Magali Ferrari Grando

Transformation of bahiagrass (Paspalum notatum Flugge)

Abstract. Bahiagrass (Paspalum notatum Flugge), a forage species widely used in the southeastern United States, and from Central Mexico to Argentina, was targeted for improvement through genetic engineering. Embryogenic callus, initiated from germinating seedlings, was bombarded with a vector containing the bar selectable marker/reporter gene that confers resistance to phosphinothricin (glufosinate) herbicide (trade names Liberty, Ignite and Finale). Thirty-two transgenic plants were recovered. These plants were identified by the polymerase chain reaction (PCR) and verified by Southern analysis. Transgenic plants with bar, as well as non-transgenic plants without bar, regenerated from bombarded callus and selected with glufosinate, developed strong and stable resistance to glufosinate during selection. This unusual resistance in non-transgenic plants has persisted for over a year and is passed on to new tillers. The development of resistance in non-transgenic cells reduced the herbicide selection efficiency and made it necessary to identify transgenic plants by PCR where the 32 transgenic plants were recovered from 674 glufosinate-resistant plants, giving a very low selection efficiency.

Two point deterministic model for acquisition of in vitro pollen grain androgenetic capacity based on wheat studies

This article discusses, from the standpoint of cellular biology, the deterministic and indeterministic androgenesis theories. The role of the vacuole and of various types of stresses on deviation of the microspore from normal development and the point where androgenetic competence is acquired are examined. Based on extensive literature review and data on wheat studies from our laboratory, a model for androgenetic capacity of pollen grain is proposed. A two point deterministic model for in vitro androgenesis is our proposal for acquisition of androgenetic potential of the pollen grain: the first switch point would be early meiosis and the second switch point the uninucleate pollen stage, because the elimination of cytoplasmatic sporophytic determinants takes place at those two strategic moments. Any abnormality in this process allowing the maintenance of sporophytic informational molecules results in the absence of establishment of a gametophytic program, allowing the reactivation of the embryogenic process

RESGATE VEGETATIVO POR ALPORQUIA DE GENÓTIPOS ADULTOS DE URUCUM (Bixa orellana L.)

This study aimed to evaluate the usefulness of an air layering technique for vegetative rescue of annatto ( Bixa orellana L.) genotypes and obtainment of propagules for vegetative propagation. Ten 12 year-old annatto stock plants, whose genotypes are derived from artificial crossing between “Hairy-green capsule” X “Smooth-red capsule” were used in this study. Air layering adventitious roots were induced in branches (1-2 cm diameter) using as substrate a mixture of vermiculite and moss. The effects of (1) the type of girdling (total or partial), 1 cm in length, (2) IBA (indole-3-butyric acid at 0 and 4.92 mM) solution in paper filter and (3) the covering with either transparent or dark plastic films and tencel were evaluated. The technique of air layering provided rooted branches for the 10 genotypes evaluated, with efficiency ranging from 20 to 100%, and a genotype effect was observed on rooting frequency. Under greenhouse conditions, the survival of layers derived from totally girdled stems treated with IBA and protected with transparent plastic was 100%. Established layers provided vigorous branching from which shoot apexes or nodal segment explants were obtained for in vitro establishment and propagation of this species.

AVALIAÇÃO DE GENÓTIPOS DE URUCUM ( Bixa orellana L.) POR MEIO DA CARACTERIZAÇÃO MORFOLÓGICA DE FRUTOS, PRODUTIVIDADE DE SEMENTES E TEOR DE BIXINA

Bixa orellana L. (urucum) e considerada uma fonte importante de corantes naturais para as industrias alimenticia, farmaceutica e textil, sendo o Brasil um dos maiores produtores e exportadores do corante extraido das sementes desta planta. O objetivo deste trabalho foi a caracterizacao morfologica de frutos, a avaliacao da produtividade de sementes e do teor do carotenoide bixina presente nas sementes recem- colhidas e armazenadas por periodos de 30, 60, 90 e 120 dias, realizada em 10 genotipos de urucum, a fim de fornecer subsidios para a selecao de genotipos superiores para estas caracteristicas. Houve variacao em todas as caracteristicas avaliadas entre os 10 genotipos. Os frutos, de todos os genotipos, sao recobertos por cerdas ou pelos, apresentam coloracao avermelhada, verde, amarela ou vermelha e duas formas basicas, oval ou lanceolada. Os frutos apresentaram comprimentos medios que variaram entre 3,1 a 4,8 cm e larguras entre 2,4 a 3,1 cm. O numero medio de sementes por fruto variou de 41,5 a 50,6. A massa de 100 sementes variou de 1,1 a 2,24 g. Houve significativa diferenca na concentracao de bixina entre os genotipos, sendo possivel identificar genotipos com altos niveis deste carotenoide (5,05 %), superior aos ja relatados por outros estudos. O teor de bixina decresceu com o aumento no periodo de armazenamento das sementes.

