suppression of the lignin biosynthetic...
TRANSCRIPT
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SUPPRESSION OF THE LIGNIN BIOSYNTHETIC GENE 4-COUMARATE COA LIGASE (4CL) IN SUGARCANE BY RNA INTERFERENCE
By
YUAN XIONG
A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE
UNIVERSITY OF FLORIDA
2012
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© 2012 Yuan Xiong
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To my parents, Zhiyao, for their support and unfaltering faith in me
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ACKNOWLEDGMENTS
I am very grateful to my major advisor, Dr. Fredy Altpeter, for his continual
guidance, patience, encouragement, and financial support. His knowledge of research
and his enthusiasm for work has been a great source of inspiration for me. I thank my
committee members for their time, effort, and guidance. I also wish to thank Dr. Maria
Gallo and Dr. Robert Gilbert for their encouragement, their keen observations, and
helpful suggestions. Sincere thanks to Dr. Wilfred Vermerris for his instruction in lignin
analysis.
I thank all of my lab members for their assistance and camaraderie that made
the lab an enjoyable working environment. I would especially like to thank Je Hyeong
Jung for his valuable suggestions and demonstrations. I would like to thank Dr. Janice
Zale for her assistance in editing my thesis. Sincere thanks to Dr. Qianchun Zeng for
his instructions in tissue culture, Dr. Jae Yoon Kim for his instructions in molecular
biology, and Dr. Walid Fouad for his instruction in vector construction. I especially
appreciate the friendship of Dr. Hao Wu, Dr. Hangings Zhang, Yang Zhao, Yogesh
Taparia, and Elizabeth Mayers.
There were other people outside of the lab who supported my research project. I
would like to thank Dr. Max Teplitski for his help with the MUG assays, and Drs. John
Erickson and Lynn Sollenberger for providing specific laboratory equipment and
materials. I am grateful to Dr. Jerry M. Bennett for his moral support. Most of all, I
would like to express sincere appreciation to Zhiyao Luo for her continued love, support
and inspiration.
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TABLE OF CONTENTS page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES ............................................................................................................ 7
LIST OF FIGURES .......................................................................................................... 8
ABSTRACT ..................................................................................................................... 9
CHAPTER
1 LITERATURE REVIEW .......................................................................................... 11
Introduction ............................................................................................................. 11
Sugarcane Breeding ............................................................................................... 11
In Vitro Culture of Sugarcane .................................................................................. 13
Genetic Transformation of Sugarcane .................................................................... 14
RNA Interference (RNAi) ........................................................................................ 16
Biofuels ................................................................................................................... 17
Lignin Biosynthesis ................................................................................................. 19
2 COMPARISON OF PROCEDURES FOR DNA COATING OF MICRO-CARRIERS IN THE TRANSIENT AND STABLE BIOLISTIC TRANSFORMATION OF SUGARCANE ................................................................. 22
Introduction ............................................................................................................. 22
Materials and Methods............................................................................................ 24
Tissue Culture and Gene Transfer ................................................................... 24
Selection and Regeneration of Transgenic Plants ............................................ 25
Analysis of Reporter Gene Activity ................................................................... 25
Statistical Analysis ............................................................................................ 26
Results .................................................................................................................... 26
Discussion .............................................................................................................. 27
3 RNAI SUPPRESSION OF 4-COUMARATE COA LIGASE (4CL) IN SUGARCANE ......................................................................................................... 33
Introduction ............................................................................................................. 33
Materials and Methods............................................................................................ 36
Cloning of Sc4CL and Vector Construction ...................................................... 36
Tissue Culture, Biolistic Gene Transfer, and Plant Regeneration ..................... 37
NPTII ELISA ..................................................................................................... 38
Northern Blot Analysis ...................................................................................... 38
Small RNA Northern Blot Analysis ................................................................... 39
Southern Blot Analysis ..................................................................................... 39
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Quantitative Real-Time RT-PCR Analysis ........................................................ 39
Acetyl Bromide (AcBr) Lignin Analysis and Visual Observations ...................... 40
Statistics ........................................................................................................... 41
Results .................................................................................................................... 41
Cloning and Expression Pattern of Sc4CL_2 ................................................... 41
Suppression of Sc4CL_2 by RNAi .................................................................... 41
Analysis of Lignin Content and Evaluation of Phenotype ................................. 42
Discussion .............................................................................................................. 43
4 CONCLUSION ........................................................................................................ 53
APPENDIX LABORATORY PROTOCOLS USED IN MOLECULAR BIOLOGY, BIOLISTICS TISSUE CULTURE, AND IDENTIFICATION/CHARACTERIZATION OF TRANSGENIC
SUGARCANE PLANTS .............................................................................. 56
LIST OF REFERENCES ............................................................................................... 74
BIOGRAPHICAL SKETCH ............................................................................................ 88
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LIST OF TABLES
Table page 2-1 Stable transformation efficiency for three coating treatments. ............................ 29
3-1 Means ± S.E. for plant height, fresh weight, node diameter, AcBr lignin content, and qRT-PCR expression for four transgenic plants and non transgenic sugarcane CP88-1762 control .......................................................... 46
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LIST OF FIGURES
Figure page 2-1 Schematic representation of the uidA expression cassette and the nptII
expression cassette. ........................................................................................... 30
2-2 Transient GUS expression level in sugarcane callus 48 hours after bombardment. .................................................................................................... 31
2-3 Southern blot analysis of the stable transformed sugarcane lines. ..................... 32
3-1 Schematic representation of the sugarcane 4CL partial genomic sequence, Sc4CL_2 cDNA clone and the RNAi suppression cassette. . ............................ 47
3-2 The expression pattern of Sc4CL_2 in non transgenic control plants. ................ 48
3-3 Vegetative progeny of transgenic sugarcane lines with RNAi suppression of Sc4CL-2 grown under controlled environment conditions. ................................. 49
3-4 Northern blot analysis. ........................................................................................ 50
3-5 Southern blot analysis of independent sugarcane transformants. ...................... 51
3-6 Small RNA northern blot.. ................................................................................... 52
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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science
SUPPRESSION OF THE LIGNIN BIOSYNTHETIC GENE
4-COUMARATE COA LIGASE (4CL) IN SUGARCANE BY RNA INTERFERENCE
By
Yuan Xiong
August 2012
Chair: Fredy Altpeter Cochair: Maria Gallo Major: Agronomy
Sugarcane (Saccharum spp.) is one of the most productive herbaceous monocots
used as the main source of table sugar and as a feedstock in the production of ethanol.
In this study, DNA coating protocols for biolistic gene transfer were compared, and were
used to produce transgenic sugarcane with decreased lignin content, presumably more
amenable to fermentation into cellulosic ethanol. Biolistic gene transfer is a relatively
rapid, genotype-independent method of producing transgenic plants. Three methods of
precipitating DNA onto gold microparticles were compared in biolistic gene transfer:
spermidine free base, protamine sulfate, and the proprietary Seashell DNAdelTMGold
Carrier. For stable transformation using minimal, linear expression cassettes, protamine,
spermidine and the Seashell DNAdelTMGold carrier were equally effective. For transient
gene expression, spermidine resulted in significantly higher efficiencies compared to the
other two protocols. Stable transgenic sugarcane lines were produced using biolistic
gene transfer of an RNAi hairpin construct, targeted to downregulate the 4-coumarate
CoA Ligase (4CL) gene involved in lignin biosynthesis. The transgenic sugarcane lines
were characterized by Southern blot, northern blot, siRNA northern blots, and
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quantitative Real-time RT-PCR. Three lines with strong suppression of 4CL and one line
with moderate suppression of 4CL were selected for lignin analysis. A trend of lignin
reduction was observed in all four transgenic sugarcane lines, with two of them
producing significantly less lignin (5-9%) than non transgenic control. Lignin reduction of
5% in transgenic sugarcane did not compromise biomass production under controlled
environment conditions. The results presented here indicate successful RNAi
suppression of the targeted 4CL gene of the highly polyploid sugarcane and its potential
prospects for crop improvement.
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CHAPTER 1 LITERATURE REVIEW
Introduction
In 2010, the Food and Agriculture Organization (FAO) reported that a total of 1.7
billion tons of sugarcane were produced on 23.8 million hectares (ha) of cultivated land
worldwide (FAO 2010). The five largest sugarcane producing countries, in decreasing
order, are Brazil, India, China, Thailand and Mexico (FAO 2010). The average
sugarcane yield in Brazil was 79.1 tons/ha. This yield was higher than the sweet
sorghum yield in China (65 tons/ha) and the corn yield in the United States (95 tons/ha)
(FAO 2010; Dal-Bianco et al. 2012). Sucrose in sugarcane is the source of
approximately 70% of the world’s sugar production, and in 2011, approximately 54% of
the industrialized sugarcane production was dedicated to sucrose-based biofuel
production to generate 27.67 billion liters of bioethanol (Dal-Bianco et al. 2012). It has
been recognized that the use of sugarcane residues will be a valuable source of
cellulosic materials for bioethanol production (Altpeter and Oraby 2010).
Sugarcane Breeding
The genus Saccharum is composed of six species Saccharum officinarum,
Saccharum spontaneum, Saccharum robustum, Saccharum barberi, Saccharum
sinense and Saccharum cinarum (Tew and Corbill, 2008). Cultivated sugarcane
(Saccharum spp. hybrids) is a highly polyploid interspecific hybrid with genetic
contributions from these species and other related grass genera (Brandes 1958;
Purseglove 1972; Daniels and Roach 1987; Altpeter and Oraby 2010). Current
sugarcane cultivars are derived from crosses between S. officinarum and S.
spontaneum to incorporate traits for higher yield and pathogen resistance. The
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interspecific hybrids are backcrossed to S. officinarum several times to reconstitute the
S. officinarum genetic background (Matsuoka et al. 2009). Traditional breeding of
sugarcane is impeded by a narrow gene pool and complex genome because most
cultivars are derived from a relatively small group of progenitor clones (Jackson 2005).
Other factors such as infertility, long breeding cycles, and cold intolerance also limit
breeding advancements of some sugarcane lines.
Despite these limitations, conventional breeding of sugarcane has significantly
improved yield, sugar content, ratooning ability, and disease resistance over the last
three to four decades (Jackson 2005; Lakshmanan et al. 2006; Ming et al. 2006). In
Brazil, sugarcane yield and sugar content have increased 69% and 34%, respectively,
during the last 35 years (FAO 2010; Dal-Bianco et al. 2012).
Biotechnology and molecular breeding have become important tools for
introducing new traits and accelerating sugarcane breeding. Through genetic
engineering and marker-assisted selection, sugarcane lines with improved pathogen
resistance, herbicide tolerance, increased sucrose yields, and floral inhibition have been
regenerated and evaluated in the field (Matsuoka 2009). Because sugarcane is
vegetatively propagated, this prevents the problem of segregating progeny and reduces
the risk of introducing transgenes into the environment (Bonnett et al. 2008). However,
The complexity of the sugarcane genome also can impede the use of certain molecular
tools (Arruda 2011). For example, it is difficult to sequence sugarcane because of its
highly polyploid and aneuploid genome (Dal-Bianco et al. 2012). Other limitations
include relatively low transformation efficiencies and transgene silencing (Hota et al.
2011). Therefore, strategies such as the use of molecular markers, whole genome shot-
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gun sequencing (WGS) and BAC-to-BAC map-based cloning are expected to contribute
to sugarcane molecular breeding in the future (Dal-Bianco et al. 2012). Sugarcane
genome databases like the SUCEST-database also have proven useful in molecular
studies of sugarcane (Nishiyama et al. 2010).
In Vitro Culture of Sugarcane
In vitro culture is a valuable tool for the genetic engineering of sugarcane.
Substantial progress has been made in utilizing young meristematic tissues which have
the ability to develop into whole plants via embryogenesis or organogenesis (Altpeter
and Oraby 2010).
The first reports of in vitro culture in sugarcane were published 1969 (Barba and
Nikell 1969; Heinz and Mee 1969). Somatic embryos can be formed via indirect
embryogenesis, with a callus stage, or direct embryogenesis, without a callus stage.
During indirect embryogenesis, somatic embryos are derived from pro-embryogenic
masses induced from single cells (Vasil and Vasil 1994). Plant regeneration from calli
derived from immature inflorescences (Gallo and Irvine 1996), basal shoot meristems
(Arencibia et al. 1998) and immature leaves (Snyman et al. 2001) have been reported.
The embryogenic calli induced from these protocols have been widely used as starting
materials for transformation experiments. Nevertheless, limitations include long periods
of callus culture increasing the possibility of somaclonal variants and genotypic
restrictions (Snyman et al. 2006). Alternately, direct somatic embryos derived from
single cells also have been reported (Ho and Vasil, 1983). Compared to indirect
embryogenesis, direct embryogenesis generally produces plants that are more
genetically uniform (Vasil et al. 1987), require no time in the callus phase, and as a
consequence, produce fewer somaclonal variants (Burner 1995). Protocols for direct
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embryogenesis in sugarcane have been established with immature leaf whorls (Snyman
et al. 2006; Taparia et al. 2012) and segments of immature inflorescence (Desai et al.
