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Development of novel antibacterial and antiviral transgene vectors and techniques for their application and analysis in sugarcane.Pepper, Timothy Bryan. January 2002 (has links)
Sugarcane is challenged by a number of phytopathogenic bacteria and viruses that are best
managed by the development of resistant varieties. Genetic engineering is a promising
strategy in such breeding efforts, as it allows novel mechanisms of resistance not available in
any parent germplasm to be introduced into the crop.
DNA sequences encoding cystatin from papaya (Carica papaya), and pleurocidin from the
winter flounder (Pleuronectes americanus) were envisaged as transgenes in this work due to
their theoretical potential to increase sugarcane resistance to viruses and pathogenic or
opportunistic bacteria, respectively. Cystatin is a cysteine proteinase inhibitor. Cysteine
proteinases are used by potyviruses to cleave the polyprotein gene product, an essential step in
the viral life cycle. Constitutive expression of cystatin may therefore lend the host plant
resistance to a range of potyviruses, including the economically important pathogen sugarcane
mosaic virus (SCMV). Pleurocidin is an amphipathic, α-helical, cationic peptide, with broadspectrum anti-bacterial activity at physiological pH. By binding to the cell membranes of both Gram positive and Gram negative bacteria, pleurocidin disrupts the membrane potential,
causing it to become more permeable, especially to cations, leading to death of the bacterial
cell. Initial microbiological bioassays showed that pleurocidin has inhibitory and bactericidal
effects on the organisms which cause leaf scald (Xanthomonas albilineans), gumming disease
(Xanthomonas campestris pv. vasculorum) and post-harvest sucrose conversion in sugarcane,
as well as inhibitory effects against Leifsonia xyli ssp. xyli, which causes ratoon stunting
disease (RSD).
For transformation vector construction, the cystatin and pleurocidin coding sequences were
altered so that their start codons were in the most favourable consensus context for expression
in monocotyledonous plants. In the case of pleurocidin, an extracellular peroxidase signal
sequence was attached. The prepared sequences were spliced into the vector pUBI510 in
which the gene of interest is driven by the CaMV 35S promoter linked in tandem to a
derivative of the maize ubiquitin promoter. The constructs generated were named pUBI510-cys3
and pUBI510-pleur08 respectively. The plasmid structures were confirmed using
restriction endonuclease analysis and DNA sequencing.
Since the transformation of sugarcane is known to be inefficient, two routes of morphogenesis
for the production of somatic embryos were compared in the transformation procedure. These
were (1) indirect embryo production via callus and (2) the direct and indirect production of
embryos from transverse sections of leaf roll. Field grown sugarcane varieties N12 and
NCo376 were the source of explant material. Plasmids pUBI510-cys3 and pUBI510-pleuro8
were respectively co-delivered by microprojectile bombardment with the antibiotic resistance
selection plasmid pUBIKN containing the neomycin phosphotransferase gene (npt-II).
Cultures were maintained in the dark on selection medium containing various concentrations
of the antibiotic geneticin (G418) for several weeks before being allowed to regenerate in the
light. Plantlets coming through selection were hardened off in the glasshouse when
approximately 100mm high.
Primer pairs for amplification of the cystatin insert were designed in various ways. The primer
pair which ultimately proved most useful was designed to be complementary to the 5' and 3'
ends of the papaya cystatin nucleotide sequence. Primer Premier analysis of a sorghum
cystatin sequence provided additional possible primers. A further pair for potential future use
was devised based on complementarity to conserved regions on maize cystatins 1 and 2,
sorghum, rice, and papaya cystatins. The nucleotide sequence was constructed using the most
common monocotyledon codon permutations for each amino acid. Pleurocidin primers were
designed to be complementary to 5' and 3' regions of the nucleotide sequence encoding the
pleurocidin pre-pro-protein. PCR and RT-PCR protocols for the detection of transgenes and
transcript production in putative transgenic plants were developed using these primers.
No plants survived selection via the callus route, although some were regenerated via direct
embryogenesis. Putative transformed plants were analysed using PCR to test for the presence
of integrated transgenes and Southern hybridization to determine transgene copy number.
