluding the awns), 87

tiller number, spike number, spike length (cm; measured from the base of the spike to the tips, including the awns), spikelet number spike-1, seed number spike-1, percent fertility

(number of filled spikelets/total number of spikelets x 100) (Sharma et al. 2018), awn length (cm), 100 seed weight (g), grain yield (g) plant-1.

Microsoft Excel 2016 was used to record and organize the data of all morphological traits. Illustrative analysis including; mean, standard deviation (SD), and coefficient of variability (CV) were calculated. Significant mean separation among genotypes was determined using family mean + 1 standard deviation (x̅ + 1 SD) and family mean + 2 standard deviation (x̅ + 2 SD), these units approximate α = 0.32 and

0.05, respectively. Correlation coefficient among morphological traits was determined using PROC CORR in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Sigma Plot

13.0 (Systat Software, Inc, San Jose, CA, 2015) was used to present the mean of grain yield of all families in graphic form.

Progenies of interspecific crosses between tetraploid H. vulgare and tetraploid H. bulbosum:

A flow cytometer was used to screen ploidy level of the adult progeny of individual crosses. During September 2017 hybrids were grown with their parents in a greenhouse at 25 ± 4/17° C under long day conditions using supplemental lighting. At flowering, spikes were covered with pollination bags to ensure self-pollination.

Morphological characteristics were measured and recorded (same as in 2x H. vulgare crosses), and seed collected.

Microsoft Excel 2016 was used to record and organize the data of all morphological traits. Illustrative analysis including mean, standard deviation (SD), and coefficient of variability (CV) were calculated. Significant mean separation among genotypes was determined using family mean + 1 standard deviation (x̅ + 1 SD) and family mean + 2 standard deviation (x̅ + 2 SD), these units approximate α = 0.32 and

0.05, respectively. Correlation coefficient among morphological traits was determined using PROC CORR in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Sigma Plot

13.0 (Systat Software, Inc, San Jose, CA, 2015) was used to present the mean of morphological traits of all genotypes in graphic form.

Results and Discussion

Genotypes of diploid H. vulgare crosses

With trans-species crosses such as those performed here, there are different degrees of compatibility prior to and during the mitotic divisions following fertilization.

In this experiment, each diploid H. vulgare (1, 2, 3, 4, and 5) genotype was used as a female and top crossed to 4x H. bulbosum (6, 7, 8, 10, and 11) and the single 2x (9).

Assessment for general combining ability and compatibility can be made by evaluating the number of crosses that successfully produced embryos (that were successfully rescued; Table 5.3). For Family 1, female parent 1, crosses were attempted with all six males. Crosses with males 7, 8, and 11 resulted in nonviable crosses. Crosses with male parents 6, 9, 10 produced 14 embryos that were rescued. Of six possible top crosses, three

(50%) were successful as determined by embryo production. Two (14%) of the dihaploid

progeny show grain yields greater than the mean of all crosses within this family. Cross

1x9(3) and 1x9(6) exceed mean grain yield of all progeny by +2 SD and +1 SD, respectively. It is not a surprise that rescued progeny of male parent 9 were relatively prolific. While 9, a H. bulbosum, is diploid, as such ploidy level issues are minimized; however, there are still species compatibility issues that were reflected by low values for other characteristics measure in 1x9 progeny.

In the real world, yield and traits associated with yield are most important.

Correlation coefficient is widely used by plant breeders to identify the nature of complex associations among yield components and determine the traits that directly affect the grain yield (Babaiy et al. 2011). Consequently, this information is useful for plant breeders to formulate their breeding and selection strategies to improve yield (Khaiti

2012). The correlation coefficient among yield and its component traits of Family 1 genotypes are presented in Table 5.2. Grain yield showed positive association with tiller number, fertility, and 100-seed weight (cSW). This result justified the possibility of correlated response to select these associated traits to improve grain yield. Similar association was also reported by Pal et al. (2010), Drikvand et al. (2011), Distelfeld et al.

(2014), Dorostkar et al. (2015), and Sunil et al. (2017).

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 1 crosses (2x H. vulgare crosses) for yield and related traits are presented in Table 5.3. Other traits summarized in Appendix C. Plant height of crosses varied between 49 and 78 cm with a mean of 61.5 cm. Crosses 1x6(3), 1x9(2), 1x9(3) and

1x9(6) exceeded the x̅ + 1 SD for plant height. In contrast, cross 1x9(9), 1x9(10), and

1x10(3) had a shorter stature (49, 51, and 51 cm), which less than the x̅ – 1 SD.

Depending on how they segregate, these crosses could be used in breeding program to develop lodging-resistant varieties; however, they had low grain yield. As nitrogen fertilizer is essential to increasing yield, it also promotes plant height, and concurrently lodging. However, short stature varieties were more resistant to lodging even when higher amounts of nitrogen fertilizer were applied (Okuno et al. 2014). Pal et al. (2010) found negative relationship between plant height and grain yield. In contrast, other studies found that plant height showed positive association with grain yield (Al-Tabbal and Al-Fraihat 2012; Aynewa et al. 2015). Our data for this family shows no relationship between plant height and grain yield.

Table 5.2 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 1 crosses (2x H. vulgare crosses).

PH TN FLA F cSW GY PH 1.00 0.26 0.20 -0.25 0.24 0.16 TN 1.00 -0.20 0.24 0.44 0.84*** FLA 1.00 -0.21 0.31 0.27 F 1.00 0.07 0.52* cSW 1.00 0.65** GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

Table 5.3 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 1 65.5 73 10.22 92.36 2.901* 15.75 6 93.0 39 15.67* 41.44 1.488 2.64 9 52.4† 123* 6.92 84.61 0.620 16.11 10 102.8* 76 13.62 29.22 1.691 3.72 (x̅ ) 78.43 77.75 11.61 61.91 1.675 9.56 SD 23.46 34.52 3.85 31.25 0.940 7.38 1x6(1) 68.0 53 5.43 83.15* 3.405 13.85 1x6(2) 55.0 62 10.66 58.47 2.536 3.72 1x6(3) 71.0* 48 6.89 61.01 2.532 4.56 1x9(1) 54.0 29 8.49 50.19 1.823 1.07 1x9(2) 71.5* 61 20.26** 83.42* 2.259 13.14 1x9(3) 72.0* 132** 8.03 48.99 4.664** 25.49** 1x9(6) 78.0* 93* 3.43 58.78 4.424* 17.54* 1x9(9) 49.0† 27 11.88 51.99 1.658 2.27 1x9(10) 51.0† 38 14.54* 36.11 1.934 2.83 1x9(12) 60.5 36 10.89 35.89 2.465 2.59 1x10(1) 60.0 35 5.98 55.19 1.252 3.73 1x10(2) 61.0 39 10.43 72.55 1.953 9.42 1x10(3) 51.0† 35 11.49 56.08 1.472 2.02 1x10(4) 59.0 36 3.62 67.76 2.043 6.46 (x̅ ) 61.50 51.71 9.43 58.54 2.459 7.76 SD 9.20 28.94 4.52 14.54 1.032 7.24 CV (%) 14.96 55.96 47.90 24.84 41.98 93.31 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. † Value lower than x̅ – 1 SD.

-1 25

Grain yield, g plant g yield, Grain

1 6 9

1x6(1) 1x6(2) 1x6(3) 1x9(1) 1x9(2) 1x9(3) 1x9(6) 1x9(9)

1x9(10) 1x9(12) 1x10(1) 1x10(2) 1x10(3) 1x10(4)

Genotypes

Figure 5.1 Mean grain yield (GY), g plant-1 of parental genotypes and Family 1 crosses (2x H. vulgare crosses). 1x6 1x9 A wide range1x10 of values were observed in tiller number of Family 1 crosses. Tiller 1 6 number ranged from 27 to 132 with a mean of 51.71. Cross 1x9(3) exceeded mean tiller 9 10 number by + 2 SD, cross 1x9(6) exceeded mean tiller number by + 1 SD (123, and 93 tillers, respectively), while cross 1x9(9) had a lower tiller number (27 tillers = x̅ – 1 SD) even though all three crosses were from the same two parents. Chen et al. (2011) indicated that because haploids lack homologous chromosomes, only two types of genotypes occur. For the gene “A” and its allele “a”, three genotypes would normally occur in a diploid; ¼ AA, ½ Aa, ¼ aa; however, a haploid is either A or a, when

converted to a dihaploid there are only two genotypes possible ½ AA and ½ aa. It appears crosses 1x9(3) and 1x9(6) have the alleles for greater mean tiller number, while cross 1x9(9) has alleles for fewer mean tillers. Tiller number as well as grain yield are important and have an economic value because barley is often used as animal feed

(Shakhatreh et al. 2010). We also found a strong positive association between tiller number and grain yield. Therefore, crosses 1x9(3) and 1x9(6) would be desirable for improving barley varieties.

Flag leaf area (Table 5.3) of Family 1 crosses showed wide range of variation ranging between 3.43 cm2 to 20.26 cm2 with a mean of 9.43 cm2. Crosses 1x9(2) and

1x9(10) had a higher flag leaf area (20.26 cm2) and exceeded the x̅ + 2 SD and the x̅ + 1

SD, respectively. Yadav et al (2015) also reported a wide range of variation for flag leaf area (12.1 - 47.1 cm2) when assessing progeny of their crosses.

Individual crosses of Family 1 (Table 5.3) showed wide variation in fertility percentage ranging from 35.89 to 83.42% with a mean of 58.54%. Among all crosses, only crosses 1x6(1) and 1x9(2) had higher fertility (83.15 and 83.42%, respectively), exceeding the x̅ + 1 SD. Fertility associated positively with grain yield (Table 5.2), so these crosses could be used in barley breeding program to improve grain yield. Parent 10 had lowest fertility (29.22%), which was lower than the x̅ – 1 SD. H. bulbosum is a wild self-incompatible species (Kakeda et al. 2008). Self-incompatibility frequency is typically higher in perennial compared to annual species (Baumann et al. 2000). Most crosses of Family 1 showed low fertility, which were affected by their male parents. Low fertility reduced the number of seed spike-1 (Appendix C) and thus grain yield of these crosses.

A wide range of values were observed in cSW trait, which ranged from 1.252 to

4.664 g with a mean of 2.459 g. Crosses 1x9(3) and 1x9(6) had greatest cSW (4.664 and

4.424 g, respectively) which exceeded the x̅ + 2 SD and the x̅ + 1 SD, respectively; while their male parent (9) showed lowest value (0.62 g; lower than the x̅ – 1 SD. Seed weight and seed number reflect the potential field grain yield (Distelfeld et al. 2014).

Additionally, cSW showed a high positive association with grain yield (Table 5.2), so these crosses with high cSW would be desirable in a barley breeding.

High yield is the most important characteristic in any barley breeding program. In this work, crosses demonstrated a very wide variation of grain yield; ranging from 1.07 to

25.49 g plant-1 with a mean of 7.76 g, with a high coefficient variation of 93.31% (Table

5.3 and Figure 5.1). These results indicate the high genetic variation among genotypes.

Crosses 1x9(3) and 1x9(6) had greatest grain yield (25.49 and 17.54 g) and exceeded the x̅ + 2 SD and the x̅ + 1 SD, respectively. In contrast, cross 1x9(1) showed lowest grain yield (1.07 g) even though shared both parents with crosses 1x9(3) and 1x9(6). Although crosses 1x9(3) and 1x9(6) showed low fertility, they had greater yield due to a high tiller number and cSW as well as plant height, which confirmed the high positive relationship among these two traits and their relationship with final grain yield also reported by

Drikvand et al. (2011) and Ruzdik et al. (2015).

Family 2, female parent 2, crosses were attempted with all six H. bulbosum males.

Crosses with males 6 and 10 were nonviable. Crosses with male parents 7, 8, 9, 11 produced eight embryos that were rescued (Table 5.5). Of six possible top crosses, four

(66.67%) were successful as determined by embryo production. Three (37.5%) of the

progeny show grain yields greater than the mean of all crosses within this family. Crosses

2x7(2), 2x9(1), and 2x9(2) exceeded mean grain yield of all progeny by + 1 SD.

The correlation coefficient among yield and its component traits of Family 2 genotypes are presented in Table 5.4. In this family of crosses, plant height showed negative correlation with fertility. Tiller number negatively correlated with flag leaf area, indicating the larger the number of tillers, the smaller each flag leaf was. Babaiy et al.

(2011) observed the number of fertile tillers and infertile tillers had significant negative correlation with the flag leaf area. In contrast, grain yield showed a high positive association with tiller number and fertility as it did in Family 1. Interestingly, cSW was not related to grain yield as it was in Family 1 crosses. These results provide information about interrelationship between grain yield and its component traits (tiller number and fertility). Similar finding has been observed in barley by Dadashi (2010), Pal et al.

(2010), Babaiy et al. (2011), Hossain and Akhtar (2014), and Sunil et al. (2017).

Table 5.4 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 2 crosses (2x H. vulgare crosses).

PH TN FLA F cSW GY PH 1.00 0.07 -0.02 -0.57* 0.00 -0.07 TN 1.00 -0.73** 0.29 0.05 0.72** FLA 1.00 0.00 0.19 0.26 F 1.00 0.03 0.70** cSW 1.00 0.37 GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

Table 5.5 summarized the mean, standard deviation, and coefficient of variation values of parental genotypes and Family 2 crosses (2x H. vulgare crosses) for yield and related traits.

Table 5.5 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 2 65.3 71 14.39* 86.41* 3.088* 14.13 7 92.5 69 10.13 32.21 1.273 2.71 8 96.0 69 8.10 41.64 1.721 5.54 9 52.4† 123* 6.92 84.61* 0.620 16.11* 11 85.5 69 8.11 43.12 1.363 4.43 (x̅ ) 78.34 80.20 9.53 57.60 1.613 8.58 SD 18.75 23.94 2.95 25.83 0.915 6.09 2x7(2) 84.0* 56 15.34 86.00* 2.953 24.20* 2x8(2) 69.0 30 16.03 54.56 3.386 7.73 2x9(1) 79.0 177* 3.80 61.55 3.750 25.40* 2x9(2) 84.0* 153* 3.90 63.55 4.485* 23.72* 2x9(3) 75.0 32 17.53* 43.89 4.121* 7.31 2x9(4) 76.0 36 13.10 38.08 3.497 3.82 2x11(1) 65.0 47 5.78 49.73 2.905 5.42 2x11(3) 55.0† 54 8.00 76.45* 2.694 9.85 (x̅ ) 73.38 73.13 10.44 59.23 3.474 13.43 SD 9.96 57.87 5.70 16.18 0.624 9.29 CV (%) 13.57 79.14 54.59 27.31 17.97 69.18 * Value greater than x̅ + 1 SD. † Value lower than x̅ – 1 SD.

Plant height exhibited the lowest variability among crosses with coefficient variation (13.57%) compared to all other traits. Plant height ranged between 55 to 84 cm with a mean of 73.38 cm. Crosses 2x7(2) and 2x9(2) had greatest value for plant height

(84 cm) and exceeded the x̅ + 1 SD. In contrast, cross 2x11(3) had shorter stature (55 cm; less than the x̅ – 1 SD. Al-Tabbal and Al-Fraihat (2012) also reported wide variation 39 -

91 cm for plant height with a mean of 64.11 cm among 86 barley genotypes they tested.

-1 25

Grain yield, g plant g yield, Grain 10

2 7 8 9

2x7(2) 2x8(2) 2x9(1) 2x9(2) 2x9(3) 2x9(4)

2x11(1) 2x11(3)

Genotypes

Figure 5.2 Mean grain yield (GY), g plant-1 of parental genotypes and Family 2 crosses2x7 (2x H. vulgare crosses). 2x8 2x9 2x11 2 In contrast7 to plant height, tiller number showed greatest variability among 8 crosses with coefficient9 variation (79.14%) compared to all other traits. Tiller number 11 ranged from 30 to 177 with a mean of 73.13 tiller plant-1. Crosses 2x9(1) and 2x9(2) showed greatest tiller number (177 and 153, respectively) and exceeded the x̅ + 1 SD.

These crosses were affected by male parent 9 (123) which similar to Family 1, resulting in a high number of spikes, seed number and thus grain yield (Singh et al. 2015). Because tiller number associated positively with grain yield (Table 5.4), these crosses could be a good source of genes to improve barley grain yield as well as forage yield.

Flag leaf area is one of the most essential components in determining grain yield potential in barley and other cereal crops (Shahraki and Fakheri 2016). Flag leaf area of

Family 2 crosses ranged between 3.8 to 17.53 cm2 with a mean of 10.44 cm2 (Table 5.5).

Among all the crosses, cross 2x9(3) had the greatest flag leaf area (17.53 cm2) and exceeded the x̅ + 1 SD. Mahmood et al. (1991) reported that removing the flag leaf from wheat significantly reduced plant height, spike length, seed number spike-1, seed weight and grain yield. However, among Family 2 crosses we show no relationship (Table 5.4).

Additional research indicated that the flag leaf area showed best positive correlation with forage yield components (Rahal-Bouziane et al. 2018). Consequently, crosses with a high flag leaf area could be used in breeding programs for improving barley forage and grain yield.

Crosses of Family 2 showed a wide range of fertility, ranging from 38.08 to 86% with a mean of 59.23%. Crosses 2x7(2) and 2x11(3) had greatest fertility (86 and 76%, respectively) and exceeded the x̅ + 1 SD. Cross 2x7(2) showed a positive relationship between plant height and fertility as well as grain yield, so this cross could be use in barley breeding program to improve grain yield. However, we found a negative relationship between plant height and fertility among Family 2 crosses (Table 5.4), this result confirmed by cross 2x11(3) (shorter stature with high fertility). Although the two parents (2 and 9) had a high fertility (86.41 and 84.61%, respectively); their progenies

showed a low fertility. These progenies were sterile (haploid), but fertility was restored by a colchicine treatment.

Mean cSW of Family 2 crosses varied between 2.694 to 4.485 g with a mean of

3.474 g. Crosses 2x9(2) and 2x9(3) showed greatest cSW (4.485 and 4.121 g, respectively) and exceeded the x̅ + 1 SD; while their male parent (9) had lowest value

(0.620; less than x̅ – 1 SD). Cross 2x9(2) could be used in breeding program for improving seed weight and thus grain yield. Sarkar et al. (2014) also found a wide range of variation for cSW among barley genotypes (1.2 - 6.3 g).

Grain yield of Family 2 crosses showed the greatest variability among genotypes

(Table 5.5 and Figure 5.2). Grain yield ranged from 3.82 to 25.4 g, plant-1 with a mean of

13.43 g. Crosses 2x7(2), 2x9(1), and 2x9(2) had greatest grain yield (24.2, 25.40 and

23.72 g, respectively) and exceeded the x̅ + 1 SD. A high grain yield of cross 2x7(2) was positively influenced by a high fertility and plant height, 2x9(1) by a high tiller number, and 2x9(2) by a high plant height, tiller number, and cSW. These results confirmed that tiller number, flag leaf area, fertility, and cSW are the most important yield components.

Family 3, female parent 3, crosses were attempted with all six H. bulbosum males.

Crosses with males 7 and 11 were nonviable. Crosses with male parents 6, 8, 9, 10 produced seven embryos that were rescued (Table 5.7). Of six possible top crosses, four

(66.67%) were successful as determined by embryo production. Only one (14%) of the progeny show grain yield greater than the mean of all crosses within this family. Cross

3x9(2) exceed mean grain yield of all progeny by + 1 SD.

The correlation coefficient analysis among grain yield and yield components of

Family 3 genotypes are presented in Table 5.6. Plant height had a strong negative

100

association with fertility and grain yield, most likely linked to lodging. This relationship was affected by H. bulbosum parents (very low fertility) and triploid progeny (sterile), which are both taller than typical domesticated barley varieties. Pal et al. (2010) found a high negative correlation between plant height and grain yield. On the other hand, Singh et al. (2015) reported that plant height was significantly correlated with grain yield and directly impacted yield. In contrast, grain yield was positively correlation with fertility and cSW. Similar result also reported by Singh et al. (2014), Ruzdik et al. (2015), and

Lodhi et al. (2015).

Table 5.6 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 3 crosses (2x H. vulgare crosses).

PH TN FLA F cSW GY PH 1.00 0.03 0.26 -0.79** -0.21 -0.77** TN 1.00 -0.04 -0.31 -0.16 0.08 FLA 1.00 0.23 0.54 0.12 F 1.00 0.55 0.80** cSW 1.00 0.66* GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 3 crosses (2x H. vulgare crosses) for yield and related traits are presented in Table 5.7. Plant height had a lower level of variability among crosses with a coefficient variation of 14.1% compared to all other measured traits. Plant height varied between 71 and 104.3 cm with a mean of 82.19 cm. Cross 3x8(1) had greatest value of

101

plant height (104.3 cm) and exceeding the x̅ + 1 SD. This progeny was triploid and was similar to H. bulbosum in growth habit (Appendix B). In contrast, cross 3x10(3) showed shorter stature (71 cm), which less than the x̅ – 1 SD. Previous studies also reported a wide range of plant height among barley genotypes 60.6 - 124.2 cm (Yadav et al. 2015).

Table 5.7 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 3 81.3 47 17.74* 93.77* 4.511* 13.21 6 93.0 39 15.67 41.44 1.488 2.64 8 96.0 69 8.10 41.64 1.721 5.54 9 52.4† 123* 6.92 84.61 0.620 16.11* 10 102.8 76 13.62 29.22 1.691 3.72 (x̅ ) 85.10 70.80 12.41 58.14 2.006 8.24 SD 19.86 32.91 4.72 28.97 1.470 6.04 3x6(1) 72.0 82 3.30 70.46 2.639 10.36 3x8(1) 104.3* 146* 8.03 0.00 0.000 0.00 3x9(1) 84.0 112 16.88 62.35 6.657 12.20 3x9(2) 84.0 134* 25.16* 68.13 6.541 18.28* 3x9(3) 86.0 51 25.15* 75.45 4.829 8.58 3x10(3) 71.0† 38 10.52 72.22 4.998 12.24 3x10(5) 74.0 58 5.70 62.70 5.252 16.85 (x̅ ) 82.19 88.71 13.53 58.76 4.417 11.22 SD 11.59 42.54 9.01 26.35 2.360 6.02 CV (%) 14.10 47.95 66.55 44.84 53.42 53.64 * Value greater than x̅ + 1 SD. † Value lower than x̅ – 1 SD.

Tiller number showed a wide variation among Family 3 crosses. Tiller number ranged from 38 to 146 with a mean of 88.71 tiller plant-1. Crosses 3x8(1) and 3x9(2) had greatest tiller number (146 and 134, respectively) and exceeded the x̅ + 1 SD. Cross

102

3x8(1) exceeded the tiller number of both parents (2 and 9) by 210.6% and 111.6%, respectively.

-1 25

Grain yield, g plant g yield, Grain

3 6 8 9

3x6(1) 3x8(1) 3x9(1) 3x9(2) 3x9(3)

3x10(3) 3x10(5)

Genotypes

Figure 5.3 Mean grain yield (GY), g plant-1 of parental genotypes and Family 3 crosses3x6 (2x H. vulgare crosses). 3x8 A wide range3x9 of values were observed in flag leaf area of Family 3 crosses. Flag 3x10 leaf area ranged3 between 3.3 and 25.16 cm2 with a mean of 13.53 cm2. Pal et al. (2010) 6 also found a wide8 variation of flag leaf area among barley genotypes, ranging between 9 8.99 and 33.18 cm10 2. Crosses 3x9(2) and 3x9(3) showed greatest flag leaf area (25.2 cm2) and exceeded the x̅ + 1 SD, which exceeded the mean of family by 86%. Because cross

103

3x9(2) had greatest tiller number, flag leaf area, and grain yield, this cross could be used in breeding program for improving barley forage and grain yield.

Fertility of Family 3 crosses varied between 0 and 75.45% with a mean of 58.76%

(Table 5.7). No cross exceeded the fertility x̅ + 1 SD. Cross 3x8(1) was sterile and flow cytometry determined it to be triploid (3x). This cross backcrossed as a female with its H. vulgare parent (3) as male, but it was also infertile. Pickering (1991) reported that triploid hybrid (VBB) and amphiploid (VVBBBB) progeny from interspecific cross between H. vulgare (2x) and H. bulbosum (4x) were infertile. This hybrid was similar to H. bulbosum species in growth habit.

Hundred seed weight showed a wide variation among Family 3 crosses ranging from 0 to 6.657 g with a mean of 4.417 g cSW-1. No cross exceeded cSW x̅ + 1 SD.

Grain yield also revealed a wide variation ranging between 0 and 18.28 g, plant-1 with a mean of 11.22 g (Table 5.7 and Figure 5.3). Among all crosses of Family 3, only cross

3x9(2) had greater grain yield plant-1 (18.28 g) and exceeded the grain yield x̅ + 1 SD.

High grain yield of cross 3x9(2) was likely affected by a high tiller number, flag leaf area, and cSW; however, the contribution of flag leaf area is not correlated across all the families. This result confirmed the association among final grain yield and these yield trait components.

Family 4, female parent 4, crosses were attempted with all six H. bulbosum males.

Crosses with males 7, 9, 10, and 11 were nonviable. Crosses with male parents 6 and 8 produced eight embryos that were rescued in tissue culture (Table 5.9). Of six possible top crosses, two (33.3%) were successful as determined by embryo production. Only one

104

(12.5%) of the progeny showed grain yields greater than the mean of all crosses within this family. Cross 4x8(5) exceed mean grain yield of all progeny by + 1 SD.

The correlation coefficient among yield and its component traits of Family 4 genotypes are presented in Table 5.8. Fertility was associated positively with cSW. Also, grain yield showed a strong positive association with fertility and cSW. Similar association was also reported by Al-Tabbal and Al-Fraihat (2012), Mawati (2014), Singh et al. (2015), and Sunil et al. (2017).

Table 5.8 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 4 crosses (2x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 0.50 0.53 -0.45 0.00 -0.25 TN 1.00 0.07 -0.08 0.10 0.34 FLA 1.00 0.03 0.39 0.13 F 1.00 0.78** 0.80** cSW 1.00 0.76** GY 1.00 ** Significant at the 0.01 probability level.

Table 5.9 summarized the mean, standard deviation, and coefficient of variation values of parental genotypes and Family 4 crosses (2x H. vulgare crosses) for yield and related traits. Plant height revealed the lowest level of variability among genotypes with a coefficient variation of 17.35% compared to all other traits. Plant height varied between

66 and 106 cm with a mean of 75.25 cm. Derbew et al. (2013) also observed high variation among 225 barley genotypes in plant height ranging from 44.95 to 94.10 cm.

105

Cross 4x8(6) had greatest plant height (106 cm) and exceeded the x̅ + 2 SD. This cross was triploid and similar to H. bulbosum parent (Appendix B). Cross 4x6(3) was also triploid, but was short (66 cm).

Table 5.9 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 4 77.0† 56 11.98 87.93* 1.957* 8.59* 6 93.0 39 15.67 41.44 1.488 2.64 8 96.0 69 8.10 41.64 1.721 5.54 (x̅ ) 88.67 54.67 11.92 57.00 1.722 5.59 SD 10.21 15.04 3.79 26.78 0.234 2.98 4x6(2) 76.0 42 10.19* 56.31 1.020 3.73 4x6(3) 66.0 29 5.70 0.00 0.000 0.00 4x8(1) 70.0 22 5.56 67.75 0.881 2.96 4x8(2) 71.0 52 3.74 65.46 1.189 5.31 4x8(3) 70.0 36 4.90 55.37 1.021 1.60 4x8(4) 77.0 71 6.24 45.66 0.973 3.07 4x8(5) 66.0 71 7.04 72.75 1.198 9.10* 4x8(6) 106.0** 79* 8.89* 0.00 0.000 0.00 (x̅ ) 75.25 50.25 6.53 45.41 0.785 3.22 SD 13.06 21.42 2.12 29.25 0.496 2.99 CV (%) 17.35 42.63 32.41 64.42 63.16 92.88 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. † Value lower than x̅ – 1 SD.

Family 4 crosses had a lower mean tiller number (50.25 tiller plant-1) compared to all other families (Table 5.9). It ranged between 22 and 79. Cross 4x8(6) exceeded the x̅

+ 1 SD with 79 tiller plant-1. However, this cross was sterile (3x), and it backcrossed as a female with its H. vulgare parent (8) as male but was infertile.

