Novel dye for detection of callus embryo by confocal laser scanning fluorescence microscopy.

In the present study a new luminescent dye 3-N-(2-pyrrolidinylacetamido)benzanthrone (AZR) was synthesized. Spectroscopic measurements of the novel benzanthrone 3-aminoderivative were performed in seven organic solvents showing strong fluorescence. The capability of the prepared dye for visualization has been tested on flax, red clover and alfalfa to determinate the embryo in plant callus tissue cultures. Callus cells were stained with AZR and further analysed utilizing confocal laser scanning fluorescence microscopy. Performed experiments show high visualization effectiveness of newly synthesized fluorescent dye AZR that is efficient in fast and relatively inexpensive diagnostics of callus embryos that are problematic due to in vitro culture specificity.

Identification of a potential bottleneck in branched chain fatty acid incorporation into triacylglycerol for lipid biosynthesis in agronomic plants.

In plant, unusual fatty acids are produced by a limited number of species. The industrial benefits of these unusual structures have led several groups to study their production in transgenic plants. Their research results led to very modest accumulation in seeds which was largely due to a limited knowledge of the lipid metabolism and fatty acid transfer in plants. More specifically we need to better understand the substrate specificity and selectivity of acyltransferases which are required for the incorporation of these unusual fatty acids into storage triacylglycerols. In our studies we have compared the incorporation of [(14)C] Oleoyl-CoA and Branched Chain Acyls-CoA into [(3)H] LPA-C18:1 by the Lysophosphatidic acid Acyltransferase (LPAAT) from developing seeds of agronomic plants (flax (Linum usitatissimum) and rape (Brassica napus)) and from a plant capable of producing high amounts of hydroxy fatty acids (castor bean (Ricinus communis)). Our assays demonstrate that LPAATs of the three studied species (1) incorporated preferentially oleyl-CoA, (2) could incorporate cyclopropane acyl-CoA when added alone as a substrate, however very weakly for rapeseed and castor bean seeds, (3) presented a low capacity to incorporate methyl branched acyl-CoA when added alone as a substrate (4) weakly incorporated cyclopropane acyl-CoA and was unable to incorporate methyl branched acyl-CoA when presented with an equimolar mix of oleyl-CoA and branched chain acyl-CoA. In all cases, the LPAAT had a low affinity for branched chain acyl-CoAs. The results show that LPAAT activity from agronomic plants constitutes a bottleneck for the incorporation of branched Chain acyl-CoA into PA.

In vivo 13C NMR determines metabolic fluxes and steady state in linseed embryos.

The dynamics of developing linseed embryo metabolism was investigated using (13)C-labelling experiments where the real-time kinetics of label incorporation into metabolites was monitored in situ using in vivo NMR. The approach took advantage of the occurrence in this plant tissue of large metabolite pools - such as sucrose or lipids - to provide direct and quantitative measurement of the evolution of the labelling state within central metabolism. As a pre-requisite for the use of steady state flux measurements it was shown that isotopic steady state was reached within 3 h at the level of central intermediates whereas it took a further 6h for the sucrose pool. Complete isotopic and metabolic steady state took 18 h to be reached. The data collected during the transient state where label was equilibrated but the metabolic steady state was incomplete, enabled the rates of lipid and sucrose synthesis to be measured in situ on the same sample. This approach is suitable to get a direct assessment of metabolic time-scales within living plant tissues and provides a valuable complement to steady state flux determinations.

Pinoresinol-lariciresinol reductase gene expression and secoisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds.

The transcription activity of the pinoresinol-lariciresinol reductase (PLR) gene of Linum usitatissimum (so-called LuPLR), a key gene in lignan synthesis, was studied by RT-PCR and promoter-reporter transgenesis. The promoter was found to drive transcription of a GUSint reporter gene in the seed coats during the flax seed development. This fitted well with the tissue localization monitored by semi-quantitative RT-PCR of LuPLR expression. Accumulation of the main flax lignan secoisolariciresinol diglucoside was coherent with LuPLR expression during seed development. This three-way approach demonstrated that the LuPLR gene is expressed in the seed coat of flax seeds, and that the synthesis of PLR enzyme occurs where flax main lignan is found stored in mature seeds, confirming its involvement in SDG synthesis.

The onset of gravisensitivity in the embryonic root of flax.

Vertical orientation of emerging roots typically is the first response of plants to gravity. Although root gravitropism has been studied extensively, no conclusive data on the onset of gravisensing exist. We determined the inception of gravisensitivity in flax (Linum usitatissimum) roots by clinorotating germinating seeds after various periods of static orientation (gravistimulation) of imbibed seeds. Gravitropic competency was established about 8 h after imbibition, 11 h prior to germination. The time was determined based on 50% of the newly emerged roots curving in the direction of the gravity vector during static imbibition, despite subsequent clinorotation. The threshold value was affected by the orientation of the seeds. Upward orientation of the micropyle/radicle reduced the number of graviresponding roots to about one-half. Prolonged clinorotation weakened the graviresponse. Gravisensing was accompanied by the development of amyloplasts, but the actin cytoskeleton was not involved because imbibition in Latrunculin B did not affect the onset of gravisensitivity or germination, and the development of F-actin in untreated controls was observed only after the onset of gravisensitivity.

