Engineering Drought Tolerance in Tropical Maize

BIOEGIEERIG DROUGHT TOLERACE I TROPICAL MAIZE
(Zea mays L.) THROUGH IDUCIBLE EXPRESSIO OF
ISOPETEYLTRASFERASE GEE
LETA TULU BEDADA (BSc., MSc.)
I84F/12347/2009
A THESIS SUBMITTED I FULFILMET OF THE REQUIREMETS
FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY
I PLAT BIOTECHOLOGY I THE SCHOOL OF PURE AD
APPLIED SCIECES OF KEYATTA UIVERSITY
February, 2014

ii
DECLARATIO
This thesis is my original work and has not been presented for a degree in any
other University or any other award.
Signature___________________________Date_______________________
Leta Tulu Bedada
Department of Biochemistry and Biotechnology
Kenyatta University,
Nairobi, Kenya.
We confirm that the work reported in this thesis was carried out by the candidate
under our supervision.
Signature___________________________Date_______________________
Prof. Jesse Machuka, Ph.D
Kenyatta University
Nairobi, Kenya.
Signature___________________________Date_______________________
Dr. Steven Maina Runo
Kenyatta University
Nairobi, Kenya.
Signature___________________________Date_______________________
Dr. Wondyifraw Teffera
Ethiopian Institute of Agricultural Research
P. O. Box 2003
Addis Ababa, Ethiopia.

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DEDICATIO
To the Almighty God

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ACKOWLEDGMET
Having been trained as a plant scientist (BSc.) and an agronomist (MSc.), I never
had the dream that I would raise a plant from a single cell in a test tube leave
alone inserting a foreign gene in it from another unrelated organism to effect
genetic transformation. I am very grateful to all who have contributed to this
momentous professional transformation. My foremost thanks go to my supervisor
the late Professor Jesse Machuka, for admitting me to his Plant Transformation
Laboratory (PTL) and for his close supervision and constant encouragement in the
course of my work. May the Almighty God rest his soul in eternal peace. I also
thank Dr. Charless Mugoya, manager of the AGROBIO program of the
Association for Strengthening Agricultural Research in Eastern and Central Africa
(ASARECA), to support my admission to this Ph.D study. My special
acknowledgment goes to Dr. Steven Maina Runo for his encouragement,
motivation and guidance he showed me at all levels of my work. His contributions
were so great and immense without which I would not have accomplished most of
my molecular works mainly molecular cloning and analyses of the transgenic
maize using Southern blot and RT-PCR. Thank you so much Dr. Runo. I am
different from how I was when I joined Kenyatta University because of your
assistance and kindness. My thanks also go to Dr. Wondyifraw Teffera for
dedicating his time to edit my thesis and articles published in different journals. I
thank Dr. Eduardo Blumwald, in the Department of Plant Sciences, University of
California, Davis, CA, USA for availing the gene construct. The tropical maize
germplasm used in this study was obtained from CIMMYT-Nairobi and Ethiopian
Institute of Agricultural Research (EIAR). I thank both research institutions for
their kind and generous support. I would like to take this opportunity to express
my utmost appreciation to the World Bank and ASARECA for the financial
support they provided for my Ph.D study.
I am grateful to my wife, Meseret Tesfaye, for being supportive of my education
despite her ill health. I avail this opportunity to thank my brother Worku Gachena
and my sister Mulu Guddissa who took all the responsibility of taking care of my
wife and our children. It is indeed a pleasure to thank Mr. Gebresilassie Hailu for
paying frequent visit to my family and carrying the monthly subsistence to them
from Jimma Agricultural Research Centre. I am indebted to my children Natnael
Leta, Talile Leta and Iyuel Leta who missed me for the whole four years. Thank
you dear Natnael, Talile and Iyuel, I was really feeling at home all the time you
were chatting with me on the phone and Skype.
I am also pleased to express my deepest appreciation to all my colleagues and
friends with whom I stayed and worked at the PTL of Kenyatta University. I am
indeed grateful to my friend Dr. Miccah Songelael Seth for his support and
assistance at times when I have had problems, providing me constructive
comments at every step of my work. My sincere thanks go to Dr. Allan Jalemba
Mgutu, Dr. Omwoyo Ombori, Dr. Amos Alakoyna, Dr. Richard Okoth Oduor,
Mr. Eric Kimani Kuria, Dr. Ngugi Mathew Piero, Dr. Rasha Omer Abdella, Mr.
Jonathan Mutie Matheka, Mr. Nzaro Makenzi, Mr. Akoy Jossek Nabongo, Mr.

v
Wycliffe Luasi, Mr. Dankan Odiambo, Mrs. Olive Sande, Mrs. Sylvia Nawiri,
Miss. Asami Pauline and Miss. Dinah Karimi for their help and assistance in the
laboratory.
Finally, I would like to wish peace, prosperity and love to the community of
Kenyatta University and the whole people of Kenya who hosted me for the last
four years. Thank you, God bless you all.

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TABLE OF COTETS
DECLARATIO .............................................................................................II
DEDICATIO ............................................................................................... III
ACKOWLEDGMET................................................................................IV
TABLE OF COTETS ...............................................................................VI
LIST OF TABLES........................................................................................ XII
LIST OF FIGURES.....................................................................................XIII
LIST OF PLATES.......................................................................................XIV
ABBREVIATIOS AD ACROYMS ...................................................XVI
ABSTRACT .................................................................................................. XX
CHAPTER OE............................................................................................... 1
GEERAL ITRODUCTIO ....................................................................... 1
1.1 Importance of maize to African economy ................................................ 1
1.2 Leaf senescence ........................................................................................... 4
1.2.1 Drought induced leaf senescence in maize............................................ 5
1.2.2 Approaches to delay leaf senescence..................................................... 6
1.3 Problem statement and justification....................................................... 10
1.4 Hypotheses................................................................................................. 12
1.5 Objectives .................................................................................................. 12
1.5.1 General objective................................................................................. 12
1.5.2 Specific objectives............................................................................... 12
CHAPTER TWO............................................................................................ 14
LITERATURE REVIEW .............................................................................. 14
2.1 Constraints to maize production in Africa............................................. 14

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2.1.1 Drought................................................................................................ 14
2.1.2 Poor soil fertility.................................................................................. 16
2.1.3 Diseases ............................................................................................... 16
2.1.4 Insect pests........................................................................................... 17
2.1.5 Weeds .................................................................................................. 17
2.2 Maize genetic improvement for drought stress tolerance..................... 18
2.2.1 Conventional breeding......................................................................... 18
2.2.2 Genetic engineering............................................................................. 20
2.3 Agrobacterium tumefaciens-mediated maize transformation ............... 22
2.3.1 Advantages of Agrobacterium tumefaciens-mediated transformation 24
2.3.2 Factors influencing Agrobacterium tumefaciens-mediated maize
transformation…………………………………………………………....... 25
2.3.2.1 Genotypes ..................................................................................... 26
2.3.2.2 Explant types ................................................................................ 27
2.3.2.3 Growth condition of donor plants................................................. 28
2.3.2.4 Agrobacterium tumefaciens strains and vectors ........................... 28
2.3.2.5 Media composition ....................................................................... 30
2.4 Delaying leaf senescence through genetic engineering.......................... 31
2.4. 1 Factors driving leaf senescence .......................................................... 31
2.4.2 Metabolic changes during drought-induced leaf senescence............... 33
2.4.3 Plant growth regulators and leaf senescence ....................................... 35
2.4.3.1 Role of cytokinins in delaying leaf senescence............................ 36
2.4.4 Molecular genetic manipulation of leaf senescence............................ 39
2.4.4.1 Expressing ipt gene using inducible promoters............................ 39
2.4.4.1.1 Heat shock inducible promoters ................................................ 39
2.4.4.1.2 Promoters inducible by external environments ......................... 41
2.4.4.1.2.1 Senescence inducible promoter .............................................. 42
2.4.4.1.2.2 Drought inducible promoter ................................................... 44
CHAPTER THREE........................................................................................ 46
REGEERATIO OF ELITE AD COMMERCIAL TROPICAL MAIZE
(Zea mays L.) GEOTYPES.......................................................................... 46
3.1 ITRODUCTIO .................................................................................... 46
3.2 MATERIALS AD METHODS............................................................. 47
3.2.1 Experimental design ............................................................................ 47
3.2.2 Plant materials and explants preparation............................................. 48
3.2.3 Media for maize regeneration.............................................................. 50
3.2.4 Callus initiation and maintenance........................................................ 50
3.2.5 Embryo maturation and plant regeneration ......................................... 52
3.2.6 Acclimatization and growth of primary regenerants ........................... 52
3.2.7 Statistical analysis of data on regeneration.......................................... 53

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3.3 RESULTS.................................................................................................. 54
3.3.1 Callus initiation.................................................................................... 54
3.3.2 Somatic embryo maturation and plant regeneration............................ 59
3.3.3 Acclimatization and growth of regenerants......................................... 60
3.4 DISCUSSIO............................................................................................ 63
CHAPTER FOUR .......................................................................................... 72
AGROBACTERIUM TUMEFACIES-MEDIATED GEETIC
TRASFORMATIO OF TROPICAL MAIZE (Zea mays L.) GEOTYPES
WITH ipt GEE ............................................................................................. 72
4.1 ITRODUCTIO .................................................................................... 72
4.2 MATERIALS AD METHODS............................................................. 75
4.2.1 Construct preparation........................................................................... 75
4.2.1.1 Sub-cloning PSARK:: IPT::OST expression cassette into pNOV2819
binary vector…………….…….………………………………………….75
4.2.1.2 Digestion of the PSARK::IPT::OST PCR product and pNOV2819
vector with HindIII and AscI restriction enzymes……………………….78
4.2.1.3 Ethanol precipitation of the digested products ............................. 79
4.2.1.4 Quantification of the insert and the vector DNA.......................... 80
4.2.1.5 Ligation of the insert into the vector............................................. 81
4.2.1.6 Media for growing Escherichia coli and A. tumefaciens.............. 81
4.2.1.7 Transformation of Escherichia coli strain DH5α cells with the
ligated product………………………………………………… .............. 82
4.2.1.7.1 Preparation of competent Escherichia coli strain DH5α cells... 82
4.2.1.7.2 Transformation of competent E. coli strain DH5α cells............ 83
4.2.1.7.3 Plasmid DNA extraction from transformed Escherichia coli.... 84
4.2.1.7.4 Transformed Escherichia coli strain FDH5α cells selection and
construct confirmation………………………………………………….. 85
4.2.1.7.5 Sequencing the sub-cloned PSARK::IPT::OST cassette............ 86
4.2.1.8 Introducing the pNOVIPT1 construct into Agrobacterium
tumefaciens……………………………………………………… .................... 88
4.2.1.8.1 Preparation of competent Agrobacterium tumefaciens cells ..... 88
4.2.1.8.2 Transformation of competent A. tumefaciens cells.................... 89
4.2.1.8.3 Testing transformed A. tumefaciens colonies through colony
PCR………………………………………………....................................90
4.2.1.8.4 Plasmid DNA extraction from Agrobacterium tumefaciens and
confirmation of transformed colonies…………………………………...91
4.2.2 Media for maize transformation ...................................................... 92
4.2.3 Procedures for maize transformation............................................... 93
4.2.3.1 Preparation of Agrobacterium tumefaciens for infecting immature
zygotic embryos……………………………………………………….... 93
4.2.3.2 Growth of source of explants, cob sterilization and preparation of
immature zygotic embryos for infection………………………………... 94

