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LIBRARY
Michigan State
University

This is to certify that the
dissertation entitled

TRANSGENIC IMPROVEMENT OF OAT FOR SALINITY
TOLERANCE AND RICE FOR ALCOHOL FUEL
PRODUCTION AND REDUCED AIR POLLUTION

presented by

Hesham Farouk Oraby

has been accepted towards fulﬁllment
of the requirements for the

Doctoral degree in Plant Breedmg and Genetics

 

 

Crop and Soil Sciences Dept.

 

 

w~39~ng

 

Major Professors Signatur

Date

MSU is an Afﬁrmative Action/Equal Opportunity Institution

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE

DATE DUE

DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

2/05 p:ICIRC/DaieDue.indd-p.1

 

 

TRINSGEN
RICE ‘

TRANSGENIC IMPROVEMENT OF OAT FOR SALINITY TOLERANCE AND
RICE FOR ALCOHOL FUEL PRODUCTION AND REDUCED AIR
POLLUTION
BY

Hesham Farouk Oraby

A DISSERTATION

Submitted to
Michigan State University
In partial fulﬁllment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Plant Breeding and Genetics Program
Department of Crop and Soil Sciences

2006

tittVSGENlC
FOR ALC

Enxironmental
most limiting 1
yield loss is C
third generatit
tolerance, B-g
plants shone
herbicide resi
Stable expres
transgenic K
lﬂCTEaSe in IQ
0f days to he
Silkelets’pan
and kernel yi

Rj

ABSTRACT

TRANSGENIC IMPROVEMENT OF OAT FOR SALINITY TOLERANCE AND RICE
FOR ALCOHOL FUEL PRODUCTION AND REDUCED AIR POLLUTION

By

Hesham Farouk Oraby

Environmental stresses such as drought, salinity and air pollution represent some of the
most limiting factors for agricultural productivity worldwide. A major cause of oat crop
yield loss is osmotic stress due to drought and/or salinity. This study investigated the
third generation of transgenic oat (Avena sativa L.) expressing barley H VA! stress
tolerance, B-glucuronidase (uidA; gus) and bar herbicide resistance genes. Transgenic
plants showed normal 9:7 third generation inheritance for glufosinate ammonium
herbicide resistance. Molecular and histochemical studies conﬁrmed the presence and
stable expression of all three genes. Compared with the non-transgenic control,
transgenic R3 plants exhibited greater growth and showed a signiﬁcant (P < 0.05)
increase in tolerance to salt stress conditions (200 mM NaCl) for traits including number
of days to heading, plant height, ﬂag leaf area, root length, panicle length, number of
spikelets/panicle, number of tillers/plant, number of kemels/panicle, IOOO-kemel weight,
and kernel yield/plant.

Rice is the major food crop of the world with minimal use for its straw. A successﬁil
strategy for producing biologically active hydrolysis enzymes (molecular farming) in rice
for alcohol fuel production provides an environmentally superior technology that
substitutes the wasteful and polluting practice of rice straw burning. The catalytic domain

of Acidothermus cellulolyticus thermostable endoglucanase gene (encoding for endo-1,4-

B—glueanase enzy
mediated transfot
Rl plants conlirn
accounted for up
apparent deleteri
iii of the cellu
biomass was con
results of these I
to solving two

technologies pro

B-glucanase enzyme or E1) was constitutively expressed in rice using the Agrobacterium-
mediated transformation system in an apoplast-targeting manner. Molecular analyses of
R1 plants conﬁrmed presence and expression of the transgene. The amount of E1 enzyme
accounted for up to 4.9% of the plant total soluble proteins, and its accumulation had no
apparent deleterious effects on plant growth and development. Approximately 22 and
30% of the cellulose in the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize
biomass was converted into glucose using rice E1 heterologous enzyme respectively. The
results of these two studies provide effective transgenic-based means that can contribute
to solving two of the most complex problems for which traditional plant breeding

technologies provide only limited answers.

DEDICATION

This thesis is dedicated to
The memory of my father

My family and friends, and my lovely country Egypt

-IV-

The WOT
challenging- but

Iwould
opportunity l0 \'
and guidance th
for my other co:
has made him
Stadent and a
suggestions and
Deehun Wang \

hlany tI
discussion and
Veronica and S
idrice, which it
and Dr Balan
ileUAbIC. Algg
Cal, Donna, R

[hIQUEhOUt in

bi.

DIWOIOEE’ (
l0 Ste -

\e for hell

ACKNOWLEDGMENT

The work on this thesis has been mostly inspiring, oﬁen exciting, sometimes
challenging, but always interesting. It has been made possible by many other people.

I would like to thank my major advisor, Dr. Mariam Sticklen, for giving me the
opportunity to work in her lab, develop new skills in the ﬁeld of biotechnology and, help
and guidance throughout the years. Also, I would like to express my sincere appreciation
for my other commettiee members. The scientiﬁc intution and humor of Dr. Jim Hancock
has made him a continuous oasis of thoughts which enriched my development as a
student and a researcher. Dr. Russel Freed was always willing to provide valuable
suggestions and crucial contributions, which helped me pass through the diffeculties. Dr.
Dechun Wang was a source of encauragement, valuable experience and advice.

Many thanks go to my colleague Callista for her enormous help, scientiﬁc
discussion and pleasure working together. I was extraordinarily fortunate to have
Veronica and Susanah who always showed their willingness to share bright thoughts and
advice, which were very inﬂuential on my ideas and research. The help of Dr. Bruce Dale
and Dr. Balan Venkatesh, in the Department of Chemical Engineering at MSU, was
invaluable. Also my thanks go to Malin, Halima, Guo—Qing, Ann, Cholani, Han, Karolyn,
Cal, Donna, Rita, Kristi and Dr. Taylor Johnston for their sincere help and support
throughout my stay at MSU. In addition, I would like to thank Dr. Ken Sink and Dr.
Dave Douches for giving me the opportunity to participate in their undergraduate
biotechnology class and helping me strengthen my teaching skills. Thanks are extended

to Steve for help formatting and critical comments about the thesis.

Where \
Essatti, Rita l
hospitality and
Ahmad Omar 2
ideas about res:

Withou
mention My f2
generousity an;
showed me the
My Mother is
bean) indivisih
She will may
past, and “=7th
nith kind Unde

.t -
Sll

tiling daUgh
been trying to
dedlcalion’ fa
pat

ml‘ Strength \

Where would I be without my off campus friends? I would like to thank Farouk
Essawi, Rita, Mohamed, Eman, Fathy, Marwa, Abody, Ahmad and Hassan for their
hospitality and kindness during the stay in Lansing. I convey special acknowledgement to
Ahmad Omar at the University of Florida for sharing various thoughts, discussions, and
ideas about research during the time we spent together attending different conferences.

Without my family I could not have done it. My parents deserve extraordinary
mention. My father is the person who had the strength of a mountain and was a source of
generousity and support. He established my personality, founded my learning ability and
showed me the value and enjoyment of intellectual pursuit. Thanks Dad for everything.
My Mother is the one who raised me with her tender care, endless love of her golden
heart, indivisible support and prayers. I hope that when she thinks of me, a part of her,
she will always see. Thanks Mom for being there. My sisters, Manal and Maha, are my
past, and write my future. I recognize myself in their eyes, and they accepted me as I am
with kind understanding. Thanks sisters for caring. Special thanks to Medhat, my brother
in law, for help and support. I appreciate the “pleasant” atmosphere created by my happy,
shining daughter Nagham and my energetic, bright son Bassel. Since long time, I have
been trying to express my deepest gratefulness and appreciation to my wife Ola whose
dedication, faith and conﬁdence in me were absolute. Her intelligence, enthusiasm,
patience and ambition were the lift when I couldn't reach. I want to thank her for being
my strength when I was weak, for all those times she stood by me, and for all the
happiness she brought to my life. Really, I'm everything I am because she loved me.
Finally, I would like to owe my success to my gorgeous country, Egypt, for education,

preparation for life and ﬁnancial support.

—vi-

LIST OF TAB:
LIST OF FlGl

Cllhl’IER l: '
GENERAL 1?
REFERENCI

CHIPTER ll

INTRODL'C'
Definition
it Genomics
if Bioinforn'
; Genetic T
r Molecular
"' Diagnosti
i’ Vaccine]
P lam BiOlecl
ARTURO C'
D€ﬁniti0n
MO‘PhOgene
Explant ”Hp,
Media COm;
GENETICA
efll’llllon ar

TABLE OF CONTENTS

LIST OF TABLES ....................................................................................................... x
LIST OF FIGURES ..................................................................................................... xi
CHAPTER I: GENERAL INTRODUCTION ............................................................... 1
GENERAL INTRODUCTION .................................................................................... 2
REFERENCES ............................................................................................................ 6
CHAPTER II: BIOTECHNOLOGY AND THE GENERATION OF TRAN SGENIC
PLANTS ............................................................................................ 9
INTRODUCTION ..................................................................................................... 10
Deﬁnition .................................................................................................................. 11
> Genomics ............................................................................................................. 11
> Bioinformatics ...................................................................................................... 12
> Genetic Transformation ........................................................................................ 12
> Molecular Breeding .............................................................................................. 13
> Diagnostics ........................................................................................................... 13
> Vaccine Technology ............................................................................................. 14
Plant Biotechnology .................................................................................................. 14
IN VIYRO CULTURE TECHNOLOGY .................................................................... 15
Deﬁnition .................................................................................................................. 15
Morphogenesis Methods ............................................................................................ 18
Explant Type ............................................................................................................. 19
Media Composition and Physical Factors .................................................................. 26
GENETICALLY MODIFIED CROPS ...................................................................... 28
Deﬁnition and Importance ......................................................................................... 29
Current Status ............................................................................................................ 37
Developing the GM Crops (Methods of Transformation) ........................................... 30
Direct Gene Transfer Systems ................................................................................... 32
Agrobacterium-Mediated Transformation System ...................................................... 35
GM Crops: Concerns and Possible Solutions ............................................................. 38
Engineering and Breeding ......................................................................................... 40
REFERENCES .......................................................................................................... 46
CHAPTER III: BARLEY H VA] GENE CONFERS SALT TOLERANCE IN R3
TRANSGENIC OAT ........................................................................ 58
INTRODUCTION ..................................................................................................... 59
REVIEW OF LITERATURE .................................................................................... 60
The Importance of Oat ............................................................................................... 60
Effect of Abiotic Stresses on Oat ............................................................................... 62
In Vitro Culture of Oat ............................................................................................... 63
Genetic Transformation of Oat .................................................................................. 69

-vii-

l'se of Stress 7
MATERIALS
Plant Material
Segregation ol
Salinity Treatr
PC R Analysis
Histochemica'
DNA Isolatioi
RVA lsolatioi
Measurement
Statistical An
RESULTS A
Segregation c
Histochemica
Southern Blo
Nonhem Blo
Salinit} Effc‘
SLMMARY
REFERENCT

CHAPTER IV
1

mRootc
REWEW 0}
”muggm
BlOﬁleI Ejha
Ce Impone
Ce Tissue q
MATERI‘ \l
Tr ranSfOrmati
Selection of
Hetic Tran
Rochemic
A'nal} 5i

Use of Stress Tolerance Genes in Transgenic Plants .................................................. 73

MATERIALS AND METHODS ............................................................................... 75
Plant Materials .......................................................................................................... 75
Segregation of Herbicide Resistance of R3 Progeny .................................................. 75
Salinity Treatments .................................................................................................... 76
PCR Analysis ............................................................................................................ 77
Histochemical Analysis of GUS ................................................................................ 77
DNA Isolation and Southern Blot Hybridization Analysis ......................................... 78
RNA Isolation and Northern Blot Hybridization Analysis .......................................... 78
Measurements of Parameters ..................................................................................... 79
Statistical Analyses .................................................................................................... 80
RESULTS AND DISCUSSION ................................................................................ 81
Segregation of Herbicide Resistance of R3 Progeny .................................................. 81
Histochemical Analysis of GUS ................................................................................ 82
Southern Blot Analysis of the H VAI Gene in R3 Progeny Plants ............................... 82
Northern Blot Analysis of the H VAI Gene in R3 Progeny Plants ............................... 85
Salinity Effects on Plant Growth, Yield and Its Components ..................................... 85
SUMMARY .............................................................................................................. 95
REFERENCES .......................................................................................................... 97

CHAPTER IV: ENHANCED CONVERSION OF BIOMASS CELLULOSE INTO
GLUCOSE USING TRANSGENIC RICE-PRODUCED CELLULASE

............................................................................................................. 107
INTRODUCTION ................................................................................................... 108
REVIEW OF LITERATURE .................................................................................. l10
Plants as Biofactories (Molecular Farming) ............................................................. 110
Bioﬁrel Ethanol ....................................................................................................... l 12
Rice Importance and Straw Burning ........................................................................ 114
Rice Tissue Culture and Genetic Transformation ..................................................... 116
MATERIALS AND METHODS ............................................................................. 122
Transformation Vector ............................................................................................. 122
Selection of Transformants Using Glufosinate Herbicide ......................................... 122
Genetic Transformation ........................................................................................... 122
Histochemical Analysis of GUS .............................................................................. 124
PCR Analysis .......................................................................................................... 124
DNA Isolation and Southern Blot Hybridization Analysis ....................................... 125
RNA Isolation and Northern Blot Hybridization Analysis ........................................ 126
Protein Extraction and Western Blot Analysis ......................................................... 126
Immunoﬂuorescence Microscopic Analysis ............................................................. 128
The Biological Activity Assays of Heterologous E1 Enzyme ................................... 128
Cellulose Hydrolysis Assay ..................................................................................... 129
RESULTS ............................................................................................................... 13 1
Transgene Construct and Genetic Transformation .................................................... 131
Molecular Analysis of the Transgenic Plants ........................................................... 131
Localization of the E1 Enzyme in the Apoplast ........................................................ 134
High-Level Production of Biologically Active E1 Enzyme ...................................... 136

- viii -

Cellulose to t
DISC LTSSIO
SUNARY
REFERENCE

CHAPTER 1‘
CONCLUSR
ELTLRE RE
REEERENCl

Cellulose to Glucose Conversion ............................................................................. 136

DISCUSSION ......................................................................................................... 140
SUMMARY ............................................................................................................ 143
REFERENCES ........................................................................................................ 144
CHAPTER IV: CONCLUSION AND FUTURE RESEARCH ................................. 153
CONCLUSIONS ..................................................................................................... 1 54
FUTURE RESEARCH ............................................................................................ 157
REFERENCES ........................................................................................................ 159
- IX -

 

TableZ l. The g6
Tahlell Examr
1ahle23 Major
Table23 (Cont'
Table24 Exam}
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Table 3 2 Mean
chara
contr

Table 3.3 Diffe
unde
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Idblf‘ll Expla

rice r

LIST OF TABLES

Table 2.1. The general media composition and its function in plant tissue culture. ......... 28

Table 2.2. Examples of research on genetically modiﬁed crops for different purposes. .. 31

Table 2.3. Major public concerns over the GM crops and some solutions. ..................... 42
Table 2.3. (Cont’d) ........................................................................................................ 43
Table 2.4. Examples of GM crop failures ....................................................................... 44
Table 3.1. Segregation of herbicide resistance of R3 transgenic Ogle cultivar. ............... 83

Table 3.2. Mean squares for kernel yield and its components and some other agronomic
characters for the R3 transgenic lines expressing the H VA! gene and the
control .......................................................................................................... 88

Table 3.3. Differences between the R3 transgenic lines and the non-transgenic control
under different salinity levels for the number of tillers/plant, number of
kernels/panicle, lOOO-kemel weight and kernel yield/plant ........................... 94

Table 4.1. Explants and methods used in rice genetic transformation. .......................... 1 18

Table 4.2. The amount of heterologous E1 enzyme in different independent transgenic
rice events determined by the MUCase activity assay (average of 3 reps)... 135

 

 

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LIST OF FIGURES

Figure 2.1. Developing transgenic crops using tissue culture and plant transformation
techniques ................................................................................................. 45

Figure 3.1. R3 transgenic seedlings after in vitro selection ............................................. 84

Figure 3.2. PCR ampliﬁcation of (a) the bar (0.59 kb) and (b) HVAI (0.7 kb) genes
shows the presence of the transgenes in R3 for the 5 lines. Lane 1: 100 bp
Ladder marker, Lane 2: Plasmid (positive control), Lane 3: Non-transformed
(negative control), Lanes 4-8: Transgenic lines ......................................... 84

Figure 3.3. GUS expression in R3 transgenic oat seed husks (a), seeds (b) root segments
(c) and leaf tissue cells ((1) ......................................................................... 84

Figure 3.4. Top; pBY520 partial map, bottom; Southern blot (a) and Northern blot (b)
analyses showing bands for R3 of transgenic oat plants digested with
HindIII, M: Ladder marker; P: plasmid BY520; Lanes 1-5: Ogle BRA-82,
Ogle BRA-17, Ogle BRA-8, Ogle BRA-l9, Ogle BRA-41 respectively; C:
non-transgenic control. .............................................................................. 87

Figure 3.5. The effect of increasing NaCl concentrations on number of days to heading
(a), plant height (b), ﬂag leaf area (c), root length (d), panicle length (e) and
number of spikelets/panicle (f) for the transgenic lines and the non-
transgenic control. ..................................................................................... 93

Figure 3.6. R3 transgenic oat (a) and non-transgenic (b) at 150 mM NaCl stress. ........... 96
Figure 3.7. R3 transgenic (a) and non-transgenic (b) oat roots at 100 mM NaCl stress. ..96

Figure 4.1. Schematic representation of ApoEl binary vector containing the
Acidothermus cellulolyticus E1 catalytic domain driven by Cauliﬂower
Mosaic Virus 358 Promoter (CaMV 35S), tobacco Mosaic Virus
translational enhancer (Q), and the sequence encoding the tobacco
pathogenesis-related protein 1a (Prla) signal peptide for apoplast-targeting
of the E1 enzyme, and the polyadenylation signal of nopaline synthase
(nos) ........................................................................................................ 132

Figure 4.2. Figure 4.2. (a) Mature derived calli of rice cultivar Taipei 309, (b) selection of
transgenic calli on 15 mg/L glufosinate ammonium, (c and d) regeneration
of transgenic plants, (e) grth chamber grown E1 transgenic rice plants, (f
and g) greenhouse fertile grown A. cellulolyticus E1 transgenic rice plants
and (h and i) gus expression in calli and plantlets of transgenic rice as
compared to the non-transgenic control. .................................................. 133

-xi-

relf

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re~l

 

Figure 4.3. PCR (a), Southern (b), Northern (c) and Western ((1) blot analyses show the
presence of the transgenes in ﬁve transgenic rice lines. ........................... 135

Figure 4.4. Immunoﬂuorescence confocal microscopy for the transgenic (a) and non-
transgenic (b) rice showing apoplast localization of the E1 enzyme in
transgenic rice leaves. ............................................................................. 137

Figure 4.5. Detection of the E1 enzyme activity using CMCase activity assay. Zones of
CMC hydrolysis were decolorized with washing leaving yellow regions in
the transgenic as compared to red background in the control. .................. 137

Figure 4.6. (a) The amount of glucose released from the enzymatic hydrolysis of CMC
(1%, 5%, 10%) and Avicel (1%, 5%, 10%) using total protein extracted
from E1 expressed rice straw. (b) Comparison of percentage of glucan
converted in the enzymatic hydrolysis of corn stover (CS) and rice straw
(RS). CE, commercial enzyme, UT, untreated biomass, CS1, RS1, C82, and
R82 represent, reaction done using 0.5 ml and 4 ml of total soluble protein
(with 4.9% of E1) and commercial B-glucosidase (6.5 mg/ 15 ml)
respectively. ............................................................................................ 138

-xii-

CHAPTER I

GENERAL INTRODUCTION

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GENERAL INTRODUCTION

Environmental stresses such as drought, salinity and air pollution represent some
of the most limiting factors for agricultural productivity worldwide (Boyer, 1982; Roy
and Wu, 2002). Not only do they greatly decrease the potential yield of current crop
species, but these stresses also restrict the area where production can take place and
hinder the introduction of crop plants into new areas (Epstein et al., 1980). Many
varieties have been developed and released in different crops but very few of them could
c0pe with changes in the environmental conditions.

Traditional breeding has limitations and cannot solve the environmental problems
alone. This could be, perhaps, due to the inefﬁciency of selection methods, the lack of the
genetic variability in a crop’s background, or the complexity of traits studied (Cushman
and Bohnert, 2000). Crop improvement strategies that are based on the use of new
technologies, such as biotechnology, can be used in conjunction with traditional breeding
efforts (Abebe et al., 2003; Epstein et al., 1980; Ribaut and Hoisington, 1998), offering a
responsible way to enhance agricultural productivity. Biotechnology could help eliminate
many obstacles limiting crop production in developing countries. Also, modem plant
genetic engineering presents the possibility of rapid and precise introduction of desirable
traits from closely related plants without associated deleterious genes (Richards, 1996)
because it avoids the transfer of unwanted chromosomal regions (Cushman and Bohnert,
2000; Sharma et al., 2002).

Salinity in particular is a major problem, affecting crop production on nearly one-
third of the world’s irrigated agricultural land (Apse et al., 1999; Schachtman and Lui,

1999). There is a shortage of arable land, and this area is steadily decreasing, as poor

farming
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farming practices cause cultivated land to become more saline (Qadir et al., 1998).
Farmers are, therefore, being forced to cultivate in salinity-prone areas, but plants grown
in these areas experience severe salinity stress. This situation will only worsen in the
coming decades (Cherry et al., 1999).

Osmotic stress due to drought and salinity is a major cause of global oat crop yield
loss (Frey, 1998; Martin et al., 2001; Tamm, 2003). Oat is an important cereal crop used
in human and animal diets, and soil conservation (Welch, 1995). Although, compared
with the other cereals, oat is considered a moderately salt tolerant crop (Mtu et al.,
1984), soil salinity is responsible for decreasing oat seed germination and stunting
subsequent development in a cultivar-dependent manner (Mtu et al., 1984; Verma and
Yadava, 1986). The development of transgenic crops, such as oat, with the internal
capacity to withstand abiotic stresses such as salinity, would help reduce the use of water
(F A0, 1999), thus promoting sustainable yields (Sharma et al., 2001).

In addition to osmotic stresses, for the last few decades, air pollution caused by
agricultural burning has affected agricultural production (Emerson et al., 2004).
Agricultural burning is the practice of the open burning of waste materials produced from
growing and harvesting crops such as wheat, corn and rice for weed and disease control
(Canadian Lung Association, 2003). It reduces plant productivity both directly in
decreasing plant growth and yield (Schenone et al., 1992; Fuhrer et al., 1997), and
indirectly in signiﬁcant yield losses caused by chemical and physiological changes that
increase plants’ sensitivity to different stresses (Emerson et al., 2004). Moreover, post-
harvest burning is often at a time when wind ﬂows bring the smoke into populated areas.

