Sugarcane (Saccharum spp.).

We describe the procedures for recovering transgenic sugarcane from co-cultivation of both calli and in vitro plants with Agrobacterium tumefaciens. The correct tissue culture strategies and the use of super-binary vector or super-virulent strain are crucial for the successful sugarcane transformation. Both plant regeneration via calli culture and micropropagation strategies can be optimized to a wide spectrum of sugarcane genotypes, thus the procedures presented here could be applied to genetic engineering of Saccharum spp. after minor modifications. For the case of sugarcane transformation using in vitro plants, four selective micropropagation steps must be sufficient to eliminate chimera plants.

Identification of genes induced upon water-deficit stress in a drought-tolerant rice cultivar.

Among the abiotic stresses, the availability of water is the most important factor that limits the productive potential of higher plants. The identification of novel genes, determination of their expression patterns, and the understanding of their functions in stress adaptation is essential to improve stress tolerance. Amplified fragment length polymorphism analysis of cDNA was used to identify rice genes differentially expressed in a tolerant rice variety upon water-deficit stress. In total, 103 transcript-derived fragments corresponding to differentially induced genes were identified. The results of the sequence comparison in BLAST database revealed that several differentially expressed TDFs were significantly homologous to stress regulated genes/proteins isolated from rice or other plant species. Most of the transcripts identified here were genes related to metabolism, energy, protein biosynthesis, cell defence, signal transduction, and transport. New genes involved in the response to water-deficit stress in a tolerant rice variety are reported here.

Identification of sugarcane genes induced in disease-resistant somaclones upon inoculation with Ustilago scitaminea or Bipolaris sacchari.

To understand the molecular basis of a specific plant-pathogen interaction, it is important to identify plant genes that respond to the pathogen attack. Amplified fragment length polymorphism (AFLP) analysis of cDNA was used to identify sugarcane genes differentially expressed in disease-resistant but not in susceptible sugarcane somaclones in response to inoculation with either Ustilago scitaminea or Bipolaris sacchari (also known as Helminthosporium sacchari or Drechslera sacchari), causal agents of smut and eyespot respectively. In total 62 differentially regulated genes were identified, of which 10 were down-regulated and 52 were induced. Of these 52, 19 transcript derived fragments showed homology to known plant gene sequences, most of them related to defense or signaling. The total set of differentially expressed sugarcane genes can be an important resource for further studies aimed at understanding sugarcane pathogen defense.

Hydrogen peroxide induced phenylpropanoids pathway eliciting a defensive response in plants micropropagated in Temporary Immersion Bioreactors (TIBs).

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.

Differential expression analysis by cDNA-AFLP of Saccharum spp. after inoculation with the host pathogen Sporisorium scitamineum.

A differential expression study on the Saccharum spp.-Sporisorium scitamineum pathogenic interaction was carried out in the susceptible Ja60-5 and the resistant M31/45 genotypes. Using cDNA-AFLP analysis, a total of 64 transcript-derived fragments (TDFs) was found to be differentially expressed, with the majority (67.2%) of the differential TDFs up-regulated in the resistant M31/45 cultivar. The plant response against S. scitamineum infection was complex, representing major genes involved in oxidative burst, defensive response, ethylene and auxins pathways during the first 72 h post-inoculation. Results obtained suggest a key role for genes involved in the oxidative burst and the lignin pathways in the initial sugarcane defense against the S. scitamineum infection.