Alain David

Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis

SummaryCallus production along with caulogenesis was obtained from leaf explants of micropropagated clonal Eucalyptus grandis after six to twelve weeks of culture. Out of eight clones tested, six were amenable to shoot production using simple media containing naphthaleneacetic acid and either 6-benzyladenine or zeatin. Differences in growth regulator requirements for organogenesis were observed between different clones. These shoots were then elongated on a medium containing gibberellic acid and rooted using media derived from the micropropagation medium. Light conditions were also found to be important for regeneration. This protocol is the first published on regeneration from nonseedling material and it will facilitate the Agrobacterium tumefaciens -mediated transformation of selected clonal Eucalyptus grandis.

A Basic Chitinase-like Protein Secreted by Embryogenic Tissues of Pinus caribaea acts on Arabinogalactan Proteins Extracted from the same Cell Lines

Summary Embryogenic cell lines of Pinus caribaea consist of a high number of somatic embryos. We have previously characterized the embryogenic state by studying the proteins and glycoproteins ionically bound to the cell surfaces of preglobular somatic embryos. The embryogenic tissues and nonembryogenic calli produce proteins of 48 kDa or 56 and 25 kDa, respectively. All of these proteins cross-react with several classes of tobacco chitinases (CHs). These CH-like proteins express a potential chitinase activity on SDS-PAGE gels overlaid with glycol chitin as a synthetic substrate. When an arabinogalactan protein (AGP) fraction from embryogenic tissues substitutes for glycol chitin on gels, only the 48 kDa embryo-specific CH-like protein acts on this substrate, indicating that an interaction between this protein and a specific set of AGPs might exist within embryogenic tissues of Carribean pine.

Recovery of plants from cryopreserved embryogenic cell suspensions of Pinus caribaea

SummaryEmbryogenic cell suspension cultures of Pinus caribaea var. hondurensis have been cryopreserved in liquid nitrogen for up to four months, using sucrose and dimethylsulfoxide as cryoprotectants. Post-thaw growth was obtained after a short lag phase. Removal of the remaining liquid around the cells using a filter disc favoured subsequent regrowth of the cells. These reestablished cultures maintained an embryogenic potential similar to non-frozen cultures. The embryos produced were able to regenerate into plants, which are now growing in a greenhouse.

cDNA sequence, genomic organization and differential expression of three Arabidopsis genes for germin/oxalate oxidase-like proteins

Wheat germin is a protein expressed during germination which possesses an oxalate oxidase activity. Germin-type oxalate oxidases have been extensively studied in monocotyledons (wheat and barley) where they are thought to have important functions for development, stress response and defence against pathogens. In contrast, almost nothing is known about the germin-like proteins found in dicotyledons, gymnosperms and myxomycetes. In this work, cDNA clones for three genes (ATGER1, ATGER2 and ATGER3) encoding germin-like proteins, initially characterized as expressed sequence tags (ESTs), from Arabidopsis thaliana cDNA libraries were further characterized. In addition, we isolated and sequenced a Brassica napus cDNA which was strongly homologous to the cDNA for ATGER1. Sequence analysis and secondary structure predictions of the proteins encoded by these cDNAs showed that they possess all the characteristic features of members of the germin family and of the germin/seed globulins/sucrose binding protein superfamily. Sequence comparisons and mapping demonstrated the existence of at least two different gene families in the A. thaliana genome encoding a minimum of three genes for germins. These three genes have been mapped in three different location on the Arabidopsis genome. By northern blot hybridizations we found that these genes are differentially regulated. ATGER1 was expressed during germination, like wheat germin, but also in leaves whereas ATGER2 transcripts were exclusively found in developing embryos, like wheat pseudo-germin. ATGER3 mRNAs were found in leaves and flowers and their abundance was shown to vary during the circadian cycle.

Cryopreservation does not affect the expression of a foreign sam gene in transgenic Papaver somniferum cells

Abstract Transgenic cell lines of Papaver somniferum have been obtained via Agrobacterium tumefaciens. Papaver somniferum is known to be genetically unstable in in vitro culture conditions. Cryopreservation at ultra-low temperature is an appropriate strategy for long-term preservation of germplasm. We have studied the effects of osmotic stress due to cryoprotectants during pretreatment and of storage at –196 °C on the stability and the expression of the foreign sam1 gene from Arabidopsis thaliana. We established that the integrity, transcription of the transgene and enzymatic activity of its product were not affected by cryopreservation in liquid nitrogen

Antisense transgenesis of Linum usitatissimum with a pectin methylesterase cDNA

Abstract A cDNA of a flax ( Linum usitatissimum ) pectin methylesterase (PME) gene, Lupme3 , was isolated by RACE-PCR. A partial sequence of this cDNA was inserted in antisense orientation downstream the cauliflower mosaic virus 35S promoter and introduced into the flax genome via Agrobacterium tumefaciens . Transgenic calli derived from the cocultivated explants were analysed for the antisense sequence expression, and for their PME activity as well as their degree of pectin methylesterification and level of bound cations in the cell wall. Expression of the antisense sequence was correlated with a decrease of sense transcripts and a decrease of the PME enzyme activity of cell extracts at pH 8.5. In transgenic cells, a slight increase of activity was observed at acidic pH (5.5), possibly due to a compensation phenomenon and a moderately basic isoform appeared on IEF of transgenic lines. A decrease of the bound potassium level was also noted.

