MULTIPLE TRANSGENE ENGINEERING FOR MAIZE (Zea mays L.) DROUGHT
AND SALT TOLERANCE
By
Thang Xuan Nguyen

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Crop and Soil Sciences
2012

ABSTRACT
MULTIPLE TRANSGENE ENGINEERING FOR MAIZE (Zea mays L.) DROUGHT
AND SALT TOLERANCE
By
Thang Xuan Nguyen
Maize (Zea mays L.) is an important basic food and feed grain, and its stover is used for
animal feed and biofuel. Increased soil-salinity and water-deficiency are the two major factors
limiting the maize plant growth and development and subsequently affecting its grain and
biomass yields, and the yield. The research in this dissertation is focused on multiple transgene
engineering (transgene pyramiding) for drought and salt tolerance in maize.
In the research presented in this dissertation, genetic transformation of maize was
performed via the gene gun bombardment of embryogenic immature embryos of maize using
single and multiple constructs, pBY520 containing the barley (Hordeum vulgare) HVA1 and the
JS101 containing the bacterial mannitol 1 phosphate dehydrogenase (mtlD), both genes regulated
by rice actin promoter and potato protease inhibitor II terminator. There were two gene linked
cassettes in each of these two constructs, one cassette containing the abiotic stress tolerance gene
of interest (HVA1 or mtlD) and the other containing the bar herbicide resistance gene regulated
by cauliflower mosaic virus 35S promoter and nos terminator.
The pBY520 and JS101 constructs were co-bombarded in 1:1 ratio into maize genome
for transgene pyramiding. The confirmation of transgene integration and expression were made
via molecular techniques, including polymerase chain reaction (PCR) for transgene integration,
and reverse transcriptase (RT) PCR and Northern blotting for transgene transcription. Southern
blotting was performed to find the number of copies of each transgene in transgenic plants.
Results showed stable integration and expression of the HVA1, mtlD and HVA1-mtlD in
transgenic maize plants. Up to fourth generation transgenic (T3) plants were produced, with all
progenies showing the co-integration of abiotic stress tolerance genes of interests and the bar
gene with a frequency of 100%. The single HVA1 or mtlD transgenic plants showed higher leaf

relative water content (RWC) and higher percent of plant survival as compared to their wild-type
non-transgenic control plant counterparts under water withholding condition for 15 days
followed by 7 days of re-watering. When exposed to different salt concentrations (0, 100, 200
and 300 mM NaCl) for 10 days, the HVA1 and mtlD transgenic plants showed higher fresh and
dry shoot and dry root biomass matter as compared to their wild-type non-transgenic control
plants. The research also demonstrated that the mtlD transgenic plants that were salt tolerant, also
accumulated mannitol in their cells. More research is needed on mannitol accumulation in
transgenic plants to see whether the mannitol level measured by gas chromatography (GC) was
not partially or totally representing sorbitol accumulation. Considering that mannitol and sorbitol
are both osmoprotectants, the salt tolerance of the mtlD transgenic plants might be due to the
accumulation of one or both osmoprotectants in mtlD transgenic plants.
Co-transformation of two transgenes (HVA1+mtlD) in maize plants also demonstrated the
co-integration and co-expression of these two stacked genes in up to T3 plants, resulting in
improved plant survival rate under 15 and 20 days of water withholding and enhanced shoot and
root biomass weight at 100mM NaCl as compared to single transgene (HVA1 or mtlD) transgenic
plants and as compared to the wild-type control plants.
This dissertation also covers genetic transformation of maize plants with a construct
containing the sorghum dehydration responsive element binding 2 (DREB2) transcription factor
regulated by the Arabidopsis rd29 drought inducible promoter and nos terminator. The
preliminary results showed the integration of DREB2 into maize plants via PCR analysis. The
DREB2 transgenic maize research will be continued in Sticklen’s laboratory by another
researcher in the near future.
Although salt stress is predictable, drought is not predictable in most counties on earth.
The dissertation research presented here is a step towards production of maize plants that can
tolerate the harsh abiotic stress conditions of drought and salt.

© COPYRIGHT BY
THANG XUAN NGUYEN
2012

DEDICATION
I dedicate this work to my wife, my son, and my daughter for their patience, love,
encouragement and tireless support throughout my studies.

v

ACKNOWLEDGEMENTS
I would like to thank my major advisor Dr. Mariam Sticklen for all her guidance and
support, providing me with great scientific advices in my research project and for providing
financial assistance when I was in financial struggling situation. I also wish to sincerely thank
Dr. Russell Freed, Dr. James Hancock, Dr. Dechun Wang, and Dr. Janet Lewis for accepting to
be members of my PhD guidance committee and for their valuable advices and comments in my
research. I also would like to thank Dr. Wayne Loescher for giving me opportunities to work in
his Laboratory, and for his collaboration and guidance in the mannitol section of my research. I
would like to thank my parents Mr. and Mrs. Nguyen, my wife Phan Huyen, my son Xuan Minh,
my daughter Anh Thu, and all my brothers and sisters for their love, encouragement, and tireless
support throughout my studies. My PhD program would not have been possible without their
support. I also give my sincere appreciation to AST project, MAD and NMRI of Vietnam for
giving me the opportunity to pursue my PhD program at MSU and providing me with a full
scholarship for my Ph.D. research. I would like to thank Mr. Chris E. and Mrs. Judith M.
Rossman Foundation, CANR Dean’s office, Dr. James Kells, Dr. Taylor Johnston, as well as
Plant, Soil and Microbial Department for their help and financial supports. I also thank my
research laboratory team members and colleagues Kingdom Kwapata, Sang Park, Jason Matakas,
Hussien Alameldin., Truong, Dung, and Mingzhang for their excellent collaboration and
discussions. I would also like to thank Dr.Guo-Qing Song, Veronica, Ann and Jesse for their
help and comments on molecular works. My sincere thanks also go to Mrs. Robab Sabzikar, our
senior lab technician for her guidance on using lab equipment. Last but certainly not least, I
would like to acknowledge and thank Darlene Johnson, Sandie Litchfield, Therese Ladipaolo,
Calvin Bricker and my best friends Vy, Tuan, Lenh, Trung, and Uy for all their moral supports.

vi

TABLE OF CONTENTS

LIST OF TABLES

xiii

LIST OF FIGURES

xiv

KEY TO SYMBOLS AND ABBREVIATIONS

xviii

CHAPTER I

5

BARLEY GENE HVA1 CONFERS DROUGHT AND SALT TOLERANCES IN
TRANSGENIC MAIZE (Zea mays. L)

5

1.1. LITERATURE REVIEW ........................................................................................................ 5
1.1.1. Introduction ............................................................................................................... 5
1.1.2. LEA – Late Embryogenesis Abundant proteins ....................................................... 6
1.1.3. Expression of the Barley HVA1 gene conferring abiotic stress tolerance in plants. . 7
1.2. RESEARCH GOAL AND OBJECTIVES ............................................................................ 11
1.3. MATERIALS AND METHODS ........................................................................................... 11
1.3.1. Explants................................................................................................................... 11
1.3.2. Gene construct ........................................................................................................ 11
1.3.3. Transformation of plasmid vector into E. coli competent cells .............................. 12
1.3.4. BiolisticTM bombardment ........................................................................................ 13
1.3.5 Confirming the integration and expression of transgenes in putatively transgenic
plants ................................................................................................................................. 14
1.3.6. Biological activity tests ........................................................................................... 16
1.4. RESULTS AND DISCUSSIONS .......................................................................................... 18
1.4.1. Maize tissue culture and regeneration..................................................................... 18
1.4.2. Integration of HVA1 and bar transgenes via PCR and Southern blot analysis ....... 20
1.4.3. HVA1 transcription confirmed by RT-PCR and Northern blot analysis ................. 23
1.4.4. Confirmation of herbicide resistance in bar transgenic plants ............................... 24
1.4.5. Confirmation of drought stress tolerance in T3 plants............................................ 26
1.4.6. Salt stress tolerance in T3 plants ............................................................................. 30
CHAPTER II

38

BACTERIAL MTLD GENE CONFERS SALT TOLERANCE AND ACCUMULATION OF
MANNITAL IN TRANSGENIC MAIZE (Zea Mays. L)
38
2.1. LITERATURE REVIEWS .................................................................................................... 38
vii

2.1.1. Introduction ............................................................................................................. 38
2.1.2. Biosynthesis of compatible solutes in plants for drought and salt tolerance ......... 38
2.1.3. Expression of bacterial mtlD gene in transgenic plants .......................................... 41
2.2. GOAL AND OBJECTIVES .................................................................................................. 43
2.3. MATERIALS AND METHODS ........................................................................................... 43
2.3.1. Gene constructs ....................................................................................................... 43
2.3.2. Conformation of gene integration and expression .................................................. 44
2.3.3. Salinity stress tolerance test .................................................................................... 45
2.3.4. Mannitol analysis .................................................................................................... 46
2.3.5. Analysis of photosynthetic gas exchange ............................................................... 47
2.4. RESULTS AND DISCUSIONS ............................................................................................ 47
2.4.1. Integration of mtlD transgene confirmed by PCR and Southern blot analysis, and
confirmation of mtlD transcription via northern blotting ................................................. 47
2.4.2. Salt stress tolerance of T3 mtlD plants ................................................................... 49
COEXPRESSION OF A COMBINATION OF HVA1 AND MTLD TRANSGENES CONFERS
DROUGHT AND SALT TOLERANCE IN TRANSGENIC MAIZE PLANTS
58
3.1. LITERATURE REVIEWS .................................................................................................... 58
3.1.1. Introduction ............................................................................................................. 58
3.1.2. Strategies for transgene stacking via breeding of transgenic plants ....................... 59
3.1.3. Strategies for transgene stacking via re-transformation of transgenic plants ......... 60
3.1.3. Strategies for transgene stacking via co-transformation of transgenic plants......... 61
3.2. RESEARCH GOAL AND OBJECTIVES ............................................................................ 64
3.3 MATERIALS AND METHODS ............................................................................................ 65
3.4. RESULTS AND DISCUSSIONS .......................................................................................... 65
3.3.1. Confirmation of integration of the stacked HVA1-mtlD transgenes via PCR ......... 65
3.3.2. Drought stress tolerance in T3 transgenic maize plants that were co-transformed
with two constructs ........................................................................................................... 68
3.3.3. Salinity stress tolerance in T3 transgenic plants ..................................................... 73
3.3.4. Accumulation of mannitol and other soluble sugar contents after 7 days exposing to
different salinity concentrations. ....................................................................................... 76
CHAPTER IV

80

SORGHUM TRANSCRIPTION FACTOR DREB2 TRANSGENIC MAIZE PLANTS FOR
DROUGHT AND SALT TOLERANCE

80

viii

4.1. LITERATURE REVIEW ...................................................................................................... 80
4.2. GOALS AND OBJECTIVES: ............................................................................................... 83
4.3. MATERIALS AND METHODS ........................................................................................... 84
4.3.1. Gene construct ........................................................................................................ 84
4.3.2. Conformation of SbDREB2 transgene via PCR analysis ....................................... 84
4.4. RESULTS AND DISCUSSIONS .......................................................................................... 85
CHAPTER V

86

CONCLUSIONS AND FUTURE PERSPECTIVES

86

APPENDICES

90

APPENDIX 1: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF WATER STRESS ON LEAF RELATIVE WATER CONTENT (%) OF
HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ............................................... 91
APPENDIX 2: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT LENGTH
(cm) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS................................. 92
APPENDIX 3: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT LENGTH (cm)
OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ......................................... 93
APPENDIX 4: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (gr) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ................. 94
APPENDIX 5: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY
WEIGHT (gr) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ................. 95
APPENDIX 6: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT FRESH
WEIGHT (gr) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ................. 96
APPENDIX 7: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY
WEIGHT (gr) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS ................. 97
APPENDIX 8: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT LENGTH REDUCTION (%) OF T3
HVA1 TRANSGENIC AND WILD-TYPE PLANTS ...................................................... 98
ix

APPENDIX 9: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT LENGTH REDUCTION (%) OF T3
HVA1 TRANSGENIC AND WILD-TYPE PLANTS ...................................................... 99
APPENDIX 10: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%)
OF T3 HVA1 TRANSGENIC AND WILD-TYPE PLANTS ........................................ 100
APPENDIX 11: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF
T3 HVA1 TRANSGENIC AND WILD-TYPE PLANTS .............................................. 101
APPENDIX 12: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT FRESH WEIGHT REDUCTION (%) OF
T3 HVA1 TRANSGENIC AND WILD-TYPE PLANTS .............................................. 102
APPENDIX 13: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF
T3 HVA1 TRANSGENIC AND WILD-TYPE PLANTS .............................................. 103
APPENDIX 14: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON PLANT HEIGHT
GROWTH RATE (cm/day) OF MTLD TRANSGENIC AND WILD-TYPE PLANTS 104
APPENDIX 15: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (gr) OF MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS .............. 105
APPENDIX 16: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY
WEIGHT (gr) OF MTLD TRANSGENIC AND WILD-TYPE PLANTS ..................... 106
APPENDIX 17: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY
WEIGHT (gr) OF MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS .............. 107
APPENDIX 18: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%)
OF T3 MTLD TRANSGENIC AND WILD-TYPE PLANTS ....................................... 108
APPENDIX 19: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF
T3 MTLD TRANSGENIC AND WILD-TYPE PLANTS.............................................. 109
APPENDIX 20: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF
T3 MTLD TRANSGENIC AND WILD-TYPE PLANTS.............................................. 110

x

APPENDIX 21: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON MANNITOL
ACCUMULATION (µmol/gr FW) OF MTLD TRANSGENIC AND WILD-TYPE
PLANTS ......................................................................................................................... 111
APPENDIX 22: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF WATER STRESS ON LEAF RELATIVE WATER CONTENTS (%) OF
HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS 112
APPENDIX 23: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT LENGTH
(cm) OF HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS ......................................................................................................................... 114
APPENDIX 24: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (gr) OF HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILDTYPE PLANTS .............................................................................................................. 115
APPENDIX 25: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY
WEIGHT (gr) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS ......................................................................................................................... 116
APPENDIX 26: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY
WEIGHT (gr) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS ......................................................................................................................... 117
APPENDIX 27: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT LENGTH REDUCTION (%) OF HVA1,
MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS ........................... 118
APPENDIX 28: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%)
OF HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS ......... 119
APPENDIX 29: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF
HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS ............... 120
APPENDIX 30: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF
HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS ............... 121
APPENDIX 31: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT OF
DIFFERENT SALINITY CONCENTRATIONS ON MANNITOL CONTENTS
xi

(µmol/gr FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS ......................................................................................................................... 122
APPENDIX 32: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT OF
DIFFERENT SALINITY CONCENTRATIONS ON GLUCOSE CONTENT (µmol/gr
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
......................................................................................................................................... 123
APPENDIX 33: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT OF
DIFFERENT SALINITY CONCENTRATIONS ON SUCROSE CONTENTS (µmol/gr
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
......................................................................................................................................... 124
APPENDIX 34: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT OF
DIFFERENT SALINITY CONCENTRATIONS ON FRUCTOSE CONTENTS (µmol/gr
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
......................................................................................................................................... 125
APPENDIX 35: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT OF
DIFFERENT SALINITY CONCENTRATIONS ON INOSITOL CONTENTS (µmol/gr
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
......................................................................................................................................... 126
REFERENCES

127

xii

LIST OF TABLES
Table 1: The expression of HVA1 gene in different transgenic crops ......................................... 10
Table 2: Reduction in shoot and root length and their biomass affected by salt treatments......... 34
Table 3: Effect of salinity stress on number of leaves per plant after 6 days of NaCl treatment . 36
Table 4: Expression of bacterial mannitol-1-phophate dehydrogenase (mtlD) confers biochemical
changes resulting in drought and salinity tolerance in different crop species. ..................... 42
Table 5: Effect of salt stress treatments on the absolute plant growth rate in T3 mtlD plants ..... 50
Table 6: Biomass growth reduction of T3 mtlD plants versus the wild-type control plant treated
with different NaCl concentrations for 10 days .................................................................... 52
Table 7: Effects of salt stress treatment on T3 plant photosynthesis and stomatal conductance.
Values are means ± SEs (n=3) .............................................................................................. 55
Table 8: Effect of drought tolerance on the percentage of survival of T3 lines versus wild-type
control plants that were exposed to water deficit for 15 days followed by 7 days of recovery
from water deficit. ................................................................................................................. 70
Table 9: Effects of salt treatment on the percentage reduction of shoot length, shoot fresh weight,
and shoot and root dry weight of each set of three transgenic plants (HVA1, mtlD, and
HVA1-mtlD) affected by different concentrations of salt treatments. Number in this table
represent the percentage of reduced shoot length, shoot fresh weight, or shoot and root dry
weight as compared to those of transgenic and wild-type plants at 0mM NaCl (non salt
treatment). ............................................................................................................................. 74
Table 10: Effects of different salt concentrations on mannitol sucrose, glucose, fructose and
inositol contents in transgenic and wild-type plants treated with 0, 100, 200, or 300mM
NaCl. ..................................................................................................................................... 76

xiii

LIST OF FIGURES
Figure 1: Linear map of pBY520 construct. Rice actin promoter (Act1), Barley (Hordeum
vulgare or HVA1 LEA3 gene, Cauliflower Mosaic Virus 35S (35S) promoter, Bar Liberty
herbicide resistance gene (bar) and nopaline synthase terminator (Nos). For interpretation of
the references to color in this and all other figures, the reader is referred to the electronic
version of this dissertation. ................................................................................................... 12
Figure 2: Maize in vitro culture, plant regeneration and breeding of transgenic plants: (A)
Highly embryogenic Hi II immature embryos (B) Highly embryogenic immature embryos
after transgene bombardment; (C) Embryogenic tissue growth in selection medium; (D) and
(E) embryogenic tissues in regeneration media; (F) plantlet growth in rooting media; (G)
Acclimatization of plantlets in growth chamber; (H) plants growing in greenhouse; (I) Plant
breeding, and (J) breeding of mature plants. ......................................................................... 19
Figure 3: PCR analysis of T0 HVA1 transgenic plants confirming the correct expected band size
of 680bp. ............................................................................................................................... 21
Figure 4: PCR analysis of T1 HVA1 and bar transformants (line#132) showing the expected
band size of 680 bp for HVA1 and 484bp for bar gene. ....................................................... 21
Figure 5: PCR analysis of T2 of HVA1 transformants (line#132) with correct expected band size
(680bp). ................................................................................................................................. 21
Figure 6: Southern blot showing integration of HVA1 gene in T0 transgenic plants. P: pBY520
plasmid as positive control. WT: wild-type. Digestion was performed using the HindIII
restriction enzyme. ................................................................................................................ 22
Figure 7: RT-PCR showing expression of HVA1 transgene in T1 transgenic plants; + pBY520
plasmid positive control, wt: wild-type. The expected band size for HVA1 gene is 680bp.
The lower portion of this figure shows the expression of maize endogenous actin1 gene with
an expected band size of 430bp is loaded as control to show that the RT-PCR has been
working well. ........................................................................................................................ 23
Figure 8: Northern blot expression of HVA1 transgene in T0 transgenic lines; WT: wild-type
(non-transgenic plant). .......................................................................................................... 24
Figure 9: Northern blot expression of HVA1 transgene in T1 transgenic lines; WT: wild-type
(non-transgenic plant). .......................................................................................................... 24
Figure 10: Kill curve for herbicide selection: Young leaves of wild-type non-transgenic plants
painted with 5 different concentrations of commercial Liberty herbicide containing 18.2%
glufosinate anominum. The concentration of 350mg/L glufosinate anominum was used to
apply for transgenic plants. ................................................................................................... 25
Figure 11: Leaf painting (at 350mg/L glufosinate ammonium after 7 days) showing herbicide
injury symptom on non-transgenic plant versus T1 transgenic plant confirmed resistant. .. 26

xiv

Figure 12: Percentage of leaf relative water content (% RWC) of HVA1 transgenic line #132
after 10 and 15 days of water deficit (withholding). ............................................................. 27
Figure 13: Percentage of survival rate of transgenic versus wild-type control plant after 7 days of
water recovery treatment ....................................................................................................... 28
Figure 14: Effect of drought stress on plant growth and mature root system of HVA1 transgenic
maize versus its wild-type control plants. The pictures were taken for drought stress
tolerance of T3 HVA1 transgenic plants after one week of water recovering (A), and mature
roots (B) ................................................................................................................................ 29
Figure 15: Effect of salt treatment on shoot length of T3 plants. ................................................. 31
Figure 16: Effect of salt treatment on root length of T3 plants. ................................................... 31
Figure 17: Effect of salt treatment on shoot fresh weight of T3 plants. ....................................... 32
Figure 18: Effect of salt treatment on shoot dry weight of T3 plants. .......................................... 32
Figure 19: Effect of salt treatment on root fresh weight of T3 plants........................................... 33
Figure 20: Effect of salt treatment on root dry weight of T3 plants. ............................................ 33
Figure 21: Effect of salt stress on seedling growth (A) and root system (B) of HVA1 transgene
and wild-type plants after 10 days of NaCl treatment. ......................................................... 35
Figure 22: Effect of salinity stress on the development of leaves in transgenic and wild-type
plants. Picture was taken after 6 days of NaCl treatment ..................................................... 37
Figure 23: A summary of the pathway for the synthesis of mannitol. .......................................... 40
Figure 24: pJS101 plasmid constructs containing a bacterial mtlD gene which is driven by Actin
Rice Promoter (Act1) and potato proteinase inhibitor terminator (Pin); and selectable
marker bar gene with Cauliflower Mosaic Virus (CaMV) 35S promoter and nopaline
synthase terminator (Nos). .................................................................................................... 44
Figure 25: T3 and wild-type plants at 4-leaf stage growth before being exposed to four different
salinity concentrations. ......................................................................................................... 45
Figure 26: PCR analysis of T0 mtlD gene. The expected band size was 431 bp. ........................ 47
Figure 27: PCR analysis confirmed the mtlD transgene in T1 plants, with the expected band
size of 431 bp. ....................................................................................................................... 48
Figure 28: PCR analysis confirmed the stable co-integration of mtlD gene and bar gene in T2
plants. +: positive DNA from JS101 plasmid; - H20 and wt: wild-type loading as negative
control; 1-5: genomic DNA of transgenic plants. The expected band size is 431 bp. .......... 48
Figure 29: Northern blot hybridization showing the expression of mtlD gene in T1 plants. ....... 49
xv