The increased levels of smoke pose threats to human health (Golshan et al., 2002).

that ﬂl't‘ﬁ

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One of the most promising uses proposed for agricultural residues and biomass
that avoids burning, disposal wastes and air pollution is ethanol ﬁtel production. Fuel
ethanol is currently a substitute for, as well as additive to, the traditional fossil fuels
(Talebnia et al., 2004). In 2004, US. ethanol production capacity reached 3.4 billion
gallons, (Farrell et al., 2006), about 600 million gallons more than 2003 (Bothast and
Schlicher, 2005). Another production increase of more than 1.6 billion gallons is
projected for 2012 (Bothast and Schlicher, 2005). An ambitious goal is taking shape to
make energy production a primary objective of US agriculture. It is expected that US
agriculture will provide 25% of the total energy consumed in the United States by 2025,
while continuing to produce abundant, safe and affordable food and ﬁber (Hillgren,
2005)

For some crops, particularly rice, seed is considered the most useful portion of the
plant while the remaining biomass has only limited use. Burning of straw or stubble is the
most efﬁcient and effective way to control major fungal disease problems such as stem
rot and aggregate sheath spot. Residents in rice farm areas are exposed to smoke from
burning of its straw and may have serious health problems affecting their respiratory
systems (Jacobs et al., 1997; Torigoe et al., 2000; Golshan et al., 2002). Increased
pollution levels obliged the California Legislature, for example, to pass the Straw
Burning Act of 1991 which phased down, and eventually out in 2001, rice straw burning
except in cases of crOp disease. This act and other similar acts worldwide will result in
accumulation of excessive piles of rice straw biomass, as rice is grown globally on more
than 148 million hectares worldwide (Chandra Babu et al., 2003) with a total production

of about 731 million tons of straw (Kim and Dale, 2004). Therefore, transgenic rice is a

strong c:

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strong candidate for producing biomolecules required for ethanol production that may
signiﬁcantly improve proﬁt margins and lower pollution levels at the same time.

The application of biotechnology in the production of crops which are tolerant to
environmental stresses and plants that produce active commodity products
(biomolecules) may offer beneﬁts in terms of sustainable agriculture, resource supply and
economic security. Once transgenic plants are successfully created to address these
speciﬁc needs, several challenges and concerns remain to be met in terms of
environmental impact, biosafety and risk assessment. Those issues have a large inﬂuence
on the release, commercial success, and large scale cultivation of transgenic plants
(Twyman et al., 2003). Therefore, by overcoming the challenges related to the production
and acceptance of transgenic crops, it ultimately might be possible to mitigate the
problem of global food and ﬁiel security in the ﬁature (Sharma et al., 2002).

The general objective of this study was to investigate the transgenic improvement
of oat for salinity tolerance and rice for alcohol fuel production-and reduced air pollution.
Chapter I is a general introduction about theme of the work. Chapter 11 presents an
extended discussion on biotechnology, in vitro culture, genetic manipulation (theory and
techniques) and concerns over genetically modiﬁed crops. Chapter 111 represents the
potential of using the barley H VAI gene to confer salt tolerance in R3 transgenic oat to
promote sustainable oat yields in salinity-prone areas. Chapter IV examines high-level .
production of biologically-active Acidothermus cellulolyticus endo-l , 4-B-glucanase (E1)
enzyme in transgenic rice plants for ethanol production and possible reduction of air

pollution. Chapter V is a conclusion and gives an insight on further research.

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REFERENCES

Abebe, T., A.C. Guenzi, B. Martin, and 1C. Cushman. 2003. Tolerance of mannitol-
accumulating transgenic wheat to water stress and salinity. Plant Physiol.
131:1748-1755.

Apse, M.P., G.S. Aharon, W.A. Snedden, and E. Blumwald. 1999. Salt tolerance
conferred by over expression of a vacuolar NaVIF antiport in Arabidopsis.
Science 285: 1256-1258.

Bothast, RJ. and MA. Schlicher. 2005. Biotechnological processes for conversion of
corn into ethanol. Appl. Microbiol. Biotechnol. 67: 19—25.

Boyer, J .S. 1982. Plant productivity and environment. Science 218:444—448.

Canadian Lung Association. 2003. Burning unwanted crop residues (stubble burning).
C.O.N. DO fact sheet. [Online] http://wwwlung.ca/cando/content/FS-stubble.pdf.

Chandra Babu, R, et al. 2003. Genetic analysis of drought resistance in rice by molecular
markers association between secondary traits and ﬁeld performance. Crop Sci. 43,
1457-1469.

Cherry, J.H., RD. Locy, and A. Rychter. 1999. Proc. NATO Adv. Res. Workshop,
Mragowa, Poland. 13-19 June 1999. Kluwar Academic Publishers. Amsterdam,
The Netherlands.

Cushman, J .C., and H]. Bohnert. 2000. Genomic approach to plant stress tolerance. Curr.
Opin. Plant Biol. 3: 1 17-124.

Emberson, L., M. Ashmore, and F. Murray. 2004. Air pollution impacts on crops and
forests: A global assessment. p. 3-4. In L. Emberson, M. Ashmore, and F. Murray.
Air Pollution Reviews, vol 4. Imperial College Press, London, UK.

Epstein, E., J. Norlyn, D. Rush, R. Kingsbury, D. Kelley, G. Cunningham, and A. Wrona.
1980. Saline culture of crops: a genetic approach. Science 210:399-404.

FAO. 1999. Biotechnology in food and agriculture [Online] http://www.fao.org/unfao/
bodies/COAG/COAGI 5/X0074E.htm.

Farrell, A.E., R.J. Plevin, B.T. Turner, A.D. Jones, M. O’Hare, and D.M. Kammen. 2006.
Ethanol can contribute to energy and environmental goals. Science 311:506 —
508.

Frey, K. 1998. Genetic responses of oat genotypes to environmental factors. Field Crops
Res. 56:183-185.

Fuhrer, J., L. Skarby, and MR Ashmore. 1997. Critical levels for ozone effects on
vegetation in Europe. Environ. Pollut. 97:91—106

Golshan, M., et al. 2002. Early effects of burning rice farm residues on respiratory
symptoms of villagers in suburbs of Isfahan, Iran. Int. J. Environ. Health Res.
12:125-131.

Hillgren, S. 2005. An ambitious energy goal. Farm Journal, Jan, 2005, p.58.

Jacobs, J., R Kreutzer, and D. Smith. 1997. Rice burning and asthma hospitalizations,
Butte County, California, 1983-1992. Environ. Health Perspect. 1052980-985.

Kim, S., and BE. Dale. 2004. Global potential bioethanol production from wasted crops
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Agronomy Conference, Hobart, Australia.

Murty, A.S., P.N. Misra, and M.M. Haider. 1984. Effect of different salt concentrations
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Qadir, M., RH. Qureshi, and N. Ahmad. 1998. Horizontal ﬂushing: A promising
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Ribaut, J.M., and DA. Hoisington. 1998. Marker assisted selection: new tools and
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Growth Regul. 20:157-166.

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in rice increases polyamine level and enhances sodium chloride-stress tolerance.
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Schachtman, D., and W. Lui. 1999. Molecular pieces to the puzzle of the interaction
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Shanna.
r"
l

lalehni:

I
a .
(1L

lamni l
a

 

longoe.

Vilma. t.
V

l'elcli s
T

 

Shanna, H.C., J.H. Crouch, K.K. Sharma, N. Seetharama, and CT. Hash. 2002.
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Welch, RW. 1995. Oats in human nutrition and health. p. 433—471. In R.W. Welch (ed).
The oat crop: Production and utilization. lst ed. Chapman and Hall, London.

BlO'l

 

CHAPTER II

BIOTECHNOLOGY AND THE GENERATION OF TRAN SGENIC PLANTS

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INTRODUCTION

During the 21St century, humankind will be confronted with an extraordinary set
of challenges (Borlaug, 1997). By 2050, the United Nations projects that approximately
nine billion people will populate the world, which is an increase of 2.5 billion people
from today’s population- an amount equal to the world’s total population in 1950
(UNFPA, 2004). Therefore, hunger, poverty, social demands, health requirements,
environmental stresses, and ecological considerations around the globe must be
addressed. Moreover, food production must increase proportionally, while the life-
support systems provided by the world’s natural environment are maintained (Sharma et
al., 2005). To meet these challenges, it will be necessary to generate new knowledge and
techniques through continued scientiﬁc advances in the existing technologies and
emerging new technologies such as biotechnology. It will also be necessary to spread this
knowledge, technology, and the capacity to use both wisely throughout the world (N AS,
2000). The merging of conventional plant breeding with improved agricultural practices
and modern biotechnology is now a reality, and contributes to dramatic crop
improvements. Moreover, these technologies are expected to continue providing ﬁiture
beneﬁts by fostering the yield of crops that feed the world (Sharma et al., 2005).

This chapter will discuss the general deﬁnition of biotechnology, in vitro culture
and genetic engineering (theory and techniques) from the educational perspective with an

eye toward building understanding of the new technology beneﬁts and risks.

-10-

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BIOTECHNOLOGY

Definition

Biotechnology as deﬁned by Persley and Doyle (1999) is “any technique that uses
a living organism or substances from those organisms to make or modify a product,
improves plants or animals or develops microorganisms for speciﬁc uses”. The key
components of modern biotechnology are described below:

> Genomics
Genomics is the study of DNA sequences on the whole-genome scale involving

high throughput data acquisition and computational tools for analysis (Bazer and
Spencer, 2005). Gutterson and Zhang (2004) broadly divided genomics into three
overlapping branches:

0 Structural genomics concerns the identiﬁcation of proteins’ three-dimensional
structures based on their amino acid sequence. This technology has a potential
impact on drug discovery (Dry et al., 2000; Linhart and Valenta, 2005).

0 Comparative genomics is the analysis and comparison of genomes from
different species to understand how species have evolved and to determine the
functions of genes. This approach has been applied recently to analyses of
kinases, phosphatases and transcription factors (Gutterson and Zhang, 2004).

0 Functional genomics is the attempting to make use the ample of data produced
by genome sequencing to describe the function and interactions of genes. This
resulted in sequencing of Arabidopsis genome and development of tens of
thousands of molecular markers for different important traits. To do so, functional

genomics uses high-throughput techniques such as DNA microarrays,

-11-

 

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transcriptomics, proteomics, metabolomics, reverse genetics, map-based cloning

and mutation analysis. Because of the huge amount of data and the desire to

analyze and determine gene ﬁrnctions and interactions, bioinforrnatics is crucial.

> Bioinformatics

Bioinformatics is the assembly of biological data from genomic analysis into
accessible forms using advanced computing techniques (Fadiel et al., 2005). Research in
bioinforrnatics includes the development of method for collection, storage, retrieval,
sharing and analysis of the data (Fadiel et al., 2005), using techniques and concepts from
informatics, statistics, mathematics, chemistry, biochemistry, physics and computer
science (King, 2004). Bioinformatics has many practical applications in different areas of
biology and establishes the foundation for many aspects of genetic and genomic research
(Rhee, 2005). Most recently, bioinforrnatics is being used to automatically assign putative
functions to different DNA sequences (Toldo and Rippmann, 2005). Before the existing
data processing systems, it was too complex, time-consuming and expensive to collect
and analyze the genetic and genomic research outcome data (Bansal, 2005).

> Genetic Transformation

Genetic transformation is the genetic alteration of a cell resulting from the
introduction, uptake and expression of foreign genetic material (one or more genes)
conferring potentially useﬁrl traits into different species (Fink, 2005). Biolistic
bombardment, Agrobacterium tumefaciens, electroporation, microinjection and
polyethelyneglycol are common methods for plant transformation (Veluthambi et al.,
2003). Currently, the production of genetically modiﬁed plants is a central part of applied

plant science and a practical component of agricultural biotechnology (Taylor and

-12-

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Fauquet, 2002). More details about the concept of genetic transformation and examples
will be elaborated upon in later sections.
> Molecular Breeding

Molecular Breeding is the identiﬁcation and evaluation of desirable traits in
breeding programs by the use of marker assisted selection for different species (Persley
and Doyle, 1999). The marker assisted selection concept assumes that there are genes with
signiﬁcant effects, which may be speciﬁcally targeted in selection. Single genes control some
traits but most of the economically important traits are quantitatively controlled by a large
number of genes (Meredith, 2005). The ability to identify molecular markers linked to
quantitative traits has given the breeder a useﬁrl tool for testing these complex traits.
Hancock (2004) explained that the analysis is performed by constructing a genetic
linkage map of molecular markers and searching for a signiﬁcant association between
phenotypic differences in a population and the presence of those markers. This signiﬁcant
linkage indicates that the markers are genetically associated to one or more of the
quantitative trait loci (QTLs) (regions of the genome that account for a portion of the
total genotypic variation of a quantitative trait). For example, loci that are associated with
NaCl tolerance have been identiﬁed in Arabidopsis using this QTL approach (Quesada et al.,
2002). Following the pattern of inheritance at QTLs assists in selection.

> Diagnostics

Diagnostics is the rapid and more accurate identiﬁcation of pathogens and other
organisms using molecular characterization technology (Pfaller, 2001). For example,
technological advances such as PCR (polymerase chain reaction) allowed quick, precise

detection and quantiﬁcation of several wheat pathogens. This information could be used

-13-

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to improve disease control regarding the choice and use of ﬁrngicides and resistant
cultivars (McCartney et al., 2003).
> Vaccine Technology

Vaccine technology is the characterization of eliciting allergens and development
of vaccines to prevent or therapeutically treat allergies and infections resulted from
pathogens and lethal diseases using modern immunology techniques. Marker allergens
have been identiﬁed for some allergen sources such as diseases and insects which are
very crucial for allergen-speciﬁc immunotherapy success (Linhart and Valenta, 2005).
Plant Biotechnology

Plant biotechnology offers novel schemes and techniques applicable to
agriculture. It uses concepts, theories and technical approaches of plant tissue culture,
genetic modiﬁcation and molecular biology to develop commodities of commercial
medical and industrial values. The rapid development and achievements in the ﬁeld of
plant biotechnology strengthened the agriculture as a science-based industry (Sharma et
al., 2005). For example, due to the ability to reprogram, control tissue speciﬁcity and
expression level of genes, and the capacity to introduce them into plants; it is possible to
produce cr0ps tolerant to biotic and abiotic stresses (Sharma et al., 2002) as well as plants
that produce active commodity products (molecular farming) (Fischer et al., 2004). These
two aspects are expected to enhance sustainable agriculture and economic security
(Sharma et al., 2002). Moreover, success of applied plant biotechnology research,
commercialization and environmental safety of the products will be a driving force
behind fundamental scientiﬁc progress and is expected to have a signiﬁcant impact in

various aspects of human civilization in the ﬁrture.

-14-

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IN VIT R0 CULTURE TECHNOLOGY

Definition

Plant cell/tissue in vitro culture refers to the sterilized in vitro growing and
multiplication of cells, tissues and organs on deﬁned solid or liquid nutrient media under
aseptic conditions in a controlled environment (Uyoh et al., 2003). The large scale
commercial production of tissue culture-produced plants was started in the United States
and became one of the foremost agriculture technologies worldwide during the past 30
years (Ahloowalia et al., 2004). The major advantage of the tissue culture technology
relies on the use of seeds or plant pieces to produce quality and uniform plants. These
novel plants can be multiplied under pathogen-free conditions anywhere, regardless of
the time of the year and climate (Martinez-Gomez et al., 2003). Other advantages and
applications of the tissue culture technology include:

o It is often easy to select desirable traits directly from the in vitro culture such as
the selection of freezing tolerance in wheat, salt tolerance in rice, and disease and
herbicide tolerance in tobacco (Wenzel and Foroughi-Wehr, 1993). This
decreases the space required for ﬁeld trials and accelerates the selection process
as there is no need to wait for the whole seed life cycle development.

0 It enables rapid propagation of recalcitrant crop varieties and endangered species
such as woody plants (Benson, 2000; Cho et al., 1998; Ha et al., 2001).

o It allows for preservation and reduces the labor costs for maintenance of in vitro
cell collections that are used for plant propagation (Panis and Lambardi, 2005).

o It reduces the space required for the cryopreservation of large numbers of viable

plants (Martinez-Gomez et al., 2003).

-15-

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0 It ensures the production of pathogen-free stocks for the international germplasm
exchange, thus simplifying quarantine procedures (Engelmann, 1998).

o It helps the recovery of hybrids ﬁom incompatible species through either embryo
or ovule culture (Taji et al., 2002).

o It enables the production of haploid plants through anther culture. Haploid plants
may be used to recover recessive mutations in breeding programs. Subsequent
regeneration of double haploids provides homozygous and thus pure-breeding
lines (Taji et al., 2002).

0 It is the most efﬁcient method for the production of genetically modiﬁed plants
(Uyoh et al., 2003).

Even though there are distinct advantages to in vitro culture technology, there are
concerns worth addressing. There are always the problems of genetic instability, and the
risk of losing germplasm stocks and accessions due to contamination and human errors
(Panis et al., 2001). Also, donor plants can mutate and generations can become
contaminated with the mutation before the mutation is discovered. These mutations
observed in plants produced by tissue culture are known as somaclonal variations (Larkin
and Scowcroft, 1981). These variations are heritable, i.e. transmitted through meiosis and
are usually irreversible (Tremblay et al., 1999). Somaclonal variations can be genotypic
or phenotypic, which in the later case can be either genetic or epigenetic (i.e. factors that
affect the development or function of an organism other than changes in its primary DNA
sequences). The means of genetic alterations include changes in chromosome structure
(translocations, deletions and duplications), chromosome numbers (polyploidy and

aneuploidy) and DNA sequences. The epigenetic events involve gene ampliﬁcation and

-16-

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methylation (Soniya et al., 2001). In addition to the somaclonal variation, tissue culture
technology is expensive, laborious, energy intensive and is technically difﬁcult for
developing cultures of a number of important plants. This makes the implementation of
such technology difﬁcult in some developing countries as the necessary resources (most
importantly trained personnel and equipment) are often not readily available. However,
tissue culture technology has created new opportunities in the global trades and improved
rural economies (Ahloowalia, 2004).

For better understanding of plant tissue culture and regeneration, some concepts
are worth deﬁning such as plasticity, totipotency, and explants. Plasticity refers to a
plant’s ability to endure a range of environmental conditions and predation (Miner et al.,
2005), which permits alteration of metabolism, growth and development to adapt with an
environment. When plant cells and tissues are cultured in vitro, they generally exhibit a
very high level of plasticity, which allows the initiation of one type of tissue or organ
from another (Miner et al., 2005; Zhong et al., 1992a,b) and subsequent regeneration of
whole plants occurs. This regeneration depends on the ability of plant cells to express the
total genetic potential of the donor plant after suitable stimulation. The presence of this
genetic potential is called totipotency (Walden and Wingender, 1995). Thus, plant tissue
cultures are initiated from tiny pieces, known as explants, removed surgically from any
part of a plant such as leaves, stems, roots, meristems, ﬂowers, pollen and ovules
(Sticklen and Oraby, 2005). In practice, these isolated explants are surface sterilized and
placed on the nutrient medium to initiate the basic culture that is multiplied repeatedly by
subculture. The resulting regenerated plants can then be potted in soil and transferred to

the glasshouse/ﬁeld.

-17-

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Morphogenesis Methods

Generally, there are two methods of plant morphogenesis or regeneration (i.e. the
development of organs such as shoots, roots, or ﬂowers, and overall plant shape and
structure), that are commonly used especially in plant tissue culture studies, i.e. somatic
embryogenesis and organogenesis (Ramage and Williams 2002). In somatic
embryogenesis, somatic (non-sexual) cells form embryo-like structures which develop
into whole plants in a similar manner to a zygotic embryo from the seed (Meinke, 1995).
These somatic embryos can be produced either directly or indirectly. In direct somatic
embryogenesis, the embryo is developed directly from a cell(s) without callus production
(Paiva Neto et al., 2003). In indirect somatic embryogenesis, which is much more
common, embryos are produced from callus tissues or from a cell suspension of that
callus (Paiva Neto et al., 2003).

Organogenesis is the formation of adventitious organs, either directly from plant
pieces (explants) or indirectly from a callus culture, eliminating the involvement of an
embryo (Sudha et al., 2000, Sugiyama, 1999; Zhao et al., 2003). In addition, a whole
plant can be produced from the formation and growth of axillary buds (Martin, 2002;
Sudha et al., 1998). Generally, regeneration of plants via somatic embryogenesis is

Preferred to organogenesis because of the single cell origin of embryoids. This originates
genetically uniform plants instead iof chimeric ones, thereby making such embryogenic
Cells preferable for genetic manipulation. Organogenesis depends on the plasticity of the

Plant tissue and is controlled by altering culture medium components (Kumar et al.,

2001),

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The developmental pathway that the regenerating tissue will follow is determined
by the auxin to cytokinin balance (Sugiyama, 1999). For example, a high auxin signal is
usually necessary to induce somatic embryogenesis, whereas a high cytokinin is typically

required to induce shoot organogenesis (Phillips, 2004).

Explant Type

Explant type is a major factor that affects the success of any tissue culture process
(Ozgen et al., 1998). Many features of the explant are known to affect the efficiency of
culture initiation, shoot formation and regeneration processes, and consequently genetic
transformation (Azad et al., 2004). Some of the types of explants that are most commonly
used for different purposes are summarized below:
- Callus Cultures: A callus is an undifferentiated, unorganized mass of growing plant
tissue produced at the edge of a wound (Bottino, 1981) that can be grown in vitro and
induced to differentiate by balancing the ratios of the auxin and cytokinin in the culture
medium (Gamborg et al., 1969, Mineo, 1990). Even though a callus is an undifferentiated
mass of cells, some degree of dedifferentiation can occur (Carrillo-Castaﬁeda and Mata,
2001). The dedifferentiation is the capacity of mature cells to return to meristematic
condition and development of a new growing point, followed by redifferentiation which
is the ability to reorganize into new organs (Taji et al., 2002). This dedifferentiation
causes a loss of the photosynthetic ability. Thus, calli usually grow in the dark as light
Iinduces certain differentiation processes, including the activation of genes not expressed
in the dark (Zhao et al., 2001). This lack of photosynthetic ability causes differences in

Me metabolic behavior between the callus cultures and the parent plant which

-19-

 

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necessitates the addition of vitamins and carbohydrates. Explants from different parts of a
plant (preferably young tissues) can be used to initiate calli when cultured on the
appropriate medium. Initially, the cells of the callus proliferate and after its biomass
increases several-fold, it is divided and placed on fresh medium for callus multiplication.
Such multiplied calli can be maintained indeﬁnitely by repeated subculture. By
increasing the cytokinin concentration and decreasing auxin content in the culture media,
multiplied calli can be stimulated to form shoots which can be separated and transferred
to a rooting medium. After rooting, regenerated plants can be acclimatized in growth
chambers and then transferred to greenhouses. Regardless of the fact that the regeneration
from calli may be relatively genotype-dependent (Wan and Lemaux, 1994), the most
important advantage of callus cultures is that they are used to initiate transgenic plants for
a variety of plant transformation studies.