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4.2.3.3 Agrobacterium tumefaciens infection of immature zygotic embryos
.................................................................................................................. 94
4.2.3.4 Co-cultivation of immature zygotic embryos with Agrobacterium
tumefaciens…………………………………………………………….. ........... 94
4.2.3.5 Callus initiation............................................................................. 95
4.2.3.6 Selection of putatively transformed callus events ........................ 95
4.2.3.7 Maturation of somatic embryos.................................................... 96
4.2.3.8 Regeneration of putative transgenic maize plants ........................ 96
4.2.3.9 Acclimatization and growth of putative transgenic maize plants. 96
4.3 RESULTS.................................................................................................. 97
4.3.1 Response of tropical maize genotypes to Agrobacterium tumefaciens-
mediated transformation…………………………………………………... 97
4.3.1.1 Callus initiation and survival on mannose selection .................... 97
4.3.1.2 Somatic embryo maturation and regeneration of transgenic plants
................................................................................................... ……….102
4.3.1.3 Acclimatization and growth of putative transgenic plants in soil
........................................................................................................... ….103
4.4 DISCUSSIO.......................................................................................... 104
CHAPTER FIVE .......................................................................................... 111
AALYSES OF TRASGEIC MAIZE (Zea mays L.) GEOTYPES
USIG MOLECULAR TECHIQUES .................................................... 111
5.1 ITRODUCTIO .................................................................................. 111
5.2 MATERIALS AD METHODS........................................................... 112
5.2.1 DNA extraction.................................................................................. 112
5.2.2 PCR analyses of putative transgenic maize plants ............................ 113
5.2.3 Southern blot analysis........................................................................ 114
5.2.3.1 Preparation of probes.................................................................. 115
5.2.3.2 Labelling probes ......................................................................... 115
5.2.3.3 Transfer of DNA to membrane, hybridization and detection..... 115
5.2.4 Reverse transcription-polymerase chain reaction (RT-PCR) ............ 116
5.2.4.1 RNA extraction, DNase I treatment and cDNA synthesis......... 116
5.2.4.2 PCR amplification of the ipt mRNA transcript .......................... 118
5.3 RESULTS................................................................................................ 119
5.3.1 Detection of transgene in putatively transformed plants using PCR. 119
5.3.2 Detection of stable transformation and gene integration using Southern
blot analysis……………………………………………………………….123
5.3.3 Gene expression analysis using RT- PCR ......................................... 124
5.4 DISCUSSIO……………………………………………………………125

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CHAPTER SIX............................................................................................. 129
EVALUATIO OF PSARK::IPT TRASGEIC TROPICAL MAIZE
(Zea mays L.) FOR TOLERACE TO DROUGHT STRESS ................. 129
6.1 ITRODUCTIO .................................................................................. 129
6.2 MATERIALS AD METHODS........................................................... 130
6.2.1 Experimental design .......................................................................... 130
6.2.2 Determination of optimum amount of water for each plant .............. 131
6.2.3 Growth condition and establishment of plants .................................. 132
6.2.4 Management of drought stress........................................................... 132
6.2.5 Physiological parameters measured during drought experiment....... 133
6.2.5.1 Leaf relative water content ......................................................... 133
6.2.5.2 Chlorophyll pigment content...................................................... 134
6.2.6 Induction of leaf senescence in dark.................................................. 135
6.2.7 Important agronomic parameters monitored ..................................... 135
6.2.7.1 Days to anthesis.......................................................................... 135
6.2.7.2 Days to silking............................................................................ 135
6.2.7.3 Anthesis-silking interval............................................................. 136
6.2.7.4 Plant height................................................................................. 136
6.2.7.5 Ear height.................................................................................... 136
6.2.7.6 Leaf number per plant................................................................. 136
6.2.7.7 Leaf length.................................................................................. 136
6.2.7.8 Leaf width................................................................................... 137
6.2.7.9 Plant fresh weight ....................................................................... 137
6.2.7.10 Plant dry weight........................................................................ 137
6.2.7.11 Root fresh weight...................................................................... 137
6.2.7.12 Root dry weight ........................................................................ 138
6.2.8 Grain yield and major yield components........................................... 138
6.2.8.1 Ear length.................................................................................... 138
6.2.8.2 Seed yield per plant .................................................................... 138
6.2.8.3 Hundred seeds weight................................................................. 138
6.2.8.4 Seed number per plant ................................................................ 138
6.2.9 Statistical analysis of physiological and agronomic data ................. 139
6.3 RESULTS................................................................................................ 139
6.3.1 Response of transgenic and non-transgenic plants to drought stress. 139
6.3.1.1 Development of stress and leaf senescence................................ 139
6.3.1.2 Effect of drought stress on leaf relative water content ............... 144
6.3.1.3 Effect of drought stress on total chlorophyll content.................. 146
6.3.1.4 Effect of drought stress on chlorophyll a content....................... 147
6.3.1.5 Effect of drought stress on chlorophyll b content....................... 147
6.3.1.6 Effect of drought stress on total carotenoids content.................. 148
6.3.2 Induction of leaf senescence in dark.................................................. 149

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6.3.3 Effect of drought stress on growth and agronomic performance of
transgenic and non-transgenic plants.......................................................... 151
6.4 DISCUSSIO.......................................................................................... 156
CHAPTER SEVE ...................................................................................... 164
GEERAL DISCUSSIO, COCLUSIOS AD RECOMMEDATIOS
........................................................................................................................ 164
7.1 DISCUSSIO.......................................................................................... 164
7.2 COCLUSIOS..................................................................................... 167
7.3 RECOMMEDATIOS AD FUTURE STUDIES .......................... 168
REFERECES ............................................................................................. 170
APPEDICIES ............................................................................................. 192

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LIST OF TABLES
Table 1.1. Maize area, production and productivity in ECA countries ...................2
Table 3.1. Primary callus induction frequencies of ten tropical maize genotypes
evaluated in response to four concentrations of 2,4-D ..........................................55
Table 3.2. Embryogenicb
callus frequencies of ten tropical maize genotypes
evaluated in response to four concentrations of 2,4-D ..........................................57
Table 3.3. Regeneration efficienciesa
of ten tropical maize genotypes evaluated in
response to four concentrations of 2,4-D...............................................................61
Table 4.1. PCR reaction to amplify the PSARK::IPT::OST expression casset......77
Table 4.2. Double digestion reaction of the insert and the pNOV2819 binary .....79
Table 4.3. Primers designed for use in PCR, RT-PCR and sequencing................87
Table 4.4. Summary of callus induction of tropical maize immature zygotic
embryos post Agrobacterium tumefaciens infection, ............................................98
Table 6.1. Important phenological and agronomic characters of non-transgenic
and transgenic plants............................................................................................154
Table 6.2. Seed yield and major yield components of non-transgenic and
transgenic plants ..................................................................................................154

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LIST OF FIGURES
Figure 2.1. Trends of maize area, production and productivity in East and
Central Africa………………………………………………………………. .......15
Figure 3.1. Total, type I and type II embryogenic callus formed in ten tropical
maize genotypes………………………………………………………………….59
Figure 4.1. Gene constructs used in the study. ......................................................76
Figure 5.1. Transformation efficiency of tropical maize genotypes....................123
Figure 6.1. Number of leaves affected by senescence in PSARK::IPTCML216
transgenic and non-transgenic plants after watering/drought/rewatering
experiment………………………………………………………………………144
Figure 6.2. Leaf relative water content measured in 8 weeks old
PSARK::IPTCML216 transgenic and non-transgenic plants at different time points
during watering/drought/rewatering experiment in the glasshouse……………145
Figure 6.3. Chlorophyll content measured in 8 weeks old PSARK::IPTCML216
transgenic and non-transgenic plants at different time points during
watering/drought/rewatering experiment in the glasshouse. ..............................147
Figure 6.4. Concentration of chlorophyll a and b, and their total measured in non-
transgenic and PSARK::IPTCML216 transgenic leaves before dark assay and after
12 days of dark assay…………...……………………………………………… 151
Figure 6.5. Growth parameters of PSARK::IPTCML216 transgenic and non-
transgenic plants as influenced by watering/drought/rewatering
treatments……………………………………………………….………………152

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LIST OF PLATES
Plate 3.1. Tissue culture procedures applied in in vitro regeneration of the tropical
maize inbred line CML442....................................................................................51
Plate 3.2. Somaclonal variations observed in R0 regenerants ……………….......62
Plate 4.1. Amplification of PSARK:: IPT::OST expression cassette using Pfu DNA
polymerase……………………………………………………………………….78
Plate 4.2. Different colonies of Escherichia coli strain DH5α cells transformed
with the ligated product growing on LBA medium supplemented with 100 mg/l
spectinomycin…………………………………………………………………… 84
Plate 4.3. Confirmation of pNOVIPT1 construct in five transformed DH5α cell
colonies through double digestion of plasmid DNA with restriction enzymes...86
Plate 4.4. Analysis of 8 EHA101 colonies transformed with pNOVIPT1 construct
through colony PCR……………………………………………………………...91
Plate 4.5. Transformation and regeneration profile of transgenic tropical maize
CML216 using Agrobacterium tumefaciens………………………………………...101
Plate 4.6. Aberrant phenotypes observed among T0 transgenic plants................104
Plate 5.1. PCR analysis of putative T0 PSARK::IPTCML216 plants using primers
specific to the PSARK::IPT::OST expression cassette…………………… ........120
Plate 5.2. PCR analysis of putative T0 PSARK::IPTCML216 plants using primers
specific to the pmi gene…………………………………………………… .......120
Plate 5.3. PCR analysis of putative T0 PSARK::IPTMelkassa-2 plants using primers
specific to the pmi gene…………………………………………………………121
Plate 5.4. PCR analysis of T1 plants of PSARK::IPTCML395. ............................122
Plate 5.5. Southern blot analysis of nine independent events of
PSARK::IPTCML216…………………………………………………….. ....................124
Plate 5.6. RT-PCR analysis of ipt gene expression in drought stressed
PSARK::IPTCML216 transgenic plants ………………………………….............125
Plate 6.1. Responses of PSARK::IPTCML216 transgenic and non-transgenic tropical
maize plants to drought stress at different time points during the drought assay in
the glasshouse…………………………………………………………………...141