Polyamine content and somatic embryogenesis in Papaver somniferum cells transformed with sam-1 gene

Summary Papaver somniferum calluses transformed with the sam-1 gene from Arabidopsis thaliana , which encodes a SAM-synthetase, were subcultured over a 4-year period. The stability of the expression as well as the level of SAM synthetase activity were evaluated in 5 transgenic cell lines (STSI, STSII, STSIII, STSIV, STSV) and in the control. All transgenic cell lines exhibited a level of SAM-synthetase activity higher than that of the control. The enhancement of the enzyme activity has been confirmed by Northern blot analyses. The level of polyamines (putrescine, spermidine, spermine and 1,3-diaminopropane) was also evaluated in cell lines cultured either in Linsmaier and Skoog medium containing growth regulators (LS) or on a hormone free medium (LSHF). Cell lines cultured in LS medium showed a variable yield of the polyamine content. Putrescine was the major polyamine. After transfer to a hormone free medium, all of the cell lines, except STSIV, were able to form embryo-like structures. In this condition, the polyamine level decreases about 4-fold, spermidine being the most abundant.

Pectins in walls of protoplast-derived cells imbedded in agarose and alginate beads

SummaryFlax hypocotyl protoplasts were embedded in agarose and alginate beads. The pectin molecules of the formed colonies were observed in electron microscopy using 2F4 antibody specific of a calcium-induced supramolecular conformation of homopolygalacturonic acid. Little pectin, mostly methylesterified, was present in agarose-entrapped colonies. The regenerating cells immobilized in alginate secreted much higher amounts of methylesterified pectins in their walls. De-esterification of the pectins was clearly seen after 6 days of culture. These results illustrate the importance of the external matrix on wall differentiation.

How plant regeneration from Mentha × piperita L. and Mentha × citrata Ehrh. Leaf protoplasts affects their monoterpene composition in field conditions

Summary A procedure to regenerate plants from leaf protoplasts of two micropropagated hybrid species of mint, Mentha × piperita L. and Mentha X citrata Ehrh., has been developed in order to determine whether the in vitro treatment could influence the monoterpene composition. Purified protoplasts were first plated in liquid media containing 3.5 μmol/L BA, 1.25 μmol/L zeatin, 2.5μmol/L NAA and 2.25 or 5.5μmol/L 2,4-D as growth regulators to induce initial divisions. In these conditions, high percentages of protoplast-derived cells divided, especially for M. × piperita clones (ADF ranging from 17 % to 31 % at day 6). Reduction of both medium osmolality and 2,4-D concentration resulted in a sustained development of microcalli for both species. Calli were then transferred onto solidified regeneration media. The first regenerated shoots of M. × piperita were observed 3 months after protoplast isolation on media containing 8.9μmol/L and 13.2μmol/L BA or 1.8μmol/L and 4.5 μmol/L TDZ. Regeneration frequency did not exceed 4%. Regeneration medium sequences were required for shoot regeneration on M. × citrata calli. At first, they were cultured on a 1.8 μmol/L TDZ containing-medium for 1 week. They were then transferred onto media supplemented with various concentrations of TDZ or BA. The highest frequencies of shoot regeneration were around 10 %. Shoot morphology was affected by TDZ for both species, but not by BA. In a field trial, the amount of menthone, menthol and carvone in the regenerated plants of M. × piperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of carbone levels in all protoplast-derived plants.

Supporting matrix influences protoplast-derived colony formation: Structural analysis

SummaryFlax (Linum usitatissimum) protoplasts were immobilized in agarose and in Ca-alginate, medium (MV) and high viscosity (HV) grades. Protoplast viability was markedly decreased, probably as a result of the entrapment procedure itself. On the other hand, mitotic activity of surviving protoplasts was not influenced by the type of matrix agarose or alginate HV grade. Light and electron microscopical observations revealed compact heterogeneous cell colonies in agarose surrounded by cells in a more or less advanced state of lysis. In Ca-alginate (MV and HV) cell colonies were compact and spherical with poorly vacuolated cells. In this matrix, cell walls were intensely stained and sinuous, separated from the plasma membrane by a large periplasmic space.