2004). Embryogenesis is also efficient in eliminating viruses from the explants
(Goussard and Wiid 1992; Strandberg 1993). This is because the vascular system
between somatic embryos and explants are not interconnected (Newton and Goussard
1990).
Organogenesis includes three steps: dedifferentiation, re-entry of quiescent cells
into the cell cycle, and determination of specific organ formation (Sugiyama 1999;
Altpeter and Oraby 2010). Organogenesis has been extensively used in the commercial
propagation of sugarcane, primarily using nodal buds which also eliminates viruses
(Lakshamanan et al. 2005).
Genetic Transformation of Sugarcane
Agrobacterium-mediated and biolistic transformation protocols are the most
common gene delivery methods in plant genetic engineering (Vain 2007). In general,
Agrobacterium-mediated transformation generates transgenic plants with simple
transgene integration patterns, but this procedure may be genotype dependent. In
contrast, biolistic gene transfer generally produces more complex transgene integration
patterns. However it is less genotype dependent, and vector construction is more facile
as multiple unlinked transgene expression cassettes can be co-bombarded, which is
useful in pathway engineering (Wu et al. 2002; Datta et al. 2003; Altpeter et al. 2005).
Improvements of, and modifications to, sugarcane transformation protocols have been
reported (Khanna et al. 2008; Kim et al. 2012; Taparia et al. 2012).
Biolistics is the preferred method of gene delivery because a variety of target
tissues can be used and a wide range of genotypes are amenable to transformation
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(Lakshmanan et al. 2006). Pioneering studies in biolistic gene transfer to sugarcane
were reported in the 1990’s (Bower and Birch 1992; Gallo-Meagher and Irvine 1993;
Gallo-Meagher and Irvine 1994; Gallo and Irvine 1996; Meagher et al. 1996). Following
these reports, biolistics has been used to deliver novel genes into sugarcane. Various
traits such as insect or disease resistance (Nutt et al. 1999; Setamou et al. 2002; Falco
and Silva-Filho 2003; Weng et al. 2006; Gilbert et al. 2009), herbicide tolerance (Falco
et al. 2000; Leibbrandt and Snyman 2003), and drought tolerance (Correa Molinari et al.
2007; Wu et al. 2008). Also the accumulation of sucrose or sucrose isomers (Ma et al.
2000; Groenewald and Botha 2008; Hamerli and Birch 2011; Basnayake et al. 2012),
cellulolytic enzymes (Mark et al. 2011) and other valuable molecules (Lars A Petrasovits
et al. 2012) have been goals of transformation experiments. Embryogenic callus is the
preferred tissue for biolistic transformation due to the high plant regeneration potential.
Successful transformation of sugarcane has also been achieved using cell suspension
cultures which readily regenerate plants (Chowdhury and Vasil 1992), apical meristems
(Gambley et al. 1993), leaf whorls, and inflorescences (Elliott et al. 2002).
The first reports of Agrobacterium-mediated sugarcane transformation were
published in 1998 (Arencibia et al. 1998; Enriquez-Obregon et al. 1998). Arencibia et al.
(1998) generated transgenic sugarcane by co-cultivation of calli with Agrobacterium
strains LBA4404 and EHA101. Enriquez-Obrigon et al. (1998) co-cultivated immature
leaf explants with Agrobacterium strain GV2260. Agrobacterium-mediated
transformation of sugarcane was also reported using axillary buds as explants
(Manikavasagam et al. 2004). Agrobacterium-mediated transformation has been widely
used in sugarcane improvement, such as for pathogen resistance, protein expression,
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and herbicide tolerance (Enriquez et al. 2000; Manickavasagam et al. 2004; Kalunke et
al. 2009). In addition, Agrobacterium-mediated transformation has been a tool in
functional genomics and to alter metabolic regulatatory mechanisms in sugarcane
(Melotto-Passarin 2009; Wang et al. 2009).
RNA Interference (RNAi)
RNA interference (RNAi) is an RNA-dependent gene silencing phenomenon
conferred by double stranded RNA (dsRNA) that occurs in plants, worms, insects, and
mammals. It is a mechanism that induces resistance to pathogens, genetic regulation,
and gene silencing (Kusaba 2004). The phenomenon of RNAi was first reported as
coat-protein-mediated virus protection in transgenic plants (Ecker and David 1986) and
since then, the mechanisms of RNAi have been elucidated (Fire and Mello 1998).
The common mechanism for all RNAi phenomena is the endonucleolytic
fragmentation of long dsRNAs into small interfering RNAs (siRNAs) by a ribonuclease
enzyme, Dicer (Kusaba 2004; Watanabe 2011). Dicer binds to the long dsRNA and this
triggers cleavage into 19-25 bp siRNA fragments (Berstain et al. 2001). The siRNAs
are then unwound into single stranded RNAs (ssRNAs) that bind to an enzyme complex,
the RNA-induced silencing complex (RISC) (Kusaba 2004). The activated RISC-siRNA
complex binds to complementary mRNAs within the cytoplasm to trigger additional
endonucleolytic cleavage reactions until the majority of the target mRNAs are degraded.
This process silences gene expression by preventing translation of a gene or gene
family.
MicroRNAs (miRNAs) are endogenous siRNA-like, non-coding RNAs involved in
genetic regulation of developmental growth stages and in physiology responses
(Kusaba 2004; Poethig et al. 2006). They are structurally similar to siRNAs and share
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similar mechanisms of targeted mRNA degradation. Primary miRNAs (pri-miRNAs) are
transcribed and folded into 70-nucleotide stem-loop RNA structures, pre-mRNAs
(Gregory et al. 2006). The dsRNA portion of this structure is recognized by Dicer and is
cleaved into fragments as mature miRNAs. These miRNAs are less specific in base
pairing with target mRNAs than siRNAs.
siRNAs and miRNAs are important mechanisms of silencing gene expression.
The former can be applied in genetic engineering and functional genomics to
knockdown, rather than knockout, gene function, while the latter is an essential, innate
mechanism of gene silencing and gene regulation during plant growth and development
(Voorhoeve and Agami 2003; Watanabe 2011).
Biofuels
The depletion of fossil fuels and society’s growing energy demands have forced
technological developments in the renewable energy sectors such as plant biomass,
plant oils, wind, hydro-electric, solar, and geothermal energy. Cellulosic bioethanol and
biodiesel produced by the utilization of lignocellulosic feedstocks and plants oils,
respectively, could potentially replace part of petroleum consumption (Sarkar et al.
2012). In addition, biofuels are forecast to reduce 50% of greenhouse gas emissions
compared to emissions from fossil fuels (EPA 2010).
Currently, the bioethanol produced from starch and sucrose-based technologies
are the most widely used biofuels in motor vehicles. Global production of bioethanol
reached approximately 88 billion liters in 2011 (RFA 2011a). The US and Brazil are the
two largest producers, responsible for 85% of global bioethanol production (RFA 2011b).
In the US, significant quantities of bioethanol are produced from starch derived from
corn, whereas in Brazil, bioethanol is produced from sucrose found in sugarcane.
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In order to increase bioethanol production, and avoid the competition of food
versus fuel commodities grown on limited farmland at escalating prices, it is desirable to
produce bioethanol from lignocellulosic biomass (Schubert 2006). Lignocellulosic
biomass is an inexpensive, abundant, and renewable source material. This feedstock
includes grasses, wood chips, sawdust, and crop residues such as sugarcane bagasse,
and straw from corn, rice, or wheat (Sarkar et al. 2012). It is estimated that by using
lignocellulosic biomass, ethanol yields could be drastically increased (Kim et al. 2004;
Vermerris 2008).
Due to its inherent strength and diverse polymeric structure, there are limitations to
the utilization of lignocellulosic feedstocks. Lignocellulose is a complex structure in
which the polysaccharides (cellulose and hemicellulose) are crosslinked with the lignin
via ester and ether linkages. Lignin imparts rigidity to the cell wall, provides stiffness
and strength to the stem, defense against pathogens, and water transport within the
xylem (Boerjan et al. 2003). Lignin is the most recalcitrant component in the cell wall
and it impedes enzymatic hydrolysis of cellulose and hemicellulose. Altering the lignin
composition or lignin content, as well as the type of bonds within the lignin, cellulose
and hemicellulose polymers are promising strategies to produce high quality biomass
feedstock (Vermerris et al. 2007; Hisano et al. 2009).
The production of ethanol from lignocellulosic feedstocks essentially consists of
three steps: pretreatment, enzymatic hydrolysis or saccharification, and fermentation.
Pretreatment breaks down the cell wall matrix, rendering it more accessible to
hydrolysis. Different pretreatments such as steam explosion, liquid hot water extraction,
acid and alkali treatments, physical methods, a wet oxidation method, and biological
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degradation have been tested on different lignocellulosic materials (Sarkar et al. 2012).
In enzymatic hydrolysis, cellulases convert the polysaccharides present in cellulose and
hemicellulose to simple monomeric sugars, primarily glucose and xylose, respectively,
in addition to other minor sugars. These monomeric sugars are fermented to ethanol by
microorganisms in the final step.
There is room for improvement in almost every step of bioethanol production from
lignocellulosic feedstocks (Schubert 2006). Current research to optimize the processes
include developing novel cellulases, bioengineering microbes for fermentation with
broader substrate specificities and higher efficiencies, reducing the inhibitors of
fermentation, designing higher-quality biomass feedstocks, and implementing novel
pretreatment technologies. It is believed that these efforts will eventually decrease the
costs associated with cellulosic ethanol production.
Lignin Biosynthesis
Lignin is a phenolic polymer mainly present in the cell walls of vascular plants
(Zhao and Dixon, 2012). It is primarily composed of three hydroxycinnamyl alcohols:
coniferyl alcohol, sinapyl plants is variable depending on cell type and species.
During the last decade, revisions to the lignin biosynthetic pathway have
continually been made because of new results obtained from mutant analyses and
reverse genetics in a variety of species (Vanholme et al. 2010). Monolignols are
biosynthesized from the amino acid phenylalanine via the phenylpropanoid and the
monolignol pathways (Umezawa 2009). It is currently thought that twelve major gene
families are involved in the lignin biosynthetic pathway (Zhao and Dixon, 2012). The
phenylpropanoid pathway is also important for the biosynthesis of plant secondary
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compounds such as coumarins, suberins, flavonoids, isoflavonoids, and stilbenes (Boud
et al. 2007; Umezawa 2009).
Recent data suggest that lignin biosynthesis is regulated by various transcription
factors. AC-rich promoter elements, which are the binding motifs for MYB transcription
factors, are found in most lignin biosynthetic genes except ferulic acid 5-hydroxylase
(F5H) (Zhao and Dixon, 2012). Various transcription factors appear to act differently.
For example, transcription factors MYB46 andMYB83 can activate the entire formation
of secondary cell walls, while AtMYB85, AtMYB58 and AtMYB63 are lignin specific
(Zhong et al. 2008; McCarthy et al. 2009; Zhou et al. 2009; Zhao and Dixon, 2012).
Additionally, lignin deposition in response to various stimuli may be regulated by the
binding of activators such as NAC transcription factor (NST) or its homolog VND, or it
can be downregulated by lignin repressors such as AtMYB32, which acts in non-lignified
floral tissue (Zhao and Dixon, 2012). Additionally, the activities of lignin transcriptional
factors are closely related to the developmental stage of the plant, the environment
condition, and the hormone status of the plant (Zhao and Dixon, 2012).
To date, the exact mechanism of transport of monolignols to the cell wall where
they become polymerized into lignin, remains largely unknown (Ehlting et al. 2005;
Lanot et al. 2006; Kaneda et al. 2008). Lignin polymerization starts with the oxidization
of monolignol phenols, generating phenolic radicals. Subsequently, single monomer
radicals couple to form dimers by establishing covalent bonds between the subunits
(Vanholme et al. 2010). Following dimerization, they are dehydrogenated by
peroxidases/laccases into phenolic radicals that connect to other monomer or dimer
radicals to form the lignin polymers (Vanholme et al. 2010).
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The depletion of fossil fuels and society’s growing energy demands have
stimulated technological developments in the renewable energy sectors such as plant
biomass, plant oils, wind, hydro-electric, solar, and geothermal energy. Cellulosic
bioethanol and biodiesel produced by the utilization of lignocellulosic feedstocks and
plants oils, respectively, could potentially replace petroleum (Sarkar et al. 2012). In
addition, biofuels are forecast to reduce 50% of greenhouse gas emissions compared to
emissions from fossil fuels (EPA 2010).
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CHAPTER 2 COMPARISON OF PROCEDURES FOR DNA COATING OF MICRO-CARRIERS IN THE TRANSIENT AND STABLE BIOLISTIC TRANSFORMATION OF SUGARCANE
Introduction
Biolistic transformation (also termed microparticle bombardment) is a direct plant
transformation method (Klein et al. 1987). With this technique, plasmid DNA is
precipitated onto particles in the presence of positively charged polyamine and buffer
followed by bombardment of plant cells with DNA-coated particles. Biolistic
transformation has become an important tool for studying gene expression and for crop
improvement (Taylor et al. 2002). Compared to Agrobacterium-mediated transformation,
biolistic transformation is less genotype dependent (Altpeter et al. 2005) and vector
construction is simplified since multiple unlinked transgene expression cassettes can be
co-bombarded (Datta et al. 2002; Wu et al. 2002).