106

Family 4 crosses also had lowest mean flag leaf area (6.53 cm2) compared to all other families. Flag leaf area varied among crosses from 3.74 to 10.19 cm2. Crosses

4x6(2) and 4x8(6) had the greatest flag leaf area (10.19 and 8.89 cm2, respectively) and exceeded the x̅ + 1 SD. Previous studies also indicated that flag leaf area varied among genotypes. Mawati (2014) reported 11.8 to 18.9 cm2 for flag leaf area of barley genotypes, while Liu et al. (2015) found 9.66 to 30.42 cm2, and concluded that the variation was due to a wide genetic diversity among barley genotypes.

-1 25

Grain yield, g plant g yield, Grain 10

4 6 8

4x6(2) 4x6(3) 4x8(1) 4x8(2) 4x8(3) 4x8(4) 4x8(5) 4x8(6) Genotypes

Figure 5.4 Mean grain yield (GY), g plant-1 of parental genotypes and Family 4 crosses (2x H. vulgare crosses). 4x6 Fertility is4x8 one of the most important traits affected seed number and thus grain 4 yield. Family 4 crosses6 showed a wide range of fertility 0 - 72.75% with a mean of 8 107

45.41% No cross exceeded the fertility x̅ + 1 SD. Crosses 4x6(3) and 4x8(6) were sterile and triploid. Crosses were likely affected by their H. bulbosum parents for fertility.

Family 4 crosses showed a low mean of cSW (0.785 g) compared to all other families. All crosses had values within 1 SD unit around the x̅ . Because crosses of this family have the lowest values in all previous traits, it had also the lowest grain yield (3.22 g plant-1) compared to all other families (Table 5.9 and Figure 5.4). Genotypes showed the widest variation for grain yield compared to all other traits (CV 92.88%). Among all crosses of Family 4, only cross 4x8(5) had greatest grain yield (9.1 g plant-1) and exceeded the x̅ + 1 SD. Grain yield of parent 4 was more affected by fertility and cSW as well as short stature. These results confirmed a positive relationship among these traits and grain yield similar to results found by Hossain and Akhtar 2014.

Family 5, female parent 5, crosses were attempted with all six H. bulbosum males.

Crosses with all male parents produced 34 embryos that were rescued (Table 5.11). All six possible top crosses were successful as determined by embryo production. This result indicates that female parent (5) has a high general combining ability and compatibility with H. bulbosum compared to all other H. vulgare parents. Six (17.6%) of the progeny show grain yields greater than the mean of all crosses within this family. Crosses 5x7(1) and 5x11(1) exceed mean grain yield of all progeny by + 2 SD. Crosses 5x8(10-1),

5x8(12), 5x9(2), and 5x11(9) exceed mean grain yield by + 1 SD.

The correlation coefficient analysis among grain yield and yield components of

Family 5 genotypes presented in Table 5.10. Plant height showed positive correlation with flag leaf area (Pal et al. 2010; Singh et al. 2015), while plant height associated negatively with fertility (Ruzdik et al. 2014). Hossain and Akhtar (2014) reported

108

positive correlation between plant height and seed number spike-1. Tiller number was negatively correlation with fertility and cSW. Similar trend has been observed in barley by Singh et al. (2015). Grain yield showed positive correlation with fertility and cSW.

Similar results were also reported by Hossain and Akhtar (2014), Lodhi et al. (2015), and

Sunil et al. (2017) and seen among the other families of this study.

Table 5.10 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 5 crosses (2x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 0.29 0.32* -0.47** -0.30 -0.17 TN 1.00 0.22 -0.41** -0.51*** 0.10 FLA 1.00 -0.24 0.15 0.01 F 1.00 0.76*** 0.74*** cSW 1.00 0.56*** GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 5 crosses (2x H. vulgare crosses) for yield and related traits are presented in Table 5.11. Plant height had the lowest level of variability among crosses

(CV 17%) compared to all other traits. Plant height ranged between 52 and 106 cm with a mean of 71.21 cm. Derbew et al. (2013) also reported a variation among barley genotypes for plant height between 44.95 and 94.10 cm, while Thahar et al. (2015) reported 81.3 and 124.3 cm, and they claimed this variation to a wide genetic variability among barley

109

genotype. Crosses 5x6(1) and 5x8(9) exceeded the x̅ + 2 SD with plant height (106 and

98 cm, respectively). These crosses were triploid hybrid and similar to their H. bulbosum parent in growth habit (Appendix B). Crosses 5x8(6) and 5x8(7-2) were also triploid hybrid and exceeded the x̅ + 1 SD for plant height, but as triploids, both were sterile.

Cross 5x7(1) also exceeded the x̅ + 1 SD. In contrast, crosses 5x7(2), 5x8(3), 5x8(4),

5x8(10-2), 5x9(1), 5x11(6-1) had shortest plant height (52.4, 52, 54, 58, 56, 59, and 58, respectively; shorter than the x̅ – 1 SD).

Table 5.11 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 5 82.2 54 9.99 73.81* 2.811* 8.25 6 93.0 39 15.67* 41.44 1.488 2.64 7 92.5 69 10.13 32.21 1.273 2.71 8 96.0 69 8.10 41.64 1.721 5.54 9 52.4†† 123* 6.92 84.61* 0.620 16.11** 10 102.8* 76 13.62* 29.22 1.691 3.72 11 85.5 69 8.11 43.12 1.363 4.43 (x̅ ) 86.34 71.29 10.36 49.44 1.567 6.20 SD 16.41 25.99 3.19 21.22 0.661 4.78 5x6(1) 106.0** 85 4.00 0.00 0.000 0.00 5x6(2) 65.0 56 3.79 75.61 2.309 7.33 5x7(1) 84.0* 65 4.79 73.75 3.000* 13.91** 5x7(2) 52.0† 39 3.85 44.79 1.217 1.37 5x7(3) 74.0 55 6.65 78.50 1.562 6.54 5x8(1) 62.0 58 5.51 55.98 1.512 3.38 5x8(3) 54.0† 36 4.99 63.55 2.743* 4.65 5x8(4) 58.0† 61 4.96 50.63 1.885 3.80 5x8(5) 67.0 63 4.35 63.38 1.807 7.04 5x8(6) 88.0* 162**** 9.12* 0.00 0.000 0.00 5x8(7-1) 73.0 41 3.62 25.99 1.328 0.45 5x8(7-2) 85.0* 88* 7.85 0.00 0.000 0.00 5x8(8) 76.0 62 3.03 54.91 2.424 9.18 110

Table 5.11 (Continued)

5x8(9) 98.0** 89* 6.30 0.00 0.000 0.00 5x8(10-1) 79.0 51 5.15 81.19* 2.568 9.70* 5x8(10-2) 56.0† 45 3.76 47.36 1.232 1.01 5x8(12) 62.0 74 10.04* 67.43 2.803* 11.85* 5x8(13) 65.0 65 12.26** 57.57 2.110 4.51 5x9(1) 59.0† 48 13.35** 35.81 1.651 3.23 5x9(2) 77.0 61 8.20 79.13* 2.502 10.05* 5x10(1) 65.0 69 6.28 50.56 1.921 5.16 5x11(1) 80.0 79 8.36* 80.53* 1.831 15.68** 5x11(2) 81.0 39 4.01 81.48* 2.741* 5.47 5x11(3) 73.0 48 3.34 67.62 2.014 4.22 5x11(4) 70.5 31 3.82 65.32 1.763 2.13 5x11(5) 61.8 71 3.12 53.94 2.027 4.50 5x11(6-1) 58.0† 39 3.98 63.75 1.706 2.10 5x11(6-2) 62.0 49 4.54 54.73 2.333 5.78 5x11(7) 75.0 63 2.73 67.43 2.131 6.73 5x11(8) 70.0 52 5.22 60.87 1.798 3.60 5x11(9) 68.0 55 5.69 71.54 2.446 10.91* 5x11(10) 74.0 58 3.16 74.15 2.972* 7.45 5x11(11) 77.0 65 2.22 61.46 2.578 7.35 5x11(12) 66.0 81 7.25 47.45 2.671 6.85 (x̅ ) 71.21 61.85 5.57 54.60 1.870 5.47 SD 12.10 23.22 2.66 24.02 0.845 4.05 CV (%) 17.00 37.54 47.75 43.98 45.19 74.01 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. **** Value greater than x̅ + 4 SD. † Value lower than x̅ – 1 SD. †† Value lower than x̅ – 2 SD.

Tiller number revealed a wide variation among Family 5 crosses, ranging from 31 to 162 with a mean of 61.85 tiller plant-1. Cross 5x8(6), triploid hybrid, had greatest tiller number (162) and exceeded the x̅ + 4 SD. Crosses 5x8(7-2) and 5x8(9), triploid hybrids, exceeded the x̅ + 1 SD for tiller number. These crosses probably contributed to the non-

111

significance of the correlation between tiller number and grain yield because they were sterile (3x), and therefore had 0 fertility, cSW and grain yield.

-1 25

Grain yield, g plant g yield, Grain

5 6 7 8 9

10 11

5x6(1) 5x6(2) 5x7(1) 5x7(2) 5x7(3) 5x8(1) 5x8(3) 5x8(4) 5x8(5) 5x8(6) 5x8(8) 5x8(9) 5x9(1) 5x9(2)

5x8(12) 5x8(13) 5x10(1) 5x11(1) 5x11(2) 5x11(3) 5x11(4) 5x11(5) 5x11(7) 5x11(8) 5x11(9)

5x8(7-1) 5x8(7-2)

5x11(10) 5x11(11) 5x11(12)

5x8(10-1) 5x8(10-2) 5x11(6-1) 5x11(6-2) Genotypes

-1 Figure 5.55x6 Mean grain yield (GY), g plant of parental genotypes and Family 5 5x7 crosses (2x H. vulgare crosses). 5x8 Flag5x9 leaf area varied among Family 5 crosses ranged between 2.22 and 13.35 cm2 5x10 with a mean5x11 of 5.57 cm2. Mawati (2014) also reported a variation among barley 5 genotype6s for a flag leaf area ranging from 11.75 to 18.86 cm2 and concluded that the 7 8 variation9 was due to genetic variability among genotypes. Crosses 5x8(13) and 5x9(1) 10 2 had the largest11 flag leaf area (12.26 and 13.35 cm , respectively) and exceeded the x̅ + 2

SD. Crosses 5x8(6), 5x8(12), and 5x11(1) exceeded the flag leaf area x̅ + 1 SD. Mawati 112

(2014) reported a variation among genotype for flag leaf area ranged from 11.75 to 18.86 cm2.

Fertility was highly variable among Family 5 crosses ranged from 0 in triploid hybrids [(5x6(1), 5x8(6), 5x8(7-2), and 5x8(9)] to 81.48% with a mean of 54.6%. Crosses

5x8(10-1), 5x9(2), 5x11(1), and 5x11(2) exceed the x̅ + 1 SD (81.19, 79.13, 80.53, and

81.48%, respectively). These crosses could be used in a barley breeding program for improving fertility, and thus grain yield, as long as grain yield was sufficient. This result confirmed the strong relationship between fertility and grain yield (Table 5.10). In contrast, along with the triploids, cross 5x8(7-1) showed fertility less than the x̅ – 1 SD

(25.99%). Fertility reduced seed number spike-1 for this cross (Appendix B) and thus grain yield.

Family 5 crosses revealed a mean of 1.870 g for cSW. Crosses 5x7(1), 5x8(3),

5x8(12), 5x11(2), 5x11(10), and 5x11(12) had cSW exceed the x̅ + 1 SD (3.0, 2.743,

2.803, 2.741, and 2.972 g, respectively). These crosses would be desirable for barley breeding.

Crosses showed a widest variation for grain yield compared to all other traits

(Table 5.11 and Figure 5.5). Grain yield ranged from 0 in triploid hybrids to 15.68 g plant-1 with a mean of 5.47 g plant-1. Among all genotypes, only crosses 5x7(1) and

5x11(1) exceeded the x̅ + 2 SD for grain yield (13.91 and 15.68 g plant-1). Crosses

5x8(10-1), 5x8(12), 5x9(2), and 5x11(9) had grain yield exceeded the x̅ + 1 SD. High grain yield of cross 5x7(1) was positively influenced by cSW as well as tall stature;

5x8(10-1) and 5x9(2) affected by fertility; 5x8(12) affected by flag leaf area and cSW;

5x11(1) affected by flag leaf area and fertility. Previous studies reported a positive

113

association between grain yield and yield trait components ( Kole 2006; Dorostkar et al.

2015, Lodhi et al. 2015).

Conclusion

The number of successful crosses varied for each family, so there is a difference in general combining ability and compatibility for each H. vulgare (♀) parent with the six

H. bulbosum (♂) parents. For Family 1, only crosses with male parents 6, 9, and 10 produced embryos that were rescued. Two of 14 progeny (11.8%) show grain yields greater than the mean of all crosses within this family. Family 2, only crosses with male parents 7, 8, 9, 11 produced embryos that were rescued. Three of eight progeny (37.5%) had grain yields greater than the mean of all crosses within this family. Family 3, crosses with male parents 6, 8, 9, 10 produced embryos that were rescued. Only one of seven progeny (14%) showed grain yield greater than the mean of all crosses within this family.

Family 4, only crosses with male parents 6 and 8 produced embryos that were rescued.

Only one of eight progeny (12.5%) had grain yields greater than the mean of all crosses within this family. In Family 5, all six possible top crosses were successful as determined by embryo production. This result indicates that female parent (5) has a high general combining ability and compatibility with H. bulbosum compared to all other H. vulgare parents. Six of 34 progeny (17.6%) show grain yields greater than the mean of all crosses within this family. Parent 9 had also a high general combining ability and compatibility with vulgare parents compared to all other H. bulbosum parents, except with parent 4.

Additionally, progeny of parent 9 exceeded the mean of the traits measured in many cases with most families. It is not surprising that rescued progeny of male parent 9 were

114

relatively prolific. While parent 9 is H. bulbosum, it is diploid, and as such ploidy level issues are minimize; however, there are still species in compatibility issues.

Grain yield was associated positively with fertility and cSW for most families as well as tiller number for Family 1 and 2. These results support the possibility of using these associated traits to select and improve grain yield. Progenies in this study showed a high level of variation for most morphological traits included in this study. As genetic variation is essential in breeding programs for improvement and development any crop, there is great potential that these progenies can be used in a breeding program for barley improvement. Additionally, these progenies may have tolerance to biotic or abiotic stress conditions, so they could be used by breeders in a barley breeding program.

Genotypes of tetraploid H. vulgare crosses

With trans-species crosses such as those performed here, there are different degrees of compatibility prior to and during the mitotic divisions following fertilization.

In this experiment, we converted the diploid H. vulgare genotypes to tetraploids using colchicine to minimize ploidy incompatibilities. Each tetraploid vulgare (1, 2, 3, 4, and 5) genotype was used as a female and top crossed to 4x H. bulbosum (6, 7, 8, 10, and 11).

Assessment for general combining ability and compatibility can be made by evaluating the number of crosses that successfully produced viable embryos (that were successfully rescued). For Family 1, female parent 1, crosses were attempted with all five H. bulbosum males. Crosses with all male parents produced 34 embryos that were rescued

(Table 5.13). All five possible top crosses were successful as determined by embryo production. This result indicates that female parent (1) has a high general combining ability and compatibility with all H. bulbosum parents. Four (11.8%) of the progeny had 115

grain yields greater than the mean of all crosses within this family. Crosses 1x6(3),

1x7(8), 1x8(1) and 1x10(5) exceeded the mean grain yield of all progeny by + 1 SD.

Grain yield is the essential and complex trait for any crop genetic improvement.

This trait was determined directly and indirectly by yield component characteristics.

Correlation coefficient analysis measures the mutual association between yield and its component traits and identifies the traits on which selection can be based for improvement (Shi et al. 2009). The correlation coefficient among yield and its component traits of tetraploid Family 1 genotypes are presented in Table 5.12. Plant height had negative correlation with fertility (Singh et al. 2014) and grain yield (Pal et al. 2010); most likely linked to lodging. This relationship was affected by H. bulbosum parents

(very low fertility) and triploid crosses (sterile) both of which are tall. However, Singh et al. (2015) found a high positive correlation between plant height and grain yield. Grain yield had a high positive correlation with tiller number, fertility, and 100-seed weight

(cSW). Tiller number was positively correlation with fertility, and they positively correlated with cSW. Similar results also reported by Hossain and Akhtar (2014), Mawati

2014, Lodhi et al. 2015, and Sunil et al. (2017).

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 1 crosses (4x H. vulgare crosses) for yield and related traits are presented in Table 5.13. Other traits summarized in Appendix C. Plant height had lower variability (CV 11.13%) among Family 1 crosses compared to all other traits. Plant height of Family 1 crosses varied between 54.5 and 102.5 cm with a mean of 84.47 cm.

Previous studies also showed a wide range of plant height among barley genotypes 50.63

- 77.63cm (Kumar and Shekhawat 2013) and between 90.3 to 127.3 cm (Haile et al.

116

2015) and claimed that variation due to genetic diversity among genotypes. Crosses

1x7(2), 1x7(3), 1x7(7), 1x10(1), 1x10(3), 1x10(5) exceeded the x̅ + 1 SD with plant height (102.5, 95, 96, 94.5, 96, and 98 cm). In contrast, crosses 1x7(5) had a shorter stature (54.5 cm; less than the x̅ – 3 SD). This cross, 1x7(5), was tetraploid (4x)

(Appendix B) and similar to H. bulbosum in growth habit. Crosses 1x6(4), 1x7(10),

1x7(11),1x8(4), and 1x10(7) showed plant height less than the x̅ – 1 SD.

Tiller number showed a wide variation among Family 1 crosses ranged from 19 to

191 with a high mean 118.79 tillers plant-1. Crosses 1x6(2), 1x6(3), 1x10(6), 1x11(3), and

1x11(4) had greatest values of tiller number and exceeded the x̅ + 1 SD (169, 172, 191,

179, and 185, respectively). Previous studies also reported a strong positive correlation between tiller number and grain yield (Singh et al. 2006) as well as fertility and cSW

(Table 5.12), so these crosses could be desirable for improving barley productivity (grain and forage yield).

Table 5.12 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 1 crosses (4x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 -0.24 0.18 -0.33* -0.27 -0.32* TN 1.00 0.04 0.40* 0.50*** 0.74***

FLA 1.00 0.23 0.21 0.23

F 1.00 0.79*** 0.76***

cSW 1.00 0.82***

GY 1.00

* Significant at the 0.05 probability level. *** Significant at the 0.001 probability level.

117

Table 5.13 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 1 70.8† 124* 7.76 93.03* 4.161** 23.10** 6 124.9 40 8.94* 36.21 1.816 4.59 7 111.3 45 6.33 26.59 1.382 3.40 8 119.9 46 7.23 35.95 1.679 5.20 10 123.1 65 6.17 21.39 1.470 3.71 11 97.8 40 8.74* 38.18 1.619 5.57 (x̅ ) 107.97 60.00 7.53 41.89 2.021 7.60 SD 20.76 32.69 1.17 25.89 1.059 7.64 1x6(1) 87.0 157 6.09 81.67 5.285 29.51 1x6(2) 77.0 169* 4.09 68.59 4.150 25.37 1x6(3) 92.0 172* 8.34 81.25 5.126 37.26* 1x6(4) 74.0† 51 8.32 79.79 4.694 9.91 1x6(5) 81.0 88 2.99 80.14 4.989 20.61 1x7(1) 79.0 82 4.37 78.94 4.062 18.45 1x7(2) 102.5* 42 13.93* 88.09 5.562 21.58 1x7(3) 95.0* 80 6.25 78.02 5.747 21.62 1x7(4) 79.0 145 2.92 76.44 4.035 23.93 1x7(5) 54.5‡ 19 1.53 0.00 0.000 0.00 1x7(6) 80.5 98 13.60* 82.24 5.235 28.20 1x7(7) 96.0* 104 8.66 67.36 5.236 23.08 1x7(8) 91.0 133 11.02 74.04 5.536 32.27* 1x7(9) 83.0 115 22.24*** 84.15 4.421 23.58 1x7(10) 74.0† 118 4.92 79.93 4.490 19.23 1x7(11) 74.0† 66 3.18 81.41 4.325 13.26 1x8(1) 82.0 115 7.10 74.61 5.222 37.65* 1x8(2) 90.0 153 6.29 86.03 4.890 30.27 1x8(3) 81.0 103 5.00 35.86 6.278* 12.07 1x8(4) 74.0† 106 9.81 83.67 4.500 24.86 1x10(1) 94.5* 113 4.68 0.00 0.000 0.00 1x10(2) 84.0 141 5.00 67.46 3.892 21.07 1x10(3) 96.0* 157 5.68 78.10 4.824 30.90 1x10(4) 93.0 77 7.71 76.06 5.327 21.65 1x10(5) 98.0* 149 9.19 51.33 5.699 32.17* 1x10(6) 83.0 191* 5.37 75.67 5.154 27.58 1x10(7) 75.0† 109 5.57 54.4 4.108 20.75 1x10(8) 79.0 133 4.73 76.73 4.640 18.97 1x11(1) 89.0 114 12.09* 83.33 4.865 28.77 1x11(2) 93.0 146 4.31 45.93 4.412 19.93

118

Table 5.13 (Continued)

1x11(3) 84.0 179* 5.78 77.53 5.074 30.02 1x11(4) 86.0 185* 18.41** 59.69 4.622 26.84 1x11(5) 85.0 121 5.39 81.37 4.905 25.01 1x11(6) 86.0 108 3.87 80.24 4.767 20.96 (x̅ ) 84.47 118.79 7.31 69.71 4.590 22.86 SD 9.40 41.22 4.46 21.28 1.287 8.59 CV (%) 11.13 34.70 61.08 30.52 28.04 37.56 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. *** Value greater than x̅ + 3 SD. † Value lower than x̅ – 1 SD. ‡ Value lower than x̅ – 3 SD.

-1 25

Grain yield, g plant g yield, Grain

1 6 7 8

10 11

1x6(1) 1x6(2) 1x6(3) 1x6(4) 1x6(5) 1x7(1) 1x7(2) 1x7(3) 1x7(4) 1x7(5) 1x7(6) 1x7(7) 1x7(8) 1x7(9) 1x8(1) 1x8(2) 1x8(3) 1x8(4)

1x7(10) 1x7(11) 1x10(1) 1x10(2) 1x10(3) 1x10(4) 1x10(5) 1x10(6) 1x10(7) 1x10(8) 1x11(1) 1x11(2) 1x11(3) 1x11(4) 1x11(5) 1x11(6)

Genotypes

Figure 5.6 Mean grain yield (GY), g plant-1 of parental genotypes and Family 1 crosses (4x H. vulgare crosses).

1x6 119 1x7 1x8 1x10 1x11 1 6 7 8 10 11

Family 1 crosses had largest variation for flag leaf area (CV 61.08%) compared to all other traits. Flag leaf area ranged between 1.53 and 22.24 cm2 with a mean of 7.31 cm2. Previous studies also found a wide variation of flag leaf area among barley genotypes ranged between 11.8 - 18.9 cm2 (Mawati 2014) and from 18.9 - 25.5 cm2

(Kumar and Shekhawat 2013). Cross 1x7(9) had greatest flag leaf area (22.24 cm2) and exceeded the x̅ + 3 SD, which exceeded the mean of this family by 204%. Cross 1x11(4) exceeded the x̅ + 2 SD, and crosses 1x7(2), 1x7(6), 1x11(1) exceeded the x̅ + 1 SD for flag leaf area.

Fertility of Family 1 crosses varied between 0 and 88.09% with a mean of 69.71%

(Table 5.13). No cross exceeded the x̅ + 1 SD for fertility. Crosses 1x7(5), tetraploid hybrid (4x), and 1x10(1), triploid hybrid (3x) were sterile (Appendix B). These hybrids were similar to H. bulbosum spike structure and growth habit. There were 13 crosses with a good fertility (80 - 88%) within 1 SD unit around the x̅ . All H. bulbosum parents had low fertility (21.39 - 38.18%) due to innate self-incompatibility systems.

Mean cSW of Family 1 crosses was 4.590 g (Table 5.13). Among all crosses, only cross 1x8(3) had greatest value of cSW (6.278 g) and exceeded the x̅ + 1 SD. All H. bulbosum parents showed low cSW (1.382 - 1.816 g). Tahar et al. (2015) also reported a range of cSW among 2.22 - 4.95 g and 3.1 - 4.72 g at two locations, and concluded that variation due to genetic variability among genotypes as well as environment conditions was the cause.

Grain yield revealed a wide variation among Family 1 crosses ranging from 0 to

37.65 g plant-1 with a high mean 22.86 g plant-1 (Table 5.13 and Figure 5.6). Crosses

1x6(3), 1x7(8), 1x8(1), and 1x10(5) had greatest grain yield plant-1 (37.26, 32.27, 37.65,

120

and 32.17 g, respectively) and exceeded the x̅ + 1 SD. Grain yield affected by yield component traits for each genotype, which were confirmed by a correlation coefficient analysis (Table 5.12) and previous studies (Dyulgerova 2012; Mehripour et al. 2014;

Aynewa et al. 2015). Since only one progeny exceeded the x̅ for cSW and only 4 of 34 progeny exceeded the x̅ for grain yield plant-1, parent 1 is not a likely candidate for incorporation in a barley breeding program.

Family 2, female parent 2, crosses were attempted with all five H. bulbosum males. Crosses with all male parents produced 56 embryos that were rescued through tissue culture (Table 5.15). All five possible top crosses were successful as determined by embryo production. This result indicates that female parent (2) has a high general combining ability and compatibility with all H. bulbosum parents. Ten (17.9%) of the progeny showed grain yields greater than the mean of all crosses within this family.

Crosses 2x6(1), 2x6(2), 2x7(1), 2x7(7), 2x8(6), 2x10(4), 2x10(5), 2x10(6), 2x10(7), and

2x11(5) exceed mean grain yield of all progeny by + 1 SD.

The correlation coefficient analysis among grain yield and yield components of

Family 2 genotypes are presented in Table 5.14. Grain yield showed a positive correlation with tiller number, flag leaf area, fertility, and cSW. Tiller number and flag leaf area were also correlated positively with fertility and cSW. Fertility showed positive association with cSW. These results support the possibility of using these associated traits to select and improve grain yield. Similar findings were also reported by Pal et al. (2010),

Al-Tabbal and Al-Farihat (2012), Singh et al. (2014), Singh et al. (2015), and Sunil et al.

(2017).

