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At least 19 recordsLinked to original sources

PubMed2006

Quantitative trait loci identified for sugar related traits in a sugarcane (Saccharum spp.) cultivar x Saccharum officinarum population.

The identification of markers linked to quantitative trait loci (QTLs) for increased sugar accumulation could improve the effectiveness of current breeding strategies in sugarcane. Progeny from a cross between a high sucrose producing cultivar, (denotes Australian plant breeding rights), and a Saccharum officinarum clone, IJ76-514 were grown in two field experiments in different years, and evaluated in the early and mid-season phases of crop maturity, to identify robust QTLs in affecting sucrose content in cane. Using an extensive genetic map constructed for with over 1,000 AFLP and SSR markers, a total of 37 QTLs were identified for brix and pol of which, 16 were detected in both experiments. Of these 37 QTL, 30 were clustered into 12 genomic regions in six of the eight homo(eo)logous groups. Each QTL explained from 3 to 9% of the phenotypic variation observed. Both positive and negative effects were identified and the location of the QTLs on linkage groups belonging to the same homo(eo)logy group suggested that a number of the QTLs were allelic forms of the same genes. Of the 37 QTLs identified, the majority were significant in both early and mature cane, but 8 were identified as early specific QTLs and 9 as mature cane QTLs. In total, 97 interactions were significant (P<10(-5)) and these were localised to 32 genomic regions of which 6 were detected with both years' data. Models including all the QTLs explained from 37 to 66% of the total phenotypic variation, depending on the trait. The results will be subsequently applied in marker assisted breeding.

Alleles↗

PubMed1995

Ecophysiological attributes of the native Acer saccharum and the exotic Acer platanoides in urban oak forests in Pennsylvania, USA.

When the exotic Acer platanoides L. (Norway maple) and the native A. saccharum Marsh. (sugar maple) grow together in the understories of urban Quercus forests in the eastern USA, average annual height growth increments are nearly twice as large in A. platanoides as in A. saccharum, 19.26 +/- 3.22 versus 10.01 +/- 1.69 cm. We examined several ecophysiological mechanisms that might be associated with the superior invasive ability and growth of A. platanoides in two urban oak forests in Pennsylvania. Leaf longevity was 12 days greater in A. platanoides than in A. saccharum. In addition, leaf mass/leaf area ratio was greater in A. platanoides than in A. saccharum (2.67 +/- 0.03 versus 2.32 +/- 0.02 mg cm(-2)); however, leaf thickness was significantly lower in A. platanoides than in A. saccharum suggesting that A. platanoides contains more dense palisade and mesophyll cell layers than A. saccharum. Field net photosynthesis (mass basis) and photosynthetic light response curves (area basis) indicated significantly greater carbon assimilation, and nitrogen and phosphorus use efficiencies in A. platanoides than in A. saccharum. Acer platanoides also exhibited higher water use efficiency than A. saccharum (0.88 +/- 0.12 versus 0.32 +/- 0.09 mmol CO(2) mol(-1) H(2)O). Acer platanoides exhibited significantly lower osmotic potentials than A. saccharum, but a similar relative water content at zero turgor. We conclude that A. platanoides utilizes light, water and nutrients more efficiently than A. saccharum.

Journal Article↗

PubMed2002

Phylogenetics of Miscanthus, Saccharum and related genera (Saccharinae, Andropogoneae, Poaceae) based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnLintron and trnL-F intergenic spacers.

DNA sequences were used to assess the monophyly and inter-relationships of Miscanthus, Saccharumand related genera in the Saccharum complex. Three DNA regions were sequenced, including the trnLintron and the trnL-F intergenic spacer of the plastid genome and the ITS region of nuclear ribosomal DNA (nrDNA). Because it was more variable, the ITS region proved most suitable for phylogenetic reconstruction at this level, and the results indicate that Miscanthus s.l. and Saccharum s.l. are polyphyletic. A set of species from Saccharum section Ripidium(clade a) do not group closely with any members of Saccharum s.l. A number of Miscanthus species from eastern or south-eastern Asia represent a monophyletic group with a basic chromosome number of 19 (clade b), but the other species from Africa and the Himalayas are clearly excluded. There is support for a monophyletic Saccharum s.s. clade including S. officinarumand S. spontaneum that is sister to Miscanthus s.s(clade c). There is no evidence to support the division of some Saccharum s.l. into the genera currently known as Erianthus and Narenga. Saccharum contortum( =Erianthus contortus), S. narenga (= Narenga porphyrocoma) and Erianthus rockii, group more closely with Miscanthus fuscus, a species from the Himalayas and also with the African Miscanthus s.l. species (= Miscanthidium, clade d).

