Laszlo Sagi

Genetic Transformation of Banana and Plantain (Musa spp.) via Particle Bombardment

We have developed a simple protocol to allow the production of transgenic banana plants. Foreign genes were delivered into embryogenic suspension cells using accelerated particles coated with DNA. Bombardment parameters were optimized for a modified particle gun resulting in high levels of transient expression of the β-glucuronidase gene in both banana and plantain cells. Bombarded banana cells were selected with hygromycin and regenerated into plants. Molecular and histochemical characterization of transformants revealed the stable integration of the transferred genes into the banana genome.

Optimisation of transformation conditions and production of transgenic sorghum (Sorghum bicolor) via microparticle bombardment

AbstractConditions for microparticle bombardment were optimised for four explant types of sorghum Sorghum bicolor (L.) Moench based on transient expression of the uidA reporter gene. The tested physical parameters included acceleration pressure, target distance, gap width and macroprojectile travel distance. The sorghum explants studied were immature and mature embryos, shoot tips and embryogenic calli. In addition, the activity of four heterologous promoters was determined both by GUS histochemical staining and enzymatic activity assay in immature embryos and shoot tips. The strength of these promoters could be placed in the following order: ubi1> act1D> adh1>CaMV 35S. The optimised bombardment conditions were applied for selecting phosphinothricin- or geneticin-resistant in vitro cultures in order to generate transgenic plants. Production of transgenic plants via phosphinothricin-selection was not successful due to the release of phenolic substances from the herbicide-resistant cultures during the regeneration process. After selection on geneticin, however, fertile transgenic sorghum plants were regenerated from immature embryos as well as from shoot tips. Stable integration and Mendelian inheritance of the neo selectable marker gene was demonstrated in all transgenic plants.

Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants

Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the β-glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots.

A promoter from sugarcane bacilliform badnavirus drives transgene expression in banana and other monocot and dicot plants

A 1369 bp DNA fragment (Sc) was isolated from a full-length clone of sugarcane bacilliform badnavirus (ScBV) and was shown to have promoter activity in transient expression assays using monocot (banana, maize, millet and sorghum) and dicot plant species (tobacco, sunflower, canola and Nicotiana benthamiana). This promoter was also tested for stable expression in transgenic banana and tobacco plants. These experiments showed that this promoter could drive high-level expression of the β-glucuronidase (GUS) reporter gene in most plant cells. The expression level was comparable to the maize ubiquitin promoter in standardised transient assays in maize. In transgenic banana plants the expression levels were variable for different transgenic lines but was generally comparable with the activities of both the maize ubiquitin promoter and the enhanced cauliflower mosaic virus (CaMV) 35S promoter. The Sc promoter appears to express in a near-constitutive manner in transgenic banana and tobacco plants. The promoter from sugarcane bacilliform virus represents a useful tool for the high-level expression of foreign genes in both monocot and dicot transgenic plants that could be used similarly to the CaMV 35S or maize polyubiquitin promoter.

SuperSAGE combined with PCR walking allows global gene expression profiling of banana (Musa acuminata), a non-model organism

Super-serial analysis of gene expression (SuperSAGE) was used to characterize, for the first time, the global gene expression pattern in banana (Musa acuminata). A total of 10,196 tags were generated from leaf tissue, representing 5,292 expressed genes. Forty-nine tags of the top 100 most abundantly expressed transcripts were annotated by homology to cDNA or EST sequences. Typically for leaf tissue, analysis of the transcript profiles showed that the majority of the abundant transcripts are involved in energy production, mainly photosynthesis. However, the most abundant tag was derived from a type 3 metallothionein transcript, which accounted for nearly 3% of total transcripts analysed. Furthermore, the 26-bp long SuperSAGE tags were applied in 3′-rapid amplification of cDNA ends (3′RACE) for the identification of unknown tags. In combination with thermal asymmetric interlaced PCR (TAIL-PCR), this allowed the recovery of a full gene sequence of a novel NADPH:protochlorophyllide oxidoreductase, the key enzyme in chlorophyll biosynthesis. SuperSAGE in conjunction with 3′RACE and TAIL-PCR will be a powerful tool for transcriptomics of non-model, but otherwise important organisms.