121

Table 5.14 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 2 crosses (4x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 0.05 0.15 0.06 0.03 0.04 TN 1.00 0.17 0.45*** 0.46*** 0.73*** FLA 1.00 0.35** 0.34** 0.31* F 1.00 0.91*** 0.79*** cSW 1.00 0.76*** GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

Table 5.15 summarized the mean, standard deviation, and coefficient of variation of parental genotypes and Family 2 crosses (4x H. vulgare crosses) for yield and related traits. Crosses with this family had lower variability (CV 15.41%) compared to all other traits. Plant height ranged between 25 to 108.5 cm with a mean of 84.29 cm. Al-Tabbal and Al-Fraihat (2012) also observed high variability among 98 barley genotypes for plant height (39 - 91 cm) and concluded that variation due to genetic diversity among genotypes. Crosses 2x7(6), 2x8(2), 2x8(5), 2x8(6), and 2x10(3) had greatest plant height

(108.5, 98, 100, 98, 99 cm, respectively) and exceeded the x̅ + 1 SD. Crosses 2x7(6) and

2x10(3) were tetraploid and triploid hybrids, respectively (Appendix B). In contrast, cross

2x6(3) had shorter stature (25 cm) and was shorter than the x̅ – 4 SD. This cross was tetraploid (4x). Crosses 2x6(5), tetraploid, and 2x7(3), triploid, showed plant height less than the x̅ – 2 SD (48 and 49 cm, respectively). Plant height of other crosses were within

1 SD unit around the x̅ . 122

Table 5.15 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 2 74.4† 121* 8.72 90.32* 4.517** 20.57** 6 124.9 40 8.94 36.21 1.816 4.59 7 111.3 45 6.33 26.59 1.382 3.40 8 119.9 46 7.23 35.95 1.679 5.20 10 123.1 65 6.17 21.39 1.470 3.71 11 97.8 40 8.74 38.18 1.619 5.57 (x̅ ) 108.57 59.50 7.69 41.44 2.081 7.17 SD 19.48 31.51 1.27 24.82 1.203 6.62 2x6(1) 88.0 91 8.19 93.79* 5.030 35.38* 2x6(2) 92.0 155* 4.21 69.55 4.897 33.51* 2x6(3) 25.0‡‡ 20 0.00 0.00 0.000 0.00 2x6(4) 77.0 87 3.70 15.50 4.350 2.07 2x6(5) 48.0†† 36 12.21 0.00 0.000 0.00 2x6(6) 95.0 106 24.84*** 85.97 5.349 27.74 2x6(7) 88.0 82 14.89* 67.37 4.760 24.63 2x6(8) 91.0 163* 10.38 85.58 4.791 29.87 2x6(9) 86.5 82 4.79 0.00 0.000 0.00 2x6(10) 74.3 59 3.02 91.21 4.947 12.44 2x6(11) 75.0 18 5.81 66.06 4.856 4.85 2x7(1) 83.0 133 8.89 71.07 5.157 32.37* 2x7(2) 81.0 91 4.21 58.80 6.036* 15.02 2x7(3) 49.0†† 12 1.97 0.00 0.000 0.00 2x7(4) 92.0 69 7.83 86.97 5.101 18.97 2x7(5) 84.0 179* 10.40 53.14 3.909 18.39 2x7(6) 108.5* 45 3.75 0.00 0.000 0.00 2x7(7) 87.0 173* 5.80 79.79 4.675 36.55* 2x7(8) 94.0 82 7.00 82.36 4.694 24.95 2x7(9) 90.0 147 10.17 85.52 4.958 28.93 2x7(10) 87.0 123 12.82* 81.68 5.327 27.03 2x7(11) 88.0 144 7.49 68.13 4.682 27.04 2x7(12) 73.0 45 5.95 88.62 5.084 11.75 2x8(1) 84.0 147 8.13 77.38 4.858 26.58

123

Table 5.15 (Continued) 2x8(2) 98.0* 96 4.66 73.95 4.204 20.66 2x8(3) 84.0 133 11.49 86.21 4.827 31.14 2x8(4) 86.0 128 7.64 78.24 4.793 21.75 2x8(5) 100.0* 90 8.12 65.48 5.750 31.38 2x8(6) 98.0* 111 11.90 81.92 4.978 33.81* 2x8(7) 97.0 68 5.90 59.61 4.276 11.75 2x8(8) 78.0 136 9.28 58.94 4.444 20.85 2x8(9) 92.0 100 6.69 0.00 0.000 0.00 2x8(10) 85.0 120 6.90 64.85 4.406 18.48 2x8(11) 84.0 126 16.68* 59.20 4.282 19.72 2x8(12) 97.0 142 9.59 51.27 4.955 24.54 2x8(13) 81.0 61 12.16 73.41 5.480 12.69 2x8(14) 85.0 95 6.19 69.61 4.451 13.70 2x8(15) 90.0 72 8.79 74.51 5.454 14.96 2x8(16) 74.0 141 13.46* 83.93 5.025 14.21 2x10(1) 80.0 206** 5.96 57.31 4.069 26.01 2x10(2) 83.0 74 6.19 83.10 5.490 16.90 2x10(3) 99.0* 102 5.42 0.00 0.000 0.00 2x10(4) 96.0 148 8.29 85.49 4.966 34.19* 2x10(5) 85.5 115 11.47 76.40 5.216 35.98* 2x10(6) 87.0 156* 4.79 82.82 4.736 35.42* 2x10(7) 86.0 184* 6.55 79.30 5.386 37.86* 2x10(8) 89.0 143 4.95 78.63 4.456 24.86 2x10(9) 82.0 155* 15.49* 66.94 4.969 23.32 2x10(10) 81.0 79 4.58 0.00 0.000 0.00 2x10(11) 81.0 91 11.13 80.10 5.246 22.82 2x10(12) 86.0 73 12.81* 83.48 5.108 13.75 2x11(1) 87.0 106 13.80* 81.74 4.668 28.58 2x11(2) 86.5 86 10.38 68.50 5.095 15.57 2x11(3) 88.0 82 7.53 82.86 5.033 19.12 2x11(4) 75.0 158* 2.98 75.00 4.592 21.17 2x11(5) 79.0 173* 4.73 86.38 4.724 33.50* (x̅ ) 84.29 107.84 8.27 63.53 4.188 19.94 SD 12.99 44.60 4.27 29.04 1.770 11.51 CV (%) 15.41 41.35 51.62 45.71 42.26 57.72 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. **** Value greater than x̅ + 3 SD. † Value lower than x̅ – 1 SD †† Value lower than x̅ – 2 SD. ‡‡ Value lower than x̅ – 4 SD. 124

-1 25

Grain yield, g plant g yield, Grain

2 6 7 8

10 11

2x6(1) 2x6(2) 2x6(3) 2x6(4) 2x6(5) 2x6(6) 2x6(7) 2x6(8) 2x6(9) 2x7(1) 2x7(2) 2x7(3) 2x7(4) 2x7(5) 2x7(6) 2x7(7) 2x7(8) 2x7(9) 2x8(1) 2x8(2) 2x8(3) 2x8(4) 2x8(5) 2x8(6) 2x8(7) 2x8(8) 2x8(9)

2x6(10) 2x6(11) 2x7(10) 2x7(11) 2x7(12) 2x8(10) 2x8(11) 2x8(12) 2x8(13) 2x8(14) 2x8(15) 2x8(16) 2x10(1) 2x10(2) 2x10(3) 2x10(4) 2x10(5) 2x10(6) 2x10(7) 2x10(8) 2x10(9) 2x11(1) 2x11(2) 2x11(3) 2x11(4) 2x11(5)

2x10(10) 2x10(11) 2x10(12)

Genotypes

Figure 5.7 Mean grain yield (GY), g plant-1 of parental genotypes and Family 2 crosses (4x H. vulgare crosses). 2x6 Plant height2x7 has been associated positively (Aynewa et al. 2015) and negatively 2x8 2x10 (Pal et al. 2010)2x11 with grain yield, but shorter plant considered positive trait for breeding 2 lodging-resistanceCol 4:cultivar. 4.59 However, among Family 2 genotypes we show no-significant Col 4: 3.4 relationship betweenCol 4: 5.2plant height and grain yield, but this could be affected by triploid Col 4: 3.71 and tetraploid hybridsCol 4: 5.57 (sterile).

Mean tiller number of Family 2 crosses was less than mean tiller number of all other families (107.84 tiller plant-1). Tiller number varied widely among genotype, ranging from 12 to 206 tiller plant-1. Among all crosses, only cross 2x10(1) exceeded the x̅ + 2 SD for tiller number (206 tillers). Crosses 2x6(2), 2x6(8), 2x7(5), 2x7(7), 2x10(6), 125

2x10(7), 2x10(9), 2x11(4), and 2x11(5) exceeded the x̅ + 1SD for tiller number. These crosses could be a good genetic source for forage yield because of their large tiller number and improving grain quality of barley because it showed a high positive association with fertility, cSW, and grain yield (Table 5.14). Shakhatreh et al. (2010) reported that tiller number and biomass yield are essential traits because barley is primarily used as animal feed (grain and forage).

Individual crosses showed a high variation of flag leaf area (CV 51.62%) with a mean of 8.27 cm2. Among all crosses, only cross 2x6(6) exceeded the x̅ + 3 SD for flag leaf area (24. 84 cm2). Crosses 2x6(7), 2x7(10), 2x8(11) 2x8(16), 2x10(9), 2x10(12), and

2x11(1) exceeded the x̅ + 1 SD for flag leaf area. Our data for this family shows positive relationship between flag leaf area and fertility, cSW, and grain yield (Table 5.14).

Previous studies reported that flag leaf area associated positively with plant height, spike length, seed number spike-1, seed weight, vegetative biomass yield and thus grain yield

(Pal et al. 2010), and it showed best positive correlation with forage yield components

(Rahal-Bouziane et al 2018).

Family 2 crosses showed a wide range of fertility 0 - 93.79% with a mean of

63.53% (Table 5.15). Among all crosses, only cross 2x6(1) exceeded the x̅ + 1 SD for fertility (93.79%). As fertility is one of the most important traits affecting seed number and thus grain yield, this specific cross shows potential for improving grain yield. There were 19 crosses with high fertility (80.1 - 91.21%), but were within 1 SD unit around the mean. Some crosses were sterile due to ploidy level (triploid or tetraploid). Crosses

2x6(9) 2x7(3) 2x8(9) 2x10(3) 2x10(10) were triploid hybrids and produced no grain

(Appendix B), while crosses 2x6(3) 2x6(5) 2x7(6) were tetraploid also producing no

126

grain. These hybrids (3x and 4x) were similar to H. bulbosum parents in growth habit, except 2x7(3) was similar to H. vulgare parent. Cross 2x6(4) had lowest fertility (15.5%; less than the x̅ – 1 SD), this cross was mixploid.

A wide range of values were observed in cSW trait which ranged from 0 to 6.036 g with a mean of 4.188 g cSW-1. Sarkar et al. (2014) also found a wide range of cSW among 220 barley genotypes ranging between 1.2 to 6.3 g and claimed this variation due to genetic variability among genotypes. Among all crosses, only cross 2x7(2) exceeded the x̅ + 1 SD with cSW (6.036 g).

Crosses of Family 2 showed widest variation for grain yield compared to all other traits of Family 2 with coefficient variation 57.72% and a mean of 19.94 g plant-1 (Table

5.15 and Figure 5.7). Crosses 2x6(1), 2x6(2), 2x7(1), 2x7(7), 2x8(6), 2x10(4), 2x10(5),

2x10(6), 2x10(7), and 2x11(5) showed highest grain yield and exceeded the x̅ + 1 SD

(32.37 - 37.86 g plant-1). High grain yield of these crosses was positively influenced by one or more of yield component traits such as tiller number, flag leaf area, fertility, or cSW. This result confirmed positive association among these traits and grain yield

(Table 5.14). Therefore, Female 2 would be good to incorporate in a barley breeding program.

Family 3, female parent 3, crosses were attempted with all five H. bulbosum males. Crosses with all male parents produced 48 embryos that were rescued (Table

5.17). All five possible top crosses were successful as determined by embryo production.

This result indicates that female parent (3) has a high general combining ability and compatibility with all H. bulbosum parents. Four (8.3%) of the progeny show grain yields

127

greater than the mean of all crosses within this family. Crosses 3x6(9), 3x6(10), 3x7(2), and 3x8(4) exceeded the mean grain yield of all progeny by + 1 SD.

The correlation coefficient among yield and its component traits of Family 3 genotypes presented in Table 5.16. Plant height showed negative association with tiller number, fertility, cSW, and grain yield. Similar result also reported by Pal et al. (2010), except fertility which associated positively with plant height. This relationship may affected by bulbosum parents, tallest stature and lowest tiller number, fertility, cSW, and thus lowest grain yield (Table 5.17). Additionally, triploid and tetraploid progeny were sterile (produced no grain) and were similar to their bulbosum parents in growth habit. In contrast, grain yield associated positively with tiller number, fertility, and cSW, similar to families 1 and 2. Tiller number showed positive association with fertility. Flag leaf area and fertility associated positively with cSW. Similar observations have been made in barley by Dadashi (2010), Pal et al. (2010), Sarkar et al. (2014), Singh et al. (2015).

Table 5.16 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 3 crosses (4x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 -0.36** -0.07 -0.39** -0.39** -0.39** TN 1.00 -0.13 0.36** 0.16 0.46*** FLA 1.00 0.26 0.38** 0.25 F 1.00 0.88*** 0.82*** cSW 1.00 0.82*** GY 1.00 ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

128

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 3 crosses for yield and related traits are presented in Table 5.17.

Table 5.17 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 3 74.0† 84* 12.09* 92.46* 6.472** 21.37** 6 124.9 40 8.94 36.21 1.816 4.59 7 111.3 45 6.33 26.59 1.382 3.40 8 119.9 46 7.23 35.95 1.679 5.20 10 123.1 65 6.17 21.39 1.470 3.71 11 97.8 40 8.74 38.18 1.619 5.57 (x̅ ) 108.50 53.33 8.25 41.80 2.406 7.31 SD 19.62 17.64 2.21 25.66 1.998 6.94 3x6(1) 90.0 226** 2.51 86.21 4.055 17.38 3x6(2) 87.0 146 5.22 71.31 5.206 27.03 3x6(3) 75.0† 28 4.65 0.00 0.000 0.00 3x6(4) 84.0 97 7.35 90.64 7.434* 28.43 3x6(5) 88.0 179* 5.22 86.13 5.163 25.50 3x6(6) 112.0** 134 8.89 0.00 0.000 0.00 3x6(7) 91.0 116 17.48* 87.79 4.864 25.78 3x6(8) 77.0 179* 2.91 48.91 3.962 16.56 3x6(9) 83.0 137 6.53 69.23 4.840 29.56* 3x6(10) 90.0 122 9.79 82.22 6.061 32.01* 3x6(11) 78.0 210* 5.39 78.72 4.142 22.67 3x6(12) 75.0† 77 4.10 76.88 6.225 16.30 3x7(1) 76.0† 141 4.84 90.32 5.928 21.20 3x7(2) 98.0* 111 8.10 93.32 5.143 32.46* 3x7(3) 79.0 204* 3.39 63.52 3.645 21.01 3x7(4) 98.0* 120 12.29 87.00 4.948 26.19 3x7(5) 81.0 215* 6.85 67.09 3.541 16.05 3x7(6) 81.0 157 12.66 77.36 4.699 28.81 3x7(7) 81.0 149 19.53* 67.34 5.079 27.55 3x7(8) 96.0 104 12.33 90.35 5.487 23.56 3x7(9) 80.0 92 18.51* 81.95 7.684* 28.03 3x7(10) 97.0* 89 8.08 81.83 5.637 24.42 3x7(11) 77.0 35 4.43 0.00 0.000 0.00 129

Table 5.17 (Continued)

3x8(1) 78.0 163 3.60 86.04 4.394 21.82 3x8(2) 77.0 90 24.53** 87.26 6.723* 20.52 3x8(3) 81.0 109 20.68** 87.68 6.180 26.20 3x8(4) 101.0* 156 7.19 77.43 5.014 31.40* 3x8(5) 91.0 122 8.49 86.73 6.187 27.97 3x8(6) 78.0 162 4.99 72.45 4.157 23.87 3x8(7) 81.0 69 10.30 69.85 6.344 20.57 3x8(8) 93.0 75 11.78 96.52 6.526* 14.79 3x8(9) 83.0 95 16.67* 78.57 6.524* 20.03 3x10(1) 80.0 188* 6.81 67.71 4.577 27.40 3x10(2) 97.0* 160 5.36 83.30 4.769 24.75 3x10(3) 111.0** 108 7.65 0.00 0.000 0.00 3x10(4) 90.0 143 15.68* 87.26 4.499 20.82 3x10(5) 88.0 168 10.28 81.37 4.428 20.66 3x10(6) 99.0* 89 8.33 88.09 5.373 23.23 3x10(7) 85.0 187* 15.16* 78.01 4.018 18.27 3x10(8) 98.0* 94 15.13* 63.30 5.323 22.30 3x10(9) 66.0† 111 9.05 93.44 4.158 15.39 3x10(10) 73.0† 200* 7.08 78.27 3.311 16.31 3x11(1) 70.0† 166 18.73* 68.55 4.391 21.00 3x11(2) 104.0* 94 10.69 84.75 5.147 28.20 3x11(3) 93.0 119 12.86 93.32 5.344 23.50 3x11(4) 91.0 97 8.55 60.90 4.767 11.59 3x11(5) 90.0 134 3.54 78.74 3.986 16.29 3x11(6) 76.0† 75 3.11 87.08 4.862 19.23 (x̅ ) 86.42 130.04 9.53 73.22 4.682 20.97 SD 10.32 46.14 5.38 24.41 1.724 8.03 CV (%) 11.94 35.48 56.50 33.34 36.84 38.30 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. † Value lower than x̅ – 1 SD.

Plant height showed lesser level of variability among Family 3 crosses (CV

11.94%). Plant height varied between 66 and 112 cm with a mean of 86.42 cm. Crosses

3x6(6) and 3x10(3) exceeded the x̅ + 2 SD with plant height (112 and 111 cm, respectively). These crosses [3x6(6) and 3x10(3)] were tetraploid and triploid hybrids,

130

respectively (Appendix B), and were sterile. Crosses 3x7(2), 3x7(4), 3x7(10), 3x8(4),

3x10(2), 3x10(6), 3x10(8), and 3x11(2) showed plant height exceeded the x̅ + 1 SD. In contrast, crosses 3x6(3), 3x6(12), 3x7(1), 3x10(9), 3x10(10), 3x11(1) and 3x11(6) had short stature (66 - 75 cm, less than the x̅ – 1 SD). Cross 3x6(3) was triploid and sterile showing similar growth habit of H. bulbosum, except plant height which was similar to its H. vulgare parent.

-1 25

Grain yield, g plant g yield, Grain

3 6 7 8

10 11

3x6(1) 3x6(2) 3x6(3) 3x6(4) 3x6(5) 3x6(6) 3x6(7) 3x6(8) 3x6(9) 3x7(1) 3x7(2) 3x7(3) 3x7(4) 3x7(5) 3x7(6) 3x7(7) 3x7(8) 3x7(9) 3x8(1) 3x8(2) 3x8(3) 3x8(4) 3x8(5) 3x8(6) 3x8(7) 3x8(8) 3x8(9)

3x6(10) 3x6(11) 3x6(12) 3x7(10) 3x7(11) 3x10(1) 3x10(2) 3x10(3) 3x10(4) 3x10(5) 3x10(6) 3x10(7) 3x10(8) 3x10(9) 3x11(1) 3x11(2) 3x11(3) 3x11(4) 3x11(5) 3x11(6)

3x10(10)

Genotypes

Figure 5.8 Mean grain yield (GY), g plant-1 of parental genotypes and Family 3 3x6crosses (4x H. vulgare crosses). 3x7 3x8 3x10 3x11 3 6 131 7 8 10 11

A wide range of values were observed for tiller number of Family 3 crosses

(Table 5.17). Tiller number ranged from 28 to 226 with a high mean (121.52 tiller plant-1). Among all the crosses, only cross 3x6(1) exceeded the x̅ + 2 SD for tiller number with (226 tillers). This cross exceeded the mean of Family 3 crosses by 73.8% and its H. vulgare parent by 169%. Crosses 3x6(5), 3x6(8), 3x6(11), 3x7(3), 3x7(5), 3x10(1),

3x10(7), and 3x10(10) had also high tiller number (179 - 215) and exceeded the x̅ + 1

SD. Tiller number as well as grain are important and have an economic value because barley is used as animal feed (Shakhatreh et al 2010). Also, tiller number associated positively with fertility and thus grain yield (Table 5.16). Consequently, this trait could be exploited in a barley breeding program to select for high forage and grain yield.

Flag leaf area showed widest variation among Family 3 crosses (CV 56.5%) compared to all other traits. Flag leaf area ranged between 2.51 and 24.53 cm2 with a mean of 9.53 cm2. Yadav et al. (2015) also found a wide range among barley genotypes for flag leaf area (12.1 - 47.1 cm2), and they concluded that variation due to genetic diversity among genotype. Crosses 3x8(2) and 3x8(3) had greatest flag leaf area (24.53 and 20.68 cm2, respectively) and exceeded the x̅ + 2 SD, while cross 3x8(1) had lowest flag leaf area (3.6 cm2 = the x̅ – 1 SD) even though all three crosses were from the same two parents. Chen et al. (2011) indication that because haploids lack homologous chromosomes, only two types of genotypes occur. For the gene “A” and its allele “a”, three genotypes would normally occur in a diploid; ¼ AA, ½ Aa, ¼ aa; however, a haploid is either A or a, when converted to a dihaploid there are only two genotypes possible ½ AA and ½ aa. It appears crosses 3x8(2) and 3x8(3) have the alleles for greater mean flag leaf area, while cross 3x8(1) has alleles for smaller mean flag leaf area.

132

Crosses 3x6(7), 3x7(7), 3x7(9), 3x8(9), 3x10(4), 3x10(7), 3x10(8), and 3x11(1) exceeded the x̅ + 1 SD for flag leaf area.

Individual crosses of Family 3 showed a wide variation in fertility percentage ranged from 0 to 96.52% with a mean of 73.22%. No cross exceeded the x̅ + 1 SD for fertility. Some crosses 3x6(4), 3x7(1), 3x7(2), 3x7(8), 3x8(8), and 3x11(3) had a high fertility (90.3 - 96.5%; within the x̅ + 1 SD). As fertility had a strong positive relationship with tiller number, cSW and grain yield (Table 5.16), the use of these traits could be exploited to select for high grain yield. Triploid hybrids [3x6(3), 3x10(3)] and tetraploid [3x6(6), 3x711] were sterile (Appendix B). These crosses had similar growth habit to H. bulbosum parents, except cross 3x7(11) was similar to H. vulgare parent.

Family 4 crosses showed a mean of 4.682 g cSW -1. Crosses 3x6(4), 3x7(9),

3x8(2), 3x8(8), and 3x8(9) showed a high cSW (7.434, 7.684, 6.723, 6.526, 6.524 g, respectively) and exceeded the x̅ + 1 SD. Seed weight and seed number reflect the potential field grain yield (Distelfeld et al. 2014).

High yield is the main aim in any barley breeding program. Family 3 crosses showed a wide range of grain yield ranged from 0 to 32.46 g with a mean of 20.97 g plant-1 (Table 5.17 and Figure 5.8). These data indicate a high genetic variation among genotypes. Crosses 3x6(9), 3x6(10), 3x7(2), and 3x8(4) had greatest grain yield (29.56,

32.01, 32.46, 31.4 g plant-1, respectively) and exceeded the x̅ + 1 SD. Grain yield affected by yield component traits, which confirmed the high positive association among grain yield and its component traits.

Family 4, female parent 4, crosses were attempted with all five H. bulbosum males. Crosses with all male parents produced 31 embryos that were rescued (Table

133

5.19). All five possible top crosses were successful as determined by embryo production.

This result indicates that female parent (4) has a high general combining ability and compatibility with all H. bulbosum parents. Three (9.7%) of the progeny show grain yields greater than the mean of all crosses within this family. Cross 4x8(2) exceed mean grain yield of all progeny by + 2 SD. Crosses 4x6(4) and 4x8(3) exceeded the mean grain yield by + 1 SD.

The correlation coefficient among yield and its component traits of Family 4 genotypes are presented in Table 5.18. Grain yield showed positive correlation with tiller number, flag leaf area, fertility, and cSW as it did in Family 2. Hundred-seed weight correlated positively with tiller number, flag leaf area, and fertility. Tiller number had positive correlation with flag leaf area and fertility. Similar finding has been observed in barley by Singh et al. (2006), Pal et al. (2010), Hossain and Akhtar (2014), Singh et al.

(2015), and Sunil et al. (2017).

Table 5.18 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 4 crosses (4x H. vulgare crosses).

PH TN FLA F cSW GY PH 1.00 -0.26 0.15 -0.04 -0.06 -0.17 TN 1.00 0.36* 0.47** 0.49** 0.64*** FLA 1.00 0.27 0.37* 0.33* F 1.00 0.90*** 0.76*** cSW 1.00 0.87*** GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level. 134

Table 5.19 summarized the mean, standard deviation, and coefficient of variation values of parental genotypes and Family 4 crosses for yield and related traits.

Table 5.19 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 4 80.0† 150* 7.82 94.24* 2.992* 21.03** 6 124.9 40 8.94* 36.21 1.816 4.59 7 111.3 45 6.33 26.59 1.382 3.40 8 119.9 46 7.23 35.95 1.679 5.20 10 123.1 65 6.17 21.39 1.470 3.71 11 97.8 40 8.74* 38.18 1.619 5.57 (x̅ ) 109.50 64.33 7.54 42.09 1.826 7.25 SD 17.55 42.97 1.18 26.37 0.591 6.80 4x6(1) 80.0 152 10.30 59.55 3.836 14.02 4x6(2) 95.0 110 5.05 0.00 0.000 0.00 4x6(3) 96.5 144 10.01 86.24 4.927 27.23 4x6(4) 88.0 168* 13.43* 75.80 5.369 34.26* 4x6(5) 87.0 148 12.43* 65.36 4.188 25.75 4x6(6) 91.0 154 5.33 69.31 4.854 22.11 4x6(7) 84.0 82 6.21 76.87 4.562 16.73 4x6(8) 50.0‡ 97 3.16 0.00 0.000 0.00 4x6(9) 72.0† 72 7.03 84.76 4.735 13.94 4x7(1) 93.0 70 6.53 87.23 4.766 14.22 4x7(2) 99.0* 147 8.20 88.76 5.156 23.99 4x7(3) 90.0 102 10.40 82.15 4.957 27.83 4x7(4) 62.0†† 73 3.68 0.00 0.000 0.00 4x7(5) 79.0 134 17.53** 68.13 4.988 21.99 4x7(6) 83.0 125 5.72 61.39 4.586 23.16 4x7(7) 79.0 152 9.20 63.48 4.971 22.25 4x7(8) 90.0 126 8.64 87.98 5.027 20.50 4x8(1) 96.0 150 11.86 89.02 4.720 18.68 4x8(2) 93.0 122 8.86 68.81 5.141 38.33** 4x8(3) 87.0 139 4.71 83.85 5.144 30.52* 4x8(4) 88.0 97 4.88 86.49 4.495 17.83 4x10(1) 89.0 221** 6.55 80.60 3.664 14.65 4x10(2) 71.0† 83 3.68 69.71 4.097 14.98 4x10(3) 90.0 90 9.53 73.12 5.086 20.38 4x10(4) 90.0 147 13.42* 68.86 4.747 23.65 4x10(5) 79.0 151 6.17 85.91 4.656 26.63 135

Table 5.19 (Continued)

4x10(6) 88.0 142 4.35 92.29 4.564 27.20 4x11(1) 90.0 97 9.27 81.16 4.910 15.97 4x11(2) 98.0* 142 4.96 68.60 5.163 28.45 4x11(3) 96.0 191* 17.23** 82.03 4.629 21.57 4x11(4) 94.0 86 6.10 92.53 4.935 19.75 (x̅ ) 86.05 126.26 8.21 70.32 4.286 20.21 SD 10.78 36.38 3.79 25.24 1.477 8.97 CV (%) 12.52 28.81 46.13 35.89 34.46 44.36 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. † Value lower than x̅ – 1 SD. ‡ Value lower than x̅ – 3 SD.

-1 25

Grain yield, g plant g yield, Grain

4 6 7 8

10 11

4x6(1) 4x6(2) 4x6(3) 4x6(4) 4x6(5) 4x6(6) 4x6(7) 4x6(8) 4x6(9) 4x7(1) 4x7(2) 4x7(3) 4x7(4) 4x7(5) 4x7(6) 4x7(7) 4x7(8) 4x8(1) 4x8(2) 4x8(3) 4x8(4)

4x10(1) 4x10(2) 4x10(3) 4x10(4) 4x10(5) 4x10(6) 4x11(1) 4x11(2) 4x11(3) 4x11(4)

Genotypes

Figure 5.9 Mean grain yield (GY), g plant-1 of parental genotypes and Family 4 crosses (4x H. vulgare crosses).

4x6 136 4x7 4x8 4x10 4x11 4 Col 8: 4.59 Col 8: 3.4 Col 8: 5.2 Col 8: 3.71 Col 8: 5.57

Plant height showed lesser level of variability among Family 4 crosses (CV

12.52%) compared to all other traits. Plan height of crosses ranged between 50 and 99 cm with a mean of 86.05 cm. Yadav et al. (2015) also reported that plant height varied among barley genotypes from 60.6 - 124.2 cm, and claimed that variation due to genetic diversity among studied genotypes. Among all Family 4 crosses, only crosses 4x7(2) and

4x11(2) exceeded the x̅ + 1 SD with plant height (99 and 98 cm, respectively). In contrast, cross 4x6(8) had short stature (50 cm, less than the x̅ – 3 SD). This cross was tetraploid and was sterile. Cross 4x7(4) showed plant height less than the x̅ – 2 SD, this cross was mixploid and was sterile. Crosses 4x6(9) and 4x10(2) had plant height less than the x̅ – 1 SD.

Tiller number revealed a wide variation among genotypes ranged from 70 to 221 tillers with a mean of 126.26 tiller plant-1. Cross 4x10(1) exceeded the x̅ + 2 SD with 221 tillers and exceeded Family 4 crosses mean by 75%. Crosses 4x6(4) and 4x11(3) exceeded the x̅ + 1 SD with 168 and 191 tillers, respectively. These crosses have cSWs similar to their H. vulgare parent indicating agronomically valuable grain size, but only in the case of 4x6(4) did grain yield exceed the mean of the family.