Journal Article↗

PubMed1998

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.

DNA, Plant↗

PubMed2002

Analysis of genetic similarity detected by AFLP and coefficient of parentage among genotypes of sugar cane ( Saccharum spp.).

Despite the economical importance of sugar cane, until the present-date no studies have been carried out to determine the correlation of the molecular-based genetic similarity (GS) and the coefficient of parentage ( f)-estimates generated for cultivars. A comprehensive knowledge of the amount of genetic diversity in parental cultivars, could improve the effectiveness of breeding programmes. In this study, amplified fragment length polymorphism (AFLP) and pedigree data were used to investigate the genetic relationship in a group of 79 cultivars (interspecific hybrids), used as parents in one of the Brazilian breeding programmes, and four species of Saccharum ( Saccharum sinense, Saccharum barberi and two of Saccharum officinarum). The objectives of this study were to assess the level of genetic similarity among the sugar-cane cultivars and to investigate the correlation between the AFLP-based GS and f, based on pedigree information. Twenty one primer combinations were used to obtain the AFLP molecular markers, generating a total of 2,331 bands, of which 1,121 were polymorphic, with a polymorphism rate, on average, of 50% per primer combination. GSs were determined using Jaccard's similarity coefficient, and a final dendrogram was constructed using an unweighted pair-group method using arithmetic average (UPGMA). AFLP-based GS ranged from 0.28 to 0.89, with a mean of 0.47, whereas f ranged from 0 to 0.503, with a mean of 0.057. Cluster analysis using GS divided the genotypes into related subgroups suggesting that there is important genetic relationship among the cultivars. AFLP-based GS and f were significantly correlated ( r= 0.42, P< 0.001), thus the significance of this r value suggests that the AFLP data may help to more-accurately quantify the degree of relationship among sugar-cane cultivars.

Journal Article↗

PubMed2002

Photosynthetic acclimation of overstory Populus tremuloides and understory Acer saccharum to elevated atmospheric CO2 concentration: interactions with shade and soil nitrogen.

We exposed Populus tremuloides Michx. and Acer saccharum Marsh. to a factorial combination of ambient and elevated atmospheric CO2 concentrations ([CO2]) and high-nitrogen (N) and low-N soil treatments in open-top chambers for 3 years. Our objective was to compare photosynthetic acclimation to elevated [CO2] between species of contrasting shade tolerance, and to determine if soil N or shading modify the acclimation response. Sun and shade leaf responses to elevated [CO2] and soil N were compared between upper and lower canopy leaves of P. tremuloides and between A. saccharum seedlings grown with and without shading by P. tremuloides. Both species had higher leaf N concentrations and photosynthetic rates in high-N soil than in low-N soil, and these characteristics were higher for P. tremuloides than for A. saccharum. Electron transport capacity (Jmax) and carboxylation capacity (Vcmax) generally decreased with atmospheric CO2 enrichment in all 3 years of the experiment, but there was no evidence that elevated [CO2] altered the relationship between them. On a leaf area basis, both Jmax and Vcmax acclimated to elevated [CO2] more strongly in shade leaves than in sun leaves of P. tremuloides. However, the apparent [CO2] x shade interaction was largely driven by differences in specific leaf area (m2 g-1) between sun and shade leaves. In A. saccharum, photosynthesis acclimated more strongly to elevated [CO2] in sun leaves than in shade leaves on both leaf area and mass bases. We conclude that trees rooted freely in the ground can exhibit photosynthetic acclimation to elevated [CO2], and the response may be modified by light environment. The hypothesis that photosynthesis acclimates more completely to elevated [CO2] in shade-tolerant species than in shade-intolerant species was not supported.

Acclimatization↗

PubMed2006

In vivo and in situ rhizosphere respiration in Acer saccharum and Betula alleghaniensis seedlings grown in contrasting light regimes.

