Microcallus formation from leaf mesophyll protoplasts in the genus Actinidia Lindl

Summary Microcallus (more than 60 cells) formation was obtained from leaf mesophyll protoplasts of 6 species and varieties in the genus Actinidia Lindl. (kiwifruit). The best results were achieved by using liquid over agarose culture for A. arguta var. arguta, liquid and agarose disc type culture for A. arguta var. purpurea, agarose disc type culture for A. arguta cv. Issaï and A. deliciosa and liquid agarose bead type- and disc type culture for A. kolomikta and A. polygama. Several factors influencing purification, browning, survival and sustained division of the protoplasts are briefly discussed.Abbreviations BAP benzylaminopurine - CPW cell and protoplast washing solution - 2,4-D dichlorophenoxyacetic acid - GA3 gibberellic acid - IAA indole-3-acetic acid - MES 2-(N-morpholino) ethanesulfonic acid - NAA 1-naphthaleneacetic acid - PVP polyvinylpyrrolidone - BT agarose bead type culture - DT agarose disc type culture     

Benzoate-driven dehalogenation of chlorinated ethenes in microbial cultures from a contaminated aquifer

Microbial dehalogenation of tetrachloroethene (PCE) and cis-dichloroethene (cis-DCE) was studied in cultures from a continuous stirred tank reactor initially inoculated with aquifer material from a PCE-contaminated site. Cultures amended with hydrogen and acetate readily dechlorinated PCE and cis-DCE; however, this transformation was incomplete and resulted in the accumulation of chlorinated intermediates and only small amounts of ethene within 60 days of incubation. Conversely, microbial PCE and cis-DCE dechlorination in cultures with benzoate and acetate resulted in the complete transformation to ethene within 30 days. Community fingerprinting by denaturing gradient gel electrophoresis (DGGE) revealed the predominance of phylotypes closely affiliated with Desulfitobacterium, Dehalococcoides, and Syntrophus species. The Dehalococcoides culture VZ, obtained from small whitish colonies in cis-DCE dechlorinating agarose cultures, revealed an irregular cell diameter between 200 and 500 nm, and a spherical or biconcave disk-shaped morphology. These organisms were identified as responsible for the dechlorination of cis-DCE to ethene in the PCE-dechlorinating consortia, operating together with the Desulfitobacterium as PCE-to-cis-DCE dehalogenating bacterium and with a Syntrophus species as potential hydrogen-producing partner in cultures with benzoate. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Improved protocols for transformation of indica rice mediated by Agrobacterium tumefaciens

A highly efficient gene transfer method mediated by Agrobacterium tumefaciens was developed for Group I indica rice, which had been quite recalcitrant in tissue culture and transformation. Freshly isolated immature embryos from plants grown in a greenhouse were inoculated with A. tumefaciens LBA4404 that harbored super-binary vector pTOK233 or pSB134, which had a hygromycin-resistance gene and a GUS gene in the T-DNA. The efficiency of gene transfer varied with the kinds of gelling agents and the basic compositions of co-cultivation media. The highest activity of GUS after co-cultivation was observed when NB medium solidified with agarose was used. For the subsequent cultures, two types of media (modified NB and CC) were chosen to recover hygromycin-resistant cells efficiently. The transformation protocol thus developed worked very well in all of the varieties tested in this study, and the transformation frequency (number of independent hygromycin-resistant and GUS-positive plants per embryo) reached more than 30% in IR8, IR24, IR26, IR36, IR54, IR64, IR72, Xin Qing Ai 1, Nan Jin 11, and Suewon 258. Most of the transformants (T₀) were normal in morphology and fertile. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the T₀ and T₁ generations. For the recovery of multiple independent transgenic events from a single immature embryo, procedures were developed to section the embryo into as many as 30 pieces after non-selective cultures following co-cultivation. Transformants were then obtained from the pieces cultured on the selective media, and, in the highest case, more than seven independent transgenic plants per original embryo (transformation frequency of 738%) were produced. Thus, the efficiency of transformation was remarkably improved.     

Supporting matrix influences protoplast-derived colony formation: Structural analysis

Summary Flax (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.     

l-Malic acid formation by immobilized Saccharomyces cerevisiae amplified for fumarase

The yeast Saccharomyces cerevisiae was amplified for the enzyme fumarase by cloning the single nuclear gene downstream of a strong promoter. The overproducing strain converted fumaric acid to l-malic acid at a rate of 65 mM g⁻¹ h⁻¹ in free cell experiments, and approximately 87% of the fumaric acid was converted to l-malic acid within 45 min. Activity was dependent on the addition of surfactant to the medium, and minimal activity was seen with the wild-type yeast strain. The constructed strain was immobilized in agarose beads (2.4 mm mean diameter) and within agarose microspheres (193 and 871 μm mean diameter). The rate of bioconversion increased with decreasing bead diameter, with similar rates observed with the 193-μm diameter microspheres to that achieved with the free cells. The presence of surfactant was essential for initial activity of the immobilized cells; however, high activity was observed in subsequent experiments in the absence of surfactant. Stable activities over a 48-h period were maintained within the large-diameter agarose beads, while decreasing activities were observed within the agarose microspheres.     