Figure 30: Northern blot hybridization showing the expression of mtlD gene in T2 plants ........ 49
Figure 31: Effect of different salinity conditions on shoot fresh weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3) ............................. 50
Figure 32: Effect of different salinity conditions on shoot dry weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3) ............................. 51
Figure 33: Effect of different salinity conditions on root dry weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3). ............................ 51
Figure 34: T3 mtlD versus wild-type control plants after 7 days of water treatment recovery from
10 days of 200mM NaCl salinity treatment. ......................................................................... 53
Figure 35: Effect of salt stress treatment on photosynthesis of T3 mtlD versus that of the wildtype non-transgenic control plants after 7 days of water treatment recovery regime from 10
days of 100mM NaCl. T: T3, NT: non-transgenic control plants. ....................................... 55
Figure 36: Accumulation levels of mannitol in leaves of mtlD transgene plants treated 7 days
with different concentration of NaCl. ................................................................................... 56
Figure 37: PCR analysis of co-integration of HVA1 and mtlD genes in T1 maize lines. ............. 65
Figure 38: Northern blot of HVA1 transgenes in T1 HVA1-mtlD plants using HVA1 probe ..... 66
Figure 39: Northern blot hybridization of mtlD transgene in T1 of HVA1-mtlD transgenes plants
using mtlD probe................................................................................................................... 66
Figure 40: Northern blot hybridization of HVA1 in HVA1-mtlD T2 plants using HVA1 probe 67
Figure 41: Northern blot hybridization of mtlD transgene in HVA1-mtlD in T2 plants using mtlD
probe ..................................................................................................................................... 67
Figure 42: PCR analysis confirming the stable integration of both HVA1 and mtlD transgenes in
T3 plants (line #51-1)............................................................................................................ 68
Figure 43: T3 transgenic and wild-type plants at four week old plants before exposing to water
withholding. .......................................................................................................................... 69
Figure 44: Percentage of leaf relative water content (% RWC) of transgenic lines after 10 and 15
days of water deficit (withholding). Bars are mean ± SE of three samples. ......................... 69
Figure 45: Effect of 15 days of drought stress followed by 7 days of water treatment recovery on
the survival of HVA1-mtlD transgenic plants (lower left) as compared with the HVA1
(upper left) and mtlD transgenic plants (upper right), and wild-type non-transgenic plants.71
Figure 46: Effect of 20 days of drought stress on the growth of HVA1, mtlD, and HVA1- mtlD
transgenic plants as compared with the wild-type non-transgenic plants. ............................ 72
xvi

Figure 47: Comparisons of the effect of 10 days of salt treatments on the growth of T3 of HVA1,
mtlD and HVA1-mtlD plants versus their wild-type control plants. .................................... 75
Figure 48: pCAMBIA1301 plasmid containing the sorghum transcription factor SbDREB2
regulated by Arabidopsis inducible promoter (rd29A), and nos terminator, cauliflower
mosaic virus 35S promoter and Hygromycin phosphotransferase (hpt) gene. ..................... 84
Figure 49: PCR analysis confirming the integration of hpt gene in the first generation (T0)
transgenes plants. .................................................................................................................. 85

xvii

KEY TO SYMBOLS AND ABBREVIATIONS
ABA: Abscisic Acid
Act1: Actin Rice Promoter
BAP: Benzylaminopurine
CaMV35S: Cauliflower Mosaic Virus (CaMV) 35S Promoter
DREB: Dehydration Responsive Binding Element
Hpt: Hygromycin phosphotransferase
Hsp: Heat Shock Protein
HVA1: Hordeum vulgaris abandant protein
IBA: Indole-3-butyric Acid
JS101: plasmid containing bar and mtlD gene
LB: Luria Bentani media
MS: Murashige and Skoog media
Nos: nopaline synthase terminator
pBY520: plasmid containing bar and HVA1 gene
pC1301: plasmid containing hpt and DREB2 gene
PCR: Polymerase Chain Reaction
PinII: potato proteinase inhibitor terminator
ROS: reactive Oxygen Species
RT-PCR: Reverse Transcription- PCR

xviii

INTRODUCTION
After wheat and rice, maize is one of the most important cereal crops in the world with a
global production of 844.4 million tons. The United States is the largest maize producer in the
world with average maize yield of 9.59 tons per ha and production of 316.16 million tons in
2010 (FAO, 2010). The yield and production of maize has been predicted to increase rapidly in
the coming decades via the continuation of the use of plant breeding and biotechnology,
including genetic transformation to meet the global increase demand for food, feed and industrial
needs (Vega et al., 2008).
Plant regeneration is the first important step which contributes to the success in plant
genetic transformation. In the last years, maize scientists have tried to use different explants
cultured and grown in different media, and then finally regenerated fertile plants (Sahrawat et al.,
2003; Shan et al., 2009).
Maize tissue culture was initiated by Green and Phillips in 1975 when they used a
compact type-I immature embryo callus from inbred line A188 in their studies. However, the
problem with this callus type was that it grew very fast and therefore did not produce large
number of somatic embryos. Then, Armstrong and Green cultured maize immature embryos on
N6 media resulting in large number of somatic embryos. In their report, they explained the type
I callus as-less embryogenic culture, but type II as highly embryogenic. In their research,
regeneration and transformation of maize Hi II derived from a cross of A188xB73 (Armstrong et
al. 1991) as it was the best choice for maize transformation due to it’s highly type II
embryogenic callus. In the author’s dissertation, he used the type II HiII maize callus for his
maize regeneration and genetic transformation.

1

Jones and Reiter (1992) regenerated maize plants from immature embryo-derived shoot
meristems. However, this organogenic system has been less used due to the production of
chimeric plants, plants that had transformed and non-transformed cells (Lowe et al., 1995).
A very efficient and genotype independent regeneration of maize was developed by the
Sticklen team based on the plasticity and manipulation ability of mature seeds germinated shoot
apical meristem primordium and their in vitro multiplication ability (Zhong et al., 1992).
Maize transformation technology was initially developed in the 1990s for production of
European corn border resistant plants. To date, the introduction of different genes into maize has
been adapted to increase resistance to pests, herbicides and other biotic and abiotic stresses
resulting in improved maize productivity for food, animal feed, biofuel and other expanding
demand.
In the last decades, the development of several plant transformation methods was adopted
to introduce genes into plant tissues. The concept of Agrobacterium-mediated transformation
was developed in the 1970s and commonly used up to now in dicotyledonous plants because
these plants could be infected by Agrobacterium (Zupan and Zambryski., 1995; Oneto et al.,
2010). However, in monocotyledonous plants including maize, this technique has shown the
limitations of Agrobacterium infection (Komari et al., 1998; Kriz and Larkins., 2009).
Biolistic or particle bombardment technology has been considered the best choice and
used widely for maize transformation (Zhong et al., 1996; Frame et al., 2000; Aulinger et al.,
2003). Sticklen team used apical shoot meristem primordium bombardment for an efficient and
relatively independent genetic transformation of maize. The success of this system is based on
the plasticity and manipulation ability of this explant and its in vitro multiplication ability
(Zhong et al., 1996). In their technique, the team bombarded the inert particles such as tungsten

2

or gold which were coated with genes of interest to penetrate into the plant meristem subepidermal cell layer (stem cell layer) because this layer has the potential to produce fertile
transgenic plants (Sticklen and Oraby, 2005) using 22 caliber shells. Other scientists used the
Biolistic device (gene gun) to bombard genes into immature embryos using helium gas instead of
22 caliber shells (Li et al., 2003; Fadeev et al., 2006).
Wu et al. (2002) used particle bombardment method and introduced up to nine
independent genes at once, including selectable markers and expressed independently in
transgenic rice. Then, the system was used for transformation of functioning artificial mini
chromosome into maize (Yu et al., 2007).
In addition to the method of DNA delivery system and explants used, other factors are
important for high efficiency in plant genetic transformation.

These include; the use of

genotype, selectable marker and types of in vitro culture media (Decima et al., 2010). Using
suitable selection markers might be the most important factor in plant transformation (Sahrawat
set al., 2003). The ideal selectable marker for dicotyledonous genetic transformation include;
neomycin phosphotransferaseII (nptII) and hygromycin phosphotransferase (hpt) genes. In
addition, the beta-glucuronidase (gus) gene has been utilized widely to select transgenics from
non transgenic events via color indication. Nevertheless, the use of those markers in maize cell
selection has not been efficient (Kriz and Larkins. 2009). However, the genes encoding a mutant
form of acetohydroxy acid synthase (AHAS) protein or the 5-enolpyruvylshikimate-3-phosphate
(EPSP) synthase have been used successfully in maize transformation (Peng et al., 2006).
However, the most successful selectable marker gene for maize transformation is the bialaphos
resistance (bar) gene, a gene encoding phosphinothricin acetyltransferase (PAT) (Zhao et al.,

3

2001; Frame et al., 2002; Li et al., 2003; Valdez et al., 2004; Huang et al., 2005; Ishida et al.,
2007; Stickle and Oraby, 2005; Decima et al., 2010).
Among other factors, salinity and drought have been two major stress factors reducing
maize seed yield, yield consistency and biomass yield. Salinity and drought play important
stress factors affecting plant growth and development. Most of reports suggest that drought and
salinity have similar effects on the plant cells as the input signal for drought is believed to be
reduction of the turgor pressure due to water loss of the cells, and the input signal for salt stress
is the high concentration of soluble salts inside cells.
The dissertation presented here is on the development of salinity and drought tolerance in
maize genotypes via the transfer of the barley HVA1, the bacterial mtlD, and a combination of
the HVA1-mtlD transgenes into maize genome. This dissertation also covers the transfer of the
Sorghum bicolor Dehydration Responsive Element Binding (sbDREB2) transcription factor
regulated by a drought inducible promoter into maize genome. The salinity and drought tolerance
of transgenic maize plants showing the integration of sbDREB2 will be completed and published
elsewhere.

4

CHAPTER I
BARLEY GENE HVA1 CONFERS DROUGHT AND SALT TOLERANCES IN
TRANSGENIC MAIZE (Zea mays. L)
1.1. LITERATURE REVIEW
1.1.1. Introduction
Maize is an important basic food and feed grain, and its stover (leaves and stems) is used
for animal feed and biofuel. Increased soil-salinity and water-deficiency are the two major
factors limiting the maize plant growth and development and subsequently affecting its yield and
the yield. Maize is considered more sensitive to salt stresses at the early stages of its growth and
development compared to all other cereal crops with the exception of rice (Cramer., 1994, US
Salinity Laboratory, 2006). Also, maize requires adequate water in all stages of its physiological
development to reach optimum productivity (Zhang et al., 2008). In the past decades, many
attempts to increase maize yield and yield stability have been taken through conventional
breeding and selection under drought and salt conditions (Duvick and Cassman, 1999; Troyer,
1996). However, conventional maize breeding for drought and salt tolerances has not been very
successful due to the complex interaction between genotype and environment (Johnson and
Geadelmann, 1989). Therefore, maize genetic engineering for drought and salt tolerance is
necessary not only to increase understanding of complex drought and salt tolerance, but also
relatively quickly create commercial varieties tolerant to drought and salt stresses in order to
meet the demands in planting maize in the salinity soil and drought stressed areas of the world.
Plant drought and salt tolerance is reported to be related to a group of late embryogenesis
abundant proteins.

5

1.1.2. LEA – Late Embryogenesis Abundant proteins
The plant hormone abscisic acid (ABA) regulates many key processes in plants including
the response to abiotic stresses (Hubbard et al., 2010), and the processes induce the expression of
genes encoding proteins involved in desiccation tolerance and dormancy in seeds (Hong et al.
1992; Gomez-Cadenas et al., 1999).
Under drought conditions, the ABA level increases in plants. Such increase in ABA
results in the closure of stomata and therefore a decrease in transpiration from leaves. The role of
ABA is regulating of multiple stress responsive genes related to drought or salt stresses in
response to drought to protect the plant cells from osmosis (Finkelstein and Rock, 2002).
Late embryogenesis abundant (LEA) proteins are encoded by ABA-induced genes, first
discovered as accumulating during late embryogenesis of wheat and cotton and subsequently
they were found in different other organs of various plant species (Chen et al., 2002).
LEA proteins are generally divided into several groups based on the structural domains or
chemical characteristics, and found to be expressed under cellular stress conditions such as
drought, desiccation, and cold, osmotic stress and heat (Wise, 2003). Many genes encoding LEA
proteins have been successfully isolated under such conditions. For example, according to
Hundertmark and Hincha (2008), 55 protein encoding genes have been isolated and classified
into nine different groups from the Arabidopsis thaliana genome. The expression of all these
genes was detected in various organs in which half of them displayed high expression levels.
The accumulation of LEA proteins in different transgenic plants has been demonstrated
to confer resistance to various abiotic stresses including water deficiency. For instance, Liu et al.
(2009) reported that in transgenic tobacco, a group of two LEA genes called BhLEA1and
BhLEA2, encoding group 4 LEA proteins were induced by dehydration and signaling molecules
including ABA, resulting in drought and salt tolerance. Transgenic tobacco plants expressing
6

BhLEA1 and BhLEA2 had higher relative water content of leaves, higher photosystem II activity,
and lower membrane permeability than their wild-type control plants. Moreover, high
accumulation of BhLEA1 and BhLEA2 proteins was found under drought tolerance. The research
by this group confirmed the important role of LEA protein accumulation in transgenic tobacco in
the protection and maintenance of plant cell membrane during dehydration.
LEA gene from Tamarix transferred to blueberry (Vaccinium corymbosum) via
Agrobacterium tumefaciens method resulted in an increase cold tolerance of transgenic plants
(Zhao et al., 2011). Transgenic plants of this experiment exposed to 0oC for 24 hrs showed
improved activities of peroxidase and superoxide dismutase, and also significantly reduced level
of lipid peroxidation as compared to non-transgenic plants.
Different LEA protein genes have been isolated and conferred cold, drought or salinity
tolerance in rice (Oryza sativa L.) (Xiao et al., 2007); lettuce (Lactuca sativa) (Park et al.,
2005a); cabbage (Brassica oleracea) (Park et al., 2005b); Arabidopsis (Figueras et al., 2004);
and tobacco (Nicotiana tabacum) (Ziat et al., 2011). The LEA protein gene from barley
(Hordeum vulgare L.) has been studied the most.
1.1.3. Expression of the Barley HVA1 gene conferring abiotic stress tolerance in plants.
HVA1 is a group 3 LEA proteins that specifically expresses and accumulates in barley
seed aleurone layer and embryo during late seed development. Several scientists initially
suggested that the expression of HVA1 gene in transgenic plants could confer various abiotic
stress tolerance including drought and salinity tolerance. Hong et al. (1992) reported that the
expression of HVA1 was rapidly induced in young seedlings under salt or ABA treatments. This
phenomenon was also associated with the HVA1 an increase in the levels of HVA1 mRNA in

7

both shoot and root of the young seedlings under cold and heat stresses, but such expression
dramatically decreased in old seedlings (7 day old seedlings).
Accumulation of HVA1 protein has been known for its important role in the
understanding of the mechanism of dehydration tolerance in plants. Babu et al. (2004)
transferred HVA1 to rice, and reported on the role of this gene as related to the dehydration
tolerance in transgenic rice plants. This group reported that relative water content of transgenic
plants maintained at high percentages (92%) after 28 days of water stress, whereas relative water
content decreased to 51% in non-transgenic plants under the same conditions. The dry shoot and
root biomass matter were more and the percentage of cell membrane leakage was less in HVA1
transgenic plants as compared to their wild-type control plants. This suggested that the
expression of the HVA1 gene encoding group 3 LEA proteins confers dehydration tolerance in
transgenic rice by protecting plant cell membranes.
Moreover, the expression of HVA1 gene in transgenic mulberry (Morus indica) plants
resulted in higher cellular membrane stability (70%) as compared to non-transgenic plants (50%)
under 48hrs of 2% polyethylene glycol (PEG) osmotic treatment (Lal et al., 2008), and
transgenic plants showed 1.5-fold higher relative water content after 10 days of water stress and
accumulated higher 5-19-fold higher proline under 400mM NaCl stress condition. Checker et al.
(2012) also reported that the expression of HVA1 gene in transgenic mulberry plants enhanced
proline accumulation, membrane stability and photosynthetic yield after 30 days of water deficit,
or when exposed to 200mM NaCl for 30 days. The authors also reported similar results under
cold stress (10oC) condition.
The expression of HVA1 in T3 transgenic rice seedlings driven either by a rice actin
constitutive (Act1) or inducible (4ABRC) promoters resulted in enhanced drought and salt

8

tolerance. In this experiment, transgenic plants showed greater seedling growth performance,
more shoot height and shoot weight, and lesser ion leakage than in wild-type non-transgenic
plants under drought and salinity stress conditions (Rohila et al., 2002).
Xu et al. (1996) studied salt stress tolerance in transgenic rice expressing the Barley
HVA1 gene regulated by the rice Act1 promoter, and reported that shoot height and root fresh
weight of transgenic plants increased significantly after totally 30 days of salt treatment at the
rotation of 10 days for 200mM-water-50mM NaCl, as compared to non-transgenic control plants
that were maintained under the same conditions. HVA1 transgenic rice plants also showed high
survival rate (60-80%) whereas all of the non-transgenic control died after extending more
number of days of salt treatment. Results also revealed accumulation of HVA1 protein in
transgenic rice leaves and roots.
Studying fourth generation (T3) HVA1 transgenic wheat, Sivamani et al. (2000) reported
an increase in crop biomass and a higher water use efficiency under moderate water deficit
condition as compared with their wild-type non-transgenic control plants. In this experiment,
both homozygous and heterozygous lines of T3 generation showed up to 55% higher root dry
biomass and up to 17% higher shoot dry weight as compared to their wild-type non-transgenic
control plants.
Similarly, Bahieldin et al. (2005) confirmed that the expression of HVA1 gene in
transgenic spring wheat lines conferred drought tolerance, and most of the transgenic plants
showed higher plant height, total biomass as well as higher grain yield, as compared to those of
HVA1 non expressing plants (Plants that had integrated, but had not expressed the HVA1 gene)
and those of the wild-type non-transgenic control plants.

9

Oraby et al. (2005) found that under different salinity levels, T3 oat plants expressing
HVA1 transgene showed better performance than non-transgenic plants on flower induction,
seedling growth, and the number of kernels per plant. Under 200mM NaCl treatment, the panicle
length and number of spikelets per panicle of control plants were less by 50% as compared to
transgenic plants. The kernel yield was also reduced by 40% and 90% for transgenic and wildtype control plants respectively. Furthermore, when HVA1 gene in transgenic creeping bentgrass
(Agrostis stolonifera var. palustris) was up-regulated under water deficit condition, plants
showed higher leaf water content, and significantly less leaf wilting (Fu et al., 2007).
The role of LEA protein accumulation in plants as well as the role of HVA1 gene
encoding group 3 LEA proteins have been well known as evidence for abiotic stress responses in
a vary of crop species. Table 1 represents the expression of the barley HVA1 gene conferring
drought and/or salt tolerance in different transgenic crops.
Table 1: The expression of HVA1 gene in different transgenic crops
Species

Phenotype
Rice
Drought and salinity tolerance
Drought and salinity tolerance
Drought tolerance and stability of cell
membrane
Increased biomass and water use efficiency
under stress
Wheat
Drought tolerance
Improved plant water status and yield under
field drought conditions
Drought tolerance
Oat
Salinity tolerance in increasing yield per plant
Creepingbentgrass Drought tolerance
Salinity and drought tolerance
Mulberry
Drought, salinity and cold tolerance

10

Reference
Xu et al., 1996
Rohila et al., 2002
Babu et al., 2004
Sivamani et al., 2000
Detvisitsakun et al., 2001
Bahieldin et al., 2005
Maqbool et al., 2002
Oraby et al., 2005
Fu et al., 2007
Lal et al., 2008
Checker et al., 2012

1.2. RESEARCH GOAL AND OBJECTIVES
The goal of this research was to express the barley HVA1 gene in maize plants and breed
transgenic plants for development of drought and salt tolerance. The following specific
objectives were to be met in order to reach the above goal.
The specific objectives of the research were:
(i)

Transfer the Barley HVA1 gene into the maize genome.

(ii)

Determine the segregation of the linked bar and HVA1 genes and verify their proper
expression and stability of transmission to up to four generations.

(iii)

Evaluate the effects of the HVA1 transgene on maize seedling development, plant
height, and fresh and dry biomass matter as compared to those of the wild-type nontransgenic control plants under drought and salt stress conditions.

1.3. MATERIALS AND METHODS
1.3.1. Explants
Maize Hi II immature zygotic embryos were ordered from Plant transformation facility,
Department of Agronomy, Iowa State University. Calli were established and maintained in an
induction media containing 4g/L of Chu N6 base media with vitamins supplemented with 30g/L
sucrose, 2.76g/L proline, and 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D). Type II calli were
transferred to fresh media every 2 weeks for proliferation and for gene gun bombardment.
1.3.2. Gene construct
Plasmid pBY520 (Figure 1) derived from pBluescript II KS (+) (Stratagene, 11011 North
Torrey Pines Rd, La Jolla, CA 92037) was generously provided to our laboratory from the late
Professor Ray Wu of Cornell University, and used for the transformation of maize. This
construct contains HVA1 coding sequences driven by 1.3 kb upstream region of the rice Act1

11

translation codon (McElory et al., 1990; Zhang et al., 1991) and terminated with the polyA 3`
terminal of potatoes proteinase inhibitor II (pin II) gene (Keil et al., 1986). The pBY520 also
contains the bacterial phosphinothricin acetyl transferase (bar) structural gene, as a selectable
marker in maize transformation. In this construct, the bar coding sequences are driven by the
cauliflower mosaic virus (CaMV 35S) promoter and terminated by the nopaline synthase nos 3`
region (Bevan et al., 1983).

