Thesis (Ph. D.)--University of Hawaii at Manoa, 1994. / Includes bibliographical references (leaves 126-140). / Microfiche. / 140 leaves, bound ill., photographs (some col.) 29 cm

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: Sugarcane, a complex polyploid, may theoretically contain up to 12 alleles for a particular gene at a single locus. The number of alleles and the extent of their variation is of particular importance due to the potential for the exploitation of genetic variation through breeding. Also, allelic variation has implications for the manipulation of gene function via genetic engineering. Pyrophosphate dependent phosphofructokinase (PFP) is considered a key regulatory enzyme involved in sucrose biosynthesis, which may provide a target for genetic manipulation to increase sucrose yields in sugarcane. The enzyme is composed of a regulatory (alpha) and catalytic (beta) protein subunit encoded by the PFP-a and PFP-p genes respectively. The PFP-p gene, which has been shown to be a single locus gene in other plant species, was used in this study as a model for allelic variation in sugarcane. Two main areas of investigation involved genomic and expression analyses to further characterise the gene. Polymerase Chain Reaction (PCR) using specific primers previously designed from conserved regions of the PFP-p gene from different plant sources, were used to amplify across exons 10 to 12 of the sugarcane PFP-p gene. Two PCR products, designated PFP-81/881250bp and PFP-81/881100bp respectively, were obtained from commercial cultivars N19 and N21. Numerous clones of the fragments were obtained and sequenced. International database searches confirmed that both amplicons were identifiable as PFP-p. Comparative sequence analysis indicated that the PFP-81/881250bP and PFP-81/881100bp fragments were poorly homologous to each other, with higher regions of homology residing in the putative exon regions (77-78%) compared to the intron regions (34- 56%). Although minor sequence variation was detected within the amplicon populations, it was evident that two major variants of the PFP-p gene are present in sugarcane. Southern hybridisation analysis revealed a simple banding pattern for PFP-p. Also, there are DNA polymorph isms for the genomic regions corresponding to the PFP-81/881250bP and PFP-81/881100bPfragments. Previous evidence indicates that both variants are also present in the ancestral sugarcane germplasm and maintain the same level of heterozygosity. The presence of both gene forms in the ancestral and commercial germplasm prompts speculation that the two variants may not segregate. This theory, together with the simple Southern hybridisation pattern obtained for PFP-p, leads to the hypothesis that the two gene forms are at separate loci in the sugarcane genome, which may be closely linked on the same chromosome. The expression of the variants was investigated during different stages of sucrose accumulation in the sugarcane culm using a Reverse Transcription (RT)- peR approach. A single, identical transcription product was isolated from these and other selected tissues of the plant. In addition, the same transcript was obtained from the ancestral species representatives of modern sugarcane, Saccharum officinarum and Saccharum spontaneum. Sequence comparison of the transcribed product and the derived exon regions of the two variants implies that the PFP-p gene represented by the PFP-B 1/B81250bpvariant is being expressed in sugarcane while the gene form characterised by the PFP-B1/B81100bp amplicon is silent. Northern hybridisation analysis indicates that PFP-p is differentially expressed at different stages of sucrose accumulation. PFP-p expression is higher in the immature culm tissue of sugarcane and low in the mature culm, which suggests that PFP-p is highly regulated during maturation. It is hypothesised that the PFP-p gene underwent duplication and that one gene form was subject to accumulative mutations evolving into a pseudogene. On the basis of present results, it can be suggested that future genetic manipulation of PFP-p should involve the gene variant characterised by the PFP-B 1/B81250bp