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Plate 6.2. Phenotype of delayed drought induced leaf senescence in
PSARK::IPTCML216 transgenic plants compared to non- transgenic plants ….. .142
Plate 6.3. PSARK::IPTCML216 transgenic and non-transgenic plants growing in
the glasshouse ………………………………………………………….............143
Plate 6.4. Induction of leaf senescence in dark in leaves detached from drought
stressed PSARK::IPTCML216 transgenic and non-transgenic plants……… .......150
Plate 6.5. Root architecture of PSARK::IPTCML216 transgenic and non-transgenic
plants after watering/drought/rewatering experiment………………………….153
Plate 6.6. Ears harvested from non-transgenic, and PSARK::IPTCML216 transgenic
plants after watering/drought/rewatering treatments………………………….. 155

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ABBREVIATIOS AD ACROYMS
ABA Abscisic acid
ACC 1-aminocyclopropane-1-carboxylic acid
ACCS 1-aminocyclopropane-1-carboxylic acid synthase
ANOVA Analysis of variance
ARCs Age-related changes
ASI Anthesis-silking interval
ATP Adenosine triphosphate
BAP 6-Benzylaminopurine
ßME ß-mercaptoethanol
bp Base pair
BSA Bovine serum albumin
cDNA Complementary DNA
CIMMYT International Maize and Wheat Improvement Centre
CIM Callus induction medium
CK Cytokinin
CKX cytokinin oxidase/dehydrogenase
CML CIMMYT maize line
CMPS Cestrium yellow leaf curling virus promoter
sequence
CMM Callus maintenance medium
cm Centimetres
CTAB Cetyltrimethylammonium bromide
C5 Colony # 5
DAP Di-ammonium phosphate
DF Dgrees of freedom
DIMBOA 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
dNTP Deoxynucleotide triphosphate
DNase I Deoxyribonuclease I
DW Dry weight
ECF Embryogenic callus frequency
ECA East and Central Africa
EDTA Ethylenediaminetetraacetic acid
EIAR Ethiopian Institute of Agricultural Research
EMM Embryo maturation medium
Fe Iron
FW Fresh weight
g Grams
g/l Grams per liter
GA Gibberellic acid
GLS Gray leaf spot
GUS ß-Glucoronidase
h Hours

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ha Hectares
HF High fidelity
hptII hygromycin phosphotransferase gene
HSP Histidine heat shock promoter
IPT/ipt Isopentenyltransferase
JA Jasmonic acid
K Potassium
K Kinetin
kb Kilo base pair
kg Kilograms
l Litres
LBA Luria-Bertani agar
LBB Luria-Bertani broth
LGB Larger grain borer
LS Linsmaier and Skoog
LSD Least significant difference
M Molar solution
MES 2-N-morpholinoethane salfonic acid
min Minutes
µM Micromolar
mM Millimolar
mm Millimeters
Mn Manganese
Mg Magnesium
mg Milligram
mg/l Milligram per litre
mg/ml mg per millilitre
mha Million hectares
mt Million tones
µg/gfw Microgram per gram fresh weight
MSV Maize streak virus
MS Murashige and Skoog
N Normal solution
N Nitrogen
NADPH Nicotinamide adenine dinucleotide phosphate
NaoAC Sodium acetate
NAA Naphthalene acetic acid
NEB New England Biolabs
ng Nanograms
NOST Nopaline syntase terminator
NPK Nitrogen-phosphate-potassium
NT Non-transgenic
nm Nanometers
N6 Chu et al. basal medium salts
OD Optical density
OPV Open-pollinated variety
P Phosphorus
PAT Phosphinothricin acetyl transferase

xviii
PCIF Primary callus induction frequency
PCD Programmed cell death
PCR Polymerase chain reaction
PEG Polyethylene glycol
PGRs Plant growth regulators
pmoles Pico moles
PMI/pmi Phosphomannose isomerase
PTL Plant Transformation Laboratory
QPM Quality protein maize
QTL Quantitative trait loci
RE Regeneration efficiency
RNA Ribonucleic acid
RT-PCR Reverse transcriptase PCR
RT Room temperature
RNase RNA endonuclease
ROS Reactive oxygen species
R0 Primary regenerants
rpm Revolution per minute
Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase
RWC Relative water content
s Seconds
S Sulphur
SA Salicylic acid
SAG12 Senescence activated gene 12
SARK Senescence-Associated Receptor Kinase
SDS Sodium dodecyl sulfate
SEE Senescence enhanced
SIM Shoot induction medium
SSC Standard sodium citrate
ST Stressed
T Transgenic
T0 Primary transgenic
T1 Secondary transgenic
t/ha Tones per hectare
TAE Tris acetate EDTA buffer
TBE Tris borate EDTA buffer
Tris Tromothamine(2-amino-2-hydroxymethyl-1,3-
propanediol
2,4-D 2,4-Dichlorophenoxy acetic acid
T-DNA Transfer DNA
TDZ Thidiazuron
TE Tris EDTA buffer
TW Turgid weight
U/µl Units per microliter
v/v Volume by volume
w/v Weight/volume
w/w Weight by weight
WUE Water use efficiency

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WW Well watered
ZmACS6 Zea mays gene coding for 1-aminocyclopropane-1-
carboxylic acid synthase
Zn Zinc

xx
ABSTRACT
Drought is one of the major abiotic constraints contributing to low productivity in
maize. In tropical region, it causes grain yield losses of as high as 70% while
complete crop failure is also common depending on the severity of drought.
Drought diminishes crop productivity mainly by causing premature leaf
senescence. It is now possible to delay drought induced leaf senescence in order to
enhance tolerance to drought and stabilize crop yield through bioengineering. The
ipt gene codes for isopentenyltransferase (IPT) enzyme, which catalyzes the rate-
limiting step in the biosynthesis of cytokinin (CK) and enhances tolerance to
drought by increasing the foliar level of CK that delays drought-induced leaf
senescence in transgenic crops. This study was designed to genetically transform
locally adapted elite and commercial tropical maize genotypes with ipt gene to
develop drought stress tolerance through Agrobacterium tumefaciens-mediated
genetic transformation. Ten maize genotypes adapted to Ethiopian and the Eastern
and Central African (ECA) countries were evaluated for in vitro regeneration
ability using immature zygotic embryos as explants. Six genotypes (Melkassa-2,
Melkassa-6Q, [CML387/CML176]-B-B-2-3-2-B [QPM], CML395, CML442 and
CML216) were identified as the best regenerating ones having potential for
improvement through genetic transformation. Subsequently, the ipt gene was sub-
cloned into the pNOV2819 binary vector to take advantage of the pmi gene as
plant selectable marker and mannose as selective agent. The pNOV2819 binary
vector carrying the ipt gene was introduced into the Agrobacterium strain
EHA101 which was subsequently used to transform immature zygotic embryos
obtained from the six genotypes. Among the six genotypes studied, transgenic
plants were successfully regenerated in Melkassa-2 and CML216 with
regeneration efficiency of 87.5 and 59.6%, respectively. Transgenic plants were
analyzed using PCR, Southern blot and RT-PCR. Based on PCR results,
transformation efficiencies were found to be 97.4 and 100% for Melkassa-2 and
CML216, respectively, indicating stringency of the pmi/mannose based selection
system for maize transformation. Southern blot analysis indicated stable
integration of the transgene into the genome of CML216 with 2-3 copy numbers
in five independent events. In drought assay carried out in the glasshouse
transgenic plants expressing the ipt gene maintained higher leaf relative water
content (RWC) and total chlorophyll concentration during the drought period and
produced significantly higher grain yield, major yield components and root dry
matter compared to the non-transgenic plants. The ipt gene was observed to
improve drought tolerance in tropical maize by delaying drought induced leaf
senescence. It was concluded that the transgenic line developed can be further
tested for tolerance to drought under contained field trials. Furthermore it can be
used in breeding programs to improve drought tolerance in other commercial
tropical maize genotypes through conventional breeding.

1
CHAPTER OE
GEERAL ITRODUCTIO
1.1 Importance of maize to African economy
Maize (Zea mays L.) is a member of the grass family, gramineae, to which all the
major cereals belong. Cultivated maize is a fully domesticated form of the wild
grass, teosinte, native to Central America (Galiant, 1988). Maize has the highest
grain yield potential among the cereals and is a wonder of efficiency in converting
solar energy into food energy. These characters combined with its elasticity to
grow in diverse environments prompted maize to spread from its centre of origin
to different parts of the world. Currently maize is an important food crop growing
globally in about 160 million hectares (ha) with more than 800 million tones (mt)
of grain production per annum (FAO STAT, 2010).
The Portuguese first brought maize to Africa at the beginning of the 16th
century
(Dowswell et al., 1996; McCann, 2005). Maize has since gained tremendous
popularity and currently it is one of the major crops having significant
contribution to the African economy, not only as a source of food but also as feed
and fuel. With the introduction of maize to new African cultures and agro-
ecologies, new varieties were selected to meet new dietary preferences and new
uses were developed to maximize its benefits. Accordingly, tens of millions of
Africans had shifted in food production and consumption patterns from traditional
sorghum and millet to maize (McCann, 2005).