Flag leaf area varied among Family 4 genotypes and ranged between 3.16 and

17.53 cm2 with a mean of 8.21 cm2. Crosses 4x7(5) and 4x11(3) showed greatest flag leaf area (17.53 and 17.23 cm2, respectively) and exceeded the x̅ + 2 SD. Crosses 4x6(4),

4x6(5), and 4x10(4) exceeded the x̅ + 1 SD for flag leaf area. Flag leaf area associated positively with cSW and grain yield (Table 5.18). Additional research indicated flag leaf area showed best positive correlation with forage yield components (Rahal-Bouziane et al

137

2018). Consequently, crosses with a high flag leaf area could be used in breeding program for improving barley forage and grain yield.

Fertility showed a wide variation among Family 4 crosses (Table 5.19). Fertility ranged from 0 to 92.53% with a mean of 70.32%. No cross exceeded the x̅ + 1 SD for fertility. Crosses 4x6(2), 4x6(8), and 4x7(4) were sterile. Cross 4x6(2) was tetraploid and similar to H. bulbosum in growth habit, while crosses 4x6(8) and 4x7(4) were tetraploid and mixploid, respectively, and similar to H. vulgare growth habit (Appendix B).

Mean of cSW of Family 4 crosses was 4.286 g. No crosses exceeded the x̅ + 1

SD. This trait showed a strong positive correlation with grain yield (Table 5.18), so the use of this trait could be exploited to select for high grain yield.

Individual crosses of Family 4 showed a wide variation for grain yield (CV

44.36%) and a mean of 20.21 g plant-1 (Table 5.19 and Figure 5.9). Among all crosses, only cross 4x8(2) exceeded the x̅ + 2 SD with a high grain yield (38.33 g plant-1). Crosses

4x6(4) and 4x8(3) exceed the x̅ + 1 SD for grain yield (34.26 and 30.52 g plant-1, respectively). High yield was affected by yield component traits, that confirmed positive association between grain yield and its component traits (Table 5.18).

Family 5, female parent 5, crosses were attempted with all five H. bulbosum males. Crosses with all male parents produced 24 embryos that were rescued (Table

5.21). All five possible top crosses were successful as determined by embryo production.

This result indicates that female parent (5) has a high general combining ability and compatibility with all H. bulbosum parents. Two (8.3%) of the progeny show grain yields greater than the mean of all crosses within this family. Crosses 5x10(3) and 5x10(4) exceeded the mean grain yield of all progeny by + 1 SD.

138

The correlation coefficient among yield and its component traits of Family 5 genotypes are presented in Table 5.20. Plant height negatively associated with tiller number, and grain yield as it did in Family 3. Similar finding also reported by Pal et al.

(2010). This relationship may be affected by H. bulbosum parents, tallest stature and lowest tiller number, fertility, cSW, and thus lowest grain yield (Table 5.21). In contrast, grain yield positively associated with tiller number, fertility, and cSW. Tiller number showed positive association with fertility and cSW. Fertility positively correlated with cSW. Similar result has been reported by Pal et al. (2010), Singh et al. (2014), Singh et al. (2015), and Sunil et al. (2017) and seen among the other families of this study.

Table 5.20 Correlation coefficient analysis among different quantitative characteristics; plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), grain yield plant-1 (GY) of parental genotypes and Family 5 crosses (4x H. vulgare crosses).

PH TN FLA F cSW GY

PH 1.00 -0.51** -0.05 -0.25 -0.21 -0.40* TN 1.00 -0.19 0.58*** 0.63*** 0.78*** FLA 1.00 -0.04 0.06 0.07 F 1.00 0.91*** 0.87*** cSW 1.00 0.88*** GY 1.00 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

The mean, standard deviation, and coefficient of variation values of parental genotypes and Family 5 crosses for yield and related traits are presented in Table 5.21.

139

Table 5.21 Mean (x̅ ), standard deviation (SD), and coefficients of variation (CV%) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) for plant height (PH), tiller number plant-1 (TN), flag leaf area (FLA), percentage fertility (F), 100-seed weight (cSW), and grain yield plant-1 (GY).

Genotypes PH TN FLA F cSW GY 5 83.4† 117* 4.32 91.30* 4.169** 20.18** 6 124.9 40 8.94* 36.21 1.816 4.59 7 111.3 45 6.33 26.59 1.382 3.40 8 119.9 46 7.23 35.95 1.679 5.20 10 123.1 65 6.17 21.39 1.470 3.71 11 97.8 40 8.74* 38.18 1.619 5.57 (x̅ ) 110.07 58.83 6.96 41.60 2.023 7.11 SD 16.43 29.96 1.74 25.21 1.063 6.46 5x6 (1) 91.0 163 5.02 78.81 3.950 23.07 5x6(2) 103.0 42 11.98* 93.74 4.789 17.58 5x6(3) 95.0 148 8.70 87.79 4.562 26.46 5x6(4) 107.0* 51 8.17 66.22 5.180 13.76 5x6(5) 94.0 115 4.56 77.68 4.606 23.62 5x7(1) 90.0 176 2.27 81.06 4.808 28.74 5x7(2) 95.0 137 14.52** 72.09 4.766 22.95 5x7(3) 104.0 161 2.46 68.44 4.498 20.65 5x8(1) 82.0† 204* 3.12 83.63 4.108 27.63 5x8(2) 102.0 122 8.11 72.47 4.574 28.38 5x8(3) 94.0 216* 4.12 83.06 4.728 21.24 5x8(4) 100.0 180 4.08 72.91 5.158 24.21 5x8(5) 97.0 144 8.74 81.68 5.112 24.46 5x8(6) 107.0* 103 3.24 79.84 4.210 21.57 5x8(7) 91.0 135 6.69 76.25 4.270 28.07 5x10(1) 93.0 141 9.13 83.31 4.598 20.22 5x10(2) 103.0 167 9.59 68.83 4.605 30.67* 5x10(3) 98.0 151 3.42 94.48* 4.939 29.39* 5x10(4) 93.0 154 7.92 83.04 4.811 25.93 5x10(5) 104.0 86 2.90 82.20 4.506 20.09 5x11(1) 104.0 178 4.66 91.30 5.232 26.13 5x11(2) 80.0† 136 13.32* 75.90 3.868 20.88 5x11(3) 79.0† 170 14.20* 61.40 4.907 20.29 5x11(4) 71.0‡ 81 3.80 0.00 0.000 0.00 (x̅ ) 94.88 140.04 6.86 75.67 4.449 22.75 SD 9.41 43.49 3.83 18.17 1.017 6.34 CV (%) 9.92 31.05 55.82 24.01 22.86 27.86 * Value greater than x̅ + 1 SD. ** Value greater than x̅ + 2 SD. † Value lower than x̅ – 1 SD. 140

-1 25

Grain yield, g plant g yield, Grain

5 6 7 8

10 11

5x6(1) 5x6(2) 5x6(3) 5x6(4) 5x6(5) 5x7(1) 5x7(2) 5x7(3) 5x8(1) 5x8(2) 5x8(3) 5x8(4) 5x8(5) 5x8(6) 5x8(7)

5x10(1) 5x10(2) 5x10(4) 5x10(5) 5x11(2) 5x11(3) 5x11(4)

5x10(3) 5x11(1)

Genotypes

Figure 5.10 Mean grain yield (GY), g plant-1 of parental genotypes and Family 5 5x6crosses (4x H. vulgare crosses). 5x7 5x8 5x10 5x11 Plant height5 of crosses ranged between 71 and 107 cm with a high mean (94.88 Col 10: 4.59 cm) comparedCol to 10:all other3.4 families. Lodhi et al. (2015) also reported a wide range 79.6 - Col 10: 5.2 122.45 cm of Colplant 10: height 3.71 with a mean of 103 cm among barley genotypes. Among all Col 10: 5.57 Family 5 crosses, only crosses 5x6(4) and 5x8(6) exceeded the x̅ + 1 SD with plant height

107 cm. In contrast, cross 5x11(4) had shorter stature (71 cm, less than the x̅ – 2 SD).

This cross was tetraploid and was sterile (Appendix B). Crosses 5x8(1), 5x11(2), and

5x11(3) showed plant height less than the x̅ – 1 SD.

141

Tiller number showed a wide range among Family 5 crosses ranged from 42 to

216 with a high mean 140.04 tiller plant -1 (Table 5.21) compared to all other families.

Crosses 5x8(1) and 5x8(3) had greatest tiller number (204 and 216, respectively) and exceeded the x̅ + 1 SD. Tiller number associated positively with spike number, fertility, seed weight, and grain yield as well as forage yield (Lodhi et al. 2015; Singh et al. 2015), so the use of this trait could be exploited in a barley breeding program to select for high forage and grain yield.

Flag leaf area showed widest variability among crosses of this family (CV

55.82%) compared to all other traits. Flag leaf area ranged between 2.27 and 14.52 cm2 with a mean of 6.86 cm2. Mawati (2014) also reported a wide range of flag leaf area among 24 barley genotypes ranged between 11.75 - 18.86 cm2. Crosses 5x7(2) showed greatest flag leaf area (14.52) and exceeded the x̅ + 2 SD. Crosses 5x6(2), 5x11(2) and

5x11(3) exceeded the x̅ + 1 SD.

Individual crosses of Family 5 showed a wide range of fertility between 0 and

94.48% with a mean of 75.67%. Among all crosses, only cross 5x10(3) exceeded the x̅ +

1 SD with fertility (94.48%). As fertility had a strong positive correlation with grain yield

(Table 5.20), this cross would be desirable in a barley breeding program. In contrast, cross 5x11(4) was tetraploid hybrid and was sterile. This cross was similar to H. bulbosum in growth habit (Appendix B).

Family 5 crosses had a range of cSW between 3.868 and 5.232 g with a mean of

4.449 g cSW-1 (except the tetraploid hybrid, no grain). No crosses exceeded x̅ + 1 SD.

Haile et al. (2015) also reported a range of cSW among barley genotypes between 3.29 to

5.04 g.

142

Grain yield showed a variation among Family 5 crosses with a mean of 22.75 g plant-1 (Table 5.21 and Figure 5.10). Crosses 5x10(2) and 5x10(3) had greatest grain yield (30.67 and 29.39 g plant-1, respectively) and exceeded the x̅ + 1 SD. High grain yield of cross 5x10(3) was positively influenced by a high fertility, confirming the strong positive association between fertility and final grain yield.

Conclusion

The number of successful crosses varied for each family, so there is a difference in general combining ability and compatibility for each H. vulgare (♀) parent with the five H. bulbosum (♂) parents. For each family, all five possible top crosses were successful as determined by embryo production. These results indicate that all H. vulgare parents have a high general combining ability and some degree of compatibility with all

H. bulbosum males. However, the number of viable crosses varied among families.

Female 2 gives rise to the best family for general combining ability and compatibility (56 progeny), and 17.9% of its progeny exceeding the mean grain yield of all progeny within this family by + 1 SD.

Grain yield associated positively with tiller number, fertility, and cSW for all families, while it was associated negatively with plant height for most families. Grain yield had a positive relationship with flag leaf area for Family 2 and 4. These results support the possibility of using these associated traits to select and improve grain yield.

There were progenies that showed a high tiller number and flag leaf area, so they may have an economic value for improving grain and forage yield. Many crosses in this study showed a very high grain yield such as, crosses 1x6(3), 1x8(1), 2x7(7), 2x10(7), and

4x8(2) with 37.3, 37.7, 36.6, 37.9, and 38.3 g plant-1, respectively. They exceeded the 143

mean of all crosses within their family by 63, 64.7, 83.3, 89.9, and 89.7%, respectively.

Progenies in this study showed a high level of variation for most morphological traits, which is supported by the literature. As genetic variation is essential in breeding programs for improvement and development any crop, there is a high valuable in these progenies for use in a breeding program for barley improvement. Additionally, these progenies may have tolerance to biotic or abiotic stress conditions, so they could be used by breeders in a barley breeding program.

144

REFERENCES

Al-Tabbal, J.A., and A.H. Al-Fraihat. 2012. Genetic variation, heritability, phenotypic and genotypic correlation studies for yield and yield components in promising barley genotypes. J. Agric. Sci. 4(3):193-210.

Aynewa, Y., T. Dessalegn, W. Bayu, and B. Lakew. 2015. Estimate genetic variability of malt barley (Hordeum vulgare L.). Int. J. Adv. Multidiscip. Res. 2(3):8-14.

Babaiy, A.H., S. Aharizad, A. Mohammadi, and M. Yarnia. 2011. Survey, correlation of yield and yield components in 40 lines barley (Hordeum vulgare L.) in region Tabriz. Middle-East J. Sci. Res. 10(2):149-152.

Baumann, U., J. Juttner, X. Bian, and P. Langridge. 2000. Self-incompatibility in the grasses. Ann. Bot. 85:203-209.

Chahal, G.S., and S.S. Gosal. 2006. Principles and procedures of plant breeding: Biotechnological and conventional approaches. Alpha Sci. Int. Ltd. Harrow, UK. p. 429-456.

Chen, J.F., L. Cui, , A.A. Malik, and K.G. Mbira. 2011. In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tissue Organ Cult. 104(3):311-319.

Derbew, S., U. Elias, and M. Hussein. 2013. Genetic variability in barley (Hordeum vulgare (L.)) landrace collections from southern Ethiopia. Intern. J. Sci. Res. 2:2319-7064.

Devaux, P. 2003. The Hordeum bulbosum (L.) method. In: M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht. p.15-19.

Devaux, P., and K.J. Kasha. 2009. Overview of barley doubled haploid production. In: A. Touraev, B.P. Forster, and S.M. Jain, editors, Advances in haploid production in higher plants. Springer, Dordrecht. p. 47-63.

Distelfeld, A., R. Avni, and A.M. Fischer. 2014. Senescence, nutrient remobilization, and yield in wheat and barley. J. Exp Bot. 65:3783–3798. doi: 10.1093/jxb/ert477

Dorostkar, S., H. Pakniyat, M.A. Kordshooli, M. Aliakbari, N. Sobhanian, R. Ghorbani, and M. Eskandari. 2015. Study of relationship between grain yield and yield components using multivariate analysis in barley cultivars (Hordeum vulgare L.). Int. J. Agron. Agri. Res. 6(4):240-250.

Drikvand, R., K. Samiei, and T. Hossinpor. 2011. Path coefficient analysis in hull-less barley under rainfed condition. Aust. J. Basic Appl. Sci. 5:277-279.

145

Dyulgerova, B. 2012. Correlations between grain yield and yield related traits in barley mutant lines. Agric. Sci. Technol. 4(3):208-210.

Gomez-Pando, L., J. Jimenez-Davalos, A.B. Eguiluz-De La, E. Aguilar-Castellanos, J. Falconi-Palomino, M. Ibanez-Tremolada, and J. Lorenzo. 2009. Estimated economic benefit of double-haploid technique for Peruvian barley growers and breeders. Cereal Res. Commun. 37(2):287-293.

Haile, J., H. Legesse, and C.P. Rao. 2015. Genetic variability, character association and genetic divergence in barley (Hordeum vulgare L.) genotypes grown at Horo District, Western Ethiopia. Sci. Technol. Arts Res. J. 4(2):01-09.

Hossain, B., and M. Akhtar .2014. Growth and yield of barley (Hordeum vulgare L.) as affected by irrigation, sowing method and phosphorus level. Acad. J. Agric. Res. 2(1):30-35.

Houben, A., M. Sanei, and R. Pickering. 2011. Barley doubled-haploid production by uniparental chromosome elimination. Plant Cell Tissue Organ Cult. 104(3):321- 327.

Jauhar, P.P., S.S. Xu, and P.S. Baenziger. 2009. Haploidy in cultivated wheats: Induction and utility in basic and applied research. Crop Sci. 49(3):737-755.

Jensen, N. 1988. Breeding and selection methods; double haploid method. Plant breeding methodology. Wiley, New York. p. 15-62.

Jha, T.B., and G. Biswajit. 2005. Plant tissue culture: Basic and applied. Universities Press India. p. 96-103.

Khaiti, M. 2012. Correlation between grain yield and its components in some Syrian barley. J. Appl. Sci. Res. 8(1):247-250.

Kakeda, K., T. Ibuki, J. Suzuki, H. Tadano, Y. Kurita, Y. Hanai, and Y. Kowyama. 2008. Molecular and genetic characterization of the S locus in Hordeum bulbosum L., a wild self-incompatible species related to cultivated barley. Mol. Genet. Genomics. 280(6):509-519.

Kindiger, B. 2012. Utilizing a dihaploid-gamete selection strategy for tall fescue development. In: G. Acquaah, Editor, Principles of plant genetics and breeding. Wiley and Sons Publ. p. 312-318.

146

Kole, P.C. 2006. Variability, correlation and regression analysis in third somaclonal generation of barley. Barley Genet. Newsl. 36:44-47.

Kumar, M. and S.S. Shekhawat. 2013. Genetic variability in barley (Hordeum vulgare L.). J. Plant Breed. 4(4):1309-1312.

Liu, L., G. Sun, X. Ren, C. Li, and D. Sun. 2015. Identification of QTL underlying physiological and morphological traits of flag leaf in barley. BMC Genet. 16(1):29. doi: 10.1186/s12863-015-0187-y

Lodhi, R., L.C. Prasad, A.M.S. Bornare, and R. Prasad. 2015. Study of genetic parameters for yield and yield contributing trait of elite genotypes of barley (Hordeum vulgare L.). Indian Res. J. Genet. Biotechnol. 7(1):17-21.

Mahmood, A., K. Alam, A. Salam, and S. Iqbal. 1991. Effect of flag leaf removal on grain yield, its components and quality of hexaploid wheat. Cereal Res. Commun. 19:305-310.

Mawati, R.K. 2014. Studies on character association and selection indices for grain yield and its components in F4 generation of barley (Hordeum vulgare l.). MSc. Thes., Jawaharlal Nehru Krishi Vishwa Vidyalaya, Jabalpur, MP.

Mehripour, H., A. Rashidi, M. Zahravi, and A. Ebrahimi. 2014. Study of relationship between related yield traits using correlation and regression in wild barley (Hordeum murynum). Int. J. Adv. Biol. Biom. Res. 2(6):2120-2126.

Niroula, R.K. and H.P. Bimb. 2009. Overview of wheat x maize system of crosses for dihaploid induction in wheat. World Appl. Sci. J. 7:1037-1045.

Okuno, A., K. Hirano, K. Asano, W. Takase, R. Masuda, Y. Morinaka, and M. Matsuoka. 2014. New approach to increasing rice lodging resistance and biomass yield through the use of high gibberellin producing varieties. PLoS One 9(2), e86870. doi: 10.1371/journal.pone.0086870

Pal, S., T.Singh, and B. Ramesh. 2010. Estimation of genetic parameters in barley (Hordeum vulgare L.). J. Crop Imp. 37(1):52-56.

Pickering, R.A. 1991. The production of fertile triploid hybrids from crosses between Hordeum vulgare L.(2n= 4x= 28) and H. bulbosum L.(2n= 2x= 14). Hereditas, 114(3):227-236.

Pickering, R., and P.A. Johnston. 2005. Recent progress in barley improvement using wild species of Hordeum. Cytogenet. Genome Res. 109(1-3):344-349.

147

Rahal-Bouziane, H., F. Bradai, F. Alane, and S. Yahiaoui. 2018. Influence of flag leaf traits on forage yield components and their ash contents in barley landraces (Hordeum vulgare L.) of south Algeria. J. Agron. 17(1):28-36. doi:10.3923/ja.2018.28.36

Ruzdik, N.M., D. Valcheva, D. Valchev, L. Mihajlov, I. Karov, and V. Ilieva. 2015. Correlation between grain yield and yield components in winter barley varieties. J. Agric. Sci. Tech. 7(1):40-44.

Sarkar, B., A. Sarkar, R.C. Sharma, R.P.S. Verma, and I. Sharma. 2014. Genetic diversity in barley (Hordeum vulgare) for traits associated with feed and forage purposes. Indian J. Agric. Sci. 84(5):650-5.

SAS Institute. 2011. SAS guide to macro processing. Vol. 11. SAS Inst., Cary, NC.

Shahraki, H., and B.A. Fakheri. 2016. QTLs mapping of morpho-physiological traits of flag leaf in Steptoe× Morex doubled haploid lines of barley in normal and salinity stress conditions. Int. J. Farm. and Alli. Sci. 5(5):356-362.

Shakhatreh, Y., N. Haddad, M. Alrababah, S. Grando, and S. Ceccarelli. 2010. Phenotypic diversity in wild barley (Hordeum vulgare L. ssp. spontaneum (C. Koch) Thell.) accessions collected in Jordan. Genet. Resour. Crop Ev. 57:131-46.

Shi, J., R. Li, D. Qiu, C. Jiang, Y. Long, C. Morgan, I. Bancroft, J. Zhao, and J. Meng. 2009. Unraveling the complex trait of crop yield with quantitative trait loci mapping in Brassica napus. Genet. 182(3):851-861.

Singh, H.L., S.K. Singh, P. Singh, and B.C. Singh. 2006. Genetic variability and characters association in barley (Hordeum vulgare L.). Int. J. Plant Sci. 1:256- 258.

Singh, J., L.C. Prasad, A.H. Madakemohekar, and S.S. Bornare. 2014. Genetic variability and character association in diverse genotypes of barley (Hordeum vulgare L.). Bioscan Int. Q. J. Life Sci. 9(2):759-761.

Singh, S., A.H. Madakemohekar, L.C. Prasad and R. Prasad. 2015. Genetic variability and correlation analysis of yield and its contributing traits in barley (Hordeum vulgare L.) for drought tolerance. Indian Res. J. Genet. and Biotech. 7(1):103- 108.

Sunil, K.D., and M. Khan. 2017. Evaluation of character association in barley (Hordeum vulgare L.) genotypes for yield and yield related traits. Curr. J. Appl. Sci. Technol. 24(1):1-9.

148

Tahar, Z., A. Assefa, S. Alamerew, and W. Gebreselassie. 2015. Genetic variability and character association in some Ethiopian food barley (Hordeum vulgare L.) genotypes. Middle-East J. Sci. Res., 23 (12):2817-2827. doi:10.5829/idosi.mejsr.2015.23.12.96188

Thomas, W.T.B., B.P. Forster, and B. Gertsson. 2003. Doubled haploids in breeding. In: M. Maluszynski, K.J. Kasha, B.P. Forster, I. Szarejko, editors, Doubled haploid production in crop plants. A manual. Kluwer, Dordrecht. p. 337-349.

Walker, C.K., J. Panozzo, R. Ford, and D. Moody. 2009. Measuring grain plumpness in barley using image analysis. In proceedings of the 14th Australian barley technical symposium, Sunshine Coast.

Wedzony, M., B.P. Forster, I. Zur, E. Golemiec, M. Szechynska-Hebda, E. Dubas, and G. Gotebiowska. 2009. Progress in doubled haploid technology in higher plants. In: A. Touraev, B.P. Forster, and S.M. Jain, editors, Advances in haploid production in higher plants. Springer, Dordrecht. p. 1-33.

Yadav, S.K., A.K., Singh, P. Pandey, and S. Singh. 2015. Genetic variability and direct selection criterion for seed yield in segregating generations of barley (Hordeum vulgare L.). Am. J. Plant Sci. 6(09):1543.

149

CHAPTER VI

EVALUATING SALINITY TOLERANCE IN PROGENY OF DOMESTIC

(HORDEUM VULGARE) AND WILD BARLEY (H. BULBOSUM) AT

GERMINATION STAGE

Introduction

Domestic barley (Hordeum vulgare) is one of the most important crops used as feed, food, and chemical industry. Production and cultivation of barley and other cereal crops are affected by several environment stress conditions such as high wind, extreme temperatures, salinity, drought, and flood (Shahbaz and Ashraf 2013). Salinity in soil or water is the most important challenge facing crop productivity around the world. It causes a major reduction in cultivated land area, productivity, and crop quality (Zhou et al.

2012). More than 20% of the world’s total cultivated land and 33% of irrigated land are impacted by salt (Machado and Serralheiro 2017). Pimentel et al. (2004) estimated that

10 million ha of the world agricultural land destroyed by salt each year. Climate changes, excessive use of groundwater, poor water and soil management practices exasperate the problem. Bartels and Sunkar (2005) reported that 50% of the world’s agricultural land would be affected by salinity in 2050.

Consequently, producing plant varieties with high water use efficiency and the ability to tolerate salinity is a primary objective of plant breeders. In breeding program for salt tolerance, there is a need to screen genotypes for salt tolerance at the germination

150

and seedling growth stages to identify genotypes with salt stress tolerance (Mano and

Takeda 1998). Additional studies have used various phenotypic parameters for screening salt tolerance such as germination percentage, radicle length, coleoptile length, relative water content, leaf chlorosis, fresh and dry weight of roots and shoots (Zhou et al. 2012

Adjel et al. 2013; Abu-El-Lail et al. 2014; El Goumi et al. 2014; Sbei et al. 2014; Abdi et al. 2016; Chikha et al. 2016; Angessa et al. 2017). Seed germination is the first stage of plant growth and takes place near the surface soil where a high level of salt accumulates, especially soil with a high water table, so screening for salinity tolerance at germination is the most important stage in breeding programs (Matsumoto 1989). In order to germinate, a seed must have greater osmotic tension than its surroundings in order to attract water into the embryo and accelerate metabolic processes. This requires the seed to be innately high in osmoticums, or to be able to increase metabolism with limited water. El Madidi et al. (2014) reported that germination percentage had a greater reduction than the other parameters of early plant growth under salt stress. Consequently, germination is the most affected stage of developement by salt stress.

Materials and Methods

Germination tolerance to saline conditions was conducted in growth chamber

(Percival Scientific, Mod. GR-37L, Boone, IA), on March 2018 to assess salt tolerance of

249 genotypes (64 genotypes from season 2015/2016 (2x H. vulgare crosses), 174 genotypes from season 2016/2017 (4x H. vulgare crosses), and the 11 parents (Table

6.1). Twelve sets of 20 seed of each genotype were placed in nylon mesh bags and surface-sterilized (1% NaOCl) solution for 10 min, followed by three rinses with distilled water. Seed were placed on 2 layers of WhatmanTM filter paper (GE Healthcare, 85cm, 151

Buckinghamshire, UK) in plastic Petri dishes (Fisher Scientific, 90 x 15 mm, Suwanee,

GA). There were three replications for each genotype. Experimental design was a completely randomized design (CRD). Seed were irrigated with 8 ml each NaCl treatment- solutions of 0, 100, 200, and 300 mM. Germination tests were carried out at

20° C in darkness for 10 days according to methods described by Angessa et al. (2017).

Germination counts (seed with roots longer than 2 mm) were made on day 2, 4, 6, 8 and

10 after incubation.

Table 6.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study

The term, “2x H. vulgare crosses” is used to identify crosses between diploid (2x)

H. vulgare lines serving as females and diploid or tetraploid (2x, 4x) H. bulbosum lines serving as males. The term, “4x H. vulgare crosses” is used to identify crosses between tetraploid (4x) H. vulgare lines as females and tetraploid (4x) H. bulbosum as males.

Family name depends on H. vulgare female number given in table 6.1. For example,

“Family 1” means H. vulgare (♀) parent 1 crossed with all other H. bulbosum lines (♂).

152

The final germination percentage (FG%) was calculated from the formula described by Scott et al. (1984):

FG% = Final number of germinated seed (6.1) Total number of seed planted

Germination index (GI) was calculated by using the formula of Arnold et al.

(1991):

GI= (10 × n2) + (8 × n4) + (6 × n6) + (4 × n8) + (2 × n10) (6.2)

Where:

n2, n4, n6, n8, and n10 are the number of germinated seed on the 2nd, 4th, 6th, 8th, and

10th day, respectively.

Numbers 10, 8, 6, 4, 2 are weights given to the number of germinated seed on the

second, fourth, sixth, eighth and tenth day, respectively.

Corrected germination percentage (CG%) was used to identify physiological limitations of salt concentration on seed germination (Abdi et al. 2016). It was calculated by using the formula of Smith and Dobrenz (1987):

CG% = (Nx/No) × 100 (6.3)

Where,

Nx is the germination percentage in x mM of NaCl treatment

No is germination percentage in the control.

The data were statistically analyzed as a CRD using PROC MEANS and PROC

GLM in SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Significant mean separation among genotypes was determined using Fisher’s least significant difference (LSD) at α =

153

0.05 and 0.01. Graphs were assembled using SigmaPlot version 13 (Systat Software Inc.,

San Jose, CA).