fragment. / AFRIKAANSE OPSOMMING: Suikerriet, 'n komplekse poliploïede organisme, kan teoreties tot 12 allele vir 'n spesifieke geen by 'n enkele lokus bevat. Die aantal allele en hul mate van variasie is veral van belang, weens die potensiaal vir die benutting van hierdie genetiese variasie deur teëling. Alleelvariasie het ook implikasies vir die manipulering van geenfunksie via genetiese inginieurswese. Pirofosfaat-afhanklike fosfofruktokinase (PFP) is 'n belangrike regulatoriese ensiem vir sukrose biosintese en kan geteiken word vir genetiese manipulasie met die oog op verhoogde sukroseproduksie in suikerriet. Die ensiem bestaan uit 'n regulatoriese (alfa) en katalitiese (beta) proteïen subeenheid wat deur die PFP-a en PFP-~ gene respektiewelik gekodeer word. Die PFP-~ geen, wat in ander plantspesies as 'n enkellokusgeen aangedui is, is in hierdie studie gebruik as 'n model vir alleelvariasie in suikerriet. Die twee hoofroetes van ondersoek wat gevolg is, behels genoom- en uitdrukkingsanalises om die geen verder te karakteriseer. Eksons 10 tot 12 van die suikerriet PFP-~ geen is geamplifiseer met behulp van die Polimerase Ketting Reaksie (PKR), bemiddel deur die gebruik van spesifieke voorvoerders wat voorheen ontwerp was vanaf gekonserveerde areas van die PFP-~ geen uit verskillende plantbronne. Twee PKR produkte, genoem PFP-B1/B81250bPen PFP-B1/B81100bPrespektiewelik, is deur die kommersiële kultivars N19 en N21 opgelewer. Verskeie fragmentklone is gekonstrueer en hul DNA basisvolgorde is bepaal. Soektogte in internasionale databasisse het bevestig dat beide amplikons PFP-~ was. Vergelykende DNA basisvolgorde analise het aangedui dat PFP-B1/B81250bP en PFP-B1/B81100bP fragmente swak homologie toon, terwyl 'n hoër mate van homologie in die oënskynlike ekson areas (77-78%), vergeleke met die intronareas (34-56%) gevind is. Alhoewel klein basisvolgordeverskille opgemerk is binne die amplikonpopulasies, was dit duidelik dat twee hoofvariante van die PFP-~ geen in suikerriet teenwoordig is. Southern hibridisasie analisie het 'n eenvoudige band patroon vir PFP-~ onthul. Daar is ook DNA polimorfismes vir die genoomstreke wat met die PFP-B1/B81250bPen PFP-B1/B81100bPfragmente ooreenstem. Vorige bewyse het aangetoon dat beide variante ook in die voorvaderlike suikerriet kiemplasma voorkom en dat dieselfde vlak van heterosigositeit gehandhaaf word. Die voorkoms van beide geenvorme in die voorvaderlike asook kommersiële kiemplasmas stel voor dat hierdie twee variante miskien nie segregeer nie. Hierdie teorie, tesame met die eenvoudige Southern hibridisasiepatroon vir PFP-p, lei tot die hipotese dat die twee geen vorme by verskillende lokusse in die suikerrietgenoom voorkom, en dat hierdie lokusse nou gekoppel mag wees op dieselfde chromosoom. Die uitdrukking van die variante is ondersoek gedurende verskillende stadia van sukrose akkumulasie in die suikerrietstingel deur van Trutranskripsie (TT)-PKR gebruik te maak. 'n Enkele, identiese transkripsie produk is hieruit en uit ander geselekteerde plantweefsels geïsoleer. Dieselfde transkrip is ook verkry vanaf die voorouerlike spesieverteenwoordigers van hedendaagse suikerriet, Saccharum officinarum en Saccharum spontaneum. 'n DNA basisvolgordevergelyking tussen die getranskribeerde produk en die ekson-areas van die twee variante impliseer dat die PFP-p geen verteenwoordig deur die PFP-B1/B81250bPvariant uitgedruk word in suikerriet, terwyl die geen verteenwoordig deur die PFP-B1/B81100bpamplikon stil is. Northern hibridisasie analise toon aan dat PFP-p verskillend uitgedruk word gedurende verskillende stadia van sukrose akkumulasie. PFP-p uitdrukking is hoër in die onvolwasse stamweefsel van suikerriet en laag in die volwasse stam, wat voorstel dat PFP-p hoogs gereguleer word gedurende maturasie. Daar word gehipotetiseer dat die PFP-p geen duplikasie ondergaan het en dat een geenvorm onderworpe was aan akkumulerende mutasies wat deur evolusie tot 'n pseudogeen gelei het. Dit word voorgestel, gebaseer op die huidige resultate, dat toekomstige genetiese manipulasie van die PFP-p geen, die geenvariant gekarakteriseer deur die PFP- B1/B81250bPfragment moet behels.

