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Regeneration of plants from tobacco protoplasts and some factors affecting the plant differentiation.

Protoplasts were isolated from the cell suspension culture derived from leaf and stem calli of tobacco (Nicotiana tabacum cv. Ko Hsin No. 1) haploid pollen-plants. After 12 hr in the liquid culture medium, the protoplasts became oval-shaped, and produced a new cell wall. The first division of the newly formed cells was completed after 24 hr in culture. After 4 weeks in culture, the yellowish calli reached 1 mm in size were then transferred to an auxophyton. 18 days later, the calli became 3-4 mm in size. After the calli were transferred to a differentiation culture medium, shoots and roots soon turned up. Regeneration of whole plants was obtained thereafter. The division and differentiation of regenerated cells were affected not only by the calli originated from different organs and their, age, but also by the basic components of the differentiation culture medium and the type of cytokinin used.

Cell Differentiation↗

PubMed1998

Early events of tomato spotted wilt transcription and replication in protoplasts.

A protoplast transfection system for tomato spotted wilt tospovirus (TSWV) was established by PEG-mediated infection of tobacco protoplasts. Analysis of viral RNA synthesis revealed an asymmetric production of viral (v) and viral-complementary (vc) strands of all three genomic RNA segments and a separation in time of subgenomic transcription during the first 72 hr post infection (p.i.). Synthesis of vc-RNA was detectable 8-10 hrs prior to v-RNA production. During the first 72 h of infection the accumulation of S-RNAs exceeded M- and L-RNAs and the amount of vc-RNAs was larger than that of genomic v-RNAs. The subgenomic N-mRNA was first detected 5 hr p.i., followed by vc-S RNA and the mRNA for the nonstructural protein (NSs) 15 hr later. The two subgenomic mRNA species of the S RNA appeared to be regulated independently from each other and from other viral mRNA species. Defective interfering (DI) elements from L RNA associated with the virus inoculum, were not preferentially replicated at the expense of full-sized L-RNA.

Immunohistochemistry↗

PubMed1998

Permeabilized Arabidopsis protoplasts provide new insight into the chromatin structure of plant alcohol dehydrogenase genes.

New data from permeabilized protoplasts have expanded our view of the 5'DNase I hypersensitive area of the Arabidopsis Adh gene derived from nuclei. DNase I hypersensitivity analyses conducted with permeabilized protoplasts from Arabidopsis cell cultures indicates that there are four distinct sites of hypersensitivity centered around positions -425, -325, -200, and -60. The hypersensitive site at -200 coincides with an in vitro hypersensitive site created by purified transcription factors bound to a G-box element. The G-box is a functional cis element that plays a role in the signal transduction of hypoxia and other stresses in Adh. The data presented in this paper support the notion that G-box-related elements may also play a role in defining chromatin structure. The new Arabidopsis data are discussed within the context of what is known about the chromatin structures and regulation of two other plant Adh genes; maize Adh1 and Adh2. The chromatin of the maize Adh1 promoter is divided into a region that is constitutively hypersensitive to DNase I (-700 to -160) and an inducibly hypersensitive region (-140 and -40). There are several sequence elements within the hypersensitive regions bound by proteins in vivo. The anaerobic response element is the most well characterized and functions in the detection of hypoxia. The maize Adh2 gene promoter is constitutively hypersensitive to DNase I, with the exception of a small region that extends to include the TATA box as the gene becomes active. Several cis elements in the Adh2 promoter are bound by factors in vivo. One, at -160, is a functional element that acts as an activator in vascular tissue. The overall goal of our research with the Adh genes from maize and Arabidopsis is to gain further insight into the relationships between the regulation of gene transcription and chromatin structure in plants as it is clear that all the necessary components that characterize regulated gene activity may not be found simply by elucidating the linear sequence of nucleotides that lie 5' to the protein coding regions and finding proteins capable of binding the promoter in vitro.

Alcohol Dehydrogenase↗

PubMed1985

Liposome-mediated transformation of tobacco mesophyll protoplasts by an Escherichia coli plasmid.

An Escherichia coli plasmid, pLGV23neo, carrying a kanamycin resistance gene expressed in plant cells, was encapsulated into negatively charged liposomes prepared by the reverse phase evaporation technique. These liposomes were induced to fuse with tobacco mesophyll protoplasts by polyethyleneglycol treatment. Kanamycin-resistant clones were reproducibly isolated from transfected cultures at an average frequency of 4 X 10(-5). Plants regenerated from these resistant colonies were confirmed to be transformed according to three criteria. Protoplasts isolated from their leaves were resistant to 100 micrograms/ml kanamycin. The enzyme aminoglycoside 3'-phosphotransferase II encoded by the plasmid pLGV23neo was detected in leaf extracts. Approximately 3-5 copies of the gene encoding for kanamycin resistance were inserted in the genome of at least one of the studied transformants. The restriction pattern of inserted DNA was best explained by assuming a tandem integration of the pPLGV23neo sequences, implying an homologous recombination event between these sequences during transformation. Kanamycin resistance was transmitted as a single dominant nuclear marker to the progeny of resistant plants after selfing or cross-pollination with the wild-type.