In preparation for biolistic transformation, plasmid DNA is typically precipitated
onto gold or tungsten micro-particles in the presence of spermidine and CaCl2 (Klein
et.al 1987; Finer et.al 1992; Sanford et al. 1993). Spermidine is a cationic polyamine,
naturally produced in all eukaryotes and most prokaryotes and plays an important role
in germination, root development, flowering and abiotic/biotic stress resistance (Minois
et al. 2011; Wimalasekera et al. 2011). Spermidine stabilizes the DNA double helix
(Tabor 1962) and leads to its compaction and precipitation following electrostatic
interactions (Razin and Rosansky 1959). Spermidine also prevents activation of
endonucleases and DNA fragmentation (Brune et al. 1991). The use of spermidine for
biolistic transformation was first described by Sanford and collaborators in order to
promote DNA precipitation onto microparticle (Klein et.al 1987). It has become the
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standard coating reagent in biolistic transformation (Sivamani et al. 2009). Although Perl
et al. (1992) reported higher transformation efficiency without spermidine, subsequent
studies concluded that spermidine increases transformation efficiency (Rasco-Guant et
al. 1999; Sivamani et al. 2009). The lack of spermidine during DNA coating resulted in
decreased precipitation efficiency and loss of DNA during the washing steps (Rasco-
Guant et al. 1999).
As an alternative to spermidine, other cationic polymers have been used to
condense and stabilize DNA for delivery in animal studies (Wagner et al. 1991). More
recently protamine was used for precipitation of DNA onto gold particles prior to biolistic
gene transfer into maize and rice (Sivamani et al. 2009). Protamine is a small arginine-
rich protein produced in spermatids where it contributes to condensation of the genome
(Balhorn 2007). The protamine-coated plasmid DNA resisted degradation by DNase
longer than spermidine-coated plasmid DNA (Sivamani et al. 2009).
Seashell DNAdelTM Gold Carrier Particles (Seashell Biotechnology, La Jolla, CA)
have a -COOH terminal modification that has a neutral charge in the binding buffer and
is negatively charged at physiological pH. The proprietary precipitation buffer supplied
with the DNAdelTM Gold Carrier Particles is free of spermidine (free base) or protamine
used in the other DNA precipitation methods. In this study, transient and stable
transformation efficiencies for sugarcane were evaluated following the precipitation of
minimal transgene expression cassettes onto gold particles using protamine,
spermidine or Seashell DNAdelTM (Seashell) protocols.
24
Materials and Methods
Tissue Culture and Gene Transfer
Sugarcane embryogenic callus was induced from immature leaf cross sections
on modified MS basal medium (CI3) (Chengalrayan and Gallo-Meagher 2001) at 28ºC
under illumination (150 µmol m-2 s-1) with a 16 h light/8 h dark regime.Transient GUS
expression was assayed as described by Allen et al. (1993). Embryogenic callus was
bombarded with uidA (Jefferson et al. 1987) under the transcriptional control of the
maize ubiquitin promoter (Christensen et al. 1992) with first intron (Toki et al. 1992) and
NOS 3’UTR (Bevan 1984) (Fig. 2-1 A), following 4 hours of osmotic treatment on CI3
medium (72.7 g L-1 D-sorbitol). Bombardments were performed with the PDS-1000/He
Particle Delivery System (Bio-Rad, Hercules, CA) according to the protocol described
by Sandhu and Altpeter (2009). Calli (25-30) were placed in the center of the petri dish
to cover a circular area of 4.6 cm2 per bombardment. Gold particles (1 µm diameter,
Bio-Rad) were suspended in 50% glycerol (v/v). The suspension of gold particles was
vortexed and 1.8 mg 30 µL-1 were transferred to a 1.5 mL Eppendorf tube (Eppendorf,
Hamburg, Germany). The uidA cassette DNA (1 µg 30 µL-1) was transferred to the
suspension of gold particles and mixed by vortexing for 10 sec. Then 20 µL of 0.1 M
spermidine (Sigma, St. Louis, MO) or 20 µL of protamine (1 mg mL-1) (Sigma) and 50
µL of 2.5 M CaCl2 were added for precipitation of DNA onto the gold particles during
continuous vortexing (1 min). Spermidine and protamine were used in concentrations
that resulted in the highest transformation efficiency in earlier experiments by Sivamani
et al. (2009). The mixture was centrifuged briefly and washed 2 times with absolute
ethanol. The DNA coated gold particles were resuspended in 90 µL absolute ethanol by
sonication for 2 s (Branson, Danbury, CT). Alternatively, DNA was precipitated onto
25
Seashell DNAdelTM gold particles using the protocol provided by the manufacturer
(http://www.seashelltech.com/protocols.shtml). In brief, 1.5 mg of Seashell DNAdelTM
gold particles (s1000d) were suspended in 50 µL binding buffer (20 mM sodium acetate,
pH 4.5) and mixed with 1 µg of the uidA cassette DNA in a 1.5 mL tube by vortexing for
10 s. An equal volume of precipitation buffer was added and incubated at room
temperature for 3 min. The DNA-particle mixture was washed and resuspended
following the same protocol described for spermidine/protamine coating. Following
bombardment, callus was transferred to CI3 medium.
Selection and Regeneration of Transgenic Plants
For stable transformation, callus induction and particle bombardment were carried
out with the same procedure described for transient transformation. Callus was
bombarded with a nptII expression cassette (Fraley et al. 1983) under the control of the
maize ubiquitin promoter with first intron and CaMV 35S 3’UTR (Dixon et al. 1986) (Fig.
2-1 B). Callus selection and plant regeneration were performed as described by Kim et
al. (2012). In brief, bombarded callus was transferred to CI3 medium without selective
agent for 1 week followed by three biweekly subcultures on CI3 medium containing 20
mg L-1 geneticin. Callus regeneration occurred after two to three biweekly subcultures
on hormone free medium containing 20 mg L-1 paromomycin and formed roots after 2-4
additional biweekly subcultures on paromomycin containing hormone free medium.
Analysis of Reporter Gene Activity
All calli from each bombarded plate were transferred 48 h after bombardment
into one 2 mL tube (Eppendorf) followed by protein extraction (Jefferson et al. 1987).
Protein concentration of each sample was determined using CoomassiePlus Protein
Assay reagent (Thermo Fisher Scientific Inc., Rockfort, IL) and bovine serum albumin
26
standards. Sample fluorescence was measured with a VICTORTM X3 Multilabel Plate
Reader (Perkin Elmer, Waltham, MA) using 365 nm excitation; 460 nm emission filters
after 0, 30 and 60 min incubation with MUG assay buffer (Sigma) in the dark. GUS
activity was normalized by subtracting the average fluorescence reading of negative
control samples (bombarded without uidA).
Statistical Analysis
Statistical analysis was performed using the SAS system (SAS 2009) and the
general linear model (GLM) procedure with the t-test was applied.
Results
Spermidine resulted in 3 times higher transient GUS expression than protamine
and 2.65 times higher expression than the Seashell method on average over 10 coating
reactions per treatment (Fig. 2-2). Protamine coating did not result in significant different
transient GUS activity than Seashell coating (p< 0.05).
Initially, the transgenic nature of the regenerated lines was confirmed by PCR and
NPTII ELISA (data not shown). From a total of three independent experiments and 30
bombardments per treatment 53, 48 and 41 independent transgenic sugarcane lines
were produced following spermidine, protamine or Seashell coating reactions,
respectively (Table 2-1). The three coating reactions did not differ significantly in stable
transformation efficiency (p<0.05).
For Southern blot hybridization analysis, six lines derived from the spermidine
treatment, four lines from the protamine treatment and four lines from the Seashell
treatment were randomly selected. In contrast to genomic DNA from non-transgenic
sugarcane, all of the transgenic lines displayed hybridization signals following digestion
with EcoRI and hybridization with nptII. The pattern of the hybridization signals differed
27
for all the evaluated lines. The number of nptII hybridization signals ranged from one to
more than 10. A trend for increased complexity following protamine coating compared to
Seashell and spermidine was observed with this relatively small number of evaluated
samples (Fig. 2-3).
Discussion
The procedure that is used for precipitation of DNA onto gold micro-particles prior
to biolistic transformation may impact transformation success and efficiency (Rasco-
Guant et al. 1999; Sivamani et al. 2009). Typically DNA is precipitated onto micro-
particles in the presence of spermidine and CaCl2 for biolistic transformation of plants
(Klein et al. 1987; Finer et al. 1992; Sivamani et al. 2009) including sugarcane (Bower
and Birch 1992). While the earlier publications utilized full plasmids for biolistic gene
transfer, recently minimal expression cassettes without vector backbone were
introduced into sugarcane (Kim et al. 2012) and other crops (Fu et al. 2000; Romano et
al. 2003; Lowe et al. 2009). Sivamani et al. (2009) reported that by using a cationic
polymer protamine, instead of spermidine for biolistic transfer of full plasmids to rice and
maize resulted in a over 5-fold increase in transient transformation efficiency and 3.3-
fold increases in stable transformation efficiency. Our observations in sugarcane
suggest that for stable transformation of minimal, linear expression cassettes by biolistic
transfer to sugarcane protamine, spermidine and Seashell protocols are equally
effective. In contrast to the earlier report the current study utilized linear DNA molecules.
Interestingly in our observations, spermidine resulted in significantly higher transient
expression than the other two precipitation protocols. This may suggest that spermidine
may be more efficient in precipitation of the linear minimal expression cassette DNA.
The fact that this does not translate into significantly more stable transgenic events
28
compared to the two other methods could be explained by inferior protection by
spermidine against degradation compared to the other coating agents. Sivamani et al.
(2009) found that protamine coating protects plasmid DNA longer from degradation by
DNase than spermidine. Southern blot results suggest that protamine coating may
contribute to increased copy number when compared to Seashell and spermidine. The
copy number following biolisitic gene transfer depends on the quantity of the DNA that is
delivered to the nucleus (Sandhu and Altpeter 2008, Lowe et al. 2009, Kim et al. 2012).
Further experiments should clarify if differences observed after the different coating
reactions are associated with the precipitation efficiency, release of the DNA from the
gold particle or stability of the delivered DNA in the cellular environment. Further
optimizations of the coating protocols may be possible by changing the concentration of
DNA, cationic polyamine or CaCl2 (Zlatanova et al.1998; Makita et al. 2009).
In conclusion, we observed that DNA precipitations using spermidine, protamine or
the Seashell DNAdelTM protocol are similarly effective for stable genetic transformation
of sugarcane by biolistic gene transfer.
29
Table 2-1. Stable transformation efficiency for three coating treatments.
Treatment Experiment Number of
bombardments
Number of transgenic plants
Number of transgenic plants/ bombardment
Spermidine
I 8 13 1.63 II 8 15 1.88 III 14 25 1.79
Spermidine total 30 53 1.76 ± 0.07a
Protamine
I 8 23 2.88 II 8 9 1.13 III 14 16 1.14
Protamine total 30 45 1.71 ± 0.58a
Seashell DNAdelTM
I 8 7 0.88 II 8 9 1.13 III 14 25 1.79
Seashell DNAdelTM total 30 41 1.26 ± 0.27a
30
Figure 2-1. A, Schematic representation of the uidA expression cassette used for transient transformation assays. B, Schematic representation of the nptII expression cassette for stable transformation analysis. The black line underneath nptII gene represents the nptII ORF region (703 bps) that was used as probe in Southern blot hybridization.
31
Figure 2-2. Transient GUS expression in sugarcane callus 48 hours after bombardment.
Gold particles were coated with expression cassette DNA using spermidine or protamine or following the Seashell DNAdelTM protocol. Each column represents the mean value of 10 different coating reactions per treatment. The standard error bar is shown. Significant differences at P<0.05, are indicated by different letters.
32
Figure 2-3. Southern blot analysis of stable transformed sugarcane lines. Genomic DNA was hybridized with a radiolabeled 703 bp nptII probe following restriction digestion with EcoRI. M, 2-Log DNA ladder (0.1-10.0kb); wt, non transgenic CP88-1762 sugarcane control; 1-6, sugarcane transformed with spermidine; 7-10, sugarcane lines transformed with protamine; 11-14, sugarcane transformed with Seashell DNAdelTMGold carrier.
33
CHAPTER 3
RNAI SUPPRESSION OF 4-COUMARATE COA LIGASE (4CL) IN SUGARCANE
Introduction
The US and Brazil produce the vast majority of the world’s fuel ethanol by utilizing
corn and sugarcane as feedstocks, respectively (Valdes 2011). These first-generation
energy crops can be efficiently converted to biofuels, because the sucrose stored in
sugarcane internodes can be directly fermented into ethanol and the corn starch can be
readily converted to fermentable glucose (Bai et al. 2008; Ra et al. 2012).