Thesis (MSc)--University of Stellenbosch, 2001. / ENGLISH ABSTRACT: The goals of this project were to (i) determine maximum extractable neutral invertase (NI) activity in the sugarcane culm, (ii) sequence a cDNA encoding for the sugarcane NI (SNI), (iii) determine SNI copy number in the genome, (iv) describe SNI transcript and protein expression patterns throughout the plant, and (v) attempt to determine the contribution of hydrolysis to sucrose accumulation. SNI and sugars were extracted from the developing culm tissues of sugarcane, commercial variety N19. Tissues were divided according to developmental stage (internodes 3, 6 and 9) and anatomical differentiation (enriching for elongating, vascular or storage tissues). The lowest sucrose content was found in the core of the bottom of each of the internodes. The ratio between hexoses and sucrose was highest in the young internodes. In these internodes hexose content was higher in the bottom than the top. There was a significant correlation between sucrose content and NI. Fluxes involved in sucrose synthesis and hydrolysis were investigated. The hexoses glucose and fructose were supplied as a carbon source for tissue discs of young and maturing internodal tissues of sugarcane, varieties N19 and US6656-15. Sucrose content was 10-fold higher in maturing internodes of N19 than US6656-15. Calculated sucrose hydrolysis rates via invertase were higher in maturing internodes of US6656-15 than N19. Taking metabolic compartmentation into account, hydrolysis of sucrose via invertase made a significant contribution to the net turnover of sucrose. Along with this, it would appear that the ability to partition sucrose between the vacuole and cytosol causes a significant difference in sucrose content between varieties. A full-length cDNA for SNI was sequenced. This expressed gene showed significant homology to known NI sequences on both nucleic and amino acid levels. The SNI sequence did not contain the putative invertase catalytic amino acid sequence, suggesting it developed separately from the other classes of invertases. Approximately 1.8 kb of the SNI cDNA was incorporated into a vector suitable for direct bombardment into sugarcane tissue. Southern blot analysis showed the enzyme has a low copy number. SNI transcript expression was observed in all tissues of the sugarcane plant: roots, internodes, leaf roll and leaves. In culm tissues where sucrose content was low and hexose contents were high, SNI transcript and protein levels were high. This suggests that SNI is involved in growth metabolism. / AFRIKAANSE OPSOMMING: Die doel van die projek was om (i) maksimum ekstaheerbare neutrale invertase (NI) aktiwiteit in die suikerriet stingel te bepaal, (ii) die volgorde van 'n eDNA wat vir suikerriet NI (SNI) kodeer te bepaal, (iii) die SNI kopie-getal in die genoom te bepaal, (iv) SNI m- RNA en proteïenuitdrukkingspatrone deur die plant te beskryf, en (v) te poog om die bydrae van hidrolise op sukrose akkumulering te bepaal. SNI en suikers is geëkstraheer uit 'n kommersiële varieteit, N19. Weefsels was volgens ontwikkelingstadiums (internodes 3, 6 en 9) en anatomiese verskille (verryking vir groeiende, vaat- en bergings-weefsels) verdeel. Die laagste sukrose inhoud is in die middel van die onderste helfte van elke internode gevind. Die verhouding van heksoses tot sukrose was die hoogste in die jong internodes. Die inhoud heksoses was hoër in die onderste deel van die internode as die boonste deel. 'n Betekenisvolle korrelasie tussen sukrose inhoud en SNI is gevind. Flukse betrokke by sukrose sintese en hidrolise is ondersoek. Glukose en fruktose is as koolstofbron aan stingelweefsel van twee variëteite (US6656-15 and N19) toegedien. Sukrose-inhoud het tienvoudig tussen volwasse weefsels van die twee variëteite verskil. Hidrolise via invertase was hoër in ouer weefsels van US6656-15 as N19, en het In noemenswaardige bydrae tot sukroseomset gemaak. Die verdeling van sukrose tussen die vakuool en die sitosol kan moontlik 'n groot rol speel in die vermoë van die sel om sukrose te akkumuleer. Die volgorde van 'n volledige SNI eDNA is bepaal. The uitgedrukte geen het, op beide In nukleïen- en aminosuur vlak, betekenisvolle ooreenkoms getoon met ander bekende plant NI volgordes. Die SNI volgorde bevat nie die kenmerkende invertase katalitiese setel nie, wat daarop kan dui dat dit onafhanklik van ander klasse invertases ontwikkel het. Min of meer 1.8 kb van die SNI eDNA is in 'n vektor geskik vir bioliestiese transformering van suikerrietweefsel, geïnkorporeer. Southern klad analise het gewys dat die ensiem 'n lae kopiegetal op geen vlak het. SNI mRNA uitdrukking is waargeneem in elke weefseltipe van die suikerriet plant: wortels, internodes, blaarrol en blare. In stingelweefsels met lae sukrose- en hoë heksose-inhoud, was die vlakke van beide SNI-mRNA en -proteïen hoog. Dit dui daarop dat SNI moontlik betrokke is by groei-metabolisme.