Figure 1: Linear map of pBY520 construct. Rice actin promoter (Act1), Barley (Hordeum
vulgare or HVA1 LEA3 gene, Cauliflower Mosaic Virus 35S (35S) promoter, Bar Liberty
herbicide resistance gene (bar) and nopaline synthase terminator (Nos). For interpretation of the
references to color in this and all other figures, the reader is referred to the electronic version of
this dissertation.
1.3.3. Transformation of plasmid vector into E. coli competent cells
The pBY520 plasmid was transferred into E. coli for multiplication by heat shocking for
0

45 seconds in a water bath at a temperature of 42 C containing 2 µl of plasmid DNA mixed with
50 µl of DH5α E. coli competent cells (Sigma). The mixture was then immediately placed on ice
for 2 min and added 700 µl of Luria Bertani (LB) medium into the tubes. This mixture was then
0

incubated for 1 hour at 37 C with 250 rpm shaking. The cultures were placed on solid LB
0

selection media contained 100mg/L ampicilin, and incubated overnight at 37 C. Single colonies
12

were taken and placed into glass flask containing 50 ml of LB media. This was then incubated at
0

37 C with 250 rpm shaking overnight. The plasmid DNAs were isolated and purified using
Qiagen plasmid purification kit (Cat. No.12123) as per manufacturer’s instruction. Purified
plasmid was then used for biolistic bombardment.
1.3.4. Biolistic

TM

bombardment

For gene gun bombardment, the embryogenic type II friable calli from one week callus
cultures were selected and placed together in a 1x1 cm area on top of the osmotic induction
media at least 4 hours prior to bombardment. About 6l of the plasmid mixture containing DNA
coated tungsten particle (M10) were used for bombardment as described by Frame et al. (2000).
The bombardments were carried out twice per plate by using a Helium PDS 1000HE device
(Biotechnology Systems Division, Wilmington, DE) with 1100 psi acceleration pressure.
Bombarded calli were then cultured on the induction medium for 1 week while maintained in
0

darkness at 27 C before transferring them to selection medium contained 2.5 mg/L of glufosinate
ammonium. Cultures were sub-cultured at two-week intervals.
All DNA precipitation and bombardment steps were performed under sterile conditions
under a laminar flow hood at room temperature. For plant regeneration, the selected embryonic
calli were transferred to Murashige and Skoog (1962) or MS medium containing 1 mg/L 6benzylaminopurine (BAP). Regenerated shoots were rooted on MS medium containing 1 mg/L
Indole-3-butyric acid (IBA). Plantlets were transferred into small square container pots
containing BACCO Professional Planting Soil Mix, and covered using transparent plastic bags
for keeping the humidity for acclimation into their new environment. The plastic covers were

13

removed gradually over a period of 4 days and the plantlets were transferred into 30cm diameter
pots and grown to maturity in a greenhouse.
1.3.5 Confirming the integration and expression of transgenes in putatively transgenic
plants
1.3.5.1. Polymerase Chain Reaction (PCR) analysis
Genomic DNA was isolated from young leaf tissues using CTAB method. HVA1 F, 5`ACC AGA ACC AGG GGA GCT AC-3` (forward primer) and HVA1 R, 5`-TGG TGT TGT
CCC CTC CCA TG-3` (reverse primer) were used to detect HVA1 gene for T0-T3 plant
regenerations. DNA amplifications were performed in a thermo cycler (PerkinElmer/ Applied
0

Biosystem, Forster City, CA). Optimized PCR conditions were 94 C for 3 min for initial
0

0

0

denaturation, 35 cycles of 30 s at 94 C, 30 s at 56 C, 45s at 72 C and a final 10 min extension at
0

72 C. The PCR product was loaded directly onto a 1% (weight/volume) agarose gel stained with
2ul ethidium bromide and visualized under UV light.
1.3.5.2. Southern Blot Hybridization Analysis
Southern blot hybridization analysis was carried out to determine the stability of
transgenic event and gene copy number of HVA1 and bar transgenic plants. 15 g of maize
genomic DNA from each putatively transgenic line was digested with HindIII or BamHI
enzymes in the proper buffer [500 mM Tris-HCl (pH 6.5), 1 mM EDTA] in a total volume of
0

0

250 l overnight at 37 C. The digested DNA was precipitated with 70% ethanol at –20 C
overnight, pelleted by centrifugation at 13,000 rpm for 10 min, allowed to dry and then redissolved in sterile distilled water before electrophoresis. The digested DNA was run on
electrophoresis at 70 v on 0.8% agarose gel and transferred to a Hybond-N+ membrane

14

(Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV Crosslinker
1800, Stratagene, CA ) at an energy level of 2,000 J. Gene-specific probes were synthesized
using HVA1 forward and reverse primers and purified using the DNA clean and concentrator -5
32

Kit (ZYMO, RESEARCH, D4004), and labeled with -[ P]-dCTP using Random

Prime

labeling kit (GE Healthcare) according to the manufacturer’s instructions. Membranes were
0

hybridized at 68 C overnight with the radiolabled probe and autoradiographed on premium
0

autoradiography films (Hyblot CL, Denville, Scientific INC, E3018) at –80 C for overnight.
1.3.5.3. Reverse Transcription-PCR (RT-PCR)
PCR positive plants of HVA1 gene were used in RT-PCR analysis. A total of 200 mg
young leaf tissues were grinded into liquid nitrogen for each 1 ml Trizol Reagent (Invitrogen,
Carlsbad, CA). 0.2 ml chloroform was added and vortexed for a few seconds. The tubes were
0

placed into a centrifuge and spun at maximum speed for 15 min at 4 C. An aqueous phase was
then transferred into fresh tubes and added 0.5 ml of cold isopropanol. Samples were incubated
0

0

at -20 C for 1 hour and centrifuged at maximum speed for 10 min at 4 C. The supernatant was
thrown away leaving the RNA pellet. This was washed with 700ul of 70% ethanol then spun in a
0

centrifuge at 12,000prm for 5 min at temperature of 4 C. The RNA pellets were dried at room
temperature and dissolved in RNase-free water and quantified using a spectrophotometer. 2g
RNA obtained was used for cDNA synthesis using the Superscript™ First-Strand Synthesis
System for RT-PCR (Invitrogen, Carlsbad, CA) as per manufacturer’s instructions. The same
primers and PCR conditions for HVA1 as described above were used.

15

1.3.5.4. Northern Blot
Northern blot analysis was performed using Random Prime labeling kit (GE Healthcare)
as per described in southern blot to assay the HVA1 gene expression of the transgenic plants. A
total of 15 µg of RNA per sample was loaded onto a 1.2% (w/v) agarose-formaldehyde
denaturing gel as described by Sambrook et al. (1989) and transferred to an Hybond-N+
membrane (Amersham-Pharmacia Biotech) and fixed with a UV crosslinker (Stratalinker UV
32

Crosslinker 1800, Stratagene, CA) at an energy level of 1200 J. Same DNA -[ P]-dCTP
labeled probe as in southern was used for detection of transcripts.
1.3.6. Biological activity tests
1.3.6.1. Biological assay for bar
Five different concentrations (200, 250, 300, 350, and 400mg/L) of glufosinate
ammonium (Aventis, Strasboug, France) corresponding to 200, 300, 350, and 400 µl/L of
Liberty herbicide dilutions (1.67 pounds of active ingredient glufosinate ammonium per gallon
of Liberty herbicide) were applied for leaf painting using a cotton swab to determine the kill
curve (lowest concentration that could kill maize leaf). The level of 350mg/L of glufosinate
ammonium solution was used and applied to leaves using brush to find the segregation ratios of
the transgenic progenies. The tips of young leaves of 3-leaf stage seedlings were selected and
painted (Lee et al., 2007). The leaf injury caused by herbicide application was observed one
week after painting treatment.
1.3.6.2. Drought tolerance test
A total of 30 seeds from the fourth generation (T3) transgenic line and a non-transgenic
line were planted in the greenhouse in 15 cm diameter pots containing BACCTO High Porosity
Professional Planting Mix (Michigan Peat Company, Houston, TX) under greenhouse conditions
16

0

of 29 and 25 C corresponding to day and night temperature; and 16-hour day length with light
-2 -1

intensity of 600-700 mu mole m

s . A dilution of commercial 20-20-20 fertilizer solution was

applied to seedlings twice per week. Four week old seedlings were selected and used for drought
tolerance test. Seedlings were exposed to 15 days of no water treatment (drought) followed by 7
days of re-watering. Data were collected for leaf relative water content (RWC) at 10 days and 15
days after water stress (before re-watering for recovery) and percentage of plant survival was
determined 7 days after re-watering.
1.3.6.3. Salt tolerance stress test
A total of 40 seeds of T3 of a transgenic line and a non-transgenic line were sown in the
round clay pots containing BACCTO High Porosity Professional Planting Mix (Michigan Peat
Company, Houston, TX). This experiment was replicated in two locations of the same
greenhouse. To test the herbicide resistance of transgenic plants to assure the presence of HVA1
gene (linked to the bar gene cassette), the transgenic seeds were allowed to germinate under
15mg/L of glufosinate ammonium. The surviving seedlings were then daily treated with equal
volume of four different concentrations (0, 100, 200 and 300 mM) of NaCl by gradually
increasing 50mM per day to reach the final concentrations in 10 days. The commercial 20-20-20
fertilizer was supplemented into salinity solution for nutritional needs. Seedling samples were
taken after 10 days of salt treatment for shoot and root length, and shoot and root fresh and dry
weights data collections. After weighting for fresh shoot and root biomass, these tissues were
0

dried out in an oven at 80 C for two days and weighed to determine for shoot and root dry
weights.

17

1.3.6.4. Measurement of leaf relative water content (RWC)
The leaf samples from transgenic and non-transgenic plants were taken for the
determination of leaf RWC after 10 days and 15 days of water withholding. In each of the two
repeated experiments, about 10 cm2 leaf discs of three randomly selected plants were collected at
8AM, their mid-ribs were cut and discarded, and the rest of the leaves were immediately placed
in plastic bags to limit water evaporation. Then, each leaf section was removed from the plastic
bag and weight for its fresh weight (FW), and then leaf samples were soaked into distilled water
for four hrs to full turgidity at normal room condition. After hydration, the samples were placed
between two tissue papers to remove any water on the leaf surface and then immediately
0

weighed to obtain fully turgid weight (TW). Samples were then dried in an oven at 80 C
overnight, placed in a desicator for 15 min to cool down, and then weighed to determine their dry
weight (DW). Leaf relative water content (RWC) was calculated by the following formula
(Gaxiola et al., 2001)
RWC (%) = [(FW-DW) / (TW-DW)] x 100
1.3.6.5. Statistical analysis
All data were collected and statistically analyzed using a completely randomized design
(CRD) using PROC GLM (SAS version 9.2 software package). Analysis of variance (ANOVA)
was conducted to test the statistical significance at an alpha level of 0.05.
1.4. RESULTS AND DISCUSSIONS
1.4.1. Maize tissue culture and regeneration
Results of maize in vitro culture and plant regeneration, followed by self pollinator are
shown on Figure 2 below.

18

Figure 2: Maize in vitro culture, plant regeneration and breeding of transgenic plants: (A)
Highly embryogenic Hi II immature embryos (B) Highly embryogenic immature embryos after
transgene bombardment; (C) Embryogenic tissue growth in selection medium; (D) and (E)
embryogenic tissues in regeneration media; (F) plantlet growth in rooting media; (G)
Acclimatization of plantlets in growth chamber; (H) plants growing in greenhouse; (I) Plant
breeding, and (J) breeding of mature plants.
Transformation experiments were performed by using Hi II immature embryos-derived
calli via Biolistic

TM

bombardment. The bombarded calli grew well in the N6 osmotic medium at

0

25 C in complete darkness for the first two weeks after bombardment (Figure 2B). Selection of
herbicide resistant cultures took place in 8-10 months, when cultures were selected on a culture
medium containing 2.5mg/L of glufosinate ammonium, where the transgenic embryogenic calli
proliferated rapidly and displayed somatic embryos in form of white and fast growing granular
sectors. There were also some non-proliferating and partially necrotic mother calli (brown
19

sectors) that did not survive the selection. Under fluorescent light condition of 16hr light/ 8 hr
0

dark and 25 C, somatic embryos quickly proliferated in regeneration medium containing 1mg/L
of Benzyl amino purine (BAP) and regenerated into normal shootlets which produced primary
and secondary roots 4 weeks after they were placed in rooting medium containing 1mg/L of
Indol butyric acid (IBA). The rooted plantlets were transferred to soil, and pots were covered
with plastic bags for one week to allow plantlets to be acclimated to normal culture room
conditions. Plantlets were then transferred to 30 cm diameter soil pots and placed in our maize
greenhouses where they were grown to maturity (Figure 2 F-J above).
The optimum conditions for maize transformation via particle bombardment using
immature zygotic embryos vary in literature. Scientists (Petrillo et al., 2008, Oneto et al., 2010)
reveal that using 2ug of DNA plasmid mixed with particle of 1um in size, 6 cm distance between
the loaded gene construct and target, and twice shot with pressure of 1100Psi are optimum
conditions for maize genetic transformation via the Biolistic

TM

device. In this candidate’s

research, the bombarded calli were transferred from the N6 medium to MS medium where they
regenerated within four weeks (Amstrong et al., 1994, Oneto et al., 2010). The purpose of N6
osmotic treatment was to minimize the damages of the cell walls caused by bombardment (Wang
et al., 2003a). Such osmoticum media assists in an increase in transformation efficiency by
decreasing the damage made to the cells (Vain et al., 1993; El-Itriby et al., 2003).
1.4.2. Integration of HVA1 and bar transgenes via PCR and Southern blot analysis
The PCR results showed positive integration of HVA1 and bar transgenes in T0, T1, and
T2 generations (Figure 3, 4, 5).

20

100bp

+ WT

4

13

16 19

50

51

71

72

131 132 141 161 164 166

680bp

Figure 3: PCR analysis of T0 HVA1 transgenic plants confirming the correct expected band size
of 680bp.
100bp +

- WT 1

2 3

4

5

6

7

8

9 10 11 12 13 14 15

16

680bp

484bp

Figure 4: PCR analysis of T1 HVA1 and bar transformants (line#132) showing the expected
band size of 680 bp for HVA1 and 484bp for bar gene.
100bp + WT

1

2

3

4

5

6

7

8

9

10 11

12 13 14 15

16

680bp

Figure 5: PCR analysis of T2 of HVA1 transformants (line#132) with correct expected band size
(680bp).

21

The results of the PCR analysis confirm the integration of HVA1 gene across T0-T2
generations (Figure 3, 4 and 5). The co-existence of HVA1 and bar genes in the transgenic plants
is due to the transcriptional linked HVA1 and bar gene cassettes, therefore transgenic plants
showed the integration of both HVA1 and bar genes together at 100% frequency (Figure 4).
Similar observations were reported by Maqbool et al. (2002), Oraby et al. (2005) and Kwapata et
al. (2012) who reported stable co-integration of two linked genes in the subsequent transgenic
progenies with a frequency of 100%.
Southern blot hybridization confirmed the integration of HVA1 transgene in T0
transgenic maize plants (Figure 6 below). Southern blot also revealed the copy number of HVA1
transgene in transgenic plants (described below).
P

131

71

72

4

161

141

166

WT

8kb

Figure 6: Southern blot showing integration of HVA1 gene in T0 transgenic plants. P: pBY520
plasmid as positive control. WT: wild-type. Digestion was performed using the HindIII
restriction enzyme.
Southern blot analyses results showed the integration of HVA1 gene with two to three
inserted gene copies (Figure 6), except for the transgenic line 141 that shows one inserted
transgene copy. Although the T0 line #4 plant showed the integration of HVA1 transgene using
the PCR technology, Southern blot did not confirm the same because the amount of transgene

22

might have not been sufficient to show in Southern blotting, or plant may have been chimerically
transformed (the piece taken for PCR analysis, but not the whole plant contained the transgene).
Several studies have shown that the use of particle bombardment for genetic
transformation can result in multiple integrated transgene copies in transgenic plants, and also
can cause the rearrangement of the transgenes (Pawlowski et al., 1996; Kohli et al., 1998; Dai et
al., 2001).
1.4.3. HVA1 transcription confirmed by RT-PCR and Northern blot analysis
Figure 7 represents the HVA1 transgene transcription in T1 plants confirmed via RT-PCR
analysis.
100bp

+

WT

131

132

161

164

166

680bp

Actin 430bp

Figure 7: RT-PCR showing expression of HVA1 transgene in T1 transgenic plants; + pBY520
plasmid positive control, wt: wild-type. The expected band size for HVA1 gene is 680bp. The
lower portion of this figure shows the expression of maize endogenous actin1 gene with an
expected band size of 430bp is loaded as control to show that the RT-PCR has been working
well.
Northern blotting of plants confirmed the transcription of HVA1 gene in T0 and T1
transgenic plants (Figure 8 and 9).

23

131

71

72

161

141

166

WT

Figure 8: Northern blot expression of HVA1 transgene in T0 transgenic lines; WT: wild-type
(non-transgenic plant).
131

132

161

164

166

WT

Figure 9: Northern blot expression of HVA1 transgene in T1 transgenic lines; WT: wild-type
(non-transgenic plant).
The above RT-PCR analysis shows the transcription of HVA1 transgene in T1 plants
(Figure 7) and confirmed by northern blot analysis (Figure 9). Figure 8 and 9 shows that HVA1
transgene show different levels of transcriptions, while T1 show higher transcriptions. The
reason for various levels of transcription might be due to different site of insertion of transgene
in maize genome, and the reason that T1 plants show higher level of transcription is because the
T0 plants were self bred towards homozygosity.
1.4.4. Confirmation of herbicide resistance in bar transgenic plants
We developed a kill curve test painting the wild-type non-transgenic maize leaves with
different concentrations of commercial Liberty herbicide (containing 18.2% glufosinate
24

anominum). This experiment showed that the minimum concentration of glufosinate anominum
that killed the leaf tissues was 350 mg/L (Figure 10).

WT200mg

WT250mg

WT300mg

WT350mg

WT400mg

Figure 10: Kill curve for herbicide selection: Young leaves of wild-type non-transgenic plants
painted with 5 different concentrations of commercial Liberty herbicide containing 18.2%
glufosinate anominum. The concentration of 350mg/L glufosinate anominum was used to apply
for transgenic plants.
By using the herbicide kill curve test (Figure 10), this candidate found the optimum
concentration of herbicide for leaf painting to be 350mg/L glufosinate ammonium. This kill
curve was used in transgenic plants to determine whether the bar gene segregation follows the
Mendelian inheritance. For this purpose, the Chi-square was used to determine the transgenes
(linked) segregation ratio (3:1) in T1 progeny. However, Chi-square test result showed that the
bar gene segregation did not follow Mendelian segregation. This might be because most
transgenic plants had integrated more than one transgene inserts, and therefore the multiple
copies of transgene insertion have caused the transgene not to follow the Mendelian segregation.
Figure 11 (below) represents the effect of 350 mg/L glufosinate anominum on leaf
painting of a T1 bar transgenic line versus that of a wild-type non-transgenic control plant.

25

WT
Non-transgeneic

Transgeneic

Figure 11: Leaf painting (at 350mg/L glufosinate ammonium after 7 days) showing herbicide
injury symptom on non-transgenic plant versus T1 transgenic plant confirmed resistant.
1.4.5. Confirmation of drought stress tolerance in T3 plants
Among all transgenic T1 plants that showed HVA1 transcription, line #132 was selected
for drought tolerance test because other lines did not produce sufficient seeds or did not produce
kernels for self breeding. The T1 line transcribed the HVA1 gene (Figure 9), and the Northern
blot positive of its line 132 produced over 150 seeds (which became T2 plants), each producing
sufficient kernels for self breeding.
The percentage of leaf RWC of transgenic line #132 and wild-type non-transgenic control
plant was calculated after 10 and 15 days of withholding of the water irrigation are shown on
Figure 12.

26

100.00

RWC (%)

80.00

60.00

WT

40.00

HVA1
20.00

Normal
watering

Water
withholding

Normal
watering

10 days

Water
withholding

15 days

Figure 12: Percentage of leaf relative water content (% RWC) of HVA1 transgenic line #132
after 10 and 15 days of water deficit (withholding).
After withholding the water for 10 days, non-transgenic plants started wilting whereas
most of the transgenic plants showed normal growth performance. However both transgenic and
non-transgenic control plants showed symptoms of drought stress i.e. leaves wilted, burned at the
leaf edge and tip, and the upper leaves turned yellow after 15 days of water withholding. The
wild-types showed more severe symptoms of drought stress with most of the leaves being wilted
and dehisced compared to transgenic plants after 15 days of water withholding treatment.
Figure 12 also shows that the leaf RWC measured in wild-type leaves was decreased to
81.7% whereas leaf RWC in transgenic plants still maintained as high as 94.9%. After 15 day of
water withholding, leaf RWC in the wild-type control plants was as low as 53.9% as compared to
the leaf RWC in transgenic plants that was 73.2%. There was no significant difference in leaf
27

RWC between the control and transgenic plants that were watered regularly, and the transgenic
plants after 10 days of water withholding. However, the leaf RWC of HVA1 transgenic line was
less after 15 days of water withholding as compared to that of 10 days water withholding, but far
less in the leaves of the wild-type control plants.
Figure 13 represents the survival rate of transgenic versus wild-type control plant after 7
days of water recovery treatment.
80

Percentage of survival (%)

70
60
50
40
30
20
10
0
HVA1

WT

Figure 13: Percentage of survival rate of transgenic versus wild-type control plant after 7 days of
water recovery treatment
Results show that when plants were re-watered for 7 days after 15 days of water
withholding for drought stress recovery, transgenic plants showed a quick (2-3 days) recovery
and performed high percentage of survival (65% recovery) compared to wild-type (30%
recovery).