2
Maize has been attractive to African farmers because of its yield potential and
diverse uses even in situations of land scarcity and high population pressure.
Currently maize covers about 25 million ha in sub-Saharan Africa mainly grown
by small-scale farmers that produce 38 million metric tons primarily for food
(Shiferaw et al., 2011). South Africa alone grows maize in 2.8 million ha in large-
scale commercial production, the lion’s share of which is put to animal feed.
Recent statistics for ten selected East and Central African (ECA) countries (Table
1.1) shows that maize is grown in about 9.6 million ha with a total production of
15 million metric tones (FAO STAT, 2010) .
Table 1.1. Maize area, production and productivity in ECA countries
Countries
Area
('000 ha)
Production
('000 t)
Productivity
(t/ha)
Burundi 125.6 126.4 1.1
Ethiopia 1,772.3 3,897.2 2.2
Kenya 2,008.4 3,222 1.6
Madagascar 371.2 411.9 1.1
Rwanda 184.7 432.4 2.3
Tanzania 3,100 4,736.2 1.5
Uganda 890 1,373 1.5
Sudan 26.5 35 1.3
Eritrea 20 18 0.9
Democratic
Republic of Congo
1,156.4 1,484.8 1.2
Total 9,654.9 15,736.7 1.63
Source: FAO STAT, 2010.
Tanzania, Ethiopia and Kenya are the leading maize producers in this region.
Maize ranks first in production and yield in Ethiopia (Tolesa et al., 1996). In
Kenya, it stands first in yield per acre. In these countries maize is grown by 80%

3
of the rural population and dominates the diets of the rural and urban
communities. It is grown from 0 to 2,400 meters above the mean sea level mainly
under rain fed condition using traditional technologies (Morris, 1998). Small-scale
farmers account for 85% of the total maize production with medium-scale farmers
producing the remaining 15%.
Maize forms a dominant source of food to meet the nutritional requirement of
millions of people in Africa. At present more than 300 million people in the
Eastern and Southern Africa, depend on maize as a staple food. Contrary to high-
income countries where 70% of maize is used as feed, more than two thirds of
maize is used as food in sub-Saharan Africa, excluding South Africa. Even here,
maize is not just limited to human consumption. Estimated amount of 18-20% is
used as animal feed. Considerable amount is also sold to meet cash requirements;
although most small-scale farmers do not get adequate maize for household
consumption (Odendo et al., 2001; Shiferaw et al., 2011).
In the ECA region, the highest amount of maize is consumed in Kenya and
Tanzania with annual per capita consumption of more than 120 kg, which is the
highest in Africa and among the highest in the world (Odendo et al., 2001). It is
also sustaining life of millions of people in Ethiopia (Tolesa et al., 1996). Maize is
the main source of calorie contributing 45% of the share of all staple cereals in
Eastern and Southern Africa and 21% in West and Central Africa. Its contribution
as a source of protein is very similar to its contribution of calories (Shiferaw et al.,
2011). The most significant role of maize as a source of food is the consumption

4
of its immature but well grown cob at dough stage that can be cooked or roasted
and consumed as fresh maize. Maize is often called “hunger killer” because of this
fresh cob that is ready for harvest the earliest in the hungriest season.
1.2 Leaf senescence
Leaf senescence is a developmental stage in plants characterized by a sequence of
physiological and biochemical processes that mark the end of growth and
development and onset of disassembly of photosynthetic plant organs with the
ultimate purpose of providing nutrients needed to support the growth of grains for
subsequent generation (Grabau, 1995; Gan and Amasino, 1996; Buchanan-
Wollaston, 1997). It is a type of programmed cell death (PCD) that takes place
through an active and highly regulated process. However, Gan and Amasino
(1997) and Munné-Bosch and Alegre (2004) reported that PCD in leaf senescence
differs from other PCDs in three different aspects. First, PCD in leaf senescence is
a slow process, taking place functionally to enhance efficient recycling of
nutrients that are translocated from the senescing cells to other parts of the plant
such as young leaves, developing flowers, fruits and storage tissues rather than
being a simple degenerative process. Second, leaf senescence, involves an organ-
level cell death that eventually encompasses the entire leaf, whereas other PCDs
involve rather localized cell death or occur in limited tissues and cell types. Third,
senescence can be reversed if inducing stress conditions are relieved before it has
progressed beyond a certain point (Stoddart and Thomas, 1982).

5
1.2.1 Drought induced leaf senescence in maize
Drought, among others, is a major environmental factor that induces leaf
senescence in maize. Under the real field condition where multiple stress factors
operate, drought usually accentuates the effects of other stresses such as high
temperature and high solar intensity. Under dry and hot environment, excessive
irradiation causes leaves to turn yellow because of photoinhibition and
chlorophyll bleaching (Smart et al., 1991). Temperature as high as 47-50˚C was
reported to inhibit leaf photosynthetic activity and leaf chlorophyll accumulation
in maize (Caers et al., 1985). It was also reported that the CK level in the leaves
decreased considerably because of the heat shock. It is possible that high
temperature contributed to the decline in CK level interfering with its biosynthesis
in the roots that might have triggered the senescence symptoms seen in the leaves
(Van Staden et al., 1988). It is also evident that drought causes leaf senescence by
limiting the uptake of nutrients. If there is deficiency of elements, such as nitrogen
or phosphorus, senescence of older leaves occurs presumably because of nutrient
remobilization to the younger leaves.
Generally, plants have evolved mechanisms by which leaf senescence can be
induced by drought stress to reallocate nutrients to reproductive organs and to
eliminate water consumption by older, less productive leaves. Maize launches
such adaptation strategies to avoid drought by reducing canopy size and to
mobilize nutrients like nitrogen to support the growth of grains for subsequent
generation (Grabau, 1995). This regulation of leaf senescence has an obvious
adaptive value, allowing the plant to complete its life cycle even under stressful

6
conditions. However, it greatly affects photosynthetic capacity, dry matter
production and allocation in plants. While it ensures continuity of generation in
wild plants, accelerated leaf senescence is often undesirable in food crops as it
diminishes grain productivity (Gan and Amasino, 1996). This brings the need to
delay senescence so that leaves remain green for longer period thereby carrying
out photosynthesis and maintaining dry matter accumulation that can lead to
higher crop yield.
1.2.2 Approaches to delay leaf senescence
Delayed leaf senescence is positively correlated with grain yield under drought
and has, therefore, been proposed as a suitable indicator of drought tolerance in
maize (Bänziger et al., 2000). Hence, delaying drought induced leaf senescence
has become a prime target as a crop improvement option to enhance drought
tolerance with substantial improvement in grain yield (Thomas and Howarth,
2000; Bhatnagar-Mathur et al., 2008). List of options, ranging from agronomic
practices to genetic engineering, can be brought on board to delay leaf senescence
in maize. Agronomically leaf senescence can be delayed by adequate supply of
nutrients mainly nitrogen (Grabau, 1995; Bänziger et al., 2000) and by
maintaining optimum level of soil moisture and plant population per unit area of
land. Diversity among maize genotypes in grain yield is often ascribed to
differences in staying green to support extended photosynthetic activity (Watson,
1952). Stay green variants of maize have been used in breeding programs to
enhance yield (Thomas and Smart, 1993) indicating possibility of exploiting the

7
natural maize genetic resource diversity to delay leaf senescence through
conventional breeding methods.
Genetic engineering for delaying drought-induced leaf senescence in maize
capitalizes on either blocking the biosynthesis or perception of ethylene or
modifying the genome with transgenes that enhance the biosynthesis of
endogenous CK in transgenic maize. The senescence-enhancing role of ethylene
has been deactivated by repressing the expression of the maize gene (ZmACS6)
coding for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (ACS)
enzyme involved in ethylene biosynthesis that has been shown to delay drought-
induced leaf senescence in temperate maize (Young et al., 2004a). The ipt gene
from Agrobacterium tumefaciens (Akiyoshi et al., 1984) coding for IPT enzyme
which catalyzes the rate-limiting step in the biosynthesis of CK has been shown to
play significant role in promoting drought tolerance in transgenic crops by
delaying drought-induced leaf senescence (Rivero et al., 2007).
Extended greenness accompanied by a delay in senescence induced by nitrogen
stress has been reported in temperate maize transformed with ipt gene driven by
native promoter of the senescence enhanced (SEE) gene (Robson et al., 2004).
Under this system, the transgenic line, however, failed to recycle internal nitrogen
from senescing lower leaves, which has accounted for significant chlorosis in
emerging younger leaves when plants were grown in low nitrogen stress condition
(Robson et al., 2004). Such undesirable trait had already been observed in
transgenic tobacco plants transformed with this gene driven by promoters from

8
senescence-activated genes (Jordi et al., 2000). Then scientists came up with the
concept that other promoters targeting CK production spatially and temporally
should be driving the ipt gene, while maintaining the minimum level required to
delay senescence (Gan and Amasino, 1995; Rivero et al., 2007).
Among the strategies used to achieve this target was to express ipt gene during
plant maturation but before the onset of senescence (Rivero et al., 2007). This
approach avoided confining CK production to older leaves to the extent that it
would not interfere with nitrogen mobilization within the plant. They also
proposed a promoter that induced the ipt gene at the onset of stress signalling in
the plant, allowing the production of CK in all tissues facing water-induced stress.
A Senescence-Associated Receptor Kinase (SARK) gene of which expression is
up-regulated at the earliest stage of leaf senescence before any visible sign like
leaf yellowing was identified in haricot bean (Phaseolus vulgaris L.) (Hajouj et
al., 2000). This was corroborated with appearance of the SARK transcripts at the
earliest stages of senescence both in the attached and detached leaves, which gave
a clue on the regulatory role of this gene in the senescence process. The temporal
variation in the magnitude of the SARK protein also followed the same trend as
that of the RNA and substantiating the concept that the SARK protein is also
associated with the senescence processes. The upstream region of 5'-end of the
SARK gene was isolated as an 830-nucleotide length promoter to which the
Agrobacterium tumefaciens ipt gene was linked and PSARK::IPT construct was
created (Rivero et al., 2007).

9
In tobacco lines transformed with this PSARK::IPT construct, nitrogen mobilization
was not affected as the basal leaves displayed chlorophyll degradation during
drought period (Rivero et al., 2007). In general, the result from this particular
study indicated that the expression of the PSARK::IPT in plants could facilitate the
development of transgenic crops, which can be cultivated in water-limited
environment without significant yield penalties. This created interest to use the
PSARK::IPT construct to improve drought tolerance in tropical maize.
This study aimed to investigate if ipt gene driven by the drought inducible SARK
promoter can be useful in enhancing tolerance to drought stress by delaying
drought induced leaf senescence in locally adapted tropical maize genotypes. For
this purpose a construct carrying PSARK::IPT::OST cassette and hygromycine
phosphotransferase (hptII) gene as plant selectable marker was received from Dr.
Eduardo Blumwald, Department of Plant Sciences, University of California, Davis, CA,
USA. The expression cassette was sub-cloned to the binary vector, pNOV2819, to
avoid use of antibiotic resistance gene as plant selectable marker. Ten well
adapted and farmer preferred elite and commercial tropical maize genotypes were
evaluated for their regeneration capacity using immature zygotic embryos to
establish in vitro regeneration system as a prerequisite in the application of
genetic transformation techniques to enhance their tolerance to drought stress
(Chapter Three). The best six regenerable genotypes were further investigated for
their genetic transformability with ipt gene using Agrobacterium tumefaciens-
mediated transformation technique (Chapter Four).