Thesis (MSc)--University of Stellenbosch, 2002. / ENGLISH ABSTRACT: For the expression of transgenes in plant cells, appropriate promoter sequences have to be introduced upstream of the gene to ensure efficient transcription. While to date the maize ubiquitin (Ubi1) promoter has been the most effective transgene promoter for sugarcane, there is a high demand for tissue and stage specific promoters for localised transgene expression in the mature culm. The present study sought to characterise genes preferentially expressed in the core and peripheral tissues of the mature culm, which can further be used as research tools for specific promoter isolation. cDNA expression arrays containing 3840 clones from a late stage cDNA library representative of the core and peripheral tissues of the mature culm were prepared. The cDNA expression arrays were then differentially screened in independent hybridisation experiments with radioactively-labeled cDNA representations of core and peripheral tissues of internode 7, and peripheral tissues of internode 10. Comparison of the expression profiles of the arrayed cDNA targets in the three probes led to the identification of 60 tissue-specific, 17 stage-specific and 50 selectively expressed cDNAs within the mature sugarcane culm. ~ESTs of 33 chosen selectively expressed cDNAs with a relatively stronger pattern of expression in the core than in the peripheral tissues revealed sequence homology to a diverse collection of genes in the mature culm. These included genes associated with general cellular metabolism such as protein synthesis, protein modification and structural protein. Also identified were stress-responsive genes. The putative translational products of some of these clones had homologs that are involved in cell-wall structure in other species. These included the [acalin homolog, a lectin, hydroxyproline rich glycoprotein and structured polyprotein C. Many of the cDNAs thought to be involved in cell wall structure or stress related responses also accumulate in a developmental manner in other plants. These may indicate that specific mature culm mRNAs accumulate in response to stresses such as rapid cell expansion or as part of the late developmental program. An unexpected observation was that only one gene associated with sucrose metabolism was identified, namely sucrose synthase. These results confirmed that culm maturation was not controlled by sucrose metabolism despite its distinct physiological characteristic of storing high levels of sugars. ESTs analysis further revealed that sequence homology was not obtained for all the cDNAs exhibiting stage and tissue specific expression in the core and peripheral tissues of the mature culm. These could represent novel genes not only from sugarcane but all plants. Northern analysis demonstrated that 9 putatively identified selectively expressed genes tested so far accumulated specifically in the core and peripheral tissues of the mature culm. No expression was detected in root, leaf, leafroll and internode 3. However, their selective expression in a single internode as observed on the arrays (i.e hybridisation signal intensity being higher in the core than in the peripheral tissue) was not detected on the northern blots. These showed that cDNA expression arrays were not a highcapacity gene expression assay since they were prone to false expression analysis. The validity of results obtained through array screening should always be verified in an independent manner, preferably by the northern hybridisation analysis. Hence, the present study shows that the combination of differential screening, northern blot and DNA sequence analysis permits the rapid characterisation of differentially expressed genes in the core and peripheral tissues of the mature sugarcane culm. These can further be used as research tools for mature culm - specific promoter isolation in the sugarcane. / AFRIKAANSE OPSOMMING: Die doeltreffende uitdrukking van transgene in plantselle is afhanklik van 'n gepaste promotorvolgorde wat stroomop van die geen ingevoeg word. Die Ubi1-promotor van mielies was tot dusver die doeltreffendste transgeenpromotor in suikerriet, maar daar is 'n groot behoefte aan promotors wat weefsel- en ontwikkelingstadium-spesifieke geenuitdrukking kan beheer. Hierdie studie het op die isolering en karakterisering van gene wat selektief in die kern- of periferale stingelweefsel van suikerriet uitgedruk word, gefokus. Hierdie gene sal verder benut kan word om promotors te isoleer. eDNA uitdrukkingsreekse ("expression arrays") van 'n volwasse stingel eDNA biblioteek is voorberei. Hierdie reekse, wat 3840 klone bevat het, is in onafhanklike hibridiseringseksperimente met radioaktiefgemerkte eDNA van onderskeidelik kern- en periferale stingelweefsel van lit 7 en periferale stingelweefsel van lit 10 afgetas. 'n Vergelyking van die uitdrukkingsprofiele van die eDNA teikens in dié drie peilergroepe het tot die identifisering van 60 weefsel-spesifieke-, 17 ontwikkelingstadium-spesifiekeen 50 selektief uitgedrukte eDNAs in die volwasse suikerrietstingel gelei. Uitdrukkingsvolgordemerkers ("ESTs") van 33 geselekteerde eDNAs wat in hoër vlakke in die kern uitgedruk is, se volgordes toon homologie aan 'n wye verskeidenheid gene in die volwasse stingel. Hierdie groep sluit gene in wat met algemene sellulêre ..metabolisme soos proteïensintese, proteïenmodifisering en strukturele proteïene geassosieer is. Spanningsverwante gene is ook hier geïdentifiseer. Die transleringsprodukte van sommige klone het homoloë wat by selwandstruktuur in ander spesies betrokke is, soos die jaealin-homoloog, 'n lektien, hidroksiprolien-ryke glikoproteïen en gestruktureerde poliproteïen C. 