Elisabeth Mansur

Efficient shoot organogenesis of eggplant (Solanum melongena L.) induced by thidiazuron

Abstract Morphogenesis from several explant types excised from in-vitro-grown plantlets of a Brazilian eggplant (Solanum melongena L.) variety (F-100) was evaluated in response to thidiazuron (TDZ). Leaves and cotyledons were found to be the most responsive explants. Optimal shoot bud induction rates (75–100 buds/explant) were achieved in the presence of 0.2 μm TDZ. Organogenic calli were transferred to growth regulator free MS medium before shoot excision. Rooting was induced on half-strength MS medium supplemented with 0.6 μm IAA.

Anthocyanin production in callus cultures of Cleome rosea: modulation by culture conditions and characterization of pigments by means of HPLC-DAD/ESIMS.

Leaf and stem explants of Cleome rosea formed calluses when cultured on MS medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) or 4-amino-3,5,6-trichloropicolinic acid (PIC). The highest biomass accumulation was obtained in the callus cultures initiated from stem explants on medium supplemented with 0.90 microM 2,4-D. Reddish-pink regions were observed on callus surface after 6-7 months in culture and these pigments were identified as anthocyanins. Anthocyanins production was enhanced by reducing temperature and increasing light irradiation. Pigmented calluses transferred to MS1/2 with a 1:4 ratio NH(4)(+)/NO(3)(-), 70 g L(-1) sucrose and supplementation with 0.90 microM 2,4-D maintained a high biomass accumulation and showed an increase of 150% on anthocyanin production as compared with the initial culture conditions. Qualitative analysis of calluses was performed by high performance liquid chromatography coupled to diode array detector and electrospray ionization mass spectrometry (HPLC-DAD/ESIMS). Eleven anthocyanins were characterized and the majority of them were identified as acylated cyanidins, although two peonidins were also detected. The major peak was composed by two anthocyanins, whose proposed identity were cyanidin 3-(p-coumaroyl) diglucoside-5-glucoside and cyanidin 3-(feruloyl) diglucoside-5-glucoside.

Eggplant (Solanum melongena L.): tissue culture, genetic transformation and use as an alternative model plant

A berinjela e uma especie solanacea nao tuberosa de importância agronomica, cultivada principalmente por seus frutos. Na medicina popular, a berinjela e indicada para o tratamento de varias doencas, incluindo diabetes, artrite, asma e bronquite. A berinjela e suscetivel a varias doencas e pragas que causam perdas economicas significativas. Esse problema tem sido abordado com tecnicas convencionais de melhoramento, utilizando especies silvestres resistentes de Solanum, que possuem uma grande diversidade genetica e sao fontes de genes de interesse agronomico. A aplicacao de metodologias in vitro a berinjela tem resultado em sucesso consideravel. Os tecidos de berinjela apresentam um alto potencial morfogenetico, sendo uteis para estudos de desenvolvimento e para o estabelecimento de abordagens biotecnologicas para a producao de variedades melhoradas, tais como o resgate de embrioes, a selecao in vitro, a hibridizacao somatica e a transformacao genetica. O conjunto dessas caracteristicas tambem torna a berinjela um modelo completo para estudos em diferentes areas de pesquisa, incluindo o controle da expressao genica e a avaliacao da estabilidade de somaclones derivados de diferentes processos morfogeneticos. Neste trabalho, sao analisados fatores importantes que afetam a eficiencia dos processos de regeneracao in vitro por meio de organogenese e embriogenese, assim como de transformacao genetica, explorando ainda o potencial da especie como planta modelo para o estudo de varios aspectos da genetica e fisiologia vegetais.

Transient expression of GUS and the 2S albumin gene from Brazil nut in peanut (Arachis hypogaea L.) seed explants using particle bombardment

The effect of parameters involved in the transformation efficiency of peanut (Arachis hypogaea L.) seed tissues by direct gene transfer using a helium inflow particle bombardment device was evaluated. Transient gene expression was affected by both particle and DNA amounts, and was positively correlated with gene copy number, as determined byβ-glucuronidase (GUS) activity assays. No influence of plasmid size on GUS gene expression was observed. Transcriptional control of GUS by either the CaMV 35S or the 2S promoter from Brazil nut 2S albumin gene varied with the developmental stage of the seed and was approximately tenfold greater under the influence of the 35S promoter than under the 2S promoter. The gene products of both the Brazil nut methionine-rich 2S albumin and GUS genes under the transcriptional control of the 35S promoter were detected by ELISA assays.

In vitro regeneration of peanut (Arachis hypogaea L.) through organogenesis : Effect of culture temperature and silver nitrate

SummaryThe effect of culture temperature on the morphogenetic response of Arachis hypogaea was studied. Cotyledons were cultivated on MS medium supplemented with 110 µM 6-benzyladenine. Leaf explants were cultivated in the presence of the same growth regulator at 22 µM. Cultures were incubated at temperatures of 25, 28, and 35±5° C. Both direct organogenesis from cotyledons and development of organogenic calluses from leaves showed optimal rates at 35±5° C. The highest frequency of elongation of buds into shoots from leaf-derived calluses occurred in the presence of 5 µM AgNO3. At the best culture temperature, an average of 95% of shoots formed roots on growth-regulator-free MS medium. Plants were successfully transferred to soil, showing normal phenotypes.