Both types of transgene were reproducibly detectable by PCR in DNA from some immature
plants, but results were negative in DNA from those same plants when mature. Southern
hybridization analysis detected the cystatin transgene in DNA from immature plants but no
transgenes were detected in up to 20 µg DNA from mature plants. Single copy constructions
of the transgenes in backgrounds of non-transformed DNA were detectable by both PCR and
Southern hybridization analysis. Overall, PCR, RT-PCR and Southern hybridization results
indicated that the plants regenerated fell into two categories: non-transformed plants that had
survived selection (escapes) and chimaeric individuals with a component of both transformed
and non-transformed cells, in which the transgene had probably become diluted during plant
development under non-selective conditions.
A method for extracting leaf exudates was tested, in conjunction with a cysteine proteinase
assay to detect the presence of cystatin transgenes in the intracellular spaces of sugarcane
leaves of confirmed transformants. Although it could not be applied within the scope of this
project, this assay will prove useful in future work. / Thesis (M.Sc.)-University of Natal, Durban, 2002.
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The development of an in situ hybridisation technique to determine the gene expression patterns of UDP-Glucose dehydrogenase, pyrophosphate-dependent phosphofructokinase and UDP-Glucose pyrophosphorylase in sugarcane internodal tissuesRamoutar, Rakeshnie 12 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2003. / ENGLISH ABSTRACT: The cellular expression of the enzymes implicated in regulating sucrose metabolism and
accumulation in sugarcane is poorly understood. The present study was therefore aimed at the
development of an in situ hybridisation (ISH) technique to study differential gene expression
among the various cell types of the sugarcane culm. This technique in conjunction with
northern and western blotting was then used to determine the sites of cellular and tissue
specific expression of the cytosolic enzymes, UDP-Glc dehydrogenase, pyrophosphate
dependent phosphofructokinase and UDP-Glc pyrophosphorylase, involved in sucrose
metabolism.
This study revealed that the determination of the influencing parameters associated with the
development of an ISH protocol was essential for the successful detection of the endogenous
RNA sequences in sugarcane internodal tissues. The parameters that were investigated
included the type of embedding medium, duration of fixation period, pre-treatment procedures
and hybridisation temperature. It further revealed that fresh internodal tissue sections, fixed
for a period of 24 h and thereafter exposed to pre-treatment and hybridisation, facilitated the
analysis of cytological gene expression at all stages of sugarcane development.
The second part of this study revealed very localised transcript expression for UDP-Glc DH,
PFP and UGPase in the different internodal tissue and cell types. The UDP-Glc DH and
UGPase transcripts were localised to the phloem elements, whilst xylem tissue only expressed
the UDP-Glc DH transcript. Transcripts of UDP-Glc DH, PFP and UGPase were all
expressed in the parenchyma cells that were associated with the vascular bundles and the stem
storage compartment, suggesting that the parenchyma cells distributed throughout the stem in
the different tissue types complement each other in function for the purposes of phloem
loading, unloading and assimilate transport processes.
Complimentary northern and western hybridisations demonstrated that internode 7 represents
a shift in the sink from utilisation to storage. This is evident by the observed decline in both
the relative transcript and protein abundances of UDP-Glc DH, PFP and UGPase at this stage
of development. The relative mRNA and protein abundances for the three enzymes showed a
similar trend. Higher levels of the gene transcripts and translated products were observed in
the younger sucrose importing tissues, than in the older sucrose accumulating internodes. At
a cellular level, it was found that the sites of cellular UDP-Glc DH, PFP and UGPase
expression differed marginally. Whilst UDP-Glc DH was expressed in the phloem, xylem and parenchyma cells of the vascular complex and in storage parenchyma cells, PFP was
expressed exclusively in parenchyma cells that were associated with the vascular bundles and
those serving a storage function in the stem pith and UGPase was found to be localised in the
phloem and parenchyma of the vascular bundles and the storage parenchyma cells. Such
findings have demonstrated an increase in resolution with which gene expression can be
examined at a cellular level. Hence, the results from this study have demonstrated that the
knowledge of metabolic compartmentation between different tissue and cell types is a
requisite to understanding the function(s) of individual enzymes within complex structures
such as the sugarcane culm. / AFRIKAANSE OPSOMMING: Die sellulêre lokalisering van die ensieme wat geïmpliseer word in die regulering van sukrose
metabolisme is onbekend. Met dit in gedagte, was hierdie studie gefokus op die ontwikkeling
van 'n in situ hibridisasie (ISH) tegniek om differensiële geenuitdrukking in die verskillende
seltipes van die suikerrietstingel te ondersoek. Hierdie tegniek, tesame met RNA-en proteïen
gel blots, is volgens aangewend om die areas van sellulêre-en weefselspesifieke uitdrukking
van die sitosoliese ensieme UDP-glukose dehydrogenase, pirofosfaat-afhanklike
fosfofruktokinase en UDP-glukose pirofosforilase, wat almal betrokke is by
sukrosemetabolisme, te bepaal.