Histological study of embryo-like structures initiated from hypocotyl segments of flax (Linum usitatissimum L.).

Cultivation of flax hypocotyl segments on MS medium supplemented with auxin (2,4-D, NAA) and combination of auxin (NAA) and cytokinin (BAP, zeatin) resulted in production of callus on the cut ends of segments and prolonged cultivation in globular structures resembling early stages of somatic embryos. Embryo-like structures protruded on the surface directly from the subepidermal layers of hypocotyl segments. Despite these globular structures closely resembling somatic embryos, histological observations did not reveal their embryogenic character-organogenesis was the predominant developmental morphogenic pathway. Based on our experiments, as well as on critical revision of existing reports on flax somatic embryogenesis, we conclude, that there has not yet been convincing histological proof of somatic embyogenesis from flax hypocotyl segments.

[Effect of culture medium composition on callus formation and regeneration in oil flax anther culture]. / Vliianie sostava sredy na protsessy kallusogeneza i regeneratsii v kul'ture pyl'nikov l'na.

Regeneration ability in callus cultures from anthers of two hybrid genotypes of oil flax was studied on N6 and LMA-1 nutrient media at various concentrations of cytokinine 6-benzylamynopurine (BAP). It was shown that callus grew and developed better at BAP concentrations of 2 mg/l, comparing with 4 and 6 mg/l. Shoot and root regeneration was observed in F1 genotype 6-8-gnezdny x M22 only and did not depend on BAP concentration in the medium and on the medium composition itself. Transfer onto fresh medium often stimulated dedifferentiation of the regenerated structures.

Regeneration of flax ( Linum usitatissimum L.) plants from anther culture and somatic tissue with increased resistance to Fusarium oxysporum.

The aim of this study was to establish a protocol for the efficient production of flax plants of microspore origin. The results were compared to those obtained for plants regenerated from somatic explants from hypocotyls, cotyledons, leaves, stems and roots. All the plants obtained during the experiments were regenerated from callus that was grown for periods from a few weeks to a few months before the regeneration was achieved. Anther cultures were less effective in plant regeneration than somatic cell cultures. However, regenerants derived from anther cells showed valuable breeding features, including increased resistance to fungal wilt. The age of the donor plants and the season they grew in had a noticeable effect on their anther callusing and subsequent plant regeneration. Low temperature had a negative effect and dark pre-treatment a positive effect on callusing and plant regeneration. Different media were most effective for callus induction, shoot induction and rooting. For callus induction two carbon sources (2.5% sucrose and 2.5% glucose) were most effective; for shoots, only sucrose at lower concentration (2%) was effective. Rooting was most efficient in 1% sucrose and reduced (50%) mineral concentration in the medium. It was found that the length of in vitro cultivation significantly increases the ploidy and affects such features as regenerant morphological characteristics, petal colour, and resistance to Fusarium oxysporum-induced fungal wilt. The established plant regeneration system provides a basis for the creation of transgenic flax.

Biosynthetic pathway to the cancer chemopreventive secoisolariciresinol diglucoside-hydroxymethyl glutaryl ester-linked lignan oligomers in flax (Linum usitatissimum) seed.

Application of stable and radioisotope precursor/tracer experiments resulted in the identification of various phenylpropanoid, monolignol, and lignan metabolites involved in the biosynthesis of the cancer chemopreventive secoisolariciresinol diglucoside (SDG; 1)-containing ester-linked "polymer(s)" in flax (Linum usitatissimum) seed. Individual analysis of size-segregated flax seed capsules at five early stages of their development provided a metabolic profile of intermediates leading to "biopolymer" biosynthesis. The use of (1)H and (13)C NMR and HRMS analyses resulted in the identification of 6a-HMG (hydroxymethyl glutaryl) SDG (17) and 6a,6a'-di-HMG SDG (18) as the two major components of the ester-linked "biopolymer(s)". Based on metabolic tracer analyses and relative radioisotopic incorporations throughout each of these five stages of seed development, a biochemical pathway is proposed from phenylalanine to SDG (1), with subsequent mono- and di-substitutions of SDG (1) with HMG CoA. These metabolites then serve as precursors for formation of the SDG-HMG ester-linked oligomers. Results from this study will facilitate future isolation and characterization of the proteins and enzymes involved in biosynthesis of the SDG-HMG ester-linked oligomers in flax seed.