'n Wye verskeidenheid eDNAs wat by selwandstruktuur of spanningsverwante reaksies betrokke is, akkumuleer ook in 'n ontwikkelingsafhanklike wyse in ander plante. Dit mag 'n aanduiding wees dat spesifieke mRNAs in die volwasse stingel in reaksie op spanning wat met vinnige seluitsetting gepaardgaan, versamel. Slegs een geen wat met sukrose metabolisme geassosieer is, nl. sukrosesintase, is in hierdie studie geïdentifiseer. Hierdie onverwagte waarneming het bevestig dat, ondanks suikerriet se kenmerkende vermoë om hoë konsentrasies suiker te berg, stingelveroudering nie net met sukrose metabolisme geassosieer kan word nie. Nie al die eDNA-fragmente wat geïsoleer is, het homologie aan ander gene in die internasionale databasisse getoon nie, wat moontlik kan aandui dat nuwe gene suksesvol geïsoleer is. Nege ontwikkelingstadium-spesifieke gene wat slegs in die volwasse stingelweefsels uitgedruk word, is dmv noordelike oordraganalises geïdentifiseer. Geen transkripte van hierdie gene is in die wortels, blaarrol, blare of jong stingel waargeneem nie. Die weefselspesifisiteit wat met die uitdrukkingsreekse waargeneem is, kon nie mbv noordelike orrdraganalises bevestig word nie. Dit mag 'n aanduiding wees dat die uitdrukkingsreekse vals positiewe resultate kan oplewer en dit is raadsaam om voortaan altyd die verkrygde profiele met ander, meer sensitiewe tegnieke, te bevestig. Die studie het aangetoon dat 'n kombinasie van differensiële aftasting, noordelike oordraganalise en DNA-volgordebepaling gebruik kan word om gene wat differensieel uitgedruk word in die volwasse suikerrietstingel, te identifiseer. Hierdie geenfragmente kan nou vir promotorisoleringsdoeleindes aangewend word.

Sugarcane is an economically important crop and its high demand has necessitated the use of biotechnology methods to produce and accelerate the production of desirable genotypes. One such method is genetic transformation. However, as sugarcane is a highly polyploid crop, which originated from interspecific crosses between Saccharum spontaneum and S. officinarum, efforts to transform it are inhibited by transgene promoter silencing. As ancestral lines have a simpler genetic makeup than modern varieties, they may be useful to test promoter function. Intrinsic to the generation of transgenic plants is the ability to produce plants from specific species and varieties, for which an indirect method of regeneration is needed. Consequently, the first objective of this study was to determine a high yielding protocol for somatic embryogenic calli. The second was to transform such calli and produce regenerated plants to assess transgene expression. A preliminary study was conducted using eight ancestral varieties to determine which were the most responsive in culture. Leaf roll disks were cultured on 5 mg.1¯¹ 2, 4-D and callus production was assessed. Based on these results and the availability of plant material, S. spontaneum Nigeria 1, S. spontaneum Nigeria 2, S. spontaneum Coimbatore, S. officinarum NG 77-69, and S. officinarum Black Cheribon and the commercial polyploid variety NCo376 were selected and tested on 11 different callus induction media. The S. spontaneum variety that generated the highest percentage of leaf disks that produced callus and plant yield was Nigeria 1 (61 % and 259 plants/10 disks, respectively), whilst the S. officinarum variety was Black Cheribon (75 % and 90 plants/10 disks, respectively). The best media for both comprised of MS salts and vitamins, 20 g.1¯¹ sucrose, 0.5 g.1¯¹ casein hydrolysate 5 mg.1¯¹ 2, 4-D and 8 g.1¯¹ agar. NCo376 produced the most amount of callus (93 %) when cultured on media containing 3 mg.1¯¹ 2, 4-D and gave a final yield of 450 plants/10 disks. Based on the yields obtained above and the availability of plant material, the varieties S. spontaneum Nigeria 1 and S. officinarum NG77-69 were selected for genetic transformation studies. Calli of these varieties as well as that of NCo376 were microprojectile bombarded with either pEmuKN + pAHC27 or pEmuKN + pR₁₁F¯. Following bombardment, the calli were cultured onto paromomycin-containing (1 ml.1¯¹) selection media and regenerated plants were obtained after 8-12 weeks. Transgene integration into the plant genome was assessed using PCR and qPCR techniques, and indicated that all NCo376 plantlets contained the GUS and npt II transgenes. However, only 4 out of 5 and 2 out of 3 S. officinarum NG77-69 plants transformed with pAHC27 and pR₁₁F¯- respectively, and 6 out of 10 S. spontaneum Nigeria 1 plants transformed with pR₁₁F¯- contained these transgenes. The transformation efficiencies achieved for NCo376, for the constructs pAHC27 and pR₁₁F¯- was 0.27 and 0.33 transgenic plants/blast, respectively. For NG77-69 it was 0.27 and 0.13 transgenic plants/blast, whilst that of Nigeria 1 was 0.20 and 0.40 transgenic plants/blast. Stable transgene expression in acclimatized plants was then assessed using a histochemical GUS assay and none of the plants expressed the GUS gene. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2013.