The Environmental Protection Agency (EPA) considers ethanol produced from
sucrose in sugarcane to be an advanced biofuel that reduces greenhouse gas
emissions by more than 50% compared to gasoline (EPA, 2010). The energy yield per
land area of sugarcane ethanol is four to six times greater than that of corn based
ethanol (von Blottnitz and Curran, 2006; Macedo and Seabra, 2008; Shapouri et al.,
2010; Valdes 2011). To further increase the sugarcane-derived ethanol yield per land
area, it is desirable to include the structural polysaccharides, cellulose and
hemicellulose from the abundant lignocellulosic sugarcane residues in the conversion
process (Byrt et al. 2011). Cellulose and hemicellulose represent approximately 70-
80% of the total biomass in sugarcane bagasse (Peng et al. 2009).
Lignin is the most recalcitrant component of the plant cell wall, impeding the
saccharification of cellulose and hemicellulose. Lignin content and composition
influence the conversion efficiency to biofuels. In grasses, ferulic and p-coumaric acids
are esterified to hemicelluloses and lignin, respectively (Jeffries 1990). Lignin is
essential for the structural integrity of the cell wall, the stiffness and strength of the stem,
34
enabling water transportation through the vascular system, and it contributes to
pathogen defense (Boerjan et al. 2003).
From a biofuels perspective, high-quality biomass refers to a composition that can
be easily and inexpensively converted to liquid transport fuels. This depends on the
accessible yield of monosaccharides and disaccharides, and the amount of easily
extractable polysaccharides (Byrt et al. 2011). Lignin is one of the most important
factors in the recalcitrance of plant cell walls against polysaccharide utilization (Withers
2012). Therefore, genetic modifications resulting in reduced lignin content or altered
composition will likely improve the quality of lignocellulosic biomass (Hisano et al. 2009).
Lignin is a phenolic polymer primarily synthesized from three p-hydroxycinnamyl
alcohols (monolignols), p-coumaryl alcohol, coniferyl alcohol, and sinapyl alcohol via
combinatorial radical coupling reactions (Ralph et al. 2004a). In the current view of
lignin biosynthesis, 12 gene families are involved in this pathway: phenylalanine
ammonia lyase (PAL); cinnamate 4-hydroxylase (C4H); 4-coumarate-CoA ligase (4CL);
cinnamoyl CoA reductase (CCR); hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl
transferase (HCT); coumarate 3-hydroxylase (C3H); caffeoyl CoA 3-O-
methyltransferase (CCoAOMT); ferulate 5-hydroxylase (F5H); caffeic acid 3-O-
methyltransferase (COMT); cinnamyl alcohol dehydrogenase (CAD); peroxidase (PER);
and laccase (LAC) (Zhao and Dixon 2012).
The suppression of lignin biosynthetic genes has been successful in increasing
pulping efficiency, producing forages with better digestibility, or promoting
saccharification efficiency of lignocellulosic biofuel feedstocks (Pilate et al. 2002; Chen
et al. 2003; Fu et al. 2011). Recalcitrance to both acid pretreatment and enzymatic
35
digestion is directly proportional to the amount of lignin present in alfalfa biomass (Chen
and Dixon 2007).
4-Coumarate CoA ligase (4CL) catalyzes thioester formation from
hydroxycinnamic acid in the last step of the general phenylpropanoid pathway (Wagner
et al. 2009), providing precursors for various monolignols and plant secondary
metabolic compounds such as coumarins, suberins, flavonoids, isoflavonoids and
stilbenes (Dixon and Reddy 2003; Boudet 2007; Umezawa 2010). The 4CL gene family
has multiple isoforms with different substrate specificities, directing metabolic flux from
the general phenylpropanoid pathway into various branch pathways (Ehlting et al. 1999).
Transgenic suppression of 4CL has been reported in several dicots. In tobacco,
suppression of 4CL resulted in a 25% decrease in lignin content and brown color in the
xylem of transgenic plants (Kajita et al. 1996). Antisense suppression of 4CL in
Arabidopsis caused a 20-30% reduction of lignin without inducing changes in plant
phenotype (Lee et al. 1997). In transgenic aspen, up to a 45% reduction of lignin and
accumulation of cellulose was observed as a result of the depletion of 4CL activity (Hu
et al. 1999; Li et al. 2003). In contrast to aspen, a strong decrease in lignin content in
Pinus radiata resulted in dwarf trees and the collapse of the water conducting vessels
(Wagner et al. 2009).
While there are numerous studies describing 4CL suppression via RNAi and other
methods in dicots, only a few studies have been reported in monocots. RNAi
suppression of 4CL in switchgrass resulted in less lignin, no significant alteration in
biomass yield, and improved yields of fermentable sugars (Xu et al. 2011). In rice,
suppression of 4CL resulted in less lignin, reduced fertility, reduced plant height,
36
decreased 1000 grain weight, and increased cellulose expression in the walls of
sclerenchyma cells (Gui et al. 2011).
Materials and Methods
Cloning of Sc4CL and Vector Construction
Sequences of 4-coumarate: coenzyme A ligase (4CL) from Arabidopsis thaliana
(Q42524 and Q9S725), Nicotiana tabacum (O24145 and O24146), and Oryza sativa
(P17814 and Q42982) were retrieved from GenBank and aligned to identify conserved
regions for the design of degenerate primers. Degenerate primers were designed to
anneal to the conserved regions 5’-CARGGITAYGGIATGACNGARGC-3’ and 5’-
ACRAAIGCIACIGGNAYYTCNCC-3’ for amplification of a partial 4CL sequence from
genomic DNA of sugarcane cv. CP88-1762. Amplifications were carried out with 35
cycles of denaturation at 94°C for 45 s, 60 s of annealing at 58°C, and extension of
72°C for 2 min and produced a 743-bp product in which 4 exons are interrupted by three
introns (Fig. 3-1A).. Following gel electrophoresis the PCR band was gel extracted
(QiaQuick Gel Extraction Kit; Valencia, CA), cloned into a TOPO 2.1 TA Cloning vector
(Invitrogen, Carlsbad, CA), and sequenced for confirmation.
A blast search was performed with the Sc4cl_2 sequence as a query to identify
the ORF. Amplification of exon 1 was carried out using specific primers Sc4Cle1 F (5’-
caagggtacgggatgacggaagcc-3’) and Sc4Cle1 R (5’-ccctttcatgatctgcgggc-3’). The
amplified fragment was sequenced for confirmation, and subcloned into compatible
cloning sites of vector pWF_BG_4CL RNAi (Fouad et al. 2009). This resulted in an
inverted repeat of the 208 bp Sc4CL_2 of exon I with both repeats being separated by a
94 bp intron from Bahiagrass (Paspalum notatum) 4CL, under transcriptional control of
37
the OsC4H promoter and CaMV35S 3’UTR (Fig. 3-1C) (Yang et al. 2005; Dixon et al.
1986). For biolistic gene transfer, the RNAi cassette including OsC4H promoter,
inverted repeats of Sc4CL_2 exon I, and CaMV35S 3’UTR (Dixon et al. 1986) were
released from the vector by restriction digestion with XmnI (New England Biolabs Inc.,
Ipswich, MA, USA), gel purified (QIAquick Gel Extraction Kit; Qiagen) and used for
biolistic gene transfer.
Tissue Culture, Biolistic Gene Transfer, and Plant Regeneration
Tops of sugarcane cultivar CP88-1762 were collected from the UF-IFAS
Everglades Research and Education Center in Belle Glade, Florida. Embryogenic calli
were induced from cross sections of immature leaf whorls (Kim et al. 2012). Two to
three mm cross-sections of the immature leaf whorl were cultured on modified MS basal
medium (CI3) (Chengalrayan and Gallo-Meagher 2001) at 28 °C under 16 h
photoperiod (30 μmol m-2 s-1). Tissue cultures were transferred to fresh medium every 2
wks for 2-3 mos. Calli were transferred to CI3 medium containing 0.4M D-sorbitol for 4 h
of osmotic treatment prior to bombardment. Biolistic transformations were performed as
described by Sandhu and Altpeter (2009). Two μg of the RNAi cassette and 1 μg of the
nptII selection cassette were co-precipitated onto 1.0 µM diameter gold microparticles
(Sigma, St. Louis, MO, USA) with 0.96 M CaCl2 and 0.04 M spermidine free base
(Sigma). The particles were delivered into embryogenic calli using the PDS-1000/He
Particle Delivery System (Bio-Rad, Hercules, CA, USA). Following a 6 d resting period
on CI3 medium the calli were transferred to CI3 medium supplemented with 20 mg L-1
geneticin. After three biweekly subcultures, actively growing calli were transferred to MS
medium (Murashige and Skoog 1962) containing 2.5 μM thidiazuron (TDZ) and 20 g L-1
sucrose and placed in a growth chamber with 100 μmol m-2 s-1 light intensity with a 16 h
38
photoperiod at 28 ± 2 °C for 1 wk. Regenerating calli were placed on hormone free
medium with 20 mg L-1 paromomycin for selection of transgenic plants. Regenerated
shoots were transferred to soil and maintained in an air conditioned greenhouse under
natural photoperiod at 28/22 ± 4 °C day/night temperature, respectively, until maturity.
NPTII ELISA
The expression of nptII in putative sugarcane lines was tested using a
commercially available NPTII ELISA assay (Agdia, Elkhart, IN, USA). Protein was
extracted from 100 mg of mature leaf tissue using the extraction buffer supplied with the
kit. Protein concentrations were measured spectrophotometrically (Evolution 300 UV-
VIS) at 595nm using the Coomassie Plus (Bradford) Protein Assay reagent (Pierce,
Rockford, IL, USA). Twenty μg of soluble protein extract were used in ELISA assays,
and the samples were visually examined for color intensity and compared against
positive and negative controls.
Northern Blot Analysis
Secondary tillers of sugarcane, approximately 15 cm in length, were collected and
immersed in liquid nitrogen before storage at -80 °C. Total RNA was extracted from
frozen samples (Shirzadegan et al. 1991). Thirty μg of RNA was denatured in a 65 °C
water bath for 10 min, separated by denaturing gel electrophoresis, and transferred to
the Hybond-N+ membrane according to the manufacturer’s instructions (GE Healthcare
Biosciences, Pittsburgh, PA, USA). For the probe, the 730 bp open reading frame (ORF)
of Sc4CL_2 was PCR amplified from a cDNA clone according to the conditions
previously described, and labeled with 32P dCTP using the Prime-a-Gene Labeling
System (Promega, San Luis Obispo, CA, USA). The hybridization procedure was
carried out as described by Jeffreys and Flavell (1977). After hybridization, the
39
membrane was washed with 0.1X SSC and 0.1% SDS three times at 65 °C, 20 min per
wash, and exposed to X-ray film according to the manufacturer’s instructions (Phenix
Research Products, Asheville, NC, USA) at -80 °C for 2 d.
Small RNA Northern Blot Analysis
For siRNA northern blot analysis, total RNA was extracted (Shirzadegan et al.
1991) using tissue from the third youngest internode. Small RNA was separated from
total RNA as described by Lu et al. (2007). Thirty μg of small RNA was loaded in each
lane of a 15% polyacrylamide TBE/urea gel (Bio-Rad, Hercules, CA, USA), separated
by electrophoresis, and transferred to Hybond-N+ membrane (GE Healthcare
Biosciences) according to the directions of the manufacturer. The 730 bp Sc4CL_2
exon I was used as a probe in hybridization. Washing and detection were carried out as
previously described.
Southern Blot Analysis
High molecular weight genomic DNA was extracted from leaf tissue using a
modification of the hexadecyltrimethylammonium bromide (CTAB) method (James et al.
2008). For each sample, 30 μg of genomic DNA was digested to completion with EcoRI
(New England Biolabs Inc., Ipswich, MA, USA), separated by agarose gel
electrophoresis (0.8%), and transferred to Hybond-N+ membrane according to the
manufacturer’s instructions (GE Healthcare Biosciences). The probe used in
hybridization was the 803 bp fragment of the OsC4H promoter sequence obtained by
PCR.
Quantitative Real-Time RT-PCR Analysis
Total RNA was extracted from the second youngest internode using Trizol reagent
(Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. RNase-
40
Free RQ1 DNase (Promega) was added to the RNA extract to remove DNA. For the
cDNA synthesis step, 500 ng of total RNA was reverse-transcribed into cDNA by using
iScript cDNA Synthesis Kit (Bio-Rad). Quantitative Real-time PCR was performed with
the MyiQ cycler (Bio-Rad) with iQ SYBR Green supermix (Bio-Rad). Sugarcane
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the control.
Acetyl Bromide (AcBr) Lignin Analysis and Visual Observations
Insoluble lignin content was measured with the acetyl bromide (AcBr) lignin
method described by Hatfield et al. (1999) with the following modifications. Stalks with
12 internodes were collected from clonally propagated transgenic or non transgenic
control plants, and the mature internode # 6 was selected for analysis. Color of leaf
veins of fully expanded leaves was evaluated before sampling of the internodes.
Internode cross-sections were examined for color immediately after cutting. Internodes
were dried at 55°C for 2 d, and ground to a powder using a coffee grinder. The powder
was suspended in 50% ethanol, sonicated three times (Branson, Danbury, CT, USA) for
30 min each. The samples were dried at 55°C for 2 d, the powder was passed through
a 0.25 mm sieve, and collected. For each transgenic line, three biological and three
technical replications were evaluated by transferring 2 mg of powder into a 1.5 mL tube
with 1 mL of 25% v/v AcBr in glacial acetic acid. The tubes were incubated at 50 °C for
4 h, with vortexing every 15 min, followed by 30 min incubation on ice. Samples were
prepared for spectrophotometry by adding 1.7 mL of 25% v/v AcBr in glacial acetic acid,
200 μL 2M NaOH, and 100 μL sample to Sub-Micro Cells (ThermoFisher,Waltham, MA,
USA), and mixed thoroughly. The absorption was measured on a spectrophotometer
(Evolution 300, ThermoFisher) at 280 nm.
