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The relation between the oxidative burst and phenylpropanoid pathways has been studied using the sugarcane cultivar C86-56, which does not release phenolics in agar-base micropropagation systems. In stationary liquid culture, a significant production of phenolic compounds and plant survival were determined in sugarcane plants treated with 5mM H(2)O(2). The spectrophotometer determinations and the gene expression analysis corroborated that releasing of phenolics and soluble θ-quinones was induced during the first 24h of treatment. In comparison with the control treatments, sugarcane plants treated with H(2)O(2) demonstrated differences in the micropropagation-related variables when multiplied in Temporary Immersion Bioreactors (TIBs) supplemented with polyethyleneglycol (PEG 20%). Expression of selected genes related to photosynthesis, ethylene, auxins, oxidative burst, and defense pathways were confirmed during the entire PEG 20% stress in the plants coming from the 5mM H(2)O(2) treatment; whereas, much more heterogeneous expression patterns were evidenced in plants stressed with PEG but not previously treated with H(2)O(2). RT-PCR expression analysis supports the hypothesis that while H(2)O(2) induces the oxidative burst, the phenylpropanoids pathways elicit and maintain the defensive response mechanism in micropropagated sugarcane plants.

The production of phenolic metabolites has been optimized in parallel to sugarcane micropropagation in temporary immersion bioreactors (TIBs). Culturing micropropagated plants in 0.4% CO2-rich air induced their photosynthetic activity by enhancing the change from a heterotrophic to a photomixotrophic metabolic stage. Under 0.4% CO2 enrichment, the transcript levels of both phenylalanine ammonia-lyase (PAL EF189195) and ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco CF576750) increased and were correlated to a 4.6- and 6.3-fold increase in the phenolic levels when plants were multiplied in 20 or 30 g/l sucrose, respectively. A novel application of plant phenolic metabolites as elicitors of resistance to tomato bacterial wilt in the Solanum lycopersicum–Ralstonia solanacearum pathosystem has been identified. The culture media was collected, and the phenolics were sprayed onto tomato plants infected with R. solanacearum, eliciting and/or maintaining an early defense signaling mechanism that resulted in the protection of the plant against the tomato bacterial wilt disease. RT-PCR analyses confirmed that selected genes from defense-related pathways were differentially expressed between plants treated with sugarcane metabolites, non-treated pathogen-free plants, and non-treated plants inoculated with R. solanacearum.

The genomic characterization of sugarcane plants has been achieved by suppressing key genes of the phenylpropanoid pathway; as a result, a new function of phenolic metabolites has been characterized during micropropagation in TIBs. Genes related to cell metabolism and development (10), plant defenses (9), phenylpropanoids (7), methyl jasmonate response (5), ethylene (5), oxidative burst (3) and, auxins (3) pathways, among others (8) were found to be induced in sugarcane plants micropropagating in TIBs with phenolic metabolites, supporting that phenylpropanoids might act as elicitor molecules of others biochemical pathways. During adaptation to natural conditions, plants micropropagated in TIBs with highest levels of phenolics displayed an increased number of functional roots, a high growth rate and, an early ability to be colonized by the natural sugarcane endophytic Gluconacetobacter diazotrophicus.

For the first time both priming and biopriming approaches have been integrated into the sugarcane micropropagation technology by temporary immersion bioreactors (TIBs). Sugarcane micropropagation in CO2-rich TIBs induces a mixotrophic condition adequate for the production of natural phenolic metabolites. Scaling up has been conducted in the C86-56 and C90-317 commercial genotypes. While phenolics demonstrate to act as priming molecules during the in vitro culture, vitroplantlets growing and shooting in the presence of phenolic metabolites display an enhanced vigour (measure as plant size), emitted functional roots and increase adaptability to the natural environment. Additionally, when combined with the inoculation of the endophytic Gluconacetobacter diazotrophicus during transplanting, a significant improvement of the percentage of survival has been attached through this critical step. Altogether, results indicate a promising potential for diversification of the sugarcane micropropagation industry by the production of useful metabolites as byproducts.

Inductions of systemicresistance in sugarcane-G. diazotrophicus interactions which protect the plants againstX. albilineans attack have been demonstrated.G. diazotrophicus activates the sugarcane defense response resulting in the plant resistance toX. albilineans, in this particular case controlling the pathogen transmission to emerging agamic shoots. A total of 47 differentially expressed transcript derived fragments (TDFs) were identified by cDNA-AFLP. Transcripts showed significant homologies to genes of the ethylene signaling pathway (26%), proteins regulates by auxins (9%), β-1,3 Glucanase proteins (6%) and ubiquitin genes (4%), all major signaling mechanisms. A new role for the plant growth-promoting nitrogen-fixing endophytic bacteriaGluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions.

Replicated Saccharum in vivo-plants from cv. Ja60-5 genealogy were evaluated for pre-existing genomic variability by AFLP. By using six different copy primer combinations, 29 bands were found polymorphic within asexually propagated plants, covering approximately 6265 bp of the Saccharum genome. A total of 14 bands were polymorphic in at least four genotypes, representing hypervariable regions of the sugarcane genome. UPGMA analysis illustrates the presence of a natural pre-existing variability in all vegetative-propagated Saccharum genotypes, which do not have a significative effect on the classification of the studied genotypes. Southern blot demonstrates that integration pattern of the variable sequences were not high-complex and its copy number ranged one to six per genome. Sequencing of hypervariable bands showed novel non-repetitive elements in Saccharum with quite low homologies to both genomic DNA sequences reported in public databases and Expressed Sequences Tags in sugarcane. Results support hypothesis about the role of natural mechanisms in plants capable of modifying genome structure, which promote a potential source of variability and could be related to the genome plasticity.

A new role for the plant growth-promoting nitrogen-fixing endophytic bacteria Gluconacetobacter diazotrophicus has been identified and characterized while it is involved in the sugarcane-Xanthomonas albilineans pathogenic interactions. Living G.diazotrophicus possess and/or produce elicitor molecules which activate the sugarcane defense response resulting in the plant resistance to X. albilineans, in this particular case controlling the pathogen transmission to emerging agamic shoots. A total of 47 differentially expressed transcript derived fragments (TDFs) were identified by cDNA-AFLP. Transcripts showed significant homologies to genes of the ethylene signaling pathway (26%), proteins regulates by auxins (9%), beta-1,3 Glucanase proteins (6%) and ubiquitin genes (4%), all major signaling mechanisms. Results point toward a form of induction of systemic resistance in sugarcane-G. diazotrophicus interactions which protect the plant against X. albilineans attack.