Ariel D. Arencibia

An efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium tumefaciens

This is the first successful report of the recovery of morphologically normal transgenic sugarcane plants from co-cultivation of calluses with Agrobacterium tumefaciens. Transformation frequencies (total of transgenic plants/number of cell clusters) were between 9.4 × 10−3 and 1.15 × 10−2. In our experiments, both LBA4404 (pTOK233) and EHA101 (pMTCA3IG), carrying a super-binary vector or supervirulent strain, respectively, were successful for sugarcane transformation. We found that three main factors: (1) the use of young regenerable calluses as target explants; (2) induction and/or improvement of the A. tumefaciens virulence system with sugarcane cell cultures and (3) pre-induction of organogenesis or somatic-embryogenesis-like sexual embryos, seem to be crucial in order to increase the cells competence for T-DNA transfer process. Patterns generated by Southern hybridization confirmed that T-DNAs were randomly integrated into sugarcane genome without th e persistence of A. tumefaciens in the transgenic plants

Transgenic sugarcane plants resistant to stem borer attack

A truncated cryIA(b) gene encoding the active region of the Bacillus thuringiensis δ-endotoxin was expressed in transgenic sugarcane plants (Saccharum officinarum L.) under the control of the CaMV 35S promoter. Genetic transformation was accomplished by electroporation of intact cells. The levels of recombinant toxin were established and biological activity tests were performed against neonate sugarcane borer (Diatraea saccharalis F.) larvae. Transgenic sugarcane plants showed significant larvicidal activity despite the low expression of CryIA(b).

Somaclonal variation in insect‐resistant transgenic sugarcane (Saccharum hybrid) plants produced by cell electroporation

A population of 42 transgenic sugarcane ( hybrid, cv. Ja60‐5) clones expressing a truncated cryIA(b) gene from Bacillus thuringiensis was evaluated in field trials under artificial borer (Diatraea saccharalis Fab.) infection. Five clones displaying the highest borer tolerance were selected and analysed with molecular tools (RAPD, AFLP and RAMP) to verify genomic changes. Results of field trials provided evidence both for the expression of the resistance trait and for the occurrence of limited but consistent morphological, physiological and phytopathological variation, as compared with control plants regenerated from dedifferentiated culture without transformation (C1‐control) or with plants that were clonally propagated in the field (C2‐control). The five elite transgenic clones, selected for consistent borer‐resistance and good agronomic traits, were further evaluated in a large scale field trial. It was found that the majority of agronomic and industrial traits were those of the original cv. Ja60‐5, but that a small number of qualitative traits was different. DNA changes were verified in the five selected clones. A total of 51 polymorphic DNA bands (out of the 1237 analysed bands) was identified by extensive AFLP and RAMP analysis, thus showing rare but consistent genomic changes in the transgenic plants, as compared with C1‐ and C2‐control plants. It is proposed that the increased variability verified in transgenic plants by field trials and DNA analysis is essentially correlated with cell growth in the dedifferentiated state during the transformation procedure. The results, which are consistent with those published in the case of other transgenic plant populations, are discussed in the context of selecting approaches to gene transfer that minimize somaclonal variation. This is important especially in cases, such as that of sugarcane, where success of backcrosses to restore the original genotype is made difficult by the complex ploidy state of the plant.

Molecular analysis of the genome of transgenic rice (Oryza sativa L.) plants produced via particle bombardment or intact cell electroporation

In the present work we utilised some of the most discriminative molecular tools, such as RAPD, AFLP, AFRP and RAMP, to analyse the genome of independently derived transgenic plants from three elite Italian cultivars (cv. Lido, Carnaroli and Thaibonnet) and found that two methods for direct gene transfer, namely particle bombardment and intact cell electroporation (the latter being a procedure set up in this work), result in transgenic rice (Oryza sativa L.) plants that exhibit negligible genomic changes. This is in contrast with recently published results showing relevant changes in the DNA of transgenic rice plants generated through protoplasts electroporation and of transgenic poplar plants engineered through Agrobacterium tumefaciens infection. Implications of these findings are discussed in the context of selecting appropriate gene transfer methodologies to produce transgenic plants expressing genes of interest while retaining their genomic integrity and, thus, their superior agronomic and/or industrial traits.

Gluconoacetobacter diazotrophicus Elicitate a Sugarcane Defense Response Against a Pathogenic Bacteria Xanthomonas albilineans

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 posse 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%), ß-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.

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.

Molecular and Field Analysis of Somaclonal Variation in Transgenic Plants

Transgenic plants are expected to integrate a foreign gene in an otherwise unmodified genome. Indeed, the foreign gene is stably integrated in the genome of selected plants and is transmitted through the sexual progeny (Bellini et al, 1992). On the other hand, there is no reason to exclude that genomic changes may have occurred in transgenic plants and that these determine visible or cryptic phenotypic and metabolic changes. In fact, all transformation protocols foresee cell culture in the dedifferentiated state before plant differentiation and this has been demonstrated to be a powerful mutagenic treatment (Walbot, Cullis, 1983 and 1985). Changes in phenotypic traits, accompanied by decrease in agronomic potential have already been verified in transgenic rice (Shun et al., 1993) and potato plants (Dale, McPartland, 1992). In general, it is normal practice to disregard transgenic plants with undesirable agronomic traits and to select those that show the highest expression of the transgene and are apparently normal for other traits.

Priming and biopriming integrated into the sugarcane micropropagation technology by Temporary Immersion Bioreactors (TIBS)

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.

Genetic and Symptomatic Characterization of Sugarcane mosaic virus (SCMV) in Cuba

Sugarcane mosaic resistance is established as a key criterion for the breeding program in Cuba. However, molecular characterization of Sugarcane mosaic virus (SCMV) strains occurring in Cuba has not been reported so far. Leaf samples from commercial sugarcane cultivars were collected for SCMV screening in three testing sites (the Matanzas, Camagüey and Holguin provinces). Reverse-Transcriptase polymerase chain reaction was used to identify the virus. The nucleotide sequence comparison grouped the isolates into SCMV subgroups. Sugarcane cultivars inoculated with SCMV from three testing sites displayed different symptoms. Results should be decisive to generate the highest selection pressure, increasing the efficiency of the sugarcane breeding program to the SCMV resistance in the commercial cultivars.

Induced-resistance in sugarcane against pathogenic bacteriaXanthomonas albilineans mediated by an endophytic interaction

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.