41
Statistics
Replicated samples were grown in randomized complete blocks with 8 replications.
Data were collected for variables such as plant height (cm), fresh weight (g), node
diameter (cm), lignin content, and analyzed by ANOVA using SASTM Version 9.3.
Mean comparisons were performed using LSD at the 5% level. For AcBr determinations,
sample size was nine for each replication (three biological replications x three technical
replications).
Results
Cloning and Expression Pattern of Sc4CL_2
The expression pattern of Sc4CL_2 in four different tissues of field grown
sugarcane was analyzed by northern blot analyses. Sc4CL_2 showed the highest
expression levels in the youngest internode and emerging tiller. There was barely
detectable expression in the youngest node, leaf whorl (immature leaf), and youngest
expanded leaf (Fig. 3-2).
Suppression of Sc4CL_2 by RNAi
Co-transformation of embryogenic calli with the linearized, minimal RNAi cassette
and the minimal nptII selectable marker cassette using biolistic gene transfer generated
66 transgenic lines as identified by PCR and NPTII ELISAs (data not shown) (Fig. 3-3).
Reduced transcript levels of Sc4CL_2 (Fig. 3-4) were observed in 12 transgenic
sugarcane lines (21; 22; 23; 31; 32; 41; 44; 53; 55; 64; 71; 74) as determined by
northern blot analysis using the Sc4CL_2 ORF as a probe (Fig. 3-1B). The independent
transgenic nature of these 11 lines was confirmed by Southern blot analysis using the
OsC4H promoter sequence as a probe (Fig. 3-5). Different patterns of hybridization
signals were observed for all 11 lines, and the number of hybridization signals ranged
42
from two (line 71) to six for the majority of lines (Fig. 3-5). RNAi suppression of
Sc4CL_2 was confirmed in four of these sugarcane lines (41, 44, 71 and 74) by small
RNA northern blot analysis (Fig. 3-6). Quantitative Real-time RT-PCR analysis of these
four transgenic lines displayed 4.1%, 12.1%, 11.6% and 16.1% expression of the
Sc4CL_2 transcript in lines 41, 44, 71 and 74 compared to non transgenic sugarcane
control, respectively (Table 3-1).
Analysis of Lignin Content and Evaluation of Phenotype
The four primary transformants were vegetatively propagated, grown in the
greenhouse in randomized complete blocks with eight replications, and lignin content
was measured using samples derived from basal internodes. The AcBr lignin content of
the four lines (41, 44, 71, 74) was used in comparisons with the non transgenic control
(Table. 3-1). Lines 41 and 71 had a 9.3% and 5.2% reduction in lignin, respectively. No
significant reductions of lignin were observed for lines 44 and 74.
Variables such as biomass, plant height, node diameter, and color were measured
from greenhouse grown replicates and compared with non transgenic control (Table 3-
1). Transgenic lines 71 and 44 accumulated similar amounts of biomass as non
transgenic control, while lines 74 and 41 accumulated significantly less biomass, 24%
and 49%, respectively, compared to the non transgenic control. In terms of plant height,
lines 44 and 74 were not significantly different from non transgenic control. However,
line 71 was 21% taller than non transgenic control and line 41 was 22% shorter than
non transgenic control. Stalk diameters of lines 71, 44, 74 and 41 were reduced by 12,
13, 15 and 36%, respectively, compared to non transgenic control. The color of the
mature internode tissues and the leaf veins did not differ from non transgenic control as
determined by visual assessments.
43
Discussion
In this study RNAi was used for the first time successfully to suppress a 4-
coumarate coA ligase (4CL) gene in the complex and highly polyploid sugarcane
genome. A single earlier report demonstrated the utility of RNAi for targeted
downregulation of candidate genes in sugarcane using the phytoene desaturaturase
(PDS) gene which resulted in photo-bleaching (Osabe et al. 2009). RNAi allows one to
evaluate the correlation between the level of 4CL suppression or other lignin
biosynthetic genes, lignin content and plant performance (Carrol and Somerville, 2009;
Xu et al. 2011). Four transgenic sugarcane lines with 4CL suppression were studied in
detail. Moderate reduction of lignin (5%) did not compromise biomass production under
controlled environmental conditions. However, the line with the strongest suppression of
4CL displayed a 9% reduction in lignin content along with a severe reduction (49%) of
biomass. It cannot be ruled out that part or all of the observed reduction on plant
biomass may have been due to the tissue culture or transformation process. For
example, line 74 which did not have a significant reduction in lignin content also
displayed reduced biomass content. Some deleterious effects of tissue culture on
sugarcane plant performance can be eliminated during vegetative propagation (Lourens
and Martin, 1986). Sexual reproduction is very effective in eliminating somaclonal
variation (Bregitzer et al. 2008), but will cause significant segregation in sugarcane.
Reducing the time in tissue culture by utilizing direct embryogenesis in sugarcane is
expected to reduce the occurrence of somaclonal variation (Taparia et al. 2012).
Sc4CL_2 is primarily expressed in the internodes of the immature stem and the
newly emerging tillers and barely detectable in leaf tissue. This expression profile is
consistent with other monocot 4CLs of the monolignol pathway (Gui et al. 2011; Xu et al.
44
2011). In contrast, 4CLs of the flavonoid pathway display low and similar expression
levels in internodes and leaves (Gui et al. 2011; Xu et al. 2011).
Suppression of Sc4CL_2 in transgenic sugarcane lines by RNAi is confirmed in
the present study, by mRNA northern blot, quantitative Real-time RT-PCR and the si-
RNA northern blot. The level of siRNA expression, target gene suppression, and lignin
reduction were consistent in the analyzed sugarcane lines. The general trend for lignin
reduction was observed in all transgenic sugarcane lines and two of them were
statistically significant. Line 71, with 5.2 % suppression of lignin did not display a
significant reduction in biomass compared to non transgenic control. In COMT
suppressed transgenic sugarcane lines, a 4% reduction of lignin increased
saccharification efficiency by 30% (Jung et al. 2011).
Suppression of Os4CL_3 in diploid rice and Pv4CL1 in tetraploid switchgrass
resulted in approximately 29% and 21% reduction of lignin, respectively (Gui et al. 2011;
Xu et al. 2011). The sorghum brown mid rib 2 mutant (bmr2) showed a 20% reduction
in lignin in both stover and leaf tissue (Saballos et al. 2012). In the present study, the
reduction of lignin caused by suppression of Sc4CL_2 is 4-9% which is less than
reported for other plant species (Ehlting et al. 1999; Kajita et al. 1996; Hu et al. 1999;
Wagner et al. 2009). The highly polyploidy sugarcane contains several 4CL loci with
significant sequence diversion, and more drastic reduction of the lignin content may
require co-suppression of multiple alleles. Different genetic backgrounds in various
plant species may also influence plant performance and biomass in response to
reduced lignin content (Oliver et al. 2005). Future work should identify and co-suppress
additional 4CL loci. Replicated field testing of transgenic sugarcane lines will enable a
45
better estimation of genotype x environment interactions, saccharification efficiency of
lignocellulosic biomass and its conversion efficiency to ethanol.
46
Table 3-1. Means ± S.E. for plant height, fresh weight, node diameter, AcBr lignin
content, and qRT-PCR expression for four transgenic plants and non transgenic sugarcane CP88-1762 control
Lines Plant height
(cm) Fresh weight
(g) Node diameter
(cm) AcBr lignin
(%)
Sc4CL_2 expression (% of WT)†
WT 91.5 ± 3.9 525.1 ± 34.4 2.2 ± 0.1 20.5± 0.1 100.0 41 71.4 ± 5.9* 267.1 ± 34.1* 1.4 ± 0.1* 18.5±0.2* 4.1 44 83.7 ± 6.3 435.4 ± 31.2 1.9± 0.1* 19.8±0.2 12.1 71 110.7 ± 5.4* 479.8 ± 36.1 1.9 ± 0.1* 19.3±0.3* 11.6 74 97.5 ± 5.5 399.4± 15.2* 1.9 ± 0.0* 19.9 ±0.5 16.1
* Significantly different from wild type at P<0.01 as determined by LSD † WT; non transgenic sugarcane CP88-1762 control
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Figure 3-1. A, Schematic representation of the sugarcane 4CL partial genomic sequence of Sc4CL_2. The 743 bp of the partial genomic Sc4CL_2 sequence include 4 exons and 3 introns. B, Schematic representation of Sc4CL_2 cDNA clone. The 730 bp probe derived from Sc4CL_2 ORF and 3’UTR is shown. C, Schematic representation of the RNAi suppression cassette. Inverted repeats of the 208 bp Sc4CL_2 exon1, separated by the BG4CL intron, under transcriptional control of the OsC4H promoter and CaMV35S 3’UTR. The 803 bp probe derived from the OsC4H promoter and used in Southern blot hybridizations is shown.
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Figure 3-2. The expression pattern of Sc4CL_2 in non transgenic sugarcane CP88-1762 control plants. Upper panel: Total RNA was extracted from different tissues of non transgenic sugarcane plants. RNA was separated in electrophoresis and hybridized to a 730 bp probe derived from the Sc4CL_2 cDNA clone. 1: immature leaf whorl; 2: young, expanded leaf; 3: Immature node; 4: Immature internode; 5: young, secondary tiller; Lower panel: Total RNA
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Figure 3-3. Vegetative progeny of transgenic sugarcane lines with RNAi suppression of Sc4CL-2 grown under controlled environmental conditions.
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Figure 3-4. Northern blot analysis. Upper panel: Total RNA was separated by electrophoresis and hybridized to a 730 bp probe derived from the Sc4CL_2 cDNA clone. WT: non transgenic sugarcane CP88-1762 control; lanes 2-13: independent transgenic events; Lower panel: total RNA was used as a control.
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Figure 3-5. Southern blot analysis of independent sugarcane transformants. Genomic DNA was extracted from leaf rolls, digested with EcoRI, and the blots were hybridized with a 803 bp open reading frame of OsC4H promoter. WT: non transgenic sugarcane CP88-1762 control; 22; 31; 32; 41; 42; 44; 52; 64; 71; 72; 74 represent independent transgenic sugarcane lines with co-integration of the 4CL construct ; 23; 53 represent nptII transgenic sugarcane lines without co-integration of the 4CL construct. PC: plasmid control.
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Figure 3-6. Small RNA northern blot analysis. The small RNAs were separated by electrophoresis and hybridized to a 730 bp probe derived from the Sc4CL_2 cDNA clone. PC: plasmid control; 11 numerically labeled lanes: 11 transgenic sugarcane lines.
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CHAPTER 4 CONCLUSION
Sugarcane is a perennial C4 grass in the genus Saccharum, and it is one of the
highest yielding biomass crops in the world (FAO 2010). Modern sugarcane cultivars
are highly polyploid, interspecific hybrids derived from crosses among S. spontaneum,
S. officinarum, S barberi, S. robustum, S. sinense, Miscanthus, and other related grass
genera (Altpeter and Oraby 2010; Brandes 1958; Purseglove 1972; Daniels and Roach
1987). Approximately 70% of the world’s sugar production comes from sugarcane.
Since the 1970s, the sugar derived from sugarcane has been used for sucrose-based
bioethanol production to replace fossil fuels, primarily in Brazil. It has been suggested
that by the inclusion of the structural polysaccharides of the residues, cellulose and
hemicellulose, into the conversion process, ethanol yields can be substantially
increased (Kim et al. 2004).
Genetic transformation provides a relatively rapid method to improve the biomass
quality of sugarcane for cellulosic ethanol production. Biolistics is an important gene
transfer method for both transient gene expression studies and for stable genetic
transformation in crop improvement. During the last two decades, protocols for biolistic
gene transfer have been improved (Sanford et al. 1993; Sivamani et al. 2009). The
precipitation of plasmid DNA onto gold or tungsten micro-particles in the presence of
spermidine and CaCl2 is an important step in the procedure. Protamine, a small
arginine-rich protein, has been substituted for spermidine, in order to increase
transformation efficiencies in rice and maize (Sivamani et al. 2009). The present study
compared three different DNA coating procedures in the biolistic transformation of
sugarcane: spermidine free base, protamine sulfate, and the Seashell DNAdelTMGold
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Carrier with a proprietary precipitation buffer derived from Seashell Technology. For
stable transformation using minimal, linear expression cassettes, protamine, spermidine
and the Seashell were equally effective. Differences compared to the earlier report by
Sivamani et al. (2009) may be associated with the difference in size and form (linear
versus circular) of the precipitated DNA molecules. Further optimizations of the coating
protocols may be possible by changing the concentration of DNA, cationic polyamine or
CaCl2 concentrations (Zlatanova et al.1998; Makita et al. 2009).