A perfusive method combined with an open-system carbon dioxide measurement system was used to assess rhizosphere respiration of Acer saccharum Marsh. (sugar maple) and Betula alleghaniensis Britton (yellow birch) seedlings grown in 8-l pots filled with coarse sand. We compared in vivo and in situ rhizosphere respiration between species, among light regimes (40, 17 and 6% of full daylight) and at different times during the day. To compute specific rhizosphere respiration, temperature corrections were made with either species-specific coefficients (Q10) based on the observed change in respiration rate between 15 and 21 degrees C or an arbitrarily assigned Q10 of 2. Estimated, species-specific Q10 values were 3.0 and 3.4 for A. saccharum and B. alleghaniensis, respectively, and did not vary with light regime. Using either method of temperature correction, specific rhizosphere respiration did not differ either between A. saccharum and B. alleghaniensis, or among light regimes except in A. saccharum at 6% of full daylight. At this irradiance, seedlings were smaller than in the other light treatments, with a larger fine root fraction of total root dry mass, resulting in higher respiration rates. Specific rhizosphere respiration was significantly higher during the afternoon than at other times of day when temperature-corrected on the basis of an arbitrary Q10 of 2, suggesting the possibility of diurnal variation in a temperature-independent component of rhizosphere respiration.

Acer↗

PubMed1998

Isolation and characterization of a satellite DNA family in the Saccharum complex.

EaCIR1, a 371-bp Erianthus-specific satellite DNA sequence, was cloned from TaqI restricted genomic DNA after agarose-gel electrophoresis. This sequence has 77% homology with a 365-bp satellite of Helictotrichon convolutum and 72% homology with a 353-bp tandem repeat sequence from Oryza sativa. PCR primers defined in the conserved regions of these repetitive sequences were used to isolate other satellite DNAs in different representatives of the Saccharum complex: SoCIR1 in Saccharum officinarum, SrCIR1 in Saccharum robustum, SsCIR1 and SsCIR2 in Saccharum spontaneum, and MsCIR1 in Miscanthus sinensis. EaCIR1 and SoCIR1 were localized to subtelomeric regions of the chromosomes by fluorescence in situ hybridization. Southern hybridization experiments, using two representatives of this repeat sequence family as probes, illustrated contrasting species-specificity and demonstrated the existence of similar repetitive elements in sorghum and maize.

Base Sequence↗

PubMed2005

Very close relationship of the chloroplast genomes among Saccharum species.

We recently determined the complete sequence of the sugarcane chloroplast genome. Here, we have used the information for a comprehensive phylogenetic analysis of the genus Saccharum, using all six species (13 accessions). The polymorphisms between sugarcane and maize in 26 chloroplast genome regions were used for the analysis. In 18 of the 26 regions (a total of 5,381 bp), we found 41 mutations involving 17 substitutions, three inversions, six insertion/deletion mutations, and 15 simple sequence repeat length polymorphisms. Based on these results, we calculated a phylogenetic tree of the genus Saccharum, in which all six species are clearly separated. By the analysis, (1) S. sinense and S. barberi, which have identical sequences, belong to the same clade, whereas the other four species, S. officinarum, S. robustum, S. edule, and S. spontaneum, form an independent clade; (2) S. spontaneum has a paraphyletic relationship with the other five species; and (3) no or very low intraspecific variation was observed in S. officinarum, S. robustum, S. sinense, S. barberi, and S. edule, whereas higher intraspecific variation was observed in S. spontaneum. Based on the number of nucleotide substitutions, the divergence time between S. officinarum and S. spontaneum, and between S. officinarum and maize were calculated to be about 730-780 thousand years ago and about 5.9 million years ago, respectively. These results suggest that the cytoplasm of Saccharum species are very closely related.

Base Sequence↗

PubMed2026

Genome-wide identification of HCT gene family in sugarcane (Saccharum spp. hybrid) and characterization of putative cis-elements in gene regulation.