Results and Discussion

Genotypes of diploid H. vulgare crosses

Analysis of variance of barley diploid crosses (2x H. vulgare crosses) of all families (Table 6.2) indicated highly significant (P ≤ 0.001) effects for the genotype x salinity interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl. The significant effects among the genotypes and genotype x salinity interaction under four salinity levels indicate that there was high genetic variation among the screened barley genotypes. Previous studies have also reported highly significant differences among barley genotypes and interactions with salinity for germination (Bagci et al. 2003; El

Madidi et al. 2004; Kook et al. 2009; Yousofinia et al. 2012; El Goumi et al. 2014; Abdi et al. 2016; Angessa et al. 2017).

The mean final germination percentage (FG%), corrected germination percentage

(CG%), and germination index (GI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl are presented in Table 6.3. Data for FG%, CG%, and GI of genotypes at 100 and 200 mM NaCl are presented in Appendix D. Mean final germination percentage ranged from 0 to 15.56% with a mean of 4.32% at 300 mM NaCl.

Among all genotypes, only cross 1x9(6) had greater FG% (15.56%) compared to family mean (P ≤ 0.01). Also, parent 9 and cross 1x9(3) showed FG% (13.33 and 14.07%, respectively) greater than family mean (P ≤ 0.05). These two crosses show the affects of 154

their bulbosum parent (9), which confirms this parent (9) contributed substantial tolerance to salinity, but only for these two crosses.

Table 6.2 Analysis of variance of barley genotypes (2x H. vulgare crosses), salinity, and their interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl.

Family Source of variance FG% CG% GI Genotypes (G) *** *** *** Family 1 Salinity (S) *** *** *** G x S *** *** *** Genotypes (G) *** *** *** Family 2 Salinity (S) *** *** *** G x S *** *** *** Genotypes (G) *** *** *** Family 3 Salinity (S) *** *** *** G x S *** *** *** Genotypes (G) *** *** *** Family 4 Salinity (S) *** *** *** G x S *** *** *** Genotypes (G) *** *** *** Family 5 Salinity (S) *** *** *** G x S *** *** *** *** Significant at the 0.001 probability level.

155

Table 6.3 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 1 53.33 1.67 3.13 0.67 6 81.32** 1.67 2.05 0.67 9 91.67** 13.33* 14.55 12.00* 10 76.67* 5.00 6.52 6.00 1x6(1) 70.61* 0.00 0.00 0.00 1x6(2) 22.73 0.00 0.00 0.00 1x6(3) 86.78** 11.11 12.80 4.44 1x9(1) 45.61 1.67 3.65 0.67 1x9(2) 44.72 1.96 4.38 1.57 1x9(3) 60.95 14.07* 23.09* 10.74* 1x9(6) 79.82** 15.56** 19.49 12.72* 1x9(9) 52.20 2.38 4.56 0.95 1x9(10) 10.18 0.00 0.00 0.00 1x9(12) 20.51 5.13 25.00* 4.10 1x10(1) 12.50 0.00 0.00 0.00 1x10(2) 0.00 0.00 0.00 0.00 1x10(3) 40.28 0.00 0.00 0.00 1x10(4) 60.42 4.17 6.90 2.50 Mean (x̅ ) 50.57 4.32 7.01 3.17 Range 0.00 - 91.67 0.00 - 15.56 0.00 - 25.00 0.00 - 12.72 LSD (0.05) 19.743 8.3828 13.995 7.26 LSD (0.01) 26.474 11.231 18.985 9.735 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Generally, regardless of the genotype, mean final germination percentage decreased as the level of NaCl increased (Table 6.3 and Appendix D). Mean corrected germination (CG%) was used to identify physiological limitations of salt concentration on seed germination (Abdi et al. 2016). Mean corrected germination was 7.01%, which indicated salt treatment 300 mM NaCl reduced germination in this family by 93%

156

compared to control (0 mM). Crosses 1x9(3) and 1x9(12) had CG% (23.09 and 25%, respectively) greater than family mean (P ≤ 0.05). These crosses were less affected by high level of salinity than other genotypes. However, cross 1x9(12) showed low germination even under control conditions due to what we speculate to be seed dormancy

[i.e., it germinated later after the seed exposure to low temperature (3° C) for a week

(chilling)]. Adjel et al. (2013) reported low seed germination of some barley genotypes under their control conditions and related this to poor seed quality and primary dormancy.

Germination index (GI) used to describe the germination percentage:speed relationship (Kader 2005). It ranged between 0 and 12.72 with a mean of 3.17. Similar to the final germination, germination index was reduced with increasing salinity for all the studied genotypes (Appendix D). These results are confirmed by Begum et al. (2010).

They reported that high salt concentration induced delay germination and reduced final germination due to low water uptake and high accumulation of Na+ and Cl-. Among genotypes, only parent 9 and crosses 1x9(3) and 1x9(6) showed GI (12, 10.74, and 12.72, respectively) greater than family mean (P ≤ 0.05). Thus confirming those genotypes had greater percentage and speed of germination. These genotypes would have an advantage for improving barley salt tolerance at the germination stage, but it is unknown what a final grain yield would be. Previous studies have also reported significant variation in salt tolerance among barley genotypes at germination stage (Tajbakhsh et al. 2006; Kook et al. 2009). Seed germination under salinity is reduced due to osmotic stress, high levels of

NaCl reduce the bioavailability of water (caused by Na+) resulting in what is effectively a water deficit (Mano et al. 1996; Parida and Das 2005). Additionally, Na+ show toxic effects on the germinating embryo as it displaces essential K+ (Mazher et al. 2007),

157

interfering with enzyme functions in the seed, which decreased activity of polyphenol- oxidases and amylases (Khemiri et al. 2004). These findings were confirmed by Othman et al. (2006); they found that increasing salt concentration significantly reduced K concentration in barley seed after 24 hr of imbibition.

Table 6.4 summarized the mean of measured parameters (FG%, CG%, and GI) of

Family 2 genotypes at 0 and 300 mM NaCl. Mean final germination percentage among genotypes ranged from 0 to 31.98% with a mean of 7.54% under 300 mM NaCl. Among all genotypes, only cross 2x9(4) had greater FG% (31.98%) compared to family mean (P

≤ 0.01). This cross also showed high germination at 0, 100, 200 mM NaCl (96.1, 95.35, and 71.1%, respectively) (Appendix D). Also, parent 8 showed FG% (18.33%) greater than family mean (P ≤ 0.05). Cross 2x9(4) had greatest FG% while other crosses showed lowest FG% or failed to germinate under 300 mM NaCl even though they were from the same two parents (2 and 9). Chen et al. (2011) indicated that because haploids lack homologous chromosomes, dihaploids generated from haploids have only two classes in the segregating array. For the gene “A” and its allele “a”, three genotypes would normally occur in a diploid; ¼ AA, ½ Aa, ¼ aa; however, a haploid is either A or a, when converted to a dihaploid there are only two genotypes possible ½ AA and ½ aa. It appears cross 2x9(4) has the alleles for salt tolerance, while other crosses do not have.

158

Table 6.4 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 2 58.33 11.67 20.00 10.67 7 93.33** 1.67 1.79 1.33 8 83.33 18.33* 22.00 9.33 9 91.67* 13.33 14.55 12.00 11 83.91 11.84 14.11 4.74 2x7(2) 48.33 0.00 0.00 0.00 2x8(2) 100.00** 0.00 0.00 0.00 2x9(1) 58.33 5.09 8.72 3.40 2x9(2) 37.19 0.00 0.00 0.00 2x9(3) 45.79 0.00 0.00 0.00 2x9(4) 96.08** 31.98** 33.28** 29.41** 2x11(1) 50.89 4.17 8.19 2.50 2x11(3) 56.25 0.00 0.00 0.00 Mean (x̅ ) 69.50 7.54 9.43 5.65 Range 37.19 - 100 0.00 - 31.98 0.00 - 33.28 0.00 - 29.41 LSD (0.05) 16.68 10.058 12.993 9.5223 LSD (0.01) 22.548 13.596 17.674 12.873 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean corrected germination (CG%) was 9.43%, which indicated salt treatment under 300 mM NaCl reduced germination in this family by 90.57% compared to the control (0 mM). Only cross 2x9(4) had a higher CG% (33.28%) than the family mean (P

≤ 0.01). This cross could be important in breeding program for salt tolerance because it showed less germination reduced than other genotypes (0.76, 26.01, and 66.72% under

100, 200, and 300 mM, respectively). In contrast, cross 2x8(2) had 100% germination under control but failed to germinate at all under 300 mM NaCl. Adjel et al. (2013) reported that germination percentage of twelve barley varieties decreased linearly (from 159

86 to 50%) as salinity increased from 0 to 150 mM NaCl. High salinity reduced and delayed the germination through low water uptake (osmotic pressure) and high accumulation of Na+ and Cl- (toxicity) (Begum et al., 2010).

Mean germination index (GI) ranged between 0 and 29.41 with a mean of 5.65 under 300 mM NaCl, while mean GI of this family was 117.36 under 0 mM NaCl. Thus, germination percentage and speed germination decreased sharply as the level of salinity increased. Only cross 2x9(4) had greater GI (29.41) compared to the family mean (P ≤

0.01). This confirmed this cross had greater percentage and speed of germination than family genotypes. This cross confirms the effect of its H. bulbosum parent (9) similar to

Family 1.

The means of measured parameters (FG%, CG%, and GI) of Family 3 genotypes at 0 and 300 mM NaCl are presented in Table 6.5. Mean final germination percentage ranged from 0 to 14.81%, with a mean of 3.48% under 300 mM NaCl. Among all crosses, only cross 3x9(1) germinated under high level of salinity with 14.81%. This result indicates that crosses of this family were affected by the dormancy of their H. vulgare parent (3), which also failed to germinate even under control condition, but also the positive affect of parent 9 in the 3x9(1) cross itself. Final germination was greatly reduced under highest level of salt (300 mM NaCl). This result confirmed by the mean corrected germination (CG%) of this family (4.6%) under 300 mM NaCl, which indicated germination in this family reduced by (95.4%) compared to control (0 mM). Only cross

3x9(1) had greater CG% (22.86%) compared to family mean (P ≤ 0.05), which showed less germination reduced than other genotypes (77.14%).

160

Table 6.5 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 3 0.00 0.00 0.00 0.00 6 81.32** 1.67 2.05 0.67 9 91.67** 13.33 14.55 12.00* 10 76.67** 5.00 6.52 6.00 3x6(1) 42.13 0.00 0.00 0.00 3x9(1) 64.81* 14.81 22.86* 5.93 3x9(2) 12.29 0.00 0.00 0.00 3x9(3) 6.67 0.00 0.00 0.00 3x10(3) 38.61 0.00 0.00 0.00 3x10(5) 39.32 0.00 0.00 0.00 Mean (x̅ ) 45.35 3.48 4.60 2.46 Range 0.00 - 91.67 0.00 - 14.81 0.00 - 22.86 0.00 - 12.00 LSD (0.05) 18.984 11.796 13.803 9.5044 LSD (0.01) 25.895 16.09 18.976 12.964 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Othman et al. (2006) found that germination percentage greatly reduced under

300 mM NaCl; germination of some genotypes decreased from 84.5% (control) to 3.7%

(under 300 mM). Germination index (GI) ranged between 0 and 12 with a mean of 2.46.

Only parent 9 showed greater GI (12) compared to family mean (P ≤ 0.05). These data indicated that H. vulgare is more affected by salinity than H. bulbosum (Tavili and Biniaz

2009).

Table 6.6 summarized the mean of measured parameters (FG%, CG%, and GI) of

Family 4 genotypes at 0 and 300 mM NaCl. Mean final germination ranged from 0 to

18.33% with a mean of 2.41% under 300 mM NaCl. All crosses of this family failed to

161

germinate under high level of salinity (300 mM NaCl) as well as under 200 mM NaCl

(Appendix D). This result indicates this family more susceptible to salinity. Among parental genotypes, only parent 8 had greater FG% (18.33%) compared to family mean

(P ≤ 0.01).

Table 6.6 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 4 5.00 1.67 33.33** 1.33 6 81.32** 1.67 2.05 0.67 8 83.33** 18.33** 22.00** 9.33** 4x6(2) 50.00** 0.00 0.00 0.00 4x8(1) 0.00 0.00 0.00 0.00 4x8(2) 0.00 0.00 0.00 0.00 4x8(3) 1.85 0.00 0.00 0.00 4x8(4) 10.42 0.00 0.00 0.00 4x8(5) 11.86 0.00 0.00 0.00 Mean (x̅ ) 27.09 2.41 6.38 1.26 Range 0.00 - 83.33 0.00 - 18.33 0.00 - 33.33 0.00 - 9.33 LSD (0.05) 12.032 2.859 8.767 1.9808 LSD (0.01) 16.485 3.9171 12.937 2.7138 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean corrected germination (CG%) of this family was 6.38%, which indicated that salt treatment under 300 mM NaCl reduced germination by 93.62% compared to control. Parents 4 and 8 had CG% (33.33 and 22%, respectively) greater than family mean (P ≤ 0.01). However, parent 4 showed low germination even under control condition. Previous studies reported low seed germination of some barley genotypes

162

under control condition and related this to poor seed quality and primary dormancy

(Adjel et al. 2013; Nakamura et al. 2017). Mean germination index (GI) ranged between

0 and 9.33 with very low mean 1.26. Only parent 8 had GI (9.33) greater than family mean (P ≤ 0.01). Yousofinia et al. (2012) reported that salinity decreased and delayed germination of barley cultivars under a low level of salinity (50 and 100 mM NaCl).

Houle et al. concluded the inhibition of seed germination by salinity is due to osmotic stress.

The mean of measured parameters (FG%, CG%, and GI) of Family 5 genotypes at

0 and 300 mM NaCl are presented in Table 6.7. Mean final germination percentage

(FG%) of Family 5 genotypes ranged from 0 to 18.33% with very low mean 1.81% under

300 mM NaCl. Among all genotypes, only H. bulbosum parents 8, 9, and 11 showed greater FG% (18.33, 13.33, and 11.84%, respectively) compared to family mean (P ≤

0.01). In contrast, all crosses of H. vulgare parent 5 with H. bulbosum parents 6, 7, 8, 9, and 10 failed to germinate under 300 mM NaCl, which affected by their H. vulgare parent. Among all crosses of 5x11, only cross 5x11(9) had FG% (7.14%) greater than family mean (P ≤ 0.05). These data confirm H. vulgare is more sensitive to salinity compared to H. bulbosum (Tavili and Biniaz 2009). There were some crosses that had great FG% under control and 100 mM NaCl. For example, cross 5x8(4) showed FG%

(100%) under control and 100 mM, which was not affected by low salinity (Appendix D).

However, this cross failed to germinate at 300 mM NaCl. This cross would have an advantage for use in soils with a low level of salinity.

163

Table 6.7 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 5 19.07 0.00 0.00 0.00 6 81.32** 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33** 18.33** 22.00** 9.33** 9 91.67** 13.33** 14.55** 12.00** 10 76.67* 5.00 6.52 6.00* 11 83.91** 11.84** 14.11** 4.74 5x6(2) 36.37 0.00 0.00 0.00 5x7(1) 19.31 0.00 0.00 0.00 5x7(2) 13.25 0.00 0.00 0.00 5x7(3) 53.89 0.00 0.00 0.00 5x8(1) 31.67 0.00 0.00 0.00 5x8(3) 57.87 0.00 0.00 0.00 5x8(4) 100.00** 0.00 0.00 0.00 5x8(5) 60.10 0.00 0.00 0.00 5x8(7-1) 35.71 0.00 0.00 0.00 5x8(8) 24.56 0.00 0.00 0.00 5x8(10-1) 34.56 0.00 0.00 0.00 5x8(10-2) 70.37 0.00 0.00 0.00 5x8(12) 33.48 0.00 0.00 0.00 5x8(13) 18.73 0.00 0.00 0.00 5x9(1) 33.54 0.00 0.00 0.00 5x9(2) 30.79 0.00 0.00 0.00 5x10(1) 27.47 0.00 0.00 0.00 5x11(1) 84.39** 0.00 0.00 0.00 5x11(2) 51.11 0.00 0.00 0.00 5x11(3) 54.85 0.00 0.00 0.00 5x11(4) 57.14 4.17 7.29 3.33 5x11(5) 43.17 0.00 0.00 0.00 5x11(6-1) 75.19* 3.70 4.93 1.48 5x11(6-2) 55.98 0.00 0.00 0.00 5x11(7) 30.56 0.00 0.00 0.00 5x11(8) 41.37 0.00 0.00 0.00 5x11(9) 75.00* 7.14* 9.52* 1.90 164

Table 6.7 (Continued)

5x11(10) 13.98 0.00 0.00 0.00 5x11(11) 23.15 0.00 0.00 0.00 5x11(12) 27.27 0.00 0.00 0.00 Mean (x̅ ) 49.84 1.81 2.24 1.10 Range 13.25 - 100 0.00 - 18.33 0.00 - 22.00 0.00 - 12.00 LSD (0.05) 20.578 5.1944 5.7168 4.8438 LSD (0.01) 27.305 6.8924 7.6543 6.4272 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean corrected germination (CG%) of this family was very low (2.24%) which indicated salt treatment under 300 mM NaCl reduced germination in this family by

97.76% compared to control. Parents 8, 9 and 11 had CG% (22, 14.55, and 14.11%, respectively) greater than family mean (P ≤ 0.01). Among all crosses, only cross 5x11(9) showed greater CG% (9.52%) compared to family mean (P ≤ 0.05). Mean germination index (GI) ranged between 0 and 12 with very low mean 1.1. Only parents 8, 9, and 10 had GI (9.33, 12, 6, respectively) greater than family mean. Within this family of crosses parent 9, which has been beneficial with other females, except female 4, did not cross well with female 5.

Generally, mean final germination percentage of all families were sharply decreased as the level of NaCl increased (Figure 6.1). Family 2 showed higher FG% under all levels of NaCl compared to all other families. In contrast, Family 4 had lower

FG% under 0, 100, and 200 mM NaCl compared to all other families. Under high level of salinity (300 mM), Family 5 showed lower FG% compared to all other families. Previous studies reported that salinity decreased and delayed germination of barley genotypes.

165

This decrease was expressed with the increase of NaCl concentration (Bagci et al. 2003;

El Goumi et al. 2014; Abdi et al. 2016; Askari et al. 2016; Angessa et al. 2017).

80 Family 1 70 Family 2 Family 3 Family 4 60 Family5

%FG 30

0 0 mM 100 mM 200 mM 300 mM NaCl solution

Figure 6.1 Mean final germination percentage (FG%) of barley diploid crosses (2x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl.

Genotypes of tetraploid H. vulgare crosses

Analysis of variance of barley tetraploid crosses (4x H. vulgare crosses) of all families (Table 6.8) indicated highly significant (P ≤ 0.001) effects for the genotype x salinity interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl. The significant effects among the genotypes and genotype x salinity interaction under four 166

salinity levels indicate that there was high genetic variation among the screened barley genotypes. Previous studies have also reported highly significant differences among barley genotypes and interactions with salinity for germination (Mano and Takeda 1998;

Naseer et al. 2001; Othman et al. 2006; Tavili and Biniaz 2009; Adjel et al. 2013; Abdi et al. 2016; Askari et al. 2016).

Table 6.8 Analysis of variance of barley genotypes (4x H. vulgare crosses), salinity, and their interaction for final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) at 0, 100, 200, and 300 mM NaCl.

The mean final germination percentage (FG%), corrected germination percentage

(CG%), and germination index (GI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl are presented in Table 6.9. Data for FG%, CG%, and GI of genotypes at 100 and 200 mM NaCl are presented in Appendix D.

167

Table 6.9 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 1 53.33 1.67 3.13 0.67 6 81.32** 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33** 18.33** 22.00 9.33 10 76.67** 5.00 6.52 6.00 11 83.91** 11.84 14.11 4.74 1x6(1) 56.67* 0.00 0.00 0.00 1x6(2) 5.38 1.75 32.60* 2.11 1x6(3) 75.24** 22.02** 29.26* 22.39** 1x6(4) 2.78 0.00 0.00 0.00 1x6(5) 25.00 0.00 0.00 0.00 1x7(1) 22.04 0.00 0.00 0.00 1x7(2) 1.67 0.00 0.00 0.00 1x7(3) 35.40 6.67 18.83 5.33 1x7(4) 24.97 0.00 0.00 0.00 1x7(6) 43.94 0.00 0.00 0.00 1x7(7) 88.07** 35.00** 39.74** 34.67** 1x7(8) 77.49** 25.26** 32.60* 30.32** 1x7(9) 15.18 0.00 0.00 0.00 1x7(10) 5.00 0.00 0.00 0.00 1x7(11) 9.18 0.00 0.00 0.00 1x8(1) 20.00 0.00 0.00 0.00 1x8(2) 21.25 0.00 0.00 0.00 1x8(3) 14.70 0.00 0.00 0.00 1x8(4) 83.33** 20.61** 24.74 23.33** 1x10(2) 48.33 3.33 6.90 5.33 1x10(3) 20.00 0.00 0.00 0.00 1x10(4) 79.82** 17.28* 21.65 18.63* 1x10(5) 55.88 18.42** 32.97** 16.04* 1x10(6) 50.00 0.00 0.00 0.00 1x10(7) 57.72* 11.93 20.67 10.95 1x10(8) 23.95 1.67 6.97 3.33 1x11(1) 15.00 0.00 0.00 0.00 1x11(2) 40.70 0.00 0.00 0.00 168

Table 6.9 (Continued)

1x11(3) 37.37 1.67 4.46 1.33 1x11(4) 12.30 0.00 0.00 0.00 1x11(5) 31.67 1.67 5.26 1.33 1x11(6) 18.33 1.67 9.11 1.33 Mean (x̅ ) 41.85 5.50 8.83 5.24 Range 1.67 - 93.33 0.00 - 35.00 0.00 - 39.74 0.00 - 34.67 LSD (0.05) 14.592 9.1498 17.833 10.427 LSD (0.01) 19.358 12.138 23.794 13.832 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean final germination percentage ranged from 0 to 35% with a mean of 5.5% under 300 mM NaCl. Parent 8 and crosses 1x6(3), 1x7(7), 1x7(8), 1x8(4), and 1x10(5) had greatest FG% (18.33, 22.02, 35.0, 25.26, 20.61, and 18.42%, respectively) compared to family mean (P ≤ 0.01). Also, cross 1x10(4) showed FG% (17.28%) greater than family mean (P ≤ 0.05). For all genotypes, mean final germination percentage was decreased as the level of NaCl increased. Mean corrected germination was 8.83%, which indicated salt treatment 300 mM NaCl reduced germination in this family by 91.17% compared to control (0 mM). Crosses 1x7(7) and 1x10(5) had CG% (39.74 and 32.97%, respectively) greater than family mean (P ≤ 0.01). Crosses 1x6(2), 1x6(3), and 1x7(8) also showed CG% greater than family mean (P ≤ 0.05). These crosses seem to be less affected by high level of salinity than other genotypes. However, cross 1x6(2) showed low germination even under control conditions, likely due to poor seed quality and primary dormancy (Adjel et al. 2013). Germination index ranged between 0 and 34.67 with a mean of 5.24 under 300 mM NaCl compared to 68.92 under control (Appendix D).

These reduction in speed and percentage of germination (GI) due to osmotic pressure

(lower water uptake) toxicity (higher accumulation of Na+ and Cl-) (Begum et al., 2010). 169

Crosses 1x6(3), 1x7(7), 1x7(8), and 1x8(4) showed greater GI (22.39, 34.67, 30.32, and

23.33, respectively) compared to family mean (P ≤ 0.01). Crosses 1x10(4) and 1x10(5) also had GI greater than family mean (P ≤ 0.05). Thus, confirming those crosses had greater percentage and speed germination compared to all other genotypes of this family.

These crosses would have an advantage for improving barley salt tolerance at germination stage, but it is unknown what the final grain yield would be. Previous studies have also reported significant variation in salt tolerance among barley genotypes at germination stage (Abdi et al. 2016; Askari et al. 2016; Angessa et al. 2017). Seed germination under salinity is reduced as well as delayed due to osmotic stress, high levels of NaCl reduce the bioavailability of water (caused by Na+) resulting in what is effectively a water deficit (Mano et al. 1996; Parida and Das 2005; Begum et al., 2010).

Additionally, Na+ show toxic effects on the germinating embryo as it displaces essential

K+ (Mazher et al. 2007) interfering with enzyme functions in the seed, which decreased activity of polyphenol-oxidases and amylases (Khemiri et al. 2004).

Table 6.10 summarized the mean of measured parameters (FG%, CG%, and GI) of Family 2 genotypes at 0 and 300 mM NaCl. Mean final germination percentage ranged from 0 to 51.41% with a mean of 9.15% under 300 mM NaCl. Crosses 2x6(1), 2x6(2),

2x7(11), 2x8(2), 2x8(10), 2x10(1), and 2x10(11) had greater FG% (44.12, 25.0, 51.41,

29.68, 26.67, 30.53, and 34.30%, respectively) compared to family mean (P ≤ 0.01).

Cross 2x8(11) showed FG% (22.7%) greater than family mean (P ≤ 0.05). There were some crosses had greatest FG% while other crosses showed lowest FG% or failed to germinate under 300 mM NaCl even though they were from the same two parents. The lack of intermediate types can be attributed to the lack of heterozygotes in dihaploids

170

(Chen et al. 2011). It appears crosses with high FG% have the alleles for salt tolerance, while other crosses do not have.

Table 6.10 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 2 58.33 11.67 20.00 10.67 6 81.32* 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33* 18.33 22.00 9.33 10 76.67 5.00 6.52 6.00 11 83.91* 11.84 14.11 4.74 2x6(1) 89.82** 44.12** 49.12** 36.67** 2x6(2) 64.47 25.00** 38.78** 25.33* 2x6(6) 77.98 1.67 2.14 1.33 2x6(7) 94.17** 3.33 3.54 4.00 2x6(8) 83.65* 0.00 0.00 0.00 2x6(10) 31.62 0.00 0.00 0.00 2x6(11) 80.56* 0.00 0.00 0.00 2x7(1) 86.67** 1.67 1.92 2.00 2x7(2) 20.51 0.00 0.00 0.00 2x7(4) 53.42 0.00 0.00 0.00 2x7(5) 79.65 1.67 2.09 1.33 2x7(7) 41.67 6.84 16.42 6.18 2x7(8) 82.45* 17.22 20.89 16.00 2x7(9) 76.67 6.67 8.70 6.00 2x7(10) 76.67 18.33 23.91 15.33 2x7(11) 91.67** 51.41** 56.09** 56.12** 2x7(12) 59.92 14.14 23.60 16.06 2x8(1) 60.19 0.00 0.00 0.00 2x8(2) 95.00** 29.68** 31.24** 15.62 2x8(3) 71.23 5.26 7.39 5.61

171

Table 6.10 (Continued)

2x8(4) 74.30 2.22 2.99 0.89 2x8(5) 78.33 18.33 23.40 20.67 2x8(6) 61.67 3.51 5.69 2.81 2x8(7) 2.38 0.00 0.00 0.00 2x8(8) 98.33** 8.10 8.23 7.08 2x8(10) 84.46** 26.67** 31.57** 22.67* 2x8(11) 89.91** 22.70* 25.25* 29.05** 2x8(12) 30.00 0.00 0.00 0.00 2x8(13) 50.80 3.33 6.56 5.33 2x8(14) 48.72 0.00 0.00 0.00 2x8(15) 64.05 2.22 3.47 0.89 2x8(16) 42.61 0.00 0.00 0.00 2x10(1) 73.33 30.53** 41.63** 46.18** 2x10(2) 62.18 0.00 0.00 0.00 2x10(4) 61.67 0.00 0.00 0.00 2x10(5) 91.83** 20.00 21.78 24.67* 2x10(6) 25.35 0.00 0.00 0.00 2x10(7) 73.33 10.73 14.63 11.47 2x10(8) 23.68 0.00 0.00 0.00 2x10(9) 37.46 5.00 13.35 6.00 2x10(11) 93.25** 34.30** 36.78** 46.70** 2x10(12) 83.81* 0.00 0.00 0.00 2x11(1) 52.54 1.67 3.17 1.33 2x11(2) 42.55 1.85 4.35 0.74 2x11(3) 22.72 1.75 7.72 1.40 2x11(4) 86.67** 13.33 15.38 16.67 2x11(5) 38.07 1.67 4.38 2.67 Mean (x̅ ) 65.83 9.15 11.75 9.20 Range 2.38 - 98.33 0.00 - 51.41 0.00 - 56.09 0.00 - 56.12 LSD (0.05) 13.933 11.916 13.19 12.795 LSD (0.01) 18.434 15.765 17.589 16.928 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Final germination was greatly reduced under highest level of salt (300 mM NaCl).

