Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

Abstract Low transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22°C and 25°C, and a 400 mM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciens overgrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future

Acetosyringone, pH and temperature effects on transient genetic transformation of immature embryos of Brazilian wheat genotypes by Agrobacterium tumefaciens

AbstractLow transformation efficiency is one of the main limiting factors in the establishment of genetic transformation of wheat via Agrobacterium tumefaciens. To determine more favorable conditions for T-DNA delivery and explant regeneration after infection, this study investigated combinations of acetosyringone concentration and pH variation in the inoculation and co-cultivation media and co-culture temperatures using immature embryos from two Brazilian genotypes (BR 18 Terena and PF 020037). Based on transient expression of uidA, the most favorable conditions for T-DNA delivery were culture media with pH 5.0 and 5.4 combined with co-culture temperatures of 22 °C and 25 °C, and a 400 μM acetosyringone supplement. These conditions resulted in blue foci in 81% of the embryos. Media with more acidic pH also presented reduced A. tumefaciensovergrowth during co-culture, and improved regeneration frequency of the inoculated explants. BR 18 Terena was more susceptible to infection by A. tumefaciens than PF 020037. We found that it is possible to improve T-DNA delivery and explant regeneration by adjusting factors involved in the early stages of A. tumefaciens infection. This can contribute to establishing a stable transformation procedure in the future

Developmental changes in abundance of the VSPβ protein following nuclear transformation of maize with the Soybean <it>vspβ </it>cDNA

Abstract Background Developing monocots that accumulate more vegetative tissue protein is one strategy for improving nitrogen-sequestration and nutritive value of forage and silage crops. In soybeans (a dicotyledonous legume), the vspA and B genes encode subunits of a dimeric vegetative storage protein that plays an important role in nitrogen storage in vegetative tissues. Similar genes are found in monocots; however, they do not accumulate in leaves as storage proteins, and the ability of monocot leaves to support accumulation of an ectopically expressed soybean VSP is in question. To test this, transgenic maize (Zea Mays L. Hi-II hybrid) lines were created expressing soybean vspB from a maize ubiquitin Ubi-1 promoter. Results From 81 bombardments, 101 plants were regenerated, and plants from five independent lines produced vspB transcripts and VSPβ polypeptides. In leaves from seven-week-old plants (prior to flowering), VSPβ accumulated to 0.5% of the soluble leaf protein in primary transgenic plants (R0), but to only 0.03% in R1 plants. During seed-filling (silage-stage) in R1 plants, the VSPβ protein was no longer detected in leaves and stems despite continued presence of the vspB RNA. The RNA transcripts for this peptide either became less efficiently translated, or the VSPβ protein became unstable during seed-fill. Conclusion Developmental differences in the accumulation of soybean VSPβ when transgenically expressed in maize show that despite no changes in the vspB transcript level, VSPβ protein that is readily detected in leaves of preflowering plants, becomes undetectable as seeds begin to develop.</p

Caracterização citogenética, viabilidade de pólen e hibridação artificial em gérbera Chromosome number, pollen viability and gerbera hybridization

Este trabalho foi conduzido com o objetivo de confirmar o número de cromossomos em cultivares de Gerbera hybrida Hort., determinar o número de cromossomos em acessos não comerciais de Gerbera sp., avaliar a viabilidade de pólen e a possibilidade de cruzamentos entre cultivares e acessos não comerciais. Foram coletados ápices de raízes e pólen de seis cultivares e de sete acessos não comerciais. O material coletado foi corado com carmim acético a 45%. A contagem dos cromossomos foi realizada em células metafásicas intactas e a estimativa de viabilidade de pólen realizada por meio da contagem do número de grãos de pólen viáveis e não viáveis. A possibilidade de cruzamento entre as cultivares e entre as cultivares e acessos não comerciais foi avaliada por meio da hibridação entre os genitores femininos, cv. Terra Fame e acesso A8, e masculinos, cvs. Cariba e Azteca. Todos os acessos contiveram cinqüenta cromossomos, indicando que a variação morfológica nos capítulos (simples, semidobrado e dobrado) não é devida a mutações cromossômicas numéricas ou a poliploidia. A viabilidade do pólen variou de 87,67% a 99,27%. A formação de sementes foi de 4,46% nos cruzamentos entre cultivares, e de 50% entre o A8 e as cultivares. A compatibilidade genômica entre os acessos, a alta viabilidade do pólen e o sucesso na obtenção de sementes entre acessos comercias e não comerciais, revela a possibilidade de produção de híbridos com novas combinações alélicas e transferência de caracteres desejáveis dos acessos não comerciais para os comerciais<br>This work was conducted to confirm the chromosomes number of Gerbera hybrida Hort. cultivars, to determine the chromosomes number in the non commercial accessions of Gerbera sp., and to estimate the pollen viability and the possibility of crossings among different accessions. Root-tip and pollen were collected from six cultivars and seven non commercial accessions. The collected material was stained with acetic carmim at 45%. The chromosome counting was performed in metafasic intact cells and the pollen viability estimated by counting the number of viable and non viable pollen grains. The crossing possibility among cultivars and among cultivars and non commercial accessions was evaluated through the hybridization between the feminine cv. Terra Fame and A8 and masculine genitors, cvs. Cariba and Azteca. All accessions presented fifty chromosomes, indicating that the morphological variation in the capitulum (simple, semidouble and double) is not due to the chromosome number mutation or polyploidy. The evaluated accessions displayed high pollen viability, varying from 87.67% to 99.27. The seed formation was 4.46% in the crossings among cultivars, and 50% among cultivar and non commercial accession of gerbera. The genomic compatibility among the accessions, the high pollen viability and the success in the seed formation among commercial and non-commercial accessions, reveal the possibility of hybrid production with new allelic combination and transference of desirable traits from the non-commercial to the commercial accessions