BACKGROUND: Sugarcane (Saccharum spp. hybrid) is a globally important crop, and its bagasse can be converted into bioethanol and other industrial products. Lignin, a core component of sugarcane cell walls, plays a crucial role in bagasse quality and lodging resistance. Shikimic acid hydroxycinnamyl transferase (HCT) is the key enzyme in lignin biosynthesis. However, the HCT gene family in sugarcane and its regulatory roles in sugarcane remain poorly understood. RESULTS: A total of 663 HCT genes (including alleles) were identified in the Saccharum hybrid R570 genome, which were classified into six groups (I-VI) and were unevenly distributed across 77 chromosomes. Bioinformatics analysis revealed that the subgroups of R570HCTs had similar gene structures, suggesting conserved functions. Moreover, the different subgroups presented unique putative cis‑element distribution patterns. Transcriptome data indicated that some R570HCTs exhibited significant spatiotemporal and tissue‑specific expression patterns. Further Pearson correlation analysis between putative cis‑element distribution and normalized expression values at the subgroup level revealed that light-responsive elements (L‑box and GA‑motif) were positively correlated with R570HCT expression, and different subgroups formed a complex regulatory network by integrating hormone response and stress elements. Importantly, this subgroup-level correlation was cross-validated by comparing the cis‑element clustering heatmap with the expression heatmap, revealing consistent enrichment patterns. CONCLUSIONS: The study's findings provide novel insights into the correlation among motifs, putative cis‑elements, and gene expression, and propose a cross-validated framework for understanding regulatory divergence among HCT subfamilies in polyploid sugarcane, serving as a hypothesis generating resource for future research on R570HCT expression.

Saccharum↗

PubMed2026

Establishment of an efficient Agrobacterium-mediated genetic transformation protocol for Saccharum officinarum using Black Cheribon as a model genotype.

Efficient Agrobacterium-mediated transformation (AMT) is vital for the biotechnological improvement of sugarcane (Saccharum spp.). Saccharum officinarum is the main ancestor of all modern cultivars, yet little research has been conducted on its AMT system. In this work, an efficient AMT protocol for S. officinarum was developed, with Black Cheribon as the model genotype owing to its superior tissue culture performance and regeneration capacity. The optimized agro-infection protocol comprised the following main parameters: concentration of acetosyringone (AS) in Agrobacterium culture, concentration of AS for infection, Agrobacterium concentration at OD600 = 0.4, infection time of 30 minutes, vacuum infiltration time of 10 minutes and co-cultivation time of 3 days. To further improve transformation efficiency, 0.5 mg/L thidiazuron and 200 mg/L citric acid were added to the regeneration medium, which enhanced the regeneration of shoots. A modified stage-dependent selection strategy (FlexII) was established by using glufosinate-ammonium at concentrations of 2.0, 1.0, and 0.75 mg/L in the callus proliferation, shoot regeneration, and rooting stages, respectively. This strategy was more successful than the minimum inhibitory concentration-based strategy in S. officinarum transformation. The optimized protocol further boosted the transformation efficiency of Black Cheribon from 1.12% to 7.17%. The resulting transgenic lines were confirmed by PCR amplification of T-DNA regions and immunochromatographic detection of Bar protein expression in primary transformants, respectively. These results provide a sound technical foundation for the functional genomics and biotechnological optimization of S. officinarum germplasm, and may serve as a reference for future transformation studies in other sugarcane germplasm.

Agrobacterium↗

PubMed2000

Characterisation of microsatellite markers from sugarcane (Saccharum sp.), a highly polyploid species.

Cultivated sugarcane varieties (Saccharum spp) are derived from complex interspecific hybridisations between the species S. spontaneum (2n=40-128) and S. officinarum (2n=60 or 80). To analyse this complex genome, the potential of microsatellite repeats as genetic markers in sugarcane with respect to their abundance, variability and ability to detect polymorphisms was investigated. A set of microsatellite markers for genome analysis in cultivated sugarcane was identified from an enriched genomic DNA library constructed from Saccharum sp. cv Q124. Sequencing of 798 sugarcane genomic DNA clones from an enriched microsatellite library, yielded 457 inserts containing microsatellite repeat motifs. Just over 84% of the microsatellites contained dinucleotide or trinucleotide repeats averaging 15 and 13 repeat motifs, respectively. Primer sets were designed and synthesised for over 100 microsatellite sequences and tested on a set of five sugarcane cultivars. Both, heterozygosity as witnessed by the number of alleles, and length polymorphisms as seen in the differences in PCR product size for a particular allele were observed. Microsatellite markers are likely to have many applications in sugarcane genetics and breeding including germplasm analysis, cultivar identification, parent evaluation and marker assisted breeding.