This result confirmed by the mean corrected germination (CG%) of this family (11.75%) under 300 mM NaCl, which indicated germination in this family reduced by (88.25%)

172

compared to control (0 mM). Othman et al. (2006) found that germination percentage sharply reduced under 300 mM NaCl; germination of some genotypes decreased by

95.6% under 300 mM NaCl. Crosses 2x6(1), 2x6(2), 2x7(11), 2x8(2), 2x8(10), 2x10(1), and 2x10(11) had greater CG% (49.12, 38.78, 56.09, 31.24, 31.57, 41.63, and 36.78%, respectively) compared to the family mean (P ≤ 0.01), which showed less germination reduced than other genotypes. Cross 2x8(11) showed CG% (25.25%) greater than the family mean (P ≤ 0.05). In contrast, some crosses were very susceptible even to the low level of salinity (100 mM) (Appendix D). For example, cross 2x6(7) had FG% (94.17%) under control while showed FG% (28.89%) under 100 mM NaCl (decreased by 69.32%).

Germination index (GI) ranged between 0 and 56.12 with a mean of 9.2. Crosses 2x6(1),

2x7(11), 2x8(11), 2x10(1), and 2x10(11) had greater CG% (36.67, 56.12, 29.05, 46.18, and 46.7%, respectively) compared to family mean (P ≤ 0.01). Crosses 2x6(2) and

2x8(10) showed GI greater than family mean (P ≤ 0.05). These results confirm those genotypes had greater percentage and speed of germination. Consequently, these genotypes would have an advantage for improving barley salt tolerance at germination stage, but it is unknown their tolerance at more mature growth stages especially grain fill.

The mean of measured parameters (FG%, CG%, and GI) of Family 3 genotypes at

0 and 300 mM NaCl are presented in Table 6.11.

173

Table 6.11 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 3 0.00 0.00 0.00 0.00 6 81.32** 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33** 18.33** 22.00** 9.33 10 76.67** 5.00 6.52 6.00 11 83.91** 11.84 14.11 4.74 3x6(1) 80.78** 10.00 12.38 10.67 3x6(2) 35.83 5.00 13.95 6.00 3x6(4) 16.65 0.00 0.00 0.00 3x6(5) 25.00 0.00 0.00 0.00 3x6(7) 47.54 0.00 0.00 0.00 3x6(8) 94.91** 48.33** 50.92** 60.67** 3x6(9) 20.00 1.75 8.77 2.81 3x6(10) 18.77 0.00 0.00 0.00 3x6(11) 93.07** 46.67** 50.14** 58.07** 3x6(12) 26.67 0.00 0.00 0.00 3x7(1) 2.08 0.00 0.00 0.00 3x7(2) 11.93 0.00 0.00 0.00 3x7(3) 88.99** 0.00 0.00 0.00 3x7(4) 43.33 0.00 0.00 0.00 3x7(5) 88.33** 15.00** 16.98* 14.00* 3x7(6) 65.40** 1.67 2.55 2.00 3x7(7) 80.74** 3.92 4.86 1.57 3x7(8) 13.60 0.00 0.00 0.00 3x7(9) 6.41 0.00 0.00 0.00 3x7(10) 11.75 0.00 0.00 0.00 3x8(1) 28.59 0.00 0.00 0.00 3x8(2) 0.00 0.00 0.00 0.00 3x8(3) 1.75 0.00 0.00 0.00 3x8(4) 71.67** 13.33* 18.60** 15.33** 3x8(5) 0.00 0.00 0.00 0.00 3x8(6) 11.85 0.00 0.00 0.00 3x8(7) 5.36 0.00 0.00 0.00 3x8(8) 0.00 0.00 0.00 0.00

174

Table 6.11 (Continued)

3x8(9) 0.00 0.00 0.00 0.00 3x10(1) 20.00 0.00 0.00 0.00 3x10(2) 25.00 0.00 0.00 0.00 3x10(4) 68.33** 12.02 17.59* 10.98 3x10(5) 53.73 1.75 3.27 2.81 3x10(6) 59.30** 9.29 15.67* 11.82 3x10(7) 88.20** 13.33* 15.12* 10.67 3x10(8) 15.26 0.00 0.00 0.00 3x10(9) 10.57 0.00 0.00 0.00 3x10(10) 91.48** 45.23** 49.45** 50.18** 3x11(1) 27.37 0.00 0.00 0.00 3x11(2) 61.67** 15.56** 25.23** 10.44 3x11(3) 32.93 0.00 0.00 0.00 3x11(4) 17.78 0.00 0.00 0.00 3x11(5) 60.00** 0.00 0.00 0.00 3x11(6) 5.00 0.00 0.00 0.00 Mean (x̅ ) 40.92 5.63 7.04 5.80 Range 0.00 - 94.91 0.00 - 48.33 0.00 - 50.92 0.00 - 60.67 LSD (0.05) 12.866 6.9368 7.9242 6.37 LSD (0.01) 17.029 9.1812 10.566 8.43 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean final germination percentage (FG%) of Family 3 genotypes ranged from 0 to 48.33% with a mean of 5.63% under 300 mM NaCl. Parent 8 and crosses 3x6(8),

3x6(11), 3x7(5), 3x10(10), and 3x11(2) showed greater FG% (18.33, 48.33, 46.67, 15.0,

45.23, and 15.56%, respectively) compared to the family mean (P ≤ 0.01). Crosses

3x8(4) and 3x10(7) had FG% greater than the family mean (P ≤ 0.05). Many crosses of this family failed to germinate under 300 mM NaCl. This result indicates that these crosses were affected by their vulgare parent which also failed to germinate even under control. These data confirm H. vulgare species more affected by salinity than H. bulbosum (Tavili and Biniaz 2009). Final germination was greatly reduced under highest

175

level of salt (300 mM NaCl). This result confirmed by the mean corrected germination

(CG%) of this family (7.04%) under 300 mM NaCl, which indicated germination in this family reduced by (92.96%) compared to control (0 mM). Parent 8 and crosses 3x6(8),

3x6(11), 3x8(4), 3x10(10), and 3x11(2) had greater CG% (22.0, 50.92, 50.14, 18.6,

49.45, 25.23%, respectively) compared to the family mean (P ≤ 0.01). Crosses 3x7(5),

3x10(4), 3x10(6) and 3x10(7) showed CG% greater than the family mean (P ≤ 0.05).

Cross 3x10(10) showed a minimal reduction in germination under 200 mM NaCl

(18.12%) with FG% (74.90%) (Appendix D). This cross would have an advantage under this level of salinity as well as 300 mM NaCl. The mean germination index (GI) ranged between 0 and 60.67 with a mean of 5.80. Crosses 3x6(8), 3x6(11), 3x8(4), and 3x10(10) had greater GI (60.67, 58.07, 15.33, and 50.18%, respectively) compared to family mean

(P ≤ 0.01). Cross 3x7(5) showed GI greater than family mean (P ≤ 0.05). These results confirm those genotypes had greater percentage and speed of germination compared to all other genotypes of this family. Generally, like the final germination, germination index was reduced with increasing salinity for all the studied genotypes (Abdi et al. 2016;

Askari et al. 2016).

Table 6.12 summarized the mean of measured parameters (FG%, CG%, and GI) of Family 4 genotypes at 0 and 300 mM NaCl. Mean final germination percentage ranged from 0 to 36.67% with a mean of 6.35% under 300 mM NaCl. Parent 8 and crosses

4x6(7), 4x6(9), and 4x10(6) had greater FG% (18.33, 30.0, 25.87, and 36.67%, respectively) compared to family mean (P ≤ 0.01). Cross 4x7(5) showed FG% greater than family mean (P ≤ 0.05).

176

Table 6.12 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 4 5.00 1.67 33.33** 1.33 6 81.32** 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33** 18.33** 22.00* 9.33 10 76.67* 5.00 6.52 6.00 11 83.91** 11.84 14.11 4.74 4x6(1) 54.58 0.00 0.00 0.00 4x6(3) 60.17 1.67 2.77 0.67 4x6(4) 50.00 1.67 3.33 3.33 4x6(5) 72.89 5.00 6.86 3.33 4x6(6) 72.81 1.67 2.29 1.33 4x6(7) 93.16** 30.00** 32.20** 25.33** 4x6(9) 95.24** 25.87** 27.17** 26.29** 4x7(1) 88.62** 15.48* 17.46 12.22 4x7(2) 13.68 0.00 0.00 0.00 4x7(3) 59.30 0.00 0.00 0.00 4x7(5) 84.26** 16.84* 19.99* 18.18** 4x7(6) 75.76* 3.72 4.90 2.19 4x7(7) 86.05** 1.67 1.94 0.67 4x7(8) 90.00** 11.67 12.96 10.00 4x8(1) 30.53 0.00 0.00 0.00 4x8(2) 40.69 0.00 0.00 0.00 4x8(3) 59.12 1.67 2.82 0.67 4x8(4) 73.33 0.00 0.00 0.00 4x10(1) 33.22 0.00 0.00 0.00 4x10(2) 25.04 1.75 7.01 1.40 4x10(3) 61.05 1.85 3.03 0.74 4x10(4) 64.30 0.00 0.00 0.00 4x10(5) 38.33 3.33 8.70 4.00 4x10(6) 98.33** 36.67** 37.29** 36.00** 4x11(1) 21.67 0.00 0.00 0.00 4x11(2) 74.12 10.00 13.49 7.33 4x11(3) 25.35 3.42 13.49 2.74 4x11(4) 63.77 1.67 2.61 1.33 177

Table 6.12 (Continued)

Mean (x̅ ) 62.62 6.35 8.83 5.33 Range 5.00 - 98.33 0.00 - 36.67 0.00 - 37.29 0.00 - 36.00 LSD (0.05) 12.733 8.2566 10.187 7.1177 LSD (0.01) 16.91 10.965 13.824 9.4526 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean corrected germination (CG%) was 8.83%, which indicated that salt treatment under 300 mM NaCl reduced germination in this family by 91.17% compared to control (0 mM). Parent 4 and crosses 4x6(7), 4x6(9), and 4x10(6) had greater CG%

(33.33, 32.20, 27.17, and 37.29%, respectively) compared to the family mean (P ≤ 0.01).

However, parent 4 had showed low germination even under control condition. Previous studies reported low seed germination of some barley genotypes under control condition and related this to poor seed quality and primary dormancy (Adjel et al. 2013; Nakamura et al. 2017). Parent 8 and cross 4x7(5) had GI greater than family mean (P ≤ 0.05). These genotypes could be used in breeding program for salt tolerance because showed less germination reduced than other genotypes in this family. Mean germination index (GI) ranged between 0 and 36.0 with a mean of 5.33 under 300 mM NaCl, while mean GI of this family was 110.85 under 0 mM NaCl (Appendix D). Thus, germination percentage and speed germination decreased sharply as the salinity level increased (El Madidi et al.

2004; Tavili and Biniaz 2009; Adjel et al. 2013). Crosses 4x6(7), 4x6(9), 4x7(5), and

4x10(6) had greater GI (25.33, 26.29, 18.18, and 36.0%, respectively) compared to the family mean (P ≤ 0.01). These data confirm the greater percentage and speed of germination for these crosses compared to the other genotypes of this family.

178

The mean of measured parameters (FG%, CG%, and GI) of Family 5 genotypes at

0 and 300 mM NaCl are presented in Table 6.13.

Table 6.13 Mean final germination percentage (FG%), corrected germination percentage (CG%), and germination index (GI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for FG%, CG%, and GI of genotypes at 0, 100, and 200 mM NaCl Appendix Table D).

FG% FG% CG% GI Genotypes 0 mM 300 mM 300 mM 300 mM 5 19.07 0.00 0.00 0.00 6 81.32** 1.67 2.05 0.67 7 93.33** 1.67 1.79 1.33 8 83.33** 18.33** 22.00** 9.33 10 76.67** 5.00 6.52 6.00 11 83.91** 11.84** 14.11** 4.74 5x6(1) 30.00 1.67 5.56 0.67 5x6(2) 14.51 0.00 0.00 0.00 5x6(3) 19.51 0.00 0.00 0.00 5x6(4) 31.25 0.00 0.00 0.00 5x6(5) 59.30 0.00 0.00 0.00 5x7(1) 94.58** 21.67** 22.91** 27.33** 5x7(2) 93.33** 23.33** 25.00** 28.00** 5x7(3) 56.93 0.00 0.00 0.00 5x8(1) 25.93 0.00 0.00 0.00 5x8(2) 13.60 0.00 0.00 0.00 5x8(3) 55.00 0.00 0.00 0.00 5x8(4) 49.30 0.00 0.00 0.00 5x8(5) 35.00 0.00 0.00 0.00 5x8(6) 54.12 0.00 0.00 0.00 5x8(7) 38.25 1.67 4.36 1.33 5x10(1) 46.67 6.67 14.29** 7.33 5x10(2) 91.30** 16.67** 18.26** 17.33** 5x10(3) 41.67 0.00 0.00 0.00 5x10(4) 63.24 1.67 2.64 1.33 5x10(5) 51.27 0.00 0.00 0.00 5x11(1) 56.93 0.00 0.00 0.00 5x11(2) 10.45 0.00 0.00 0.00 5x11(3) 58.33 0.00 0.00 0.00 Mean (x̅ ) 52.69 3.86 4.81 3.63 179

Table 6.13 (Continued)

Range 10.45 - 94.58 0.00 - 23.33 0.00 - 25.00 0.00 - 28.00 LSD (0.05) 17.148 4.9488 6.064 5.8632 LSD (0.01) 22.815 6.5844 8.1508 7.801 * Value significantly greater than family mean at 0.05 probability level. ** Value significantly greater than family mean at 0.01 probability level.

Mean final germination ranged from 0 to 23.33% with a mean of 3.86% under

300 mM NaCl. Parents 8 and 11, and crosses 5x7(1), 5x7(2), and 5x10(2) had FG%

(18.33, 11.84, 21.67, 23.33, and 16.67%, respectively) greater than family mean (P ≤

0.01). Many crosses of this family failed to germinate under high level of salinity (300 mM NaCl) similar to their H. vulgare parent (5). These data confirm H. vulgare species more affected by salinity than H. bulbosum (Tavili and Biniaz 2009). Mean corrected germination (CG%) of this family was 4.81%, which indicated that salt treatment under

300 mM NaCl reduced germination by 95.19% compared to control. Othman et al. (2006) reported that high salinity (300 mM NaCl) decreased germination percentage of some barley genotypes sharply by 95.62%. Parents 8 and 11, and crosses 5x7(1), 5x7(2),

5x10(1), and 5x10(2) had CG% (22.0, 14.11, 22.91, 25.0, 14.29, and 18.26%, respectively) greater than family mean (P ≤ 0.01). Mean germination index (GI) ranged between 0 and 28.0 with a mean of 3.63. Crosses 5x7(1), 5x7(2), and 5x10(2) had GI

(9.33) greater than the family mean (P ≤ 0.01). These results confirm those crosses had greater percentage and speed of germination compared to other genotypes of this family.

Therefore, these crosses would have an advantage for improving barley salt tolerance in terms of germination. Yousofinia et al. (2012) reported that salinity decreased and delayed germination of barley cultivars under low level of salinity (50 and 100 mM

180

NaCl) due to low water uptake (osmotic pressure) and high accumulation of Na+ and Cl-

(toxicity) (Begum et al., 2010), these crosses are able to overcome these negative issues.

Generally, mean final germination percentage of all families were sharply decreased as the level of NaCl increased (Figure 6.2). Family 2 and Family 4 showed higher FG% under 0, 100, and 200 mM NaCl. Under high level of salinity (300 mM),

Family 2 had higher FG% compared to all other families. Previous studies reported that salinity decreased and delayed germination of barley genotypes. This decrease was expressed with the increase of NaCl concentration (Naseer et al. 2001; Othman et al.

2006; Tavili and Biniaz 2009; Adjel et al. 2013; Abdi et al. 2016).

80 Family 1 70 Family 2 Family 3 Family 4 60 Family 5

%FG 30

0 0 mM 100 mM 200 mM 300 mM NaCl solution

Figure 6.2 Mean final germination percentage (FG%) of barley tetraploid crosses (4x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl.

181

Conclusion

The mean of all measured parameters (FG%, CG%, and GI) of all families were sharply decreased as the level of NaCl increased. This impact decreases barley production. Furthermore, there was a high genetic variability to salt tolerance among genotypes. These responses among genotypes for studied parameters under four levels of salinity can be used to identify genotypes with salt stress tolerance potential for germination under saline conditions.

Families varied in the number of crosses that showed germination percentage greater than family mean at 300 mM NaCl. For diploid crosses (2x H. vulgare crosses), females 1 and 2 show greatest promise for ability to cross with H. bulbosum males especially male 9. Progeny of these two females was 14.3% and 12.5% saline tolerant, respectively. Tetraploid crosses (4x H. vulgare crosses), generated 174 progeny from five families. Of the 174, 29 were superior for germination under 300 mM saline conditions.

The greatest percentage of superior progeny came from Family 1 (crosses of female parent 1); while the greatest number of superior individuals came from Families 2 and 3, eight and seven, respectively. These progenies would have an economic value for improving barley tolerance for salinity. Yet, success is still dependent upon grain yield at maturity. Furthermore, many studies reported that there was no relationship between salinity tolerance at germination and tolerance at the seedling growth stage, so there is a need to screen these genotypes for salt tolerance at seedling growth stages to identify genotypes with salt stress tolerance.

182

REFERENCES

Abdi, N., S. Wasti, M.B. Salem, M. El Faleh, and E. Mallek-Maalej. 2016. Study on germination of seven barley cultivars (Hordeum vulgare L.) under salt stress. J. Agric. Sci. 8:88-97.

Abu-El-lail, F.F.B., K.A. Hamam, K.A. Kheiralla, and M.Z. El-Hifny. 2014. Salinity tolerance in 280 genotypes of two-rows barley. Egypt. J. Plant Breed. 18:331-345.

Adjel, F., H. Bouzerzour, and A. Benmahammed. 2013. Salt stress effects on seed germination and seedling growth of barley (Hordeum vulgare L.) genotypes. J. Agric. Sustain 3:223-237.

Angessa, T.T., X.Q. Zhang, G. Zhou, S. Broughton, W. Zhang, and C. Li. 2017. Early growth stages salinity stress tolerance in CM72 x Gairdner doubled haploid barley population. Plos. One. 12:1-16.

Arnold, R.L.B., M. Fenner, and P.J. Edwards. 1991. Changes in germinability, ABA content and ABA embryonic sensitivity in developing seeds of Sorghum bicolor (L.) Moench. induced by water stress during grain filling. New Phytol. 118(2):339-347.

Askari, H., S.K. Kazemitabar, H.N. Zarrini, and M.H. Saberi. 2016. Salt tolerance assessment of barley (Hordeum vulgare L.) genotypes at germination stage by tolerance indices. Open Agric. 1(1):37-44.

Bagci, S.A., H. Ekiz, and A. Yilmaz. 2003. Determination of the salt tolerance of some barley genotypes and the characteristics affecting tolerance. Turk. J. Agric. For. 27:253-260.

Bartels, D., and R. Sunkar. 2005. Drought and salt tolerance in plants. Crit. Rev. Plant Sci. 24:23-58.

Begum, F., I.M. Ahmed, A. Nessa, and W. Sultana. 2010. The effect of salinity on seed quality of wheat. J. Bangladesh Agric. Univ. 8(1):19-22.

Chen, J.F., L. Cui, , A.A. Malik, and K.G. Mbira. 2011. In vitro haploid and dihaploid production via unfertilized ovule culture. Plant Cell Tissue Organ Cult. 104(3):311-319.

Chikha, M.B., K. Hessini, R.N. Ourteni, A. Ghorbel, and N. Zoghlami. 2016. Identification of barley landrace genotypes with contrasting salinity tolerance at vegetative growth stage. Plant Biotechnol. 33:287-295.

El Madidi, S., B. El Baroudi, and F.B. Aameur. 2004. Effects of salinity on germination and early growth of barley (Hordeum vulgare L.) cultivars. Int. J. Agric. Biol. 6(5):767-770.

Houle, G., L. Morel, C.E. Reynolds, and J. Seigel. 2001. The effect of salinity on different developmental stages of an endemic annual plant, Aster laurentianus (Asteraceae). Am. J. Bot. 88(1):62-67

Kader, M.A. 2005. A comparison of seed germination calculation formulae and the associated interpretation of resulting data. J. Proc. Roy. Soc. New South Wales. 138:65-75.

Kook, H.S., T. Park, A. Khatoon, S. Rehman, and S.J. Yun. 2009. Avoidance of sodium accumulation in the shoot confers tolerance to salt stress in cultivated barley. Pak. J. Bot. 41:1751-1758.

Machado, R.M.A., and R.P. Serralheiro. 2017. Soil salinity: Effect on vegetable crop growth, management practices to prevent and mitigate soil salinization. Hortic. 3(2):1-30.

Mano, Y., and K. Takeda. 1998. Genetic resources of salt tolerance in wild Hordeum species. Euphytica. 103:137-141.

Mano, Y., H. Nakazumi, and K. Takeda. 1996. Varietal variation in and effects of some major genes on salt tolerance at the germination stage in barley. Breed. Sci. 46:227-233.

Matsumoto, S. 1989. Controversial points from soil diversity for arid land developments. Sand Dune Res. 36:1-10.

Mazher, A.A.M., F.E.M. EL-Quensi, and M.M. Farahat. 2007. Responses of ornamental plants and woody trees to salinity. World J. Agric. Sci. 3:386-395.

Nakamura, S., M. Pourkheirandish, H. Morishige, M. Sameri, K. Sato, and T. Komatsuda. 2017. Quantitative trait loci and maternal effects affecting the strong grain dormancy of wild barley (Hordeum vulgare ssp. spontaneum). Frontiers Plant Sci. 8:1840.

Naseer, S., A. Nisar, and M. Ashraf. 2001. Effect of salt stress on germination and seedling growth of barley (Hordeum vulgare L.). Pak. J. Biol. Sci. 4(3):359-360.

184

Othman, Y., G. Al-Karaki, A.R. Al-Tawaha, and A. Al-Horani. 2006. Variation in germination and ion uptake in barley genotypes under salinity conditions. World J. Agric. Sci. 2(1):11-15.

Parida, A.K., and A.B. Das. 2005. Salt tolerance and salinity effects on plants: A review. Ecotox. Environ. Safe. 60:324-349. http://dx.doi.org/10.1016/j.ecoenv.2004.06.010 (accessed 29 July 2018).

SAS Institute. 2011. SAS guide to macro processing. Vol. 11. SAS Inst., Cary, NC.

Scott, S.J., R.A. Jones, and W. Williams. 1984. Review of data analysis methods for seed germination 1. Crop Sci. 24(6):1192-1199.

Shahbaz, M., and M. Ashraf. 2013. Improving salinity tolerance in cereals. Crit. Rev. Plant Sci. 32:237-249.

Smith, S.E., and A.K. Dobrenz. 1987. Seed age and salt tolerance at germination in alfalfa 1. Crop Sci. 27(5):1053-1056.

Tavili, A., and M. Biniaz. 2009. Different salts effects on the germination of Hordeum vulgare and H. bulbosum. Pak. J. Nutr. 8:63-68.

Yousofinia, M., A. Ghassemian, O. Sofalian, and S. Khomari. 2012. Effects of salinity stress on barley (Hordeum vulgare L.) germination and seedling growth. Int. J. Agric. Crop Sci. 4(18):1353-1357.

Zhou, G., P. Johnson, P.R. Ryan, E. Delhaize, and M. Zhou. 2012. Quantitative trait loci for salinity tolerance in barley (Hordeum vulgare L.). Mol. Breed. 29(2):427-436.

185

CHAPTER VII

EVALUATING SALINITY TOLERANCE IN PROGENY OF DOMESTIC

(HORDEUM VULGARE) AND WILD BARLEY (H. BULBOSUM) AT

SEEDLING GROWTH STAGE

Introduction

Salinity is the major abiotic stresses facing the productivity of crops over the world. More than 20% of the world’s total agricultural land area is impacted by salt. In addition, the global economic losses due to saline soils amount to US$27.3 billion annually (FAO 2013). By 2050, 50% of the world’s agricultural land will be affected by salinity (Bartels and Sunkar 2005). Human population growth has caused a lack of available fresh water for agriculture. Climate change and population growth have exacerbated desertification, salinity, drought, and corresponding water shortages. Thus, there is a need to breed barley varieties with high water use efficiency and the ability to tolerate salinity. Additionally, the increase in areas affected by salinity and climate changes require new varieties with higher tolerance than in current varieties.

Salt tolerance is a complex trait controlled by many genes and involving different mechanisms. Thus, there is slow progress in developing salt tolerant crops (Bagci et al.

2003; Colmer et al. 2006). Salinity affects plant growth due to water stress, changes in nutrient uptake and translocation, and ion toxicities of NaCl which can deactivate cellular functions and physiological processes (Marschner 1995; Mano et al. 1996; Widodo et al.

186

2009; Kook et al. 2009). Osmotic stress reduces cell expansion in root tips and young leaves, and causes stomatal closure and a corresponding reduction in the rate of photosynthesis (Munns and Tester 2008).

The continuous intensive cultivation of barley has led to a narrowing of its genetic base. Also, a breeding program for salinity tolerance requires crossing between new genetic resources, selection between genotypes and evaluation under saline conditions (Zhu

2002; Abu-El-lail et al. 2014). Consequently, the wild species are a valuable genetic resource for inclusion in breeding programs to obtain tolerance to stress conditions (Ellis et al. 2000; Knezevic et al. 2004). Additionally, there is a need to screen genotypes for salt tolerance to identify those with satisfactory growth and yield under saline conditions

(Mano and Takeda 1998). Various screening methods based on physiological traits can be used in a barley breeding program, such as; germination percentage, radicle length, coleoptile length, degree of leaf injury, relative water content, plant height, root length, fresh and dry weight of roots and shoots, plant survival under salinity, and accumulation of Na+ and K+ in leaves and roots (Bagci et al. 2003; Chen et al. 2005; Adjel et al. 2013;

Chalbi et al. 2013; Abu-El-lail et al. 2014; El Goumi et al. 2014; Sbei et al. 2014; Abdi et al. 2016; Chikha et al. 2016; Shahraki and Fakheri 2016; Angessa et al. 2017, Jamshidi and Javanmard 2017). Naseer et al. (2001) reported that germination and seedling growth under saline environment are the most widely used screening criteria to select the salt tolerant germplasm. Barley genotypes showing tolerance to salinity at germination can be sensitive at the seedling stage, then tolerant at maturity (Epstein et al. 1980; Munns

2002). Consequently, screening genotype to salinity at the seedling stage is one of the most important stage in a barley breeding program.

187

Materials and Methods

Screening the effect of salinity on seedling growth was conducted in greenhouse during April 2018. One hundred sixty-one genotypes (34 genotypes from season

2015/2016, 116 genotypes from season 2016/2017, and the 11 parents (Table 7.1) were exposed to saline conditions during the seedling growth stage. Due to limited seed availability, seedlings from the control treatment of the seed germination experiment

(Chapter VI) were used to test salt tolerance at the seedling stage.

Table 7.1 Barley (Hordeum) accessions obtained from GRIN, USDA-ARS- NSGC (https://npgsweb.ars-grin.gov/gringlobal/search.aspx) used in this study

Germinated seedlings were placed on two layers of moist seed germination paper

(Anchor Paper Company; St. Paul, MN) placed in clear deli plastic containers 18.4L x

16.2W x 5.7H cm (Genpak AD24; Glens Falls, New York). Containers were placed in a

3° C dark growth chamber (Conviron, Mod. E15, Controlled Environments. Winnipeg,

MB, Canada) to limit growth and obtain uniform seedlings. Uniform seedlings were transplanted into plug trays containers (T.O. Plastics Company; Code 720700C,

Clearwater, MN). Container measured 54.4L x 27.7W x 12.7H cm and contained 50 cells

188

(5 x 10). Each cell measured (5 x 5 cm top, 3 x 3 cm bottom, 12.7 cm depth). Trays container were filled with a mixture (1:2 V/V) of fine sand and Sungro® Mix (Sun Gro

Horticulture, Agawam, MA) and placed in greenhouse under ambient April photoperiod at 25 ± 4/17° C. Seedlings were irrigated with tap water (0 mM NaCl) until the third leaf stage. At, the 3rd leaf stage, seedlings were irrigated with half-strength Hoagland’s nutrient solution (Hoagland and Arnon, 1950). Twenty-four hours later, seedlings of each flat (50 seedlings) were irrigated with 4 L of each NaCl treatment to fully wet the soil profile. Solutions of 0, 100, 200, and 300 mM NaCl were applied as needed; approximately twice a week. The salt treatments were sub-applied by applying 2 L of saline solution to a standard unperforated flat tray (T.O. Plastics Company; Code

710240C, Clearwater, MN) measured 54.5L x 27.8W x 6.2H and placing tray container of seedlings inside the flat tray. Seedlings were fertilized with 100 ppm of urea- ammonium nitrate (UAN) after ten days of saline treatment. The experiment was arranged as a randomized complete block design (RCBD) with three replications

(containers) with five plants for each replication (5 cells) at each NaCl level (4) = 60 plants. After 21 days of salt treatment, plants were harvested. Shoot dry weight was measured after oven-drying at 65° C until constant weight was obtained. At harvest time, shoots of five plants from each replication were harvested. The shoot dry weight was measured to identify salinity impact on each genotype.