- Cell Suspension: Calli are divided into two types: compact calli which are aggregated
and condensed cells and ﬁiable calli which are breakable, as individual cells are loosely
associated with each other. The ﬁiable calli provide the initial source to generate cell
suspensions when they are placed in suitable liquid or semi-solid medium (same solid
medium components with less gelling agent) with agitation. The loose cells continue to
grow and divide producing the cell-suspension culture. After the initiation, cultures are
ﬁltered periodically to eliminate large tissue masses. These cultures can be maintained by
subculture as they enter the stationary phase, i.e. when nutrients in the medium are
ﬁnished and/or toxic products accumulate to inhibitory levels. Although, cell suspension

is easy to initiate on a large scale, it still has some disadvantages such as slow growth

-20-

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. rates, formation of aggregates, the need for constant maintenance, and the rapid loss of
regenerative competence and somaclonal variation (Hellwig et al., 2004).

- Protoplasts: Protoplasts (known as naked cells) are plant cells with removed cell walls
where the plasma membrane is the only barrier between the cytoplasm and its immediate
external environment (Davey et al., 2005). Two different methods can be used to remove
the cell wall. The ﬁrst is mechanical isolation which always results in very poor yields
and low performance in subsequent cultures due to the toxins released from damaged

cells. This method is now rarely employed for protoplast isolation, unless limited

numbers of protoplasts are required (Davey et al., 2005). The second method, most
favorable when large populations of protoplasts are required, is enzymatic isolation using
cell-wall degrading enzymes in solution of a high osmoticum salt. These enzymes include
cellulases that digest the cell wall and pectinases that breakdown the pectin which hold

the cells together (Davey et al., 2003). Protoplast isolation is now a routine in many
species and they can be isolated from shoots (Keskitalo, 2001), leaf mesophyll and

scutellum (Mliki et al., 2003), and seedlings including radicles, hypocotyls, cotyledon
tissues, roots, and root hairs, and embryogenic cell suspensions (Davey et al., 2005).

Protoplasts are totipotent when given the correct stimuli and have the capability of
generating new walls and producing new cells from which fertile plants may be
regenerated. Although protoplasts are fragile and easy to damage, it has. been an ideal

target for genetic transformation as plants can be regenerated by organogenesis or
somatic embryogenesis (Davey et al., 2005).

- Root Cultures: The in vitro culture of root tips excised from the primary or lateral

roots of young seedlings on nutritional media was one of the ﬁrst achievements in plant

-21-

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tissue culture. Root tissues are genetically and biochemically stable, capable of fast and
unlimited growth in culture media and can be easily cloned to produce a large supply of
experimental tissue (Shanks and Morgan, 1999). Although roots were the ﬁrst tissues
established in plant tissue culture, they are not widely used in genetic engineering studies.
- Embryo Culture: Embryo culture is the excision and cultivation of mature and
immature zygotic embryos under aseptic conditions in a nutritional medium (Raghavan,
2003). Immature zygotic embryo has been the most commonly used explant source to
develop embryogenic callus lines, cell suspensions and protoplasts cultures for different
crops. However, the lack of competence of immature embryos in certain elite lines is still
a barrier to routine production of transgenic cereal crops in certain commercial cultivars.
In addition, a great deal of effort is required to produce immature embryos, manipulate its
cultures or their cell suspensions, and cryopreserve them for further use. In addition,
undifferentiated cells may have reduced regenerability after a few months of in vitro
culture (Sticklen and Oraby, 2005). Therefore, mature embryos have been suggested as a
source of friable embryogenic calli, which can be produced within approximately 2
months in a genotype-independent manner (Torbert et al., 1998). The great advantage of
using mature embryos as an explant is that they are less physiologically variable
compared to immature embryos. Moreover, they are cheap, convenient, and available
year-round in large quantities (Birsin et al., 2001). While embryo culture is time
consuming and requires a great deal of manipulative skill (Raghavan, 2003) compared to
some other explants, the use of embryo culture has several advantages:

0 It can be used to determine the essential requirements of embryos for growth,

differentiation, and morphogenesis.

-22-

o It eases the manipulation of the embryo outside the seed.

0 It is widely used to obtain plants from interspeciﬁc or intergeneric crosses where
the hybrid embryo dies due to the failure of developing a normal endosperm. The
embryo can then be rescued by early excision and subsequently cultured in vitro
on nutritional media. Thereaﬁer a whole plant can be regenerated.

o In some plant species, it overcomes sterility, incompatibility, breaks seed
dormancy, and allows for the propagation of recalcitrant plants.

0 It is very adaptable to a wide range of DNA delivery systems and the recovery of
fertile transgenic plants.

- Shoot Tip and Meristem Culture: The shoot apex or shoot tip is a microscopic
structure that is enclosed in the apical bud that produces the whole shoot including leaves
and ﬂowers. It consists of the shoot apical meristem, the formation region of lateral organ
primordia, the subapical enlargement region of shoot and primordia, and several leaf
primordia. The shoot apical meristem is the distal-most portion of the shoot and is
involved in forming the shoot by cell division and differentiation (Medford, 1992). It is
comprised of the initial cells and the subepidermal cells that initiate the germline cells of
tissue and organ (Medford, 1992). The meristem of the shoot apex region can be seen in
shoot apicesof mature plants (Medford, 1992) and seedlings (Chen and Dale, 1992). The
shoot apical meristem has a number of unique features that made it highly useful for
tissue culture manipulation and genetic transformation. For example, while a shoot in
planta will create only a speciﬁed number of leaves before forming reproductive
structures, a shoot apex in culture can be ‘reset’ by removing the leaf primordia that

supply the developmental signals (Irish and Jegla, 1997). This gives physiological

-23-

exider
Slums
aﬁer l
the m
1991 .
nature
of the

plants

 

 

 

 

 

evidence that a meristematic region can be maintained indeﬁnitely in culture (Smith and
Murashige, 1970). Moreover, shoot apical meristems can rebuild the meristematic region
after being damaged or sectioned (Bommineni et al., 1995) providing more evidence of
the in vitro sustainability and plasticity of the meristematic region (Irish and Nelson,
1991; Irish, 1998). In addition, a meristem cell has the ability to pass its meristematic
nature on to its daughter cells (Zhang et al., 1998). Because of the above characteristics
of the shoot apical meristem, scientists have cultured shoot apical meristems of different
plants in vitro for different application including:

0 Production of pathogen-free plants.

0 Identically propagate plants for long-term storage without the impact of
environmental conditions.

0 Understanding the morphogenesis of meristems and developing a unique in vitro
regeneration system that could be used in plant genetic modiﬁcation in a
genotype- independent manner.

0 The susceptibility of shoot meristems for foreign DNA delivery is perhaps equal
to (if not better than) most other desirable targets, such as immature embryos
(Sticklen and Oraby, 2005).

- Pollen and Anther Culture: Pollen containing the microspores (male gametophyte)
and anthers (somatic tissue that surrounds and contains the pollen) could be used as
haploid explants for in vitro culture (Roy and Manda], 2005). Immature pollen extracted
from developing anthers can be cultured, although this is a very time-consuming process.
Pollen released from mature anthers and cultured on nutritional medium can produce

embryos with low plant regeneration efﬁciency. The advantage of using microspore

-24-

culture
ellicier
Furthe:
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culture rather than anther culture is that microspore culture can be ﬁve to ten times more
efficient for embryo production than anther culture (Davies and Morton, 1998).
Furthermore, it is possible to monitor the pollen development pathway (Kumlehn and
Lorz, 1999). Moreover, it is easy to determine the tissue culture responsive genotypes and
use the direct access to transformation approaches (Deutsch et al., 2004). Doubling the
haploid chromosome number can occur naturally during culture or by using chemicals
such as colchicine (Smith and Drew, 1990). In general, the process has the disadvantages
of producing a large percentage of albino plants (Y amagishi, 2002) and it is tedious and
time consuming to remove the anthers and culture them in a speciﬁc orientation to get a
desired response. On the other hand, it has the advantages summarized below:
0 It is easy to handle the selection for desirable traits directly from the in vitro
anther culture for genetic improvement (J anhe et al., 1991).
o It enables the production of homozygous doubled haploid plants that are
homozygous for all loci which is useful for gene mapping (Wan et al., 1992).
. The technique is simple and quick and can reduce the time required to develop
basic germplasm and new varieties (Roy and Manda]. 2005).
- Ovary or Ovule Culture: In this process surface sterilized ovaries are harvested at
deﬁned intervals of between 6 and 24 h after pollination, on solid or liquid nutrient
medium followed by the regeneration of fertile plants (Kumlehn et al., 1997). Ovule
culture is used to produce haploid plants, overcoming the abortion of embryos of hybrids
at very early stages of development due to incompatibility barriers (Tomasi et al., 2002).
Also, it has been used as a means of in vitro fertilization for the production of distant

hybrids avoiding style and stigmatic incompatibility that inhibits pollen germination and

-25-

pollen tube growth (Kumlehn et al., 1997). Despite its advantages, this method is not

widely adopted by tissue culture laboratories because it is difﬁcult, time consuming and
labor intensive.

Regardless of the signiﬁcant effect of the explant type on the success of any tissue

culture system, the tissue culture conditions and media composition required to foster the

cell totipotency have a signiﬁcant impact and are responsible for great differences in

regeneration.

Med ia Composition and Physical Factors

The choice of a speciﬁc media to use is very important when developing a tissue
culture system. Nutrient media for plant tissue culture were created to maintain plant
tissues in a synthetic environment similar to nature. Many different kinds of media have
been developed for different tissue culture purposes. For example, MS (Murashige and
Skoo g, 1962), one of the most successful and wide-.spread media, was developed for
tobacco tissue culture based on the mineral compounds present in tobacco tissues.
White's medium (White, 1943), which contains a low salt formulation, was developed for
the on lture of tomato roots. Nitsch and Nitsch (1969) medium, which also contains lower
salt concentrations, is considered one of the most successﬁil media known for anther
cultLll‘es. Nutrient media contain macro- and micronutrients, and organic compounds such
as carbon sources, vitamins and plant grth regulators. The general media composition
is slltilrnarized in Table 2.1. In addition to media composition, the manipulation

(“busting or completely changing) of culture environmental factors such as temperature,

hum ~ - . . . . . .
l(llty, pH, light (quality, quantity and duration), gaseous envrronment and osmotic

-26-

pressu'

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pressure within certain levels is one of the most important factors in optimizing the
growth and morphology of in vitro plants (Phillips, 2004).

Modiﬁcation of the plant genome using genetic engineering methods would
facilitate rapid development of new varieties with new traits that ﬁilﬁll human needs. An
efficient in vitro plant regeneration method is often considered a prerequisite for plant

genetic modiﬁcation (Gassama-Dia et al., 2004).

-27-

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-23-

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GENETICALLY MODIFIED CROPS

Deﬁnition and Importance

Generally, crop improvement can rely on classical recombination breeding
methods and/or molecular techniques for speciﬁc goals (Sharma et al, 2005).
Traditionally, crop improvement occurs when a plant breeder alters the genetic
composition of a variety to enhance or decrease expression of important crop traits.
Conventional breeding methods transfer genes into crops by pollination. Because of some
constraints related to pre- and post-fertilization incompatibility, moving genes across
sexual barriers proved very difﬁcult (Dale, 2001). With the application of in vitro
techniques such as ovary and young single ovule culture and embryo rescue in plant
breeding, it became possible to obtain crop hybrids that were almost impossible to
achieve in the past (Chi, 2002). However, this process is time consuming, requires
intensively skilled labor and the regenerated plants often exhibit some sterility problems.

Currently, plant recombinant DNA techniques are enabling scientists to pursue
novel strategies for plant improvement, most of which would be difﬁcult using traditional
plant breeding methodologies (Potenza et al., 2004). The terms recombinant DNA,
genetic engineering (GE), transgenic modiﬁcation, or genetic modiﬁcation (GM) refer to
methods of recombinant DNA technology by which scientists transfer genes from one or
more species (any living organism) into the DNA of any crop plant to transfer desired
genetic traits (Rommens, 2004). The transferred genes need to be modiﬁed ﬁrst by
attaching different regulatory sequences. These regulatory sequences, such as promoters
(sequences that turn on genes), enhancers (DNA binding sites for gene-speciﬁc

transcriptional regulatory proteins that regulate the transcription of one or a subset of

-29-

genes), and terminators (sequences that turn off genes), control gene expression in the
new crop plant. The temporal (a type of regulation of gene expression in which a gene is
only expressed at a speciﬁc time in the plant’s development) and spatial (a type of
regulation of gene expression in which a gene is only expressed in a speciﬁc location in
the plant) expression of introduced genes are both necessary to enhance effective and
efﬁcient production of crop plants (Dale, 2001).

These genetically modiﬁed (GM) crops have a great deal of promise and potential
not only in increasing yield and quality and enhancing the environmental safety through
heavy metal removal, but also can be used as biofacton'es for the production of industrial
and human health-related biomolecules such as antibodies, vaccines and enzymes. Table

2.2 represents examples of the research on GM crops for diﬁ‘erent purposes.

Developing the GM Crops (Methods of Transformation)

Currently, the production of transgenic crops using genetic transformation
techniques is an essential aspect of applied plant science and a major component of
agricultural biotechnology (Sharma et al., 2005). There are two major steps involved in
plant genetic transformation. First, DNA must be transferred into the cell. Second, the
added DNA must integrate into the plant genome. The latter is difﬁcult and much less
efﬁcient resulting in the stable transformation of only a small percentage of the cells that
are initially used. In the remaining cells, the non-integrated DNA shows transient
expression that does not require plant regeneration, and degrades by nucleases shortly

after entering the cell (Altpeter et al., 2005).

-30-

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-31-

div-J
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Therefore, transient expression, that is much more frequent than the stable one,
can be used to optimize and assess the efﬁciency of transformation protocols. In that
regard, the two reporter genes gus and gp were used widely and efﬁciently to serve that
purpose (Sticklen and Oraby, 2005).

Methods of transferring and integrating foreign DNA into plant cells can be
divided into two categories. First, direct gene transfer systems involve using physical or
chemical principles to deliver naked DNA into plant cells. Those systems such as
electroporation, polyethylene glycol (PEG)-mediated DNA transformation,
microinjection, silicon carbide ﬁbers (silicon whiskers), and microprojectile
bombardment have been successﬁilly used to produce transgenic plants especially in
cereals. Second, indirect gene transfer systems involve using vectors as carriers of the
targeted DNA. One such system is Agrobacterium-mediated gene transfer which is used

today to routinely transform many agronomic and horticulture important species.

Direct Gene Transfer Systems

1- Electroporation and Polyethylene Glycol (PEG)—Mediated DNA Transformation.
Electroporation involves the creation of short electrical shocks that cause temporary
pores in membranes and facilitates the DNA delivery from a buffer into cells. It was used
to transform all major cereals including rice and maize (D’Halluin et al., 1992; Xu and
Li, 1994). One of the advantages of electroporation is that all cells are in the same
physiological stage aﬁer transformation. However, optimization of tissue culture
(treatments), DNA preparation (linear versus supercoiled) and field strength (voltage and
resistance) are all questionable factors in the efficiency of electroporation (Slater et al.,

2003). The polyethylene glycol (PEG) technique uses PEG and divalent calcium cations

-32-

llm
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that disturb the plasma membrane and increase permeability for DNA. It held great
promise for the transfer of genes into cereals and other monocots, which are difﬁcult to
transform by Agrobacterium tumefactions. However, while it is possible to produce
transgenic plants using this system, extreme care of cell handling is needed because of
the delicacy of cells used (Slater et al., 2003).

These two methods are simple, quick, inexpensive, and relatively non-toxic to the
transformed cells. One of the ﬁrst attempts to produce transgenic plants was by DNA
uptake through the protoplast plasma membrane using electroporation and PEG
(Songstad et al., 1995). Although the production of enough high-quality protoplast ready
for transformation is technically complicated, laborious and practically difficult, the
frequencies of producing high numbers of normal and fertile plants were signiﬁcantly
higher than what was achieved using other types of cells (Taylor and F auquet, 2002).

2- Silicon Carbide Fibers (Silicon Whiskers). These are microﬁbers 10—100 pm in
length and approximately 0.3-0.6 pm in diameter (Slater et al., 2003). When mixed with
plant cells and DNA carrying genes of interest, they penetrate the cells and permit the
delivery and integration of the DNA into the genomic chromosomes (Song et al., 2002).
Scientists were able to regenerate fertile transgenic plants (maize and other monocots)
using this system (Frame et al., 1994; Dalton et al., 1999). Although it is easy and cheap,
the requirements of careful ﬁber handling, the availability of suitable tissues and the need
to match speciﬁc cell types with the ﬁbers to achieve successful transformation are major
obstacles for the widespread use of this technology (Petolino, 2002).

3- Microinjection. In this technique, glass micropipettes with very small diameter at the

tip are used to transfer DNA into plant cells (Crossway et a1. 1986). Although this

-33-

technique yields high rates of success in transforming maize and wheat meristimatic
tissue (Chen and Dale, 1992; Escudero et al., 1996), it is very slow, expensive and
requires highly skilled and experienced labor.

4- Microprojectile Bombardment (Gene Gun). Since the ﬁrst particle delivery device
was developed almost two decades ago (Sanford, 1988), microprojectile bombardment
has received wide acceptance by plant researchers as a highly effective and efﬁcient
method for gene transfer to plant cells (Altpeter et al., 2005). Microprojectile
bombardment is a technique by which micron sized metal particles (tungsten or gold) are
coated with nucleic acid sequences of interest and are accelerated (currently by a
discharge of helium gas under high pressure) at velocities sufﬁcient to penetrate the cell
wall without lethal damage. Those particles locate themselves randomly in the cellular
organelles, the DNA dissociates by the action of the cellular liquid, and the process of
integrating the exogenous DNA in the genome occurs (Altpeter et al., 2005).

Initial uses of the gene gun were limited to the transient expression of marker
genes in corn, soybean, wheat, and rice (Klein et al., 1992), but were soon followed by
the production of transgenic maize plants (Fromm et al., 1990; Gordon-Kamm et al.,
1990). Through the 1990s, the application of this technology increased rapidly, and was
successfully used to produce transgenic plants in a wide range of different plant species
(Sticklen and Oraby, 2005). Compared to Agrobacterium, microprojectile bombardment,
which is similar to all direct methods of DNA transfer, leads to higher frequencies of
transgene rearrangements and copy numbers that result in gene silencing (Slater et al.,

2003). In spite of the cell damage that it causes and the need for the optimization of

-34-

Agn

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various parameters, this technology is currently one of the most important tools in the

ﬁeld of plant genetic engineering (Altpeter et al., 2005).

Agrobacterium-Mediated Transformation System

Agrobacterium tumefaciens is a soil bacterium that causes the crown gall (tumor-
like swellings) disease in a wide range of dicotyledonous (broad-leaved) plants. Most of
the genes involved in crown gall disease are not borne on the bacterium chromosome but
on a large plasmid, termed the Ti (tumour-inducing) plasmid. A. tumefaciens has the
ability to transfer and integrate a particular DNA segment (T-DNA) of the tumor-
inducing (Ti) plasmid into the nucleus of infected cells where it is then transcribed
causing the disease (Gelvin, 2003). The T-DNA contains two types of genes: the
oncogenic genes, encoding for enzymes involved in the tumor formation by the synthesis
of auxins and cytokinins; and the genes encoding for the synthesis of opines. These
compounds are used by the bacteria as carbon and nitrogen sources. Moreover, the 25-bp
direct repeats at the right and the leﬁ borders of the T-DNA act as a cis element signal for
the transfer apparatus and are needed for DNA integration (Gelvin, 2003). Outside the T-
DNA are located other functional parts for virulence (vir) (a region of about 30 kb that
encodes at least 10 operons), conjugation (con) (genes involved in bacterium-bacterium
plasmid conjugative transfer), the origin of its own replication (on) and antibiotic
resistant genes. The processing and transfer of T-DNA from Agrobacterium to plant cells
is regulated by the activity of the vir genes. Virulence gene activity (such as virA and

virG) is induced by plant wound-induced phenolic compounds such as acetosyringone

-35-

and rel

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and related molecules. Then, T-DNA transfer complex is generated and transferred to
plant cells and T-DNA integrates into plant genome. (Wei et al., 2000)

Biotechnologists capitalized on the Agrobacterium ’s natural ability to act as a
“genetic engineer” by disarming the tumor inducing genes and replacing them with genes
of agronomic interest for transfer into crop species (Slater et al, 2003). A wide range of
dicotyledonous species has been successfully transformed with Agrobacterium.
Monocots, especially cereals, were generally considered to be outside the host-range of
the bacteria (Wei et al., 2000); however, considerable progress has been achieved for the
last two decades using Agrobacterium transformation techniques in the successful
recovery of transgenic plants of all major cereals and crops which were previously
transformed by particle bombardment (Hiei et al., 1997; Ishaida et al., 1996). In addition
to the reduced operating costs, one of the most attractive features of using Agrobacterium
is its ability to insert a complete single copy of the transgene compared to the multiple
copy rearrangements and broken transgene integrations common in particle
bombardment. This feature is very important for stable and high expression of the genes
of interest (Taylor and Fauquet, 2002). Nevertheless, despite the advantages of the
Agrobacterium transformation system, there are challenges that remain. These include
frequent multiple transgene insertions, the presence of viable Agrobacterium cells within
the regenerated tissues (Christou, 1996), and the integration of unnecessary DNA
segments from outside the T-DNA borders into the plant genome (Repellin et al., 2001;
Smith et al., 2001). Regardless, current trends indicate that Agmbacterium and particle

bombardment transformation systems are likely to continue playing important roles in

-36-

plant biol

Slater e1

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plant biology and crop biotechnology for many years in the future (Altpeter et al., 2005; i

Slater et al, 2003).

Current Status

In the mid-1990's, GM crop technology made it possible to transfer a genetic trait
from any organism in the living world into a crop, instead of being restricted to those
species that were sexually compatible. In 1994, the nonperishable FlavrSavr® tomato
was the ﬁrst GM crop introduced to the market. It was resistant to bruising because it
ripened more slowly than non—engineered tomatoes. As a result, farmers were able to
leave the tomato on the vine longer and to transport the ﬁ'uit with less risk of damage.