28

WT
Transgenic WT
Normal
Under
Under
watering drought
drought

WT
Normal
watering

Transgenic
Under
drought

WT
Under
drought

Figure 14: Effect of drought stress on plant growth and mature root system of HVA1 transgenic
maize versus its wild-type control plants. The pictures were taken for drought stress tolerance of
T3 HVA1 transgenic plants after one week of water recovering (A), and mature roots (B)
Maize is very susceptible to drought and requires water for cell elongation across all
stages of its growth and development. However, the amount of water requirement will vary
depending on the growth stages in which increases during vegetative stages and reduces during
reproductive stage. It has been reported that drought stress during early vegetative stage of
maize growth cause a constant leaf wilting resulting in 5 to 10% yield losses (Thelen, 2012).
There are several mechanisms explained for drought tolerance ability of plants (including
maize) under water deficit conditions. One of these mechanisms is the high accumulation of
abscisic acid (ABA) found in drought tolerant lines as compared with drought sensitive lines
(Wang et al., 2003b). ABA is known to regulate stomata opening and other responses resulting
in tolerance to drought in plants. In the results [presented here, it is possible that accumulation of
ABA may have played a role.
29

The expression of HVA1 gene encoding the Barley LEA3 protein in this study is in
agreement with several other reports on other HVA1 transgenic crop species showing higher leaf
RWC, significantly increased plant growth, and drought tolerance of transgenic plants as
compared to their non-transgenic control plant counterparts (Xu et al., 1996; Babu et al., 2004;
Fu et al., 2007).
1.4.6. Salt stress tolerance in T3 plants
The salt treatment was conducted after seed germination (i.e. seven days after sowing the
seeds). Four different salt concentrations (0, 100, 200, and 300mM NaCl) were applied daily as
250 ml solution per pot while increasing the salt concentration at 50mM per day until it reached
its final concentration. Transgenic and wild-type plants showed normal development up to one
week after being exposed to 100mM NaCl, but wild-type leaves started showing injurious
symptom after the NaCl treatment. After increasing the NaCl concentration to 200 mM, the wildtype plants were severely affected as compared to those treated with 100mM. At 300mM NaCl
concentration, both transgenic and wild-type plants showed severe leaf injury after one week of
salt treatment.
Figures 15-20 (below) display the results of the effect of different salt concentrations on
T3 HVA1 transgenic versus non-transgenic control plants on shoot and root length, and shoot and
root fresh and dry weight biomasses.

30

35.0

Shoot length (cm)

30.0
25.0
20.0

WT

15.0
10.0
5.0
0.0
0mM

100mM
NaCl concentration

200mM

Figure 15: Effect of salt treatment on shoot length of T3 plants.
45.0
40.0

Root length (cm)

35.0
30.0
25.0

WT
HVA1

20.0
15.0
10.0
5.0
0.0
0mM

100mM
NaCl concentration

Figure 16: Effect of salt treatment on root length of T3 plants.

31

200mM

3.0

Shoot fresh weight (gr)

2.5
2.0
1.5

WT
HVA1

1.0
0.5
0.0
0mM

100mM
NaCl concentration

200mM

Figure 17: Effect of salt treatment on shoot fresh weight of T3 plants.
0.3

Shoot dry weight (gr)

0.2

0.2

WT
HVA1

0.1

0.1

0.0
0mM

100mM
NaCl concentration

200mM

Figure 18: Effect of salt treatment on shoot dry weight of T3 plants.

32

2.5

Root fresh weight (gr)

2

1.5

WT
HVA1

1

0.5

0
0mM

100mM
NaCl concentration

200mM

Figure 19: Effect of salt treatment on root fresh weight of T3 plants.
0.160

Root dry weight (gr)

0.140
0.120
0.100

WT
HVA1

0.080
0.060
0.040
0.020
0.000
0mM

100mM
NaCl concentration

200mM

Figure 20: Effect of salt treatment on root dry weight of T3 plants.

33

Table 2 shows that the reduction in shoot length of both transgenic and wild-type plants
were respectively at 25% and 26% when exposed to 100 mM NaCl. However at 200mM,
percentage of shoot and root length reduction in wild-type plants increased significantly to
55.7% and 47.7%, respectively (Appendix 8 and 9) whereas the reduction of shoot and root
length were respectively 34.6% and 19.5% in transgenic plants. Similarly, fresh and dry shoot of
wild-type control plants were reduced by 42% and 45% at 100mM and up to 80% and 72% at
200mM NaCl concentration. Salt stress also reduced by 85% of root dry weight in wild-type
compared to 63% reduction in transgenic plants at 200mM NaCl. Over all, the results show that
the percentage of reduction of shoot and root length and their fresh and dry biomass were
affected by different salinity concentrations, with a maximum reduction at 200 mM NaCl
treatment.
In general, the growth of seedlings and fresh and dry biomass of transgenic plants were
less affected by different NaCl levels as compared to their wild-type control plants.
Table 2: Reduction in shoot and root length and their biomass affected by salt treatments
Salt concentration
Genotype

100mM

200mM

T3 HVA1

WT

T3 HVA1

WT

% shoot length reduction

25.5b

26.4b

34.6b

55.7a

% root length reduction

20.0b

18.9b

19.6b

47.7a

% shoot fresh weight reduction

15.3b

41.5b

39.2b

79.8a

% shoot dry weight reduction

22.4c

44.9b

29.4c

71.7a

% root fresh weight reduction

20.0d

43.7c

49.0b

82.2a

% root dry weight reduction

44.9c

57.8b

62.8b

84.9a

34

Shoot and root length, and shoot and root fresh and dry biomass weights of both
transgenic and wild-type seedlings (10 days after salt treatment) were decreased while increasing
salt concentrations. However, wild-type seedlings showed more severe injury on leaves and roots
resulting in reduced shoot and root length and fresh and dry matters significantly compared with
transgenic plants.
A

WT

B

Transgenic

0mM

100mM

200mM

300mM Transgenic Transgenic
WT
0mM
200mM
200mM

WT
0mM

Figure 21: Effect of salt stress on seedling growth (A) and root system (B) of HVA1 transgene
and wild-type plants after 10 days of NaCl treatment.
To determine the further effect of the different salinity concentrations on the growth of
plants and on the number of leaves per plants, additional study was performed with 3 -leaf stage
plants exposed to four different NaCl concentrations (0, 100, 200, 300mM) for 6 days (after all
treatments reached to the final NaCl concentration). The results are shown on the Table 3
(below)

35

Table 3: Effect of salinity stress on number of leaves per plant after 6 days of NaCl treatment
Number of leaf (leaf/plant)

NaCl levels

0mM

100mM

200mM

300mM

HVA1

4.8 ± 0.01a

3.9 ± 0.01b

3.7 ± 0.04c

3.4 ± 0.07d

WT

4.8 ± 0.04a

3.3 ± 0.07d

3.1 ± 0.01e

3.0 ± 0.00e

Means within columns followed by the same letter are not significantly different (P<0.05)
Table 3 indicated that number of leaves per plants in the wild-type was significantly
reduced more than that in HVA1 transgenic plants under NaCl stress. Under 300mM NaCl level,
the youngest leaves (the fourth leaf) of wild-type plants were not produced or rolled and were
dead, whereas the fourth leaf in transgenic plants were produced and extended normally to
become a new normal leaf (Figure 22). The effect of NaCl stress resulted in decreased number of
leaves per plant in wild-type plants from 3.3 to 3.0 at 100mM and 300mM corresponding to 30%
to 37% of reduction, respectively.
The average number of leaves in transgenic plants was reduced from 3.9 to 3.4 leaves per
plant at 100mM and 300mM (19% and 29% reduction), respectively. Whereas there was no
difference of number of leaves per plant between transgenic plants and wild-type non-transgenic
control plants.

36

Figure 22: Effect of salinity stress on the development of leaves in transgenic and wild-type
plants. Picture was taken after 6 days of NaCl treatment
In the research presented here on the effect of salinity treatment of the maize seedlings
agrees with the report on HVA1 transgenic versus control non-transgenic oats, indicating that the
wild-type control plants tended to be shorter and the shortest height for most genotypes was
obtained at 200mM NaCl (Oraby et al., 2005). Similar findings were reported by Xu et al. (1996)
in transgenic rice and wheat (Checker et al., 2011) expressing the HVA1 gene. Furthermore, the
relationship between accumulation of LEA 3 proteins and abiotic stress tolerance has been
confirmed in studies for transgenic wheat (Sivamani et al., 2000; Bahieldin et al., 2005), rice
(Rohila et al. 2002; Babu et al., 2004), and Oat (Maqbool et al., 2002; Oraby et al., 2005).

37

CHAPTER II
BACTERIAL MTLD GENE CONFERS SALT TOLERANCE AND ACCUMULATION
OF MANNITAL IN TRANSGENIC MAIZE (Zea Mays. L)
2.1. LITERATURE REVIEWS
2.1.1. Introduction
Plants often face adverse effects of extreme environmental conditions such as drought,
high salinity or low temperature. One of the mechanisms against abiotic stresses is that plants
can withstand the impacts of these conditions by synthesizing and accumulating sufficient
amounts of osmoprotectants inside their cells. However, many crop species cannot naturally
synthesize and accumulate enough of these compounds in their cells. Therefore, it is possible to
transfer certain heterologous genes coding for enzymes associated with the production of these
osmoprotectants into the susceptible crop species for development of abiotic stress tolerance
(Rontein et al., 2002). The mechanisms of drought and salt tolerance are explained below.
2.1.2. Biosynthesis of compatible solutes in plants for drought and salt tolerance
Plant responses to unfavorable environments include escape, avoidance or tolerance to
the conditions, meaning that plants usually avoid or escape the harsh abiotic stresses, tolerating
and surviving the conditions. Many previous studies have focused on the mechanisms that plants
can tolerate abiotic stresses, especially the water deficit and high salt concentrations. For
instance, halophytes plants are able to live under very high salinity concentrations, via the
function of exclusion of both Na+ and Cl- ions during their water uptake or these plants may have
the ability of storing salt inside their cells in a way that such salt concentration does not affect
their growth and development (Garthwaite et al., 2005).
Abiotic stress tolerant plants have the capability to go through biosynthesis and
accumulation of compatible solutes in their cells to balance the concentration of those solutes
38

between their cells and the solutes in their surrounding environment (Mimura et al., 2003). The
compatible solutes produced inside the water stressed cells are called osmoprotectants. The
osmoprotectants prevent the cellular dehydration and maintain plant cell turgor pressure in favor
of plant survival during osmotic stresses (Valliyodan and Nguyen, 2006).
Plant osmoprotectants consist of sugars (raffinose, galactinol, sucrose; trehalose; sorbitol
and mannitol); amino acids (proline, arginine, citrulline, orthinine); glycine betaine (GB);
polyamine (putrescine); and amides (glutamine, asparagines). Although the basic roles of these
compatible solutes are to stabilize proteins and cell membranes of plants during osmotic stresses,
each of them also has other functions (Valliyodan and Nguyen, 2006).
It has been reported that increases in concentration of sugar alcohols or polyols (glucose,
mannitol, or trehalose) in chickpea tissues caused accumulation of proline, an amino acid needed
to balance carbon nitrogen under stress conditions (Sheveleva et al., 1997) and improved yield in
chickpea (Cicer arietinum L.) (Bhatnagar et al., 2009). It has also been reported that certain
osmoprotectants improve tolerance to water deficit in wheat and soybean (Vendruscolo et al.,
2007; De Ronde et al., 2004), and rice (Xu et al., 2008). In addition, the synthesis of complex
sugars such as trehalose can stabilize the plant lipid membranes, proteins and other biological
structures during the dehydration stress conditions. For example, Zhang et al. (2005) transferred
a trehalose synthase gene in tobacco and reported that the increased trehalose accumulation in
transgenic tobacco plants resulted in increased drought and salinity tolerance of transgenic
tobacco plants.
Mannitol biosynthesis pathway is begun with the photosynthetic carbon reduction cycle,
where glucose-6- phosphate is converted into fructose-6-phosphate, and the fructorse-6-Phophate
into Mannitol-1-Phophate via the mannitol phosphate dehydrogenase (mtlD) enzymatic activity.

39

Then the Mannitol-1-Phosphate is converted into mannitol via the Pase (phosphatase) enzymatic
activity (Figure 20).

Figure 23: A summary of the pathway for the synthesis of mannitol.
Mannitol is a sugar alcohol that is naturally synthesized as a natural osmotic protectant,
contributing to salt tolerance in many crops. For example, celery (Apium gravelens L.) is a salt
tolerant plant that is known to accumulate mannitol under salt stress conditions (Everard et al.,
1994).
The mtlD enzyme (encoded by mtlD gene) has been found in various bacteria, known to
help in generating NADPH. The bacterial mtlD gene was first transferred into tobacco (Nicotiana
tabacum L.) by Tarczynski et al. (1992) who reported an enhanced mannitol accumulation in
transgenic plants, along with significantly higher growth performance compared to nontransgenic plants under high salt concentration condition.
Another enzyme associated with mannitol biosynthesis in manotol-6-phosphate reductase
(M6PR). When the celery M6PR gene was transferred to Arabidopsis, transgenic plants showed
accumulation of mannitol, enhancing the transgenic plants salt tolerance at a concentration of
100mM NaCl. However, when transgenic plants were exposed to 200mM NaCl, transgenic
plants showed higher survival rate and higher dry biomass matters as compared to control nontransgenic plants, however they did not produce seeds (Abebe et al., 2003).

40

2.1.3. Expression of bacterial mtlD gene in transgenic plants
Abebe et al. (2003) demonstrated that the expression of the E. Coli mtlD gene in third
generation transgenic (T2) wheat (Triticum aestivum L. cv Bobwhite) resulted in an improved
tolerance to drought and salinity, transgenic plants also showed enhanced fresh weight, dry
weight and plant height as compared with the wild-type non-transgenic wheat plants. Another
report (Karakas et al., 1997) indicated that salinity could reduce the growth of non-transgenic
tobacco plants by 40%, whereas it did not affect the growth of the mtlD transgenic tobacco
plants. Other researchers reported that mtlD transgenic poplar trees (Populus tomentosa)
survived up to 40 days in a hydroponic culture containing 75mM NaCl solution, while nontransgenic plants only survived 25mM NaCl. The stomatal conductance and photosynthesis of
transgenic poplar were also higher than that of wild-type control plants under the same salt
condition treatments (Hu et al., 2005), meaning that stomatal conductance in transgenic plants
may have contributed towards reduced transpiration keeping the cellular turgor pressure lower.
Prabhavathi et al. (2001) reported that the expression of mtlD transgene in the first
generation (T0) transgenic eggplant (Solanum melongena L.) seeds that were germinated under
in vitro salt treatment (MS medium plus 200mM NaCl) condition, resulted in seedlings that grew
well, whereas the wild-type control seeds did not germinate under the same conditions. Also,
canola (Brassica napus L.) transgenic seeds expressing the mtlD gene, germinated and survived
up to 24 days under in vitro conditions on MS media containing 350 mM of NaCl, whereas the
wild-type control seeds failed to germinate (Motallebi and Rahnama, 2011). Motallebi and
Rahnama (2011) also reported a high level of accumulation of mannitol in mtlD transgenic
canola plants as compared to the non-transgenic control plants.
The expression of bacterium mtlD gene in transgenic potato plants revealed an increased
salt tolerance under both in vitro and hydroponic stress conditions. The effect of 100 mM NaCl
41

on potato shoot fresh weight under hydroponic condition was reduced by only 17.3% as
compared to 76.5% reduced fresh weight of control non-transgenic plants (Rahnama et al.,
2011). Maheswari et al. (2010) also reported 1.7 to 2.8-fold shoot and root performance
enhancement in transgenic sorghum plants expression the mtlD gene.
Table 4 represents the effect of expression of bacterial mtlD gene for improved salinity
and drought tolerance in different plant species.
Table 4: Expression of bacterial mannitol-1-phophate dehydrogenase (mtlD) confers
biochemical changes resulting in drought and salinity tolerance in different crop species.
Species

Tobacco

Arabidopsis
Rice
Wheat
Petunia
loblolly pine
Populus tomentosa
Cotton
Sorghum
Potato
Canola

Phenotypes
Enhanced plant height and fresh
weight under salinity stress
conditions
No contribution to sustained
growth under salinity and drought
stress.
Increased germination under
salinity stress
Salt tolerance
Drought and salinity tolerance of
calli and plants
Chilling tolerance
Accumulation of mannitol and
glucitol and high salt tolerance
Salinity tolerance
Accumulation of amino acids and
enhanced salt tolerance
Increased drought and salinity
tolerance
Enhanced salt stress tolerance
Increased germination under
salinity stress

42

references
Tarczynski et al., 1993

Karakas et al., 1997
Thomas et al., 1995
Huizhong et al., 2000
Abebe et al., 2003
Chiang et al., 2005
Tang et al., 2005
Hu et al., 2005
Momtaz et al., 2007
Maheswari et al., 2010
Rahnama et al., 2011
Motallebi and Rahnama.
2011

2.2. GOAL AND OBJECTIVES
The goal of this research is to test the salt tolerance capacity of the bacterial mtlD in
maize plants.
The specific objectives of the research were:
(i)

Transfer the bacterial mtlD gene in maize plants.

(ii)

Verify their proper expression and stability of transmission of mtlD into T0, T1, T2
and T3 plant progenies.

(iii)

Evaluate the effects of the mtlD transgene on maize seedling development, plant
height, and fresh and dry biomass matter as compared to those of the wild-type nontransgenic control plants under salt stress conditions.

(iv)

Determine the accumulation of mannitol contents in T3 mtlD transgenic versus their
wild-type counterpart plants.

2.3. MATERIALS AND METHODS
2.3.1. Gene constructs
The JS101 plasmid (Figure 24) construct was used in this dissertation containing the
bacterial mannitol-1-phophate dehydrogenase (mtlD) gene regulated by rice actin promoter
(Act1) and the potato protease II terminator, linked to the bar herbicide resistance selectable
marker gene regulated by the 35S promoter and (Nos) terminator. This construct was developed
by the late Prof. Ray Wu of Cornell University, and obtained via a material transfer agreement
between MSU and Cornell University for the purpose of research.

43

Figure 24: pJS101 plasmid constructs containing a bacterial mtlD gene which is driven by Actin
Rice Promoter (Act1) and potato proteinase inhibitor terminator (Pin); and selectable marker bar
gene with Cauliflower Mosaic Virus (CaMV) 35S promoter and nopaline synthase terminator
(Nos).
The process of maize genetic transformation is as described in the Chapter I of this
dissertation.
2.3.2. Conformation of gene integration and expression
The sequences of the primers used in PCR analysis to confirm the mtlD transgene are 5’
ATC GGT CGT GGC TTT ATC GG 3’ (forward primer) and 5’ TCG ACA AAG CCA ACG
0

TGT TC 3’ (reverse primer). The PCR program was set at 94 C for 3 min for one cycle; the
0

0

0

0

following 35 cycles of 30 s at 94 C, 30 s at 55.5 C, 45s at 72 C; one cycle at 72 C for 10 min;
0

and the final cycle at 4 C forever. The amplified 431bp segment of the mtlD gene was also used
as DNA probes for northern and southern blotting.
All the procedures for DNA extraction, RNA extraction, and northern blots were done as
described in the chapter I.

44

2.3.3. Salinity stress tolerance test
A total 40 seeds of T3 of a generation line and a non-transgenic line were sown in the
small square plastic pots containing BACCTO High Porosity Professional Planting Mix
(Michigan Peat Company, Houston, TX). This experiment was replicated in two locations of the
same greenhouse. The seedlings were watered daily with normal tap water for two weeks before
being salt treated. Salt treatments were performed on the seedlings that were at their 4-leaf stage
of growth for 10 days (Figure 25). Plant height, the distance from ground level to the tip of the
longest leaf, was measured in cm for the absolute growth rate (AGR) followed the formula: AGR
= (h2 -h1)/ (t2 -t1). Where h2 and h1 were final and initial height of plant; t2 and t1 are final and
initial days (Singh et al., 2011).

Figure 25: T3 and wild-type plants at 4-leaf stage growth before being exposed to four different
salinity concentrations.

45

Thereafter, seedlings were daily treated with equal volume of four different
concentrations (0, 100, 200 and 300 mM) of NaCl by gradually increasing 50mM per day to
reach the final concentrations within 10 days. The commercial 20-20-20 fertilizer was weekly
supplemented into salinity solution for nutritional needs. After 10 days of salt water application,
observations were made on shoot fresh weight and shoot and root dry weight. Plants were then
watered daily for one week in order to allow them to recover from salt stress injuries.
2.3.4. Mannitol analysis
Carbohydrate extraction, derivatization, and analysis procedures were followed based on
protocols devised and updated by Dr. Wayne Loescher and colleagues (2012). Fresh leaves of T3
transgenic and non-transgenic plants were collected 7 days after they were exposed to different
salt concentrations. 1 gram of fresh leaf from each sample was ground in liquid nitrogen. The
tissue powder was then transferred into 15 ml tube followed by adding 2ml of 95% alcohol, and
incubated at 650C for 30 min. The supernatants were transferred into fresh cultured tubes, and
then 2 ml of chloroform was added into each tube and kept overnight at 40C. Supernatants were
transferred into fresh cultured tubes, and the samples were dried out during overnight using the
SC 200 Speedvac (Savant, Thermo Fisher Scientific, Asheville, NC, USA). The oximation
solution was then added into the dried sample tubes and samples were agitated on the shaker tray
for 1 hr to dissolve the sugars. To convert oximes and sugars to trimethylsilyl derivatives, 1.0ml
of hexamethyldisilazane and 0.1ml of trifluoroacetic acid were added carefully in the tubes and
tubes were left under the laminar flow hood at room temperature for 1 hr. Then, the supernatants
were transferred into 2ml clear crimp seal vials (SUPELCO Analytical, Bellefonte, PA, USA,
and Cat # 27058). Then, 1.0, 0.5, and 0.1 mg of the mixture of carbohydrates were used as

46

standards. Gas chromatography was performed on a 6890 N Network GC system (Agilent
Technologies, Santa Clara, CA, USA).
2.3.5. Analysis of photosynthetic gas exchange
-2

Leaf net photosynthetic rate (µmolCO2 m
-2

(molH20 m

-1

leaf area s ), and stomatal conductance

-1

leaf area s ) were determined on the second-uppermost expanded leaf of plants

using the LI-6400XT portable photosynthesis system (LICOR, model LI6400, sr. PSC 2925) as
described by Wang et al. (2010).
2.4. RESULTS AND DISCUSIONS
2.4.1. Integration of mtlD transgene confirmed by PCR and Southern blot analysis, and
confirmation of mtlD transcription via northern blotting
PCR analysis of T0, T1 and T2 plants confirmed the integration of bacterial mtlD
transgene in maize plants (Figures 26, 27 and 28). In all progenies, the amplification products
showed the expected band size of 431bp in all transformants and in the positive control (JS101
plasmid) whereas as expected, no band was observed in the wild-type control plants.
100bp

+

WT

2

27

35

36

37

39

42

43

Figure 26: PCR analysis of T0 mtlD gene. The expected band size was 431 bp.