Salinity susceptibility index (SSI) was one of the stress indices used to identify the relative salt tolerance of barley genotypes (Jamshidi and Javanmard 2017), it was calculated by using the formulas of Fischer and Maurer (1978).

Stress intensity (SI), a component of SSI, was calculated by using the following formula:

189

SI = 1 – X̅s/X̅p (7.1)

Where,

X̅s = mean all genotypes under stress condition.

X̅p = mean of all genotypes under non-stress condition.

Salinity susceptibility index (SSI) was then calculated by using the following formula:

SSI = 1 – Xs/Xp (7.2) SI Where,

Xs = mean of plant genotype under stress condition.

Xp = mean of plant genotype under non-stress condition.

When:

SSI ≥ 1, the genotype is susceptible to salinity.

SSI < 1, the genotype is more tolerant to salinity.

The term, “2x H. vulgare crosses” is used to identify crosses between diploid (2x)

Family name depends on H. vulgare female number given in table 7.1. For example,

“Family 1” means H. vulgare (♀) parent 1 crossed with all other H. bulbosum lines (♂).

The data were analyzed as a RCBD using PROC MEANS and PROC GLM in

SAS (Version 9.4, SAS Institute, 2011, Cary, NC). Separation of means among genotypes was tested using Fisher’s least significant difference (LSD) at α = 0.05 and

0.01. Correlation coefficient of morphological traits with final germination percentage

190

(FG%) and shoot dry weight (SDW) was determined using PROC CORR in SAS

(Version 9.4, SAS Institute, 2011, Cary, NC). Data were graphed using SigmaPlot version 13 (Systat Software Inc., San Jose, CA).

Results and Discussion

Genotypes of diploid H. vulgare crosses

Analysis of variance (Table 7.2) of barley diploid crosses (2x H. vulgare crosses) indicated highly significant (P ≤ 0.001) effects for shoot dry weight (SDW) due to genotype and salinity for Family 1, Family 2, and Family 3 at 0, 100, 200, and 300 mM

NaCl. Shoot dry weight was significantly affected by salinity, genotype, and the genotype x salinity interaction for Family 4 and Family 5, indicating each genotype responded to each level of salinity independently. The significant effects among the genotypes, salinity, and genotype x salinity interaction under four salinity levels indicated that there was a high genetic variation among the screened barley genotypes. Previous studies have also reported highly significant differences among barley genotypes, salinity (Naseer et al. 2001; El Madidi et al. 2004; Angessa et al. 2017) and the genotype x salinity interaction (Bagci et al. 2003; Adjel et al. 2013; Chalbi et al. 2013; El Goumi et al. 2014;

Sbei et al. 2014) for SDW.

191

Table 7.2 Analysis of variance of barley genotypes (2x H. vulgare crosses), salinity, and their interaction for shoot dry weight (SDW) at 0, 100, 200, and 300 mM NaCl.

Family Family Family Family Family Source of variance 1 2 3 4 5 Block NS† NS† NS† NS† * Genotypes (G) *** *** *** *** *** Salinity (S) *** *** *** *** *** G x S NS† NS† NS† * * * Significant at the 0.05 probability level. *** Significant at the 0.001 probability level. † NS, significance level P > 0.05.

Correlation between final germination percentage (FG%) and shoot dry weight

(SDW) were calculated. All values were significant as expected, e.g. FG% at 0 mM was negatively associated with SDW at all saline levels. Similar association was also reported by El Madidi et al. (2004). Many studies confirmed no relationship between salinity tolerance at germination and tolerance at the seedling growth stage for barley genotypes (Mano and Takeda 1998; Munns 2002; Foolad 2004; Kook et al. 2009; Adjel et al. 2013).

Correlation coefficient of FG% and SDW with morphological traits of Family 1 genotypes are presented in Table 7.3. Shoot dry weight (SDW) under 0 and 100 mM

NaCl showed a negative association with plant height, while under 300 mM showed a positive association with cSW. Final germination percentage (FG%) under 300 mM associated positively with grain yield and tiller number. Sebi et al. (2014) found that

SDW under 0 and 250 mM NaCl showed a strong positive correlation with shoot length and leaf number. These results justified the possibility of a correlated response for selection of these associated traits to improve barley salt tolerance. Bchini et al. (2010)

192

reported that grain yield and spike number in mature plants can be used as a direct scale of selection to salinity tolerance in a saline field.

Table 7.3 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 13 genotypes of Family 1 (2x H. vulgare crosses).

FG% SDW 0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH 0.44 0.51 0.22 0.10 -0.68** -0.58* -0.54 -0.42 TN 0.30 0.45 0.53 0.72** -0.27 -0.32 -0.29 -0.07 FLA -0.29 -0.05 -0.23 -0.46 -0.51 -0.42 -0.20 -0.53 F -0.07 -0.17 -0.17 -0.08 0.35 0.27 0.28 0.30 cSW -0.10 -0.17 -0.16 0.35 0.39 0.37 0.39 0.67* GY 0.19 0.18 0.19 0.57* 0.06 0.03 0.06 0.35 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

The mean shoot dry weigh (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM

NaCl are presented in Table 7.4. Data for SDW and SSI of genotypes at 100 and 200 mM

NaCl are presented in Appendix E. Mean SDW varied among genotypes in response to salinity. Mean SDW of this family ranged from 0.2507 to 0.6113 g with a mean of 0.4370 g plant-1 under 300 mM NaCl. Crosses 1x6(1) and 1x9(6) had greater SDW (0.6113 and

0.5787 g, respectively) compared to the family mean (P ≤ 0.01). Also, cross 1x9(1) showed SDW (0.5400 g) greater than the family mean (P ≤ 0.05). Interestingly, cross

1x9(6) had also greater FG% compared to the family mean under 300 mM (Chapter VI).

193

Therefore, this cross could be a good genetic material for salt tolerance breeding.

Generally, SDW for all genotypes decreased as the level of NaCl increased (Table 7.4 and Appendix E). There was 49.6% reduction in the mean SDW of this family under 300 mM compared to control. This reduction varied among genotypes in response to salinity. El

Goumi et al. (2014) reported high reduction (49.94%) of SDW for barley genotypes under 250 mM NaCl.

Table 7.4 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300mM 1 0.9500 0.5240 0.9038 6 0.5213 0.2673 0.9820 9 0.7007 0.2947 1.1679 10 0.5780 0.2507 1.1415 1x6(1) 1.1347** 0.6113** 0.9296 1x6(2) 0.9707 0.3873 1.2113 1x6(3) 0.8653 0.3933 1.0994 1x9(1) 1.0753* 0.5400* 1.0034 1x9(2) 0.7613 0.3707 1.0343 1x9(3) 0.8593 0.5140 0.8100‡ 1x9(6) 0.9187 0.5787** 0.7460‡ 1x9(9) 1.0120 0.5067 1.0065 1x10(4) 0.9280 0.4427 1.0541 Mean (x̅ ) 0.8673 0.4370 1.0069 Range 0.5213 – 1.1347 0.2507 – 0.6113 0.7460 – 1.2113 LSD (0.05) 0.1948 0.0936 - LSD (0.01) 0.264 0.1269 - SD - - 0.1358 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

194

Salinity susceptibility index (SSI) was used to identify the relative salt tolerance of barley genotypes (Jamshidi and Javanmard 2017). Genotypes with SSI < 1 were determined to be tolerant to salinity, while with SSI ≥ 1, were susceptible to salinity.

Mean SSI of this family was 1.0541 with a range of 0.7460 - 1.2113. Crosses 1x9(3) and

1x9(6) showed SSI – 1 SD < 1, indicating these genotypes had more salt tolerance than the other genotypes of this family. Cross 1x9(6) considered more important in a breeding program due to its low SSI value (0.7460) with SDW significantly greater than the family mean at 300 mM. These results are consistent with previous findings that indicated significant differences among barley genotypes for salt tolerance and different responses to increasing [NaCl] (Bagci et al. 2003; Adjel et al. 2013).

Salinity affects plant growth through water stress, changes in nutrient uptake and translocation, and ion toxicities of NaCl, which can deactivate cellular functions and physiological processes (Marschner 1995; Mano et al. 1996; Bagci et al. 2003; Kook et al. 2009). Furthermore, osmotic stress reduces cell division and cell expansion rates in root tips and young leaves resulting in reduction of the photosynthetic area, and causes stomatal closure then reduce the rate of photosynthesis (Munns 2002; Munns and Tester

2008). Salinity inhibits plant growth through displacement of Ca+2 by Na+ from critical cell wall binding sites then disrupts cell wall synthesis, modifies the metabolic activities, and limits the cell wall elasticity (Xue et al. 2004; Khan et al. 2008). Kook et al. (2009) reported that high salt tolerance of T76 barley cultivar was due to exclusion of Na+ in the shoot. Sodium ion content was about three times higher in G41 (susceptible) than in T76 in the shoot of the seedlings treated for 5 days at 200 mM NaCl. Adjel et al. (2013) found that shoot and root fresh weight decreases as saline level increased associated with

195

increased [Na+], decreased [K+] and K+:Na+. Albacete et al. (2008) reported that salinity affects the cytokinin/auxin ratio in the leaves and roots causing decrease in leaf growth and leaf number.

The correlation coefficient of FG% and SDW with morphological traits of

Family 2 genotypes are presented in Table 7.5. Final germination percentage (FG%) under 0 and 200 mM NaCl associated negatively with cSW as well as with grain yield under 0 and 100 mM. In contrast, SDW under all saline levels showed positive association with cSW. These results support the possibility of using these associated traits to select and improve salt tolerance of barley at low and moderate saline levels.

Table 7.5 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 13 genotypes of Family 2 (2x H. vulgare crosses).

FG% SDW 0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH 0.07 0.09 0.07 0.07 -0.26 -0.33 -0.39 -0.37 TN -0.25 -0.21 -0.11 -0.10 -0.05 -0.19 -0.08 -0.30 FLA 0.14 0.06 -0.04 -0.01 0.17 0.32 0.20 0.41 F -0.38 -0.46 -0.45 -0.24 0.39 0.33 0.26 0.26 cSW -0.60* -0.54 -0.62* -0.24 0.67* 0.75** 0.77** 0.68* GY -0.55* -0.58* -0.54 -0.34 0.49 0.36 0.32 0.22 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

Table 7.6 summarized the mean of SDW, g plant-1 and SSI of Family 2 genotypes at 0 and 300 mM NaCl.

196

Table 7.6 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 2 0.7093 0.3720 0.9570 7 0.6360 0.2447 1.2382 8 0.6053 0.1847 1.3984 9 0.7007 0.2947 1.1660 11 0.5260 0.2633 1.0049 2x7(2) 1.1353** 0.5767* 0.9902 2x8(2) 0.8347 0.4433 0.9434 2x9(1) 0.8400 0.3887 1.0812 2x9(2) 1.0000* 0.4440 1.1188 2x9(3) 0.9987* 0.6887** 0.6246‡ 2x9(4) 0.7973 0.4300 0.9271 2x11(1) 0.9240 0.4847 0.9568 2x11(3) 1.0180* 0.5800* 0.8658 Mean (x̅ ) 0.8250 0.4150 1.0209 Range 0.5260 - 1.1353 0.1847 - 0.6887 0.6246 - 1.3984 LSD (0.05) 0.1487 0.1355 - LSD (0.01) 0.2016 0.1837 - SD - - 0.1891 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

Mean SDW among genotypes ranged from 0.1847 to 0.6887 g plant-1 with a mean of 0.4150 g plant-1. Chen et al. (2005) also observed a range of SDW among seven barley cultivars (0.140 - 0.440 g) under 320 mM NaCl. Cross 2x9(3) had greater SDW (0.6887 g) compared to the family mean (P ≤ 0.01). Cross 2x7(2) and 2x11(3) showed SDW

(0.5767 and 0.5800 g, respectively) greater than the family mean (P ≤ 0.05).

Interestingly, these crosses failed to germinate under 300 mM. This result confirms a lack of relationship between salt tolerance at germination and seedling growth stage (Adjel et 197

al. 20013). There was 49.7% reduction in the mean SDW of this family under 300 mM compared to control, which indicate the high effects of salinity on plant growth. Bagci et al. (2003) found 51.3% reduction in SDW under 314 mM vs. control. Family 2 had SSI ranged from 0.6246 to 1.3984 with a mean of 1.0209. Only cross 2x9(3) showed SSI – 1

SD < 1, indicating this progeny had more tolerance than all the other genotypes of this family. This progeny would have an advantage in a barley breeding program for salinity.

The correlation coefficient of FG% and SDW with morphological traits of

Family 3 genotypes are presented in Table 7.7. Final germination percentage (FG%) under 100 and 300 mM NaCl had positive association with tiller number similar to

Family 1, while under 200 mM associated negatively with cSW similar to Family 2. In contrast, SDW under control conditions associated positively with cSW.

Table 7.7 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 6 genotypes of Family 3 (2x H. vulgare crosses).

FG% SDW 0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH -0.13 -0.09 -0.12 -0.20 -0.08 0.20 0.03 0.06 TN 0.80 0.82* 0.76 0.97** -0.25 -0.17 -0.34 -0.15 FLA -0.36 -0.19 -0.20 0.09 0.46 0.46 0.33 0.37 F -0.46 -0.34 -0.27 -0.05 0.53 0.28 0.46 0.43 cSW -0.56 -0.76 -0.84* -0.20 0.88* 0.80 0.78 0.67 GY -0.22 -0.29 -0.28 0.02 0.43 0.33 0.48 0.45 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

198

The means of SDW, g plant-1 and SSI of Family 3 genotypes at 0 and 300 mM

NaCl are presented in Table 7.8.

Table 7.8 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 3 1.0927* 0.6147** 0.8218 9 0.7007 0.2947 1.0885 10 0.5780 0.2507 1.0638 3x9(1) 1.1340** 0.5373* 0.9884 3x10(3) 0.9767 0.2860 1.3284 3x10(5) 0.9093 0.5380* 0.7671‡ Mean (x̅ ) 0.8986 0.4202 1.0097 Range 0.5780 - 1.1340 0.2507 - 0.6147 0.7671 - 1.3284 LSD (0.05) 0.1423 0.1163 - LSD (0.01) 0.2024 0.1654 - SD - - 0.2026 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

Mean SDW of this family ranged from 0.2507 to 0.6147 with a mean of 0.4202 g plant-1 under 300 mM NaCl. Parent 3 had greater SDW (0.6147 g) compared to the family mean (P ≤ 0.01). Crosses 3x9(1) and 3x10(5) showed SDW (0.5373 and 0.5380 g, respectively) greater than the family mean (P ≤ 0.05). However, parent 3 and cross

3x10(5) failed to germinate under 300 mM (Chapter VI). There was 53% reduction in the mean SDW of this family under 300 mM compared to control. Sbei et al. (2014) found

50% reduction in SDW under 250 mM vs. control. Mean SSI ranged from 0.7671 to

1.3284 with a mean of 1.0097. Cross 3x10(5) had SSI – 1 SD < 1, indicating this cross

199

more tolerant than the other genotypes of this family based on performance of both tests.

Cross 3x10(5) exceeded the SDW of cross 3x10(3) by 88% under 300 mM NaCl even though they were from the same two parents (3 and 10). It appears cross 3x10(5) received the alleles for salt tolerance, while cross 3x10(3) did not.

The correlation coefficient of FG% and SDW with morphological traits of

Family 4 genotypes are presented in Table 7.9. Final germination percentage (FG%) under 100 and 200 mM NaCl had positive association with plant height. Shoot dry weight

(SDW) under 100 mM had a strong negative association with flag leaf area. These results support the possibility of using these associated traits to select and improve salt tolerance only at low and moderate saline levels, but not at high levels.

Table 7.9 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 3 genotypes of Family 4 (2x H. vulgare crosses).

FG% SDW 0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH 0.78 1.00* 1.00* 0.54 -0.98 -0.96 -0.76 -0.70 TN -0.97 -0.65 -0.57 0.35 0.74 0.34 -0.07 -0.16 FLA 0.58 0.94 0.97 0.75 -0.89 -1.00** -0.91 -0.86 F -0.99 -0.76 -0.69 0.20 0.83 0.48 0.08 -0.01 cSW -0.59 -0.02 0.08 0.87 0.14 -0.33 -0.69 -0.75 GY -0.97 -0.66 -0.58 0.34 0.75 0.35 -0.07 -0.16 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

200

Table 7.10 summarized the mean of SDW, g plant-1 and SSI of Family 4 genotypes at 0 and 300 mM NaCl. This family had only one progeny that screened for salinity because all other progenies showed very low FG% under control conditions.

Progeny 4x6(2) had SDW (0.6793 g) greater than both parents (P ≤ 0.01). This cross had

SSI – 1 SD < 1, indicating this cross had more salt tolerance even though both parents were saline susceptible based on SSI and SDW. This result is confirmed by only a slight reduction (22%) of SDW under 300 mM vs. control. Therefore, this cross could be a good genetic material for salt tolerance breeding.

Table 7.10 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 4 0.9360* 0.3507 1.4115 6 0.5213 0.2673 1.0997 4x6(2) 0.8720 0.6793** 0.4987‡ Mean (x̅ ) 0.7764 0.4324 1.0033 Range 0.5213 - 0.9360 0.2673 - 0.6793 0.4987 - 1.4115 LSD (0.05) 0.1593 0.1461 - LSD (0.01) 0.2642 0.2423 - SD - - 0.4640 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

The correlation coefficient of FG% and SDW with morphological traits of

Family 5 genotypes are presented in Table 7.11. Final germination percentage (FG%) under all saline levels (except control) showed positive correlation with tiller number, while associated negatively with cSW under 0, 100, and 200 mM. Similar results were 201

also found in Family 2 and 3. Bchini et al. (2010) reported spike number can be considered as a direct criterion of selection for salinity tolerance. Shoot dry weight

(SDW) under 200 and 300 mM associated positively with fertility.

Table 7.11 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 18 genotypes of Family 5 (2x H. vulgare crosses).

FG% SDW 0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH -0.04 0.02 0.04 0.28 -0.33 -0.27 -0.07 -0.35 TN 0.45 0.50* 0.51* 0.49* -0.18 -0.11 -0.19 -0.28 FLA 0.02 0.14 0.22 0.26 -0.24 -0.14 -0.06 -0.33 F -0.41 -0.41 -0.37 -0.20 0.40 0.25 0.47* 0.47* cSW -0.63** -0.55* -0.57* -0.42 0.22 0.11 0.10 0.13 GY 0.05 0.05 0.05 0.20 0.20 0.18 0.19 0.12 * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level.

The means of SDW, g plant-1 and SSI of Family 5 genotypes at 0 and 300 mM

NaCl are presented in Table 7.12. Genotypes of this family showed a wide range of SDW

(0.1847 - 0.4833 g plant-1) with a mean of 0.3532 g plant-1 under 300 mM NaCl. Among all genotype, only crosses 5x11(1) and 5x11(6-1) showed SDW (0.4727 and 0.4833 g, respectively) greater than the family mean (P ≤ 0.05). This family showed 51% reduction in the mean SDW under 300 mM compared to control. Similar finding reported by

Angessa et al. (2017) who found 53.4% reduction in barley lines under 150 mM NaCl.

Mean SSI of this family was 1.0013 with a range of 0.6820 - 1.3593. Crosses 5x7(3) and

202

5x8(10-2) had SSI – 1 SD < 1, indicating these progeny more tolerant to salinity than the other genotypes.

Table 7.12 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (2x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 5 0.7907 0.4307 0.8906 7 0.6360 0.2447 1.2035 8 0.6053 0.1847 1.3593 9 0.7007 0.2947 1.1334 11 0.5260 0.2633 0.9768 5x7(3) 0.6367 0.4147 0.6820‡ 5x8(3) 0.7753 0.2760 1.2597 5x8(4) 0.7240 0.3060 1.1293 5x8(5) 0.7387 0.4313 0.8138 5x8(10-2) 0.7187 0.4467 0.7403‡ 5x9(2) 0.6313 0.2913 1.0534 5x11(1) 0.9853** 0.4727* 1.0177 5x11(3) 0.6920 0.3480 0.9723 5x11(4) 0.8253 0.3847 1.0444 5x11(6-1) 0.8300 0.4833* 0.8170 5x11(6-2) 0.7673 0.3480 1.0689 5x11(8) 0.7547 0.3073 1.1594 5x11(9) 0.6693 0.4293 0.7014 Mean (x̅ ) 0.7226 0.3532 1.0013 Range 0.5260 -0.9853 0.1847 - 0.4833 0.6820 - 1.3593 LSD (0.05) 0.1349 0.119 - LSD (0.01) 0.1811 0.1598 - SD - - 0.1951 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

203

Generally, whatever the family or genotype, mean SDW was decreased as the level of NaCl increased (Figure 7.1). The severity of high salinity (300 mM) was reflected by highly reduced of the mean SDW compared to control conditions. Results from this study corroborates previous studies that indicated salinity stress inhibits barley growth and development and thus SDW (Chen et al. 2005; Kook et al. 2009; Sbei et al.

2014; El Goumi et al. 2014; Angessa et al. 2017). This suppression was expressed with the increase of [NaCl].

1.2 Family 1 Family 2 1.0 Family 3 Family 4 Family 5 0.8

-1

0.6

SDW, g plant 0.4

0.2

0.0 0 mM 100 mM 200 mM 300 mM

NaCl concentration

Figure 7.1 Mean shoot dry weight (SDW), g plant-1 of barley diploid crosses (2x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl.

204

Genotypes of tetraploid H. vulgare crosses

Analysis of variance (Table 7.13) of barley tetraploid crosses (4x H. vulgare crosses) indicated highly significant (P ≤ 0.001) effects for shoot dry weight (SDW) due to the genotype x salinity interaction for all families, except Family 4 with P ≤ 0.05, under 0, 100, 200, and 300 mM NaCl, indicating each genotype responded to each salinity level independently. The significant effects among the genotypes, salinity, and genotype x salinity interaction under four salinity levels indicate that there was high genetic variation among the screened barley genotypes. Previous studies have also reported highly significant differences among barley genotypes, salinity and their interaction (Bagci et al. 2003; Adjel et al. 2013; Chalbi et al. 2013; El Goumi et al. 2014;

Sbei et al. 2014) for SDW.

Table 7.13 Analysis of variance of barley genotypes (4x H. vulgare crosses), salinity, and their interaction for shoot dry weight (SDW) at 0, 100, 200, and 300 mM NaCl.

Family Family Family Family Family Source of variance 1 2 3 4 5 Block NS† NS† * NS† ** Genotypes (G) *** *** *** *** *** Salinity (S) *** *** *** *** *** G x S *** *** *** * *** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level. † NS, significance level P > 0.05.

Correlation between final germination percentage (FG%) and shoot dry weight

salinity tolerance at germination and tolerance at the seedling growth stage for barley genotypes (Bagci et al. 2003; Foolad 2004; Kook et al. 2009; Adjel et al. 2013).

Correlation coefficient of FG% and SDW with morphological traits of Family 1 genotypes are presented in Table 7.14.

Table 7.14 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 20 genotypes of Family 1 (4x H. vulgare crosses).

FG% SDW 0mM 100m 200m 300m 0mM 100mM 200mM 300mM M M M PH 0.50* 0.61** 0.45* 0.03 -0.56** -0.60** -0.52* -0.49* TN -0.58** -0.64** -0.49* -0.07 0.68** 0.71*** 0.63** 0.58** FLA 0.29 0.24 0.24 0.38 -0.04 0.08 0.09 -0.06 F -0.43 -0.56** -0.44 0.09 0.62** 0.67** 0.66** 0.63** cSW -0.53* -0.58** -0.42 0.22 0.70*** 0.72*** 0.71*** 0.72*** GY -0.47* -0.54* -0.36 0.22 0.66** 0.72*** 0.66** 0.62** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

Final germination percentage (FG%) under 0, 100, and 200 mM NaCl showed positive association with plant height, while had negative association with tiller number.

Also, FG% under 0 and 100 mM associated negatively with fertility, cSW, and grain yield. In contrast, SDW under all salinity levels associated negatively with plant height, while it showed a strong positive correlation with tiller number, fertility, cSW, and grain yield. Sebi et al. (2014) found that shoot length and leaf number showed a strong positive correlation with SDW under 0 and 250mM NaCl. Bchini et al. (2010) reported that grain 206

yield and spike number can be used as a direct scale of selection to salinity tolerance.

These results support the possibility of using these associated traits to select and improve salt tolerance for germination at low and moderate saline levels, and for SDW at high saline level.

The mean shoot dry weigh (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM

NaCl are presented in Table 7.15. Data for SDW and SSI of genotypes at 100 and 200 mM NaCl are presented in Appendix E. Barley genotypes responded differently to the varying salinity levels. Mean SDW among genotypes ranged from 0.1847 to 0.8200 g with a mean of 0.4538 g plant-1 under 300 NaCl. Crosses 1x7(3), 1x7(8), 1x10(6),

1x11(2), and 1x11(3) showed greater SDW (0.8200, 0.6200, 0.7087, 0.6173, and 0.7387 g, respectively) compared to the family mean (P ≤ 0.01). Interestingly, cross 1x7(8) had also greater FG% (25.26%) compared to the family mean under 300 mM (Chapter VI).

Therefore, this cross would have an advantage for improving barley salt tolerance at germination and seedling stages, but it is unknown what a final grain yield would be. The other crosses with greater SDW failed or showed low FG% under 300 mM due to dormancy or salinity effect, confirming a lack of relationship between salt tolerance at germination and seedling growth stage (Foolad 2004; Kook et al. 2009). Generally, SDW for all genotypes was decreased as the level of NaCl increased (Table 7.15 and Appendix

E). There was 48.4% reduction in the mean SDW of this family under 300 mM compared to control. This reduction varied among genotypes in response to increasing salinity. Similar finding has been observed in barley genotypes by El Goumi et al. (2014), who found

49.9% reduction in SDW under 250 mM vs. control.

207

Table 7.15 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 1 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 1 0.9500 0.5240 0.9258 6 0.5213 0.2673 1.0059 7 0.6360 0.2447 1.2703 8 0.6053 0.1847 1.4347 10 0.5780 0.2507 1.1692 11 0.5260 0.2633 1.0309 1x6(1) 0.6907 0.2900 1.1977 1x6(3) 0.8933 0.4787 0.9583 1x7(3) 1.1553** 0.8200** 0.5992§ 1x7(6) 1.0780* 0.4313 1.2384 1x7(7) 0.7647 0.4780 0.7740‡ 1x7(8) 1.1393** 0.6200** 0.9410 1x8(4) 0.9613 0.5500 0.8834 1x10(2) 0.8933 0.4140 1.1077 1x10(4) 0.8747 0.4760 0.9410 1x10(5) 0.8413 0.4560 0.9456 1x10(6) 1.2540** 0.7087** 0.8978 1x10(7) 0.6613 0.2627 1.2445 1x11(2) 1.2507** 0.6173** 1.0455 1x11(3) 1.3273** 0.7387** 0.9156 Mean (x̅ ) 0.8801 0.4538 1.0263 Range 0.5213 - 1.3273 0.1847 - 0.8200 0.5992 - 1.4347 LSD (0.05) 0.1561 0.108 - LSD (0.01) 0.2091 0.1447 - SD - - 0.1929 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD. § Value less than mean SSI – 2 SD.

