A liquid-medium-based protocol for rapid regeneration from embryogenic soybean cultures

Soybean [Glycine max (L.) Merrill] somatic embryos of the cultivar Jack underwent histodifferentiation in liquid Murashige and Skoog (MS) medium with 3% maltose, or according to the standard published procedure employing solidified MS media, permitting the recovery of an average of 8.1 and 3.9 embryos/mg of embryogenic tissue, respectively. Cotyledon-stage embryos that developed in liquid medium were ready for desiccation within 4 weeks, while the embryos from the standard procedure required a maturation step for an additional 4 weeks. Comparison of embryo development in MS medium with maltose or FN Lite-based medium without growth regulators and supplemented with maltose or an equimolar amount of sucrose revealed that sucrose promotes faster embryo histodifferentiation and maturation, and allows the recovery of up to 50% more mature, cotyledon-stage embryos within 3 weeks. The use of this liquid-medium-based protocol relative to the standard procedure led to a fourfold increase in the number of cotyledon-stage embryos recovered from other genotypes tested. In many cases, however, the percent germination was lower. Application of this new procedure also made it possible to harvest transgenic seed 9 months following biolistic bombardment, as compared to the 13 months required when the standard solid-medium-based protocol was used. Received: 1 December 1997 / Revision received: 27 April 1998 / Accepted: 20 May 1998     

Restoration of embryogenic competence in repeatedly subcultured carrot cell lines

Summary The production of somatic embryos from carrot suspension cultures invariably decreases through simple, repeated subculturing. Extracellular, concentrated compounds extracted from already established embryo culture not only recovered the embryogenic capability, but also accelerated the embryo production as much as two-fold (up to 1600 embryos/ml) compared with that of a control culture. Sugars, which were only a small portion of the total concentrate, were excluded as possible causative factors. It is likely that a protein fraction that is generated directly by competent, embryogenic cultures is important for the restoration of embryogenic potential.     

Comparative assessment of different approaches for obtaining terminally differentiated cell lines

The study of pathogenesis of muscle disorders needs an appropriate cell model. In the field of muscle research, there is no general cell line considered standard for studying muscular and neuromuscular diseases. Most cell lines are incapable of differentiation into a muscle lineage exhibiting morphological and physiological properties of mature muscle cells and can hardly be genetically modified. The goal of our study was to find an informative cell model of muscle differentiation suitable for examination of muscular pathogenesis in vitro. We assayed cultured human mesenchymal stem cells (MSCs), mature murine muscle fibers, and primary murine satellite cells. It was shown that MSCs had very low capacity for myogenic differentiation; they were able to differentiate only in the presence of C2C12 cells. Lentiviral transduction had a toxic effect in primary myofiber cultures, and positively transduced cells were unable to respond to electrical stimulation, i.e., were functionally inactive. Satellite cells proved to be the most appropriate cell model, since they were easily transduced with lentiviruses and rapidly formed myotubes in the differentiation medima. Functional analysis of induced myotubes showed their ability to react to electrical and chemical stimulation. The patch-clump technique showed the presence of potassium and calcium channels. Collectively, the results show that cultured satellite cells are the most promising cell line for further experiments to explore molecular pathways in muscle pathologies.     

A modified DNA isolation protocol for obtaining pure RT-PCR grade RNA

We provide a simple but very efficient method for RNA preparation from Saccharomyces cerevisiae based on a standard chromosomal DNA isolation protocol. The method yields DNA-free total RNA, including mRNA, rRNA, and tRNA but can easily be adjusted to considerably enrich low molecular weight RNAs, such as tRNAs and the small rRNA species (5S and 5.8S). The procedure was proven and validated by verification of cDNAs belonging to four different genes, two of which encoding polypeptides and two tRNA genes. Besides its simpleness, the method is further advantagous in terms of safety (omitting hazardous phenol) and cost efficiency.     

Arabidopsis thaliana: protocol for plant regeneration from protoplasts

We report a protocol for plant regeneration from leaf protoplasts of Arabidopsis thaliana ecotype Zürich. The protocol has been established in 1988 and has since been in routine use in our laboratory. Whereas recovery of proliferating protoplast-derived clones is routine, the success in plant regeneration from protoplast-derived clones is highly variable. In the hands of one of us (H.K.) average shoot regeneration frequency (% of calli regenerating at least one shoot) was ca. 60% and average plant regeneration frequency (% of calli yielding fertile plants in soil) was ca. 40%.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - NAA naphthaleneacetic acid - BAP benzylaminopurine - 2-iP 2-isopentenylaminopurine     

Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation

Summary Embryogenic cell lines of Picea abies are categorized into three groups (polar, solar, and undeveloped) based on the organization of the somatic embryos within the tissue and the ability of the somatic embryos to proceed through a maturation process when treated with ABA. The polar and the solar types consist of somatic embryos with densely packed embryonic regions subtended by vacuolated suspensors. Both types of tissue regenerate mature somatic embryos when treated with ABA. Almost all mature somatic embryos develop further into shoots or plantlets. The undeveloped type consists of somatic embryos comprised of only a few loosely aggregated cells in their embryonic regions. Mature somatic embryos were not observed with this tissue type.Abbreviations ABA cis-trans abscisic acid - A1 polar type - A2 solar type - B undeveloped type - BA benzyladenine - 2,4-D 2,4 di-chlorophenoxyacetic acid - LP von Arnolds medium (1987)     

Expression of the gus A gene in embryogenic cell lines of Sitka spruce following Agrobacterium-mediated transformation

Transformation of Picea sitchensis (Bong) Carr. was investigatedby incubating embryogenic cell lines, initiated from immatureand mature zygotic embryos, with a supervirulent strain of Agrobacteriumtumefaciens. The latter carried a gus A-intron gene. Transientgene expression was determined histochemically by recordingthe number of distinct areas of ß-glucuronidase (GUS)activity. Maximum expression of the gus gene was achieved witha bacterial suspension with an OD₆₀₀ of 0.8–1.1 dilutedwith an equal volume of MPM medium, Inoculation of cells withbacteria for 30 mm, 72 h co-cultivation period and exposureof Agrobacterium and plant cells to 50 µM acetosyrmngone.These results are discussed in relation to Agrobacterium-mediatedgene delivery for the stable transformation of Sitka spruceand other conifers. Key words: Sitka spruce, Agrobacterium, transformation, embryonal suspensor masses, GUS activity     

A new mutation protocol for obtaining auxotrophic mutants of the yeastPhaffia rhodozyma

Summary A new mutation strategy, which involves -irradiation of cells followed by a selective enzymatic enrichment step, was worked out to obtain auxotrophic mutants from the astaxanthin-producing yeast P. rhodozyma. Under the optimized conditions described, different mutants suitable for strain improvement were isolated.     

Plant regeneration from mesophyll-protoplasts of four different ecotypes and two marker lines from Arabidopsis thaliana using a unique protocol

A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of four ecotypes (Col C24, Per-1, Bur-0, Landsberg erecta) and two marker lines (M4 and M10) of Ardbidopsis thaliana is described. The different lines showed plating efficiencies between 1.0 and 3.9% using Nitsch medium or this medium supplemented with coconut water. For the differentiation of callus into normal shoots a single shoot regeneration medium was applicable to all ecotypes, but depending on the line other regeneration media showed to be more suitable. The results indicated that the protoplast culture procedure is applicable, with minor modifications, to all tested genotypes but the most suitable shoot regeneration medium should be established for each A. thaliana line.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzyl-aminopurine - IAA indole-3-acetic acid - IPA isopentenyladenine - IPAR isopentenyladenosine - MES 2-[N Morpholino]ethanesulfonic acid - MS Murashige and Skoog - NAA naphthaleneacetic acid     

A simple method for obtaining cell-specific cDNA from small numbers of growing root-hair cells in Arabidopsis thaliana

A simple and rapid method for cloning specific cDNAs from mRNA populations derived solely from small numbers of root-hair cells is described here. To identify genes expressed during the earliest visible stage of root-hair cell development, cell contents were aspirated from small numbers of Arabidopsis root-hair cells at or just before this stage. This material was used to make reusable solid-phase oligo-dT-primed cDNA libraries. To demonstrate that the libraries contained high quality longer cDNAs, a fragment located 2.7 kb from the 3' end of the cDNA of the single copy root-hair expressed gene RHD3 was cloned using a nested PCR strategy. This technique was also used to obtain novel gene expression information by cloning the full-length 0.85 kb cDNA of the Rop2 GTPase from this library. This approach offers a means of cloning larger cDNAs directly from small numbers of growing root-hair cells and, potentially, other epidermal cell types.     

Optimization of a working cryopreservation protocol for Pinus patula embryogenic tissue

Summary The protocol currently used to cryopreserve Pinus patula embryogenic tissue was investigated in an attempt to improve and optimize the recovery of tissue. This investigation describes two aspects that influence tissue recovery after cryopreservation: (i) the effects of precooling tissue prior to immersion into liquid nitrogen; and (ii) whether the choice of supports onto which the recovered tissue is suspended improved the recovery rate. Results indicated that precooling tissue to −70°C prior to immersion into liquid nitrogen was superior to precooling to −30°C or direct immersion in liquid nitrogen (−196°C). Tissue recovery improved when polyester grids were used as supports, and was slowest on filter paper supports.     