Biolistic transfer of an RNAi hairpin construct was used to suppress 4CL
expression in sugarcane. By using degenerate primers that annealed to the conserved
regions of the 4CL family, a 4CL partial sequence, Sc4CL_2, was amplified from the
sugarcane genome. Exon 1 of Sc4CL_2 was used to construct the RNAi expression
cassette. The transgenic nature of the transgenic sugarcane lines was confirmed by
NPTII ELISA, PCR, Southern blot analysis si-RNA Northern blot and realtime RT-PCR.
Four lines with suppression of Sc_4CL2 were selected for lignin analysis. The observed
lignin reduction was corresponding to the level of siRNA expression and target gene
suppression in the analyzed sugarcane lines. A general trend of lignin reduction was
observed in all four transgenic sugarcane lines, and two of them displayed significant
lignin reduction compared to non transgenic control. This is the first report of successful
suppression of a 4CL gene by RNAi in sugarcane.
Future research should include field evaluation of performance and conversion
efficiency to ethanol. Additional co-suppression studies targeting multiple 4CL loci
could be performed for stronger suppression of lignin content in combination with
different genetic backgrounds (high or low fiber content). In conclusion, this study
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generated and characterized transgenic sugarcane lines, with RNAi suppression of the
4CL gene.
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APPENDIX LABORATORY PROTOCOLS USED IN MOLECULAR BIOLOGY, BIOLISTICS
TISSUE CULTURE, AND IDENTIFICATION/CHARACTERIZATION OF TRANSGENIC SUGARCANE PLANTS
Molecular Cloning
Plasmid DNA Purification Using QIAGEN® Plasmid Midi Kit QIAGEN plasmid purification protocol as provided by the manufacturer
1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic. Incubate for approx. 8 h at 37°C with vigorous shaking (approx. 300 rpm).
2. Dilute the starter culture 1/500 to 1/1000 into selective LB medium. Inoculate 25 mL with 25–50 μL of starter culture. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm).
3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. 4. Resuspend the bacterial pellet in 4 mL Buffer P1. 5. Add 4 mL Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6
times, and incubate at room temperature (15–25°C) for 5 min. 6. Add 4 mL of chilled Buffer P3, mix immediately and thoroughly by vigorously
inverting 4–6 times, and incubate on ice for 15 min. 7. Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing
plasmid DNA promptly. 8. Centrifuge the supernatant again at ≥20,000 x g for 15 min at 4°C. Remove
supernatant containing plasmid DNA promptly. 9. Equilibrate a QIAGEN-tip 100 by applying 4 mL Buffer QBT, and allow the
column to empty by gravity flow. 10. Apply the supernatant from step 8 to the QIAGEN-tip and allow it to enter the
resin by gravity flow. 11. Wash the QIAGEN-tip with 2 x 10 mL Buffer QC. 12. Elute DNA with 5 mL Buffer QF. 13. Precipitate DNA by adding 3.5 mL (0.7 volumes) room-temperature isopropanol
to the eluted DNA. 14. Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully decant
the supernatant. 15. Wash DNA pellet with 2 mL of room-temperature 70% ethanol, and centrifuge at
≥15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.
16. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).
Restriction Digestion to Prepare Minimal Expression Cassette
1. Digest 100 µg of plasmid DNA with restriction enzymes. Ensure there is no restriction site inside the cassette.
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2. Confirm the completion of digestion by electrophoresis. Load 2 µL of DNA dilution on a 0.8% agarose gel along with DNA ladder. Electrophorese at 80-100 V for 1h to achieve good separation of bands. Check the gel using a UV-transilluminator. Check the band number and size to confirm that the digestion is complete.
3. Cut out the gel that contains the expression cassette and purify the DNA with QIAquick gel purification kit (Qiagen Inc., Valencia, CA).
4. Quantify the yield using nanodrop® (ND-1000 spectrophotometer, Nanodrop Technologies, Wilmington, DE).
5. Check the quality of DNA by electrophoresis.
Gel Extraction Using QIAquick® Gel Extraction Kit • QIAGEN plasmid purification protocol as provided by the manufacturer
1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. 2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume
of gel (100 mg ~ 100 μL). 3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To
help dissolve gel, mix by vortexing the tube every 2–3 min during the incubation. 4. After the gel slice has dissolved completely, check that the color of the mixture is
yellow (similar to Buffer QG without dissolved agarose). 5. Add 1 gel volume of isopropanol to the sample and mix. 6. Place a QIAquick spin column in a provided 2 mL collection tube. 7. To bind DNA, apply the sample to the QIAquick column, and centrifuge for 1 min. 8. Discard flow-through and place QIAquick column back in the same collection
tube. 9. Recommended: Add 0.5 mL of Buffer QG to QIAquick column and centrifuge for
1 min. 10. To wash, add 0.75 mL of Buffer PE to QIAquick column and centrifuge for 1 min. 11. Discard the flow-through and centrifuge the QIAquick column for an additional 1
min at 17,900 x g (13,000 rpm). 12. Place QIAquick column into a clean 1.5 mL microcentrifuge tube. 13. To elute DNA, add 50 μL of Buffer EB (10 mM Tris·Cl, pH 8.5) or water (pH 7.0–
8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μL elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min.
14. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.
Biolistic Transformation Using PDS-1000/He®
Preparation of Gold Stock (60 mg mL-1) 1. Weigh 30 mg of 1.0 µm gold particles in a sterile 1.5 mL microfuge tube.
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2. Add 1mL of 70% ethanol, vortex for 3-5 min, incubate at room temperature for 15 min.
3. Centrifuge briefly (5 s) to pellet the gold particles. 4. Discard the supernatant, wash the pellet with 1 mL autoclaved ddH2O (3 times). 5. Vortex for 1 min. 6. Centrifuge briefly (3-5 s), discard the supernatant. 7. Add 500 µL sterile 50% v/v glycerol. 8. Store the gold stock at -20°C.
Preparation of DNA Coated Microparticles i. Spermidine/ Protamine coating
1. Vortex the gold stock for 30 s. Mix 30 l of the gold stock and 30 l of DNA in a 1.5 mL sterile microfuge tube, vortex for 1 min.
2. Add 20 L 0.1 M freshly prepared spermidine (or 20 L 1 mg mL-1 protamine)
and 50 L 2.5 M CaCl2 while continuing to vortex. 3. Mix all components by vortex for 1 min. 4. Centrifuge briefly (3-5 s) to pellet the gold. 5. Discard the supernatant without disturbing the pellet and wash the pellet with 250
l absolute ethanol. 6. Centrifuge briefly (3-5 s) and remove the supernatant. 7. Repeat the washing one more time.
8. Resuspend the pellet in 90 L absolute ethanol by sonication for 2 s. 9. Keep the DNA-microparticles mixture on ice.
ii. Seashell biotechnology DNAdelTM gold carrier particles protocol • This protocol as provided by Seashell Biotechnology
1. The DNAdelTM gold particles are supplied as a 50 mg mL-1 suspension in binding buffer. Sonicate briefly to dissociate any aggregates prior to formulation. Dilute the gold into binding buffer to yield a final concentration of 30 mg mL-1 (minimum volume of 50 μL buffer; 1.5 mg gold (30 μL volume of 50 mg mL-1; 20 μL buffer)). Add the plasmid DNA (stock 1 mg mL-1) to gold at a ratio of 2-5 μg DNA per mg gold. (This concentration has yielded significantly higher expression levels compared to standard particles and protocols). Vortex briefly.
2. Add an equal volume of precipitation buffer. Vortex briefly and let stand for 3 min. Spin (10,000 rpm in Eppendorf microfuge 10 s) to pellet the DNA coated particles.
3. Remove the supernatant and add 500 μL of 100% cold ethanol. Vortex briefly and spin (10,000 rpm in Eppendorf microfuge 10 s) to pellet particles. Remove the supernatant and add 100% ethanol to the desired gold concentration. Briefly sonicate the solution to resuspend the gold particles and process for appropriate delivery device. The sonication step minimizes aggregation and is important for reproducible particle delivery.
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Biolistics
1. Turn on PDS-1000/He® Particle Delivery System and vacuum pump. 2. Sterilize the inside of PDS-1000/He® Particle Delivery System with 70% ethanol.
Autoclave macrocarrier holders, stopping screens, macrocarriers, and a device for securing macrocarriers into the macrocarrier holders.
3. Place macrocarriers into holders. 4. Prior to use, resuspend the DNA-coated microparticles by briefly vortexing.
5. Add 5 l of the DNA-microparticles mixture into the center (inner 5 mm diameter) of the macrocarrier; allow for complete evaporation of ethanol before use.
6. Place stopping screen into the macrocarrier plate and insert the inverted macrocarrier assembly on top. Secure the lid on top of the shelf assembly.
7. Place macrocarrier plate containing the macrocarrier at the highest level of the inner chamber.
8. Place tissue culture plate on shelf 2, below the macrocarrier plate (6 cm below). 9. Initiate a vacuum to 27.5 Hg; press and hold the fire button until the disc ruptures
at 1100 psi. Eye protection must be worn. 10. Vent the vacuum and remove petri dish. 11. Dismantle the assembly and prepare for the next shot.
Tissue Culture and Plant Regeneration
Embryogenic Callus Induction, Selection and Plant Regeneration
1. Obtain sugarcane cultivar, CP88-1762, from the greenhouse or field. 2. Remove outer mature leaves and sterilize the leaf whorl with 70% ethanol.
Remove additional layers with sterilized forceps and scalpel to obtain a tightly furled spindle of immature leave.
3. Cut 3-4 mm thick cross-sections from the leaf base above the apical meristem tissue with sterilized forceps and scalpel.
4. Place the cross-section on to MS basal medium (CI3) at 28ºC under illumination (30 µmol m-2 s-1) with a 16 h light/8 h dark regime.
5. Subculture the explants biweekly for 3-4 months. 6. Transfer the embryogenic calli to CI3 medium supplement with 0.4 M sorbitol 4 h
before bombardment. 7. Transfer bombarded calli to regular CI3 medium 12 h after bombardment. 8. Transfer calli onto selection medium (CI3 medium with 20 mg L-1 of geneticin) 7 d
after gene transfer. Subculture biweekly for 2 months. 9. Transfer the growing calli to regeneration medium with 2.5 μM TDZ for 1 week
and maintain at 100 µmol m-2 s-1 light intensity with 16/8 h (light/dark) photoperiod at 28ºC.
10. Regenerated calli are then transferred to rooting media containing 20 mg L-1 paromomycin. Subculture biweekly for 2-3 months.
11. Regenerated plantlets are transplanted to soil and cultivated in air conditioned greenhouse at 22/28 ºC day/night temperature and natural photoperiod.
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Molecular Characterization of Putative Transgenics
ELISA NPTII ELISA KIT protocol (Agdia, Elkhart, IN) as provided by the manufacturer a. Protein extraction
1. Harvest 100 mg of young fresh leaf tissue. Store samples on ice.
2. Add 10 mg polyvinyl pyrrolidone (PVP) and 600 μL 10 x PEB1 buffer (supplied with the nptII Agdia ELISA kit) to each sample.
3. Grind the leaf samples using a micro-pestle. Keep the samples on ice.
4. Centrifuge the samples at 20,800 x g at 4ºC for 15 min.
5. Transfer the supernatant to a new micro-centrifuge tube and store on ice. b. Protein estimation
1. Dilute Protein Determination Reagent (USB Corporation, product code 30098) 1:1 using sterile ddH2O. Prepare enough to use 1 mL per sample including standards and blank.
2. Prepare a standard dilution series using BSA (0-20 μg).
3. Add 1 mL diluted Protein Determination Reagent to each cuvette. Add 5 μL of sample to each cuvette and mix by vortexing.
4. Incubate at room-temperature while preparing the remaining samples.
5. Measure OD595 of each sample (ideally these should be between 0.2 and 0.8).
6. Plot a standard curve using BSA and use it to estimate total protein concentration of the samples.
7. Calculate the volume of each sample required for 20 μg total protein per well. c. Assay
1. Prepare the samples, including wild-type using 22 μg protein and the volume of buffer PEB1 required to make the total volume 110 μL.
2. Prepare standards as follows: 110 μL buffer PEB1 (negative control) and 110 μL of the provided positive control.
3. Prepare a humid box by putting damp paper towel in a box with a lid.
4. Add 100 μL of each sample and standard in the ELISA microplate provided with the kit. The order of samples should be noted at this time.
5. Place the plate in the humid box and incubate for 2 h at room temperature.
6. Prepare the wash buffer PBST by diluting 5 mL to 100 mL (20×) with ddH2O.
7. Prepare the enzyme conjugate diluent by mixing 1 part MRS-2 with 4 parts 1ˣ buffer PBST. Make enough to add 100 μL per well.
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8. A few minutes before the incubation ends, add 10 μL from bottle A and 10 μL from bottle B per 1 mL of enzyme conjugate diluent to prepare the enzyme conjugate.
9. When the incubation is complete, remove plate from humid box and empty wells.
10. Fill all wells with 1X buffer PBST and then empty wells gently. Repeat 5 times.
11. Ensure complete removal of wash solution by tapping the frame firmly upside down on paper towels.
12. Add 100 μL of the prepared enzyme conjugate into each well and incubate the plate in the humid box for 2 h at room temperature.