Dit het duidelik geword gedurende die studie dat die bepaling van die optimale parameters
van die ISH protokol vir suikerriet van deurslaggewende belang sou wees vir die opsporing
van endogene RNA volgordes. Die parameters wat ondersoek is het ingesluit die tipe
inbeddingsmedium, die tydsduur van fiksering, vooratbehandelings- en hibridisasiemetodes.
Dit het duidelik geword dat vars internodale weefselsnitte wat vir 24 h gefikseer is en daarna
voorafbehandeling en hibridisasie ondergaan het, die bepaling van geenuitdrukking tydens
alle fases van suikkerrietontwikkeling moontlik gemaak het.
Die tweede fase van hierdie studie het aangetoon dat al drie ensieme spesifiek gelokaliseerde
uitdrukkingspatrone gehad het in verskillende internodale weefsels en seltipes. Al drie gene is
konstitutief uitgedruk in internodes. Die UDP-glukose dehydrogenase en UDP-glukose
pirofosforilase transkripte is gelokaliseer na die floeëm elemente, terwyl xileem slegs die
UDP-glukose dehydrogenase transkripte bevat het. Al die gene is in die parenchiemselle
uitgedruk wat geassosieer is met die vaatbondels en die stingel stoorkompartement, wat
moontlik beteken dat die parenchiem selle wat deur die stingel versprei is 'n sentrale netwerk
vorm wat direk of indirek koolstofassimileringsprosesse beïnvloed.
RNA-en proteïen gel blots op dieselfde internodes het gewys dat internode sewe 'n
verskuiwing, van koolstofverbruik na berging, verteenwoordig. Dit word gerllustreer deur die
afname in beide transkrip en proteïen vlakke van die drie ensiem in hierdie stadium van
ontwikkeling. Alhoewel beide mRNA en proteïen vlakke vir al die ensieme 'n soortgelyke
tendens getoon het, het die sellulêre uitdrukking van die ensieme volgens ISH verskil, wat die
krag van die tegniek illustreer. Die resultate van hierdie studie het gedemonstreer dat begrip
van die kompartementalisasie van metabolisme tussen verskillende weefsel-en seltipes 'n voorvereiste is om die funksie/s van individuele ensieme in komplekse strukture soos die
suikerrietstingel te bepaal.
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An investigation into the proteolytic degradation of antimicrobial peptides by plant extracts and localisation of pleurocidin in transgenic saccharum hybrid species.Goredema, Wadzanayi Patience. 21 October 2013 (has links)
Two cationic antimicrobial peptides, ESF I-GR7, and pleurocidin, were assessed for their stability in plant intercellular fluid, the targeted locale for their expression in transgenic plants. Incubation of ESFI-GR7 and pleurocidin with intercellular fluid (ICF) extracted from sugarcane, tomato and tobacco leaves reduced their biotoxicity towards various pathogens, namely Camobacterium mobile DMSO and Xanthomollas campestris. It was concluded that it may be necessary to modify the aminoacid structures of the peptides in order to ensure that endogenous proteases would not degrade the peptides once expressed in a transgenic environment. The presence of pleurocidin was detected in transgenic sugarcane transformed (in a previous study) with pleurocidin gene cloned into the pUBI 510 plasmid. ICF was extracted from four month old transgenic Saccharum hybrid species (sugarcane). Western blotting verified the presence of the transgenic protein in crude protein extracts. Immunogold labelling and transmission electron microscopy were performed to investigate the
localisation of transgenic pleurocidin. The peptide was localized predominantly in the intercellular spaces and cell wall sugarcane leaves. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2001.