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.

Due to the increasing prevalence and severity of Al phytoxicity in certain regions of the South African sugar industry, a research programme has been initiated at SASEX to elucidate the molecular mechanisms by which sugarcane detects and responds to the metal. As part of this larger investigation, the current study aimed to assess the response of a reportedly Al tolerant cultivar, Saccharum spp. hybrid cv. N12, to phytotoxic levels of Al. Hydroponically-grown plants of this commercial genotype were used in Al inhibition studies, the results of which indicated that exposure of plants to 250µM Al for 24 hours resulted in maximum reduction of root elongation. Under these conditions, root growth was inhibited by approximately 36%, compared with only 4% for the 50µM Al treatment. Subsequently, this exposure regime was used to gather the terminal 5 to 10mm of root tips, the site of the primary Al lesion, of challenged and control, unchallenged plants for molecular analysis. Total RNA was extracted from the Al challenged and control root tips, from which mRNA was subsequently isolated, reverse transcribed and converted to double-stranded cDNA. The two populations of cDNA were reciprocally subtracted from each other and used to construct subtractive cDNA libraries in Lambda ZAP®II phages. Randomly selected clones, 576 representatives from each of the libraries, were screened using membrane-based array technology. Results indicated that only 33% (190) of the Al-treatment specific library cDNAs were found to be more highly expressed under conditions of Al stress than under control conditions. Of these potentially Al response-related cDNAs, 25 were sequenced and submitted to sequence databases for the assignment of putative identities. No genic sequences known to be directly associated with the Al stress response were identified, however, several were found to be related to pathogenesis or general stress pathways. Although further Northern hybridisation work is required to validate these results, they suggest that the induction of general stress response pathways may be involved in the aluminium stress response of this sugarcane cultivar. Such Al stress-related sequences could have applications in marker-assisted breeding programmes and as candidate genes for the genetic engineering of tolerant genotypes. / Thesis (M.Sc.)-University of Natal, Durban, 2002.

Inheritance studies have previously been undertaken at the South African Sugar Association Experiment Station (SASEX) under irrigated conditions. Since most sugarcane is grown in South Africa under dryland (raingrown) conditions, heritability estimates were calculated under these conditions in this study and compared to those previously obtained under irrigated conditions. A sugarcane population consisting of 12 crosses, 32 offspring in each cross, and their parents were planted in the first two selection stages of the SASEX selection programme to ascertain which stage provided the most useful information when selecting parent cultivars. Data collected from Stage 2 was more reliable than data collected from Stage 1. Variance components, narrow and broad sense heritabilities, correlations among traits, and clonal repeatabilities between seasons were determined for 11 sugarcane traits at Stages 1 and 2. These traits studied included: stalk population; stalk diameter; stalk height; cane mass; dry matter % cane; fibre % cane; brix % cane; brix % dry matter; purity; pol % cane; and ers % cane. Narrow sense heritabilities of the sugarcane traits were estimated by mid-parent offspring regression . Alternative heritability estimates were obtained through restricted maximum likelihood (REML) analysis of the unbalanced North Carolina design II at Stage 2. Although narrow sense heritabilities determined by mid-parent-offspring regression were comparable with those previously determined at SASEX and by other workers, REML was more efficient than regression. Use of REML enabled additive and non-additive genetic variance components to be estimated by allocating degrees of freedom to treatments and the interactions between the different treatments. Heritability estimates varied for different traits and compared favourably with those obtained under irrigated conditions and by other workers. Additive genetic variance was more important than non-additive genetic variance for some characters, but not for stalk population, cane mass, and dry matter % cane, for which both variances were important. Selection of parent cultivars for all sucrose-related traits, fibre % cane, and stalk diameter should be as successful under raingrown as under irrigated conditions, provided that the environmental variation is determined efficiently under raingrown conditions. Environmental correlations were observed between some traits, particularly between the yield related traits, and may have influenced heritability estimates for those traits determined by mid-parent offspring regression. Stalk diameter, fibre % cane, and brix % dry matter were the most repeatable traits between seasons. Cane mass was the least repeatable trait between Stages 1 and 2 but was highly repeatable between plant (-P) and ratoon (-R) crops of Stage 2. Stalk diameter was positively correlated with brix % dry matter (0.457-P and 0.623-R) and strongly negatively correlated with stalk population (-0.790-P and -0.711-R) and fibre % cane (-0.628-P and -0.651-R). Cane mass was strongly positively correlated with brix % dry matter (0.638-P and 0.679-R). By selecting for brix % dry matter and stalk diameter, indirect selection for cane mass would be possible. Brix % dry matter was determined as the most reliable trait on which to base parental and commercial cultivar selection because it was highly heritable, highly repeatable and highly positively correlated with stalk diameter and cane mass. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1995.