10
Two genotypes, Melkassa-2 and CML216, were successfully transformed and
fertile and normal transgenic plants were regenerated. The PCR analyses of
putative transgenic events obtained in both genotypes using primers targeting
different regions of the T-DNA indicated the presence of the transgene in the
genome of the two genotypes. Transgenic plants obtained from CML216 were
further advanced to T1 generation, which were further analysed for stable gene
integration using Southern blot. Stably transformed transgenic events were further
tested for tolerance to drought stress under a glasshouse condition (Chapter Six).
Transgenic plants showed tolerance to drought by delaying leaf senescence and
maintaining higher RWC and total chlorophyll concentration during drought
period compared to the non-transgenic plants. Expression of ipt gene in these
drought tolerant plants was confirmed using RT-PCR analysis.
1.3 Problem statement and justification
Drought is a constraint to maize production in about 20-25% of the global maize
area (Heisey and Edmeades, 1999). In tropical and subtropical environments over
60 million ha of maize experience water deficit at one or more growth stages
which causes average yield losses of 17% while values as high as 70% (10 million
tones/year) have been documented (Edmeades et al., 1994). In Eastern and
Southern Africa, where maize is the most important staple food for over 300
million people, drought causes significant crop failure, putting millions of people
to look for foreign food aid. The amount of aid to Africa has been reported to

11
range from USD 0.5 to 1.5 million to balance food deficits due to crop failure
caused by drought (World Food Program, 2006).
With the forthcoming global climate change (Battisti and Naylor, 2009) situations
will become worse as a result of which the loss could reach as high as 10 million
tones each year, affecting some 140 million people (Jones and Thornton, 2003).
To overcome the negative effects of climate change on crop yields and to avoid
food deficits it is mandatory to develop agricultural technologies adapted to such
changing environment. New crop varieties having enhanced water use efficiency
(WUE) and improved tolerance to drought stress rank higher in the list of such
technologies (Rivero et al., 2007). The contribution of conventional breeding
towards this goal has become inadequate because of limited genetic diversity
(Hardy, 2010) and lack of suitable selection criteria for tolerance to drought stress
(Nigussie et al., 2002) indicating the need to diversify the genetic basis of the
locally adapted germplasm by introgressing genes responsible for improving
tolerance to drought stress.
The ipt gene is currently gaining increasing popularity in genetic engineering of
crop plants for improved tolerance to drought stress. Crops genetically engineered
with this gene showed increased level of CK and enhanced tolerance to drought
stress as a result of delayed leaf senescence (Rivero et al., 2007). Therefore,
transformation of locally adapted tropical maize genotypes with the ipt gene
seems to be the right choice to develop drought tolerant genotypes for the ECA
countries.

12
1.4 Hypotheses
I. Regeneration of tropical maize genotypes can be achieved by using
immature zygotic embryos as explants.
II. Transformation of tropical maize genotypes with ipt gene can be
achieved using Agrobacterium tumefaciens-mediated transformation.
III. The ipt gene driven by the drought inducible SARK promoter improves
tolerance to drought stress by delaying leaf senescence in tropical maize
genotypes.
1.5 Objectives
1.5.1 General objective
To evaluate and select the best regenerable elite and commercial tropical
maize genotypes and to develop drought stress tolerant tropical maize
genotypes through Agrobacterium tumefaciens-mediated genetic
transformation with ipt gene.
1.5.2 Specific objectives
I. To evaluate and select the best regenerable genotypes among 10 elite
and commercial tropical maize genotypes.

13
II. To genetically transform regenerable maize genotypes with ipt gene
under drought inducible promoter through Agrobacterium tumefaciens
mediated transformation.
III. To analyze transgenic maize genotypes using molecular techniques.
IV. To evaluate the performance of transgenic and non-transgenic maize
under contained condition for tolerance to drought stress.

14
CHAPTER TWO
LITERATURE REVIEW
2.1 Constraints to maize production in Africa
2.1.1 Drought
Despite the importance of maize to African economy, its productivity is very low
in this part of the world compared to the average grain yield of 6.2 and 2.5 t/ha for
the industrialized and developing countries, respectively. In tropical Africa in
general and the ECA region in particular erratic and irregular distribution of
rainfall, in combination with high atmospheric evaporative demand, caused by
high temperature and high solar radiation, contribute to low soil moisture that is
insufficient to meet the crop water demand termed “drought stress” (Munné-
Busch and Alegre, 2004). This forms a major abiotic stress affecting productivity
of maize in Africa causing up to 70% crop loss and in certain cases total crop loss
(Edmeades et al., 1994).
More than 25% of land area in Tanzania is threatened by drought either annually
or once in every four years (Nkonya et al., 1998). In Ethiopia, drought affects
maize in 40% of the total maize growing areas with impact of diminishing
production to less than 20% of the country’s total maize production (Nigussie et
al., 2002). Similarly over 70% of the total land area in Kenya is under arid and
semi-arid agroecologies.

15
Since the last eleven years maize yield has never been more than 2 t/ha in the
ECA region (Fig. 2.1), despite the potential of producing more than 10 t/ha
(Morris, 1998).
0
5
10
15
20
25
2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010
Years
Area
(mha),
production
(mt)
and
productivity
(t/ha)
Area (mha) Production (mt) Productivity(t/ha)
Source: FAO STAT, 2010.
Figure 2.1. Trends of maize area, production and productivity in East and
Central Africa.
This rendered maize production unprofitable enterprise discouraging farmers to
invest in productivity enhancing inputs like fertilizer and improved seeds that
ended up in this yield stagnation. Hence, the major increase in production might
have come only from horizontal increase in production as farmers respond to
drought by planting more areas of land with low input in order to ensure food
security at least for home consumption (Shiferaw et al., 2011). Therefore, drought

16
apart from its direct negative influence on crop performance also causes low
productivity by indirectly influencing farmers’ decision to invest in maize
production. To change this scenario, tropical maize has to undergo genetic
transformation for enhanced tolerance to drought stress.
2.1.2 Poor soil fertility
Poor soil fertility was also reported as a serious constraint to maize production in
most environments in Africa, due to very low use of fertilizer and the decreasing
trends of crop rotation practices to replenish soil fertility (Odendo et al., 2001).
Nitrogen is one of the most important nutrients needed for maize production yet
deficient in most of tropical soils. African farmers, who are mainly smallholders
use on average, less than 10 kg of fertilizer per hectare of crop land because of
high price of fertilizer.
2.1.3 Diseases
Maize diseases of economic importance to Africa are common rust (Puccinia
sorghi), gray leaf spot (GLS) (Cercospora zeaemaydis), stalk and ear rots caused
by Diplodia and Fusarium, and seed and ear rots caused by several Fusarium and
Aspergillus species, which also contaminate grain with mycotoxins thereby
reducing grain quality and safety. Maize streak virus (MSV) is another important
disease limited to Africa. In Africa, yield reduction of 30-60% has been attributed
to gray leaf spot, depending on germplasm and environmental conditions (Ward et
al., 1997). Ear and seed rots of maize, caused by a variety of fungi, are prevalent

17
in warm, humid, tropical and subtropical maize growing environments. About
55.9% of the area under maize in sub-tropical, mid-altitude, transition zone and
highland zones experience economic losses due to ear rots, and up to 44% of
maize grown in tropical lowlands are lost to ear rots (Shiferaw et al., 2011).
2.1.4 Insect pests
Among insect pests, stem borers are the most damaging in maize cultivation
causing an estimated average annual loss of 18% (De Groote, 2001). Two species
of stem borers, Chilo partellus and Busseola fusca, are common constraints to
maize in Eastern and Southern Africa, while Sesamia calamistis, Eldana
saccharina, and Mussidia nigrivenella are the dominant pests in West and Central
Africa. In addition to pre-harvest losses, insect pests also cause substantial amount
of post-harvest losses. Grain weevils (Sitophilus zeamais) and the larger grain
borer (LGB) (Prostephanus truncatus) have been reported to cause up to 80%
losses in the tropics (Demissew et al., 2004). Storage insect pests, mainly the
maize weevil (Sitophilus zeamais), (LGB) (Prostephanus truncatus), Angoumois
grain moth (Sitotroga cereallela) and the lesser grain weevil (Sitophilus oryzae)
cause an estimated 20-30% loss of maize, thus negatively affecting food security
and income generation.
2.1.5 Weeds
Competition of maize with annual and perennial grass and broadleaf weeds is
responsible for grain yield reduction in maize (Yihun et al., 2002) depending on

18
the level of infestation, time of occurrence, and types of weeds. Maize grain yield
reduction of up to 58% was reported in Africa (Fessehaie, 1985). Three species of
striga; namely, S. hermonthica, S. asiatica and S. aspera exist in Africa. Among
these S. hermonthica is the most important biological constraint to maize
production causing more than 50% of the yield loss in the region, thereby
affecting the livelihood of about 300 million people, who depend on maize for
their food, in sub-Saharan Africa (Parker, 1991).
2.2 Maize genetic improvement for drought stress tolerance
2.2.1 Conventional breeding
The International Maize and Wheat Improvement Centre (CIMMYT) initiated
genetic improvement of tropical maize for drought tolerance in the 1970s through
full-sub recurrent selection. Later studies indicated low frequency of alleles
conferring drought tolerance (Monneveux et al., 2005), and additive (Betran et al.,
2003) and polygenic (Ribaut et al., 2002) nature of genes controlling grain yield
in maize. This established possibility of improving tolerance to drought stress by
increasing gene frequency through recurrent selection. This approach was
complimented with selection under drought stress imposed by growing progenies
during dry season and using irrigation to manage timing and intensity of the
drought stress. Drought is applied during flowering and grain filling to the extent
that average grain yield in the trials is reduced to 30-60% (intermediate stress
level, grain filling stress) or to 15-30% (severe stress level, combined flowering
and grain-filling stress), respectively, of unstressed yields (Banziger et al., 2000).

19
The same progenies were additionally grown under well-watered (WW) condition
during the main season. Selection was based on an index involving grain yield
under drought and WW conditions and reduced anthesis-silking interval (ASI) and
barrenness, delayed leaf senescence, and reduced canopy temperature under
drought (Bolaños and Edmeades, 1993; Edmeades et al., 1999). This methodology
was used to develop drought tolerant versions of several elite lowland tropical
populations (Edmeades et al., 1999). Several publications documented selection
gains under a range of environmental conditions using this approach (Bolaños and
Edmeades, 1993; Bolaños et al., 1993; Edmeades et al., 1999) but the level of
yield gains were not substantial indicating limitation of conventional breeding to
improve maize productivity in drought-affected environment.
Limited genetic diversity and the complex biochemical response plants have to
drought stress are presented as the major barriers to conventional breeding in
enhancing drought tolerance (Hardy, 2010). The major drawback lies in that,
while conventional or molecular breeding techniques involving the identification
and use of molecular markers have the potential to enhance the effectiveness of
breeding programs, the introgression of genomic portions (QTLs) involved in
stress tolerance is often linked to and brings along undesirable agronomic
characteristics from the donor parents (Bhatnagar-Mathur et al., 2008). This
emanates from lack of a precise knowledge of the key genes underlying the QTLs.
Genetic engineering makes better option to develop drought tolerant crop
circumventing this limitation by insertion of a single gene that codes for a specific
protein involved in drought stress response pathways.