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The potential use of sugarcane varieties for the identification of genetic markers.Barnes, Julie Megan. 14 January 2014 (has links)
The use of genetic markers that are linked to specific traits in sugarcane has the potential to
increase the efficiency of the selection of improved varieties. Conventionally, markers are
identified by analysing the segregation of potential markers and traits in the progeny of single
crosses. However, this approach is not practical for sugarcane breeding programmes where
replicated, well characterized progenies do not exist. The objective of this project was to
investigate the potential of using commercial varieties for identifying markers associated with
some of the important traits in sugarcane. This approach would be far more effective than
dealing with single progenies since the traits of commercial varieties have already been
characterized.
The DNA of fifty commercial varieties of sugarcane was amplified by RAPD PCR using forty-one
arbitrary decamer primers. Analysis of the resulting banding profiles, obtained by agarose
gel electrophoresis, yielded fifty-four reliable polymorphic fragments. Two approaches were
used to identify putative markers linked to the traits of resistance to eldana, sugarcane mosaic
virus, and smut: (1) a correlation approach which attempted to identify whether the presence
of any polymorphisms could be used to imply the existence of a particular phenotypic state,
and (2) multiple regression analysis, in order to determine whether polymorphisms could be
used to predict the performance of the varieties for each of the traits. Both approaches
appeared to identify associations between polymorphisms and the traits, although multiple
regression analysis yielded the most informative results and was able to assign statistical values
to the associations.
Using multiple regression, the best predictive model was obtained for sugarcane mosaic virus
resistance. This model consisted of four polymorphisms and had an r² of 0.40l. By dividing the
resistance ratings into three groups (resistant, intermediate and susceptible), 52% of the varieties
were correctly classified and only 2% of the varieties were predicted in opposite groups (i .e.
predicted susceptible when actually resistant, and vice versa). The predictive model for eldana
resistance consisted offour polymorphisms and had an r² of 0.347. This model classified 30% of
the varieties in the correct group of three while none of the varieties were predicted in opposite groups. The predictive model for smut resistance consisted of three polymorphisms and had an
r² of 0.316. This model classified 30% of the varieties in the correct group of three while 2% of
the varieties were predicted in opposite groups.
Further analysis of sugarcane varieties using additional polyrnorphisrns has the potential to identify
markers linked to important traits. These markers could be used for marker-assisted selection to
increase the efficiency of selecting for improved sugarcane genotypes for commercial release. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1996.
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Comparision of two promoters driving transgene expression in water-stressed sugarcane.Cassim, Tasmien Nadine. January 1999 (has links)
For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. Tissue- or signal-responsive promoters are in high demand in practical plant biotechnology. The present study sought to characterise the activities of two promoters in sugarcane, namely the UBI (ubiquitin) promoter and the SUC-1 promoter (UBI linked in tandem to the cauliflower mosaic virus 35S promoter). It was hypothesised that the activity of UBI would be maintained or even increased under conditions of environmental stress, since it is well documented that ubiquitin is a stress-related protein. A further hypothesis was that SUC-1 might enhance overall gene expression since the CaMV 35S component is a constitutive promoter widely and successfully used in plant transformation. Plants of the sugarcane variety NC0310, containing the cry1A(c) (Bt) gene from Bacillus thuringiensis, were used as models in a system in which the plants were stressed by withholding water supply in a controlled manner. Since large numbers of clones of both transgenic and wild-type plants were needed for the water stress and expression experiments, three micropropagation techniques, namely, shoot tip-, callus- and node culture, were optimised and compared. The objective was to propagate genetically stable plants rapidly. Compared to shoot tip culture, node and callus culture proved slow and inefficient. Shoot tip culture was thus chosen as the most suitable for the regeneration of experimental material. Relative Water Content (RWC) determination, leaf elongation measurements and Infra Red Gas Analysis (IRGA) were compared in order to find the most appropriate method of measuring plant water status. In addition to being destructive, no observable differences were evident between the control (non-stressed) and water-stressed plants when using RWC as a measure. Results