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

BackgroundDeveloping 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.ResultsFrom 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.ConclusionDevelopmental 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.

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 μM 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.

Indução de embriogênese somática em diferentes explantes de aveia (Avena sativa L.)

Three experiments were carried out to evaluate the embryogenic callus formation potential of three types of explants in oat (Avena sativa L.) (immature embryo, mature embryo and coleptile segments). Each experiment was conduced with one explant, five cultivars (UPF15, UPF16, UPF18, UPFA20 and OR3) and two induction callus medium (M1 = MS + 2 4-D and M2 = MS + 2 4-D + BAP), being analyzed the frequency of embryogenic calli at 60 days of cultivation, callus fresh weight (mg) at 90 and 120 days and callus growth rate during 30 days period. The influence of genotype on the embryogenic callus frequency was detected only when using the immature embryo explant. The culture medium did not influence the frequency of callus in any of the three tested explants. The mature embryo was not suitable for the induction of oat somatic embryogenesis. The coleoptile and immature embryo explants present a similar embryogenic capacity, producing 24 and 29% of embryogenic calli, respectively. However, the coleoptile can be considered the ideal explant due to its genotypic independence, high embryogenic callus growth rate (328% in 30 days), as well as easy and quick availability.

In vitro shoot induction and multiplication from nodal segments of adult Ginkgo biloba plants

The in vitro performance of herbaceous and woody nodal segments from adult plants and the effect of hydrolyzed casein (HC 500 mg L-1), kinetin (KIN; 6-furfurylaminopurine 0.46 and 4.65 µM) and activated charcoal (AC 1.5 g L-1) were evaluated upon new shoots induction and development, and to establish a system of in vitro propagation from adult plants of Ginkgo biloba. Woody nodal segments did not produce axillary shoots and presented 100% of bacterial and fungal contamination in culture. However, nodal segments from herbaceous shoots were successfully disinfected and displayed high in vitro morphogenic capacity. The HC was essential for the axillary shoots induction and further multiplication, stimulating shoot formation in 85% of the cultured nodal segments and multiple shoots induction in 35% of them at establishment stage. During the multiplication stage, 66.6% of propagules formed new shoots and 33.3% of them formed multiple shoots when cultured with HC. The KIN and AC inhibited the organogenic process in ginkgo. Two distinct patterns of sprouts development were observed in vitro, similar to what occurs in vivo: 1) short shoots with crowded internodes and expansion of only a few leaves and slow growth; 2) long shoots with separated nodes and marked apical growth. This is the first report of multiple shoots in vitro formation in nodal segments obtained from adult plants of Ginkgo biloba.

Proposal for commercial classification of the gerbera capitulum based in the flower overlap index

The aim of this work was to propose a methodology for commercial classification of gerbera´s capitulum based on the trans flowers overlap coefficient. Three to eight capitula of twenty-eight cultivars of gerbera cut flower and seven non-commercial accesses were used. The width measurement of the trans flowers group (internal ray flower) as well as the sum of the width of the trans and ray flowers (external ray flower) groups were taken from each capitulum. The average of each access and the overlap coefficient were calculated (CS = width of the trans flowers group / Σ of the width of the trans and ray flowers groups) and submitted to the multivariate analysis, to verify the grouping by the average Euclidian distance matrix. It is proposed that gerberas with CS= 0.01 to 0.15 be classified as simple, CS= 0.16 to 0.40 as semi-double and CS superior to 0.41 as double. The overlap coefficient (CS) can be used to systematize the classification of the gerbera capitula, reducing the divergences in the classification and the subjectivity in the decisions of producers, florists and breeders.

Androgenic response of wheat genotypes resistant to fusariosis

The objective of this work was to assess the androgenic response, via microspore culture, of wheat genotypes with different levels of resistance to Gibberella zeae. The number of androgenic embryos per spike, and of green and albino plants was counted for the BRS 179 (moderately resistant), Frontana and Sumai 3 (resistant), and BRS 194, Embrapa 27, and Fielder (susceptible) genotypes. The degree of interference by the Fielder, Pavon 76, and Sumai 3 ovary-donor genotypes, used for co-culture with the microspore cells, was also assessed regarding androgenic response. Induction efficiency ranged from 0.33 embryo per spike for Embrapa 27 to 109.8 embryos for Frontana. Sumai 3 presented the second best response, and Embrapa 27 behaved as highly recalcitrant. The co-culture of ovaries from the Fielder genotype stands out from the others and positively interferes in the embryo induction rate. No significant differences were observed for the regeneration frequency of green plants; however, for albino plants, BRS 194 produced the highest rate. Frontana and Sumai 3 present the highest androgenic response rates and can be used in breeding programs for the rapid development of cultivars resistant to Gibberella zeae.