47

52

70

100bp

+

WT

21

22

27-1 27-2

43-1

43-2

43-3

431bp

Figure 27: PCR analysis confirmed the mtlD transgene in T1 plants, with the expected band
size of 431 bp.

100bp +

WT

mtlD transgene
1 2 3 4 5

+ WT

bar transgene
1 2 3
4 5

484bp

431bp

Figure 28: PCR analysis confirmed the stable co-integration of mtlD gene and bar gene in T2
plants. +: positive DNA from JS101 plasmid; - H20 and wt: wild-type loading as negative
control; 1-5: genomic DNA of transgenic plants. The expected band size is 431 bp.
Northern blot hybridization revealed the transcription of transgenes in T1 and T2 plants
(Figure 29 and 30). The specific PCR amplification product of mtlD sequence was used as a
probe for Northern blot hybridization.

48

2-1

2-2

27-1

27-2

43-1

43-2

43-3

WT

Figure 29: Northern blot hybridization showing the expression of mtlD gene in T1 plants.
2a

2b

2c

2d

27a

27b

27c

27d

WT

Figure 30: Northern blot hybridization showing the expression of mtlD gene in T2 plants
2.4.2. Salt stress tolerance of T3 mtlD plants
Under salinity stress conditions, there was a significant reduction in plant height growth
rate (cm growth per day) with increasing salinity levels in both T3 and wild-type control plants.
However, the wild-type plants were more retarded by salt stress than transgenic plants. At 200
mM NaCl, the average plant growth rate of the wild-type control was 1.5 cm per day and
reduced by 1.1 cm per day at 300 mM of NaCl, whereas the corresponding data was 1.6 and 1.4
cm respectively in the transgenic plants. However, there was no significant difference of plant
growth rate between transgenic and wild-type plants under different salinity concentrations
(Table 5 and Appendix 14).

49

Table 5: Effect of salt stress treatments on the absolute plant growth rate in T3 mtlD plants
Genotype

NaCl treatment

LSMEAN

mtlD

0mM

2.6a

WT

0mM

2.6a

mtlD

100mM

2.0b

WT

100mM

2.0bc

mtlD

200mM

1.6dc

WT

200mM

1.5d

mtlD

300mM

1.4d

WT

300mM

1.1d

LS-means with the same letter are not significantly different.
Figures below represents the shoot fresh weight (Figure 31), shoot dry weight (Figure
32), and root dry weight (Figure 33) 10 days after being treated with different concentration of
NaCl.

Shoot fresh weight (gr)

25.00
20.00
15.00
WT

10.00

mtlD
5.00
0mM

100mM
200mM
NaCl concentration

300mM

Figure 31: Effect of different salinity conditions on shoot fresh weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3)

50

Shootdry weight (gr)

2.50
2.00
1.50
WT
1.00

mtlD

0.50
0mM

100mM
200mM
NaCal concentration

300mM

Figure 32: Effect of different salinity conditions on shoot dry weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3)

Root dry weight (gr)

0.60
0.50
0.40
0.30

WT

0.20

mtlD

0.10
0mM

100mM
200mM
NaCl concentration

300mM

Figure 33: Effect of different salinity conditions on root dry weight of transgenic and nontransgenic control plants. Data are expressed as the means ± SEs (n=3).
Table 6 (below) shows that the salinity stress treatment has affected on the wild-type
control seedling growth leading to reduce shoot fresh weight, and shoot and root dry weights.
However, the mtlD transgenic plants were less affected by salt treatments as compared to the
wild-type control plants. At 100mM NaCl treatment, the shoot fresh weight of transgenic plants
51

was reduced by 27%, shoot dry weight by 17%, and root dry weight by 6% as compared to the
no salt treated control plants. However, the reduction of shoot fresh weight, shoot, and root dry
weights in wild-type was 45%, 40% and 19% respectively.
When exposed to 300mM NaCl, the effect of stress was more severe on wild-type
reducing up to 74% of shoot fresh weight, 62% of shoot dry weight, and 44% of root dry weight.
The Table 6 also shows that T3 plants exhibited greater salt tolerance at the same salt stress
condition than wild-type plants as they reduced shoot fresh weight, shoot dry weight and root dry
weight, by 52%, 40%, and 24% for respectively. The effect of salinity stress was also reduced
the absolute plant height rate (centimes plant height per day) of both transgenic and wild-type
plants. The plant height rate of transgenic and wild-type plants was no significant difference in
the non salt stress condition. Overall, the increase in salt concentration has more negative effects
on wild-type control plants as compared to the T3 plants.
Table 6: Biomass growth reduction of T3 mtlD plants versus the wild-type control plant treated
with different NaCl concentrations for 10 days

Treatment (NaCl)
100mM
200mM
300mM

Percentage of fresh and dry biomass reduction (%)
Shoot fresh
Shoot dry
Root dry
Genotypes
weight
weight
weight
WT
45.7
40.4b
19.0bc
27.5e
17.4d
5.8d
mtlD
WT
67.4b
61.1a
36.8a
46.8
33.4c
12.9cd
mtlD
WT
74.7a
62.3a
43.7a
51.7c
39.8b
23.7b
mtlD

Means within columns followed by the same letter are not significantly different (P<0.05)
Figure 34 (below) represents the T3 mtlD versus wild-type control plants after 7 days of
water treatment recovery from 10 days of 200mM NaCl salinity treatment.

52

Figure 34: T3 mtlD versus wild-type control plants after 7 days of water treatment recovery
from 10 days of 200mM NaCl salinity treatment.

The work presented here agrees with the work of other scientists indicating that the
expression of mtlD gene confers salt tolerance and enhanced fresh and dry biomass of transgenic
plants in sorghum (Maheswari et al., 2010) and potato (Rahnama et al., 2011). In this study, we
did not find any abnormal phenotypes such as dwarfed or stunted growth of transgenic plants
that expressed the bacterial mtlD transgene as compared to their wild-type control plants under
no salt conditions. The same results have been shown in mtlD transgenic eggplant (Praghavathi
et al., 2002)
In the research presented here, salt stress experiments demonstrated significant
differences in shoot fresh and dry weight, and root dry weight in transgenic as well as the wildtype control plants under high salinity concentrations. Also the mtlD transgene plants showed
higher plant height growth rate and less shoot and root reduction under salt stress than wild-type
control plants. Similar results were reported that the expression of mtlD transgene in transgenic
53

plants resulted in higher root growth as compared to their wild-type control plants under salinity
stress treatments in tobacco (Tarczynski et al., 1993), egg plant (Prabhavathi et al., 2002), and
wheat (Abebe et al., 2003).
The effects of salinity stress on photosynthesis and stomatal conductance of T3 mtlD and
wild-type control plants are shown in Table 7 and Figure 35. Table 7 shows that the
photosynthesis rate and stomatal conductance declined with increased salinity concentrations in
wild-type non-transgenic plants and were significantly lower compared to transgenic plants at
100mM NaCl. However, the photosynthesis rate and stomatal conductance were not significantly
different in transgenic plants after 7 days of water treatment recovery from 100 and 200mM of
NaCl treatment.
Figure 35 shows the effect of salinity on photosynthesis and stomatal conductance in
wild-type plants was more severe than those of transgenic plants after 7 days of water treatment
recovery from 100mM NaCl. All wild-type plants were dead with shrunken and dried leaves
after 7 days of water treatment, not being able to recover from 10 days of 200mM of NaCl
treatment. The data presented in table 7 confirm that the mtlD transgenic plants exhibited higher
photosynthesis and stomatal conductance than wild-type control plants at 100 mM NaCl,
agreeing with Hu et al. (2005) report indicating that there were higher stomatal conduction,
transpiration rates and photosynthetic rates in poplar transgenic plants as compared to their wildtype plants treated with 50 mM NaCl after 21 days of salinity treatment.
Table 7 (below) represents the photosynthesis rate and stomatal conductance of T3 versus
wild-type non-transgenic plants 7 days after water treatment recovery from 10 days of different
NaCl treatments.

54

Table 7: Effects of salt stress treatment on T3 plant photosynthesis and stomatal conductance.
Values are means ± SEs (n=3)
Genotype/Treatment
(NaCl)

Photosynthesis
-2 -1
(µmol CO2 m s )

Stomatal conductance
-2 -1
(mol H20 m s )

mtlD transgenic plants
0 mM
23.60 ± 1.18a
0.147 ± 0.014a
100 mM
19.12 ± 0.6b
0.124 ± 0.011a
200 mM
18.06 ± 0.37b
0.113 ± 0.004ab
Wild-type control plants
0 mM
22.63 ± 1.03a
0.148 ± 0.009a
100 mM
14.90 ± 0.55c
0.086 ± 0.005b
200 mM
Means within columns followed by the same letter are not significantly different (P<0.05)
Figure 35 (below) represents the effect of salt stress treatment on photosynthesis of T3
mtlD versus that of the wild-type non-transgenic control plants after 7 days of water treatment
recovery regime from 10 days of 100mM NaCl.

Figure 35: Effect of salt stress treatment on photosynthesis of T3 mtlD versus that of the wildtype non-transgenic control plants after 7 days of water treatment recovery regime from 10 days
of 100mM NaCl. T: T3, NT: non-transgenic control plants.

55

Mannitol level (µmol/g FW)

0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00

WT
mtlD

0mM

100mM
200mM
NaCl concentration

300mM

Figure 36: Accumulation levels of mannitol in leaves of mtlD transgene plants treated 7 days
with different concentration of NaCl.
Mannitol (or sorbitol) accumulation: The results from the Figure 36 indicate that there
was an increase in detectable mannitol in transgenic plants. Mannitol has increased significantly
in transgenic plants under 100mM and 200mM of NaCl stress conditions as compared to wildtype control plants (Appendix 21). Under no salt stress condition (0mM), mannitol also
accumulated significantly higher in transgenic plants as compared to wild-type non-transgenic
plants. There was no difference of mannitol accumulation in wild-type control when increased
concentrations of NaCl (Figure 36, Appendix 21).
The accumulation of mannitol in transgenic plants usually acts as osmotic adjustment
compound. In this study, it was found that T3 plants showed mannitol accumulation; however
the wild-type control plants also had a low level of mannitol. The presence of mannitol in wildtype control plants are under study by Professor Wayne Loescher to see whether the wild-type
control and T3 plants have shown “sorbitol” accumulation rather than mannitol accumulation
because the wild-type control maize plants should not contain mannitol. Since sorbitol is

56

sometimes found in maize tissues (Carey et al., 1982; Shaw et al., 1984), confirmation of these
results awaits a mass spectrometric analysis of the samples to see whether the results shown here
were for sorbitol or for mannitol
Hu et al. (2005) reported the accumulation of mannitol of 0.003-0.035mg/g fresh weight
could improve salt tolerance in transgenic poplar plants. Similarly, mannitol content was
0.04mg/g dry weight in transgenic canola (Motallebi and Rahnama 2011), and 0.8-2.3µmol/g
fresh weight in Arabidopsis (Zhifang and Loescher 2005). Their results demonstrated that the
small amount of mannitol accumulated in the transgenic plants were not enough to act as
osmolyte and suggested that the present of mannitol may indirectly enhance osmotic adjustment
and salt tolerance in transgenic plants. Another possible reason is that the integration and
expression of a transgene accumulating mannitol in transgenic plants may activate and express
other stress inducible genes conferring drought and salt tolerance. Several trehalose synthesized
genes, ABA receptor genes, and raffinose genes that known to reduce the oxidative damage in
plants under salt stress were up-regulated under salt stress in the mannose-6-Photphatse (M6PR)
transgenic Arabidopsis accumulating mannitol (Chan et al., 2011).
In general, because mannitol has stress-protective functions in plants (Rahnama et al.,
2010) it is possible that drought and salt tolerance of transgenic maize in this study is partially
due to mannitol accumulation in them. In a report presented by Abebe et al. (2003) shows the
effect of drought stress on the mannitol content of transgenic leaves is shown to be 150% higher
than in the wild-type control plants, but this experiment was not repeated. This report also
indicated that transgenic wheat plants expressing the mtlD transgene showed significantly
different shoot dry weight, plant height, and leaf length compared to non-transgenic control
under salt conditions.

57

CHAPTER III
COEXPRESSION OF A COMBINATION OF HVA1 AND MTLD TRANSGENES
CONFERS DROUGHT AND SALT TOLERANCE IN TRANSGENIC MAIZE PLANTS
3.1. LITERATURE REVIEWS
3.1.1. Introduction
Previous two chapters of this dissertation consecutively described the effect of barley
HVA1 and the bacterial mtlD on drought and/or salt tolerance in maize. This chapter follows the
concept that “stacking” or “pyramiding” of these two stress tolerance transgenes might prove
more effective than each of the above two transgenes alone on drought and/or salt tolerance in
maize.
Gene stacking has been commercially performed for improving a combination of
different traits such as insect and herbicide resistance. The transfer of a combination of multiple
transgenes or transgene stacking to improve multiple traits at commercial level has become one
of the promising approaches in plant genetic engineering and biotechnology in the last decade.
Therefore in 2007, 40% of Biotech crops in the US carried multiple transgenes. Furthermore, the
60% of the total biotech maize Biotech crop in the US carried transgenes stacked for herbicide
and insect resistance (James, 2007).
Another concept is the use of a transcription factor as a transgene that can activate a
multiple number of naturally existing genes in plants resulting in improvement of a trait such as
salt or drought tolerance (Su et al. 2010). This concept will be described more in Chapter IV
where this candidate transferred sorghum drought tolerance transcription factor into the maize
genome.

58

3.1.2. Strategies for transgene stacking via breeding of transgenic plants
One strategy in gene stacking is to combine two or more transgenes into a crop species
via the intercrossing of two parental transgenic lines each containing one transgene, followed by
the selection of an ideal trait until all desirable transgenes are assembled into the progeny. Gene
stacking for insect, disease and weed resistance has been used widely to improve maize, cotton,
and tobacco. For example, cross-bred combination of different Bacillus thuringiensis (Bt) genes
along with phosphinothricin acetyltransferase (PAT) herbicide resistance gene have enhanced
corn borer and rootworm resistance along with herbicide tolerance in maize (Jain and Brar,
2010).
By breeding of two different transgenic tobacco plants, one expressing the betA gene
encoding choline dehydrogenase and the other expressing a vacuolar Na+/H+ antiporter
(AtNHX1)gene, enhanced salt tolerance of double transgene plants as compared to transgenic
plants that expressed only betA or AtNHX1 gene (Duan et al., 2009) was demonstrated. More
specifically, at 150mM NaCl stress condition, seeds of T0 plants expressing both of these genes
showed a significantly higher germination rate when compared to plants that expressed only betA
or AtNHX1. Also, the two-transgene stacked transgenic seedlings performed much better than the
wild-type non-transgenic control seedlings under 342 mM NaCl stress for 10 days, displaying no
injury symptoms. From these results, Duan et al (2009) recommended that the stacking of
transgenic tobacco via breeding might be a better approach than developing transgenic plants
expressing a single salt tolerance transgene.
Similarly, Wei et al. (2011) cross bred two maize transgenic parents, one expressing the
BetA gene and the other expressing the H+-PPase (TsVP) gene (encoding the vacuolar H+
pyrophosphaatase of Thellungiella halophila), and demonstrated that the expression of both
transgenes in pyramided plants could further improve drought tolerance as compared to
59

transgenic plants expressing just one of the two transgenes. In the second experiment, after 21
days of withholding water, the pyramided plants showed less cell membrane damage, higher
amount of soluble sugars, proline content, greater growth of root system and higher biomass as
compared with single BetA or TsVP transgenic plants, and non-transgenic control plants. These
maize pyramided transgenic plants also showed significant differences in phenotypic
performance including greater tassel and silk synchronization after drought stress. Similarly,
percentage of pollen viability in pyramided plants was much higher as compared to single
transgenic plants and the wild-type control plants. They concluded that a number of different
pathways might be involved in drought tolerance as the reason why their transgene-stacked
plants performed much better than transgenic plants expressing each of the two transgenes,
meaning that each transgene might be able to go through one pathway.
3.1.3. Strategies for transgene stacking via re-transformation of transgenic plants
The method of cross breeding of two more transgenic plants to stack genes is relatively
labor intensive and time consuming. Another gene stacking strategy is called re-transformation
for transgene stacking. This strategy is to produce a transgenic plant and then re-transform it with
a second transgene. This method has also been proven to improve traits in several crop species.
For instance, the independent expression of either glyoxalase I (glyI) or glyoxalase II (glyII) gene
in transgenic tobacco was reported to enhance salinity tolerance as compared to their nontransgenic control plants (Singla-Pareek et al., 2003). However, when transgenic plant
expressing the glyI was re-transformed with the glyII gene, the double transformants (glyII +
glyI) were found to be show higher level of salt tolerance. This report indicates that under
400mM and 800mM NaCl treatment for 5 days, the treated leaf discs of double transgenic plants
(glyII + glyI) showed highly significant chlorophyll content in comparison with each of the

60

single transgene transgenic plants, as well as, those of non-transgenic control plants. Similarly,
the double-transgene seedlings showed lowest injury symptoms at 400mM NaCl concentration
as compared to all other plants. When tested under the same (400mM) salinity conditions, the
seedlings and leaves of single gene transformants (glyII or glyI) showed no injury, but the wildtype seedlings showed severe injury and died after 25 days.
The simultaneous re-transformation of tobacco with three transgenes (encoding
dehydroascorbate reductase -DHAR, copper zinc superoxide dismutase -CuZnSOD, and
ascorbate peroxidase -APX) was reported to confer highly salt tolerance in the transgenic
tobacco (Lee et al., 2007). The shoot and root dry weight of triple-transgene transgenic plants
showed significant salt tolerance than that of single or double-transgene transgenic plants after
10 days of 100mM NaCl treatment.
Moreover, transgene stacking via re-transformation is reported to delay the emergence of
Bt resistance in insects of broccoli (Cao et al., 2002) and delay the tolerance of both pest and
disease in rice (Datta et al., 2002).
With all advantages of transgene stacking approach via re-transformation, this method
has the limitation that the two or more transgenes are not linked on the same construct, and
therefore they will segregate apart from each other in crop progenies. Re-transformation is also
labor intensive and time consuming.
3.1.3. Strategies for transgene stacking via co-transformation of transgenic plants
Co-transformation is to genetically engineer plants at once with a mixture of two or more
transgene constructs, each construct containing a specific transgene regulated under specific
regulatory sequences. The ratios of mixing of constructs vary, but usually are 1:1 for two
constructs or 1:1:1 for three constructs.

61

Co-transformation can be performed via Agrobacterium or the gene gun technique. The
co-transformation approach is also less time consuming and less labor intensive than that of retransformation approach. The Agrobacterium system can also be used in co-transformation when
multiple gene cassettes are placed in one binary vector for transformation. Using this concept,
Zhu et al. (2012) transferred an Agrobacterium binary vector containing three linked broadspectrum potato disease resistance genes in potato, and found that 23 putative transgenic plants
contained all three genes. Those triple-gene transformants were reported tolerant to all three
selected isolates of Phytophthora infestans.
Tang et al. (2005) co-transferred a mixture of two Agrobacterium into loblolly pine, one
Agrobacterium containing the gene encoding mannitol-1-photphate dehydrogenase (mtlD) and
the other containing the glucitol-6-photphate dehydrogenase (GutD). These researchers
demonstrated the simultaneous integration and expression of these two genes in transgenic plants
resulting in an increased accumulation of both mannitol and glucose. The transgenic plants also
maintained higher survival rates when grown under 85mM and 120mM NaCl as compared to
their wild-type control plants.
When a single expression vector construct containing six linked expression cassettes
(cassettes including two selective marker genes pat and als, two reporter genes LUC and GUS,
and two defensin genes RsAFP2 and DmAMP1) were transformed into Arabidopsis via
Agrobacterium, all six genes were integrated into plants (Goderis et al. 2002) because all six
genes were linked together. Also, Su et al. (2011) delivered four different constructs containing
five different genes (SacB, JERF36, vgb, and BtCry3A +OC-I) into poplar plants and confirmed
that the stacking of all five transgenes in two poplar transgenic lines D5-20 and D5-21 resulted in
their tolerance to drought, salinity, water logging, and insects, respectively.

62

Transgenic rice plants co-transformed with two Suaeda salsa genes encoding Glutathione
S-transferase (GST) and Catalase (CAT1) showed significantly increased salt tolerance (200 mM
NaCl for 10 days in greenhouse) as compared to non-transgenic rice plants (Zhao and Zhang
2006). Catalase and glutathione S-transferase are known to play critical roles in plant defense
mechanisms and in the detoxification of xenobiotics (Anderson et al., 1998).
Chen et al. (1998) co-bombarded a mixture of 14 plasmids into embryogenic rice tissues,
and regenerated plants. The results showed that using the PCR method to confirm transgene
integration, over 70% of the T0 plants were found to contain from two to eight transgenes, 17%
of T0 plants contained more than 10 transgene, and 2.4% contained 13 transgenes. The stability
of transgenes integration and co-expression in transgenic rice plants were also inherited through
three generations (T2). This multiple transgene transformation method sounds to be a promising
strategy for gene stacking via co-bombardment. Similarly, Agrawal et al. (2005) simultaneously
co-transformed five minimal cassettes (without vector backbone) into rice genome via the gene
gun method and found all transgenic plants had integrated two of the transgenes, but only 16% of
had integrated all five transgenes, likely because genes were not linked in one construct.
Transgenic plants also showed the stability of co-integration and co-expression of those
transgenes in the subsequent generations
The gene gun particle bombardment is considered a superior method for multiple gene
co-transformations over the Agrobacterium mediated transformation system because one can
simply mix constructs rather than placing of several gene cassettes on one binary vector.
However, in case of the gene gun method, the size of the construct must be relatively small (less
than 10 kb), otherwise, it might break during bombardment.