obtained from leaf elongation measurements compared favourably to the more sophisticated IRGA readings, showing that leaf elongation is as sensitive a measure of water stress. On the basis of preliminary studies with untransformed plants using the latter two techniques, water regimes for stress-induction in the final experiments were designed. Leaf elongation measurements, which are simple and non-destructive, were ultimately chosen to measure plant water status. In the final water stress experiment non-transgenic NCo310 and clonal populations of six transformants were used (three containing the UBI promoter; three the SUC-1 promoter). Exactly half of the plants of each type were stressed by withholding water supply, while the other half (controls) were watered manually twice a day. Leaf elongation measurements were made at the same time daily on the third youngest leaf of 6 plants from each population per treatment. At the same time, leaf samples were taken daily for molecular analysis. The stress regime led to marked differences in leaf elongation between control and water-stressed plants. In terms of physiological response (leaf rolling and senescing), plants containing the SUC-1 promoter appeared least affected. The reverse transcription-polymerase chain reaction (RT-PCR) and Northern hybridisation were used to assay UBI and SUC-1 activity. RT-PCR revealed that both promoters drove Bt gene expression in controls and experimentals throughout the stress period, although differences in signal intensity were not observed. The extent of expression occurring in each type of plant was revealed in Northern blots probed with two genic sequences (1) the transgene and (2) sugarcane EST ME42, homologous to heat shock protein 82 in rice. Individual transformants showed overall levels of transgene expression that were variable, possibly due to insert position in the plant genome, as well as variations in relation to the application of stress. SUC-1 seemed superior to UBI in terms of driving transgene expression under stressful environmental conditions, since UBI promoter activity appeared to decrease under stress, while SUC-1 promoter activity remained constant. In addition to the expected 2.0 kb Bt transcript, transcripts of smaller than expected size were also obtained, leading to the suggestion of premature polyadenylation signals in the coding region of the wild-type Bt234 gene. Upon inspection of the transgene sequence, a number of motifs rarely present in plant genes were observed, namely A/T rich sequences, ATTTA motifs and numerous potential polyadenylation sites. / Thesis (M.Sc.)-University of Natal, Durban, 1999.
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Manipulation of pyrophosphate fructose 6-phosphate 1-phosphotransferase activity in sugarcaneGroenewald, Jan-Hendrik 03 1900 (has links)
Thesis (PhD (Genetics. Plant Biotechnology))--University of Stellenbosch, 2006. / The main aim of the work presented in this thesis was to elucidate the apparent role of
pyrophosphate fructose 6-phosphate 1-phosphotransferase (PFP) in sucrose accumulation
in sugarcane. PFP activity in sugarcane internodal tissue is inversely correlated to the
sucrose content and positively to the water-insoluble component across varieties which
differ in their capacities to accumulate sucrose. This apparent well defined and important
role of PFP seems to stand in contrast to the ambiguity regarding PFP’s role in the general
literature as well as the results of various transgenic studies where neither the downregulation
nor the over-expression of PFP activity had a major influence on the phenotype
of transgenic potato and tobacco plants. Based on this it was therefore thought that either
the kinetic properties of sugarcane PFP is significantly different than that of other plant
PFPs or that PFP’s role in sucrose accumulating tissues is different from that in starch
accumulating tissues.
In the first part of the study sugarcane PFP was therefore purified and its molecular and
kinetic properties were determined. It consisted of two subunits which aggregated in
dimeric, tetrameric and octameric forms depending on the presence of Fru 2,6-P2. Both the
glycolytic and gluconeogenic reactions had broad pH optima and the kinetic parameters for
all the substrates were comparable to that of other plant PFPs. The conclusion was therefore
that sugarcane PFP’s molecular and kinetic characteristics do not differ significantly from
that of other plant PFPs.
The only direct way to confirm if PFP is involved in sucrose accumulation in sugarcane is
to alter its levels in the same genetic background through genetic engineering. This was
therefore the second focus of this study. PFP activity was successfully down-regulated in
sugarcane. The transgenic plants showed no visible phenotype under greenhouse and field
conditions and sucrose concentrations in their immature internodes were significantly
increased. PFP activity was inversely correlated with sucrose content in the immature internodes of the transgenic lines. Both the immature and mature internodes of the
transgenic plants had significantly higher fibre contents.