Family 1 genotypes showed a wide range of SSI (0.5992 - 1.4347) with a mean of

(1.0263). Crosses 1x7(3) and 1x7(7) showed SSI – 2 SD < 1 and SSI – 1 SD < 1,

208

respectively, indicating these crosses had more salt tolerance as measured by SSI than the other genotypes of this family. Cross 1x7(3) was considered more tolerant due to its low value (0.5992) and its relatively high SDW at 300 mM. Additionally, cross 1x7(7) had also greater FG% (35%) at 300 mM (Chapter VI). Therefore, these two crosses would be a good genetic material for salt tolerance breeding at germination and seedling growth stages. These results are in agreement with previous studies that indicated significant differences among barley genotypes for salt tolerance and different responses by SDW to increasing salt concentration (Chen et al. 2005; Angessa et al. 2017).

The correlation coefficient of FG% and SDW with morphological traits of

Family 2 genotypes are presented in Table 7.16.

Table 7.16 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 45 genotypes of Family 2 (4x H. vulgare crosses).

FG% SDW

0mM 100mM 200mM 300mM 0mM 100mM 200mM 300m M PH 0.24 0.42** 0.25 -0.06 -0.52*** -0.51*** -0.54*** -0.47** TN -0.23 -0.23 -0.18 0.06 0.30* 0.33* 0.43** 0.35* FLA 0.09 -0.16 -0.18 -0.18 0.11 0.07 0.01 0.17 F -0.32* -0.48*** -0.40** -0.01 0.37* 0.38** 0.43** 0.39** cSW -0.24 -0.46** -0.38** 0.03 0.41** 0.46** 0.43** 0.41** GY -0.10 -0.18 -0.10 0.18 0.30* 0.40** 0.40** 0.38** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

209

Final germination percentage (FG%) under 100 mM NaCl showed a strong positive association with plant height, while under 0, 100, and 200 mM had negative association with fertility and cSW. In contrast, SDW under all salinity levels had a strong negative association with plant height, while associated positively with tiller number, fertility, cSW, and grain yield. Similar trend was also observed in Family 1.

Table 7.17 summarized the mean of SDW, g plant-1 and SSI of Family 2 genotypes at 0 and 300 mM NaCl. Genotypes responded differently to the varying salinity levels that reflected by reduction of SDW. Mean SDW among Family 2 genotypes ranged from 0.1847 to 0.9813 g with a mean of 0.5290 g plant-1. Crosses

2x6(7), 2x7(1), 2x7(5), 2x7(7), 2x7(8), 2x8(4), 2x8(10), 2x8(16), and 2x11(1) had greater

SDW (0.9280, 0.8300, 0.7773, 0.9573, 0.9813, 0.7113, 0.7333, 0.7340, and 0.9160 g, respectively) compared to the family mean (P ≤ 0.01). Crosses 2x6(6), 2x7(12), 2x8(8),

2x10(5), and 2x10(9) also showed SDW greater than the family mean (P ≤ 0.05). All these crosses were with low FG% or failed to germinate under 300 mM due to salt stress, except cross 2x8(10) which showed greater FG% (26.67%) under 300 mM (Chapter VI).

This result confirm a lack of relationship between salt tolerance at germination and seedling growth stage (El Madidi et al. 2004; Adjel et al. 2013). This family showed

45.5% reduction in the mean SDW of this family under 300 mM compared to control, which indicate the high effects of salinity on plant growth. Fricke et al. (2006) reported

68% and 64% biomass reduction in Clipper and Arivat cultivars, respectively under 250 mM vs. control.

210

Table 7.17 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 2 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 2 0.7093 0.3720 1.0459 6 0.5213 0.2673 1.0715 7 0.6360 0.2447 1.3532 8 0.6053 0.1847 1.5283 10 0.5780 0.2507 1.2455 11 0.5260 0.2633 1.0982 2x6(1) 0.7047 0.3273 1.1777 2x6(2) 0.8460 0.4327 1.0745 2x6(6) 1.0380 0.6667* 0.7868‡ 2x6(7) 1.5000** 0.9280** 0.8386 2x6(8) 0.9613 0.4600 1.1469 2x7(1) 1.3713** 0.8300** 0.8682 2x7(4) 0.7720 0.4587 0.8926 2x7(5) 1.2493** 0.7773** 0.8309 2x7(7) 1.1700* 0.9573** 0.3998§ 2x7(8) 1.6313** 0.9813** 0.8763 2x7(9) 0.9833 0.6173 0.8186 2x7(10) 0.8920 0.5627 0.8120 2x7(11) 0.6247 0.2593 1.2862 2x7(12) 1.3973** 0.6807* 1.1280 2x8(1) 0.9360 0.4893 1.0495 2x8(2) 0.7387 0.3393 1.1889 2x8(3) 0.9333 0.4887 1.0478 2x8(4) 1.0553 0.7113** 0.7169‡ 2x8(5) 0.6267 0.2153 1.4436 2x8(6) 0.7827 0.4327 0.9835 2x8(8) 1.1553* 0.6840* 0.8972 2x8(10) 1.2460** 0.7333** 0.9049 2x8(11) 0.6533 0.2820 1.2500 2x8(14) 1.2893** 0.5093 1.3305 2x8(15) 0.6287 0.3047 1.1334 2x8(16) 1.1760* 0.7340** 0.8266 2x10(1) 0.8060 0.3727 1.1824 2x10(2) 0.9347 0.4993 1.0243 2x10(4) 0.9727 0.4953 1.0793

211

Table 7.17 (Continued) 2x10(5) 1.1380* 0.6800* 0.8851 2x10(7) 1.0340 0.6440 0.8295 2x10(8) 1.0847 0.5813 1.0206 2x10(9) 1.3307** 0.6727* 1.0875 2x10(11) 0.8693 0.4060 1.1721 2x10(12) 0.6993 0.3067 1.2349 2x11(1) 1.4053** 0.9160** 0.7658‡ 2x11(2) 1.1587* 0.6053 1.0503 2x11(4) 1.1353 0.6007 1.0357 2x11(5) 1.1493* 0.5800 1.0894 Mean (x̅ ) 0.9701 0.5290 1.0335 Range 0.5213 - 1.6313 0.1847 - 0.9813 0.3998 - 1.5283 LSD (0.05) 0.1675 0.1362 - LSD (0.01) 0.2219 0.1805 - SD - - 0.2110 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD. § Value less than mean SSI – 2 SD.

Family 2 had SSI ranged from 0.3998 to 1.5283 with a mean of 1.0335. Cross

2x7(7) had SSI – 2 SD < 1, indicating this cross had more tolerance than the other genotypes of this family due to its extremely low SSI value (0.3998). Crosses 2x6(6),

2x8(4), and 2x11(1) also showed SSI – 1 SD < 1. Female parent 2 was highly compatible with all males, resulting in 39 progeny (Table 7.17). Furthermore, Family 2 is highly desirable in a barley breeding program for saline tolerance. Twelve of the crosses

(30.8%) show SDW significantly greater than the family mean and SSI below 1.

Some crosses showed a big gap between them for SDW even though they were from the same two parents. For instance, cross 2x7(8) exceeded the SDW of cross

2x7(11) by 278% under 300 mM NaCl even though they were from the same two parents.

212

The lack of intermediate types can be attributed to the lack of heterozygotes in dihaploids

(Chen et al. 2011).

The correlation coefficient of FG% and SDW with morphological traits of

Family 3 genotypes are presented in Table 7.18. Final germination percentage (FG%) under 0 and 100 mM correlated negatively with flag leaf area as well as with fertility, cSW, and grain yield under 200 mM. Shoot dry weight (SDW) had negative correlation with plant height under 0 mM, while associated positively with flag leaf area under 300 mM. In addition, SDW under all salinity levels had a strong positive association with fertility, cSW, and grain yield. These traits (FG% and SDW) could be used as a direct criterion for screening and selecting tolerant genotype.

Table 7.18 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 27 genotypes of Family 3 (4x H. vulgare crosses).

FGP SDW

0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH 0.16 0.24 0.09 -0.26 -0.43* -0.20 -0.19 -0.26 TN 0.21 -0.01 -0.01 0.37 0.29 0.05 0.00 0.05 FLA -0.43* -0.47* -0.33 -0.38 0.23 0.27 0.30 0.43* F -0.53** -0.68*** -0.54** -0.09 0.58** 0.49** 0.58** 0.67*** cSW -0.66*** -0.78*** -0.60*** -0.15 0.63*** 0.63*** 0.69*** 0.73*** GY -0.44* -0.65*** -0.47* -0.08 0.65*** 0.65*** 0.65*** 0.70*** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

213

The means of SDW, g plant-1 and SSI of Family 3 genotypes at 0 and 300 mM

NaCl are presented in Table 7.19.

Table 7.19 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 3 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 3 1.0927** 0.6147** 0.8345 6 0.5213 0.2673 0.9294 7 0.6360 0.2447 1.1737 8 0.6053 0.1847 1.3256 10 0.5780 0.2507 1.0803 11 0.5260 0.2633 0.9525 3x6(1) 0.6553 0.3407 0.9159 3x6(2) 0.9160 0.4600 0.9496 3x6(7) 1.2000** 0.5793* 0.9866 3x6(8) 0.8307 0.2813 1.2615 3x6(11) 1.0807* 0.3140 1.3533 3x7(3) 1.0100 0.3060 1.3296 3x7(4) 0.9873 0.5340 0.8758 3x7(5) 0.6847 0.2680 1.1608 3x7(6) 1.0053 0.4927 0.9727 3x7(7) 0.6480 0.3360 0.9184 3x7(8) 0.9960 0.4247 1.0942 3x8(4) 0.8747 0.5033 0.8098‡ 3x10(4) 0.6293 0.3053 0.9820 3x10(5) 1.3600** 0.6213** 1.0360 3x10(6) 0.8940 0.5153 0.8080‡ 3x10(7) 0.7153 0.3667 0.9298 3x10(10) 0.9067 0.3987 1.0688 3x11(1) 0.9660 0.5307 0.8596 3x11(2) 1.0920** 0.7153** 0.6580‡ 3x11(3) 0.9193 0.4947 0.8811 3x11(5) 0.4920 0.2447 0.9589 Mean (x̅ ) 0.8453 0.4021 1.0039 Range 0.4920 - 1.3600 0.1847 - 0.7153 0.6580 - 1.3533 LSD (0.05) 0.1814 0.1438 -

214

Table 7.19 (Continued) LSD (0.01) 0.2417 0.1916 - SD - - 0.1735 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

Mean SDW of this family ranged from 0.1847 to 0.7153 g with a mean of 0.4021 g plant-1 under 300 mM NaCl. Parent 3 and crosses 3x10(5), 3x11(2) had greater SDW

(0.6147, 0.6213, and 0.7153 g, respectively) compared to the family mean (P ≤ 0.01).

Cross 3x6(7) also showed SDW (0.5793 g) greater than the family mean (P ≤ 0.05).

However, parent 3 and cross 3x6(7) failed to germinate under 300 mM (Chapter VI), confirming the lack of significant association between germination and seedling growth for salt tolerance. There was 52% reduction in the mean SDW of this family under 300 mM compared to control. Similar finding has been observed in barley genotypes by Sbei et al. (2014), who found 50% reduction in SDW under 250 mM vs. control. Genotypes of

Family 3 showed a wide range of SSI, ranging from 0.6580 to 1.3533 with a mean of

1.0039. Crosses 3x8(4), 3x10(6), and 3x11(2) had SSI – 1 SD < 1, indicating these genotypes more tolerant than the other genotypes of this family. Cross 3x11(2) considered more important in a breeding program due to its extremely low SSI value

(0.6580) with SDW significantly greater than the family mean at 300 mM. These results are consistent with previous findings which reported that SDW affected by salt stress in all studies genotypes ( Kook et al. 2009; Chalbi et al. 2014; Angessa et al. 2017).

The correlation coefficient of FG% and SDW with morphological traits of

Family 4 genotypes are presented in Table 7.20. Final germination percentage (FG%)

215

under most salinity levels (except 300 mM) associated negatively with all studied traits except plant height. Shoot dry weight (SDW) under all the salinity levels associated negatively with plant height, while showed positive association with all the other traits.

Similar associations have been observed in the other families.

Table 7.20 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 32 genotypes of Family 4 (4x H. vulgare crosses).

FG% SDW

0mM 100mM 200mM 300mM 0mM 100mM 200mM 300mM PH 0.17 0.30 0.23 -0.11 -0.46** -0.40* -0.39* -0.48** TN -0.53** -0.66*** -0.55** -0.27 0.70*** 0.65*** 0.64*** 0.71*** FLA -0.28 -0.36* -0.26 -0.20 0.45** 0.35* 0.49** 0.35* F -0.38* -0.50** -0.45** 0.07 0.42* 0.43* 0.43* 0.44* cSW -0.18 -0.47** -0.39* -0.02 0.57*** 0.58*** 0.62*** 0.62*** GY -0.31 -0.55*** -0.45** -0.13 0.66*** 0.73*** 0.69*** 0.69*** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

Table 7.21 summarized the mean of SDW, g plant-1 and SSI of Family 4 genotypes at 0 and 300 mM NaCl. Genotypes responded differently to the varying salinity levels, and SDW for all genotype was decreased as the level of NaCl increased

(Table 7.21 and Appendix E). Mean SDW among genotypes ranged from 0.1847 to

0.8900 g with a mean of 0.5371 g plant-1 under 300 NaCl. Crosses 4x6(4), 4x10(4), and

4x10(5) showed greater SDW (0.7653, 0.7387, and 0.8900 g, respectively) compared to the family mean (P ≤ 0.01). Crosses 4x6(1), 4x6(3), 4x6(5), 4x7(3), 4x7(6), and 4x8(2)

216

also showed SDW greater than the family mean (P ≤ 0.05). This family showed 45% reduction in the mean SDW under 300 mM compared to control, which indicate the high effects of salinity on plant growth.

Table 7.21 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 4 crosses (4x H. vulgare crosses) at 0 and 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 4 0.9360 0.3507 1.3804 6 0.5213 0.2673 1.0755 7 0.6360 0.2447 1.3582 8 0.6053 0.1847 1.5340 10 0.5780 0.2507 1.2501 11 0.5260 0.2633 1.1023 4x6(1) 1.0973 0.7060* 0.7872‡ 4x6(3) 1.1773* 0.6907* 0.9125 4x6(4) 1.1753* 0.7653** 0.7700‡ 4x6(5) 1.2467** 0.7153* 0.9408 4x6(6) 0.8440 0.5060 0.8840 4x6(7) 0.5967 0.2100 1.4305 4x6(9) 0.5933 0.2420 1.3071 4x7(1) 0.8167 0.4320 1.0397 4x7(3) 1.3307** 0.7360* 0.9865 4x7(5) 1.1233 0.5593 1.1083 4x7(6) 1.1627* 0.7240* 0.8328 4x7(7) 1.1773* 0.6680 0.9550 4x7(8) 0.9827 0.5413 0.9914 4x8(1) 1.1060 0.6833 0.8436 4x8(2) 1.4140** 0.7173* 1.0876 4x8(3) 0.6727 0.4400 0.7635‡ 4x8(4) 1.0607 0.6133 0.9310 4x10(1) 1.2453** 0.6700 1.0198 4x10(3) 1.1993* 0.6473 1.0160 4x10(4) 1.2087* 0.7387** 0.8584 4x10(5) 1.4107** 0.8900** 0.8147 4x10(6) 1.0033 0.6660 0.7422‡ 4x11(1) 1.1513 0.6300 0.9995 217

Table 7.21 (Continued) 4x11(2) 0.9380 0.4873 1.0606 4x11(3) 1.3200** 0.6187 1.1728 4x11(4) 0.5667 0.3287 0.9271 Mean (x̅ ) 0.9820 0.5371 1.0276 Range 0.5213 - 1.4140 0.1847 - 0.8900 0.7422 - 1.5340 LSD (0.05) 0.1796 0.1504 - LSD (0.01) 0.2388 0.1999 - SD - - 0.2057 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

Mean SSI among genotypes ranged from 0.7422 to 1.5340 with a mean of 1.0276.

Crosses 4x6(1), 4x6(4), 4x8(3), and 4x10(6) showed SSI – 1 SD < 1, indicating these genotypes had more salt tolerance than the other genotypes of this family. Crosses 4x6(1) and 4x6(4) with greater SDW, and cross 4x10(6) with FG% (36.67%) greater than the family mean as well as their SSI – 1SD < 1, these crosses would be more desirable than other crosses in a barley breeding program for improving salt tolerance.

The correlation coefficient of FG% and SDW with morphological traits of

Family 5 genotypes are presented in Table 7.22. Final germination percentage (FG%) under 0 and 100 mM NaCl had a positive association with plant height as well as with flag leaf area under 100 mM. In contrast, FG% under most salinity levels (except 300 mM) associated negatively with tiller number, fertility, cSW, and grain yield. Shoot dry weight (SDW) under all the saline levels associated negatively with plant height, while had a strong positive association with tiller number, fertility, cSW, and grain yield.

Similar associations have been observed in the other families. Interestingly, FG% under

218

100 mM associated positively with flag leaf area, while it showed negative association in

Family 3 and 4.

Table 7.22 Correlation coefficient analysis among germination and seedlings of different quantitative characters; plant height (PH), tiller number (TN), flag leaf area (FLA), fertility (F), 100-seed weight (cSW), grain yield (GY); at control condition, final germination percentage (FG%), and shoot dry weight (SDW) at different level of salinity of 22 genotypes of Family 5 (4x H. vulgare crosses).

FG% SDW

0mM 100mM 200mM 300m 0mM 100mM 200mM 300mM M PH 0.46* 0.49* 0.31 0.07 -0.47* -0.58** -0.64** -0.68*** TN -0.35 -0.55** -0.44* -0.10 0.78*** 0.78*** 0.72*** 0.78*** FLA 0.37 0.45* 0.37 0.39 -0.13 -0.03 -0.07 0.07 F -0.62** -0.79*** -0.67*** -0.22 0.69*** 0.69*** 0.71*** 0.68*** cSW -0.45* -0.66*** -0.57** -0.15 0.74*** 0.73*** 0.74*** 0.76*** GY -0.39 -0.61** -0.48* -0.04 0.68*** 0.65*** 0.69*** 0.72*** * Significant at the 0.05 probability level. ** Significant at the 0.01 probability level. *** Significant at the 0.001 probability level.

The means of SDW, g plant-1 and SSI of Family 5 genotypes at 0 and 300 mM

NaCl are presented in Table 7.23. Mean SDW among genotypes ranged from 0.1847 to

0.6540 g with a mean of 0.4430 g plant-1. Crosses 5x10(1) and 5x11(3) had greater SDW

(0.6393 and 0.6540 g, respectively) compared to the family mean (P ≤ 0.01). Crosses

5x8(3) and 5x10(2) also showed SDW greater than the family mean (P ≤ 0.05). This family showed 48% reduction in the mean SDW under 300 mM compared to control, which indicate the high effects of salinity on plant growth. Chen et al. (2005) found

54.3% reduction in SDW of barley genotypes under 320 mM vs. control.

219

Table 7.23 Mean shoot dry weight (SDW), g plant-1 and salinity susceptibility index (SSI) of parental genotypes and Family 5 crosses (4x H. vulgare crosses) at 300 mM NaCl (Data for SDW and SSI of genotypes at 0, 100, and 200 mM NaCl Appendix Table E).

SDW (g plant-1) SDW (g plant-1) SSI† Genotypes 0 mM 300 mM 300 mM 5 0.7907 0.4307 0.9491 6 0.5213 0.2673 1.0156 7 0.6360 0.2447 1.2826 8 0.6053 0.1847 1.4486 10 0.5780 0.2507 1.1805 11 0.5260 0.2633 1.0409 5x6(1) 0.9860* 0.5607 0.8992 5x6(5) 0.7747 0.4840 0.7821† 5x7(1) 0.7787 0.4287 0.9370 5x7(2) 0.7607 0.4333 0.8970 5x7(3) 0.9867* 0.5320 0.9606 5x8(3) 1.0667** 0.6213* 0.8703 5x8(4) 0.9187 0.3867 1.2071 5x8(6) 0.9333 0.5267 0.9082 5x8(7) 1.0273** 0.5367 0.9956 5x10(1) 1.1820** 0.6393** 0.9570 5x10(2) 0.9387 0.5973* 0.7580† 5x10(3) 0.9893* 0.4433 1.1504 5x10(4) 0.6827 0.4213 0.7980† 5x10(5) 0.7493 0.3920 0.9940 5x11(1) 1.2807** 0.4467 1.3575 5x11(3) 1.0187** 0.6540** 0.7462† Mean (x̅ ) 0.8514 0.4430 1.0062 Range 0.5213 - 1.2807 0.1847 - 0.6540 0.7462 - 1.4486 LSD (0.05) 0.1178 0.1388 - LSD (0.01) 0.1575 0.1856 - SD - - 0.1922 * Value significantly greater than the family mean at 0.05 probability level. ** Value significantly greater than the family mean at 0.01 probability level. † A value < 1.0 indicates saline tolerance. ‡ Value less than mean SSI – 1 SD.

Family 5 genotypes showed a wide range of SSI (0.7462 - 1.4486) with a mean of

(1.0062). Crosses 5x6(5), 5x10(2), 5x10(4), and 5x11(3) had SSI – 1SD < 1, indicating 220

these progeny had more salt tolerance than the other genotypes of this family. Crosses

5x10(2) and 5x 11(3) considered more tolerant due to low SSI values (0.7580 and 0.7462, respectively) with SDW significantly greater than the family mean at 300 mM. Therefore, these two crosses would be desirable in a barley breeding program for saline tolerance.

Barley genotypes of tetraploid crosses (4x H. vulgare crosses) of all families responded differently to the varying salinity levels. Generally, the mean SDW was decreased as the level of NaCl increased in all families (Figure 7.2).

1.2 Family 1 Family 2 1.0 Family 3 Family 4 Family 5 0.8

-1

0.6

SDW, g plant 0.4

0.2

0.0 0 mM 100 mM 200 mM 300 mM

NaCl concentration Figure 7.2 Mean shoot dry weight (SDW), g plant-1 of barley tetraploid crosses (4x H. vulgare crosses) of all families at 0, 100, 200, and 300 mM NaCl.

More reduction of SDW was observed under 300 mM NaCl. Similar observations were also reported by Garthwaite et al. (2005), Adjel et al. (2013), Chalbi et al. (2013), El

221

Goumi et al. (2014), and Angessa et al. (2017). This reduction in SDW with increasing salt concentration may be due to limited the plant ability to take up water, absorption and the transport of the major elements, supply of metabolites to young growing tissues, cell division and elongation, cell wall elasticity, induced Na+ and Cl- accumulation and K+ reduction, reduced the rate of photosynthesis due to leaf area reduction and stomatal closure (Munns et al., 2006; Khan et al. 2008; Kook et al. 2009; Widodo et al. 2009;

Adjel et al. 2013).

Conclusion

Different salinity levels led to different responses of barley genotypes, which were observed as mean SDW comparison. These results confirm the high genetic variation among the screened barley genotypes. The wide variation in genotypes responses to four levels of salinity could be used to provide the best information about initial selection of individual barley genotypes for salt tolerance.

In most families, FG% had positive association with plant height, while showing a negative association with tiller number, fertility, cSW, and grain yield. The exception was in diploid crosses that FG% associated positively with tiller number. In contrast, SDW showed negative association with plant height while it associated positively with tiller number, fertility, cSW, and grain yield. These traits, FG% and SDW, could be used as an efficient tool for screening and selecting salinity tolerance genotypes based on the results obtained here and others.

Families varied in the number of crosses that showed SDW greater than the family mean and SSI less than 1.0 at 300 mM NaCl. Families 2 and 4 of tetraploid crosses (4x H. vulgare crosses) had 12 of 39 and eight of 26 crosses (30.8%) show SDW 222

significantly greater than the family mean and SSI value below1. Therefore, females 2 and 4 are highly desirable in a barley breeding program for saline tolerance.

Although, there was a negative relationship between FG% and SDW under different salinity levels, there were some progeny that showed FG% and SDW greater than the family mean. These crosses could be valuable for using as a genetic material in a breeding program to improve salinity tolerance. Nevertheless, our results need to be confirmed with data from field studies in saline soil.

223

REFERENCES

Abdi, N., S. Wasti, M.B. Salem, M. El Faleh, and E. Mallek-Maalej. 2016. Study on germination of seven barley cultivars (Hordeum vulgare L.) under salt stress. J. Agric. Sci. 8:88-97.

Abu-El-lail, F.F.B., K.A. Hamam, K.A. Kheiralla, and M.Z. El-Hifny. 2014. Salinity tolerance in 280 genotypes of two-rows barley. Egypt. J. Plant Breed. 18:331-345.

Adjel, F., H. Bouzerzour, and A. Benmahammed. 2013. Salt stress effects on seed germination and seedling growth of barley (Hordeum vulgare L.) genotypes. J. Agric. Sustain 3:223-237.

Albacete, A., M.E. Ghanem, C. Martinez-Andujar, M. Acosta, J. Sanchez-Bravo, V. Martinez, ... and F. Perez-Alfocea. 2008. Hormonal changes in relation to biomass partitioning and shoot growth impairment in salinized tomato (Solanum lycopersicum L.) plants. J. Exp. Bot. 59(15):4119-4131.

Angessa, T.T., X.Q. Zhang, G. Zhou, S. Broughton, W. Zhang, and C. Li. 2017. Early growth stages salinity stress tolerance in CM72 x Gairdner doubled haploid barley population. Plos. One. 12:1-16.

Bagci, S.A., H. Ekiz, and A. Yilmaz. 2003. Determination of the salt tolerance of some barley genotypes and the characteristics affecting tolerance. Turk. J. Agric. For. 27:253-260.

Bartels, D., and R. Sunkar. 2005. Drought and salt tolerance in plants. Crit. Rev. Plant Sci. 24:23-58.

Bchini, H., M.B. Naceur, R. Sayar, H. Khemira, and L.B. Kaab‐Bettaeib. 2010. Genotypic differences in root and shoot growth of barley (Hordeum vulgare L.) grown under different salinity levels. Hereditas. 147(3):114-122.

Chalbi, N., K. Hessini, M. Gandour, S.N. Mohamed, A. Smaoui, C. Abdelly, and N.B. Youssef. 2013. Are changes in membrane lipids and fatty acid composition related to salt‐stress resistance in wild and cultivated barley? J. Plant Nutr. Soil Sci. 176(2):138-147.

Chen, Z., I. Newman, M. Zhou, N. Mendham, G. Zhang, and S. Shabala. 2005. Screening plants for salt tolerance by measuring K+ flux: A case study for barley. Plant Cell Enviro. 28(10):1230-1246.

224

Colmer, T.D., R. Munns, and T.J. Flowers. 2006. Improving salt tolerance of wheat and barley: Future prospects. Aust. J. Exp. Agric. 45(11):1425-1443.

El Madidi, S., B. El Baroudi, and F.B. Aameur. 2004. Effects of salinity on germination and early growth of barley (Hordeum vulgare L.) cultivars. Int. J. Agric. Biol. 6(5):767-770.

Ellis, R.P., B.P. Forster, D. Robinson, L.L. Handley, D.C. Gordon, J.R. Russell, and W. Powell. 2000. Wild barley: a source of genes for crop improvement in the 21st Century? J. Exp. Bot. 51(342):9-17.

FAO-AQUASTAT. 2013. Area equipped for irrigation and percentage of cultivated land. Available at http://www.fao.org/nr/water/aquastat/globalmaps/index.stm (accessed 19 July 2018).

Fischer, R.A., and R. Maurer. 1978. Drought resistance in spring wheat cultivars. I. Grain yield responses. Aust. J. Agric. Res. 29(5):897-912.

Foolad, M.R. 2004. Recent advances in genetics of salt tolerance in tomato. Plant Cell Tiss. Org. 76:101-119.

Fricke, W., G. Akhiyarova, W. Wei, , E. Alexandersson, et al. 2006. The short-term growth response to salt of the developing barley leaf. J. Exp. Bot. 57(5):1079- 1095.

Hoagland, D.R., and D.I. Arnon. 1950. The water culture method for growing plants without soil. Agric. Exp. Sta. Circ. Berkeley CA. 347:1-32.

Jamshidi, A., and H.R. Javanmard. 2017. Evaluation of barley (Hordeum vulgare L.) genotypes for salinity tolerance under field conditions using the stress indices. Ain Shams Eng. J. http://dx.doi.org/10.1016/j.asej.2017.02.006 (accessed 12 June 2018).

Khan, M.J., J. Bakht, I.A. Khalil, M. Shafi, and M. Ibrar. 2008. Response of various wheat genotypes to salinity stress sown under different locations. Sarhad J. Agric. 24(1):1-21.

225