The global area of GM crops increased from 1.7 million hectares in 1996 to 90.0
million hectares in 2005, more than a 50-fold increase in less than a decade (James,
2005). This reﬂects the high rate of acceptance and adoption of the GM crops by
governmental, scientiﬁc and agricultural communities in about twenty-one developed and
developing countries worldwide, including: USA, Argentina, Brazil, Canada, China,
Paraguay, India, South Africa, Uruguay, Australia, Mexico, Romania, the Philippines,
Spain, Colombia, Iran, Honduras, Portugal, Germany, France, and the Czech Republic
(James, 2005).

The herbicide-resistant soybean was still the dominant biotech crop in 2005,
occupying 54.4 Mha (60% of the global GM crop area) followed by Bt maize, which
occupied 11.3 Mha, (13%) and Bt/Herbicide maize, grown over 6.5 million hectares
(7%). Moreover, Bt cotton was grown on 4.9 million hectares (5%) and herbicide

resistance canola occupied 4.6 million hectares (5%). In 2005, the global market value of

-37-

GM
4

pTOu

(lam

GM 1

GM'crops was $5.25 billion (representing 15% of the $34.02 billion global crop'
production market, and 18% of the $30 billion 2005 global commercial seed market)

(James, 2005).

GM Crops: Concerns and Possible Solutions

The debate over the beneﬁts and risks of GM crops is heating up, especially in
Europe, and public concerns (Table 2.3) over the safty of GM food are escalating
(Amtzen et al., 2003) due to some cases of GM failure deployed to markets during the
past decade (Table 2.4). In Europe, consumers are not embracing the beneﬁts observed in
many countries that grow GM crops including United States. They continue opposing
them despite the fact that millions of US citizens eat GM soybean and corn products
everyday in their meals, and have done so far nearly ten years without any known adverse
consequences or illness (Burke, 2004). This opposition has been in response to deep
anxiety about the potential health and environmental threats of introducing some GM
cultivars, and the desire of Europeans to be able to choose between GM and non-GM
products in the supermarket (Smyth et al., 2002). The infamy that GM crops gained in
Europe has encouraged the governmental authorities in several countries in Africa
including Zambia, Angola, Sudan, and Benin to strongly resist growing or even accepting
imports of such crops, for fear of poisoning their people and not being able to export the
GM crops to the European market (Burke, 2004). From the GM-opponent’s viewpoint,
the claim that GM is the second green revolution and it is the answer to African hunger is
false because they claim that hunger in Africa is not due to lack of food but due to the
poverty and poor purchasing power of the population. According to such critics, Africa is

in danger of becoming the dumping ground for the struggling GM industry. Furthermore,

-33-

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they fear that pe0ple will end up being forced to pay royalties to the multinational
companies that own the patents on the GM crops, on seeds that cannot be saved for the
next season’s planting. Also, contracts, agreements and conditions of the intellectual
property rights (IPR) and unrealistic biosafety systems in Africa that lacks of expertise,
equipment, infrastructure, legislation and regulatory systems to implement effective
biosafety measures for GM crops are very delicate issues in the debate over GM cr0ps.

A set of questions about basic biology and genetics, to analyze Europeans'
perceptions of biotechnology, were asked in a survey carried out in ﬁfteen member states
of the European Union in 2002 (Europeans and Biotechnology, 2002). The survey
showed that 35% of Europeans agreed that ordinary tomatoes do not have genes while
GM tomatoes do and 20% that eating genetically modiﬁed fruit could modify a person's
genes. This reﬂects the fact that education is one of the stumbling blocks to acceptance of
GM crops. Therefore, educating the consumers about the products they are eating—what
they are, how they are produced and how they are tested before being released to the
public—is very important in helping these new technologies to be accepted, insomuch as
they do not contradict the ethics, morals, religion or cultures of the consumers. Moreover,
media coverage of the GM controversy has a critical role in that situation. It should not
be biased and should reﬂect a balanced representation of the pro-GM industry and anti-
GM campaigners. Regarding the IPRs, beneﬁt distribution can be the basis for
agreements between farmer organizations and commercial producers. Agreements can
establish ownership by equalizing investments between providers of transgenes, their
research, and the costs of establishing regulatory systems for GM crops and with time

and innovation provided by developing countries creating combinations of genes in their

-39-

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own crops and genetic resources (Goodman and Carson, 2000). Moreover, it is
worthwhile to develop a cross talk conversation between the public, scientists and policy
makers to address these concerns and help overcoming public worries over of this

technology (Hails and Kinderlerer, 2003).

Engineering and Breeding

The amazing capabilities of genetic engineering to precisely incorporate foreign
DNA into plant species and develop “super traits”, using tissue culture and plant
transformation techniques (Figure 2.1), is a valuable tool in the toolbox available for
modern breeding. Furthermore, genetic engineering uses well-deﬁned genetic elements
that introgress only desirable traits avoiding allergens or toxins resulted from introducing
unwanted genes linked with traits of interest. No one can deny that feeding the world is
not an easy task to accomplish. Genetic recourses and hard efforts exerted by scientists
are very important to make this goal real. GM crops proved their competence and ability
to meet many practical challenges related to the food production process in many
countries worldwide. The rate of acceptance and adoption is increasing and scientists are
doing their best to maximize safety evaluation and seek efﬁciencies and effectiveness of
regulatory systems for public beneﬁt. To achieve the food and energy security needed
over the next 50 years, the aims of genetically engineered crops. should be directed to:

0 develop plants that can grow in less favorable conditions such as salinity-prone
areas.

0 develop high-quality protein and yield plants with minimal inputs.

-40-

0 use plants as biofactories to produce proteins and pharmaceuticals such as
enzymes needed for biomass bioﬁiel production.
0 produce cheap and available seeds away from patents and licenses.
Moreover, a fully integrated traditional breeding and biotechnology crop improvement

programs are needed to provide the sustainable agriculture required (Rommens, 2004).

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CHAPTER III

BARLEY H VAI GENE CONFERS SALT TOLERANCE IN R3 TRANSGENIC

OAT

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INTRODUCTION

This chapter will begin with a review of literature on the importance of oat, the
effect of salinity on this important cereal crop, and the use of abiotic stress genes to
enhance plant tolerance to environmental stresses. Since salinity is a major factor in
global agricultural production, this particular abiotic stress is considered in relation to
genetic modiﬁcation. Established oat in vitro culture and transformation using
microprojectile bombardment was ﬁrst reported by Somers et al. (1992). It is now
possible to produce fertile transgenic lines of major cereal crops including oat for
different studies (Makarevitch et al., 2003; McGrath et al., 1997; Pawlowski et al., 1998;
Svitashev et al., 2000, 2002; Torbert et al., 1995, 1998 a,b,c; Zhang et al., 1999). The
ability to address salinity with fertile transgenic oat is the basis for the research presented
in this chapter.

This research studies the third generation of transgenic oat expressing H VA 1, B-
glucuronidase (uidA; gus), and the bar herbicide resistance genes under greenhouse
conditions. The objectives were:

0 Determination of the segregation of the linked bar and H VAI genes and verify
their proper expression and stability of transmission to progeny.

0 Evaluation of the effects of the transgenes on plant growth.

0 Comparison of the performance of these transgenic lines against non-transgenic

controls for common important agronomic traits under salt stress conditions.

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REVIEW OF LITERATURE
The Importance of Oat

Oat (Avena sativa L.) is ranked the sixth among the seven principal cereals grown

in the world (wheat, rice, maize, barley, sorghum, oat and rye) (Zhang et al., 1996).
The world harvests 11,666,338 hectares of oats yielding a world production of
25,899,616 metric tons. The world’s oat trade is small (about 7% of the world
production) as most of the world’s oat production is consumed domestically. Oat is the
third most important grain crop in the United States (Magness et al., 1971), which
produces 6.4% of the world harvest (FAO, 2004). Within the United States, oat
production varies among states. North Dakota (with 15% of US production), Minnesota
(13%), South Dakota (10.8%), and Wisconsin (10.7%) were the largest US oat-producing
states in 2003 (Commodity Research Bureau, 2004). Outside the United States, the
importance of oat as a cash crop also varies. Russian Federation (Former Soviet Union),
Canada, Poland, Germany, Australia, and Spain produce approximately 50% of the
world’s supply of oat grains. Since the 19605, oat-grain production has declined
dramatically in the USA and the Russian Federation, which still remains the world's
largest oat producer. Canadian production declined slightly, while it remained unchanged
in Australia and increased in Spain (F A0, 2004).

There are several reasons for the importance of oat production in many
economies. Oat has become particularly important in soil conservation and animal and
human diets (Forsberg and Shands, 1989). As a cover crop, oat is used to protect soil
during short periods when it would be otherwise empty (Sustainable Agriculture

Network, 1998; Wollenhaupt et al., 1995). It controls water and wind erosion, prevents

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damage and enhances soil life (Kaspar et al., 2001). Incorporating oat in the soil as a
green manure adds organic matter which improves poor soils to the point that helps tilth,
and sustains subsequent plant grth (Langdale et al., 1990). Oat also is used as a part of
an integrated system to control weeds. It suppresses weed growth and seed production by
competing directly for light and nutrients (1on et al., 2002).

As the most important cereal fodder crop, oat is nutritious and acceptable as green
forage, silage, hay, straw and grain to all categories of livestock (Welch, 1995). Peterson
et al. (2005) suggest that the low soluble ﬁber (B-glucan), high oil and protein content,
and high groat percentage of oat grains provide maximum livestock feed energy.

Moreover, when consumed on a daily basis, oat soluble ﬁber (IS-glucan) reduces
blood cholesterol levels, which is one of the most serious risk factors of heart disease in
humans (Cervantes-Martinez et al., 2002; Kurtzweil, 1994; Welch, 1995). Along with
more traditional vitamins and minerals, oat grains are rich in phytochemicals that
ﬁinction as antioxidants, which may be beneﬁcial for animal and human health as well
(Peterson et al., 2005).

Though it is important to many economies, the importance of oat in human
societies is also mediated by cultural factors. For example, oat is not an agricultural
product of many countries that are otherwise capable of producing it. In such locations,
the cultural and historical value of more traditional crops like wheat and barley far
outweigh the possible beneﬁts of introducing oat as another versatile cash crop.
Therefore, even though cultivating oat provides so many advantages, there are more
factors limiting agricultural decisions than simple economics. Introducing genetically

modiﬁed (GM) oats into regions where non-GM oats would be otherwise overlooked,"for

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either cultural or geographic reasons, may provide an opportunity to increase the world
harvest of this valuable crop. This highlights the need for a coordinated international oat
network targeting the introduction of oat into poor growing environments and educating

local agricultural communities.

Effect of Abiotic Stresses on Oat

A wide range of abiotic stresses such as temperature, drought and salinity affect
plants’ chemical and physiological processes (Panda and Khan, 2004). Oat is not
considered as heat or cold tolerant as barley, rye, or wheat as it is susceptible to damage
by cold or hot dry weather that occurs during reproduction (Miller, 1984; Stoskopf,
1985). Aside from temperature, a number of other abiotic stresses play a role in the
growth and development of oat.

Generally, water deﬁciency and increased soil salinity are two of the major
factors hampering the yield, quality potentials and overall performance of oat genotypes
(Frey, 1998; Martin et al., 2001; Tamm, 2003). Forsberg and Reeves (1995) reported that
cool and moist climates are considered the best environments for oat grth and
performance. Murty et al. (1984) and Verma and Yadava (1986) found that soil salinity is
responsible for decreasing oat seed germination and stunting subsequent development in
a cultivar-dependent manner. Furthermore, Martin et al. (2001) found that salinity stress
has a considerable inﬂuence on oat plants, particularly during anthesis and grain-ﬁlling
resulting in reduced panicle and grain number, smaller grain weight and consequently
decreased yield. In ﬁeld experiments to study the effect of saline water on the yield of

oat, Singh et al. (1999) found that reduced salinity and improved fertility resulted in

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much better oat grth and productivity. Moreover, Yadav et al. (2004) reported a 42%

reduction in oat yield, which declined linearly with the increase in salinity.

In Vitro Culture of Oat

For the last few decades, plant tissue culture techniques have played an important
role in many innovative aspects of applied plant science (Sharma et al., 2005). Earlier,
plant tissue culture techniques were used primarly to investigate grth and development
of cells that are totipotent—the ability of plant cells to give rise to any and all cell types
and grow another complete plant (Mineo, 1990; Steward et al., 1970). More recently,
controlled manipulations of plants at the cellular level such as genetic engineering of
tissue-cultured plants have provided new insights into plant molecular biology,
developmental physiology and gene expression (Sticklen and Oraby, 2005).

Several parameters affect the cell’s ability to express its totipotency explant type
and culture medium composition of growth regulators (Sticklen and Oraby, 2005).
Explant type is one of the main factors that inﬂuence the success of any tissue culture
process (Ozgen et al., 1996; 1998). It affects shoot formation and regeneration efﬁciency
(Azad et al., 2004) as it regulates and directs secondary metabolite synthesis (Murch et
al., 2000).

The ﬁrst and the most widely used explant source to develop embryogenic callus
lines in oat tissue culture is immature embryos collected 12-18 days after pollination
(Cummings et al., 1976; King et al., 1986; Rines and McCoy, 1981; Sticklen and Oraby,
2005). This tissue culture system has the limitations of being expensive, time consuming

and laborious, as it requires growing the donor plant year-round. Also it takes at least 12

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weeks of callus initiation and grth before transformation (Torbert et al., 1998b;
Sivamani et al., 1996). Moreover it is relatively genotype-speciﬁc (Bregitzer et al., 1989;
Cummings et al., 1976; Gana et al., 1995), although limited success has been achieved
with some oat cultivars (Bregitzer et al., 1995; Kuai et al., 2001). In addition,
undifferentiated cells may have reduced their totipotency, regenerability (after few
months of in vitro culture) and fertility due to somaclonal variation and tissue culture age.
In oat cultures, the chromosome breakage increases with the age of the cultures (McCoy
et al., 1982). Also, they are subject to physiological variation depending on the conditions
to which the donor plant is exposed (Torbert et al., 1998b). Moreover, as the immature
embryos continue to age, they are not useﬁrl for long-term (over 6 month) in vitro
cultures. Therefore, cryopreservation (a process where cells or whole tissues are
preserved by cooling to sub-zero temperatures) of immature embryo-derived cells or calli
is commonly used to have explants readily available for genetic manipulation. The lack
of competence of immature embryos in certain elite lines was a barrier to routine
production of transgenic oat crops in certain commercial cultivars. Alternative explants
and regeneration systems for efﬁcient transformation of oat were needed to avoid or
reduce the above limitations (Sticklen and Oraby, 2005).

Mature embryos (Birsin et al., 2001; Bregitzer et al., 1995; Rines et al., 1992;
Torbert et al., l998a,b) have been suggested as a source of ﬁiable embryogenic oat calli,
which require approximately 8-9 weeks to be produced. They have the capacity to
regenerate fertile plants in a genotype-independent manner. Using the mature embryos,
Torbert et al. (1998b) have reported less callus formation ﬁ'equency than what was

reported for immature embryos (Somers et al., 1992). Therefore, one needs to isolate

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larger quantities of mature embryos to produce an amount of tissue culture equal to that
obtained ﬁ'om immature embryos. However, the callus formation process is essentially
the same after this point. The great advantage of using mature embryos as an explant is
that they are less physiologically variable compared to immature embryos. Moreover,
they are cheap and convenient, available year-round in large quantities (Birsin et al.,

2001).

An efﬁcient and reproducible in vitro culture system for oat using seed-derived
highly regenerative cultures was established by Cho et a1. (1999). Green shoots have been
grown from green tissues on culture medium over time. Those green shoots were highly
capable of producing fertile plants (Choi et al., 2000). The highly regenerative tissues of
oat could be maintained for more than 1.5 years with minimal loss in regenerability (Cho

et al., 1999).

Another short-terrn efﬁcient regeneration system has been established using oat
leaf bases as an explant (Chen et al., 1995a, b; Gless et al., 1998). Several sections (leaf
base, mesocotyls and mature parts of the leaves) of oat seedlings from different cultivars
have been isolated and tested for callus formation and regeneration capabilities (Chen et
al., 1995a, b; Gless et al., 1998). The callus formation frequency was highest in the
mesocotyl tissues with the lowest capacity of regeneration (Chen et al., 1995b). Callus
derived from the leaf base (containing the meristem portion) were the most potent and
efﬁcient for plant regeneration (Chen et al., 1995a,b; Gless et al., 1998). There were no
differences noticed in the callus formed from different explants except more compactness
in the leaf base-derived calli. The frequency of regenerable callus formation and plant

regeneration was correlated with the position, developmental stage and genotype of the

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explant (Chen et al., 1995a; Gless et al., 1998). The regeneration capacity of the ﬁrst
1 mm of the leaf basal region from three-day old seedlings was comparable to that of
immature embryos and the leaf regenerable calli could be subcultured for 8 months
without loss of regeneration capabilities (Chen et al., 1995a). The leaf base is an easy,
fast and convenient explant for use in oat tissue culture as regenerated plantlets could be
planted after two months (Chen et al., 1995b). This high regeneration potential could

make oat leaf base a very attractive target for transformation (Gless et al., 1998).

Friable embryogenic calli also were initiated directly from seedling mesocotyls
(Bregitzer et al., 1989, Heyser and Nabors, 1982), roots (Nabors et al., 1982), anthers
(Kiviharju et al., 2000; 2005; Rines, 1983) and axillary tiller buds (Shekhawat et al.,
1984) of different genotypes. Since different genotypes of the same explant respond at
different levels, these systems for callus induction and regeneration are still genotype-
dependent. The cultures from these explants produce regenerable callus types that could
be useful for in vitro genetic manipulation, although no such use has yet been reported for

the genetic engineering of oat.

During the last decade, scientists have successfully manipulated the shoot apical
meristems from seedlings of oat in an effort to develop a less genotype-dependent and
more efﬁcient regeneration system that can be maintained in vitro for long periods of
time without the need for cryopreservation (Sticklen and Oraby, 2005). As an initial
explant dissected from germinated seeds, oat shoot apices have been efficiently
differentiated into multiple adventitious shoots. These shoots were generated from the
apical meristems and regenerated into mature plants (Zhang et al., 1996). The use of the

shoot apical meristem as an explant has advantages over other explants:

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mature dry seeds are used to produce multiple shoot cultures (Maqbool et al.,
2002)

meristems can be sustained in vitro using a 2-week subculture process for long
periods of time.

the shoot apical meristems have high regeneration ability, and the resulting
regenerated plants have high levels of fertility and genomic stability.

the system avoids callus and cell suspension cultures that can cause somaclonal
variations (Bommineni et al., 1995).

oat morphogenesis studies using shoot apical meristem differentiation systems
could be used to study oat developmental physiology (Sticklen and Oraby, 2005).
the system avoids chimeric genetic transformants (a state in which two or more
genetically different populations of cells co-exist) as long as meristems are
multiplied in vitro for at least 2-3 months prior to rooting and plantlet formation.
because variations induced from tissue culture are reduced/absent, cultured shoot
apices may produce plants genetically identical to the parent plant (Kartha, 1985).
the system allows the production of hundreds of disease-free plants from a single

shoot apical meristem when suitable grth regulators are employed.

Even though the use of shoot apical meristem as an explant has many advantages, the

characteristics of the growth regulators used are also an important factor in any

successful tissue culture system.

Plant grth regulators (PGRs) are chemicals that are used to affect plant growth

and/or development. Successful use of PGRs involves both the science of understanding

plant grth and development, and the art of concentration adjustments using necessary

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observational and judgmental skills to improve plant performance (Latimer, 2001). While
the science of plant growth and development is well documented, the art aspect of using
PGRs deserves some ﬁrrther clariﬁcation.

Various nutrients and PGRs in tissue culture media have been successfully used
for somatic embryogenesis of cereals (Tomes, 1985). For the enhancement of
embryogenic calli induction, MS media (Murashige and Skoog, 1962) was supplemented
with auxins such as either 2,4 dichlorophenoxyacetic acid (2,4-D) or 2,4,5-
trichlorophenoxyacetic acid (2,4,5-T) combined with indoleacetic acid (1AA), or
tryptophan in conjunction with kinetin (Carman et al., 1987; Gless et al., 1998; Linsmaier
and Skoog, 1965; Mackinnon et al., 1987; Nabors et al., 1983). Production of non-
embryogenic cereal calli was promoted by media containing 2,4-D or 2,4,5-T alone

(Nabors et al., 1983).

Speciﬁc to oat, Kiviharju et al. (2005) found that media supplemented with 2,4-D,
6-Benzyl amino purine (BAP), ethephon, L-cysteine and myo-inositol produced higher
regeneration rates for callus induction of oat anther cultures than media containing only
2,4-D and kinetin. Moreover, addition of grth regulators in the culture medium
increases shoot formation, multiplication and subsequent plant regeneration frequency
from different oat. genotypes (Sticklen and Oraby, 2005). Cummings et al. (1976)
reported that tiny green buds and leaves which developed from the calli of 16 different
oat genotypes formed shoots and plants when transferred to media without 2,4-D. Also,
competent tissue cultures were initiated from axillary tiller buds and immature leaves of
two cultivars cultured on nutrient media containing 2,4-D and BAP (Shekhawat et al.,

1984)

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Zhang et al. (1996) showed that shoot tip multiplication occurs by differentiation
of the shoot apical meristem axillary buds from the leaf bases. The medium containing
lower concentrations of BAP promoted more axillary buds than adventitious buds even
after 3 months of culture. Medium with high BAP concentration ﬁrstly stimulated shoot
apical domes to enlarge, which was followed by a mixed differentiation of both axillary
and adventitious bud from the leaf axils and the enlargement of apical domes within the
ﬁrst 2—3 months of culture. A low concentration of 2,4-D was found to greatly improve
the efﬁciency of adventitious shoot formation on the medium with high BAP
concentration. Based on the best performance of multiple shoot formation of different
genotypes on different combinations of grth regulators, oat had the third highest
efficiency and third quickest response following maize (Zhong et al., 1992a, b) and
sorghum (Zhong et al., 1998), ranging from 88-96% shoot production (Zhang et al.,
1996). With the proven possibilities of these past studies, the development of a competent
and efficient regeneration system is the crucial next step for any successful genetic

transformation process.

Genetic Transformation of Oat

In the previous section successful oat tissue culture systems were discussed as a
prerequisite for plant transformation. Different explants from the oat plant can be placed
into speciﬁc grth media that foster regeneration. Though such a tissue culture system
could be an end in itself, the useﬁrlness of this process is magniﬁed when coupled with
genetic modiﬁcation. By producing genetically modiﬁed plants using optimal grth

media and transformation techniques, speciﬁc characteristics can be improved to fulﬁll

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speciﬁc human needs. Until the present day, most of these needs are primarily those of
the scientiﬁc community; however, the promise of transgenic oat plants in the
agricultural community is also worthy of consideration.