13. In the meantime, aliquot sufficient TMB substrate (100 μL per well) and allow it to warm to room temperature.
14. When the incubation is complete, wash the plate with 1x buffer PBST as before.
15. Add 100 μL of room temperature TMB substrate solution to each well, and place the plate back in the humid box for 15 min. A blue color will develop, the intensity of which will be directly proportional to the amount of NPTII protein in the sample, while negative samples will remain white.
16. To stop the reaction, add 50 μL 3M sulphuric acid to each well. The substrate color will change from blue to yellow.
17. The results must be recorded within 15 min after addition of the stop solution otherwise the reading will decline.
18. Color development can be visually scored or recorded with the help of an ELISA plate reader.
Genomic DNA Extraction (CTAB)
1. Autoclave mortars, pestles and spatulas for 20 min at 121oC and dry in an incubator oven at 60ºC.
2. Prepare enough CTAB buffer (5 mL g-1 leaf material) for use by adding β-mercaptoethanol fresh (200 μL β-mercaptoethanol per 100 mL buffer or 30 μL in 15 mL). Heat to 65°C in a water bath.
3. Harvest 3 g young leaf material and store on ice or freeze in liquid nitrogen. 4. Cool mortar and pestle by adding liquid nitrogen. Grind fresh leaf in liquid
nitrogen to a fine powder. 5. Add frozen leaf powder to 15 mL pre-heated buffer in a 50 mL disposable
polypropylene tube and mix well using a spatula or glass rod.
6. Incubate at 65°C for 1 h. Mix the contents thoroughly 1-3 times during incubation.
7. Add 3 μL RNase A to each sample at the start of incubation.
8. Cool to room temperature.
9. Add equal volume (15 mL) chloroform: iso-amyl alcohol (24:1) and mix gently to form an emulsion for approximately 30 min. Mix by hand by gently inverting the tubes and shake on a rotary shaker for 10-15 min.
10. Spin at 4000 rpm for 30 min. Transfer the top layer to a fresh 50 mL tube using a wide bore 10 mL pipet tip.
11. Repeat step 8 through 9 again.
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12. Repetition of step 8 will depend on the quantity of precipitate visible at the organo- aqueous interface.
13. Add ⅔ volumes chilled isopropanol and mix gently by inverting. The DNA will precipitate.
14. Use a blue pipette tip, with wide bore, to pipette the DNA out of the aqueous mixture.
15. Transfer DNA to a clean 15 mL tube containing 10 mL of 70% ethanol.
16. Wash one more time in 70% ethanol by inverting the tube several times.
17. Transfer washed DNA pellet to a clean 1.5 mL microfuge tube and air dry. Resuspend in 200 μL TE buffer.
18. Prepare 10x dilution of the DNA stock. Use 1 μL to check concentration and quality using the NanoDrop® spectrophotometer and by gel electrophoresis of samples in a 0.8% agarose gel (80 V, 40 min).
19. Store the dilutions and stocks at 4ºC. Southern Blotting and Hybridization a. DNA digestion and electrophoresis
1. Extract genomic DNA from transgenic lines and wild-type using the CTAB method, described above.
2. Digest 30 μg DNA from each sample with the restriction enzyme to be used for Southern blotting. Run the digested DNA on a 1% agarose gel to ensure complete restriction digestion.
3. Use 30 μg genomic DNA to set up restriction digests for Southern blotting. Estimate the volume of each sample required for 15 μg DNA and pipette it into a sterile 1.5 mL micro-centrifuge tube. Add sterile ddH2O to make up the volume to 30 μL.
4. Set up the digestion as follows:
DNA (30 μg DNA + sterile ddH2O) 10× EcoR1 buffer 3.0 μL 100× BSA 0.3 μL EcoR1 (1000 U/μL) 1.5 μL Sterile ddH2O ~15.2 μL Final volume 30 μL
5. Prepare a master mix for all samples that includes the buffer, BSA (if required for the enzyme chosen), enzyme and water. Mix by pipetting and add 30 μL of DNA. Mix well, centrifuge briefly, and incubate in a water bath at 37ºC over-night.
6. Run 2 μL from each digest on a 1% agarose gel to ensure complete digestion.
7. Prepare a 1 cm thick 1% agarose gel for Southern blotting using 0.5 × TBE.
8. Concentrate all the digests to reduce the volume by half (30 μL) in a Speed-Vac.
9. Centrifuge the samples briefly and add 6 μL loading dye (6 x). Mix well by pipetting.
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10. Load samples on the gel. Also load a molecular weight marker (1 kb ladder, New England Biolabs®) for estimating band size following hybridization.
11. Dilute the plasmid containing the transgene and load 50 pg as a positive control.
12. Run the gel at 15V over-night (750 min) in 0.5 × TBE. Remove the gel from the electrophoresis and cut excess gel using a scalpel. Measure the dimensions of the gel.
13. Stain the gel for one hour using freshly prepared ethidium bromide stain (0.05 μg L-1).
14. Wash the gel five times using de-ionized water (DI H2O).
15. Visualize the gel on a gel-documentation system to check complete digestion of all samples. Also measure and record the distance of the bands of the molecular weight marker from the top of the gel.
b. DNA blotting procedure
1. Prepare 500 mL 0.125 N hydrochloric acid (HCl), denaturation buffer and neutralization buffer.
2. Treat the gel with 0.125 N HCl on a shaker with gentle shaking for 30 min for depurination. Wash the gel three times with DI H2O.
3. Cut three pieces of filter paper to match the size of the gel and two pieces for the bridge (24 x 18 cm). Also cut a piece of the Hybond™N+ membrane (Amersham Biosciences, Piscataway, NJ) to match the size of the gel.
4. Treat the gel with denaturation buffer on a shaker with gentle shaking for 30 min, followed by 30 min treatment with neutralization buffer.
5. Assemble the tray and the platform on which the blot is to be set up. Place the filter paper bridge on top of each other on the platform and fold them so that it dips into the tray on both sides.
6. Pour 20 x SSC on the bridge to wet it completely. Roll a glass rod over it several times to remove air bubbles. Pour more 20 x SSC onto the bridge.
7. Place the gel in the center of the bridge, pour 20 x SSC over it and remove air bubbles.
8. Mark the membrane to determine the orientation of the gel following blotting. 9. Wet the membrane using 20 x SSC, place the membrane on the gel; pour
20 x SSC onto it and remove air bubbles. 10. Place three pieces of Whatmann filter paper on the membrane; remove air
bubbles after placing each piece. Pour more 20x SSC over the top to avoid drying of the filter papers.
11. Place pieces of parafilm all around the gel to cover the bridge to ensure that the movement of the transfer buffer takes place only through the gel.
12. Fill the tray with the transfer buffer to the top and cover the tray with Saran wrap to prevent evaporation of the buffer during blotting.
13. Place a stack of absorbent paper towels on the gel and place another piece of plexi-glass on top. On this, place a small weight (ca. 750 g) to ensure uniform blotting and leave over-night (16-18 h).
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14. Disassemble the blot. Wrap the membrane in a cling film and expose it to UV for 2 min to fix the DNA on the membrane. Store the membrane in a zip-loc bag at 4ºC.
15. Visualize the gel using a gel-documentation system to make sure the transfer was complete.
c. Hybridization using the Prime-a-Gene® labeling system (promega®)
1. Check the radioactive working area for previous contamination before working. Thaw blocking DNA (sheared salmon sperm DNA), labeling buffer, dNTPs (dATP, dGTP, dTTP), BSA and probe on ice. Place [α-32P] dCTP behind the plexiglass shield to thaw.
2. Pre-heat the hybridization oven and water-bath to 65ºC. Place the hybridization buffer in the water-bath.
3. Roll the membrane and place it inside the hybridization tube. Add 50 mL 5× SSC to the tube and pre-wet the membrane for 5-10 min. Make sure that there are no leaks.
4. Estimate the volume of probe required to get 25 ng of the probe and pipette it into a clean microcentrifuge tube. Make up the volume to 30 μL using the nuclease-free water provided with the labeling kit.
5. Close the tube tightly and boil the probe for 5 min. Also boil 500 μL of salmon sperm DNA in a separate tube. Place on ice for 5 min immediately after boiling.
6. Discard the 5 x SSC into the sink and invert the hybridization tubes on a paper tissue to drain.
7. Prepare the dNT
8. P mix by mixing equal parts of dTTP, dATP and dGTP. Prepare enough to use 2 μL per labeling reaction.
9. Set up the labeling reaction as follows:
10. Add the following to the denatured probe- 5xˣ labelling buffer 10 μL
Unlabeled dNTP mix 2 μL BSA 2 μL Klenow (5U/μL) 1 μL Total volume 45 μL
11. Move the mixture behind the plexiglass shield; add 5 μl [α-32P] dCTP and mix well by pipetting. Final volume of the reaction is 50 μL.
12. Incubate the mixture behind the plexiglass shield at room-temperature for 4 h.
13. For pre-hybridization, add 15 mL pre-heated (65ºC) hybridization buffer and 500 μL denatured salmon sperm DNA in the hybridization tube.
14. Place the tubes in the hybridization oven and incubate at 65ºC for 4 h.
15. Add 500 μL salmon sperm DNA to the labeled probe and boil for 5 min behind the plexiglass shield.
16. Discard the pre-hybridization solution into the sink and drain the tube on a paper towel.
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17. Before the probe is ready, add 8 mL pre-heated hybridization buffer in the hybridization tube and move the tube behind the plexiglass shield.
18. Immediately after boiling, add the probe mixture to the hybridization tube. Take care to avoid any spills.
19. Place the hybridization tube in the hybridization oven and incubate over-night at 65ºC (18 h).
20. Prepare the wash solution (0.1× SSC + 0.1% SDS). Prepare enough to use 50 mL per wash for three washes per blot.
21. Heat the solution to 65ºC using the water-bath.
22. Remove hybridization tubes from the oven and place behind the plexiglass shield.
23. Working behind the plexiglass shield, dispose the hybridization solution into the hazardous waste container using a funnel taking care to avoid any spills.
24. Pour 70 mL pre-heated wash solution (65ºC) into the hybridization tube; replace the lid tightly and perform a quick wash by shaking the tube for a few seconds.
25. Dispose the wash solution into the hazardous waste container and add another 70 mL pre-heated wash solution into the tube.
26. Place the tubes in the oven for 20 min. Work behind the plexiglass shield, dispose the wash solution into the hazardous waste container and add 70 mL preheated wash solution into the tube for the final wash.
27. Place the tube in the oven for 20 min. Remove the tubes from the oven and place them behind the plexiglass shield. Dispose the wash solution into the hazardous waste container and wrap the membrane in Saran wrap.
28. Check for radioactivity on the membrane using a Geiger counter. Also check the working area for any radioactive contamination.
29. Place the membrane with an X-ray film (Kodak) in an autoradiography cassette and allow 16-18 h for exposure depending on the intensity of the signal from the Geiger counter. Place the cassette at -80ºC during exposure.
RNA Extractions
1. Treat mortars, pestles and spatulas with 0.1% DEPC (Sigma) water over night. Autoclave for 30 min and dry in an incubator oven at 60ºC. Prepare extraction buffer.
2. Harvest secondary tillers or internode from sugarcane and immerse the tissue into liquid nitrogen immediately. Store samples at -80ºC.
3. Cool mortar and pestle by adding Liquid nitrogen. Grind the secondary tillers under liquid nitrogen.
4. Add 10 mL pre-heated (65℃) acidic phenol (pH 4.5) and 10 mL of extraction
buffer. Mix the homogenate well.
5. Incubate at 65ºC water-bath for 5 min
6. Centrifuged at 15,000 x g (4ºC) for 15 min.
7. Carefully transfer the supernatant to a new 50 mL tube.
8. Add equal volume of chloroform.
9. Mix well. Incubate at room temperature for 10 min.
10. Centrifuged at 15,000 x g (4ºC) for 15 min.
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11. Carefully transfer the supernatant to a new 50 mL tube.