Dissertation (PhD) -- University of Stellenbosch, 2002. / ENGLISH ABSTRACT: Sugarcane (Saccharum spp. hybrids) is a commercial crop plant capable of storing up to 20% sucrose on a fresh mass basis in the culm. Knowledge about gene expression during sugarcane growth and maturation is limited. The aim of this study was to assess whether an Expressed Sequence Tag (EST)-based approach towards analysis of sugarcane would reveal new information about gene expression and metabolic processes associated with sugarcane growth and development. The specific objectives were two-fold: firstly, to develop an EST database for sugarcane and secondly, to identify and analyse genes that are expressed in different sugarcane tissue types and developmental stages, with a specific focus on leaf and culm. An EST database for sugarcane was initiated to obtain information on sugarcane gene sequences. A total cDNA library was constructed from sugarcane immature leaf (leaf roll: meristematic region) tissue and 250 clones randomly selected and subjected to single-pass DNA sequence analysis. Sugarcane ESTs were identified by sequence similarity searches against gene sequences in international databases. Of the 250 leaf roll clones, 26% exhibited similarity to known plant genes, 50% to non-plant genes while 24% represented new gene sequences. Analysis of the identified clones indicated sequence similarity to a broad diversity of genes. A significant proportion of genes identified in the leaf roll were involved in processes related to protein synthesis and protein modification, as would be expected in meristematic tissues. Submission of 495 sugarcane gene sequences to the dbEST database represented the first sugarcane ESTs released into the public domain. Two subtracted cDNA libraries were constructed by reciprocal subtractive hybridisation between sugarcane immature and maturing internodal tissue. To explore gene expression during sugarcane culm maturation, partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries was performed. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases while 111 matched uncharacterised plant ESTs only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. The expression patterns of sugarcane genes were examined in different tissue sources and developmental stages to identify differentially expressed genes. cDNA arrays containing 1000 random clones from immature leaf and maturing culm cDNA libraries were hybridised with poly (At RNA from immature leaf, mature leaf, immature culm and maturing culm. All cDNAs examined hybridised to all four probes, but differences in signal intensity were observed for individual cDNAs between hybridisation events. No cDNAs displaying tissue- or developmental-stage specific expression were detected. Comparisons between hybridisation patterns identified 61 cDNAs that were more abundantly expressed in immature and mature leaf than the culm. Likewise, 25 cDNAs preferentially expressed in immature and maturing culm were detected. ESTs established for the differentially expressed cDNAs revealed sequence homology to a diverse collection of genes in both the leaf and the culm. These included genes associated with general cellular metabolism, transport, regulation and a variety of stress responses. None of the differentially expressed genes identified in the culm were homologous to genes known to be associated with sucrose accumulation. To examme differences at the level of gene transcription between low sucroseaccumulating and high sucrose-accumulating tissues, subtracted cDNA libraries were utilised. To isolate cDNAs differentially expressed during culm maturation, cDNA arrays containing 400 random clones (200 from each library) were screened with total cDNA probes prepared from immature and maturing culm poly (At RNA. Results indicated that 36% and 30% of the total number of cDNAs analysed were preferentially expressed in the immature and maturing culm, respectively. Northern analysis of selected clones confirmed culm developmental stage-preferential expression for most of the clones tested. ESTs generated for the 132 differentially expressed clones isolated exhibited homology to genes associated with cell wall metabolism, carbohydrate metabolism, stress responses and regulation, where the specific ESTs identified in the immature and maturing culm were distinct from each other. No developmentally regulated ESTs directly associated with sucrose metabolism were detected. These results suggest that growth and maturation of the sugarcane culm is associated with the expression of genes for a wide variety of metabolic processes. In addition, genes encoding enzymes directly involved with sucrose accumulation do not appear to be abundantly expressed in the culm. / AFRIKAANSE OPSOMMING: Kommersiële suikerriet variëteite (Saccharum spp. hibriede) is in staat om tot 20% sukrose op 'n vars massa basis in die stingel op te berg. Kennis oor geenuitdrukking tydens groei en rypwording is beperk. Die doel van die huidige studie was om vas te stelof 'n grootskaalse karatersisering van die geenvolgordes wat uitgedruk word "Expressed Sequence Tag (EST)-based approach" tot nuwe inligting aangaande die aard en omvang van metabolisme tydens groei en ontwikkeling van suikerriet sal lei. 