63

Due to its potential advantages, this candidate chose the co-transformation method of
transgene pyramiding in maize via gene gun bombardment for his studies of drought and salt
tolerance.
3.2. RESEARCH GOAL AND OBJECTIVES
The goal of this research is to stack the HVA1 and mtlD transgenes to increase the
transgenic plant drought and/or salt tolerance as compared to single transgene (HVA1 or mtlD)
transgenic plants.
The specific objectives of this research are listed below:
(i)

Co-bombard the barley HVA1 and the bacterial mtlD gene constructs in a 1:1 ratio
into maize genome.

(ii)

Verify the proper expression and stability of transgenes transmission into 2
3

(iii)

rd

(T2) and 4

th

nd

(T1)

(T3) progenies.

Evaluate the effects of the HVA1-mtlD transgene stacking on maize seedling
development, plant height, and fresh and dry biomass matter as compared to single
HVA1 or mtlD transgenic plants, and as compared to those of the wild-type nontransgenic control plants under drought and salt stress conditions.

(iv)

Determine the accumulation of mannitol and other soluble sugar contents in T3
(HVA1-mtlD) transgenic versus single HVA1 or mtlD transgenic and the wild-type
plants.

64

3.3 MATERIALS AND METHODS
Two DNA plasmids, pBY520 containing the barley HVA1 gene (Figure 1) and JS101
containing the bacterial mtlD gene (Figure 24) were co-bombarded with the ratio of 1:1 into
immature maize embryogenic calli as described in chapter I. Then, transgenic plants were selfed
and the T3 of HVA1-mtlD transgenic plants (line#51-1) were selected for drought and salt
tolerance as compared to single transgene HVA1 transgenic plants (line #161) and mtlD
transgenic plants (line#27-1). The drought tolerance test and salt tolerance test in this chapter
were followed as described in chapter I and II, respectfully.
3.4. RESULTS AND DISCUSSIONS
3.3.1. Confirmation of integration of the stacked HVA1-mtlD transgenes via PCR analysis,
and their transcription via northern blot hybridization
Three lines developed via co-transformation bombardment contained both the HVA1 and
mtlD transgenes. The T0 of these plants were selfed producing T1 progeny plants. Figure 37
(below) represents the co-integration of both HVA1 and mtlD transgenes in T1 plants.
1kb

+

WT 27-1 27-2 27-3 28-1 28-2 28-3 51-1 51-2 51-3

680bp

HVA1
transgene
100bp

+

WT 27-1 27-2 27-3 28-1 28-2 28-3 51-1 51-2 51-3
mtlD
transgene

431bp

Figure 37: PCR analysis of co-integration of HVA1 and mtlD genes in T1 maize lines.

65

Figure 38 and 39 (below) represent the co-transcription of the HVA1 (Figure 38) and
mtlD (Figure 39) transgenes in the same plants.

27-1

27-2

27-3

28-1

28-2

28-3

51-1

51-2

51-3

WT

Figure 38: Northern blot of HVA1 transgenes in T1 HVA1-mtlD plants using HVA1 probe
27-1

27-2

27-3

28-1

28-2

28-3

51-1

51-2

51-3

WT

Figure 39: Northern blot hybridization of mtlD transgene in T1 of HVA1-mtlD transgenes plants
using mtlD probe
Figures 40 and 41 represent the co-transcription of HVA1 and mtlD transgenes in the
same T2 plants.

66

27-2
a

28-1
b

a

51-1
b

a

51-2
b

a

WT
b

Figure 40: Northern blot hybridization of HVA1 in HVA1-mtlD T2 plants using HVA1 probe
27-2
a

28-1
b

a

51-1
b

a

51-2
b

a

WT
b

Figure 41: Northern blot hybridization of mtlD transgene in HVA1-mtlD in T2 plants using mtlD
probe
Figure 42 (below) shows the integration of both transgenes in T3 generation plants by
PCR analysis.

67

100bp+ WT 1 2

3 4 5

6 7 8 9 10 11 12 13 14 15 16 17
HVA1
transgene

680b
p

mtlD
transgene

431bp

Figure 42: PCR analysis confirming the stable integration of both HVA1 and mtlD transgenes in
T3 plants (line #51-1).
The above molecular methods confirm the co-integration and co-transcription of the
HVA1 along with mtlD in plants that were co-transformed via the 1:1 ratio co-bombardment of
the HVA1 and mtlD transgene constructs.
The above results agree with Agrawal et al. (2005) who could obtain co-integration of up
to two mixed (1:1 ratio) bombarded transgenes in plants. Co transformation methods that show
co-integration occur more likely when both transgenes are integrated in the same chromosomal
position, and that therefore may be inherited together in the progeny (Halpin, 2005). It is rare in
co-bombardment, but chemical selection of both transgenes can assist in both transgenes
transmissions into the progenies. In this case, occasionally, multiple transgenes from multiple
plasmid co-transformations can co-integrate and inherit stably in the subsequent progenies
(Halpin et al., 2001).
3.3.2. Drought stress tolerance in T3 transgenic maize plants that were co-transformed
with two constructs
Four week old plants of T3 stacked transgenic and single transgenic and wild-type plants
were used for drought tolerance test (Figure 43). Figure 44 shows the percentage of leaf relative
68

water content (RWC) after 10 and 15 days of water withholding of transgenic plants versus wildtype non-transgenic control with no water with-holding.

RWC (%)

Figure 43: T3 transgenic and wild-type plants at four week old plants before exposing to water
withholding.
100.00
90.00
80.00
70.00
60.00
50.00
40.00
30.00
20.00
10.00
-

WT
HVA1
mtlD
HVA1/mtlD
Normal
watering

Water
withholding

Normal
watering

10 days

Water
withholding

15 days

Figure 44: Percentage of leaf relative water content (% RWC) of transgenic lines after 10 and 15
days of water deficit (withholding). Bars are mean ± SE of three samples.

69

The Figure 44 results show that after 10 days of water withholding, there was no
significant differences between the leaf RWC of all three sets of transgenic plants (HVA1, mtlD,
and HVA1-mtlD), whereas, the leaf RWC of wild-type plants reduced by 81.9%. After 15 days of
water withholding, the leaf RWC in HVA1-mtlD line was 85% higher than in individual HVA1
transgenic plants (81%) and mtlD transgenic plants (77.6%) and signifiantly higher as compared
to wild-type control plants (57.1%) . Non-transgenic control plants with no water with-holding
showed no reduction in leaf RWC (Appendix22).
Table 8 represents the effect of drought tolerance on survival of T3 plants versus their
wild-type control plants.
Table 8: Effect of drought tolerance on the percentage of survival of T3 lines versus wild-type
control plants that were exposed to water deficit for 15 days followed by 7 days of recovery from
water deficit.
Genotypes
HVA1

Total number of
plants
21

Number of plant
survival
11

Percentage (%)
52

mtlD

20

9

45

HVA1-mtlD

21

14

67

WT

20

7

35

All three sets of transgenic lines showed quick recovery after watering and a higher
percentage of plants survived as compared to their wild-type non-transgenic control plants that
were grown under 15 days of water deficit followed by 7 days of watering. Table 8 shows that
the HVA1-mtlD line showed the highest percentage of survival (67%), followed by HVA1 single
transgene transgenic plants (52%) and then the mtlD transgenic plants (45%) while their wildtype non-transgenic plants showing only 35% survival.

70

A
mtlD

HVA1

HVA1/mtlD

WT

Figure 45: Effect of 15 days of drought stress followed by 7 days of water treatment recovery on
the survival of HVA1-mtlD transgenic plants (lower left) as compared with the HVA1 (upper left)
and mtlD transgenic plants (upper right), and wild-type non-transgenic plants.
Because the wild-type control plant did survive 15 days of water stress after they were
watered for 7 days, another experiment was conducted to identify the critical threshold for wildtype plants (number of drought stress needed to kill the control plants). In this experiment, when
transgenic and wild-type plants were exposed to 20 days (instead of 15 days) of water
withholding followed by 7 days of water recovery, all wild-type non- transgenic plants died with
severe symptoms of drought stress (Figure 46), whereas, the transgenic plants including HVA1,
mtlD and HVA1-mtlD survived the water stress treatment with at quick recovery just after 2-3
days after watering. The HVA1-mtlD plants showed a faster recovery as compared with the
single transgene plants. After 20 days of water deficit stress followed by 7 days of water
recovery, the percentage of plant survivals were respectively 45.8%, 41% and 33% for the
transgene stacked HVA1-mtlD, single transgene HVA1 and mtlD transgenic plants.
71

HVA1

mtlD

HVA1/mtlD

WT

Figure 46: Effect of 20 days of drought stress on the growth of HVA1, mtlD, and HVA1- mtlD
transgenic plants as compared with the wild-type non-transgenic plants.
When plants are exposed to severe water stress, they result in reduced RWC and closed
stomata. Such conditions also tend to be associated with a decrease in the accumulation of
abscisic acid (ABA) and/or sugar contents in plants (Yordanov et al., 2003). Therefore, the leaf
RWC is considered an idea symptom for water retention capacity of a tissue. It has been reported
that when the leaf RWC is reduced below the critical threshhold (below 0.3 g H2O g–1 DW),
there is inefficient water left for preferential hydrations (Crowe et al., 1990). As such, the wildtype plants of the present study were more affected by water stress and died (in case of 20 days
of drought stress followed by 7 days of water recovery).

72

The present work agrees with Su et al. (2011) work in transgenic poplar, and Wei et al.
(2011) work in transgenic maize that shows that the two-transgene stacked in a plant can further
improve drought tolerance as compared to the drought tolerance in transgenic plants expressing
only one transgene.
3.3.3. Salinity stress tolerance in T3 transgenic plants
Table 9 (below) shows that the effects of salinity stress treatment increased as the level of
salinity concentrations increased from 100mM NaCl to 300mM NaCl, resulting in reduced shoot
length, shoot fresh weight, and shoot and root dry weights of transgenic plants (HVA1, mtlD, and
HVA1-mtlD) versus wild-type control plants. In this experiment, the wild-type control plants
were more affected by salt treatments as compared to all three sets of transgenic plants (Table 9
and Appendix 23-26).
At 100mM NaCl, the transgene stacked plants showed least damage by salt stress
treatment as compared to single transgene HVA1 or mtlD plants, and the wild-type control plants.
The percentage reduction of shoot fresh weight, and shoot and root dry weights of the transgene
stacking (HVA1-mtlD) plants were 13%, 18.4%, and 21.4%, respectively. While they were
32.1% and 14.8%, and 25.0% for HVA1 transgenic plants and 25.9%, 27.0%, and 21.9% for the
mtlD transgenic plants, respectively (Table 9 and Appendix 28-30).

73

Table 9: Effects of salt treatment on the percentage reduction of shoot length, shoot fresh
weight, and shoot and root dry weight of each set of three transgenic plants (HVA1, mtlD, and
HVA1-mtlD) affected by different concentrations of salt treatments. Number in this table
represent the percentage of reduced shoot length, shoot fresh weight, or shoot and root dry
weight as compared to those of transgenic and wild-type plants at 0mM NaCl (non salt
treatment).
Percentage reduction (%) of
Shoot length

Shoot fresh

Shoot dry

Root dry

weight

weight

weight

HVA1
100mM

7.6f

32.1f

14.8h

25.0defg

200mM

17.0d

43.3e

49.2d

25.1def

300mM

24.0c

66.4c

58.5b

43.2b

100mM

8.0f

25.9g

27.0g

21.9efg

200mM

11.3e

51.3d

35.7f

27.9de

300mM

16.7d

65.6c

50.9dc

30.1cd

100mM

10.3ef

13.0h

18.4h

21.4fg

200mM

17.6d

50.3d

42.1e

18.6g

300mM

29.7b

67.7bc

54.7bc

34.6c

100mM

15.3d

45.6e

40.3ef

30.2cd

200mM

22.6c

70.0b

57.0b

44.4b

300mM

37.7a

76.3a

73.3a

51.9a

mtlD

HVA1/mtlD

WT

Means within columns followed by the same letter are not significantly different (P<0.05)
At high salinity concentration, there were no significant difference in reduction of shoot
fresh and dry weight between the transgene stacking (HVA1-mtlD) and single transgene HVA1 or
mtlD transgenic plants. Root of the transgenic plants (HVA1-mtlD and mtlD) accumulating

74

mannitol showed less affected by salt stress displaying low percentage reduction in root dry
weight relative to HVA1 transgenic and wild-type plants.
Under differential salt concentrations, the HVA1-mtlD plants showed more shoot length
reduction as compared to HVA1 or mtlD transgenic plants, but showed lower shoot reduction
than the wild-type control plants.
Figure 47 (below) compares the effect of 10 days of salt treatments on the growth of T3
of HVA1, mtlD and HVA1-mtlD plants versus their wild-type control plants.
HVA1

mtlD

0mM 100mM 200mM 300mM
HVA1/mtlD

0mM

0mM

100mM200mM300mM

WT

100mM 200mM 300mM

0mM

100mM 200mM 300mM

Figure 47: Comparisons of the effect of 10 days of salt treatments on the growth of T3 of HVA1,
mtlD and HVA1-mtlD plants versus their wild-type control plants.

75

These results are in agreement with the previous researches which indicated that the
expression of multiple resistance genes showed better shoot and root growth performance as
compared to their wild-type control poplar (Su et al., 2011), tobacco (Duan et al., 2009), and
loblolly pine (Pinus taeda L.) (Tang et al., 2005).
3.3.4. Accumulation of mannitol and other soluble sugar contents after 7 days exposing to
different salinity concentrations.
Table 10 (below) represents data collected on the effect of different salt concentrations on
sucrose, fructose, glucose, and mannitol contents of transgenic versus wild-type plants.
Table 10: Effects of different salt concentrations on mannitol sucrose, glucose, fructose and
inositol contents in transgenic and wild-type plants treated with 0, 100, 200, or 300mM NaCl.
Mannitol
NaCl

Glucose

Sucrose

Fructose

Inositol

(µmol/g FW)

(µmol/g FW)

(µmol/g FW)

(µmol/g FW)

(µmol/g FW)

mtlD transgene
0mM

0.26 ± 0.04

5.66 ± 0.54

33.3 ± 3.4

4.15 ± 1.00

0.61 ± 0.04

100mM

0.43 ± 0.05

8.54 ± 1.00

45.2 ± 1.8

2.49 ± 0.17

1.02 ± 0.01

200mM

0.56 ± 0.05

5.52 ± 0.23

42.8 ± 1.3

4.34 ± 0.17

1.22 ± 0.03

300mM

0.35 ± 0.02

7.11 ± 0.20

49.7 ± 6.0

5.94 ± 0.10

1.24 ± 0.21

HVA1-mtlD transgene
0mM

0.32 ± 0.09

7.55 ± 2.12

35.5 ± 5.3

4.70 ± 1.90

0.65 ± 0.16

100mM

0.37 ± 0.06

9.04 ± 0.99

50.9 ± 3.5

8.68 ± 0.39

0.99 ± 0.01

200mM

0.52 ± 0.13

9.41 ± 0.68

45.3 ± 3.4

7.56 ± 0.45

1.37 ± 0.19

300mM

0.55 ± 0.18

7.20 ± 0.48

48.1 ± 4.8

6.31 ± 0.15

1.55 ± 0.14

0mM

0.18 ± 0.08

3.85 ± 0.39

30.3 ± 0.6

1.83 ± 0.01

0.46 ± 0.06

100mM

0.21 ± 0.04

9.19 ± 2.11

41.0 ± 2.0

2.74 ± 0.46

0.75 ± 0.05

200mM

0.19 ± 0.02

10.65 ± 1.24

55.6 ± 6.3

4.78 ± 0.40

1.12 ± 0.04

300mM

0.14 ± 0.02

6.92 ± 1.80

36.0 ± 2.2

2.95 ± 0.45

0.76 ± 0.01

Wild-type

76

Mannitol (or sorbitol) accumulation: The results from the Table 10 indicate that there
was an increase in detectable mannitol in transgenic plants. Since sorbitol is sometimes found in
maize tissues (Carey et al., 1982; Shaw et al., 1984), confirmation of these results awaits a mass
spectrometric analysis of the samples to see whether the results shown were for sorbitol or for
mannitol. Whether mannitol or/and sorbitol, it has increased under salt stress conditions,
especially under 200mM of NaCl (Table 10 and Appendix 31).
Similar to chapter II there were various literature reports that the expression of mtlD gene
in transgenic plants conferred the accumulation of mannitol and improved drought and salt
tolerance in different plant species such as potato (Rahnama et al., 2011); sorghum (Maheswari
et al., 2010); wheat (Abebe et al., 2003); canola (Motallebi and Rahnama 2011); Arabidopsis
(Zhifang and Loescher 2005). However, the reasons for the drought and salt tolerance of
transgenic plants accumulating small amounts of mannitol are unclear. The results demonstrated
that the small amount of mannitol accumulated in the transgenic plants but significant difference
that in wild-type non-transgenic plants could be enhanced osmotic adjustment and resulted in
improved salt tolerance in transgenic plants.
Glucose accumulation: Glucose is increased in wild-type plants under salt stress of 100300mM NaCl, especially under 200nm NaCl. However no significant difference was found in
glucose accumulation between wild-type and the transgenic plants under salt treatment
(Appendix 32).
Sucrose accumulation: Similar to glucose, sucrose accumulated more in wild-type
plants that were stressed under 100-300mM NaCl, especially those under 200mM NaCl. Also,
transgenic plants under 200 mM of NaCl stress showed less sucrose accumulation as compared

77

to their wild-type control, but higher sucrose accumulation under 100mM and 300 mM of NaCl
(Appendix 33).
Fructose accumulation: Fructose accumulation increased in the wild-type control plants
under salt stress, especially under 200mM of NaCl. Transgenic plants expressing a combination
of HVA1-mtlD showed significantly higher fructose accumulation under salt stress as compared
to their wild-type control plants, especially under 100mM of NaCl (Appendix 34).
Inositol accumulation: The wild-type inositol accumulation under no salt stress was
lower than inositol accumulations in all wild-type plants subject to salt stress, but inositol
accumulation in wild-type plants increased due to salt stress, especially under 200 mM of NaCl.
There was no significant difference in inositol accumulation of transgenic plants expressing a
combination of HVA1-mtlD transgenes and mtlD transgenic plants alone (Appendix 35). In this
research, interestingly, levels of inositol in both mtlD and HVA1-mtlD transgenic plants were
1.5-2 folds than that in wild-type plants. Similarly, high level of inositol was reported in barley
leaf cultivar that was tolerant to high Na+ accumulation compared to barley salt sensitive control
plants and the high amount of inositol was suggested to involve in cellular protection against
salinity (Widodo et al., 2009).
In Chapter 1, the integration and expression of single transgene HVA1 conferred drought
and salt tolerance in transgenic maize plants. In Chapter 2, the mtlD transgenic maize plants
showed tolerant to salt tolerance (not sufficient seeds to test for drought tolerance). From the
results in this chapter, we initially conclude that the transgene stacking HVA1-mtlD showed
greater leaf RWC, higher percentage of plants survival under drought, and higher fresh and dry
biomass weight at 100mM NaCl as compared to single transgene HVA1 and mtlD and the wildtype non-transgenic control plants. However the transgene stacked plants showed reduced shoot

78

length and were more affected by high salt concentrations than those of single transgene
transgenic and the wild-type control plants.

79

CHAPTER IV
SORGHUM TRANSCRIPTION FACTOR DREB2 TRANSGENIC MAIZE PLANTS
FOR DROUGHT AND SALT TOLERANCE
4.1. LITERATURE REVIEW
In plants, stress signaling pathways lead to activation of specific drought tolerance
transcription factors (TFs). TFs play a role at a higher level of the stress response gene cascade
and a single TF can turn on a large number of downstream drought tolerance-related genes.
DNA microarrays used to study patterns of gene expression in response to drought
tolerant genotypes show modulation of a large number of genes coding for TFs (HayanoKanashiro et al. 2009). Drought stress induced TFs are activated at the transcriptional or at the
protein level by the transduced drought signal. The TFs act as master switches in triggering a
network of expression of the stress response genes.
The promoters of stress response genes are known to have several types of cis-elements
called drought responsive elements (DRE). DRE binds to the TFs of the same family or different
families (Srivastav et al., 2010).
TFs are divided into two groups, ethylene response factors (ERFs) that are induced under
biotic stress conditions, and drought response element binding (DREB) proteins that bind to
promoters of genes induced under abiotic stress conditions. Expression profiles of the genes
under different stress situations indicate that DREB1 genes are expressed mainly under cold
stress whereas DREB2 genes are expressed under dehydration due to cell osmosis. DREB2A
expression is highly inducible by high salinity and drought stress rather than cold, and the
DREB2A protein requires posttranscriptional modification for its activity/stability (Qin et al.,
2011). Overproduction of a constitutively active form of DREB2A (DREB2A-CA) protein in

80

plants led to an increase in the expression of a number of genes responsive to water deficit stress,
and enhanced drought stress tolerance in transgenic plants (Sakuma et al. 2006).
Similarly, Maruyama et al. (2009) reported that the overexpression of DREB1A and
DREB2A conferred cold and/or drought tolerance in transgenic Arabidopsis plants. Moreover,
only DREB1A overexpression was reported to enhance the accumulation of many metabolites in
transgenic plants and resulted in an improved tolerance of both cold and drought stress. Whereas
transgenic plants expressing DREB2A had strong capability of drought tolerance but produced
less tolerance under cold stress.
Dubouzet et al. (2003) found that overexpression of the DREB1A/CBF3 in transgenic
Arabidopsis plants led to the activation of many target genes, and an increase in drought and cold
tolerance. Xu et al. (2009) also reported that the HvDREB1expression up-regulated RD29A gene
and improved salt tolerance. The expression of DREB1 or OsDREB1 was reported to increase
the content of osmoprotectants and different soluble sugars and confer abiotic stress tolerance in
the transgenic rice and Arabidopsis (Ito et al., 2006, Yamaguchi-Shinozaki & Shinozaki, 2006).
Wang and Dong (2009) demonstrated the accumulation of ZmDBP3 protein, one of the
CBF/DREB from maize, activating C-repeat/DREs in the promoter regions of genes and
improving tolerant to drought and cold stress in transgenic plants.
DREB2A apparently had dual functions in modulating the expression of different sets of
downstream genes under both heat and water deficit stress. Over-expression of DREB2C in
plants significantly enhanced their thermotolerance (Lim et al., 2007). Additionally, DREB2A
and DREB2C are known to interact with AREB/ABF proteins suggesting that they may function
cooperatively to activate the transcription of ABA-responsive genes (Lee et al., 2010).

