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Forests are vital to the world's ecological, social, cultural and economic well-being yet sustainable provision of goods and services from forests is increasingly challenged by pressures such as growing demand for wood and other forest products, land conversion and degradation, and climate change. Intensively managed, highly productive forestry incorporating the most advanced methods for tree breeding, including the application of genetic engineering (GE), has tremendous potential for producing more wood on less land. However, the deployment of GE trees in plantation forests is a controversial topic and concerns have been particularly expressed about potential harms to the environment. This paper, prepared by an international group of experts in silviculture, forest tree breeding, forest biotechnology and environmental risk assessment (ERA) that met in April 2012, examines how the ERA paradigm used for GE crop plants may be applied to GE trees for use in plantation forests. It emphasizes the importance of differentiating between ERA for confined field trials of GE trees, and ERA for unconfined or commercial-scale releases. In the case of the latter, particular attention is paid to characteristics of forest trees that distinguish them from shorter-lived plant species, the temporal and spatial scale of forests, and the biodiversity of the plantation forest as a receiving environment.

Expressed sequence tags derived markers have a great potential to be used in functional map construction and QTL tagging. In the present work, sugarcane genomic probes and expressed sequence tags having homology to genes, mostly involved in carbohydrate metabolism were used in RFLP assays to identify putative QTLs as well as their epistatic interactions for fiber content, cane yield, pol and tones of sugar per hectare, at two crop cycles in a progeny derived from a bi-parental cross of sugarcane elite materials. A hundred and twenty marker trait associations were found, of which 26 at both crop cycle and 32 only at first ratoon cane. A sucrose synthase derived marker was associated with a putative QTL having a high negative effect on cane yield and also with a QTL having a positive effect on Pol at both crop cycles. Fifty digenic epistatic marker interactions were identified for the four traits evaluated. Of these, only two were observed at both crop cycles.

Microsatellites or simple sequence repeats (SSRs) are one of the most suitable markers for genome analysis as they have great potential to aid breeders to develop new improved sugarcane varieties. The development of SSR derived from expressed sequence tags (EST) opens new opportunities for genetic investigations at a functional level. In the present work, the polymorphism obtained with a subset of 51 EST-SSRs derived from SUCEST was compared with those generated by 50 genomic SSRs (gSSR) in terms of number of alleles, polymorphism information content, discrimination power and their ability to establish genetic relationships among 18 sugarcane clones including three Saccharum species (S. officinarum, S. barberi, S. sinense). The majority of EST-SSRs loci had four to six alleles in contrast to the seven to nine observed for the gSSRs loci. Approximately, 35% of the gSSRs had PIC values around 0.90 in contrast to 15% of the EST-SSRs. However, the mean discrimination power of the two types of SSR did not differ significantly as much as the average genetic similarity (GS) based on Dice coefficient. The correlation between GS of the two types of SSRs was high (r = 0.71/P = 0.99) and significant. Although differences were observed between dendrograms obtained with each SSR type, both were in good agreement with pedigree information. The S. officinarum clone IJ76-314 was grouped apart from the other clones evaluated. The results here demonstrate that EST-SSRs can be successfully used for genetic relationship analysis, extending the knowledge of genetic diversity of sugarcane to a functional level.

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.

Previously, we genetically modified two Brazilian commercial sugarcane (Saccharum sp.) varieties (SP80-3280 and SP80-1842) for resistance to the sugarcane borer Diatraea saccharalis by co-bombardment transformation with three plasmids: pCIB4421 containing the Bacillus thuringiensis gene cry1Ab regulated by a maize phosphoenol-pyruvate carboxylase promoter, pCIB4426 carrying cry1Ab controlled by a pith promoter, and pHA9 with the neo antibiotic resistance gene. The genetically modified plants were submitted to molecular characterization and insect feeding bioassays. Plants also were screened in the field for insect infestation and had their phenotypic characteristics and quantitative traits evaluated. This study presents evidence that the B. thuringiensis cry1Ab gene can be efficiently expressed in sugarcane plants over an extended growing period, and can confer resistance to the sugarcane borer under field conditions, while not measurably altering numerous material traits of the crop.

Summary Adopting the sequencing of expressed sequence tags (ESTs) of a sugarcane database derived from libraries induced and not induced by pathogens, we identified EST clusters homologous to genes corresponding to enzymes involved in the detoxification of reactive oxygen species. The predicted amino acids of these enzymes are superoxide dismutases (SODs), glutathione-S-transferase (GST), glutathione peroxidase (GPX), and catalases. Three MnSOD mitochondrial precursors and 10 CuZnSOD were identified in sugarcane: the MnSOD mitochondrial precursor is 96% similar to the maize MnSOD mitochondrial precursor and, of the 10 CuZnSOD identified, seven were 98% identical to maize cytosolic CuZnSOD4 and one was 67% identical to putative peroxisomal CuZnSOD from Arabidopsis. Three homologues to class Phi GST were 87-88% identical to GST III from maize. Five GPX homologues were identified: three were homologous to cytosolic GPX from barley, one was 88% identical to phospholipid hydroperoxide glutathione peroxidase (PHGPX) from rice, and the last was 71% identical to GPX from A. thaliana. Three enzymes similar to maize catalase were identified in sugarcane: two were similar to catalase isozyme 3 and catalase chain 3 from maize, which are mitochondrial, and one was similar to catalase isozyme 1 from maize, whose location is peroxisomal subcellular. All enzymes were induced in all sugarcane libraries (flower, seed, root, callus, leaves) and also in the pathogen-induced libraries, except for CuZnSOD whose cDNA was detected in none of the libraries induced by pathogens (Acetobacter diazotroficans and Herbaspirillum rubrisubalbicans). The expression of the enzymes SOD, GST, GPX, and catalases involved in the detoxification was examined using reverse transcriptase-polymerase chain reaction in cDNA from leaves of sugarcane under biotic stress conditions, inoculated with Puccinia melanocephala, the causal agent of sugarcane rust disease.