Aminoglycosides↗

PubMed1988

Structure of U2 snRNA genes of Arabidopsis thaliana and their expression in electroporated plant protoplasts.

We have characterized the U2 snRNA gene family in the higher plant Arabidopsis thaliana. It consists of 10-15 genes which do not appear to be closely clustered. Six of the U2 genes were sequenced and the structure of the Arabidopsis U2 RNA termini was determined in order to define the coding regions. Each of the genes codes for a distinct RNA differing from the others by 2-13 point mutations, localized in the 3' part of the 196 nt-long RNA. The upstream non-coding regions of all genes show strong sequence similarity in positions -81 to -1 and contain three highly conserved sequence elements: GTCCCACATCG (positions -78 to -68; 100% conservation), GTAGTATAAATA (-37 to -26) and CAANTC (-6 to -1). The coding regions are followed by the sequence CAN(7-9)AGTNNAA, a putative termination signal. The expression of three of the genes was studied in electroporated Orychophragmus violaceus and Nicotiana tabacum protoplasts. The genes, one of which contains a T --> C change in the Sm antigen binding site, were actively transcribed and processed into U2 RNAs of the expected size and containing trimethylguanosine caps. Deletion analysis indicates that sequences upstream of the conserved -80 to -1 region are not important for transcription in protoplasts. The 5'-terminal parts of U2 RNAs from several monocot and dicot plants were sequenced. This region, containing the sequence implicated in base-pairing with the branch point in pre-mRNA introns, is identical in all U2 RNAs examined.

Journal Article↗

PubMed1988

UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Antirrhinum majus chalcone synthase gene.

It was shown previously that parsley protoplasts retain differential responsiveness to external stimuli, e.g. UV light. This opened the way for the development of transient expression assays to identify DNA sequences acting as regulatory cis-elements in the transcriptional activation of UV-inducible plant genes. The chalcone synthase (chs) gene of Antirrhinum majus (Snapdragon) is inducible by visible and by UV light. We demonstrate that the kinetics of light induction of a chimeric A.majus chs-nptII gene observed during transient expression in the parsley protoplasts reflect the kinetics of UV-induced CHS expression in A.majus seedlings in vivo. We define three regulatory sequence regions, each differing qualitatively and quantitatively in their effects on gene expression. Immediately upstream of the TATA box (-34) a sequence, with coordinates -39 to -197, functions as an orientation independent UV-light responsive element. The complete 1.1 kb promoter or three tandem copies of this element are capable of rendering a heterologous minimal promoter UV responsive. The next upstream region (-197 to -357) contains sequences that do not by themselves cause UV-induced expression, but specifically potentiate the level of UV-induced expression when combined with the TATA-box proximal UV-responsive element. A third element (-661 to -564) has the properties of a general enhancer since it increases the level of both uninduced and UV-induced expression.

Journal Article↗

PubMed1982

Growth characteristics and ultrastructure of protoplast type L-forms from streptomycetes.

L-form colonies from S. hygroscopicus, S. griseus and S. levoris were isolated after incubation of lysozyme protoplasts on an osmotically stabilized complex agar medium. Unstable and stable L-forms grow on solid and in liquid media. L-form colonies are 5-10 times smaller than normal colonies and show a typical morphology for each species. In ultrathin sections L-form cells are characterized by nucleoid areas with typical core-like structures, by a ribosome-rich cytoplasm with different inclusion bodies, and by a cytoplasmic membrane. Because there are no cell wall structures L-forms of the three Streptomyces species belong to the protoplast type. Analysis of cell size and cell shape shows a variation in diameter and a cell propagation by regular and irregular division- and budding-like processes. Many L-form cells contain more than two chromosomes. The results are discussed with regard to the cellular organisation of streptomycetes and the nature of the stable L-form.

Cell Membrane↗

PubMed1982

Antimicrobial activities of daunorubicin and adriamycin derivatives on bacterial and protoplast type L-form cells of Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI. Structure--activity relationship.

The antibacterial activity of ten N-alkylated derivatives of daunorubicin and adriamycin as well as of 5-iminodaunorubicin has been tested by using Bacillus subtilis 170, Escherichia coli B, and Proteus mirabilis VI and their stable protoplast type L-forms in an agar diffusion test. Eight of the substances showed similar activities against B. subtilis and the L-forms of all test organisms, but no activity against the bacterial forms of E. coli and P. mirabilis. The cell wall of these gram-negative bacteria is responsible for this resistance by not allowing the antibiotics to enter the cells. The piperidino compound N-(CH2)5 daunorubicin shows 2-4 times higher activity against B. subtilis and all L-forms in comparison to daunorubicin and the other derivatives. Five of the substances were inactive against all test strains. Their inactivity seems to be associated with the larger substituents at the C-3' position. Relations between molecular structure and activity are discussed considering data about the interaction with DNA and the antitumor activity. Stable protoplast type L-forms and their bacterial forms represent a suitable and effective test system to screen for more effective substances and to get more information about their mode of action.

