RESUMO
Sugarcane and energycane (Saccharum spp. hybrids) are prominent sources of sugar, ethanol, as well as high-value bioproducts globally. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, limited germplasm resources, long breeding cycle, as well as recalcitrance to genetic transformation. Here, we present a biolistic-based transformation and bioreactor-based micro-propagation system that has been utilized successfully to transform twelve elite cane genotypes, yielding transformation efficiencies of up to 39%. The system relies on the generation of embryogenic callus from sugarcane and energycane apical shoot tissue, followed by DNA bombardment of embryogenic leaf roll discs (approximately one week) or calli (approximately 4 weeks). We present optimal criteria and practices for selection and regeneration of independent transgenic lines, molecular characterization, as well as a bioreactor-based micro-propagation technique, which can aid in rapid multiplication and analysis of transgenic lines. The cane transformation and micro-propagation system described here, although built on our previous protocols, has significantly accelerated the process of producing and multiplying transgenic material, and it is applicable to other varieties. The system is highly reproducible and has been successfully used to engineer multiple commercial sugarcane and energycane varieties. It will benefit worldwide researchers interested in genomics and genetics of sugarcane photosynthesis, cell wall, and bioenergy related traits.
RESUMO
Sugarcane (Saccharum spp. hybrids) is an economically important crop widely grown in tropical and subtropical regions for sugar and ethanol production. However, the large genome size, high ploidy level, interspecific hybridization and aneuploidy make sugarcane one of the most complex genomes and have long hampered genome research in sugarcane. Modern sugarcane cultivars are derived from interspecific hybridization between S. officinarum and S. spontaneum with 80-90% of the genome from S. officinarum and 10-20% of the genome from S. spontaneum. We constructed bacterial artificial chromosome (BAC) libraries of S. officinarum variety LA Purple (2n = 8x = 80) and S. spontaneum haploid clone AP85-441 (2n = 4x = 32), and selected and sequenced 97 BAC clones from the two Saccharum BAC libraries. A total of 5,847,280 bp sequence from S. officinarum and 5,011,570 bp from S. spontaneum were assembled and 749 gene models were annotated in these BACs. A relatively higher gene density and lower repeat content were observed in S. spontaneum BACs than in S. officinarum BACs. Comparative analysis of syntenic regions revealed a high degree of collinearity in genic regions between Saccharum and Sorghum bicolor and between S. officinarum and S. spontaneum. In the syntenic regions, S. spontaneum showed expansion relative to S. officinarum, and both S. officinarum and S. spontaneum showed expansion relative to sorghum. Among the 75 full-length LTR retrotransposons identified in the Saccharum BACs, none of them are older than 2.6 mys and no full-length LTR elements are shared between S. officinarum and S. spontaneum. In addition, divergence time estimated using a LTR junction marker and a syntenic gene shared by 3 S. officinarum and 1 S. spontaneum BACs revealed that the S. spontaneum intergenic region was distant to those from the 3 homologous regions in S. officinarum. Our results suggested that S. officinarum and S. spontaneum experienced at least two rounds of independent polyploidization in each lineage after their divergence from a common ancestor.
RESUMO
BACKGROUND: Several high-throughput molecular genetic analyses rely on high-quality genomic DNA. Copurification of other molecules can negatively impact the functionality of plant DNA preparations employed in these procedures. Isolating DNA from agronomically important crops, such as sugarcane, rice, citrus, potato and tomato is a challenge due to the presence of high fiber, polysaccharides, or secondary metabolites. We present a simplified, rapid and reproducible SDS-based method that provides high-quality and -quantity of DNA from small amounts of leaf tissue, as required by the emerging biotechnology and molecular genetic applications. RESULTS: We developed the TENS-CO method as a simplified SDS-based isolation procedure with sequential steps of purification to remove polysaccharides and polyphenols using 2-mercaptoethanol and potassium acetate, chloroform partitioning, and sodium acetate/ethanol precipitation to yield high-quantity and -quality DNA consistently from small amounts of tissue (0.15 g) for different plant species. The method is simplified and rapid in terms of requiring minimal manipulation, smaller extraction volume, reduced homogenization time (20 s) and DNA precipitation (one precipitation for 1 h). The method has been demonstrated to accelerate screening of large amounts of plant tissues from species that are rich in polysaccharides and secondary metabolites for Southern blot analysis of reporter gene overexpressing lines, pathogen detection by quantitative PCR, and genotyping of disease-resistant plants using marker-assisted selection. CONCLUSION: To facilitate molecular genetic studies in major agronomical crops, we have developed the TENS-CO method as a simple, rapid, reproducible and scalable protocol enabling efficient and robust isolation of high-quality and -quantity DNA from small amounts of tissue from sugarcane, rice, citrus, potato, and tomato, thereby reducing significantly the time and resources used for DNA isolation.