Initially, the possibility of oat transformation was tested using gibberellin-
responsive oat aleurone protoplasts. Huttly and Daulcombe (1989) analyzed the transient
expression of fusion between the reporter gene gus (uidA gene) and the ALPHA-AMY 2
wheat promoter. Following PEG-mediated transformation, transient expression of gus
was dependent on the inclusion of gibberellin in the incubation media, thus providing
evidence of a successful, albeit transient gene insertion.

Using the biolistic delivery of foreign DNA, Hiruki et al. (1993) bombarded
immature embryos of oat with genomic transcript RNA from sweet clover necrotic
mosaic dianthovirus (SCNMV) along with the gus gene. Gus gene expression in oat
seedlings was detected in apical meristems of lateral roots, root hairs and leaf tissues. The
expression of the foreign gene was not stable and the gene was lost in the absence of
selection pressure.

The ﬁrst successful stable transformation of oat was via particle bombardment of
embryogenic calli of immature embryos. Although this system was genotype-speciﬁc and
slow, fertile transgenic plants expressing herbicide resistance (bar gene) and GUS
activity (gus gene) were produced (Somers et al., 1992; Kuai et al., 2001).

Gas gene expression was also used to investigate the transgene expression
controlled by the Commelina yellow mottle virus (CoYMV) promoter in oat.
Histochemical GUS staining was localized in the vascular tissues of shoots, leaves, ﬂoral

bracts, ovaries, scutellum of mature seeds, and roots. The results conﬁrmed successful

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stable transformation and a vascular speciﬁc expression of the gus gene under the control
of the CoYMV promoter (Torbet et al., 19980). Using the same system, Torbert et al.
(1995) were able to transform oat with plasmids containing the neomycin
phosphotransferase H gene (npt H) and used paromomycin sulfate as a selective agent.
Under the conditions of this study, oat plants transformed with the npt H showed reduced
ecological risk compared to the previously used herbicide-resistance selection system.

Along with the npt H gene, the gus gene was used to select transformants
produced from the mature embryo-derived calli of oat via microprojectile bombardment.
Using this system, fertile transgenic plants were produced at frequencies similar to those
produced from immature embryos (Torbert et al., 1998a). McGrath et al. (1997)
introduced constructs containing the coat protein (CP) genes of isolates of barley yellow
dwarf virus (BYDV), together with a construct containing the bar gene for herbicide
resistance and the gus reporter gene. Oat plants in the T1 generation were resistant to
virus isolates tested.

Still employing the gus gene as a reporter, Cho et al., (1999) used seed-derived,
highly regenerative cultures to achieve high frequency transformation of oat via
microprojectile bombardment. The hygromycin phosphotransferase (hpt) and gus (uidA)
reporter genes were used to produce independent transgenic events with high fertility.
Though not frequently used in laboratories, the success of this experimental design
established a new system for the in vitro culture and transformation of oat.

Another new system, involving multi-shoot apical meristems, was successfully
developed for the in vitro culture and transformation of maize, sorghum and millet as

well as oat. Oat morphogenesis studies indicated that oat shoot apical meristems can be

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differentiated rapidly into multiple shoots, and each shoot could then be regenerated into
a mature plant (Zhang et al., 1996). This provided strong motivation to use this multi-
meristem culture system for the production of transgenic oat plants (Cho et al., 2003;
Maqbool et al., 2002; Zhang et al., 1999). Cho et al. (2003) used a similar technique to
study the expression of green ﬂuorescent protein (GFP) and its inheritance in transgenic
oat. In vitro shoot meristematic cultures induced from shoot apices of germinating mature
seeds were used as an explant source. Strong GFP expression was observed in tissues of
T0 plants and their progeny, providing further evidence to support the applicability of the
multi-meristematic cultures in successful genetic transformation.

Transformation of oat via particle bombardment also was used to investigate
transgene integration, rearrangements, and silencing. Pawlowski and Somers (1998)
characterized transgene loci in some transgenic oat lines produced by microprojectile
bombardment. The structure of the transgenic loci indicated that the transgenes were
interspersed within the genomic DNA. Later, Svitashev et al. (2000) found that different
locations hosted the transgene integration with different levels of structural complexity.
Two transgenic oat lines that exhibited simple transgene loci determined by southern blot
were used to completely sequence those loci (Makarevitch et al., 2003). These results
indicate that transgene integration and locus formation may result from chromosomal
break, rearrangements and repair mechanisms (Svitashev et al., 2002). Moreover,
Pawlowski et al. (1998) revealed irregular unpredictable patterns of transgene silencing in
expression and inheritance studies following oat transformation with bar and gus genes.

Distorted segregation due to silencing was observed in some oat transgenic lines. They

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suggested that oat may have a prevention mechanism for unwanted gene expression and
as few as two copies of the transgene may be enough to cause silencing.

The development and application of particle bombardment technologies
represented a signiﬁcant breakthrough in plant biotechnology (Altpeter et al., 2005). As a
result, it became possible to efficiently produce genetically engineered plants in a wide
range of crop species. The use of particle bombardment changed as enhanced,
Agrobacterium transformation systems are developed for cereals and other crops. Yet,
this direct gene transfer technology will continue to be an important tool, providing an

avenue for genetic engineering in different ﬁelds, including abiotic stress tolerance.

Use of Stress Tolerance Genes in Transgenic Plants

Oat, like other crops, is affected by the major abiotic stresses of drought and
salinity. To help develop varieties more tolerant to these stresses, abiotic stress tolerance
genes can be introduced via genetic engineering. Several promising candidate genes have
been identiﬁed and used for that purpose. A ﬁrll list of genes used for stress tolerance in
transgenic plants can be obtained from the plant stress website
(http://www.plantstress.com/biotech/index.asp?Flag=1). Among those are the genes
encoding Late Embryogenesis Abundant (LEA) proteins, which are ubiquitous in plants.
These proteins accumulate during the late stage of seed formation and in vegetative
tissues under drought, heat, cold and salt stress conditions or with abscisic acid (ABA)
application (Sivamani et al., 2000). LEA proteins are generally divided into ﬁve groups
and they appear to protect cellular structures from dehydration stress; however, the exact

functional role of these hydrophilic proteins remains poorly understood. Proposed roles

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include water binding or replacement, hydration buffers (Dure, 1993a; Ingram and
Bartles, 1996), ion sequestration (Dure, 1993b), osmotic adjustment or reverse
chaperones (Close, 1996), and transport of nuclear-targeted proteins during stress (Goday
et al., 1994).

HVAl, a barley group III LEA protein, is highly induced by ABA application and
other abiotic stresses. The HVAI gene (Hong et al., 1992) has been used successfully to
confer stable tolerance to osmotic stress in rice (Xu et al., 1996) and wheat (Patnaik and
Khurana, 2003). The promising successes of the abiotic stress genes, and HVAI in
particular, motivated the development of genetically engineered oat plants with the
internal capacity to withstand salinity (Maqbool et al., 2002). The following section
describes a study on salinity tolerance of the third generation of transgenic oat expressing

H VAI gene under greenhouse conditions.

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MATERIALS AND METHODS

Plant Materials

R3 seeds from ﬁve independent transgenic oat lines of the cultivar Ogle (BRA-8,
BRA-17, BRA-19, BRA-41 and BRA-82) generated in a previous study (Maqbool et al.,
2002) were used in this study. Transgenic lines were produced using a multimeristem
transformation system with two plasmids, pBY520 and pActl-D. The plasmid BY520
contained the linked selectable marker/herbicide resistance bar (Phosphinothricin acetyl
transferase) gene (driven by cauliﬂower mosaic virus 35$ promoter and the nopaline
synthase nos terminator) and the barley H VA] gene (driven by rice Act! promoter and
terminated by the potato protease inhibitor pin II). The plasmid Actl-D contained the
non-linked Escherichia coli -glucuronidase (gus) gene ﬂanked by the Act] promoter and
the nos terminator.

The lines chosen for this work had one copy of the linked H VA I-bar genes.
Bagged ﬂowers of some R0, R1 and R2 progenies were allowed to self-pollinate and
produce seeds which were collected in bulk from each line. Bulked R3 transgenic and
control (Ogle wild type cultivar) seeds were germinated; plants of R3 generation were

grown and evaluated in two greenhouses under salt treatments.

Segregation of Herbicide Resistance of R3 Progeny

Seeds were surface-sterilized with 70% ethanol for 2 minutes, followed by 20%
(v/v) commercial bleach (5.25% sodium hypochlorite, Clorox professional Products
Company, Oakland, Calif, USA) treatment for 10 minutes, rinsed with sterile distilled

water several times, and brieﬂy blotted onto sterile ﬁlter paper. Transgenic and non-

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transgenic control seeds were germinated on MS (Murashige and Skoog, 1962) basal
medium containing 15 mg/L glufosinate ammonium for selection. Control seeds were
also germinated on the same medium lacking herbicide. Cultures were maintained under
continuous ﬂuorescent light at 28° C for one week. The seedlings that survived the

selection were used for molecular and phenotypic characterization of the lines.

Salinity Treatments

Young seedlings were transferred into Baccto Professional Planting Soil Mix (70-
80 % horticultural Sphagnum peat, 20-30 % perlite, pH 5.5-6.5) in small pots (8x4x6 cm),
one plant per pot. The pots were kept in water-ﬁlled ﬂat-bottom trays for one week. Two-
week-old seedlings from each transgenic line and the control were transferred to two-
gallon pots for another week before the salt treatments. The environmental settings in the
greenhouses were maintained between 21°C and 25° C, with a relative humidity between
80% and 90% using a controlled heating and venting system. Natural illumination was
augmented for 16 h per day with ﬂuorescent light and light levels were between 150 and
185 pmol m'zs'l. The soil surface around the seedlings was covered with redwood bark
mulch to minimize the evaporation and prevent algae growth. A dilution of commercial
20: 20: 20 fertilizer solution was added twice a week for nutritional needs. Sodium
chloride (NaCl) was added in ﬁve concentrations of 0, 50, 100, 150, and 200 mM. Plants
were watered with the saline solution once per day ensuring adequate leaching, and
preventing salinity excess. Plants were given the salinity treatments for 14 days, followed
by one week of irrigation with plain water to measure plant recovery after salt stress.

Aﬁerwards, the salt treatments were resumed uninterrupted for 35 days.

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PCR Analysis

The detection of the bar and the HVA] genes in all the studied R3 transgenic
plants by PCR ampliﬁcation was carried out using leaf disk DNA as template and
REDExtract-N-AmpTM Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, Cat # XNA-P) as
per the manufacturer’s instruction using the following primers: bar F, 5’-ATG AGC
CCA GAA CGA CG-3’ (forward primer); bar R, 5’-TCA GAT crc GGT GAC GG-3’
(reverse primer) and H VA] F, 5’-TGG CCT CCA ACC AGA ACC AG-3’ (forward
primer); H VAI R, 5’-ACG ACT AAA GGA ACG GAA AT-3’ (reverse primer). DNA
ampliﬁcations were performed in a thermo cycler (Perkin Elmer/Applied Biosystem,
Foster City, CA) using initial denaturation at 94°C for 4 min, followed by 35 cycles of 1
min at 94°C, 1 min at 55°C, 2 min at 72°C, and a ﬁnal 10 minute extension at 72°C. The
reaction mixture was loaded directly onto a 0.8 % (w/v) agarose gel, stained with
ethidium bromide and visualized with UV light. The transgene product size was about

0.59 kb for the bar gene and 0.70 kb for the H VAI gene.

Histochemical Analysis of GUS

Seeds, seed husks root segments and leaf tissue cells from the transgenic and non-
transgenic plants were used to detect GUS activity by histochemical staining using 5-
bromo-4-chloro-3-indoyl-B-D-glucuronicacid salt (X-gluc). Samples were immersed in
GUS substrate mixture and incubated at 37°C (Jefferson et al., 1987). The tissues were

thoroughly washed with 70% ethanol and examined under a Zeiss SV8 stereomicroscope.

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DNA Isolation and Southern Blot Hybridization Analysis

Independence of the lines and conﬁrmation of the H VAI transgene transmission
into the oat R3 transgenic plants were performed by Southem-blot hybridization using the
HVA I-coding sequence as a probe. Genomic DNA from transgenic and non-transgenic
oat plants was isolated using the protocol of Saghai-Maroof et al. (1984). For Southern
blots, 10-15 pg of genomic DNA was digested with HindIII restriction enzyme,
electr0phoresed in 0.8% (w/v) agarose gel, transferred onto Hybond-N+ (Amersham-
Pharmacia Biotech) membranes, and ﬁxed with a UV crosslinker (Stratalinker UV
Crosslinker 1800, Stratagene, CA) as recommended in the manufacturers’ instructions.
The H VAI gene-speciﬁc probe was generated using a HindIII-BamHI digest of pBY520
to isolate a 1.0-kb fragment. The restriction fragment was puriﬁed using the QIAquick kit
(QIAGEN). Probe labeling and detection were obtained using the DIGHigh Prime DNA
Labeling and Detection Starter Kit 11 (Kit for chemiluminescent detection with CSPD,

Roche Co.) following the manufacturer’s protocol.

RNA Isolation and Northern Blot Hybridization Analysis

Assay for transcriptional expression of the H VA 1 transgene was performed by
Northern blots. Total RNA was isolated from young leaves of oat plants (transgenic and
non-transgenic) using the TRI Reagent (Sigma-Aldrich, St. Louis, MO) according to the
manufacturer's instructions. For the Northern blot, 20 pg of RNA were separated in a
1.2% (w/v) agarose-formaldehyde denaturing gels according to Sambrook et al. (1989)
and blotted onto Hybond-N+ nylon membranes (Amersham-Pharmacia Biotech).

Transcripts of HVA] were analyzed with a standard Northem-blotting method (Sambrook

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et al., 1989) using the H VA I-coding sequence as a probe labeled with a- [32P]-dCTP
using the Random Primer Labeling Kit (Invitrogen, Carlsbad, California) according to the

manufacturer's instructions.

Measurements of Parameters

Data were collected on kernel yield and its components (number of tillers/plant,
number of kernels/panicle, 1000-kernel weight). Additional measurements were taken on
number of days to heading, plant height, ﬂag leaf area, root length, panicle length and
number of spikelets/panicle during the experiment for each plant in the ﬁve replications
under each salinity concentration. The whole experiment was also repeated at the same
time in two greenhouses. Number of days to heading was recorded when panicles were
extruded from the ﬂag leaf sheath. Plant height was measured from the soil surface to the
top of the main panicle at physiological maturity. The area of each individual fully
expanded ﬂag leaf blade was computed as length x maximum width x 0.75. It was
assumed that length and width did not change after ﬁill expansion (Elings, 2000). Stems
were counted on each plant at the 6 to 7 leaf stage. The number of spikelets and panicle
length were counted and measured at maturity before harvest. Plants were harvested at
full maturity. Shoots were removed from roots at the soil surface. The soil was carefully
washed from the roots and the root length was measured. The number of kernels/panicle
was determined by counting kernels on every panicle for each plant after harvest. Mean
kernel weight was calculated from the weight of three sets of 300 kernels. Kernel

yield/plant was determined on the basis of the harvested plants.

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Statistical Analyses

Chi square (12) analysis, using the correction factor of Yates (Steel and Torrie,
1980), was performed to determine if the observed segregation ratio of the third
generation was consistent with the expected ratio of a pair of hemizygous alleles. To
study the effects of salinity, the experimental design was a split plot with salinity as a
whole-plot factor and lines as a sub-plot factor. The experiment was conducted in two
greenhouses with ﬁve replications (blocks) in each greenhouse. In each block, pots of
each line (one plant/pot) were assigned at random to receive certain salinity level. Then,
the pots were arranged in the whole plot groups with the same salinity level for each
group. The greenhouses and blocks were considered random factors. The statistical
analysis for quantitative traits was performed using PROC MIXED (SAS Institute Inc.,
2003). The signiﬁcance of the interaction between genotype and salinity was tested and

the line means were compared at each salinity level using t-tests.

Images

Images in this thesis/dissertation are presented in color.

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RESULTS AND DISCUSSION

Here, the third generation of ﬁve independent transgenic lines obtained from a
previous study (Maqbool et al., 2002) was tested for their salinity tolerance under
greenhouse conditions. The independence of the lines was conﬁrmed by Southern blot
analysis. The molecular analyses, segregation for herbicide resistance, GUS
histochemical assay, and salinity treatment were used to investigate transgene integration

and transcription, and translation in the R3 progeny of the transgenic plants.

Segregation of Herbicide Resistance of R3 Progeny

The transmission of the linked bar and HVA] genes was ﬁrst observed by
germinating a total of 100 seeds from each of the ﬁve lines of R3 progeny and the control
in the presence of a high concentration of herbicide (15 mg/L glufosinate ammonium) in
vitro. The seedlings of the transgenic lines grew in the selection medium as vigorously as
in the absence of the herbicide (Figure 3.1). No seeds of the non-transgenic control
germinated on the same selection medium. The segregation ratio of the bar gene
(glufosinate ammonium resistance) was not signiﬁcantly different from 9:7, the
segregation ratio for one pair of hemizygous alleles in the third transgenic generation
(Table 3.1). The results of the PCR analysis (Figure 3.2) were found to be consistent with
that observed in the germination test conﬁrming the existence of the intact linked bar and
H VAI genes in the R3 progeny. The herbicide-resistant progeny of the transgenic plants

were used in the subsequent molecular and agronomic analyses.

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Histochemical Analysis of GUS

GUS expression pattern in R3 transgenic seeds and seed husks were visualized by
histochemical staining with X-Gluc (Jefferson et al., 1987). GUS staining was observed
also in younger root segments and leaf tissue cells for all of the ﬁve lines with no
expression in the control plants (Figure 3.3). The results revealed constitutive expression
of the gas gene in tissues of transgenic oat and indicated successﬁrl inheritance of the

gene to the R3 progeny.

Southern Blot Analysis of the HVA] Gene in R3 Progeny Plants

Southern blot analysis was used to investigate the stable incorporation,
independence of the transgenic lines, and transmission of the H VAI gene in the R3
progeny (Figure 3.4a). The plasmid (Lane P), transgenic lines (Lanes 1—5) and non-
transgenic control of Ogle cultivar (Lane C) DNA were included in the blot. When
hybridized with the H VAI gene probe, the non-transforrned plants did not show any
hybridization band. The plasmid and the genomic DNAs of the ﬁve transgenic lines that
hybridized with the same probe showed bands of different sizes larger than 3.67 (Figure
3.4a) as expected. Line BRA-19 showed a band size of about 3.9 kb which is smaller
than its size in R0 (Maqbool et al., 2002). This could be explained by possible DNA
rearrangements resulting from deletion, translocation or crossing-over on the genomic
DNA downstream of the two transgenes cassettes. These results provide evidence that the
H VAI gene has remained stably integrated in the plant genome with ﬁve independence

events, and transmitted into the genomic DNA of the R3 progeny.

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Table 3.1. Segregation of herbicide resistance of R3 transgenic Ogle cultivar.

 

 

 

 

 

 

Line Gesrérércirged Gerrljricirtat ed x21: P-value
BRA-82 57 43 0.0240 0.8769
BRA-17 60 40 0.5783 0.4470
BRA-8 62 38 1.3434 0.2464
BRA-19 60 40 0.5783 0.4470
BRA-41 61 39 0.9168 0.3383

 

 

 

 

 

 

 

TSeeds bulked for each line through the R3 generation were germinated on MS basal
medium containing 15 mg/L glufosinate ammonium.

1N0 signiﬁcant difference between the observed versus the 9:7 expected segregation ratio
in all the lines (P>0.05)

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12345678 123 4 5 6 7 8

a b

Figure 3.2. PCR ampliﬁcation of (a) the bar (0.59 kb) and (b) H VA] (0.7 kb) genes
shows the presence of the transgenes in R3 for the 5 lines. Lane 1: 100 bp Ladder
marker, Lane 2: Plasmid (positive control), Lane 3: Non-transformed (negative
control), Lanes 4-8: Transgenic lines.

     

a b c (I

Figure 3.3. GUS expression in R3 transgenic oat seed husks (a), seeds (b) root
segments (c) and leaf tissue cells ((1)

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Northern Blot Analysis of the HVA] Gene in R3 Progeny Plants

Northern analysis was used to assay the transcription of the H VAI regulated by
the rice Act] constitutive promoter, whose expression were associated with differences in
salinity tolerance between the transgenic lines and the control plants (Figure 3.4b). RNA
isolated from young leaves of the transgenic lines was probed with the H VAI gene. A
transcript of the expected size (approximately lkb) for this gene was detected, indicating
that R3 progeny of transgenic oat lines inherited the transcriptionally-active HVA] gene
(Lanes 1-5). As a control in the RNA blotting experiment, RNA ﬁ'om non-transformed
oat leaves was included on the blot. The assay showed that the H VA] transcript was not

detectable in the control plants (lane 6).

Salinity Effects on Plant Growth, Yield and Its Components

NaCl is a common salt that negatively inﬂuences plant grth under natural
conditions. NaCl solution was used in this study, although single salt solutions do not
exist in nature (Bernstein, 1962).

The analysis of variance of the salinity levels, genotypes (lines and control) and
their interaction is displayed in Table 3.2. Greenhouses did not have a signiﬁcant effect
on all the studied traits. Signiﬁcant differences were observed among the salinity levels
and genotypes for all the traits. Although the magnitude of the interaction between
salinity and genotypes is very small compared with the main effects of the treatments, the
ANOVA mean squares revealed signiﬁcant genotype x environment interaction (P<
0.05) for all the studied characteristics. This signiﬁcant interaction arises from the

differential genotypic responses to different salinity levels during the plant life cycle.

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The magnitude of the main effect mean squares suggested that the number of days
to heading, plant height, panicle length, root length, number of kernels/panicle, 1000-
kemel weight and kernel yield were inﬂuenced by salinity more than genotype as it
showed between 20 to 85 % of the total variation. Flag leaf area, number of
spikelets/panicle and number of tillers/plant appear to be almost equally affected by
salinity levels and genotypes, which explained more than 40 % of the total variation
particularly for these traits. These results indicate that the chosen salinity levels were very
efﬁcient in imposing the stress.

Although transgenic plants showed better recovery and grew faster than control
plants aﬁer the salt-stress recovery period and also maintained more tolerance to salinity
during and after salt treatments, higher salinity levels (150 and 200 mM) signiﬁcantly
reduced growth of both transgenic and control plants (Tables 3.3 and Figure 3.5). The
differences in stress treatments revealed a progressive decrease in average number of
days to heading, plant height, ﬂag leaf area and root length (Figure 3.5a-d).

