Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium.

A procedure has been established for Agrobacterium tumefaciens-mediated genetic transformation of Hevea brasiliensis embryogenic friable calli. Precultivation of tissues on a CaCl(2)-free maintenance medium dramatically enhanced the transient activity of the reporter gene, gusA encoding beta-glucuronidase (GUS). The increase was first noticed in highly active cells (undifferentiated or/and embryogenic), in tissues precultured for 2-8 weeks. Beyond 8 weeks of preculture, GUS activity increased again, but this time in tissues consisting of differentiated cells accumulating polyphenols. Out of five Agrobacterium strains cocultivated with CaCl(2)-free precultured tissues, only inoculation with EHA105pC2301 led to high transient GUS activity. Paromomycin proved more effective than kanamycin for the selection of transformed cells, as it inhibits the growth of non-transformed cells more radically. Five paromomycin-resistant callus lines were established. The presence of gusA and neomycin phosphotransferase ( nptII) genes in the plant genome was confirmed by DNA amplification, and by Southern hybridization. These results confirmed that A. tumefaciens is an effective system for mediating stable transformation of rubber tree calli with a low copy number of transgenes. Transgenic callus lines constitute a useful tool for studying genes of interest on a cellular level and for regenerating transgenic rubber trees.

Histological analysis of indirect somatic embryogenesis in the Marsh clubmoss Lycopodiella inundata (L.) Holub (Pteridophytes).

An efficient in vitro plant regeneration method was developed for Lycopodiella inundata (L.) Holub, an endangered medicinal Lycopod (Pteridophytes). Vegetative apices were used as explant material. Nodular calluses were established after three cycles (13 weeks each) on a medium containing a few minerals and organic compounds and supplemented with 0.05 µM 3-indolebutyric acid (IBA) and 1.4 µM kinetin (Kin). Propagation was achieved every 13 weeks on this callus medium (CM). When nodular calluses were transferred on a medium supplemented with 2.5 µM IBA and 0.33 µM gibberellic acid (GA(3)) designated as embryogenic medium (EM), organized structures appeared and developed into plantlets. Development phases were characterized by histological studies. Some phases of zygotic embryogenesis previously described for Lycopods were observed in L. inundata. Histological analyses established that an indirect somatic embryo was derived from a single embryogenic cell by following the zygotic developmental pathway. As this phenomenon has not previously been reported in Lycopods, a comparison between somatic and zygotic embryos is discussed based upon morphology and histology.

Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli.

The influence of CaCl2 was investigated on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis friable calli which are usually proliferated on maintenance medium (MM) containing 9 mM CaCl2.Five A. tumefaciens strains (C58pMP90, C58pGV2260, AGL1, LBA4404 and EHA 105) and two binary vectors (pGIN and pCAMBIA2301) were tested and the strain EHA105pC2301 was selected to conduct further experiments. The calli were precultured on MM containing a range of CaCl2 concentrations, then inoculated with Agrobacterium suspension. Transfer of friable calli from MM containing 9 mM CaCl2 to calcium-free medium significantly enhanced the transient ß-glucuronidase activity. Interestingly, the use of calcium-free Agrobacterium resuspension medium to inoculate friable calli again dramatically increased the transformation efficiency. Induction of Agrobacterium's virulence with acetosyringone remained an important factor to stimulate transformation.

Root system architecture and gravitropism in the oil palm.

The oil palm (Elaeis guineensis Jacq.) has a root system consisting of primary (or order 1) roots, which are either orthogravitropic (R1 VD, with positive gravitropism) or diagravitropic (R1 H). Their statenchyma have very similar characteristics (mainly vacuolated, large cells). However, their statoliths sediment along the longitudinal wall in R1 H and along the distal wall in R1 VD (furthest cell wall from the apical meristem, opposite the proximal wall). Order 2 roots may have vertical upward (R2 VU) or downward growth (R2 VD) or even horizontal growth (R2 H). In all cases, the statoliths are located near the lower wall of the statocyte (distal in R2 VD, proximal in R2 VU and longitudinal in R2 H). Order 3 roots are usually agravitropic. When they grow upwards, R3 VU, their amyloplasts are located near the proximal wall. Likewise, the growth direction of R4 varies, but they have little or no statolith sedimentation. Roots with marked gravitropism (positive or negative) have amyloplasts that can sediment along different walls. But, irrespective of amyloplast position in the statocytes, the direction of root growth may be stable. The relation between the different reactions of roots and different sensitivity to auxin or to a curvature-halting signal is discussed.

Cloning of a wheat puroindoline gene promoter by IPCR and analysis of promoter regions required for tissue-specific expression in transgenic rice seeds.

A genomic DNA fragment containing the 5'-upstream sequence and part of the open reading frame corresponding to Triticum aestivum puroindoline-b cDNA, was isolated by inverse PCR. Promoter fragments extending to -1068, -388, -210 or -124 upstream of the translation initiation ATG codon and the sequence coding for the first 13 amino acids of the puroindoline-b, were translationally fused to the uidA reporter gene encoding beta-glucuronidase and transferred to rice calli via particle bombardment-mediated transformation. The 1068 bp and 124 bp promoters were also transcriptionally fused to the uidA reporter gene. Out of the 196 plants regenerated from transformed rice calli, 118 plants set seeds. No GUS activity was detectable in the stems, roots, leaves or pollen of the transgenic rice which had integrated the puroindoline-b promoter or its deletions; GUS activity was detected only in seeds, except in those having integrated the 124 bp promoter. Within seeds, histological localisation showed GUS activity as being restricted to the endosperm, aleurone cells and pericarp cell layers; no GUS activity was detected in the embryonic axis. Analysis of 5' promoter deletions identified the region between -388 and -210 as essential for endosperm expression, and the region between -210 and -124 as essential for expression in the epithelium of the scutellum. No difference of expression was observed between the translational and transcriptional fusion genes.

Identification and disruption of a plant shaker-like outward channel involved in K+ release into the xylem sap.

SKOR, a K+ channel identified in Arabidopsis, displays the typical hydrophobic core of the Shaker channel superfamily, a cyclic nucleotide-binding domain, and an ankyrin domain. Expression in Xenopus oocytes identified SKOR as the first member of the Shaker family in plants to be endowed with outwardly rectifying properties. SKOR expression is localized in root stelar tissues. A knockout mutant shows both lower shoot K+ content and lower xylem sap K+ concentration, indicating that SKOR is involved in K+ release into the xylem sap toward the shoots. SKOR expression is strongly inhibited by the stress phytohormone abscisic acid, supporting the hypothesis that control of K+ translocation toward the shoots is part of the plant response to water stress.

Ribosomal RNA synthesis in imbibing radish (Raphanus sativus) embryo axes : A biochemical and cytological study.

The first hours of seed germination are characterized by an increase in the rate of RNA synthesis. Although this change is most easily accounted for by changes in the ribonucleotide pool sizes, we investigated two other aspects of rRNA synthesis which are likely to contribute to the phenomenon. Using isolated radish embryo axes, we demonstrate that processing of rRNA gene transcripts is much slower during early germination than during the growth of the seedling. We also provide evidence that rRNA gene expression is sequentially reactivated in different tissues, starting in the provascular tissue and apex cells and only later in the cortical cells of the rootlet.