Somatic embryo formation in Arabidopsis and eggplant is associated with expression of a glycine-rich protein gene (Atgrp-5)

The isolation of embryogenesis-associated genes and the characterization of their roles during embryo development are important steps towards the elucidation of the molecular mechanisms controlling embryo morphogenesis. Somatic embryogenesis continues to be an effective model for studying gene expression in embryo development. We report the analysis of the transcriptional expression of a glycine-rich gene (Atgrp-5) during somatic embryo morphogenesis. Arabidopsis thaliana transgenic lines carrying chimeric constructs containing the β-glucuronidase (GUS) reporter gene under the control of the Atgrp-5 promoter were used to analyze its expression pattern during somatic embryogenesis. To evaluate whether Atgrp-5 expression observed in Arabidopsis reflects a general pattern during somatic embryogenesis, transgenic eggplant (Solanum melongena L.) was used as non-homologous embryogenic system. High promoter activity was detected in all cells of pro-embryogenic cell clusters and somatic embryos from globular to torpedo stages. During the transition from torpedo to cotyledonar stage the Atgrp-5 gene was gradually turned off and, in mature embryos, its promoter activity was restricted to the protoderm. mRNA in situ hybridization on Arabidopsis somatic embryo cultures have confirmed the expression pattern observed by GUS histochemical assays. These results indicate that Atgrp-5 is expressed in cells that undergo the first anatomical modifications leading to somatic embryo development.

In vitro plant regeneration from seed explants of wild groundnut species (Genus Arachis, Section Extranervosae)

In vitro regeneration of wild groundnut species from Section Extranervosae (Arachis villosulicarpa, A. macedoi, A. retusa, A. burchellii, A. pietrarellii, A. prostrata, A. aff. prostrata and a new species) was examined for the purpose of germplasm renewal and conservation. Seeds of different ages, stored at the seed bank of CENARGEN/EMBRAPA were either inoculated on culture medium or used as a source of embryo axis and cotyledon explants. Whole seeds failed to germinate on MS either without growth regulators (MS0) or supplemented with 10 μM TDZ. Embryo axes cultured on MS0 produced only single plants. In the presence of 8.8 μM BAP these explants showed multi-shoot formation. Cotyledons cultured on MS supplemented with 110 μM BAP developed adventitious shoots through direct organogenesis. Plant regeneration was obtained from A. villosulicarpa, A. macedoi, A. retusa, A. burchellii and A. pietrarellii both from embryo axes and cotyledons. Explants from A. prostrata and A. aff. prostrata did not produce regenerants. Rooting of shoots was induced in the presence of 5.4 μM NAA. Primary plants derived from these explants were further multiplied by culturing nodal segments on MS medium plus 2.7 μM NAA.

In vitro regeneration from leaf explants of Neoregelia cruenta (R. Graham) L.B. Smith, an endemic bromeliad from Eastern Brazil

An efficient plant regeneration system was developed for the induction of direct shoot formation from leaves derived from seedlings of Neoregelia cruenta, an endemic Bromeliaceae of Eastern Brazil. Shoot differentiation occurred directly from the leaf bases. In vitro responses were influenced by seedling age and growth regulator combinations. Highest regeneration rates were obtained from explants excised from 7-week-old seedlings cultured in the presence of 22 μM BA and 2.5 μM NAA. Shoot conversion to whole plants was most effective in shoots formed in response to 4.4 or 8.8 μM BA combined with 2.5 μM NAA.

Regulation of transformation efficiency of peanut (Arachis hypogaea L.) explants by Agrobacterium tumefaciens

Abstract Some of the factors that modulate the transformation efficiency of regenerable tissues of peanut (Arachis hypogaea L.) were examined, using a tumor induction system based on strain A281 harbouring a binary vector. Factors examined were the composition of media, the cocultivation period, tissue type, bacterial inoculum concentration and pretreatment with acetosyringone (AS). Transformation efficiency was greatest when leaf or cotyledon explants were cocultivated on solid rather than liquid medium for 48 h. Highest transformation rates were observed on leaves from 7–10-day-old seedlings, with inoculum densities of 109-5 × 109 cells/ml. Addition of AS to the bacterial culture prior to inoculation did not alter transformation frequency.

Gene transfer into peanut (Arachis hypogaea L.) by Agrobacterium tumefaciens

Introduction of foreign genes into plant tissues via Agrobacterium tumefaciens based vectors requires specific knowledge of Agrobacterium-host compatibility. Therefore, to develop a transformation protocol for peanut (Arachis hypogaea L.), five Brazilian cultivars were screened with four wild-type A.tumefaciens strains. Successful transformation was dependent on specific bacterial strain-plant cultivar interactions and strain A281 was the most effective for tumor induction. Tumors displayed hormone autonomous growth, were opine positive and contained DNA that was homologous to the T-DNA of the inciting strain. Tumors induced on seed and seedling explants by A281 (pTD02) also expressed the reporter genes gus and npt-II contained in the binary vector. These results show that peanut is a permissive host for the acceptance of genes from specific A.tumefaciens gene vectors.