High‐throughput in planta expression screening identifies an ADP‐ribosylation factor (ARF1) involved in non‐host resistance and R gene‐mediated resistance

To identify positive regulators of cell death in plants, we performed a high-throughput screening, employing potato virus X-based overexpression in planta of a cDNA library derived from paraquat-treated Nicotiana benthamiana leaves. The screening of 30,000 cDNA clones enabled the identification of an ADP-ribosylation factor 1 (ARF1) that induces cell death when overexpressed in N. benthamiana. Overexpression of the guanosine diphosphate (GDP)-locked mutant of ARF1 did not trigger cell death, suggesting that ARF1 guanosine triphosphatase (GTPase) activity is necessary for the observed cell death-inducing activity. The ARF1 transcript level increased strongly following treatment with Phytophthora infestans elicitor INF1, as well as inoculation with a non-host pathogen Pseudomonas cichorii in N. benthamiana. In addition, ARF1 was induced in the interaction between the N gene and tobacco mosaic virus (TMV) in Nicotiana tabacum. By contrast, inoculation with the virulent pathogen Pseudomonas syringae pv. tabaci did not affect ARF1 expression in N. benthamiana. Virus-induced gene silencing of ARF1 in N. benthamiana resulted in a stunted phenotype, and severely hampered non-host resistance towards P. cichorii. In addition, ARF1 silencing partially compromised resistance towards TMV in N. benthamiana containing the N resistance gene. By contrast, and in accordance with the ARF1 gene expression profile, silencing of ARF1 transcription did not alter the susceptibility of N. benthamiana towards the pathogen P. syringae pv. tabaci. These results strongly implicate ARF1 in the non-host resistance to bacteria and N gene-mediated resistance in N. benthamiana.

Chemotactic Movement and Attachment of Agrobacterium tumefaciens to Banana Cells and Tissues

Summary For the elaboration of efficient Agrobacterium -mediated transformation of banana the initial steps in the plant-bacterium interaction were investigated. Chemotaxis of agrobacteria towards tissues was studied using a swarm agar plate system. Excised leaf, rhizome, root and in vitro proliferating tissues from different banana landraces were found to be able to elicit a positive chemotactic reaction of A. tumefaciens that could be enhanced by extensive wounding. Agrobacterium attachment was examined by UV fluorescence microscopy and scanning electron microscopy, which demonstrated the presence of bacteria individually bound or massively attached to the surface of single banana cells and tissues. It can be concluded that at least during the two early steps of interaction A. tumefaciens appears to be compatible with banana, indicating the potential for genetic transformation.

Banana (Musa sp.)

Cultivated bananas are vegetatively propagating herbs, which are difficult to breed because of widespread male and female sterility. As a complementary gene transfer method in banana, the described Agrobacterium protocol relies on highly regenerable embryogenic cell cultures. Embryogenic cells are infected and co-cultivated in the presence of acetosyringone with Agrobacterium tumefaciens harboring a binary plasmid vector to obtain a mixed population of transformed and untransformed plant cells. Transformed plant cells are promoted to grow for 2 to 3 mo on a cell colony induction medium containing the antibiotics geneticin or hygromycin as selective agents, while agrobacteria are counterselected by timentin. The whole procedure, including plant regeneration, takes approx 6 mo and results in an average frequency of 25 to 50 independent transgenic plants per plate, which equals 50 mg of embryogenic cells. This method has been applied to a wide range of cultivars and to generate large populations of transgenic colonies and plants for tagging genes and promoters in banana.

Evidence for cytoplasmic control of in vitro microspore embryogenesis in the anther culture of wheat (Triticum aestivum L.)

SummaryAnthers were cultured from two sets of seven lines of hexaploid wheat (Triticum aestivum L.) with different cytoplasms, the euplasmic nucleus donors, ‘Siete Cerros 66’ and ‘Penjamo 62’, as well as their six alloplasmic lines derived from wild relative species of the genera Triticum and Aegilops. Significant cytoplasmic and nuclear effects but no cytoplasmic-nuclear interaction were found for embryogenic anther response, with the best performance of ‘Penjamo 62’ in Ae. kotschyi cytoplasm. Plant regeneration was not affected significantly by the cytoplasmic background of the lines cultured. The possible genetic implications of the observed cytoplasmic and nuclear influences on the in vitro haploid induction of wheat are discussed.

Improved T-DNA vector for tagging plant promoters via high-throughput luciferase screening.

Transferred DNA (T-DNA) tagging is a powerful tool for tagging and in planta characterization of plant genes on a genome-wide scale. An improved promoter tagging vector is described here, which contains the codon-optimized luciferase (luc+) reporter gene 31 bp from the right border of the T-DNA. Compared to the wild-type luciferase gene, this construct provides significantly increased reporter gene expression and a 40 times higher tagging frequency. The utility of the construct is demonstrated in banana, a tropical monocot species, by screening embryogenic cell colonies and regenerated plants with an ultrasensitive charged-coupled device (CCD) camera. The improved vector resulted in a luciferase activation frequency of 2.5% in 19,000 cell colonies screened. Detailed molecular analysis of flanking DNA sequences in a tagged line revealed insertion of the luciferase tag in a novel gene with near-constitutive expression.