Early heading is one of the mechanisms that plants use to escape the damage
effects caused by salinity stress (Bajji et al., 2004). In this study, mean days to heading
decreased as salinity level increased, although this decrease did not vary among the
genotypes (Figure 3.5a). Most of the transgenic lines headed signiﬁcantly later than the
control at early high salinity stress (> 50 mM NaCl). This delay in ﬂowering gives the
opportunity of late differentiation, which subsequently enables the plant to maintain

higher number of kernels/panicle (Blum et al., 1990).

-86-

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-88-

The plant height, ﬂag leaf area and root growth parameters may serve as yield
attributes (Ulery et al., 1998). The height was not signiﬁcantly different among genotypes
at 0 and 50 mM NaCl salinity. The transgenic lines were able to retain their height until
100 mM compared with control. The control plants tended to be shorter under salinity
and the shortest height for most genotypes was obtained at 200 mM NaCl (Figure 3.5b).
Similar ﬁndings were reported by Xu et al. (1996) in rice. The minimum response of
plant height to changes in salinity levels of transgenic lines may result in the ability of the
plants to preserve current assimilates as a source of carbon stored in the stem for grain
ﬁlling (Blum, 1998). In addition, this performance may improve the response to nitrogen
fertilization under salt stress conditions.

The plant ability to maintain large leaves and delay leaf senescence under water
limitation stresses is a stress tolerance mechanism called ‘non-senescence’ or ‘stay-green’
(Rosenow, 1977; Thomas and Smart, 1993). One of the most important points is whether
or not retaining a green and large leaf area under salt stress increases kernel yield. Helsel
and Prey (1978) reported a positive correlation between leaf area duration and kernel
yield in the oat crop. Leaves of transgenic lines were normal and displayed no symptoms
of wilting at 50 mM salinity. The genotypic differences under salinity-induced changes in
the ﬂag leaf area were most prominent at 100 mM and above concentrations. The ﬂag
leaf area was more severely reduced by salt stress in the control plants than the transgenic
lines (Figure 3.5c). The data presented in Table 3.3 conﬁrmed that Line BRA-41, which
retained the highest ﬂag leaf area (Figure 3.50), maintained the highest yield among the
different salinity levels. This probably resulted from increased photosynthesis when

kernel ﬁlling occurred under stress (Blum, 1983; Van Oosterom and Acevedo, 1992).

-39-

The length of oat roots was correlated with the tolerance of juvenile plants to
water stress in the ﬁeld (Larsson and Gomy 1988). Root length of the control was not
different ﬁ'om the lines in the salinity control treatment (0 mM) (Figure 3.5d). As salt
concentration increased, root length of all transgenic lines increased relative to the
control. However, when salt concentrations increased beyond 100 mM, root growth
declined. Since only the ﬁnal root length of each line and the control was measured, no
differences in root grth were detected between the lines but there is a signiﬁcant
difference between the lines and the control at 50 mM and above (Figure 3.5d) (Sivamani
et al., 2000).

The effects of salinity levels on the panicle traits are shown in Figure 3.5e and f.
In general, panicle length (Figure 3.5e) and number of spikelets/panicle (Figure 3.50
were reduced by salinity levels 50 mM and higher. While plants of transgenic lines
maintained high panicle length and number of spikelets/panicle among the salinity levels,
salinity treatments at 100 mM and 50 mM NaCl resulted in signiﬁcant differences in
panicle length and number of spikelets/panicle respectively, compared with the control.
The panicle length and number of spikelets/panicle were reduced of approximately 23-52
% and 25-77 % at 200 mM NaCl respectively (Figure 3.5e and f). The panicle length of
control plants was 50 % shorter than the best transgenic line. The lack of panicle length
extension under salinity might have contributed to plant height reduction (Milach et al.,
2002). Moreover, Giunta et al. (1993) reported that, in wheat, severe water deﬁcit around
anthesis produces serious effects on yield, reducing the number spikelets and therefore

causing a decrease in plant fertility.

-90-

It was reported that variation in kernel yield potentials in cereals under water
stress growing conditions were predominantly associated with variations and sequential
development of yield components (Fischer and Maurer, 1978; Garcia del Moral et al.,
1991, 2003; Simane et al., 1993). Kernel yield was greater under salinity stress for the
transgenic plants than the control. The higher yield was due to increased number of
tillers, number of kernels/panicle and heavier kernels (Table 3.3). Salinity stress at 200
mM NaCl for a long period of time caused reduction in kernel yield estimated at > 40 %
for transgenic lines and 90 % for the control plants. Under the conditions of this study
(Table 3.3), number of kernels/panicle was the most sensitive yield component affected
by salinity stress (reduced by > 30 and 60%) followed by 1000-kernel weight (reduced by
> 26 and 50% for the transgenic lines and the control respectively). The number of
tillers/plant was less affected by salinity for the transgenic lines (reduction of 10-20 %)
but the inﬂuence of salinity was higher on the control plants (> 60 % reduction). Among
the transgenic lines, the line BRA-41 expressed the highest yield under severe salinity
stress (200 mM) due to its lower reduction in number of tillers/plant, 1000-kernel weight
and number of kernels/panicle (Table 3.3). Evans and Wardlaw (1976) explained yield
component compensation as the allowance of subsequently occurring components of ﬁnal
kernel yield to compensate for restrictions and/or losses during earlier stages of
development, or to maximize reproductive growth in the plant life cycle.

Based on the transgenic line observations, the results indicate that number of
tillers/plant may have a negative effect on number of kernels/panicle and kernel weight
under salinity stress. The compensatory effect between tiller production and the other

components may have resulted from the negative allometry between these traits during

-91-

plant development (Hamid and Grafius, 1978). Moreover, it is well documented that
under stress environments compensation could be mainly achieved by extensive tillering
of surviving plants (Holena et al., 2001).

Overall, transgenic plants of the R3 generation developed normal ﬂowers, grew to
maturity and set seeds in a normal manner under greenhouse conditions, suggesting that
expression of the linked bar- HVA] and non-linked gus genes had no deleterious effects
on growth and fertility.

The correlation between accumulation of LEA group III proteins and stress
tolerance is well studied in wheat and rice (Ried and Simmons, 1993; Rohila et al., 2002;
Sivamani et al. 2000; Wise, 2003). Moreover, it is evident that genetic variability exists
for stress responses and this could be due to the differential expression and regulation of
stress responsive genes such as H VAI gene when the plants are exposed to stress
(Jayaprakash et al., 1998; Uma et al., 1995). Based on our experimental results, the
positive signiﬁcant relationship between the present increase in grth of the transgenic
lines under stress over controls and the presence of H VAI transcripts suggest more
evidence that constitutive expression of H VAI gene in transgenic plants can improve
growth performance under salinity stress conditions; however, the exact function of LEA

proteins remains uncertain.

-92-

 

 

a) Number of days to heading b) Plant height

 

   

 

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60
.E
'0 53 .__-...- - .
8 52 ’1 +BRA-82 2°
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9 51 ' ' BRA-17 E
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.3 g i - BRA19 f 5°
“5 47 t +3RA-41 g 40
g 46 [ +cm 9- 20
E 45 . . o .
2 0 so 100 150 200 0 so 100 150 200
NaCl Concentrations (mM) NaCl Concentrations (mM)
e) Flag leaf area d) Root length
60 50
8 50‘ . ”I :
a ,0! E
c.- r: m.
M D
3 3° 2 2°
aozo 8
.5 M 10
L3,, 10
o g . . r—O—corlrol o . . .
o 5° 10° 150 200 0 so 100 150 200 +°°'"°'
NaCl Concentrations (mM) NaCl Concentrations (mM)
9) Pmicle length 0 1) Number of spikelets/panicle
E
m + gage: a m - +BRA-32
gal 7- BRA-17 21 5° +BRA-17
E 20 o 40-
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0 r r f +Control 3 o- . .
0 so 100 150 200 g a so 100 150 200
. Z .
NaCl Concentrations (mM) NaCl Concentrations (mM)

 

 

 

 

 

Figure 3.5. The effect of increasing NaCl concentrations on number of days to
heading (a), plant height (b), flag leaf area (c), root length (d), panicle length (e) and
number of spikelets/panicle (l) for the transgenic lines and the non-transgenic
control.

93

Table 3.3. Differences between the R3 transgenic lines and the non-transgenic
control under different salinity levels for the number of tillers/plant, number of

kernels/panicle, 1000-kernel weight and kernel yield/plant.

 

 

Traits
Salinity level Line Number of Number of 1000-kemel Kernel
tillers/plant kemels/ weight (gm) yield/plant
panicle (gm)
0 BRA-82 5.70 a'l' 74.00 a 33.48 a,b 12.10 a
BRA-17 5.50 a 92.00 c 34.56 a,b 15.44 c
BRA-8 5.90 a 90.30 b,c 32.88 a 16.05 c
BRA-l9 5.20 a 75.00 a 33.21 a,b 11.75 a
BRA-41 5.20 a 94.90 d 34.64 a,b 14.31 b
Control 5.90 a 89.00 b 35.19 b 16.93 (I
50 BRA-82 5.30 b 70.10 b 31.33 b 10.29 b
BRA-17 5.30 b 92.00 f 31.51 b 14.13 d
BRA-8 5.90 b 85.30 d 31.90 b 14.45 d
BRA-19 5.90 b 73.00 c 32.70 b,c 11.74 c
BRA-41 5.20 a,b 90.00 e 34.58 c 13.95 d
Control 4.30 a 66.90 a 26.84 a 7.26 a
100 BRA-82 5.20 b 69.00 b 31.06 b 9.19 b
BRA-17 5.40b 90.00d 31.11 b 13.66d
BRA-8 5.60 b 82.70 c 31.87 b,c 13.52 (1
BRA-l9 5.40 b 70.00 b 32.64 b,c 10.59 c
BRA-41 5.30 a,b 91.00 d 33.47 c 13.73 d
Control 3.80 a 54.00 a 24.20 a 3.75 a
150 BRA-82 4.80 b 64.00 b 27.48 c 7.64 b
BRA-l7 4.50 b 72.00 c 25.41 b 10.04 (1
BRA-8 4.10 b 79.90 d 25.77 b,c 9.82 c,d
BRA-l9 4.10 b 65.00 b 26.46 b,c 8.72 c,d
BRA-41 4.70 b 79.00 d 27.46 b,c 9.23 c
Control 2.84 a 37.17 a 19.85 a 2.01 a
200 BRA-82 4.50 b 50.00 b 22.88 b 5.96 c
BRA-l7 4.90 b 54.00 c 21.49 b 5.90 d
BRA-8 4.80 b 50.40 b 22.50 b 5.14 b
BRA-19 4.50 b 51.00 b 23.31 b 6.85 d
BRA—41 4.70 b 60.00 d 25.52 c 8.37 e
Control 1.83 a 28.79 a 16.78 a 1.11 a
Standard error 0.49 0.79 0.76 0.32

 

Tln the same column under each salinity level, numbers followed by the same letter are not
signiﬁcantly different (P > 0.05).

-94-

SUMMARY

Cultivated oat (Avena sativa L.) is an important cereal crop for humans and
animals as well. A major cause of oat crop yield loss worldwide is osmotic stress due to
drought and/or salinity. This study investigated the third generation of transgenic oat
(A vena sativa L.) expressing barley H VAI stress tolerance, B-glucuronidase (uidA; gas)
and bar herbicide resistance genes. Transgenic plants showed 9:7 third generation
inheritance for glufosinate ammonium herbicide resistance. Molecular (PCR, Southern
and Northern blots) and histochemical GUS studies conﬁrmed the presence and stable
expression of all three genes. The transgenic plants developed normal seeds under
greenhouse and salinity conditions conﬁrming that there is no deleterious effect of the
transgenes on the plants’ fertility. Compared with the non-transgenic control plants,
transgenic R3 plants exhibited greater growth and showed signiﬁcant (P< 0. 05) increase
in tolerance to high salt stress conditions (200 mM NaCl) for important agronomic traits
including number of days to heading, plant height, ﬂag leaf area, root length, panicle
length, number of spikelets/panicle, number of tillers/plant, number of kernels/panicle,
thousand kernel weight and kernel yield/plant. For the transgenic plants and among the
yield components, number of kernels/panicle was the most and number of tillers/plant
was the least affected by salinity stress. Line BRA-41 experienced the least .decrease in
performance through the salinity levels for most of the studied traits (Figure 3.6 and 3.7).
The estimated reduction of oat yield-loss was about 50 % under continuous high salinity

conditions.

-95-

_.

ill
a

 

Figure 3.7. R3 transgenic (a) and non-transgenic (b) oat roots at 100 mM NaCl
stress.

-96-

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CHAPTER IV

ENHANCED CONVERSION OF BIOMASS CELLULOSE INTO GLUCOSE

USING TRAN SGENIC RICE-PRODUCED CELLULASE

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INTRODUCTION

This chapter will begin with a review of literature on the use of plants as
biofactories (molecular farming), the promise of biofuel ethanol and rice as a bio-based
energy crop candidate, and the in vitro culture and genetic transformation of rice. Since
it is well known that the United States is the biggest oil consumer in the world and the
gap between US oil production and imports is increasing dramatically, it is a must now to
seek for alternatives. One of those alternatives that show a strong potential is ethanol ﬁiel.
The vast majority of the ethanol is produced from maize seeds and from sugarcane,
which are currently used to fulﬁll human needs with food, starch and sugar. Therefore, it
is recommended to use biomass crops for ethanol production. For ethanol to be produced
commercially from plant biomass sources, enzymatic hydrolysis of cellulose to
fermentable sugars is employed. At present, these enzymes are expensively produced in
large-scale fermentation tanks. In the US biomass inventory, rice straw is one of the most
abundant sources for lignocellulosic materials. Habitually, farmers throughout the world
burn rice ﬁelds after harvest to control diseases and insects. This burning gives rise to a
lot of health concerns. Therefore, engineered rice plants that can sustainably, efficiently
and actively produce the desired hydrolysis enzymes internally, could be recommended
for better use of the biomass while reducing air pollution. The ability to produce
heterologous protein (a cellulase enzyme) in fertile transgenic rice is the basis for the
research presented in this chapter. This research studies an enhanced conversion of

biomass cellulose into glucose using transgenic rice-produced cellulase.

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The objectives were to:
0 Produce Acidothermus cellulolyticus endo-1,4-B-glucanase enzyme in transgenic
rice.
0 Verify the expression and stability of transmission of the E1 gene to the progeny.
0 Investigate the amount of the enzyme in the plant total soluble proteins.
0 Test the localization of the enzyme in the apoplast where it is targeted and stored.
0 Test the enzyme activity in conversion of chemical and biomass cellulose to

glucose.

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REVIEW OF LITERATURE

Plants as Biofactories (Molecular Farming)

Molecular farming is a term that describes growing, harvesting and using
genetically modiﬁed crops as a valuable source for production of pharmaceuticals (drugs
and vaccines) and industrial compounds (enzymes and biodegradable plastics) instead of
food, feed, or ﬁber. The idea is to use these crops as biofactories to generate those
biomolecules that are difficult or expensive to produce other ways (Horn et al., 2004).
This technique was recently used to produce different protein products such as bovine
trypsin enzyme, which is widely involved in digesting or processing other proteins,
including some therapeutic proteins (Woodard et al., 2003). The use of higher plants as
biofactories for protein production has many advantages that include:

0 Signiﬁcant reduction of the production costs compared to microbial fermentation
and mammalian cell lines.

o The availability of advanced experiences in plant production practices.

0 No contamination of the ﬁnal product, especially since plants contain no known
human pathogens such as virions.

- High activity proteins usually can be produced in plants using eukaryotic genes
because of plant’s ability to enhance correct protein folding.

- With the advances in genetic engineering, it is possible now to target the protein
products for storage in different plant compartments to minimize lethal effects to

the plant and increase stability (Horn et al., 2004).

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Currently, different methods of protein production in plants include:
0 Transient and stable nuclear and plastid transformation of crop species with plants
grown in the greenhouse and/or the ﬁeld.
0 Stable transformation of a plant species that is grown hydroponically for the
secretion and recovery of transproteins.
More details about the economics, processing and regulatory constraints related to these
systems were reported by Nikolov and Hammes (2002).

In spite of the feasibility of using plants as a viable system for pharmaceutical
production, very few products have reached the market. The ﬁrst pharrnaceutically
relevant protein made in plants was human grth hormone (nopaline synthase enzyme),
which was expressed in transgenic tobacco in 1986 (Barta et al., 1986). A few years later,
functional glycoproteins and hepatitis B virus (HBV) surface antigen were assembled and
expressed in tobacco (Hiatt et al., 1989; Mason et al., 1992). Hood et al. (1997) reported
the ﬁrst processing, extraction, puriﬁcation and commercial production of avidin protein
in transgenic maize. Avidin is a protein found in egg white, which binds biotin (a B-
complex vitamin that is required in humans) and prevents its absorption. Since then,
research with transgenic plants continues to offer the promise of large-scale production of
safe, pure and highly efﬁcacious therapeutic molecules in an increasingly diverse range
of crop species (Ma et al., 2003).

In fact the choice of plant species to be used as the production system is critical.
Most plant-derived heterologous proteins were produced and recovered from tobacco
leaves because of its high biomass yields and simplicity, and the well established

protocols available for its transformation (Fischer and Emans, 2000). Furthermore, it is

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less contaminating for animal feed and human food chains or recombinant proteins as a
non-food/feed crop. However, tobacco has very high nicotine content and toxic alkaloids,
which must be completely removed in one of the process’s steps. Although low-alkaloid
and nicotine tobacco varieties exist, interest has moved towards genetically engineering
other crops for pharmaceutical and industrial production (Fisher et al., 2004). For
example, lettuce has been used for the production of hepatitis B human vaccine (Kapusta
et al., 1999), alfalfa for the poly-B-hydroxybutyrate biodegradable plastic polymer
(Saruul et al., 2002) and maize seeds for the laccase enzyme involved in the biomass

conversion processof ethanol biofuel production (Bailey et al., 2004).

Biofuel Ethanol

Actually, the ﬁre] ethanol industry has been growing extensively worldwide,
including in the US, Brazil and European Union countries (Ragauskas et al., 2006), and
considerable efforts have been exerted towards improving ethanol yield and reducing its
production costs during the last two decades (Lynd et al., 2005). A vision to enhance US.
economic security has set a target of using plant-derived materials to meet 10% of
chemical feedstock demand by 2020—at least a ﬁvefold increase (Singh et al., 2003). In
2004, US. ethanol production capacity reached 3.4 billion gallons (Farrell et al., 2006),
about 600 million gallons more than 2003 (Bothast and Schlicher, 2005). Another
production increase of more than 1.6 billion gallons is projected for 2012 (Bothast and
Schlicher, 2005). Until now, the vast majority of US. ethanol has been produced from
maize seeds, while Brazil produces similar quantities of ethanol from sugar derived from

cane (Oliveira et al., 2005). The economic and environmental performance of maize seed

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and sugar cane ethanol would likely be improved by producing ethanol from
lignocellulosic materials. Approximately 1.3 billion tons of crop and forest residues and
energy crops are thought to be available in the United States with proper management
(Kim and Dale, 2004). The energy value of this much lignocellulosic biomass is roughly
equivalent to 3.5 billion barrels of petroleum per year; the total amount of petroleum
produced in the United States in its peak production year—1972 (Perlack et al., 2005).
Worldwide, over 1.7 billion tons of crop residues are available annually and nearly half
of this total is rice straw (Kim and Dale, 2004), which mostly could be processed as fuels
and chemicals. A recent comprehensive study prepared under the leadership of the
Natural Resources Defense Council highlights the potential economic and environmental
beneﬁts of very large scale conversion of lignocellulosic biomass to ethanol and other
fuels (Greene et al., 2004).

For ethanol to be produced ﬁ'om plant biomass sources, enzymatic hydrolysis of
cellulose to fermentable sugars is employed (Kabel et al., 2005) using microbial
hydrolysis enzymes (Ragauskas et al., 2006). These enzymes are produced in large-scale
deep fermentation tanks (Knauf and Moniruzzaman, 2004). Although the cost of
fermentation tank enzyme production was reduced by a factor of four from 1980 to 1999
(Wyman, 1999) and by another 10 fold since 2000 (Knauf and Moniruzzaman, 2004), it
still represents about $0.30-0.50 per gallon of ethanol produced—a major cost factor
(Bothast and Schlicher, 2005). However, cost reduction might be achieved by
engineering crops that can sustainably and actively self-produce the desired hydrolysis
enzymes (Ragauskas et al., 2006; Teymouri et al., 2004). The speciﬁc commercial

enzyme mixture has been developed for corn stover treated with dilute acid, and it is yet

-ll3-

 

to ‘
ma

pl'l

 

 

to be demonstrated whether or not this mixture will be suitable for other biomass
materials or other pretreatments. In biological conversion of biomass raw material, the
pretreatment and hydrolysis enzymes used after pretreatment to produce sugars must
function together as a system (Wyman et al., 2005). Enzymes developed for acidic
pretreatments likely are not suitable for pretreatments using neutral or alkaline
conditions. One set of high value hydrolysis enzymes that might be produced
alternatively within the biomass crops and utilized in fuel ethanol production in a
bioreﬁnery are the “cellulases”—enzymes that convert cellulose into fermentable sugars.
The subsequent chapter contains further discussion of cellulases’ mode of action.

The El endo-1,4-B-glucanase enzyme of Acidothermus cellulolyticus is one of the
most thermostable cellulases known (Dai et al., 2005). The expression of Elcd endo-1,4-
p-glucanase in tobacco (Ziegelhoffer et al., 2001), potato (Dai et al., 2000), and
Arabidopsis (Ziegler et al., 2000) plants demonstrates the feasibility of producing this
enzyme in plants. Several prominent crops have been recommended for this purpose,
especially those with high-lignocellulosic biomass such as maize, sugarcane, switchgrass,
and rice, some of which presently cause disposal problems (Kim and Dale, 2004; Knauf
and Moniruzzaman, 2004). Because of the importance of rice around the world,
production of superior transgenic lines with desired novel traits is a worthy goal that is

achievable with the advent of genetic engineering and biotechnology.