This study suggests that PFP plays a significant role in glycolytic carbon flux in immature,
metabolically active sugarcane internodal tissues. The data presented here confirm that PFP
can indeed have an influence on the rate of glycolysis and carbon partitioning in these
tissues. It also implies that there are no differences between the functions of PFP in starch
and sucrose storing tissues and it supports the hypothesis that PFP provides additional
glycolytic capacity to PFK at times of high metabolic flux in biosynthetically active tissue.
This work will serve as a basis to refine future genetic manipulation strategies and could
make a valuable contribution to the productivity of South African sugarcane varieties.
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Marker assisted breeding in sugarcane : a complex polyploidButterfield, Michael Keith 03 1900 (has links)
Thesis (PhD (Genetics))—University of Stellenbosch, 2007. / Association analysis was used to improve the efficiency of breeding sugarcane varieties for the
negatively correlated traits of resistance to sugarcane smut and the eldana stalk borer. 275 RFLP and
1056 AFLP markers were scored across a population of 77 genotypes representing the genetic
variation present within the SASRI breeding programme. Genetic diversity analysis did not detect
significant structure within the population. Regression analysis identified 64 markers significantly
associated with smut rating and 115 markers associated with eldana rating at r2 > 6.25%. Individual
markers with the largest effects explained 15.9% of the phenotypic variation in smut rating and 20.2%
of the variation in eldana. Five markers were significantly associated with both smut and eldana. In
each case the marker effect was negatively correlated between the two traits, suggesting that they are
genetically as well as phenotypically negatively correlated.
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Genetic manipulation of the cell wall composition of sugarcaneBekker, Jan P. I. 03 1900 (has links)
In order to understand and manipulate carbon flux to sucrose one needs to consider not only
its biosynthetic pathways, but also the competing sinks for carbon in various parts of the
plant and at different stages of development. The cell wall and sucrose is known to be the
major sinks for carbon in young and mature tissues of sugarcane. UDP-Glucose is a central
metabolite in the synthesis of both sucrose and most of the cell wall polysaccharides
(including cellulose, hemicellulose and pectic polymers) and manipulation of the flux into
either of the cell wall components could therefore cause an increase of flux toward one or
more of the competing sinks. In the present study UDP-Glucose dehydrogenase (UGD)
activity was chosen for down regulation as it catalyzes the rate limiting step in the
biosynthesis of the precursors of both hemicellulose and pectin, a major competing sink for
assimilated carbon.
Transgenic sugarcane lines with repressed UGD activity showed significantly increased
sucrose accumulation in all internodes which was highly correlated with reduced UGD
activity. Sucrose phosphate synthase had increased activation which suggests an alteration
in carbon flux toward sucrose.
The reduction of carbon flux through UGD was compensated for by an increase in the
activity of the myo-inositol oxygenation pathway (MIOP), an alternative pathway for the
synthesis of cell wall matrix precursors. The increased activity of the MIOP resulted in
increased total uronic acids and pentoses in the cell wall. Total cell wall glucose was also
increased which is a further indication of altered carbon metabolism.
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Increasing cellulosic biomass in sugarcaneNdimande, Sandile 04 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Increased demand of petroleum, declining fossil fuel reserves, geopolitical instability and the environmentally detrimental effects of fossil fuels have stimulated research to search for alternative sources of energy such as plant derived biofuels. The main feedstocks for production of first generation biofuels (bioethanol) are currently sucrose and starch, produced by crops such as sugarcane, sugarbeet, maize, and cassava. The use of food crop carbohydrates to produce biofuels is viewed as competing for limited agronomic resources and jeopardizing food security. Plants are also capable of storing sugars in their cell walls in the form of polysaccharides such as cellulose, hemicelluloses and pectin, however those are usually cross-linked with lignin, making their fermentation problematic, and are consequently referred to as lignocellulosics. Current technologies are not sufficient to degrade these cell wall sugars without large energy inputs, therefore making lignocellulosic biomass commercially unviable as a source of sugars for biofuel production. In the present study genes encoding for enzymes for cellulosic, hemicellulosic and starch-like polysaccharides biosynthesis were heterologously expressed to increase the amount of fermentable sugars in sugarcane. Transgenic lines heterologously expressing CsCesA, encoding a cellulose synthase from the marine invertebrate Ciona savignyi showed significant increases in their total cellulose synthase enzyme activity as well as the total cellulose content in internodal tissues. Elevation in cellulose contents was accompanied by a rise in hemicellulosic glucose content and uronic acid amounts, while total lignin was reduced in internodal tissues. Enzymatic saccharification of untreated lignocellulosic biomass of transgenic sugarcane lines had improved glucose release when exposed to cellulose hydrolyzing enzymes.