'n Tweeledige benadering is in hierdie studie gevolg. Eerstens is 'n data basis oor die gene wat uitgedruk word "EST" databasis opgestel. Tweedens is gene geïdentifiseer en gekarakteriseer wat spesifiek op verskillende stadiums van ontwikkeling en in spesifiek weefsel uitgedruk word. Vir die opstel van die EST-databasis is 250 klone uit 'n totale cDNA biblioteek vanaf RNA uit suikerrietblaarweefsel (blaarrol:meristematiese streek) op 'n lukraak basis gekies en aan 'n enkel eenrigting DNA volgorde analise onderwerp. Suikerrriet EST's is geïdentifiseer deur middel van homologie soektogte teen geenvolgordes in internasionale databasisse. Uit die 250 blaarrol klone het 26% ooreenkomste met bekende plant gene en, 50% met nie-plant gene getoon. Ongeveer 24% het nuwe geenvolgordes verteenwoordig. Analise van die geïdentifeseerde klone het ooreenkomste met 'n breë diversiteit van gene getoon. 'n Betekenisvolle gedeelte van gene wat in die blaarrol geïdentifiseer is, is by proteïensintese en proteïenmodifikasies betrokke. Dit is in ooreenstemming met wat van meristematiese weefsel verwag kan word. Die 495 suikerriet geenvolgordes wat in die internasionale dbEST databasis gestort is, is die eerste sodanige inligting in die publieke domein. Twee spesifieke cDNA biblioteke (subtraction libraries) wat volgordes spesifiek aan onvolwasse suikerriet en rypwordende internodale weefsel bevat is voorberei. Geenuitdrukking gedurende die rypwordingsproses van die suikerrietstingel is bestudeer deur geenvolgorde analises van onwillekeurige geselekteerde klone van die twee eDNA biblioteke te doen. Van die 337 geenvolgordes wat geanaliseer is het 167 homologie met bekende gene en net 111ooreenkomste met ongekarakteriseerde plant gene getoon. Die oorblywende geenvolgordes het geen ooreenkomste met bekende gene getoon nie en daar kan dus aanvaar word dat hulle nuwe gene verteenwoordig. Die meerderheid ESTs het ooreenkomste met verskeie gene wat met sellulêre metabolisme geassosieer word getoon. ESTs wat homoloog was aan verskeie spannings geassosieerde gene was ook goed verteenwoordig. Die analise het gene wat by voorkeur tydens stringelrypwording uitgedruk word geidentifiseer. Die geenuitdrukkingspatrone van suikerriet in weefsels van verskillende oorsprong en ontwikkelingstadia is ondersoek om differensieel uitgedrukte gene te identifiseer. Reekse wat 1000 lukrake eDNA klone van onvolwasse en rypwordende stingel eDNA biblioteke is met poli-(A)-RNA van onvolwasse blaar, volwasse blaar, onvolwasse stingel en volwasse stingel gehibridiseer. Al die eDNA klone wat ondersoek is het met al vier die peilers gehibridiseer. Die intensiteit van die seine het egter grootliks gevarieer. Die analise het gelei tot die identifisering van 61 eDNA klone wat teen hoër vlakke in onvolwasse en volwasse blaar as in die stingel uitgedruk word. Daar is ook 25 eDNA klone wat by voorkeur in onvolwasse en rypwordende stingel uitgedruk word gevind. Gene wat geassosieer word met gewone sel metabolisme, vervoer prosesse, regulering en verskeie spannings-geassosieerde reaksies, is in die twee groepe teenwoordig. Geeneen van die volgordes wat selektief uitgedruk word kan met gene wat direk met sukrose akkumulering verband hou geassosieer word nie. Ten einde eDNA klone wat differensieel tydens rypwording van die stingel uitgedruk word te isoleer, is 400 eDNA klone (200 van elke biblioteek) lukraak geselekteer en met totale eDNA peilers, wat uit onvolwasse en rypwordende stingel poli-(A)-RNA voorberei is, gesif. Resultate het aangetoon dat 36% en 30% van die totale getal eDNA klonewat geanaliseer is, by voorkeur in die onvolwasse en rypwordende stingel uitgedruk word. RNA kladanalises van geselekteerde klone het getoon dat die meeste ontwikkelingstadium spesifieke uirtdrukkingspatrone het. Daar is gevind dat 132 van die EST klone homologie met gene geassosieerd met selwand- en koolhidraatmetabolisme, spannings geassosieerde- en reguleringsreaksies, toon. Die spesifieke ESTs wat in die onvolwasse en rypwordende stingel geïdentifiseer is het van mekaar verskil. Nie een van die ESTs wat geïdentifiseer is kan direk met sukrose metabolisme geassosieer word nie. Hierdie werk toon baie duidelik aan dat groei en rypwording van die suikerrietstingel met die uitdrukking van gene geassosieerd is wat by 'n hele aantal metaboliese prosesse betrokke is. Die resultate toon ook dat die gene wat vir ensieme kodeer wat direk by sukrose akkumulering betrokke is, nie teen hoë vlakke in die stingel uitgedruk word nie.

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.