Embryogenic calli of the Brazilian sugarcane (Saccharum officinarum L.) genotype SP80-180 were transformed with two plasmids containing genes coding for neomycin phosphotransferase (neo) and phosphinotrycin acetyltransferase (bar), by particle bombardment using an apparatus developed at Copersucar Technology Center. Transformed plants were initially selected on culture medium containing Geneticin, and resistance was confirmed by localized application of a kanamycin solution to leaves of hardened plants at the nursery. A commercial formulation of ammonium gluphosinate was sprayed twice on these antibiotic-resistant plants. The resistant plants were considered co-transformed, and Southern analysis confirmed stable integration of both bar and neo genes. In addition, phosphinotrycin acetyltransferase expression was supported by RT-PCR analysis and neomycin phosphotransferase presence was demonstrated by western blotting. Similar analyses were also performed with micropropagated transformants after three cycles of subculture.

Evidence of the viral etiology of sugarcane yellow leaf disease (SCYLD), occurring in southeast Brazil, was obtained by light and electron microscopy combined with serology. Light microscopy using epifluorescence illumination showed an abnormal yellow-green fluorescing material in the phloem of SCYLD-affected plants that was rarely observed in control plants. Immunolocalization in tissue-printed (or -blotted) nitrocellulose membranes, using barley yellow dwarf virus (BYDV) serotype PAV antiserum, showed a weak but clearly positive reaction in the phloem. Isometric viruslike particles of 24 to 26 nm in diameter were found by electron microscopy both in situ and in partially purified preparations. Examination of thin sections showed that phloem companion cells contained viruslike particles and presented cytological changes apparently related to the development of virus infection. Partially purified preparations produced UV absorption spectra typical of a nucleoprotein, with high absorbance at 260 nm, as expected for isometric virus particles. Virus particles were observed in extracts and partially purified preparations using immunosorbent electron microscopy with BYDV-PAV antiserum. Plate-trapped antigen enzyme-linked immunosorbent assays with the same antiserum indicate a weak serological relationship between BYDV-PAV and SCYLD-associated virus.

Jasmonic acid (JA) and its ester methyl jasmonate (MeJA) are linolenic acid-derived signaling molecules involved in plant development and stress responses. MeJA regulates gene expression at transcription, RNA processing and translation. We investigated the changes in gene expression in sugarcane leaves exposed to MeJA using cDNA arrays. Total RNA isolated at 0, 0.5, 1, 3, 6, and 12 h following MeJA treatment was labeled with alpha-33P-dCTP and hybridized to nylon filters containing 1,536 cDNA clones. A significant increase in gene expression in response to MeJA was detected for both novel and well known stress-related genes, while genes participating in photosynthesis and carbohydrate assimilation were down-regulated. Searches for conserved domains in unknown proteins and digital mRNA expression profile analysis revealed putative new stress-related proteins up-regulated by MeJA and the tissues where the MeJA-regulated genes are preferably expressed. O ácido jasmônico (JA) e seu éster metil-jasmonato (MeJA) são moléculas sinalizadoras derivadas do ácido linolênico e estão envolvidas no desenvolvimento da planta e na resposta aos estresses. MeJA regula a expressão gênica ao nível transcricional, do processamento do RNA e da tradução. Investigamos as mudanças na expressão gênica em folhas de cana-de-açúcar expostas ao MeJA usando arranjos de cDNA. O RNA total isolado a 0, 0,5, 1, 3, 6 e 12 horas após o tratamento com MeJA foi utilizado para a síntese de sondas contendo alfa-33P-dCTP, as quais foram, posteriormente, hibridizadas em membranas de náilon contendo 1.536 clones de cDNA. Um aumento significativo na expressão gênica em resposta ao MeJA foi detectado em genes que respondem a estresses e também em genes com função desconhecida, enquanto os genes que participam da fotossíntese e da assimilação de carboidrato foram reprimidos. A busca por domínios conservados em proteínas desconhecidas e a análise digital do perfil de expressão de mRNA revelaram possíveis proteínas novas relacionadas a estresses induzidas por MeJA e os tecidos onde os genes regulados por MeJA são preferivelmente expressos.