Inorganic nutrition in pond cultivated Gracilaria conferta (Rhodophyta): nitrogen, phosphate and sulfate

The experience gained in the outdoor cultivation system of Gracilariaconferta in Israel suggests three main inorganic variables: inorganic carbon,macronutrients and micronutrients. The effects of the macronutrientsnitrogen, phosphate and sulfate on growth rate, dry weight yield, and agaryield and quality were assessed in this research. The starvation periodbetween ammonium pulse feedings affected the agar yield and its quality asa function of the water temperature. Phosphate was the secondmacronutrient in the usual 1:10 proportion. Dark starvation as well assulfate starvation decreased agar yield. The results of nitrogen or sulfatestarvation suggest a biphasic process of agar biosynthesis. Starting with anoverall growth stage richer with sulfated agarose, and turning into an agaraccumulation stage with more neutral agarose. The elucidation of theintegrated effects of starvation and seasonality on agar production of Gracilaria is the major contribution of this research.     

Stable transformation of sugarbeet protoplasts by electroporation

Conditions were optimized for the culture, antibiotic selection and stable transformation by electroporation of suspension culture protoplasts of sugarbeet,Beta vulgaris L.. Highest plating efficiencies (up to 65% at day 21) were obtained if protoplasts were cultured in PGO salts (de Greef and Jacobs, 1979) supplemented with 0.1 mg/1 2,4-D, 0.01 mg/l BAP and 9% mannitol, and in 0.6% agarose rather than in liquid medium. Sensitivity to kanamycin also depended on whether protoplasts were cultured in liquid or agarose medium. Stable transformation of protoplast-derived colonies, as determined by resistance to kanamycin and Southern blot analysis, was achieved by electroporation using both rectangular and exponentially-decaying pulses.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - npt-II neomycin phosphotransferase - EDTA ethylenediaminetetracetic acid - SDS sodium dodecyl sulphate     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of Perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Agrobacterium tumefaciens-mediated transformation system for perilla (Perilla frutescens Britt) was developed. Agrobacterium strain EHA105 harboring binary vector pBK I containing bar and γ-tmt cassettes or pIG121Hm containing nptII, hpt, and gusA cassettes were used for transformation. Three different types of explant, hypocotyl, cotyledon and leaf, were evaluated for transformation and hypocotyl explants resulted in the highest transformation efficiency with an average of 3.1 and 2.2%, with pBK I and pIG121Hm, respectively. The Perilla spp. displayed genotype-response for transformation. The effective concentrations of selective agents were 2 mg l⁻¹ phosphinothricin (PPT) and 150 mg l⁻¹ kanamycin, respectively, for shoot induction and 1 mg l⁻¹ PPT and 125 mg l⁻¹ kanamycin, respectively, for shoot elongation. The transformation events were confirmed by herbicide Basta spray or histochemical GUS staining of T₀ and T₁ plants. The T-DNA integration and transgene inheritance were confirmed by PCR and Southern blot analysis of random samples of T₀ and T₁ transgenic plants.     

Protoplast production fromPorphyra linearis using a simplified agarase procedure capable of commercial application

Abalone enzymes, Cellulase R-10, Macerozyme and agarase fromPseudomonas atlantica were tested for activity on agarose, cellulose, xylan, the cell wall matrix and porphyran isolated fromPorphyra linearis. Agarase, and to a lesser extent Macerozyme, digested both agarose and porphyran. Abalone enzymes and Cellulase R-10 reacted only weakly with porphyran. A simple standardized protocol for making protoplasts fromPorphyra linearis was developed using 0.025% agarase in seawater without added organic osmoticants. Protoplasts prepared with agarase remained viable for at least 24 h in the digestion medium. Regeneration of the protoplasts followed the normal pattern for this species. Agarase can be used to obtain large number of protoplasts which could substitute for conchospores in seeding nets for the aquaculture ofP. linearis Author for correspondenceIssued as NRCC no. 34897     

Extraction of high-molecular-weight DNA from dry root tissue of<Emphasis Type="Italic">Berberis lycium</Emphasis> suitable for RAPD

A simple protocol for DNA isolation from dry roots ofBerberis lycium is described. Four-year-old dry roots are used, and the isolated DNA is suitable for analysis by means of restriction enzyme digestion and random amplification of polymorphic DNA (RAPD). The method involves a modified CTAB procedure using 1% PVP to remove polysaccharides and purification using low-melting-temperature agarose. DNA is amplified by means of PCR using 10-mer random primers from Operon Biotechnologies, Inc. (USA), and DNA samples are digested withTaq I,Hind III andEcoR I and examined on agarose gels. The RAPD reaction is performed according to the 1990 protocol by Williams et al.     