Journal Article↗

PubMed1997

Comparative mapping of Andropogoneae: Saccharum L. (sugarcane) and its relation to sorghum and maize.

Comparative genetic maps of Papuan Saccharum officinarum L. (2n = 80) and S. robustum (2n = 80) were constructed by using single-dose DNA markers (SDMs). SDM-framework maps of S. officinarum and S. robustum were compared with genetic maps of sorghum and maize by way of anchor restriction fragment length polymorphism probes. The resulting comparisons showed striking colinearity between the sorghum and Saccharum genomes. There were no differences in marker order between S. officinarum and sorghum. Furthermore, there were no alterations in SDM order between S. officinarum and S. robustum. The S. officinarum and S. robustum maps also were compared with the map of the polysomic octoploid S. spontaneum 'SES 208' (2n = 64, x = 8), thus permitting relations to homology groups ("chromosomes") of S. spontaneum to be studied. Investigation of transmission genetics in S. officinarum and S. robustum confirmed preliminary results that showed incomplete polysomy in these species. Because of incomplete polysomy, multiple-dose markers could not be mapped for lack of a genetic model for their segregation. To coalesce S. officinarum and S. robustum linkage groups into homology groups (composed of homologous pairing partners), they were compared with sorghum (2n = 20), which functioned as a synthetic diploid. Groupings suggested by comparative mapping were found to be highly concordant with groupings based on highly polymorphic restriction fragment length polymorphism probes detecting multiple SDMs. The resulting comparative maps serve as bridges to allow information from one Andropogoneae to be used by another, for breeding, ecology, evolution, and molecular biology.

Journal Article↗

PubMed1996

RFLP mapping in cultivated sugarcane (Saccharum spp.): genome organization in a highly polyploid and aneuploid interspecific hybrid.

Sugarcane cultivars are polyploid, aneuploid, interspecific hybrids between the domesticated species Saccharum officinarum and the wild relative S. spontaneum. Cultivar chromosome numbers range from 100 to 130 with approximately 10% contributed by S. spontaneum. We have undertaken a mapping study on the progeny of a selfed cultivar, R570, to analyze this complex genome structure. A set of 128 restriction fragment length polymorphism probes and one isozyme was used. Four hundred and eight markers were placed onto 96 cosegregation groups, based on linkages in coupling only. These groups could tentatively be assembled into 10 basic linkage groups on the basis of common probes. Origin of markers was investigated for 61 probes and the isozyme, leading to the identification of 80 S. officinarum and 66 S. spontaneum derived markers, respectively. Their distribution in cosegregation groups showed better map coverage for the S. spontaneum than for the S. officinarum genome fraction and occasional recombination between the two genomes. The study of repulsions between markers suggested the prevalence of random pairing between chromosomes, typical of autopolyploids. However, cases of preferential pairing between S. spontaneum chromosomes were also detected. A tentative Saccharum map was constructed by pooling linkage information for each linkage group.

Aneuploidy↗

PubMedDate not supplied

On-line identification of further flavone C- and O-glycosides from sugarcane (Saccharum officinarum L., Gramineae) by HPLC-UV-MS.

HPLC-UV-MS (APCI-MS/MS and CID/MS) was utilised for the identification of eight additional flavone glycosides from sugarcane (Saccharum officinarum L., Gramineae) extracts of bagasse, leaves and juice ('garapa'). Relevant information about substitution patterns was obtained through UV detection using post-column addition of shift reagents, while tandem MS provided structural information confirming the proposed structures of the C-glycosides vitexin, orientin, luteolin-8-C-(rhamnosylglucoside), 4',5'-dimethyl-luteolin-8-C-glycoside and the isomeric pair schaftoside-isoschaftoside, besides the O-glycosides tricin-7-O-neohesperidoside and tricin-7-O-glycoside.

Chromatography, High Pressure Liquid↗

PubMed2005

Development of an integrated genetic map of a sugarcane (Saccharum spp.) commercial cross, based on a maximum-likelihood approach for estimation of linkage and linkage phases.