20
2.2.2 Genetic engineering
Tolerance to insect and herbicide were the first major traits addressed through
genetic engineering in maize (James, 2003). Recently genetic engineering went
further to include more complex traits like drought tolerance. Currently several
transgenic research programmes have led to increased drought tolerance in maize
using different strategies (Quan et al., 2004a; Shou et al., 2004a; Nelson et al.,
2007; Castiglioni et al., 2008 ).
Efforts were made to exploit the role of osmoprotection that involves up
regulation of compatible solutes (osmolytes) that function principally to maintain
cell turgor, but also involve scavenging of free radicals, and chaperoning through
direct stabilization of membranes and/or proteins (Diamant et al., 2001). The
genes responsible for synthesis of osmoprotectants are lacking in many crop
plants while they are available in organisms that are stress tolerant. Therefore,
engineering osmolytes in crops that lack these genes was adopted as a strategy to
develop stress-tolerant crops (Bhatnagar-Mathur et al., 2008). Glycine betaine is a
compatible solute that has been extensively studied for its role in drought stress
response and increasing the level of glycine betaine in plants via genetic
engineering has enhanced tolerance to drought stress (Sakamoto and Murata,
2000).
This strategy was applied in maize transformation with the betA gene from
Escherichia coli encoding choline dehydrogenase, a key enzyme in the choline–
betaine aldehyde reaction and reportedly improved tolerance to drought stress at

21
seedling stage and increased grain yields as a result of glycine betaine
accumulation (Quan et al., 2004a). Transgenic lines reportedly yielded 10-23%
higher grain yield than the non-transgenic plants after three weeks of drought
stress.
A tobacco mitogen-activated protein kinase kinase (Nicotiana PK1) was
expressed in maize with a modified constitutive promoter, p35S (Shou et al.,
2004a). The gene (PK1) has been shown to have a significant effect on
photosynthetic rates under drought stress where transgenic maize plants
reportedly had significantly higher photosynthetic rates and produced 40-60%
higher seed weights than the non-transgenic controls.
Constitutive expression of transcription factors to enhance drought stress
tolerance was reported by Nelson et al. (2007) in Arabidopsis using transcription
factor from the nuclear factor (NF-Y) family, AtNF-YB1, which belongs to the
CCAAT-binding transcription factor family. Considering the improved
performance of the transgenic Arabidopsis under drought conditions, transgenic
maize lines were developed through constitutive expression of an orthologous
maize transcription factor gene, ZmNF-YB2. Transgenic maize lines were also
reported to show less wilting and faster recovery from drought than non-
transgenic lines under both glasshouse and field experiments. These stress
adaptation responses contributed to a yield advantage in transgenic maize grown
within drought environments. Later, Castiglioni et al. (2008) demonstrated that
transgenic maize lines with bacterial RNA chaperones resulted in not only abiotic

22
stress tolerance but also improved grain yield under water-limited conditions.
They reported greater than 20% increase in maize grain yield under water-limiting
conditions in field trials.
Active transport of solutes into the cell and cellular organelles, particularly the
vacuole, is another means of cell turgor maintenance as increased solute potential
facilitates the passive movement of water into cells and cellular compartments (Li
et al., 2008). Transgenic plants expressing the potassium-dependent vacuolar H+-
pyrophosphatase (V-H+-PPase) (TsVP) gene from the halophyte T. halophyta
under the control of the maize ubiquitin promoter showed better seed germination,
better root development, more biomass, and increased solute accumulation, less
cell membrane damage, less growth retardation, shorter ASI, and much higher
grain yields than non-transgenic plants. Transgenic breeding has, therefore, a
great potential of improving maize for tolerance to drought stress.
2.3 Agrobacterium tumefaciens-mediated maize transformation
Agrobacterium-tumefaciens is a soil living plant pathogenic bacterium that can
transfer DNA into a broad variety of organisms (Gelvin, 2003). Transformation
technique using Agrobacterium tumefaciens is called indirect transformation
method as a microorganism is used to deliver a certain gene of interest to a plant
cell. Maize transformation using this technique was started almost 30 years back.
The first Agrobacterium tumefaciens-mediated transformation of maize targeted
live seedlings with Agrobacterium tumefaciens that yielded expression of an
Agrobacterium tumefaciens opine gene in the inoculated plants (Graves and

23
Goldman, 1986). Subsequently, transformation of apical meristems of maize
plants with an Agrobacterium tumefaciens strain carrying T-DNA in which maize
streak virus DNA had been inserted resulted in symptoms of systemic infection of
the virus in the transformed maize (Grimsley et al., 1987). Then Gould et al.
(1991) inoculated apical meristems of maize with Agrobacterium tumefaciens and
confirmed stable integration of the gene by Southern blot analysis and the
expression of the GUS reporter gene in some seeds obtained from the resulting
plants.
While these early attempts did not offer reproducible results for maize
transformation, they played significant role in paving ways for further
investigations on Agrobacterium tumefaciens-mediated transformation of maize.
However, successful Agrobacterium tumefaciens-mediated maize transformation
was reported ten years later (Ishida et al., 1996), after the first effort made by
Graves and Goldman (1986). Within four years, Agrobacterium tumefaciens-
mediated transformation technique was reported as a highly efficient method of
transforming different monocot crops such as rice (Hiei et al., 1994), wheat
(Cheng et al., 1997), barely (Tingay et al., 1997) and Sorghum (Zhao et al.,
2000).
Transgenic plants were recovered from immature zygotic embryos obtained from
greenhouse grown plants of the temperate maize inbred line A188 with an
Agrobacterium tumefaciens strain that carried a super binary vector (in which
extra copies of vir genes assisted the DNA transfer), at a frequency ranging from

24
5 to 30% of the infected embryos (Ishida et al., 1996). The study showed that
immature zygotic embryos require attaining a specific developmental stage and
had to be obtained from non-stressed healthy plants of tissue culture responsive
genotypes, in order to be used as target explants for transformation. Having
optimized all culture conditions that contributed to their success they came up
with a conclusion explaining the complexity of the multiple factors involved, and
have revealed that narrow ranges of optimal parameters in the process were the
main reasons for the inefficiency experienced in maize transformation using
Agrobacterium tumefaciens. However, types of plant materials used for infection
with Agrobacterium, choice of vectors and Agrobacterium, and optimization of
tissue culture techniques were among the key factors identified in determining the
rate of achievements. Irrespective of all these, transformation mediated by A.
tumefaciens has now become a highly recommended technique for maize varieties
having good tissue culture responses.
2.3.1 Advantages of Agrobacterium tumefaciens-mediated transformation
Agrobacterium tumefaciens-mediated transformation has been advocated as a
method superior to other direct transformation methods in crop genetic
engineering. Preferential integration of defined T-DNA into transcriptionally
active regions of the chromosome (Koncz et al., 1989; Lee et al., 2001), with
exclusion of vector DNA (Hiei et al., 1997) and unlinked integration of co-
transformed T-DNA (Komari et al., 1996; Hamilton, 1997), are some of its merits
over the direct transformation techniques. Agrobacterium tumefaciens-mediated
transformation also yields fertile transgenic plants in which the foreign genes are

25
inherited in a Mendelian manner (Rhodora and Thomas, 1996). In comparison
with biolistic gun, this approach yields greater proportion of stable, low copy
number of transgenic events (Ishida et al., 1996 and 2007), which lead to fewer
problems with transgene co-suppression (gene silencing) and instability (De la
Riva et al., 1998; Slater et al., 2003). It also offers the possibility of transferring
larger DNA segments into recipient cells (Hamilton et al., 1996), with minimal
rearrangement, resulting in transgenic plants of high quality.
Comparing particle bombardment with that of Agrobacterium tumefaciens-
mediated transformation, Shou et al. (2004b) had reported maize transformation
using the latter technique to give higher proportion of transgenic events with low
copy number and high expression of the transgene, as well as more stable
transgene expression over generation. Moreover, due to the fact that this method
is a single-cell transformation system, it does not result in mosaic plants (De la
Riva et al., 1998). It was with all these considerations that the Agrobacterium
tumefaciens-mediated transformation technique was used in this particular study
to transform tropical maize genotypes selected for their better responses to tissue
culture, whereby immature zygotic embryo explants and MS medium
supplemented with 2,4-D as a growth hormone were used (Chapter Three).
2.3.2 Factors influencing Agrobacterium tumefaciens-mediated maize
transformation
Though Agrobacterium tumefaciens-mediated transformation is a system of
choice for genetic engineering of several crop plants including maize, several

26
factors influence the transfer and integration of the T-DNA into the plant genome.
These include plant genotypes, explant types, Agrobacterium tumefaciens strains
and vectors, addition of vir-gene inducing synthetic phenolic compounds, culture
media composition, tissue damage, suppression and elimination of A. tumefaciens
infection after co-cultivation (Hiei et al., 1994 and 1997; Komari et al., 1996;
Cheng et al., 2004; Alimohammadi and Bagherieh-Najjar, 2009). However, only
the most important factors are briefly discussed here.
2.3.2.1 Genotypes
Maize genotypes differ dramatically in their competence to Agrobacterium-
tumefaciens infection. Genotype-specific factors such as availability of vir gene-
inducing substances, endogenous hormone concentrations of the immature zygotic
embryos, embryo size and the availability of receptors for a productive attachment
of Agrobacterium tumefaciens to the surface of meristematic cells are reportedly
responsible for the differences in competence (Schläppi and Hohn, 1992).
Successful attachment of Agrobacterium tumefaciens and T-DNA transfer into
plant cell requires specific plant cell receptor in the cell wall (Neff and Binns,
1985; Gurlitz et al., 1987), possibly a glycoprotein, which may not be sufficiently
produced in some genotypes, implying genotype-dependent response of maize to
Agrobacterium tumefaciens-mediated transformation. Two plant cell wall
proteins, vitronectin-like and rhicadhesin-binding proteins, have been proposed to
mediate bacterial attachment (Gelvin, 2000). This could be the ground why
Agrobacterium tumefaciens-mediated transformation protocols differ from one
plant species to the other and from one cultivar to the another (De la Riva et al.,