12. Add equal volume of isopropanol. Mix by inverting the tube gently.
13. Centrifuged at 15,000 x g (4ºC) for 30 min.
14. Carefully remove the supernatant without disrupting the pellet.
15. Wash the pellet with 70% ethanol (prepare with DEPC-treated ddH2O) two times.
16. Air dry the pellet for 10 min until the pellet turn into transparent color. Do not over dry the pellet.
17. Dissolve the pellet in 50 μL DEPC-treated ddH2O.
18. Use 2 μL to check concentration and quality using the NanoDrop® spectrophotometer.
19. Store the dilutions and stocks at -80ºC. Quantitative Real-Time RT-PCR Analysis
a. DNase treatment
1. Total RNA is extracted from internode as described above. 2. Set up the DNase digestion reaction as follows:
RNA in water (1 μg) 8 μL RQ1 RNase-Free DNase 10X Reaction Buffer 1 μL RQ1 RNase-Free DNase 1u/μg Nuclease-free water to a final volume of 10 μL
3. Incubate at 37ºC for 30 min. 4. Add 1 μL of RQ1 DNase Stop Solution to terminate the reaction. 5. Incubate at 65 ºC for 10 min to inactive the DNase. 6. Add portion of the treated RNA to RT-PCR reaction.
b. IScriptTM cDNA synthesis kit •Protocol as provided by the manufacturer
1. Prepare the reaction solution as described below. 5 x iScript reaction mix 4 μL iScript reverse transcriptase 1 μL Nuclease-free water 4 μL RNA template (1 μg) 11 μL
Total Volumn 20 μL
2. Reaction protocol Incubate the complete reaction mix 5 min at 25ºC 30 min at 42ºC 5 min at 85ºC
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Hold at 4ºC
c. qReal-Time PCR
1. End point PCR Reaction mix 10 x Buffer 5 μL 5 x Q solution 10 μL dNTP (2 mM stock) 1 μL Primer F 1 μL Primer R 1 μL Hotstart Polymerase 0.25 μL cDNA template (50 ng) 1 μL ddH2O 30.8 μL Total 50 μL
2. Check the end-point PCR production by electrophoresis. 3. PCR was performed in the MyiQ cycler (Bio-Rad) with iQ SYBR Green supermix
(Bio-Rad). Sugarcane GAPDH was used as an internal control. Northern Blot Analysis
1. Extract total RNA from transgenic lines and wild-type using the method described above.
2. A total of 30 μg RNA for each transgenic sugarcane line was denatured in 65 °C water bath for 10 min.
3. Load the RNA samples into 18% formaldehyde agarose gel. 4. Total RNA were separated by electrophoresis at 50 V for 4 h. 5. Seperated RNA are blotted as described in Southern Blotting. 6. Hybridization using the Prime-a-Gene® Labeling System (Promega®) as
described in Southern Blotting. SiRNA Northern Blot Analysis
1. Extract total RNA from transgenic lines as well as wild-type using the method as described above.
2. Add 0.1 Volume of 5 M NaCl and 50% PEG (w/v, M.W. 8,000) to the RNA solution. Mix thoroughly.
3. Incubate on ice for 1 h. 4. Centrifuge at 20,000 x g (4°C) for 10 min. 5. Transfer the supernatant to a new tube. 6. Precipitate small RNA with equal volume of isopropanol at -20oC overnight. 7. Centrifuge 20,000 x g (4°C) for 30 min. 8. Wash the pellet with 70% ethanol 2X. 9. Air dry the pellet.
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10. Dilute the pellet in DEPC-treated H2O. 11. Load 30 μg of small RNA into a 15% polyacrylamide TBE/urea gel (Bio-Rad) and
run gel according to manufacturer’s instructions. 12. RNAs are blotted as described in Southern Blotting. 13. Hybridization using the Prime-a-Gene® Labeling System (Promega®) as
described in Southern Blotting. AcBr Lignin Measurement
1. Harvest the mature internode and dry in 55°C for 2 d. 2. Ground the dry tissue using family using commercial coffee grounder. 3. Add 50 mL of 50% ethanol. Sonicate the mixture at 55°C for 30 min x 3. 4. Filter the tissues and discard the flow through. 5. Dry the powders in 55 °C oven for 2 d. 6. Apply the powder to 0.2 mm sieve and collect the fine powders that pass through
the sieve. 7. For each line, load 2 mg sample into 1.5 mL Eppendorf (Eppendorf, Hamburg,
Germany) tube with 3 technical replications. 8. Add 1mL of 25% AcBr in acetic acid into the tube. 9. Incubate at 50°C for 3 h. 10. Vortex the tube for 10 s. Incubate at 50°C for another 15 min. 11. Repeat 3X. 12. Incubate the tube on ice for 30 min. 13. Mix 1.7 mL 25% AcBr in acetic acid, 200 μL 2M NaOH and 100 μL sample in
Sub-microCell (ThermoFisher, Waltham, MA) and mix thoroughly. 14. Read the absorption at 280 nm by Evolution 300 (ThermoFisher).
MUG assays a. Protein extraction
1. Collect calli and put into 2 mL eppendorf tube. 2. Grind the tissue into fine powders with liquid nitrogen.
3. Add 500 l protein extraction buffer 3.
4. Centrifuge at 13000 rpm in a microfuge at 4C for 15 min. 5. Remove supernatant to a fresh 1.5 mL tube.
6. Centrifuge again at 13000 rpm in microfuge at 4C for 15 min. 7. Remove supernatant to a fresh 1.5 mL tube, store on ice.
b. Protein concentration
1. Dilute 200 µL protein assay reagent in 800 µL water for each sample. 2. Aliquot 1 mL per plastic cuvette. 3. Add 4 µL of sample per cuvette. Invert to mix. 4. Add 4 µL of extraction buffer to one extra cuvette as a control. 5. Measure absorbance at 595nm.
c. Assay
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1. Place 180 µL of MUG assay buffer in clear 96 well plate (4.932 mg MUG / 14 mL / plate) using multichannel pipette.
2. Transfer clear plate to 37C water bath.
3. Place 180 µL of stop buffer in each of the corresponding 96 well black fluoro-plate (T0 min, T30 min, T60 min) using multichannel pipette.
4. Add 20 µL of sample to the 3 appropriate wells in the clear plate (with plate still in
37C water bath). Place 20 µL of water in A2. Place 20 µL of MU 50 µM in B2.
5. At timed intervals (0 30, 60 min), remove 20 µL from each well of the clear plate (in
37C bath) and add to the corresponding well in the black fluoro-plate using multichannel pipette. Keep fluoro-plates in the dark. For time 0, add one row of samples to clear plate.
d. Plate reading Once all the required samples have been collected, read the fluoro-plates using the Titertek Fluoroscan II. Save slope and coefficient.
Calculate pmol MU/min/mg protein. (Average slope of triplicate sample / 2) x 1000 / ug protein µL-1 Subtract average background fluorescence (35 ± 4 pmol MU min-1 mg-1 protein (p<0.05), measured in more than 50 negative control samples over several independent assays) from all fluorometric GUS activity values.
Due to the variation associated with background measurements, samples with less than twice the background value are considered as negligible.
Include at least one negative control sample in each fluorometric assay.
Buffers and Solution
Macro-stock solution dd H2O 800 mL Ammonium nitrate 16.5 g Potassium nitrate 19.0 g Calcium chloride dihydrate 4.4 g Magnesium sulfate heptahydrate 3.7 g Potassium phosphate, monobasic 1.7 g Mix all ingredients with constant stirring. Bring final solution up to 1 Liter and store in bottle in refrigerator. Micro-stock solution dd H2O 400 mL Potassium iodide 0.04150 g Boric acid 0.31000 g Manganese sulfate 0.64000 g
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Zinc sulfate heptahydrate 0.43000 g Sodium molybdate dehydrate 0.01250 g Cupric sulfate pentahydrate 0.00125 g Cobalt chloride hexahydrate 0.00125 g Mix all ingredients under constant stirring. Bring final solution up to 500 mL and store at 4oC in refrigerator. Iron-stock solution dd H2O 400 mL Na2EDTA 0.93 g FeSO4 ∙7H2O 0.65 g dd H2O Fill up to 500 mL Heat 400 mL ddH2O in beaker, but do not boil water. Add Na2EDTA to hot water with constant stirring. Once it dissolves, remove from heat before adding ferrous sulfate, and continue stirring. Bring final solution up to 500 mL and store in bottle wrapped in aluminum foil (to protect from light) in refrigerator. Paromomycin (30 mg mL-1) Dissolve 0.3 g paromomycin sulphate in 10 mL ddH2O. Filter sterilize and store in aliquots at -20°C. Use 1 mL L-1 media. CuSO4 (12.45 mg mL -1) 0.6225 g CuSO4.5H2O dissolved in 50 mL ddH2O. Filter sterilize by using 1 micron filter fitted to a syringe. Store in aliquots at - 20°C. Use 100 µL L-1 media. B5G-Vitamin-Stock solution, filter-sterilized: dd H2O 90 mL Nicotinic Acid 0.10 g Thiamine HCl 1.00 g Pyridoxine HCl 0.10 g Glycine 0.20 g Myo-inositol 10.0 g Bring final solution up to 100 mL. Filter sterilize with syringe into sterile Eppendorf tubes. Freeze tubes in -20°C freezer and only open in clean bench when preparing media. Before adding to media, thaw tubes completely and vortex. 2,4-Dichlorophenoxyacetic acid (3 mg mL-1) 0.15 g powder dissolved in 500 μL 1N NaOH. Make up to 50 mL with ddH2O. Store in aliquots at -20°C. Use 1 mL L-1 media. Media i. Embryogenesis Media (IEM) (500 mL) Pre-Autoclave dd H2O 400 mL Sucrose 10 g
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Macro-stock 50 mL Micro-stock 5 mL Fe-stock 10 mL 2,4-Dichlorophenoxyacetic acid-stock 500 μL CuSO4 50 μL dd H2O 500 mL pH-Measurement 5.8 for each bottle Phytagel (Gelrite) 1.5 g per bottle Post-Autoclave B5G-Vitamin-stock 500 μL per bottle ii. Osmotic pretreatment medium for particle bombardment (500 mL) dd H2O (400 mL) Sucrose 10 g Macro-Stock 50 mL Micro-Stock 5 mL Fe-Stock 10 mL Phytohormone stock solution 500 μL CuSO4 50 μL Sorbitol 36.44 g dd H2O Fill up to 500 mL pH-Measurement 5.8 for each bottle Phytagel (Gelrite) 1.5 g per bottle Post-autoclave B5G-Vitamin-Stock 500 μL per bottle iii. Regeneration medium (500 mL) dd H2O 400 mL Sucrose 10 g Macro-Stock 50 mL Micro-Stock 5 mL Fe-Stock 10 mL CuSO4 50 μL TDZ 2.5 μM dd H2O Fill up to 500 mL pH-Measurement 5.8 for each bottle Phytagel (Gelrite) 1.5 g per bottle Post-autoclave B5G-Vitamin-Stock 500 μL per bottle CTAB buffer (500 mL) 50 mL 1M Tris-HCl
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20 mL 0.5M EDTA (disodium salt) 40.91 g NaCl 10g CTAB Make up the volume to 500 mL with ddH2O and autoclave for 20 min. 3M Sodium acetate (50 mL) Dissolve 4.1 g sodium acetate in 50 mL ddH2O. Adjust pH to 5.2 with glacial acetic acid. 0.5M EDTA disodium salt, pH 8.0 (100 mL) Dissolve 18.61 g EDTA disodium salt in 100 mL ddH2O. 6x Loading dye (100 mL) 0.25% bromophenol blue 0.25% xylene cyanol FF 15% Ficoll Dissolve 15 g Ficoll in 60 mL ddH2O while stirring constantly. Add 20 mL ddH2O and warm the mixture. Add 0.25 g of both dyes, dissolve completely, and make up volume to 100 mL with ddH2O. Autoclave for 20 min, and store at room temperature. 50x TAE (1L) 242 g Tris base 57.1 mL glacial acetic acid 100 mL 0.5M EDTA (pH 8.0) Combine all components in ddH2O, make the final volume to 1000 mL. Autoclave for 20 min. Use 1x TAE as running buffer. 5X TBE (1L) 54 g Tris base 27.5 g Boric acid 20 mL 0.5M EDTA (pH 8.0) Combine all components in ddH2O, make the final volume to 1000 mL. Autoclave for 20 min. Use 0.5x TBE as running buffer. Hybridization Buffer (500 mL) 125 mL 1M Na2HPO4 (pH 7.4) 1 mL 0.5M EDTA (pH 8.0) 5 g BSA 175 mL 20% SDS
Make up the volume to 500 mL with ddH2O. Autoclave for 20 min, and store at 4C. 1M Na2HPO4, pH 7.4 (1L). Dissolve 142 g Na2HPO4 in 800 mL ddH20. Adjust the pH to 7.4 with phosphoric acid and make up the volume to 1L. 20x SSC, pH 7.0 (1L) 175.3 g NaCl
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88.2 g sodium citrate Dissolve in 800 mL ddH2O. Adjust pH to 7.0 with 1N HCl, make up the volume to 1L, and autoclave for 20 min. 0.125N HCl 11mL conc HCl 898 mL dd H20 Denaturation buffer 87.66g NaCl 20g NaOH Dissolve in 800 mL ddH20 and make final volume to 1000 mL. Neutralization Buffer 87.66g NaCl 60.5g Trizma base Dissolve in 800 mL ddH20. Adjust pH to 7.5 with concentrated HCl. Make final volume to 1000 mL with dd H20. RNA Extraction Buffer 0.1 M LiCl 0.1 M Tris-HCl (pH 8.0) 0.01M EDTA (pH 8.0) 1% SDS (w/v), and 0.1% PVP (w/v) Protein Extraction Buffer 3 250mL 0.2M NaPO4 (pH 7) 10mL 1M DTT, 2mL 0.5M EDTA 10mL 10% SLS, 10mL 10% Triton X-100
Make up to 1L volume with dH20. Solution filter sterilized and stored at 4C.
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BIOGRAPHICAL SKETCH
Yuan Xiong was born in Cheng Du, China, to parents Xue Ou Xiong and Xin Qian.
He graduated from Shenzhen Experimental School, China in 2004, and graduated with
a Bachelor of Science from Sun Yat Sen University, Guangzhou, China in 2008. In
2009-2012, he attended the University of Florida and earned a Master of Science in
Department of Agronomy.