Plant regeneration from cotyledon protoplasts of Brassica carinata

Protoplasts isolated from cotyledons of Brassica carinata, underwent sustained division when cultured at 5.0 × 10⁴ ml^-1 in modified 8p medium (KM8P) with 1.0% (w/v) Seaplaque agarose. Cell colonies produced callus when agarose droplets, in which the protoplasts were embedded, were transferred to K8 medium with 0.6% (w/v) Sigma Type I or Type VII agarose at day 16, giving a plating efficiency of 1.6%. Seventy percent of the protoplast derived-tissues produced shoot buds after subculture to MS medium containing 3.0% (w/v) sucrose, 1.125 mgl^-1 BAP, 0.035 mgl^-1 GA and 0.6% (w/v) Type I agarose, resulting in shoot formation from 1.1% of the protoplasts originally plated. Protoplast-derived colonies transferred to hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose produced roots. The latter gave rise to shoots after excision from the parent callus and culture on MS medium with 3.0% sucrose, 0.225 mgl^-1 BAP, and 0.6% (w/v) Type I agarose. Shoots regenerated directly from protoplast-derived calli, or indirectly from roots, developed prolific root systems when placed on hormone-free MS medium with 1.0% (w/v) sucrose and 0.6% (w/v) Type I agarose.Abbreviations BAP 6-benzylaminopurine - CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - GA gibberellic acid - K kinetin - NAA -naphthaleneacetic acid - MES 2(N-morpholino)ethanesulphonic acid, 2,iP-6(,-dimethylallyamino) purine - IAA indole-3-acetic acid - Z zeatin - ZR zeatin riboside     

Piercing and vacuum infiltration of the mature embryo: a simplified method for <Emphasis Type="Italic">Agrobacterium-</Emphasis>mediated transformation of <Emphasis Type="Italic">indica</Emphasis> rice

Traditional transformation methods are complex and time consuming. It is generally difficult to transform indica rice varieties using traditional transformation methods due to their poor regeneration. In this contribution, a simple method was developed for the transformation of indica rice. In this method, the mature embryos of soaked seeds were pierced by a needle, and then soaked in the Agrobacterium inoculum under vacuum infiltration. The inoculated seeds germinated and grew to maturation (T ₀) under nonsterile conditions. The herbicide or antibiotic analysis and molecular analysis were conducted on T ₀ plants. The results showed that although the efficiency of transformation was about 6.0%, it was easier to transform indica rice using the proposed method, and the transformation process was significantly shortened. The success of transformation was further confirmed by the genetic and molecular analyses of T ₁ transformants.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

Improvement of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in Hi-II maize (<Emphasis Type="Italic">Zea mays</Emphasis>) using standard binary vectors

High-frequency transformation of maize (Zea mays L.) using standard binary vectors is advantageous for functional genomics and other genetic engineering studies. Recent advances in Agrobacterium tumefaciens-mediated transformation of maize have made it possible for the public to transform maize using standard binary vectors without a need of the superbinary vector. While maize Hi-II has been a preferred maize genotype to use in various maize transformation efforts, there is still potential and need in further improving its transformation frequency. Here we report the enhanced Agrobacterium-mediated transformation of immature zygotic embryos of maize Hi-II using standard binary vectors. This improved transformation process employs low-salt media in combined use with antioxidant l-cysteine alone or l-cysteine and dithiothreitol (DTT) during the Agrobacterium infection stage. Three levels of N6 medium salts, 10, 50, and 100%, were tested. Both 10 and 50% salts were found to enhance the T-DNA transfer in Hi-II. Addition of DTT to the cocultivation medium also improves the T-DNA transformation. About 12% overall and the highest average of 18% transformation frequencies were achieved from a large number of experiments using immature embryos grown in various seasons. The enhanced transformation protocol established here will be advantageous for maize genetic engineering studies including transformation-based functional genomics.     

Floral Spray Transformation Can Efficiently Generate Arabidopsis

In this study, floral spray and floral dip were used to replace the vacuum step in the Agrobacterium-mediated transformation of a superoxide dismutase (SOD) gene into Arabidopsis. The transgene was constructed by using a CaMV 35S promoter to drive a rice cytosolic CuZnSOD coding sequence in Arabidopsis. The transgene construct was developed in binary vectors and mobilized into Agrobacterium. When Arabidopsis plants started to initiate flower buds, the primary inflorescence shoots were removed and then transformed by floral spray or floral dip. More than 300 transgenic plants were generated to assess the feasibility of floral spray used in the in planta transformation. The result indicates that the floral spray method of Agrobacterium can achieve rates of in planta transformation comparable to the vacuum-infiltration and floral dip methods. The floral spray method opens up the possibility of in planta transformation of plant species which are too large for dipping or vacuum infiltration.     