27
1998) unavoidably calling for the optimization of Agrobacterium tumefaciens-
mediated transformation methodologies considering all the factors that are known
to be detrimental to gain positive results.
2.3.2.2 Explant types
Different tissues, organs and cell types within a plant differ in their susceptibility
to Agrobacterium tumefaciens infection. Mesocotyle segments originating from
the intercalary meristem region (Ritchie et al., 1993), and leaves and coleoptile
regions of shoots (Shen et al., 1993) were reported as highly competent for
Agrobacterium tumefaciens transformation, as revealed by transient expression of
GUS activity as a reporter of transient transformation. In line with this, Schläppi
and Hohn (1992) had used agro-inoculation of maize streak virus as an indicator
of transformation to demonstrate the differences in competence of maize embryos
during transformation. The required competence was observed only in embryos of
which shoot apical meristem had begun to differentiate. They also reported the
differences observed among the three maize genotypes evaluated were in relation
to the timing of this window. The common feature of these cells is the induction
of competence in response to wounding or phytohormone treatment (Gelvin,
2000).
Since Agrobacterium tumefaciens was first reported as a highly efficient method
of maize transformation (Ishida et al., 1996), the dominant explants that were
reported to be highly competent for Agrobacterium tumefaciens infection in maize
were freshly isolated immature zygotic embryos (Negrotto et al., 2000; Zhao et

28
al., 2001; Frame et al., 2002; Gordon-Kamm et al., 2002; Zhang et al., 2003).
Numerous studies had indicated that dividing plant cells are more efficiently
transformed than quiescent cells (McCullen and Binns, 2006). The main driving
factor behind the suitability of immature zygotic embryos is, therefore, the
presence of large number of actively dividing competent cells in their scutellum
for somatic embryogenesis (Ishida et al., 2007; Ombori et al., 2008).
2.3.2.3 Growth condition of donor plants
The same genotype may exhibit different regeneration capacity in response to
variations in environmental condition under which the donor plants are grown (Lu
et al., 1983). Hence, the efficiency of Agrobacterium tumefaciens-mediated
transformation can also be affected depending on condition under which the donor
plants of the explants are grown. To this end, Ishida et al. (1996) emphasized the
use of immature zygotic embryos at a specific stage of development from non-
stressed healthy plants of tissue culture responsive genotypes grown in a well-
conditioned greenhouse. Likewise, based on the findings from their three
consecutive season transformation studies of temperate maize, Frame et al. (2006)
had reported higher frequency of success from embryos obtained from
greenhouse-grown than field-grown mother plants.
2.3.2.4 Agrobacterium tumefaciens strains and vectors
Three different Agrobacterium tumefaciens strains, i.e., LBA4404, disarmed C58,
and EHA101; and its derivatives (EHA105, AGL0 and AGL1) have been used to
successfully transform maize (Cheng et al., 2004). A strong genotypic interaction

29
between maize genotypes and the Agrobacterium tumefaciens strain has remained
a serious limitation, calling for proper identification of the best strain for a
specific genotype, and to supplement specific signalling molecules for the
induction of vir genes during co-cultivation.
Following the success stories of rice transformation using a standard binary vector
in a super virulent strain and a super binary vector (extra copy of virB, virC and
virG on the binary vector) in a regular strain, reproducible protocols for
Agrobacterium tumefaciens-mediated maize transformation have used super
binary vectors to infect immature zygotic embryos (Ishida et al., 1996; Negrotto et
al., 2000; Zhao et al., 2001). With a standard binary vector in a super virulent
strain EHA101, Frame et al. (2002) reported a transformation frequency of 5.5%,
which is low even with improved co-culture conditions as compared to the
transformation efficiency (30%) reported by Ishida et al. (1996) and Negrotto et
al. (2000), revealing the enhanced efficiency of super binary vectors in maize
transformation. The combination of a super binary vector in LBA4404 was also
found to be especially important in transformation of a difficult cultivar of rice
(Hiei et al., 1994; Dong et al., 1996). In contrast to this the stated super binary
vector system was not required in sugar cane transformation (De la Riva et al.,
1998) in which conventional genetic vectors and the vir genes naturally carried by
A. tumefaciens PG2260 were sufficient to initiate the infection and transformation
processes confirming applicability of binary vector system also to transform
monocots.

30
2.3.2.5 Media composition
Composition of culture medium is known to affect the embryogenic response of
maize tissue culture and hence transformation efficiency. Tomes and Smith
(1985) and Hodges et al. (1986) had reported higher embryogenic callus induction
frequency and number of maize inbreds responding to tissue culture on culture
medium based on MS salts (Murashige and Skoog, 1962) than on N6 (Chu et al.,
1975) salts though the response was genotype dependent. The same medium was
reported to support better callus induction and somatic embryo formation from
immature zygotic embryos (Armstrong and Green, 1985; Shohael et al., 2003).
N6 salts and vitamins (Chu et al., 1975) were dominantly used for callus
induction, maintenance and somatic embryo maturation in tropical maize
regeneration studies (Proli and Da Silva, 1989; Bohorova et al., 1995; Binott et
al., 2006; Ombori et al., 2008). They were also used for the same process in
Agrobacterium tumefaciens-mediated (Valdez-Ortiz et al., 2007) and particle
bombardment transformation (Bohorova et al., 1999). In both cases the
embryogenic calli were transferred to MS based salts (Murashige and Skoog,
1962) for plant regeneration.
There were instances where tropical maize was also regenerated successfully
using MS based medium for callus induction, maintenance, somatic embryo
maturation and plantlet regeneration (Bedada et al., 2011, 2012; Seth et al., 2012).
Higher transformation frequency was reported in Agrobacterium-tumefaciens-
mediated transformation of Hi II immature zygotic embryos (Armstrong et al.,
1991) cultured on N6 salts or a combination of N6 and MS salts instead of MS

31
salts alone (Zhao et al., 2001). However, transformation of inbred line A188 was
achieved using LS salts and LS vitamins (Linsmaier and Skoog, 1965) (Negrotto
et al., 2000) but not N6 salts and vitamins (Ishida et al., 1996).
Using silver nitrate in solid-culture steps and replacing the antibiotic cefataxime
with carbenicillin for bacteria counter-selection increased the transformation
frequency of Hi II embryos on both N6 and MS media (Zhao et al., 2001) and
inbred line H99 on MS medium (Ishida et al., 2003). Using the Agrobacterium
tumefaciens-mediated standard binary vector system (Frame et al., 2002)
improved transformation frequency was reported for inbred lines B104 and B114
when MS instead of N6 salts were used in tissue culture medium (Frame et al.,
2006). Medium with reduced salts was also reported to enhance T-DNA delivery
into maize (Armstrong and Rout, 2001). Use of half-strength MS salts in co-
culture medium in maize transformation was reported by Zhang et al. (2003). Use
of chemicals such as acetosyringone has been recommended for virulence gene
induction in most protocols for monocot transformation (Cheng et al., 1997;
Tingay et al., 1997; Zhao et al., 2000).
2.4 Delaying leaf senescence through genetic engineering
2.4. 1 Factors driving leaf senescence
Leaf senescence is controlled by the developmental age. Hence, the onset of leaf
senescence is often related to a consequence of age-related changes (ARCs).
Varied cellular mechanisms were reported to determine the age of a cell, tissue

32
and organ for onset of leaf senescence (Lim et al., 2003). Leaf senescence is also
driven by various internal and environmental signals that are incorporated into the
age information. Among endogenous factors that cause leaf senescence in plants,
growth regulators, reproductive development, developmental age and reactive
oxygen species (ROS) are indicated to be of great importance. On top of these,
genetic factors are also reported to influence senescence particularly in maize
(Gungula et al., 2005).
The environmental factors that influence leaf senescence include several abiotic
and biotic stresses. The abiotic stresses include drought, water logging, high or
low solar radiation, excessive soil salinity, inadequate mineral nutrients in the soil,
extreme temperature and oxidative stress (Gan and Amasino, 1997; Lime et al.,
2003). The biotic stresses include pathogen infection and shading by other plants.
Among the environmental factors, drought and low nitrogen stresses adversely
affect crop performance including maize (Gan and Amasino, 1997; Gungula et al.,
2005) by causing premature leaf senescence.
During leaf ageing, developmental signals lead to reduced action of the
senescence-retarding hormones such as auxin, gibberellic acid (GA) and CKs, as
well as the concomitant strengthening of the action of senescence enhancing
hormones such as ethylene, jasmonic acid (JA), abscisic acid (ABA) and salicylic
acid (SA). The action of the different hormones during the initiation of leaf
senescence does not change suddenly but gradually, allowing a gradual
integration of all the hormones controlling the process.

33
2.4.2 Metabolic changes during drought-induced leaf senescence
Leaf senescence affects metabolism of several cellular organelles. However,
chloroplasts have been the focal point for studies on changes in plant metabolism
during the progression of leaf senescence in drought-stressed plants since
chloroplasts are the main intracellular generators of ROS (Asada, 1999) which
cause oxidative stress that plays a major role in the progression of leaf senescence.
Chloroplasts are some of the first organelles to be targeted for breakdown as
senescence proceeds, while nuclei and mitochondria maintain their integrity until
the latest stages of leaf senescence (Smart, 1994). The degradation of chlorophyll
occurs through the concerted action of several enzymes located in different
intracellular compartments, starting with the thylakoids and inner envelope
membrane of chloroplasts and ending in the vacuole (Matile and Hörtensteiner,
1999).
In contrast to chlorophyll degradation associated with rapid and large
accumulation of ROS, drought-induced leaf senescence is characterized with
progressive and slow decline in chlorophylls, loss in photoprotection and nutrient
remobilization (Munné-Bosch and Alegre, 2004). Chlorophylls are degraded not
because their products are reusable but primarily because they would otherwise
block access to more reusable materials. Synthesis of chloroplastic proteases,
increases during drought induced leaf senescence even before chlorophyll
degradation is apparent (Pic et al., 2002), which in turn allows the remobilization
of as much as 75% of the total cellular nitrogen present in the leaves. In this case,
most of the remaining nitrogen present in chloroplasts is taken from the enzyme

34
ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and other stromal
photosynthetic enzymes (Hörtensteiner and Feller, 2002).
Besides chlorophyll degradation, oxidative metabolism may also play a role in
progression of drought-induced leaf senescence. Synthesis of low-molecular-
weight antioxidants such as α-tocopherol had been reported in drought-stressed
plants (Munné-Bosch and Alegre, 2002). Oxidative stress and jasmonic acid
activate the expression of genes responsible for the synthesis of tocopherols in
plants (Sandorf and Holländer-Czytko, 2002). The α-tocopherol that inhibits the
propagation of lipid peroxidation and is an efficient single oxygen quencher and
scavenger may, therefore, contribute to the photoprotection of the photosynthetic
apparatus during the first stages of leaf senescence, when chloroplasts are still
retaining photosynthetic activity.
The same report has shown significant decrease in α-tocopherol during later
stages of leaf senescence, which have been associated with increases in lipid
peroxidation. Although production of α-tocopherol may be activated during this
stage, enhanced formation of ROS in a second oxidative burst may overwhelm the
turnover of α-tocopherol. Other low molecular weight antioxidants change in
parallel with α-tocopherol during the progression of leaf senescence in drought-
stressed plants. Levels of ascorbate and glutathione, which participate in the
recycling of α-tocopherol, are kept constant or even increase during the first stage
of leaf senescence and decrease later as senescence progresses further and α-
tocopherol decreases (Munné-Bosch and Peñuelas, 2003).