Rice Importance and Straw Burning

Rice is the primary source of caloric intake for over half of the world’s human

population. It is grown on over 148 million hectares worldwide (Chandra Babu et al.,

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2003) with a total production of about 606 million tons of seeds (F AOSTAT, 2005) and
731 million tons of straw (Kim and Dale, 2004). Due to the projected increase in world
population in the coming decades, there is a pressing need to increase rice yields and
maximize the use of all plant parts (Khush, 1997). While rice seed is usually the useful
portion of this important crop, its remaining biomass has, to date, shown limited value.
Traditionally, farmers throughout the world burn rice straw in the ﬁeld after harvest.
Burning is inexpensive and mitigates rice diseases. However, the emitted smoke gives
rise to health concerns including the increased incidence of asthma (Kayaba et al., 2004).
These concerns, for example, resulted in California legislation that limits rice straw
burning to the lesser of 125,000 acres or 25% of rice area in 2001, and even then burning
is allowed only if evidence of disease is present (California Rice Commission, 2005).
California harvested 528,000 acres of rice in 2005, down from a peak of 590,000 acres in
2004 but nearly ten percent higher than the 465,000 acres harvested a decade earlier
(U SDA-National Agricultural Statistical Service, 2005). As a result, California produces
an excess of one million tons of rice straw each year. Of this produced amount, only
about 3-4% is used in commercial applications and the rest must be incorporated into the
soil for decomposition. The cost of this soil incorporation is about $43/acre, for a total of
$15-18 million per year (California Rice Commission, 2005).

Therefore, the production of bioﬁiel from rice straw is poised to become a strong,
viable bio-based energy source with the potential to lower pollution levels at the same
time. Moreover, the production of cellulase enzymes, used for biomass conversion, in
transgenic rice straw may potentially be a powerful tool to facilitate a reduced cost

conversion of cellulose to glucose in the commercial production of ethanol. Furthermore,

-1lS-

this approach may prove fruitful for the manufacture of other valuable bio-based
industrial enzymes and protein pharmaceuticals, while solving the problems associated
with accumulated agricultural waste biomass (Knauf and Moniruzzaman, 2004). Because
of its central role in food supply, signiﬁcant advances have already been made in the
development of rice tissue culture and genetic transformation methods, and the

incorporation of genes conferring important traits (N ishimura et al., 2005).

Rice Tissue Culture and Genetic Transformation

Generally, the major obstacle to the genetic manipulation of many plant species,
especially cereals, is the lack of an efﬁcient tissue culture system for the regeneration of
whole plants from tissue explants. Therefore, tissue culture is considered the common
crucible for most genetic engineering techniques (Slater et al., 2003).

Due to its global importance, especially in the most populated countries, and the
generosity of Rockefeller Foundation in funding many of its research projects, rice is
probably the most studied among cereals as a target for tissue culture and genetic
transformation (Sticklen and Oraby, 2005). Moreover, rice is the ﬁrst crop to have its
genome sequenced and became a monocotyledon model system because of its small
genome size and simple diploid character. This opens new avenues for the efforts of
improving other cereals such as maize, wheat and oat (Shimamoto and Kyozuka, 2002).

Most of the advances in tissue culture methods were demonstrated using japonica
varieties and to a lesser extent with a few responsive indica varieties (Visarada and
Sarma, 2002). Early reports indicated the possibility of regenerating rice plants from

japonica types using embryogenic cell suspensions (Abe and Futsuhara, 1986). However,

-ll6-

 

the ﬁ'equency of fertile plants regenerated from indica-type cell suspensions was
increased using high levels of maltose and sucrose in the medium, i.e. increasing the
osmoticum (Jain et al., 1996). Therefore, studies on the differences between the in vitro
response ofjaponica and indica types, as well as the dominance of japonica over indica
for transformation, are crucial (Visarada and Sarma, 2002). Other rice regeneration
protocols have been developed using embryogenic calli derived from different explants
such as pollen (Kim and Raghvan, 1988), shoot apices (Padua et al., 2001), root tips
(Khanna and Raina, 1999), immature embryos (Hoa et al., 2003), mature embryos
(LaFayette et al., 2005), protoplast (Datta et al., 1999), microspores (Balconi et al., 1998),
scutellum (Endo et al., 2002) and coleoptile (Kant et al., 2001).

Due to the signiﬁcant improvements in rice tissue culture systems during the last
decade, remarkable progress has been achieved in developing and optimizing a high
frequency and robust genetic transformation protocols for rice (see Table 4.1). Using
different tissue culture systems and either direct DNA transfer or Agrobacterium-
mediated transformation technology (Bajaj and Mohanty, 2005), a number of
experiments have been conducted to develop a broad spectrum of transgenic rice lines
that have agronomically important traits, especially increased nutritional value (Paine et
al., 2005; Tyagi et al., 1999; Tyagi and Mohanty, 2000). However, genetic engineering of
rice along with other cereals was not easy because of the initial problems related to the
introduction of genes into plant cells. The natural resistance of monocots to
Agrobacterium infection encouraged the development of direct DNA systems such as
microprojectile bombardment, electroporation and polyethylene glycol that made it

possible for foreign genes to be transferred to rice and other cereals (Koichi et al., 2002).

-117-

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-118-

 

 

 

 

 

 

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Using japonica rice, Uchimiya et al. (1986) recovered stable transformed rice
callus using PEG ﬁJsion. In addition, Zhang and Wu (1988) were able to produce
transgenic rice plants through PEG-mediated gene transfer using protoplasts while Zhang
et al. (1988) and Toriyama et al. (1988) produced transgenic rice plants through
electroporation using rice protoplasts. Furthermore, the genotype independent biolistic
particle delivery system developed for rice (Christou et al., 1991) has shown
reproducibility, overcoming limitations related to other methods, and remains one of the
best methods for introducing useful genes into rice (Ramesh and Gupta, 2005). In
addition to immature embryos, embryogenic calli and cell suspensions derived from
mature seeds of different japonica and indica elite cultivars have been successfully used
as target tissues to develop transgenic plants expressing various traits including herbicide,
insect and disease resistance (Ahmad et al., 2002; Jiang et al., 2000; Tu et al., 1998).
Also, the optimization of particle delivery system parameters for high efﬁciency
transformation was performed using gus (Ramesh and Gupta, 2005) and g)? (Vain et al.,
1998) reporter genes, and hpt (Datta, 2003) and bar (Jiang et al., 2000) as selectable
markers. However, even though they have been very successful in rice transformation,
direct-gene transfer methods have some restrictions and limitations (Koichi et al., 2002).
Therefore, a more efﬁcient technique was required; and this was found in the application
of Agrobacterium-mediated transformation. In rice, this method offered several
advantages including highly efficient and inexpensive transformation method, the ability
to transfer large pieces of DNA, reduced somaclonal variation and no protoplast
requirement (Park et al., 1996). Although Raineri et al. (1990) failed to regenerate plants

aﬁer the transfer of the T-DNA into Japonica rice via Agrobacterium in the earliest trials

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of this technique, extensive work has been done to improve the potential efficiency of
DNA transfer using Agrobacterium-mediated transformation. Most such studies focus on
the factors that limit the success of this system. One of the factors that play an important
role is the ability to regenerate plants in vitro, which seemed to be genotype independent
(Park et al., 1996). Additionally, the use of the acetosyringone, a wound induced phenolic
compound, has been reported to increase the transformation efﬁciency via Agrobacterium
(Saharan et al., 2004). Other important factors involved in the success of this technique
were genotype, age and physical state of the tissue used in the inoculation (Gould et al.,
1991), vectors and strains used (Montoro et al., 2003) and infection time and conditions
(Hiei et al., 1997).

An Agrobacterium-mediated transformation system in rice has become possible
alter the establishment of an efficient transformation protocol by Hiei et al. (1994). The
establishment of such a successful system has greatly facilitated the widespread
application of rice transformation. Soon after, Aldemita and Hodges (1996) successfully
transformed japonica and indica varieties using embryos as explants, hygromycin for
selection and high concentrations of the acetosyringone for vir genes induction.
Currently, this technique is widely used not only to introduce genes of interest into the
rice genome for the purpose of cultivar improvement such as the production of golden
rice (Al-Babili and Beyer; 2005; Sallaud et al., 2003), but also as a signiﬁcant method for
generating large mutant libraries by T-DNA insertions and testing gene functions (Lin
and Zhang, 2005).

It is worth noting that high performance rice varieties are required for continued

improvements in human welfare. Such improvements involve the introduction of an array

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of additional beneﬁcial genes from other organisms via genetic transformation into the
rice genome such as those encode for abiotic and biotic resistance, nutritional quality,
enzymes required for biofuel production and other value added traits (Vasil, 2005).
Traditional breeding programs coupled with the advent of these promising technical
advances represent an effective tool for achieving that goal.

The following section examines a speciﬁc application of these techniques: the
production of biologically active Acidothermus cellulolyticus endo-l,4-B-glucanase E1
enzyme in transgenic rice plants and the conversion of corn and rice biomass cellulose to

glucose using rice-produced El heterologous enzyme.

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MATERIALS AND METHODS

Transformation Vector

The pZM766-E1cat containing the Acidothermus cellulolyticus E1 catalytic
domain driven by the Cauliﬂower Mosaic Virus 358 promoter (CaMV 35$), tobacco
Mosaic Virus translational enhancer (Q), and the tobacco pathogenesis-related protein 1a
(Prla) signal peptide encoding sequence for apoplast-targeting of the E1 enzyme was
removed from the pUCl9 vector by digestion with XbaI. The removed cassette was
transferred to the binary vector pCAMBIA 3301 containing the bar herbicide resistance

selectable marker and the gus marker genes to generate the binary vector ApoEl.

Selection of Transformants Using Glufosinate Herbicide

A kill curve was developed to test the sensitivity of rice calli to glufosinate
ammonium. Glufosinate ammonium was used as a selection agent since the binary vector
used in this study contained the bar gene. Untransformed calli were placed on MS basal
salt mixture containing 0, 5.0, 10.0, 15.0, 20.0 and 25.0 mg/L of glufosinate ammonium
for 6-8 weeks. The calli survival was recorded. All calli turned brown and died after
being cultured on glufosinate ammonium with concentrations of 15 mg/L or more.

Therefore, 15 mg/L glufosinate ammonium was used for transgenic plant selection.

Genetic Transformation
Mature seeds of rice (Oryza sativa L. subsp. Japonica) variety Taipei 309 were
dehusked and sterilized in 70% (vol/vol) ethanol for 2—3 min and then transferred into

50% (vol/vol) Clorox solution for 20 min with gentle shaking. The sterilized seeds were

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plated for callus induction on MS salts and vitamins medium supplemented with 3%
sucrose, 300 mg/L casein enzymatic hydrolyzate, 500 mg/L proline, 2mg/L 2,4-
dichlorophenoxyacetic acid, 2.5 g/L Phytagel, pH 5.8 and grown for 21 days at 25°C in
the dark. The Agrobacterium strain LBA 4404 containing the transgenes was grown in 10
ml YM medium (containing Yeast extract 0.4 g/L, Mannitol 10.0 g/L, NaCl 0.1 g/L,
MgSO4. 7H20 0.2 g/L, K2HPO4. 3HZO 0.5 g/L, pH 7.0) supplemented with 50 mg/L of
kanamycin, streptomycin, rifampcin and 100 W acetosyringone, incubated at 28 °C and
250 rpm for 48 h. Then, the cultures were transferred to 40 ml MS medium supplemented
with 100 MM acetosyringone and incubated under the same conditions for another 24 h.
The bacterial cells were harvested by centrifugation and resuspended in 15 ml of the
same media. The cultures (cell density 0.9 at A600) were used for transformation.

Three weeks after callus induction from the scutellar region of the rice embryo,
embryogenic calli were immersed in an A. tumefaciens suspension for 20 min under
vacuum. Infected calli were co-cultivated in MS medium supplemented with MS salts
and vitamins, 3% sucrose, 1% glucose, 500 mg/L casein enzymatic hydrolyzate, 2 g/L
Gelrite, 100 [AM acetosyringone, pH 5.2. After 3-4 days of co-cultivation, calli were
washed with sterile water containing 500 mg/L cefotaxime and blotted on ﬁlter paper.
The calli were immediately plated on a selection medium, calli induction medium
supplemented with 15 mg/liter glufosinate ammonium and 500 mg/L cefotaxime, pH 5.8,
and incubated at 25°C in the dark for 3—4 weeks. The calli that had proliferated after the
initial selection were further sub-cultured for two selection cycles on the same medium
every 2 weeks. The actively dividing glufosinate ammonium-resistant calli were plated

on MS plant regeneration medium containing MS salts and vitamins, 3% sucrose, 3%

-123-

 

sorbitol, 3 mg/L N6-benzyladenine, 1mg/L naphthaleneacetic acid, 500 mg/L casein
enzymatic hydrolyzate, 3 g/L Gelrite, 15 mg/L glufosinate ammonium, pH 5.8 and grown
at 25°C for a 10-h light/14-h dark photoperiod for 4 weeks. The regenerated plantlets
were rooted on half-strength MS salts and vitamins, 4 g/L Gelrite, 15 mg/L glufosinate
ammonium, pH 5.7. Plantlets were transferred to the greenhouse after acclimatization in
grth chamber under 27°C (day), 19°C (night), 13h photoperiod (13h-light: llh-dark)

and 210-300 mE light intensity.

Histochemical Analysis of GUS

Stable expression was assayed in plantlets. from the transgenic lines and non-
transgenic control via GUS histochemical staining using 5-bromo-4-chloro—3-indoyl-B-D-
glucuronicacid salt (X-gluc). Rice calli and plantlets were immersed in the GUS substrate
mixture and incubated at 37°C, then incubated in 70% ethanol to remove the chlorophyll,

and examined under a Zeiss SV8 stereomicroscope.

PCR Analysis

PCR was used to detect bar and E1 genes in transgenic rice using leaf disk DNA
as a template and REDExtract-N-AmpTM Plant PCR Kit (Sigma-Aldrich, St. Louis, MO,
Cat # XNA-P) based on the manufacturer’s. instruction. Primers for bar included the
forward 5’-ATG AGC CCA GAA CGA CG-3’ and the reverse 5’-TCA GAT CTC GGT
GAC GG-3’), and for the the E1 included the forward S’- GCG GGC GGC GGC TAT
TG -3’and the reverse 5’- GCC GAC AGG ATC GAA AAT CG -3’. DNA ampliﬁcations

were performed in a thermo cycler (Perkin Elmer/Applied Biosystem, Foster City, CA)

-124-

using initial denaturation at 94°C for 4 min, followed by 35 cycles of 1 min at 94°C,
1 min at 55°C, 2 min at 72°C, and a ﬁnal 10 minute extension at 72°C. The reaction
mixture was loaded directly onto a 0.9 % (w/v) agarose gel, stained with ethidium
bromide and visualized with UV light. The transgene product size was about 0.59 kb for

the bar gene and 1 kb for the E1 gene.

DNA Isolation and Southern Blot Hybridization Analysis

Conﬁrmation of transgene integration into the plant genome, number of
independent transgenic lines, and transgene copy numbers were performed by Southern
blot hybridization using the EI-coding sequence as a probe. For Southern blots, 8 pg of
genomic DNA was digested with BstXI restriction enzyme, electrophoresed in 1.0 %
(w/v) agarose gel, transferred onto Hybond-N+ (Amersham-Pharmacia Biotech)
membranes, and ﬁxed with a UV crosslinker (Stratalinker UV Crosslinker 1800,
Stratagene, CA) as recommended in the manufacturers’ instructions. The E1 gene-
speciﬁc probe was generated using PCR ampliﬁcation of the E1 gene to produce a 1.0-kb
fragment. The ampliﬁed fragment was puriﬁed using the QIAquick kit (QIAGEN). Probe
labeling and detection were obtained using the DIG High Prime DNA Labeling and
Detection Starter Kit H (Kit for chemiluminescent detection with CSPD, Roche Co.),

following the manufacturer’s protocol.

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RNA Isolation and Northern Blot Hybridization Analysis

Total RNA samples of the non-transgenic and transgenic plants were isolated
from ﬁve different transgenic lines using the TRI Reagent (Sigma-Aldrich, St. Louis,
Mo) according to the manufacturer’s instructions. Aliquots of RNA (20 pg) were
fractionated in 1.2% agarose formaldehyde denaturing gel and blotted on a Hybond-N+
nylon membrane (Amersham Pharmatica Biotech) as speciﬁed by the manufacturer. The
E1 gene-speciﬁc probe was generated using PCR ampliﬁcation of the E1 gene to produce
a 1.0-kb fragment. The fragment was gel puriﬁed using the QIAquick Gel Extraction Kit
(QIAGEN Inc., Valencia, CA). Probe labeling and transcript detection were obtained
using the DIGHigh Prime DNA Labeling and Detection Starter Kit H (Kit for
chemiluminescent detection with CSPD, Roche Co.), following the manufacturer’s

protocol.

Protein Extraction and Western Blot Analysis

Plant total soluble protein was extracted using a reported protocol (Ziegelhoffer et
al., 2001) with the Invitrogen NuPAGE® Bis-Tris Discontinuous Buffer System and the
10% NuPAGE® Novex Bis-Tris Pre-Cast Gel. Total soluble protein (1 pg), NuPAGE®
LDS Sample Buffer (5 pl), NuPAGE® Reducing Agent (2 pl), and deionized water were
mixed to a total volume of 20 pl. The samples were heated at 70°C for 10 minutes prior
to electrophoresis using the XCell SureLock TM Mini-Cell with NuPageQ MES SDS
Running Buffer. The gel was run for about 45 minutes at 200 V, and blotted onto a
membrane using the XCell II® Blot Module and NuPAGE® Transfer Buffer at 30 V for

1 hour, following the manufacturer's protocol.

-126-

 

The membrane was placed into blocking buffer (1x PBS, 5% non-fat dry milk,
and 0.1 % Tween 20) immediately aﬁer transfer and incubated at room temperature for
1 hour with gentle agitation. The primary antibody (mouse anti-E1, provided by Steven
Thomas, National Renewable Energy Laboratories) was diluted in blocking buffer to a
concentration of 1 pg/ml. The blocking buffer was decanted from the membrane, 10 ml
of antibody solution was added, and the membrane was incubated at room temperature
for 1 {hour with gentle agitation. The primary antibody solution was decanted and the
membrane was washed in washing buffer (1x PBS, 0.1% Tween 20) for 30 minutes with
gentle agitation at room temperature, changing the wash solution every 5 minutes. The
enzyme conjugate anti-mouse IngHRPO (Transduction Laboratories) was diluted
1:2000 in blocking solution and added to the membrane after decanting the wash buffer.
The membrane was incubated with the secondary antibody solution for 1 hour at room
temperature with gentle agitation; the antibody solution was decanted from the membrane
and the membrane was washed in washing solution as before. For detection, 1 ml each of
Stable Peroxide Solution and Luminol/Enhancer Solution (Pierce SuperSignal® West
Pico Chemiluminescent Substrate) were mixed and incubated with the membrane for 5
minutes. The membrane was blotted slightly to remove excess substrate and placed in a
plastic envelope. Excess liquid and air bubbles were removed. The blot was exposed to
X-ray ﬁlm (Kodak BioMax XAR Scientiﬁc Imaging Film) and developed in a Kodak RP

X—OMAT Processor.

-127-

Immunoﬂuorescence Microscopic Analysis

Free-hand sections of fresh leaf tissue ﬁ'om transgenic and non-transgenic rice
plants were isolated and hydrated in NaCl/Pi buffer (0.8% NaCl, 0.02% KCl, 0.14%
Na2IIPO4-2HzO, and 0.02% KHzPO4 in water) containing 0.5% BSA (BSA/NaCl/Pi) for
2 min. Sections were incubated in primary antibody [rabbit anti-(mouse IgG)] raised
against the E1 enzyme diluted 1:250 in the same buffer, in a moist chamber for 3 hours.
The primary antibody was rinsed off with the BSA/NaCl/Pi buffer and sections were
incubated for 2 hours at room temperature with ﬂuorescein isothiocyanate (FITC)-
conjugated secondary antibody [goat anti-(rabbit whole molecule IgG)] diluted 1:250 in
the same buffer using same moist chamber. The secondary antibody was then rinsed off
with the same buffer. Intracellular localization of the FITC-labeled protein was observed
and images were taken using a confocal laser scanning microscopy Zeiss LSM 5 Pascal
(Carl Zeiss, Jena, Germany). F ITC ﬂuorescence and chloroplast autoﬂuorescence was
excited with an argon ion laser, Rex = 488 nm. Fluorescence emission was detected
through a Band Pass (BP) ﬁlter, Ln, = 530/30 nm for the FITC (images represented in
green) and Long Pass (LP) ﬁlter, 3.", = 650 nm for the chloroplast (images represented in

red). Either a 63X Plan-apochromat or a 20X Plan-neoﬂuar objective lens was used.

The Biological Activity Assays of Heterologous E1 Enzyme

The MUCase enzyme assay was conducted as reported (Ziegelhoffer et al., 2001;
Ziegler et al., 2000). E1 enzyme activity was determined by subtracting the background
contributed by Taipei 309 rice control extracts, from the spectrophotometer ﬂuorescence

readings. The resulting ﬂuorescence signals without noise were used to calculate the

-128-

activity and amount of biologically active E1 enzyme present in transgenic samples. 24-
well agar plates containing 1% carboxymethylcellulose (CMC) were exposed to leaf total
soluble protein extract. The plates were heated to 65° C for 30 min in an oven to activate
the enzyme. The plates were cooled at 4°C for 5 min and then stained with 1 mg/ml
Congo Red for 30 min. Samples were destained with l M NaCl for 5 min and ﬁxed with

10 mM NaOH.

Cellulose Hydrolysis Assay

Milled corn stover and rice straw (about 1 cm in length) were pretreated using the
Ammonia Fiber Explosion technique (AFEX). The biomass was transferred to a high
pressure Parr reactor with 60% moisture (kg water/kg dry biomass) and liquid ammonia
at a ratio of 1.0 (kg of ammonia/kg of dry biomass) was added. As the temperature was
slowly raised, the pressure in the vessel increased. The temperature was maintained at 90°
C for ﬁve minutes before explosively releasing the pressure. The instantaneous drop of
pressure in the vessel caused the ammonia to vaporize, causing an explosive
decompression and considerable ﬁber disruption. The pretreated material was kept under
a hood to remove residual ammonia and stored in a freezer until further use.

E1 activity was measured by reacting total protein extracted from E1 —expressed
rice leaves with different substrates, namely: AFEX-treated corn stover (CS), AFEX-
treated rice straw (RS), CMC and Avicel. Commercial cellulase enzyme (Spezyme CP,
Genencor International) was used in this experiment as a control. The enzyme hydrolysis

was done in a sealed scintillation vial. A reaction medium composed of 7.5 m1 of 0.1 M,

pH 4.8 sodium citrate buffer was added to each vial. In addition, 60 pl (600 pg)

-129-

tetracycline and 45 pl (450 pg) cycloheximide were added to prevent the grth of
microorganisms during the hydrolysis reaction. The reaction was supplemented with 30
CBU of B-glycosidase enzyme (Novo 188 from Sigma) to avoid inhibition by cellobiose.
Distilled water was then added to bring the total volume in each vial to 15 ml. All the
reactions were done in duplicate to test reproducibility. All hydrolysis reactions were
carried out at 50° C with a shaker speed 90 rpm. About 1 ml of sample was collected at
168 hours of hydrolysis, ﬁltered using a 0.2 pm syringe ﬁlter and kept frozen. The
amount of glucose produced in the enzyme blank and substrate blank were subtracted
from the respective hydrolyzed glucose levels. Hydrolyzate was quantiﬁed using Waters
HPLC by running the sample in Aminex HPX-87P (Biorad) column, against sugar
standards. The amount of glucose produced in the enzyme blank and substrate blank were

subtracted from the respective hydrolyzate glucose levels.