Factors influencing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of monocotyledonous species

Summary Since the success of Agrobacterium-mediated transformation of rice in the early 1990s, significant advances in Agrobacterium-mediated transformation of monocotyledonous plant species have been achieved. Transgenic plants obtained via Agrobacterium-mediated transformation have been regenerated in more than a dozen monocotyledonous species, ranging from the most important cereal crops to ornamental plant species. Efficient transformation protocols for agronomically important cereal crops such as rice, wheat, maize, barley, and sorghum have been developed and transformation for some of these species has become routine. Many factors influencing Agrobacterium-mediated transformation of monocotyledonous plants have been investigated and elucidated. These factors include plant genotype, explant type, Agrobacterium strain, and binary vector. In addition, a wide variety of inoculation and co-culture conditions have been shown to be important for the transformation of monocots. For example, antinecrotic treatments using antioxidants and bactericides, osmotic treatments, desiccation of explants before or after Agrobacterium infection, and inoculation and co-culture medium compositions have influenced the ability to recover transgenic monocols. The plant selectable markers used and the promoters driving these marker genes have also been recognized as important factors influencing stable transformation frequency. Extension of transformation protocols to elite genotypes and to more readily available explants in agronomically important crop species will be the challenge of the future. Further evaluation of genes stimulating plant cell division or T-DNA integration, and genes increasing competency of plant cells to Agrobacterium, may increase transformation efficiency in various systems. Understanding mechanisms by which treatments such as desiccation and antioxidants impact T-DNA delivery and stable transformation will facilitate development of efficient transformation systems.     

Easy determination of ploidy level in Arabidopsis thaliana plants by means of pollen size measurement

Summary Cytogenetic examination of transgenic Arabidopsis thaliana (L.) Heynh. plants obtained by Agrobacterium-mediated gene transfer to cotyledon- and root-explants or by direct gene transfer into protoplasts revealed a high percentage of tetraploid or aneuploid transformants. Depending on the transformation procedure used, 13% (root explant transformation), 33% (cotyledon explant transformation), or 38% (direct gene transfer) of the transformants showed aberrant ploidy levels. A good correlation between the ploidy level of a plant and the size of its pollen grains was observed. This allows quick and simple testing of the ploidy level of transgenic Arabidopsis plants.Abbreviations AM Arabidopsis medium - ANOVA analysis of variance - DAPI 4,6-Diamidino-2-phenylindole - PEG polyethyleneglycol     

Agars from Chilean Gelidiaceae

Cystocarpic, tetrasporic and vegetative thalli of Gelidium chilense were extracted with water at 95 °C. The contents of 3,6-anhydro-galactose and sulfate group of the hydrocolloids correspond to those of an agar-type polysaccharide. The percentages of 6-O-methyl-galactose and of pyruvic acid ranged between 5.7–6.2% and 0.42–0.54%, respectively. The gel melting and gelation temperatures of Gelidium chilense, G. rex and G. lingulatum agars were determined. A correlation between 6-O-methylgalactose content and gelation temperatures was not observed. It was found by anion-exchange chromatography that 19.8% of tetrasporic and 4.9% of vegetative G. chilense agars are unsulfated polymers. Structural studies on the neutral fraction from tetrasporic G. chilense agar by partial hydrolysis and ¹H NMR spectroscopy have shown that it is mainly composed of agarose. Methylation analysis, oxidative hydrolysis and partial hydrolysis, followed by ¹H NMR spectroscopy, have shown that the neutral fraction of the agar from tetrasporic Gelidium rex is agarose. The results obtained in this work are compared with previously reported data on studies of agars from Chilean Gelidium species.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3

Brachypodium distachyon (Brachypodium) is a small grass with biological attributes (rapid generation time, small genome, diploid accessions, small stature and simple growth requirements) that make it suitable for use as a model system. In addition, a growing list of genomic resources have been developed or are currently under development including: cDNA libraries, BAC libraries, EST sequences, BAC end sequences, a physical map, genetic markers, a linkage map and, most importantly, the complete genome sequence. To maximize the utility of Brachypodium as a model grass it is necessary to develop an efficient Agrobacterium-mediated transformation system. In this report we describe the identification of a transformable inbred diploid line, Bd21-3, and the development of a transformation method with transformation efficiencies as high as 41% of co-cultivated calluses producing transgenic plants. Conducting the co-cultivation step under desiccating conditions produced the greatest improvement in transformation efficiency.