Calli derived from transgenic sugarcane lines ectopically expressing galactomannan biosynthetic sequences ManS and GMGT from the cluster bean (Cyamopsis tetragonoloba) were observed to be capable of producing a galactomannan polysaccharide. However, after regeneration, transgenic sugarcane plants derived from those calli were unable to produce the polymer although the inserted genes were transcribed at the mRNA level. While the ectopic expression of Deinococcus radiodurans amylosucrase protein in the cytosol had a detrimental effect on the growth of transgenic lines (plants showed stunted growth through the 18 months growth period in greenhouse), contrastingly targeting the amylosucrase protein into the vacuole resulted in 3 months old transgenic lines which were having high maltooligosaccharide and soluble sugar (sucrose, glucose and fructose) levels in leaves. After 18 months growing in the greenhouse, the mature transgenic lines were morphologically similar to the untransformed lines and also contained comparable maltooligosaccharide and soluble sugar and starch amounts. The non-biosynthesis of galactomannan and amylose polysaccharides in the matured transgenic plants may be due to post-transcriptional protein processing and or protein instability, possibly explainable by other epigenetic mechanisms taking place to regulate gene expression in the at least allo-octaploid species of sugarcane under investigation in this study.
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Promoters for sugarcane transformation : isolation of specific sequences and evaluation of rolC.Groenewald, Sarita. 23 December 2013 (has links)
Increasing the sucrose yield and the disease resistance of plants are two major
objectives of the transgenic sugarcane plant programme in South Africa. The
sugarcane culm has thus been identified as one of the main target areas for
transgene expression. A shortage of reliable promoter elements as well as patent
limitations have necessitated the isolation of promoters that are preferentially
expressed in the sugarcane culm. In the present study two different approaches were
followed to isolate such promoters, and the bacterial promoter, rolC, was evaluated for tissue-specific expression in sugarcane.
Differential display is a non-directed technique that was used to identify genes that
are differentially expressed in the mature sugarcane culm. The original method was
modified, and four putative culm-preferential fragments were isolated. Sequence and
hybridisation analyses revealed that these fragments were false positives, and could therefore not be used to obtain a culm-specific promoter.
Activity of the Agrobacterium rolC promoter was evaluated by analysing expression
patterns of two reporter genes in the mature culm of transgenic sugarcane plants.
Nucleic acid analyses indicated that the foreign DNA was incorporated into the sugarcane genome, and that mRNA transcripts were produced. Histochemical
analysis was done to visualise rolC-driven GUS and GFP expression in the mature
sugarcane culm. In both cases the reporter gene expression was restricted to the vascular bundles and specifically to the phloem.
A directed approach was followed to isolate the gene and subsequently the promoter
of the β-subunit of pyrophosphate-dependent phosphofructokinase (PFP-β). An
incomplete cDNA clone was obtained from a mature culm cDNA library, and was
used for the screening of a sugarcane genomic library. Two clones containing
different parts of the PFP-β gene were isolated. A Deletion Factory™ system was
used to analyse the clone containing the 5' end of the gene. The first five exons and
1747 bp of the 5' flanking region of the gene were sequenced. Preliminary activity analysis of the promoter region was done by constructing two expression vectors, and analysing transient GUS expression in sugarcane callus. Results indicated that the promoter is capable of driving foreign gene expression in callus. Transient expression levels were lower than that of the maize Ubi-1 promoter. Further analysis of the 5' flanking region will be done to establish whether cis-acting elements outside
the analysed area have an influence on the activity of the promoter. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1997.
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