81

The expression of a transcription factor, DREB2 gene from sorghum driven by a stressinducible Arabidopsis rd29 promoter showed better seed set in transgenic rice resulting in an
increased number of pentacles relative to wild-type plants under drought stress (Bihani et al.,
2011). The overexpressing of rice DREB1G, rice DREB2B, or rice DREB1E was also reported
in transgenic rice to confer drought tolerance (Chen et al., 2008). In this experiment, transgenic
plants expressing DREB1G or DREB2B had a much higher percentage of plant survival rate
after 15 days without watering, followed by 10 days re-watering as compared to wild-type
control plants. The expression of DREB1E in transgenic rice plants showed mediated levels in
response to drought stress displaying lower plant survival rate as compared to the DREB1G or
DREB2B transgenic plants, but significantly higher than wild-type under the same water stress.
The overexpression of soybean DREB2 was demonstrated to regulate the expression of
stress inducible genes and caused an increase tolerance to drought and high salinity in transgenic
Arabidopsis (Chen et al., 2007). Moreover, the transgenic tobacco expressing this soybean
DREB2 gene was reported to accumulate large amount of proline in transgenic tobacco plants.
This research confirmed the function of soybean DREB2 for drought and salt tolerance in plants
Transgenic wheat and barley plants expressing TaDREB2 and TaDREB3 genes showed
significantly higher plant survival under drought stress as compared to their wild-type control.
Also the expression of these transcription factors in transgenic wheat and barley activated
various numbers of other genes that are known to protect cell damage against stress conditions
(Morran et al., 2011). Qiu et al. (2007) found that the overexpression of the DREB2-type
transcription factor, ZmDREB2A from maize resulted in an activation of various stress inducible
genes regarding to LEA and heat shock proteins in transgenic Arabidopsis plants. Also the
transgenic plants expressing ZmDREB2A driven by inducible or constitutive promoters showed

82

significantly higher percentage of survival compared to wild-type plants when plants exposed to
0

10 days of withholding water, or 45 C for 1hr.
This candidate transferred the sorghum DREB2 regulated by Arabidopsis thaliana
drought inducible promoter, rd29 into the maize genome. Sorghum is the major food crop of subSaharan Africa. Like maize, sorghum is also a unique plant feedstock in that all its above ground
growth components (in the form of stem sap sugar, biomass or grain starch) can be converted
into biofuel. However when compared to maize, sorghum is highly tolerant to many abiotic
stresses including drought, heat, and salinity. Therefore, it is hoped that the transfer of sorghum
DREB2 regulated by the rd29A drought inducible promoter will result in production of a maize
genotype that will resist salt and drought.
The goal and specific objectives of this research are shown below.
4.2. GOALS AND OBJECTIVES:
The goal of this research is to study the drought and tolerance of maize plants expressing
the sorghum DREB2. The specific objectives of this project are listed below:
(i)

Using a gene construct containing the sorghum drought tolerance transcription factor,
DREB2 regulated by Arabidopsis rd29 drought inducible promoter in maize genetic
transformation.

(ii)

Genetically engineering maize plants using the above construct and a construct
containing the bar herbicide resistance gene.

(iii) Confirming the sorghum DREB2 transgene integration via PCR analysis.

83

4.3. MATERIALS AND METHODS
4.3.1. Gene construct
The gene construct was provided to our laboratory under Material Transfer Agreement
between MSU and the University of Pune, Pune, India.

Figure 48: pCAMBIA1301 plasmid containing the sorghum transcription factor SbDREB2
regulated by Arabidopsis inducible promoter (rd29A), and nos terminator, cauliflower mosaic
virus 35S promoter and Hygromycin phosphotransferase (hpt) gene.
A second construct (pBY520; Figure 1) was chosen to be co-transferred into maize along
with the pCAMBIA1301 construct containing the sorghum DREB2 gene.
Maize genetic transformation was performed using a 1:1 mixture of pC1301and pBY520
co-bombardment via the gene gun as described in Chapter 1.
4.3.2. Conformation of SbDREB2 transgene via PCR analysis
Specific sequence primers for hpt gene, 5`-AAAGCCTGAACTCACCGC-3` (forward
primer) and 5`-GCTTTCCACTATCGGCGA-3` (reverse primer), were used to detect the hpt
gene in T0 plant generation. DNA amplifications were performed with optimized PCR
0

0

0

conditions 94 C for 4 min for initial denaturation, 35 cycles of 45s at 94 C, 30 s at 57 C, 1min
0

0

at 72 C and a final 10 min extension at 72 C.

84

4.4. RESULTS AND DISCUSSIONS
In this experiment, 48 transgenic lines were regenerated in the first regeneration (T0).
Because maize also contains DREB2 gene, the candidate used the hygromycin resistance
selection marker (hpt) gene to confirm the integration of sorghum DREB2 as the hpt and DREB2
cassettes were linked in the plasmid (Figure 53 below). ,
Therefore, PCR analysis was carried out to identify the integration of hpt gene in DREB2
maize plants. The results from the Figure 53 show that twelve out of 48 T0 putatively transgenic
maize plants were transgenic.

Figure 49: PCR analysis confirming the integration of hpt gene in the first generation (T0)
transgenes plants.
The initial result of the PCR analysis confirms the integration of hpt gene T0 generation.
This candidate assumes the co-existence of two transgene cassettes linked together on one
plasmid. A similar work was in reported in the various literatures for stable integration of two
transgenes at 100% frequency (Maqbool et al., 2002; Oraby et al., 2005; Kingdom et al., 2012).
More research on molecular and physiological analysis of progenies of these DREB2
transgenic plants are needed to be completed by another researcher in our laboratory.

85

CHAPTER V
CONCLUSIONS AND FUTURE PERSPECTIVES
The Biolistic bombardment method used in these studies to transfer single abiotic stress
tolerance transgenes and their combination into maize immature embryo cell lines followed by
regeneration of mature transgenic plants is a relatively fast technique for maize transformation
(Frame et al., 2000; Aulinger et al., 2003, Sticklen and Oraby, 2005). However the Hi II (i.e. a
hybrid between A188 and B73) maize chosen in these studies was not easy to work with because
the hybrid segregates and the progenies vary; but there is no maize inbred line that could be
easily transformed. The candidate concludes that the Hi II maize chosen was an ideal choice
because unlike all inbred lines, it was relatively easy to genetically transform, and because it is
non-proprietary maize.
Studies have shown that the use of gene bombardment for maize genetic transformation
can result in multiple integrated copies of a transgene in plants, and multiple copies of a
transgene could result in silencing of the transgene (Pawlowski et al., 1996; Kohli et al., 1998;
Dai et al., 2001). However, this candidate selected transgenic plants that contained no more than
2-3 copies of each transgenes. Furthermore, Northern blotting and the response to salt and
drought tolerance of transgenic plants proved that transgenes were not silenced. Therefore, here
it is concluded that the use of the gene gun for genetic transformation of maize was a good
choice, because in maize, this method is far more efficient as compared to the Agrobacterium
method of genetic transformation of maize.
Maize genetic transformation studies presented in this dissertation demonstrated that
under greenhouse conditions, transgenic maize plants expressing the HVA1, mtlD, or a
combination of HVA1-mtlD transgenes showed improved drought and salt tolerance as compared
to their wild-type non-transgenic plant counterparts. Witnessing the drought of 2012 and that of
86

2008 in the Corn Belt, corn growers do need a quick technology to protect their crop from the
unpredicted drought.
The barley HVA1 gene alone in maize genome conferred drought and salt tolerance in T3
plants, agreeing with other researchers who tested the same gene in few other transgenic crops
(Xu et al., 1996; Detvisitsakun et al., 2001; Babu et al., 2004; Fu et al., 2007). As the result of
their tolerance to these two abiotic stresses, the HVA1 transgenic lines contained a higher leaf
relative water content as compared to their control non-transgenic plants, due to possible certain
protein accumulation in transgenic plants protecting of plant cell membranes (Babu et al., 2004,
Lal et al., 2008, Checker et al., 2012).
It is important to note that the maize explants used for genetic engineering is a hybrid
(A188 x B73). This hybrid was used in this dissertation research because maize genetic
engineering is very genotype-specific, and this hybrid it highly regenerable and transformable.
Although the use of Hi II maize has the benefit of its transformability, it has a major down side,
i.e. its selfed individuals are heterogeneous populations of selfed lines that have been derived
from the Hi II hybrid.
The preliminary results from Figure 36 of the second chapter of this dissertation and
Table 10 of the third chapter (HVA1-mtlD transgenic plants) of this dissertation indicate that
there was an increase in detectable mannitol in transgenic plants. Mannitol has increased
significantly in transgenic plants under 100mM and 200mM of NaCl stress conditions as
compared to the wild-type control plants. Under no-salt stress condition (0mM), mannitol also
accumulated significantly higher in transgenic plants as compared to the wild-type nontransgenic plants. The mannitol accumulation in both mtlD and the HVA-mtlD transgenic plants
were similar because HVA1 should not contribute towards mannitol accumulation. The only

87

discrepancy in the mannitol accumulation results is the fact that mannitol also accumulated in
wild-type control plants, even thought maize plants are not thought to contain mannitol. As per
Prof. Wayne Loescher (MSU Professor of Horticulture Department), sorbitol accumulation may
have been a factor as gas chromatograph does not easily distinguish mannitol from sorbitol.
Studies are in progress in Dr. Loescher’s laboratory to see the possible contributions of sorbitol
in these studies.
The bacterial mtlD gene in maize genome conferred salt tolerance in T3 plants, resulting
in accumulation of a small amount of mannitol (and/or sorbitol) in transgenic plants. The small
amount of mannitol (and/or sorbitol) may have been the reason for the enhancement in osmotic
adjustment and salt tolerance in transgenic plants (Hu et al., 2005, Motallebi and Rahnama
2011). The combination of the two transgenes (HVA1-mtlD) improved drought and salt tolerance
as compared to plants that were expressing only one transgene. However this increase was not
accumulative or doubled, probably because plant cells have a limit in their accumulative
osmoprotectants. Similarly, a high level of inositol was shown in barley leaf cultivar that was
tolerant to high Na+ accumulation as compared to barley salt sensitive control plants, suggesting
that inositol is involved in cellular protection against salinity (Widodo et al., 2009).
The results showed that mannitol, sucrose, fructose and inositol increased significantly in
transgenic plants under salt stress conditions especially at 100mM NaCl as compared to wildtype control plants. In contrary, glucose was not different in plants under salt stress.
This candidate concludes that the method of “co-transformation” of the two abiotic stress
tolerance transgenes (HVA1-mtlD) used was an ideal choice. The method used is ideal because it
is a less time consuming and less labor intensive method as compared to the “re-transformation”

88

method, especially that the method used resulted in co-integration of both transgenes in crop
progenies, similar to the work reported by Drouglas and Halpin (2010).
Overall, the research results presented here met its promised goals and objectives of
being able to transform maize plants with two single and a combination of the two abiotic stress
tolerance transgenes resulting in an increase in stability of transgenes integration and expressions
in up to fourth progenies, and conferring an increase in fresh and dry vegetative biomass as
results of the salt and/or drought tolerance of the transgenic plants.
The candidate also developped transgenic maize plants that show the integration of the
sorghum DREB2 transgene regulated by the Arabidopsis thaiana rd29A drought inducible
promoter. This work will be completed by another researcher in our laboratory.
More studies are needed on progenies of the HVA1, mtlD, HVA1-mtlD, and DREB2
transgenic maize plants. The contradicting studies of mannitol versus sorbitol in these studies
needs to be further studied by Sticklen’s team in the near future.

89

APPENDICES

90

APPENDIX 1: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF WATER STRESS ON LEAF RELATIVE WATER CONTENT (%) OF HVA1
TRANSGENIC VERSUS WILD-TYPE PLANTS
Source

DF

day
ge
tr
Day*ge*tr

1
1
1
4

R-Square
0.988808

Type I SS
Mean Square
1784.86
1784.86
812.6302
812.6302
4448.675
4448.675
2712.983
678.2456

Root MSE
1.661789

Coeff Var
1.942808

Day

Genotype
HVA1
HVA1
WT
WT
HVA1
HVA1
WT
WT

LSMEAN
Number
1
2
3
4
5
6
7
8

LS-means with the same letter are not significantly different.

A
A
A
A
A
B
C
D

Day
15day
10day
15day
10day
10day
10day
15day
15day

Mean
85.53542

95.4
94.9
94.6
81.7
95.2
73.2
95.5
53.9

Control
stress
Control
stress
Control
stress
Control
stress

LSMEAN
95.5
95.4
95.2
94.9
94.6
81.7
73.2
53.9

Pr > F
<.0001
<.0001
<.0001
<.0001

LSMEAN

Treatment

10day
10day
10day
10day
15day
15day
15day
15day

F Value
646.33
294.27
1610.94
245.6

genotype
WT
HVA1
HVA1
HVA1
WT
WT
HVA1
WT

91

LSMEAN
treatment Number
Control
7
Control
1
Control
5
stress
2
Control
3
stress
4
stress
6
stress
8

APPENDIX 2: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT LENGTH (CM)
OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
5
30
35

R-Square
0.87427

Sum of
Squares
1335.368
192.0417
1527.41

Mean Square
267.0736
6.401389

Coeff Var
11.42113

Genotype
HVA1
HVA1
HVA1
WT
WT
WT

F Value
41.72

Root MSE
2.530097

NaCl treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
31.3
23.3
20.5
26.5
19.5
11.8

Pr > F
<.0001

Mean
22.15278

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
B
C
CD
D
E

LSMEAN
31.3
26.5
23.3
20.5
19.5
11.8

genotype
HVA1
WT
HVA1
HVA1
WT
WT

92

LSMEAN
treatment Number
0mM
1
0mM
4
100mM
2
200mM
3
100mM
5
200mM
6

APPENDIX 3: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT LENGTH (CM)
OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
5
30
35

Sum of
Squares
1251.917
518.0833
1770

Mean Square
250.3833
17.26944

F Value
14.5

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.707298

14.08696

4.155652

29.5

Genotype
HVA1
HVA1
HVA1
WT
WT
WT

NaCl treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
36.7
29.3
29.5
34.9
28.3
18.3

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
A
B
B
B
C

LSMEAN
36.7
34.9
29.5
29.3
28.3
18.3

LSMEAN
genotype treatment Number
0mM
1
HVA1
WT
0mM
4
200mM
3
HVA1
100mM
2
HVA1
WT
100mM
5
WT
200mM
6

93

APPENDIX 4: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (GR) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
5
30
35

Sum of
Squares
14.89774
2.662934
17.56067

Mean Square
2.979548
0.088764

F Value
33.57

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.848358

18.72231

0.297934

1.59133

Genotype
HVA1
HVA1
HVA1
WT
WT
WT

NaCl treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
2.3
2.0
1.4
2.2
1.3
0.4

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
AB
B
C
C
D

LSMEAN
2.3
2.2
2.0
1.4
1.3
0.4

genotype
HVA1
WT
HVA1
HVA1
WT
WT

94

LSMEAN
treatment Number
0mM
1
0mM
4
100mM
2
200mM
3
100mM
5
200mM
6

APPENDIX 5: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY WEIGHT
(GR) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
5
30
35

Sum of
Squares
0.107536
0.071716
0.179252

Mean Square
0.021507
0.002391

F Value
9

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.599914

31.42871

0.048893

0.155569

Genotype
HVA1
HVA1
HVA1
WT
WT
WT

NaCl treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
0.218
0.169
0.154
0.214
0.118
0.061

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
A
AB
B
BC
C

LSMEAN
0.218
0.214
0.169
0.154
0.118
0.061

LSMEAN
genotype treatment
Number
0mM
1
HVA1
WT
0mM
4
100mM
2
HVA1
200mM
3
HVA1
WT
100mM
5
WT
200mM
6

95

APPENDIX 6: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT FRESH
WEIGHT (GR) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS

Source
Model
Error
Corrected Total

DF
5
30
35

Sum of
Squares
12.85837
2.227612
15.08598

Mean Square
2.571674
0.074254

F Value
34.63

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.852339

20.58165

0.272495

1.323972

genotype
HVA1
HVA1
HVA1
WT
WT
WT

treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
2.114
1.691
1.077
1.759
0.990
0.313

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
B
B
C
C
D

LSMEAN
2.114
1.759
1.691
1.077
0.990
0.313

LSMEAN
genotype treatment Number
HVA1
0mM
1
WT
0mM
4
HVA1
100mM
2
HVA1
200mM
3
WT
100mM
5
WT
200mM
6

96

APPENDIX 7: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY WEIGHT
(GR) OF HVA1 TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
5
30
35

Sum of
Squares
0.059751
0.006672
0.066423

Mean Square
0.01195
0.000222

F Value
53.73

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.899554

19.97061

0.014913

0.074675

Genotype
HVA1
HVA1
HVA1
WT
WT
WT

NaCl treatment
0mM
100mM
200mM
0mM
100mM
200mM

LSMEAN
0.131533
0.0724
0.048917
0.124033
0.052383
0.018783

LSMEAN
Number
1
2
3
4
5
6

LS-means with the same letter are not significantly different.

A
A
B
C
C
D

LSMEAN
0.13153
0.12403
0.07240
0.05238
0.04892
0.01878

genotype
HVA1
WT
HVA1
WT
HVA1
WT

97

LSMEAN
treatment Number
0mM
1
0mM
4
100mM
2
100mM
5
200mM
3
200mM
6

APPENDIX 8: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT LENGTH REDUCTION (%) OF T3 HVA1
TRANSGENIC AND WILD-TYPE PLANTS
Source

Sum of
Squares
1180.832
88.1657
1268.997

DF

Model
Error
Corrected total

3
4
7

R-Square
0.988808

Source
Ge
Tr
Ge*Tr

Mean Square

F Value

Pr > F

393.6106
22.04143

17.86

0.0088

Coeff Var
1.942808

DF
1
1
1

Type I SS
242.8808000
735.7448000
202.2060500

Root MSE
1.661789

Mean Square
242.8808000
735.7448000
202.2060500

F Value
11.02
33.38
9.17

LS-means with the same letter are not significantly different.

A
B
B
B

LSMEAN
55.7
34.6
26.4
25.5

LSMEAN
treatment Number
200mM
4
200mM
2
100mM
3
100mM
1

genotype
WT
HVA1
WT
HVA1

98

Mean
85.53542

Pr > F
0.0294
0.0045
0.0388

APPENDIX 9: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT LENGTH REDUCTION (%) OF T3 HVA1
TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

3
4
7

R-Square
0.951694

Source
ge
tr
ge*tr

Sum of
Squares
1200.44
60.93145
1261.371

Mean Square

F Value

Pr > F

400.1465
15.23286

26.27

0.0043

Coeff Var
14.71485

DF
1
1
1

Type I SS
366.7986
405.1281
428.5128

Root MSE
3.90293

Mean Square
366.7986
405.1281
428.5128

F Value
24.08
26.6
28.13

LS-means with the same letter are not significantly different.

A
B
B
B

LSMEAN
47.7
20.0
19.6
18.9

LSMEAN
treatment Number
200mM
4
100mM
1
200mM
2
100mM
3

genotype
WT
HVA1
HVA1
WT

99

Mean
26.52375

Pr > F
0.008
0.0067
0.0061

APPENDIX 10: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%) OF
T3 HVA1 TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

3
4
7

R-Square
0.901387

Source
ge
tr
ge*tr

Sum of
Squares
4273.612
467.541
4741.153

Mean Square

F Value

Pr > F

1424.537
116.8853

12.19

0.0176

Coeff Var
24.6048

DF
1
1
1

Type I SS
2235.13
1935.664
102.8178

Root MSE
10.81135

Mean Square
2235.13
1935.664
102.8178

F Value
19.12
16.56
0.88

LS-means with the same letter are not significantly different.

A
B
B
B

LSMEAN
79.8
41.5
39.2
15.3

genotype

treatment

WT
WT
HVA1
HVA1

200mM
100mM
200mM
100mM

100

LSMEAN
Number
4
3
2
1

Mean
43.94

Pr > F
0.0119
0.0152
0.4014

APPENDIX 11: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF T3
HVA1 TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

3
4
7

R-Square
0.988483

Source
ge
tr
ge*tr

Sum of
Squares
2865.522
33.3877
2898.91

Mean Square

F Value

Pr > F

955.1742
8.346925

114.43

0.0002

Coeff Var
6.867783

DF
1
1
1

Type I SS
2098.224
570.8821
196.4162

Root MSE
2.889105

Mean Square
2098.224
570.8821
196.4162

F Value
251.38
68.39
23.53

LS-means with the same letter are not significantly different.

A
B
C
C

LSMEAN
71.7
44.9
29.4
22.4

genotype

treatment

WT
WT
HVA1
HVA1

200mM
100mM
200mM
100mM

101

LSMEAN
Number
4
3
2
1

Mean
42.0675

Pr > F
<.0001
0.0012
0.0083

APPENDIX 12: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT FRESH WEIGHT REDUCTION (%) OF T3
HVA1 TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

3
4
7

R-Square
0.999613

Source
ge
tr
ge*tr

Sum of
Squares
3939.993
1.525
3941.518

Mean Square

F Value

Pr > F

1313.331
0.38125

3444.8

<.0001

Coeff Var
1.266573

DF
1
1
1

Type I SS
1615.393
2279.475
45.125

Root MSE
0.617454

Mean Square
1615.393
2279.475
45.125

F Value
4237.1
5978.95
118.36

LS-means with the same letter are not significantly different.