35
With the decrease in antioxidants, concomitant increases in lipid peroxidation and
protein oxidation are observed indicating enhanced oxidative stress during the
latest stages of senescence. It is during this period that protein degradation may
occur at the highest rate, since protein oxidation seems to be a pre-requisite for
subsequent enzymatic protein degradation in senescing leaves (Buchanan-
Wollaston, 1997). Thus, it is essential that both oxidative stress and protein
remobilization be tightly controlled during the progression of drought-induced
leaf senescence. Despite these, other associated changes in plant metabolism, such
as an increase in mitochondrial respiration, nucleic acid degradation and
conversion of lipids to sugars also occur during developmental leaf senescence
but have not yet been reported in drought-induced leaf senescence (Munné-Bosch
and Alegre, 2004).
2.4.3 Plant growth regulators and leaf senescence
Among plant growth regulators (PGRs) CKs, auxins and gibberellins are known
to inhibit leaf senescence, while ethylene, ABA, brassinosteroides, JA and SA are
known to promote this process (Smart, 1994; Noodén, 2004). Effects of PGRs
vary depending on their concentrations and conditions and the plant species as
well (Gan and Amasino, 1996). Although many plant hormones have been
implicated in the senescence process, CKs and ABA have been reported
conclusively to regulate senescence (Smart, 1994). Among PGRs, which inhibit
leaf senescence, CKs are of great importance (Gan and Amasino, 1996). This
class of PGRs has been used commercially for delaying leaf senescence by either

36
exogenous application or making transgenic plants that overproduce CKs (Gan
and Amasino, 1997; Noh et al., 2004; Lim et al., 2007; Rivero et al., 2007).
2.4.3.1 Role of cytokinins in delaying leaf senescence
Cytokinins have been identified as senescence retarding growth hormones (Gan
and Amasino, 1996; Buchanan-Wollaston, 1997; Nam, 1997; Taiz and Zeiger,
2002; Lim et al., 2007). This effect was first observed with kinetin when it
delayed loss of chlorophyll and protein from detached leaves of Xanthium
(Richmond and Lang, 1957). It was later reported consecutively that CKs enhance
synthesis of protein and RNA (Osborne, 1962), chlorophyll (Fletcher et al., 1973),
chloroplast differentiation (Harvey et al., 1974) and chloroplast proteins, such as
the light-harvesting chlorophyll a/b binding protein (Axelos et al., 1984). External
application of CKs was shown to release lateral buds from apical dominance,
initiate shoots from callus cultures, stimulate pigment synthesis, inhibit root
growth and retard senescence (Medford et al., 1989; Gan and Amasino, 1996) and
also counteract the effects of heat stress. Cytokinins are also involved in the
control of gene expression (Crowell et al., 1990).
In plants, CKs are produced in the root tips, shoot apical meristem, cambium and
immature seeds. Young maize embryos synthesize CKs, as do young developing
leaves, young fruits, and possibly many other tissues. However, root apical
meristems are the major sites of synthesis of the free CKs in whole plants
(Buchenan-Wollasten, 1997) which are transported to the rest of the plant through
the xylem together with water and nutrients. Hence, all environmental factors that

37
interfere with root function, such as drought stress, salt stress, water logging or
mineral deficiency reduce the CK biosynthesis and then promote leaf senescence
(Van Staden et al., 1988). Conversely, resupply of nitrate to nitrogen-starved
maize roots resulted in an elevation of the concentration of CKs in the xylem sap
(Samuelson, 1992), which has been correlated to an induction of CK-regulated
gene expression in the shoots (Takei et al., 2001) leading to delayed leaf
senescence. It has been shown that the level of CK in the xylem sap declines when
senescence is initiated and this reduced level of CK may cause the onset of leaf
senescence (Gan and Amasino, 1996; Lim et al., 2007). The CKs involved in
delaying senescence are primarily zeatin riboside and dihydrozeatin riboside,
which may be transported into the leaves from the roots through the xylem, along
with the transpiration stream (Noodén et al., 1990).
Molecular analysis of leaf senescence disclosed that genes involved in CK
biosynthesis and signalling including the ipt gene are down regulated and the gene
for CK degradation, cytokinin oxidase, is up-regulated during leaf senescence
(Buchanan-Wollaston et al., 2005). This brought the need that either CK should
be applied externally or its synthesis should be enhanced endogenously to delay
leaf senescence.
Despite their remarkable effects in delaying leaf senescence, the underlying
molecular basis pertaining to how CKs affect this phenomenon is not clearly
known. One possible way is suggested to be through their effects in controlling
expression of some genes that link the CK response to leaf senescence (Lim et al.,

38
2003). Expression of senescence-related genes was inhibited at transcriptional
level when CKs are present beyond a certain level (Buchanan-Wollaston et al.,
1997). This has been observed in transgenic tobacco, which showed extremely
high levels of the GUS protein in senescing leaves when transformed with a
PSAG12::GUS construct alone but expressing low level of GUS when co-
transformed with PSAG12::IPT and PSAG12::GUS. This showed CKs produced as a
result of the ipt gene expression inhibited the expression of GUS from the
senescence-enhanced promoter (Gan and Amasino, 1995). This evidence is clearly
supporting the notion that CKs, either directly or indirectly, can inhibit the
transcription of senescence-related genes.
It was later reported that delay of senescence by CK is mediated by an
extracellular invertase (Cin1), an enzyme functionally linked in the apoplastic
phloem-unloading pathway. Leaf senescence was not delayed by CK when the
activity of extracellular invertase was inhibited (Balibrea Lara et al., 2004).
However, expression of an extracellular invertase under control of the senescence-
induced SAG12 promoter delayed senescence indicating that these metabolic
enzymes may substitute the role of CKs. The finding from that study clearly
demonstrated that extracellular invertase is required for delay of senescence by
CKs and that it is a key element of the underlying molecular mechanism. The
results further suggested that carbohydrate partitioning in association with
extracellular invertase activity might be involved in cytokinin-mediated delay of
leaf senescence.

39
2.4.4 Molecular genetic manipulation of leaf senescence
The most common molecular genetic approach to delay leaf senescence has been
to enable plants overproduce CKs through genetic engineering with the ipt gene
from Agrobacterium tumefaciens under the control of suitable promoters. High
levels of CK results in different physiological and morphological abnormalities in
many plants such as reduced plant and leaf size, weakened apical dominance, less
developed vascular and root systems, and can even induce cell death in some plant
species (Gan and Amasino, 1996; Carimi et al., 2003). Such abnormalities arise
from constitutive expression of the ipt gene through its native promoter and when
the native promoter is replaced with constitutive promoters like the cauliflower
mosaic virus CaMV 35S or the ubiqutin maize promoter. Actually, CaMV 35S
promoter was reported to increase ipt expression even over that of the native
promoter (McKenzie et al., 1998) to the extent of preventing studies on CK
overproduction in normal plant tissues.
2.4.4.1 Expressing ipt gene using inducible promoters
2.4.4.1.1 Heat shock inducible promoters
Because of the above mentioned reasons, efforts were made to express ipt gene in
higher plants under the control of various inducible promoters (Medford et al.,
1989; Gan and Amasino, 1996; McKenzie et al., 1998; Noh et al., 2004; Robson
et al., 2004; Luo et al., 2005; Rivero et al., 2007). Medford et al. (1989) fused a
promoter region from a maize gene encoding a heat shock protein (HSP70) to the
ipt gene to make a PHSP70-IPT construct for transforming Arabidopsis and
tobacco plants. Heat shock treatment of transgenic plants was reported to

40
substantially increase level of CKs (Medford et al., 1989). This increase in
endogenous level of CK under controlled temperature had some effects on
transgenic plants: In tobacco, the height, xylem content and leaf size of the plant
was reduced, axillary bud growth increased and an underdeveloped root system
was reported. In Arabidopsis, a reduced root system was reported. In addition, the
primary root elongated at slower rates compared to the non-transgenics.
Moreover, root hairs emerged closer to the root tip suggesting a reduction in
elongation zone in the transgenic plants (Medford et al., 1989).
A similar phenomenon was reported later by Smart et al. (1991) in tobacco plants
transformed with the ipt gene under the control of the heat shock promoter
HS6871 from soybean. Heat shock of a defined area of a single leaf still attached
to the plant resulted in a transient increase in its CK level. After four heat shocks
applied at 3-4 days intervals to the same portion of leaf, pronounced retention of
chlorophyll was seen in the treated area compared with the rest of the leaf (Smart
et al., 1991). It was further noticed that the senescence was accelerated in leaves
above the heat-shocked ones, a similar effect to that previously reported by
Leopold and Kawase (1964) after external application of CKs. Transformed plants
grown at temperature of 20ºC were shorter, had larger side shoots and remained
green for a longer time than untransformed plants.
Though plants obtained with heat shock promoters were normal, they were often
smaller and displayed a greater degree of axillary bud growth than control plants
under both inductive and non-inductive conditions (Medford et al., 1989;

41
Schmülling et al., 1989; Smart et al., 1991; Smigocki, 1991; Van Loven et al.,
1993). Even under non-heat-shock conditions, transformed plants often contained
higher levels of CK than did control plants. Thus, it seemed that the heat-shock
promoters allowed sufficient expression from the ipt gene under non-inductive
conditions to alter plant morphology.
2.4.4.1.2 Promoters inducible by external environments
To avoid use of heat-shock promoters, scientists turned to promoters that allow
temporal or spatial gene expression. These included promoters induced by
external environment, such as light (Beinsberger et al., 1991), wounding
(Smigocki et al., 1993), Cu2+
(McKenzie et al., 1998) and those related to a
particular tissue such as fruit specific (Martineau et al., 1994). Generally, a higher
level of control over ipt gene expression has been gained with these promoters
than has been provided by the heat-shock promoters. However, in many cases CK
production seemed to be dependent on external application of a particular
treatment as well as on tissue type.
The third alternative of achieving ipt gene expression was to design an
autoregulatory system in which a promoter driving the ipt gene can be activated
by environmental factors that do not depend on external application. In many
crops, environmental factors like drought induce leaf senescence, so if ipt gene is
linked to senescence or drought inducible promoters, transgenic crops can be
developed with improved tolerance to drought while CK concentration is still
maintained at the minimum level needed to delay leaf senescence.

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