Somatic embryogenesis and long term maintenance of embryogenic lines from fox grapes

In the present paper, a method for the induction and long-term maintenance embryogenic cultures for Vitis × Labruscana `Niagara' and `Fredonia' is reported. Embryogenic cultures from these two cultivars were induced in an embryogenesis establishment medium (CIM) from ovaries obtained from flowers 10–14 days pre-anthesis. The embryogenic lines obtained in this experiment have been stably maintained for more than 2 years, through repeated subcultures on a long-term maintenance medium (LTMM) without loss of embryogenic competence. Somatic embryo regeneration and maturation have been successfully achieved after 30 days of cultivating embryogenic cultures in an embryo regeneration medium (EDMM), supplemented with charcoal and polyethylene glycol. The somatic embryos were successfully germinated in two different media, `Fredonia' germination medium (FGM) and `Niagara' germination medium (NGM), and converted into normal looking plants on a conversion medium (CM). This revised version was published online in June 2006 with corrections to the Cover Date.     

Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines

Summary This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.     

A method for obtaining DNA from compost

An effective cell lysis method for extraction of bacterial genomic DNA from compost was developed in this study. Enzymatic disruption method, physical–chemical combination method, and commercial kit method were used to extract DNA from compost samples and were compared by analyzing DNA yield and efficient cell lysis. The results showed that all the three methods can be used to extract high-quality DNA from compost, but the enzymatic method had better cell lysis efficiency and DNA yields than others without the use of special equipment and expensive spending. Comparison of different methods for lysing gram-positive bacteria Bacillus subtilis indicated that the enzymatic cell lysis is superior for destroying the gram-positive cell wall. Spin-bind DNA column was used for DNA purification, and the purity of the purified sample was checked by polymerase chain reaction to amplify a region of the 16S rRNA. Results indicated that the part of 16S rRNA were amplified from all the purified DNA samples, and all the amplification products could be digested by the restriction enzyme HhaI.     

Transforming embryogenic cell lines of Gladiolus with either a bar-uidA fusion gene or cobombardment

Summary Embryogenic cell lines of Gladiolus were bombarded with the bar-uidA fusion gene under the cauliflower mosaic virus (CaMV) 35S promoter (pDM327) or cobombarded with uidA under the CaMV 35S promoter (pBCG) and bar under the CaMV 35S promoter (pDM307). Over 500 cell lines were isolated for either the fusion gene or cobombarded cells following selection on Murashige and Skoog's medium supplemented with 2 mg 1⁻¹ (9 μM) 2,4-dichlorophenoxyacetic acid and 6 mg 1⁻¹ phosphinothricin. The optimum DNA concentration for, isolating stable transformants was one-tenth that for optimal isolation of lines with gus expression, and three times as many cell lines were isolated following cobombardment as compared to bombardment with the bar-uidA fusion gene. Three times as many cell lines (72% of the cell lines) containing the bar-uidA fusion gene expressed gus as compared to cobombarded cell lines (23%) following histological staining. Gus expression ceased after 1 yr in culture for 5% of the cell lines containing the fusion gene and 3% of the cobombarded cell lines. The bifunctionality and utility of the bar-uidA fusion gene were demonstrated, accompanied by enhanced gus expression.     

Changes of shikimate pathway in glyphosate tolerant alfalfa cell lines with reduced embryogenic ability

Glyphosate tolerant cell lines were selected from highly embryogenic cell suspension culture ofMedicago sativa L. Resistant cell lines showed significant reduction of embryogenic ability and during long-term culture in the presence of glyphosate gradual loss of this ability was observed. After glyphosate treatment the increased activity of 5-enolpyruvylshikimate-3-phosphate synthase in tolerant cell lines overcame the block in aromatic amino acid synthesis which was observed in control cell lines. Glyphosate caused marked increase in the content of shikimic acid in both control and tolerant cell lines but the accumulation of shikimic acid was considerably lower in tolerant calli. Significant qualitative and quantitative differences were found in the content of individual phenolic acids. The considerable decrease in the amount of cinnamic acid derivates and broader spectrum of hydroxybenzoic acids suggest in tolerant cell lines the activation of alternative pathway not regulated by phenylalanine ammonia lyase. The possible role of altered pool of phenolic acids on the embryogenic ability is discussed.     

Plant regeneration from embryogenic cell suspension-derived protoplasts of rubber

Embryogenic cell suspensions of rubber derived from immature inflorescences and inner integuments of immature fruits released 3.1 ± 0.2 × 10⁷ protoplasts g^-1 f. wt. (mean ± s.e.m, n = 10) and 3.2 ± 0.2 × 10⁷ protoplasts g^-1 f. wt., with mean viabilities of 83 ± 2% and 77 ± 8%, respectively. Sustained mitotic division was observed only when protoplasts were cultured in KPR liquid medium on nitrocellulose membranes overlying the same semi-solid medium containing Lolium multiflorum nurse cells. Protoplast-derived cell colonies were produced within 2 months of culture. Protoplast-derived cell colonies proliferated, upon subculture to MS-based regeneration medium, with 40% of the protoplast-derived calli developing somatic embryos. The latter germinated into plants on the same medium after 3 months of culture. This revised version was published online in June 2006 with corrections to the Cover Date.