A
B
C
D

LSMEAN
82.2
49.0
43.7
20.0

genotype

treatment

WT
HVA1
WT
HVA1

200mM
200mM
100mM
100mM

102

LSMEAN
Number
4
2
3
1

Mean
48.75

Pr > F
<.0001
<.0001
0.0004

APPENDIX 13: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF T3
HVA1 TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

3
4
7

R-Square
0.984823

Source
ge
tr
ge*tr

Sum of
Squares
1661.67
25.60785
1687.278

Mean Square

F Value

Pr > F

553.8901
6.401963

86.52

0.0004

Coeff Var
4.041627

DF
1
1
1

Type I SS
611.6253
1008.23
41.81551

Root MSE
2.53021

Mean Square
611.6253
1008.23
41.81551

F Value
95.54
157.49
6.53

LS-means with the same letter are not significantly different.

A
B
B
C

LSMEAN
84.9
62.8
57.8
44.9

genotype

treatment

WT
HVA1
WT
HVA1

200mM
200mM
100mM
100mM

103

LSMEAN
Number
4
2
3
1

Mean
62.60375

Pr > F
0.0006
0.0002
0.0629

APPENDIX 14: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON PLANT HEIGHT
GROWTH RATE (CM/DAY) OF MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

Sum of
Squares
15.47793
8.602657
24.08058

DF
7
48
55

Mean Square
2.211132
0.179222

F Value
12.34

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.642755

22.84609

0.423346

1.853036

LS-means with the same letter are not significantly different.

A
A
B
BC
DC
D
D
D

LSMEAN
2.6
2.6
2.0
2.0
1.6
1.5
1.4
1.1

genotype
mtlD
WT
mtlD
WT
mtlD
WT
mtlD
WT

104

treatment
0mM
0mM
100mM
100mM
200mM
200mM
300mM
300mM

LSMEAN
Number
5
1
6
2
7
3
8
4

APPENDIX 15: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (GR) OF MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total
R-Square
0.945256

DF
7
40
47

Sum of
Squares
1515.671
87.7801
1603.451

Mean Square
216.5245
2.194503

Coeff Var
11.38666

F Value
98.67

Root MSE
1.481385

Mean
13.00983

LS-means with the same letter are not significantly different.

A
A
B
C
C
C
D
D

LSMEAN
21.460
21.357
15.488
11.658
11.364
10.327
7.004
5.421

genotype
WT
mtlD
mtlD
WT
mtlD
mtlD
WT
WT

treatment
0mM
0mM
100mM
100mM
200mM
300mM
200mM
300mM

105

Pr > F
<.0001

LSMEAN
Number
1
5
6
2
7
8
3
4

APPENDIX 16: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY WEIGHT
(GR) OF MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
7
40
47

Sum of
Squares
8.998253
0.759958
9.75821

Mean Square
1.285465
0.018999

F Value
67.66

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.922121

10.45091

0.137837

1.318896

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
2.006
1.197
0.782
0.756
1.878
1.551
1.251
1.130

Genotype
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

LSMEAN
Number
1
2
3
4
5
6
7
8

LS-means with the same letter are not significantly different.

A
A
B
C
C
C
D
D

LSMEAN
2.006
1.878
1.551
1.251
1.197
1.130
0.782
0.756

genotype
WT
mtlD
mtlD
mtlD
WT
mtlD
WT
WT

treatment
0mM
0mM
100mM
200mM
100mM
300mM
200mM
300mM

106

LSMEAN
Number
1
5
6
7
2
8
3
4

APPENDIX 17: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY WEIGHT
(GR) OF MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

Sum of
Squares
0.245573
0.105184
0.350756

DF
7
40
47

Mean Square
0.035082
0.00263

F Value
13.34

R-Square

Coeff Var

Root MSE

Mean

0.700123

13.01168

0.05128

Pr > F
<.0001

0.394104

Genotype
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
0.488
0.395
0.307
0.274
0.472
0.445
0.412
0.361

LSMEAN
Number
1
2
3
4
5
6
7
8

LS-means with the same letter are not significantly different.

A
A
AB
BC
BC
CD
DE
E

LSMEAN
0.488
0.472
0.445
0.412
0.395
0.361
0.307
0.274

genotype
WT
mtlD
mtlD
mtlD
WT
mtlD
WT
WT

treatment
0mM
0mM
100mM
200mM
100mM
300mM
200mM
300mM

107

LSMEAN
Number
1
5
6
7
2
8
3
4

APPENDIX 18: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%) OF
T3 MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source
Model
Error
Corrected total

Sum of
Squares
2840.657
31.44
2872.097

DF
5
6
11

Mean Square

F Value

Pr > F

568.1313
5.24

108.42

<.0001

R-Square

Coeff Var

Root MSE

Mean

0.989053

4.378268

2.289105

52.28333

LS-means with the same letter are not significantly different.

LSMEAN
A
74.7
B
67.4
C
51.7
CD
46.8
D
45.7
E
27.5

genotype

treatment

WT
WT
mtlD
mtlD
WT
mtlD

300mM
200mM
300mM
200mM
100mM
100mM

108

LSMEAN
Number
6
5
3
2
4
1

APPENDIX 19: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF T3
MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

5
6
11

R-Square
0.997191

Source
ge
tr
ge*tr

Sum of
Squares
2923.388
8.235
2931.623

Mean Square

F Value

Pr > F

584.6775
1.3725

425.99

<.0001

Root MSE
1.171537

Coeff Var
2.76469

DF
1
2
2

Type I SS
1783.641
1123.145
16.60167

Mean Square
1783.641
561.5725
8.300833

F Value
1299.56
409.16
6.05

LS-means with the same letter are not significantly different.

A
A
B
B
C
D

LSMEAN
62.3
61.1
40.4
39.8
33.4
17.4

genotype

treatment

WT
WT
WT
mtlD
mtlD
mtlD

300mM
200mM
100mM
300mM
200mM
100mM

109

LSMEAN
Number
3
2
1
6
5
4

Mean
42.375

Pr > F
<.0001
<.0001
0.0365

APPENDIX 20: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF T3
MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source

DF

Model
Error
Corrected total

5
6
11

R-Square
0.973544

Sum of
Squares
2070.29
56.26
2126.55

Mean Square

F Value

Pr > F

414.058
9.376667

44.16

0.0001

Root MSE
3.062134

Coeff Var
12.94771

LS-means with the same letter are not significantly different.

LSMEAN
A
43.8
A
36.8
B
23.7
BC
19.1
CD
12.9
D
5.8

genotype

treatment

WT
WT
mtlD
WT
mtlD
mtlD

300mM
200mM
300mM
100mM
200mM
100mM

110

LSMEAN
Number
3
2
6
1
5
4

Mean
23.65

APPENDIX 21: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON MANNITOL
ACCUMULATION (µMOL/GR FW) OF MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
7
8
15

Sum of
Squares
0.182546
0.02295
0.205495

Mean Square
0.026078
0.002869

F Value
9.09

Pr > F
0.0029

R-Square

Coeff Var

Root MSE

Mean

0.888321

23.73199

0.05356

0.225688

Genotype
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
0.116
0.121
0.159
0.135
0.271
0.432
0.329
0.244

LSMEAN
Number
1
2
3
4
5
6
7
8

LS-means with the same letter are not significantly different.

A
AB
BC
BCD
CDE
DE
DE
E

LSMEAN
0.432
0.329
0.271
0.244
0.159
0.135
0.121
0.116

genotype
mtlD
mtlD
mtlD
mtlD
WT
WT
WT
WT

111

LSMEAN
treatment Number
100mM
6
200mM
7
0mM
5
300mM
8
200mM
3
300mM
4
100mM
2
0mM
1

APPENDIX 22: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF WATER STRESS ON LEAF RELATIVE WATER CONTENTS (%) OF
HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS

Source
time
treatment
genotype
time*treatme*genotyp

Day
10day
10day
10day
10day
10day
10day
10day
10day
15day
15day
15day
15day
15day
15day
15day
15day

Treatment
control
control
control
control
stress
stress
stress
stress
control
control
control
control
stress
stress
stress
stress

DF
1
1
3
10

Type I SS
542.5888
1161.226
555.383
1041.022

Mean Square
542.5888
1161.226
185.1277
104.1022

Genotype
HVA1
HVA1/mtlD
WT
mtlD
HVA1
HVA1/mtlD
WT
mtlD
HVA1
HVA1/mtlD
WT
mtlD
HVA1
HVA1/mtlD
WT
mtlD

LSMEAN
95.6
95.9
94.9
95.4
94.5
94.3
81.9
92.7
94.1
95.1
94.0
95.6
81.0
85.0
57.1
77.6

112

F Value
105.74
226.29
36.08
20.29

Pr > F
<.0001
<.0001
<.0001
<.0001

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

APPENDIX 22 (Cont’d):
LS-means with the same letter are not significantly different.

A
A
A
A
A
A
A
A
A
A
A
B
BC
BC
C
D

LSMEAN
95.9
95.6
95.6
95.4
95.1
94.9
94.5
94.3
94.1
94.0
92.7
85.0
81.9
81.0
77.6
57.1

Day
10day
10day
15day
10day
15day
10day
10day
10day
15day
15day
10day
15day
10day
15day
15day
15day

Treatment
control
control
control
control
control
control
stress
stress
control
control
stress
stress
stress
stress
stress
stress

113

Genotype
HVA1/mtlD
HVA1
mtlD
mtlD
HVA1/mtlD
WT
HVA1
HVA1/mtlD
HVA1
WT
mtlD
HVA1/mtlD
WT
HVA1
mtlD
WT

LSMEAN
Number
2
1
12
4
10
3
5
6
9
11
8
14
7
13
16
15

APPENDIX 23: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT LENGTH (CM)
OF HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
15
80
95

Sum of
Squares
3631.581
489.7917
4121.372

Mean Square
242.1054
6.122396

F Value
39.54

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.881158

5.365045

2.474348

46.11979

Genotype
HVA1
HVA1
HVA1
HVA1
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
52.3
48.3
43.4
39.8
56.7
50.8
46.7
39.8
51.3
43.4
39.7
31.9
53.3
49.0
47.3
44.3

114

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

APPENDIX 24: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT FRESH
WEIGHT (GR) OF HVA1, MTLD AND HAV1-MTLD TRANSGENIC VERSUS WILDTYPE PLANTS
Source
Model
Error
Corrected Total

DF
15
80
95

Sum of
Squares
3956.901
259.4063
4216.307

Mean Square
263.7934
3.242578

F Value
81.35

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.938475

12.84632

1.800716

14.01736

Genotype
HVA1
HVA1
HVA1
HVA1
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
23.90
16.22
13.56
8.02
23.00
20.02
11.43
7.44
18.59
10.12
5.57
4.40
24.10
17.86
11.74
8.30

115

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

APPENDIX 25: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON SHOOT DRY WEIGHT
(GR) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
15
80
95

Sum of
Squares
29.55438
5.65773
35.21211

Mean Square
1.970292
0.070722

F Value
27.86

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.839324

18.49436

0.265935

1.437927

Genotype
HVA1
HVA1
HVA1
HVA1
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
1.923
1.639
0.977
0.798
2.472
2.017
1.432
1.121
1.775
1.060
0.763
0.473
2.290
1.672
1.471
1.125

116

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

APPENDIX 26: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF DIFFERENT SALINITY CONCENTRATIONS ON ROOT DRY WEIGHT
(GR) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Source
Model
Error
Corrected Total

DF
15
80
95

Sum of
Squares
2.158422
1.129157
3.287579

Mean Square
0.143895
0.014114

F Value
10.19

Pr > F
<.0001

R-Square

Coeff Var

Root MSE

Mean

0.656539

22.40445

0.118804

0.530271

Genotype
HVA1
HVA1
HVA1
HVA1
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
0.888
0.665
0.665
0.504
0.708
0.556
0.576
0.462
0.596
0.416
0.331
0.287
0.572
0.447
0.412
0.399

117

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16

APPENDIX 27: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT LENGTH REDUCTION (%) OF HVA1,
MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS
Source
Model
Error
Corrected total

Sum of
Squares
1811.129
19.46375
1830.593

DF
11
12
23

R-Square
0.989368

Source
ge
tr
ge*tr

Mean
Square
164.6481
1.621979

Type I SS
554.6645
1134.543
121.9214

Pr > F

101.51

<.0001

Root MSE
1.273569

Coeff Var
7.013209

DF
3
2
6

F Value

Mean Square
184.8882
567.2715
20.32023

F Value
113.99
349.74
12.53

LS-means with the same letter are not significantly different.

LSMEAN
A
B
C
C
D
D
D
D
E
EF
F
F

37.7
29.7
24.0
22.6
17.6
17.0
16.7
15.3
11.3
10.3
8.0
7.6

treatment

genotype
WT
HVA1/mtlD
HVA1
WT
HVA1/mtlD
HVA1
mtlD
WT
mtlD
HVA1/mtlD
mtlD
HVA1

118

LSMEAN
Number

300mM
300mM
300mM
200mM
200mM
200mM
300mM
100mM
200mM
100mM
100mM
100mM

9
6
3
8
5
2
12
7
11
4
10
1

Mean
18.15958

Pr > F
<.0001
<.0001
0.0001

APPENDIX 28: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT FRESH WEIGHT REDUCTION (%) OF
HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS

Source

DF

Model
Error
Corrected total

11
12
23

R-Square
0.998226

Source
ge
tr
ge*tr

Sum of
Squares
8504.324
15.1153
8519.439

Mean
Square
773.1203
1.259608

Coeff Var
2.217372

DF
3
2
6

Type I SS
1485.574
6468.952
549.7974

F Value

Pr > F

613.78

<.0001

Root MSE
1.122323

Mean Square
495.1913
3234.476
91.63291

Mean
50.615

F Value
393.13
2567.84
72.75

Pr > F
<.0001
<.0001
<.0001

LS-means with the same letter are not significantly different.

A
B
BC
C
C
D
D
E
E
F
G
H

LSMEAN
76.3
70.0
67.7
66.4
65.6
51.3
50.3
45.6
43.3
32.1
25.9
12.97

genotype

treatment

WT
WT
HVA1/mtlD
HVA1
mtlD
mtlD
HVA1/mtlD
WT
HVA1
HVA1
mtlD
HVA1/mtlD

300mM
200mM
300mM
300mM
300mM
200mM
200mM
100mM
200mM
100mM
100mM
100mM

119

LSMEAN
Number
9
8
6
3
12
11
5
7
2
1
10
4

APPENDIX 29: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON SHOOT DRY WEIGHT REDUCTION (%) OF
HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS

Source
Model
Error
Corrected total

Sum of
Squares
6620.224
53.60215
6673.826

DF
11
12
23

R-Square
0.991968

Source
ge
tr
ge*tr

Mean Square

F Value

Pr > F

601.8386
4.466846

134.73

<.0001

Coeff Var
4.862187

DF
3
2
6

Type I SS
1459.617
4768.923
391.6841

Root MSE
2.113491

Mean Square
486.5388
2384.462
65.28069

Mean
43.46792

F Value
108.92
533.81
14.61

LS-means with the same letter are not significantly different.

A
B
B
BC
DC
D
E
EF
F
G
H
H

LSMEAN
73.3
58.5
57.0
54.7
50.9
49.2
42.1
40.3
35.7
27.0
18.4
14.8

genotype

treatment

WT
HVA1
WT
HVA1/mtlD
mtlD
HVA1
HVA1/mtlD
WT
mtlD
mtlD
HVA1/mtlD
HVA1

300mM
300mM
200mM
300mM
300mM
200mM
200mM
100mM
200mM
100mM
100mM
100mM

120

LSMEAN
Number
9
3
8
6
12
2
5
7
11
10
4
1

Pr > F
<.0001
<.0001
<.0001

APPENDIX 30: ANOVA, LEAST SQUARES MEAN AND T COMPARISON FOR THE
EFFECT OF SALT TREATMENT ON ROOT DRY WEIGHT REDUCTION (%) OF
HVA1, MTLD, HVA1-MTLD TRANSGENIC AND WILD-TYPE PLANTS

Source
Model
Error
Corrected total

Sum of
Squares
2375.953
105.1521
2481.105

DF
11
12
23

R-Square
0.957619

Mean Square

F Value

Pr > F

215.9957
8.762675

24.65

<.0001

Coeff Var
9.490296

Source
genotype
treatment
genotype*treatment

DF
3
2
6

Type I SS
1085.153
999.4293
291.371

Root MSE
2.960182

Mean Square
361.7176
499.7146
48.56184

Mean
31.19167

F Value
41.28
57.03
5.54

Pr > F
<.0001
<.0001
0.0058

LS-means with the same letter are not significantly different.

A
B
B
C
CD
CD
ED
DEF
FDGE
FGE
FG
G

LSMEAN
51.9
44.4
43.2
34.6
30.2
30.1
27.9
25.1
25.0
21.9
21.4
18.6

genotype

treatment

WT
WT
HVA1
HVA1/mtlD
WT
mtlD
mtlD
HVA1
HVA1
mtlD
HVA1/mtlD
HVA1/mtlD

300mM
200mM
300mM
300mM
100mM
300mM
200mM
200mM
100mM
100mM
100mM
200mM

121

LSMEAN
Number
9
8
3
6
7
12
11
2
1
10
4
5

APPENDIX 31: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT
OF DIFFERENT SALINITY CONCENTRATIONS ON MANNITOL CONTENTS
(µMOL/GR FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS
Genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
0.33
0.37
0.52
0.55
0.18
0.21
0.19
0.14
0.26
0.43
0.56
0.35

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12

LS-means with the same letter are not significantly different.

A
AB
ABC
ABCD
ABCD
ABCD
ABCD
ABCD
BCD
CD
CD
D

LSMEAN
0.56
0.55
0.52
0.43
0.37
0.35
0.33
0.26
0.21
0.19
0.18
0.14

Genotype
mtlD
HVA1-mtlD
HVA1-mtlD
mtlD
HVA1-mtlD
mtlD
HVA1-mtlD
mtlD
WT
WT
WT
WT

122

Treatment
200mM
300mM
200mM
100mM
100mM
300mM
0mM
0mM
100mM
200mM
0mM
300mM

LSMEAN
Number
11
4
3
10
2
12
1
9
6
7
5
8

APPENDIX 32: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT
OF DIFFERENT SALINITY CONCENTRATIONS ON GLUCOSE CONTENT (µMOL/GR
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
7.55
9.04
9.41
7.20
3.85
9.19
10.65
6.92
5.66
8.54
5.52
7.11

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12

LS-means with the same letter are not significantly different.

A
A
A
A
AB
AB
AB
AB
AB
AB
AB
B

LSMEAN
10.65
9.41
9.19
9.04
8.54
7.55
7.20
7.11
6.92
5.66
5.52
3.85

genotype
WT
HVA1/mtlD
WT
HVA1/mtlD
mtlD
HVA1/mtlD
HVA1/mtlD
mtlD
WT
mtlD
/mtlD
WT

123

treatment
200mM
200mM
100mM
100mM
100mM
0mM
300mm
300mM
300mM
0mM
200mM
0mM

APPENDIX 33: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT
OF DIFFERENT SALINITY CONCENTRATIONS ON SUCROSE CONTENTS (µMOL/GR
FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE PLANTS
Genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mm
200mm
300mM

LSMEAN
35.51
50.87
45.26
48.13
30.31
40.97
55.62
36.02
33.30
45.21
42.80
49.67

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12

LS-means with the same letter are not significantly different.

A
AB
ABC
ABC
ABCD
ABCD
ABCD
ABCD
BCD
BCD
CD
D

LSMEAN
55.62
50.87
49.67
48.13
45.26
45.21
42.80
40.97
36.02
35.51
33.30
30.31

genotype
WT
HVA1/mtlD
mtlD
HVA1/mtlD
HVA1/mtlD
mtlD
mtlD
WT
WT
HVA1/mtlD
mtlD
WT

124

treatment
200mM
100mM
300mM
300mM
200mM
100mM
200mM
100mM
300mM
0mM
0mM
0mM

APPENDIX 34: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT
OF DIFFERENT SALINITY CONCENTRATIONS ON FRUCTOSE CONTENTS
(µMOL/GR FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS
Genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl
treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
4.70
8.68
7.56
6.31
1.83
2.74
4.78
2.95
4.15
2.49
4.34
5.94

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12

LS-means with the same letter are not significantly different.

A
AB
ABC
ABC
BCD
BCD
CD
CD
D
D
D
D

LSMEAN
8.68
7.56
6.31
5.94
4.78
4.70
4.34
4.15
2.95
2.74
2.49
1.83

genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
mtlD
WT
HVA1/mtlD
mtlD
mtlD
WT
WT
mtlD
WT

125

treatment
100mM
200mM
300mM
300mM
200mM
0mM
200mM
0mM
300mM
100mM
100mM
0mM

APPENDIX 35: LEAST SQUARES MEAN AND T COMPARISON FOR THE EFFECT
OF DIFFERENT SALINITY CONCENTRATIONS ON INOSITOL CONTENTS
(µMOL/GR FW) OF HVA1, MTLD, HVA1-MTLD TRANSGENIC VERSUS WILD-TYPE
PLANTS
Genotype
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
HVA1/mtlD
WT
WT
WT
WT
mtlD
mtlD
mtlD
mtlD

NaCl treatment
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM
0mM
100mM
200mM
300mM

LSMEAN
0.65
0.99
1.37
1.55
0.46
0.75
1.12
0.76
0.61
1.02
1.22
1.24

LSMEAN
Number
1
2
3
4
5
6
7
8
9
10
11
12

LS-means with the same letter are not significantly different.

A
AB
ABC
ABC
ABCD
ABCD
BCDE
CDE
CDE
ED
ED
E

LSMEAN
1.55
1.37
1.24
1.22
1.12
1.02
0.99
0.76
0.75
0.65
0.61
0.46

genotype
HVA1/mtlD
HVA1/mtlD
mtlD
mtlD
WT
mtlD
HVA1/mtlD
WT
WT
HVA1/mtlD
mtlD
WT

126

treatment
300mM
200mM
300mM
200mM
200mM
100mM
100mM
300mM
100mM
0mM
0mM
0mM

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127

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