~130-

RESULTS

Transgene Construct and Genetic Transformation

The ApoEl binary vector (Figure 4.1) containing the catalytic domain of the A.
cellulolyticus thermostable endoglucanase (E1 ) gene (encoding for endo-l, 4-B-glucanase
enzyme) was introduced into the nuclear genome of mature embryo-derived calli (Figure
4.2a) of the rice variety Taipei 309 (Otyza sativa L. subsp. Japonica) using the
Agrobacterium-mediated transformation system (Ahmad et al., 2002; Cheng et al., 2004).
Transformation frequency, as deﬁned in terms of percentage of glufosinate herbicide
resistant calli (Figure 4.2b) was 32%. About 78% of these glufosinate-resistant
embryogenic calli differentiated into plantlets in the presence of 15 mg/L glufosinate
ammonium (Figure 4.2 c and (1). Many transgenic plants were produced among which
ﬁve independent transgenic events were selected for further analysis. R0 and R1 plants
grew well with no apparent grth or developmental abnormalities under controlled

grth chambers (Figure 4.2e) and greenhouse environments (Figure 4.2f and g).

Molecular Analysis of the Transgenic Plants

Combining the results of stable GUS expression patterns (for the gus gene) and
PCR (for the bar and E1 genes) assays conﬁrmed the presence of intact, linked gus, bar
and E1 genes. The blue color of GUS expression patterns were observed in the transgenic
calli and plantlets (Figure 4.2 h and i). The expected PCR bands (0.59 kb for bar and 1 kb
for E1) were conﬁrmed in the plasmid and the transgenic rice lines, but not in the non-

transgenic control plants (Figure 4.3a).

-131-

355 Q Prla E] catalytic domain Nos-‘1'

K
u.._. ~).,,.._4 . _—a
" J” 319! '7
w, ..v_...-r..-- .. .7: ;... . .-
\u\

\\ /

 

 

 

Barherbicide \> 5 Reporter
resistant Gene if J5“:
LB i W051 “3
Construct =
Bacterial genes
Kan antibiotic marker \
replication origin —’ pCambia3301

 

Figure 4.1. Schematic representation of ApoEl binary vector containing the
Acidothermus cellulolyticus E1 catalytic domain driven by Cauliﬂower Mosaic Virus
358 Promoter (CaMV 35$), tobacco Mosaic Virus translational enhancer (Q), and
the sequence encoding the tobacco pathogenesis-related protein la (Prla) signal
peptide for apoplast-targeting of the El enzyme, and the polyadenylation signal of
nopaﬂnesynthase(nos)

-132-

 

(‘alli 0f Calli 0f Seedling of Seedling of
transgenic rice control rice transgenic rice control rice

 

Figure 4.2. (a) Mature derived calli of rice cultivar Taipei 309, (b) selection of
transgenic calli on 15 mg/L glufosinate ammonium, (c and d) regeneration of
transgenic plants, (e) growth chamber grown El transgenic rice plants, (1’ and g)
greenhouse fertile grown A. cellulolyticus E1 transgenic rice plants and (h and i) gus
expression in calli and plantlets of transgenic rice as compared to the non-transgenic
control.

-l33-

Southem blot analysis conﬁrmed the stable incorporation, copy number and
independence of the transgenic lines (Figure 4.3b). The genomic DNA of the ﬁve
transgenic lines shoWed bands of different sizes, as an indication of ﬁve independent
transgenic events with 1-2 copies.

When Northern blot analysis was used to conﬁrm the transcription of the E1 gene,
a transcript of approximately 1 kb for this gene was detected in the transgenic tobacco
positive control (tobacco transformed with the same construct) as well as the rice
transgenic lines, indicating that the transgenic lines possess the transcriptionally-active
E1 gene (Figure 4.3c).

Western blot analysis of leaf total soluble proteins (LTSP) using the mouse
antibody raised against the El protein conﬁrmed the expression of E1 both in transgenic
rice and transgenic tobacco positive control, with the expected molecular mass of 40-kDa
(Figure 4.3d). Furthermore, the relative amount of transcript and 40 kDa E1 polypeptide
in all ﬁve transgenic lines, judged from band intensity respectively in Northern and
Western blots, correlated well with the amount of E1 produced in the transgenic lines
using the 4-methylumbelliferyl B-D-cellobioside assay (MUCase) (Figure 4.3c,d and

Table 4.2).

Localization of the E11 Enzyme in the Apoplast

Strong green ﬂuorescent signals were detected in the apoplast of the transgenic
tissues upon the application of immunoﬂouresence scanning laser confocal microscopy,
conﬁrming accumulation of the E1 enzyme. No signals were detected in the non-

transgenic control plant tissues (Figure 4.4).

~134-

 

 

"" or
o

.
up1ia4sc upc1b2345 p1234sc

(a) PCR ampliﬁcation of the bar (0.59 kb) (a) and E1 .
(1 kb) (b) genes in 5 transgenic rice lines. M: Ladder Sifffxriltlenglsofointﬂzgizﬁxggenic
marker 100 bp (a) and 1 kb (b), P: Plasmid (positive g

. . rice lines. P: plasmid; C: non-
control), C. untransformed (negative control), 1 5. transgenic; l—S: Transgenic lines.
Transgenic lines.

 

 

(c) Northern blot analysis showing 1 kb bands ((1). Western blot analysis showing 40 kDa
for the five transgenic rice lines. +: positive bands for the ﬁve transgenic rice lines. +:
control; C: non-transgenic control; 1-5: positive control; C: non-transgenic control;
Transgenic lines. 1-5: Transgenic lines.

Figure 4.3. PCR (a), Southern (b), Northern (c) and Western ((1) blot analyses show
the presence of the transgenes in ﬁve transgenic rice lines.

Table 4.2. The amount of heterologous E1 enzyme in different independent
transgenic rice events determined by the MUCase activity assay (average of 3 reps).

 

Positive Negative

control control Lme 1 Line 2 Line 3 Line 4 Lme 5

 

E1 in the

total soluble 3.60 % 0 3.87 % 2.67 % 2.41 % 4.90 % 2.85 %
rotein

 

 

 

 

 

 

 

 

 

 

-l35-

High-Level Production of Biologically Active El Enzyme

E1 enzyme was expressed at levels of 2.4-4.9% of LTSP, as detected among the
transgenic lines. The carboxymethyl cellulase activity assay (CMCase) conﬁrmed that the
rice-produced heterologous E1 is biologically active. In this conﬁrmation, zones of
carboxymethylcellulose (CMC) hydrolyzed by the enzyme were decolorized with a
washing buffer, leaving yellow regions in the transgenic as compared with red
background in the non-transgenic control plant samples (Figure 4.5). The results suggest
that the microbial E1 enzyme remained biologically active in the transgenic rice plants

while E1 activity was not present in the non-transgenic plants (Table 4.2).

Cellulose to Glucose Conversion

The Ammonia Fiber Explosion (AFEX)-pretreated (Teymouri et al., 2004) maize
and rice biomass (lignocellulosic substrates containing both amorphous and crystalline
cellulose), as well as increasing concentrations of both CMC (amorphous cellulose) and
Avicel (crystalline cellulose) were converted into glucose using the transgenic rice plant
total soluble proteins containing the E1 enzyme. Using 10% CMC and Avicel
concentrations, approximately 0.6 and 0.2 g/L glucose was released after 168 hour of
hydrolysis, respectively (Figure 4.6a). Additionally, considerable amounts of
polyoligosaccharides were released ﬁ'om the CMC substrate blank, and the apparent
solution viscosity increased substantially. Conversely, when an aliquot of rice E1-
containing total soluble proteins was added to the CMC substrate, viscosity declined with

reduced polyoligosaccharide formation and a detectable increase in the glucose peak.

-136-

 

 

El enzyme
located in the
apoplast

autoﬂuortsrenre
of the plant
tissue

   

Figure 4.4. Immunoﬂuorescence confocal microscopy for the transgenic (a) and non-
transgenic (b) rice showing apoplast localization of the E1 enzyme in transgenic rice
leaves.

Red background in the Yellow zones in the
non- transgenic control transgenic treatment
treatment

 

Figure 4.5. Detection of the El enzyme activity using CMCase activity assay. Zones
of CMC hydrolysis were decolorized with washing leaving yellow regions in the
transgenic as compared to red background in the control.

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UT
05
05

  
  
  

 

 

 

 

100:

 

 

 

 

 

% Glucan
Converslon

 

 

Figure 4.6. (a) The amount of glucose released from the enzymatic hydrolysis of
CMC (1%, 5%, 10%) and Avicel (1%, 5%, 10%) using total protein extracted from
E1 expressed rice straw. (b) Comparison of percentage of glucan converted in the
enzymatic hydrolysis of corn stover (CS) and rice straw (RS). CE, commercial
enzyme, UT, untreated biomass, CS1, RS1, CS2, and R82 represent, reaction done
using 0.5 m1 and 4 m1 of total soluble protein (with 4.9% of E1) and commercial B-
g1ucosidase(6.5 mg/ 15 ml) respectively.

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Approximately 25% and 95% cellulose conversion was achieved for untreated
and APEX-treated corn stover respectively, when the cellulase commercial enzyme
(Spezyme CO, Genencore) along with B-glucosidase (Novo 188, Sigma) was used in
each case. Under similar conditions, untreated and APEX-treated rice straw showed 21%
and 62% cellulose conversion respectively. Since both untreated corn stover and rice
straw showed much lower conversion compared to AFEX-treated biomass (less than 2%
using El-containg rice extract and 25% and 21% using cellulase commercial
respectively), AFEX-treated biomass was used for ﬁirther experiments. When 0.5 ml of
rice extract containing 4.9% LTSP E1 along with commercial B-glucosidase were added
to the substrates, 17% and 14% of cellulose was respectively converted for APEX-treated
corn stover and AFEX-treated rice straw. When the amount of El-bearing rice extract
was increased to 4 ml, 30% and 22% were respectively converted under the same
conditions. No activity was observed when substrates were treated with non-transgenic

(NT) rice total soluble protein.

-l39-

 

DISCUSSION

Plants have been used as “green bioreactors” for the production of essential
enzymes (Chiang et al, 2005; Hong et al., 2004) and other proteins (Liu et al, 2005),
carbohydrates (Sahrawy et al., 2004) and lipids (Qi et al., 2004) while requiring minimal
inputs of raw materials and energy (Teymouri et al., 2004). Production of biomolecules in
plants, considered as molecular farming, is one approach to improve the economics and
increase the low-cost production efﬁciency of these biomolecules (Fischer et al., 2004).

Several crops have been recommended for biomass-to-ethanol conversion, among
them maize, rice, sugarcane and switchgrass (Kim and Dale, 2004; Knauf and
Moniruzzaman, 2004) —all with a high amount of lignocellulosic biomass, and some of
which have caused disposal problems. Production of enzymes in plants used for biomass
conversion is a potentially powerful tool to facilitate the conversion of cellulose to
glucose in the commercial production of ethanol while solving the problems associated
with accumulated agricultural waste biomass. It is when ethanol bioconversion enzyme
costs are decreased, that ethanol bioreﬁneries achieve ﬁnancial advantages over
petroleum reﬁneries.

There are three possible explanations why the heterologous E1 that accumulated
in apoplast did not harm transgenic plant cell walls. First, lignocellulose is difﬁcult to
hydrolyze because it is associated with hemicellulose, and surrounded by a lignin seal,
which has a limited covalent association with hemicellulose. Furthermore, it has a
crystalline structure with a potential formation of hydrogen bonds resulting in» a tightly
packed structure. Also as per Figure 4.6b, we conclude that pretreatment might be

necessary to increase the surface area and consequently accessibility of cellulases by

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removing the lignin seal, solubilizing hemicellulose and disrupting crystallinity (Demain
et al., 2005). Second, cellulases function in a synergistic enzyme complex. If only one
enzyme of the complex is expressed such as E1, this single enzyme might not be
sufﬁcient to signiﬁcantly affect the integrity of the cell wall without the pretreatment
(Ziegelhoffer et al., 2001). Third, due to the thermophilic nature of the E1, the enzyme
has limited activity under plant in vivo temperature (Dai et al, 2000).

When accumulated in cytosol, the normal level of heterologous protein
production in plants is usually no more than 0.1-0.3% of plant TSP. The high level of E1
production in this research (Table 4.2) and in Arabidopsis (Ziegler et al., 2000) might be
due to the fact that the E1 has been targeted to apoplast for accumulation. Among many
other factors, the use of the catalytic domain of the E1 gene, the use of the Tobacco
Mosaic Virus translational enhancer and the strength of the CaMV .358 promoter (Cheng
et al., 2004) might have contributed to the overall level of production of E1 in rice.

It has been well documented that cellulases work together synergistically to
decrystallize and hydrolyze the cellulose. Exo-glucanases act on crystalline cellulose (on
cellulose chain ends) and endo-glucanase (E1) acts on amorphous cellulose (interior
portions of the cellulose chain) (Bayer et al., 1998). In contrast, the results in this study
demonstrate production of glucose from atypical endoglucanase (i.e. E1) activity on a
solid substrate. This could be because E1 enzyme can cause multiple random attacks on
the same cellulose chain resulting in small fragments of cellobiose, cellotriose and
cellotetraose. These fragments can be further hydrolyzed by enzyme molecules in
solution such as E1 itself or B-glycosidase enzyme used to avoid reaction inhibition by

cellobiose (Medve et al., 1998).

-141-

 

Based on a previous study, AFEX pretreatment, although mild compared to other
pretreatment methods, can destroy as much as 2/3 of the activity of plant produced
heterologous E1 (Teymouri et al., 2004). Therefore, we recommend extracting the El
protein or the plant total soluble proteins concentrate containing the biologically active
E1 enzyme and then adding the enzyme or the crude concentrate after pretreatment, as
was performed in this study.

Production of El enzyme in plant biomass could be commercially viable, with the
caveat that additional work is needed to produce other cellulase and possibly
hemicellulase enzymes along with E1 in plants in order to maximize production of
glucose and other sugars within the crop biomass. In addition, because lignin is a biofuel-
blocking polymer in biomass conversion (Ragauskas et al., 2006), one might also
produce ligninases within the crop biomass similar to when the laccase was produced in
maize seeds (Bailey et al., 2004). When all of these enzymes are produced, especially at a
very high level of about 26% total soluble proteins (Ziegler et al., 2000) in plants, this
could well compete with the full range of commercial hydrolysis enzymes currently used
in ethanol production.

In future, transgenic methods could be used to produce hydrolysis enzymes within
the biomass crops with the use of bioconﬁnement methods to reduce seed contamination
and controversies around genetically modiﬁed plants (NRC, 2004). Further research is
needed to ensure that lignocellulosic conversion enzymes produced within the plants will

survive harvest, storage and transportation.

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":

 

SUMNIARY

The catalytic domain of Acidothermus cellulolyticus thermostable endoglucanase
gene (encoding for endo-1,4-B-glucanase enzyme or E1) was constitutively expressed in
rice using the Agrobacterium-mediated transformation system in an apoplast-targeting
manner. Molecular analyses of T1 plants conﬁrmed the presence and expression of the
transgene. The amount of E1 enzyme accounted for up to 4.9% of the plant total soluble
proteins, and its accumulation had no apparent deleterious effects on plant grth and
development. Approximately 22 and 30% of the cellulose in the Ammonia Fiber
Explosion (AFEX)-pretreated rice and maize biomass was converted into glucose using
rice E1 heterologous enzyme respectively. As rice is the major food crop of the world
with minimal use for its straw, the results may suggest a successful strategy for producing
biologically active hydrolysis enzymes in rice to help generate alcohol fuel while
substituting the wasteful and polluting practice of rice straw burning with an

environmentally superior technology.

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CHAPTER IV

CONCLUSION AND FUTURE RESEARCH

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CONCLUSIONS

The goal of the presented research was to provide effective transgenic-based
means that can contribute to solving two of the most complex problems for which
traditional plant breeding technologies provide only limited answers (W inicov, 1998).

Salinity strikes fear into the hearts of many farmers. Many call it the white death
as plants will eventually die even after reaching a certain level of tolerance. Therefore, it
limits agriculture worldwide especially on farmlands where poor irrigation practices take
place (Shannon and Grieve, 1999). This situation creates the need for salt tolerant plants.
Genetic modiﬁcation provides the opportunity to match potentially difﬁcult growing
conditions—such as high salt content—with transgenic cereal varieties better suited for
those environments.

Previously ﬁve transgenic oat lines that expressed the barley H VAI gene have
been characterized and showed increased tolerance to salinity under in vitro conditions at
the seedling stage (Maqbool et al., 2002). In this study, the molecular and agronomic
characteristics of R3 progeny of those lines were analyzed under salinity stress conditions
in greenhouses. Molecular (PCR, Southern and Northern blots), segregation for herbicide
and GUS histochemical analyses conﬁrmed the existence, transcription and translation of
the intact linked bar-HVA! and the non-linked gus genes in the R3 progeny of transgenic
oat plants.

The results showed that transgenic and non-transgenic plants performed similarly
under well-watered growing conditions (0 mM NaCl). Appearance and development of

damage symptoms caused by salinity were delayed in transgenic plants. Under salinity

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stress, differences in tolerance between transgenic lines and the control were associated
with the ﬂag leaf area growth, positive effects of plant height, panicle length and number
of spikelets/panicle and development and maintenance of extensive root system.

Kernel ﬁlling in cereals is considered to be one of the most sensitive growth
stages to stresses (Nayyar and Walia, 2004). Physiological stress expressed by salinity
during this period reduces the storage capacity of cereal kernels by decreasing the
number of endosperm cells and/or the number of amyloplasts initiated (Jones et al. 1996).
Salinity can reduce the kernel weight and number of kernels by limiting the rate and
duration of the ﬁlling process and inhibiting photosynthesis (Gupta et al. 2001;
Vishwanathan and Khanna-Chopra, 2001). As indicated by yield and yield components
(number of tillers/plant, number of kernels/panicle and 1000-kemel weight), the results
demonstrate that control plants were more sensitive to salinity and experienced more
severe water stress than did the transgenic lines.

Although transgenic lines experienced a decrease in performance at the highest
salinity stress level (200 mM), these lines were able to show better performance than non-
transgenic controls under continuous salt stress. Line BRA-41 experienced the least and
the control plants the most decrease in performance through the salinity levels for most of
the studied traits. The results provide more evidence about the role of the HV A1 protein
in water deﬁcit damage prevention and the protection of oats against salinity. This could
beneﬁt farmers with healthier plants, potentially resulting in possible contribution to
superior yield.

In addition to salinity stress, using biomass crops, especially rice, as a production

system for industrial proteins and enzymes has many advantages over bacterial

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production technology. Not only does it help by reducing the harmful practice of
agrticultural burning, but also it is considered a low cost production system for enzymes
such as cellulases used in biofuel production. This application is supported by the high
expression levels in plant systems that economically justify their use (Hood and
Woodard, 2002). In order to test the efficacy of this application, this research investigated
a system for expressing the catalytic domain of Acidothermus cellulolyticus thermostable
endoglucanase gene (encoding for endo-1,4-B-glucanase enzyme or E1) in rice plants.
Molecular (PCR, Southern, Northern and Western blots), segregation for
herbicide and GUS histochemical analyses of the ﬁrst generation plants conﬁrmed the
expression and stability of transmission of the transgene to the progeny. The results
showed enzyme accumulation of up to 4.9% of the plant total soluble proteins with no
apparent harmful effects on plant growth and fertility. This high expression could be in
part due to the use of the 358 promoter that is used in different other studied to derive
high transcription rate and gene expression (Cheng et al., 2004). Also, the use of the
thermostable E1 catalytic domain, a very truncated form of of the E1 gene, might have
added dramatic increase in gene expression specially when coupled with a strong
promoter such as 358 (Hood and Woodard, 2002). Moreover, targeting the enzyme to the
cell wall space, which is a very stable environment for accumulation and storage of
different types of proteins and enzymes, might played an important role (Zhong et al.,
1999). Using E1 heterologous enzyme extracted from rice leaves, high percentage of the
cellulose in the Ammonia Fiber Explosion (AFEX)-pretreated rice and maize biomass
was converted into glucose. This is an indication for the high activity of the enzyme,

which was not affected by using the plants in its production. These results can provide a

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successful approach for producing biologically active hydrolysis enzymes in rice for
alcohol fuel production while solving the desposal and polluting rice straw burning

problems.

FUTURE RESEARCH

The presented research successfully determined the possibility of producing
transgenic oat for salinity tolerance and rice for alcohol ﬁiel production and reduced air
pollution. However, several other questions have emerged and other experiments need to
be conducted to address those questions. It is recommended to test tolerance of the
transgenic oat lines on the ﬁeld level. Also, modern oat cultivars are not available for
poor people in marginal lands, probably because of the presence of cultural bias as well
as the absence of networks, ﬁnance and distribution mechanisms. Therefore, an
international coordinated network is needed to promote and inﬁlse salinity-tolerant GM
oat into poor environments. Regional and local international organizations and
institutions can be a starting point for ﬁnancing the introduction of GM oat and educating
local agricultural communities for its acceptance. This could also be a source of
information for the wider agricultural and scientiﬁc communities, where cultural
anxieties over oat (or any other introduced crop) might be better understood.

Moreover, a designed series of experiments and analyses should give some
insights on the economic and commercial viability of the cellulases’ production in
transgenic rice plants. Other tests are desired to guage the feasibility of either producing
all hydrolysis enzymes in one plant using different cellular compartments or in more than

one plant. Also, one of the important questions that needs to be answered experimentally

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is why production of the cellulase enzyme didn’t harm the transgenic plants, especially
since higher expression levels could be achieved by selection and breeding of the
transgenic lines (Hood and Woodard, 2002). In addition, one of the biggest challenges is
transgene containment and cross contamination with non-transgenic plants in the ﬁeld.
Risk assessment is required to test the accumulation of the cellulases in seeds. Until its
testing and approval, extra care should be taken to assure that industrial and medical
commodities produced in transgenic plants will not be present in the food supply.
Producing transgenic lines using one or more of the bioconﬁnement methods such as
chloroplast transformation, cytoplasmic male sterility, or delay in transgenic-plant

ﬂowering is needed to reduce seed contamination and contraversies over GM crops

(NRC, 2004).

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