Sugarcane (Saccharum spp.) is a clonally propagated outcrossing polyploid crop of great importance in tropical agriculture. Up to now, all sugarcane genetic maps had been developed using either full-sib progenies derived from interspecific crosses or from selfing, both approaches not directly adopted in conventional breeding. We have developed a single integrated genetic map using a population derived from a cross between two pre-commercial cultivars ('SP80-180' x 'SP80-4966') using a novel approach based on the simultaneous maximum-likelihood estimation of linkage and linkage phases method specially designed for outcrossing species. From a total of 1,118 single-dose markers (RFLP, SSR and AFLP) identified, 39% derived from a testcross configuration between the parents segregating in a 1:1 fashion, while 61% segregated 3:1, representing heterozygous markers in both parents with the same genotypes. The markers segregating 3:1 were used to establish linkage between the testcross markers. The final map comprised of 357 linked markers, including 57 RFLPs, 64 SSRs and 236 AFLPs that were assigned to 131 co-segregation groups, considering a LOD score of 5, and a recombination fraction of 37.5 cM with map distances estimated by Kosambi function. The co-segregation groups represented a total map length of 2,602.4 cM, with a marker density of 7.3 cM. When the same data were analyzed using JoinMap software, only 217 linked markers were assigned to 98 co-segregation groups, spanning 1,340 cM, with a marker density of 6.2 cM. The maximum-likelihood approach reduced the number of unlinked markers to 761 (68.0%), compared to 901 (80.5%) using JoinMap. All the co-segregation groups obtained using JoinMap were present in the map constructed based on the maximum-likelihood method. Differences on the marker order within the co-segregation groups were observed between the two maps. Based on RFLP and SSR markers, 42 of the 131 co-segregation groups were assembled into 12 putative homology groups. Overall, the simultaneous maximum-likelihood estimation of linkage and linkage phases was more efficient than the method used by JoinMap to generate an integrated genetic map of sugarcane.

Chromosome Mapping↗

PubMed2004

Structural features and transcript-editing analysis of sugarcane (Saccharum officinarum L.) chloroplast genome.

The complete nucleotide sequence of the chloroplast genome of sugarcane (Saccharum officinarum) was determined. It consists of 141,182 base-pairs (bp), containing a pair of inverted repeat regions (IR(A), IR(B)) of 22,794 bp each. The IR(A) and IR(B) sequences separate a small single copy region (12,546 bp) and a large single copy (83,048 bp) region. The gene content and relative arrangement of the 116 identified genes (82 peptide-encoding genes, four ribosomal RNA genes, 30 tRNA genes), with the 16 ycf genes, are highly similar to maize. Editing events, defined as C-to-U transitions in the mRNA sequences, were comparable with those observed in maize, rice and wheat. The conservation of gene organization and mRNA editing suggests a common ancestor for the sugarcane and maize plastomes. These data provide the basis for functional analysis of plastid genes and plastid metabolism within the Poaceae. The sugarcane chloroplast DNA sequence is available at GenBank under accession NC005878.

Base Sequence↗

PubMed2006

Prospecting the utility of a PMI/mannose selection system for the recovery of transgenic sugarcane (Saccharum spp. hybrid) plants.

For the first time, the phosphomannose isomerase (PMI, EC 5.3.1.8)/mannose-based "positive" selection system has been used to obtain genetically engineered sugarcane (Saccharum spp. hybrid var. CP72-2086) plants. Transgenic lines of sugarcane were obtained following biolistic transformation of embryogenic callus with an untranslatable sugarcane mosaic virus (SCMV) strain E coat protein (CP) gene and the Escherichia coli PMI gene manA, as the selectable marker gene. Postbombardment, transgenic callus was selectively proliferated on modified MS medium containing 13.6 microM 2,4-D, 20 g l(-1) sucrose and 3 g l(-1) mannose. Plant regeneration was obtained on MS basal medium with 2.5 microM TDZ under similar selection conditions, and the regenerants rooted on MS basal medium with 19.7 microM IBA, 20 g l(-1) sucrose, and 1.5 g l(-1) mannose. An increase in mannose concentration from permissive (1.5 g l(-1)) to selective (3 g l(-1)) conditions after 3 weeks improved the overall transformation efficiency by reducing the number of selection escapes. Thirty-four vigorously growing putative transgenic plants were successfully transplanted into the greenhouse. PCR and Southern blot analyses showed that 19 plants were manA-positive and 15 plants were CP-positive, while 13 independent transgenics contained both transgenes. Expression of manA in the transgenic plants was evaluated using a chlorophenol red assay and enzymatic analysis.

Blotting, Southern↗