Bacillus subtilis↗

PubMed2000

Formation of surface tubules and fluorescent foci in Arabidopsis thaliana protoplasts expressing a fusion between the green fluorescent protein and the cauliflower mosaic virus movement protein.

The movement protein (MP) of cauliflower mosaic virus (CaMV) is a multifunctional protein that potentiates the cell-to-cell and long distance movement of the virus. Functional domains in the CaMV MP were determined by analyzing deletions in green fluorescent protein (GFP)-MP fusions transfected into Arabidopsis thaliana leaf protoplasts. GFP-MP accumulated at fluorescent foci at the cell periphery and in tubular structures extending from the protoplast surface. A region located near the center of MP was required for targeting GFP-MP to foci, whereas a larger region extending nearly to the N-terminus was needed for tubule formation. Cytoskeletal assembly inhibitors did not disrupt tubule formation or the accumulation of GFP-MP at foci, but brefeldin A, which disrupts the plant cell endomembrane system, did interfere with the formation of tubules but not foci.

Antiviral Agents↗

PubMed1992

Herbicide-resistant Indica rice plants from IRRI breeding line IR72 after PEG-mediated transformation of protoplasts.

The commercially important Indica rice cultivar Oryza sativa cv. IR72 has been transformed using direct gene transfer to protoplasts. PEG-mediated transformation was done with two plasmid constructs containing either a CaMV 35S promoter/HPH chimaeric gene conferring resistance to hygromycin (Hg) or a CaMV 35S promoter/BAR chimaeric gene conferring resistance to a commercial herbicide (Basta) containing phosphinothricin (PPT). We have obtained so far 92 Hgr and 170 PPTr IR72 plants from protoplasts through selection. 31 Hgr and 70 PPTr plants are being grown in the greenhouse to maturity. Data from Southern analysis and enzyme assays proved that the transgene was stably integrated into the host genome and expressed. Transgenic plants showed complete resistance to high doses of the commercial formulations of PPT.

Aminobutyrates↗

PubMed1996

Genetic Nature, Stability, and Improved Virulence of Hybrids from Protoplast Fusion in Beauveria

Genetic improvement of two different strains of the entomopathogenic fungus Beauveria bassiana for more effective control of Ostrinia nubilalis and Leptinotarsa decemlineata was obtained by crosses with the insecticidal toxin-producing strain Beauveria sulfurescens. Protoplast fusion between diauxotrophic mutants resulted in the recovery of some stable prototrophic fusion products. The low levels of virulence of the wild type strain B. bassiana 28 isolated originally from L. decemlineata were enhanced both on L. decemlineata and O. nubilalis for one of the hybrids obtained (FP 8) from the cross B. bassiana 28xB. sulfurescens 2. Fusion product 25 obtained from the cross between B. sulfurescens and the highly pathogenic strain B. bassiana 147 showed a three-day reduction in the LT50 towards O. nubilalis. Southern blot hybridization with nine probe-enzyme combinations were conducted on genomic DNAs from the original wild strains, parental mutant strains, and fusion products. Additive banding patterns or unique banding pattern of either parental strain was observed in five hybrids, indicating their status as recombinant and/or partially diploid. Combination of RFLP markers indicative of both parental genomes was never observed with fusion product FP 25. The stability of the virulence following passage through insect-host and stability of molecular structure for the fusion products FP 8 and FP 25 suggest that asexual genetic recombination by protoplast fusion may provide an attractive method for the genetic improvement of biocontrol efficiency in entomopathogenic fungi.

Journal Article↗

PubMed1982

Direct selection of complementing diploids from PEG-induced fusion of Bacillus subtilis protoplasts.

A minimal medium containing horse serum is described on which Bacillus subtilis protoplasts revert to bacillary forms at high frequency (ca. 30%). Used as a plating medium for a mixture of polyethyleneglycol-treated protoplasts from two complementary polyauxotrophic parental strains, it selects the prototrophic fusion products efficiently, and also allows isolation of various auxotrophic recombinants. These prototrophs and recombinants amount respectively to 1% and 10% of the regenerated bacteria. We confirm that two types of prototrophs can be isolated after fusion: stable recombinants and complementing diploids, the latter segregating into various types of recombinants. Based on easily recognized colonial aspects, an approximate estimation of the proportion of the two types becomes possible when a spoOA mutation has been introduced in one of the parents. At least 50% of the prototrophic fusion products are complementing diploids. Incidently, the data also settle a controversy by showing the dominance of spoOA mutations in heterozygotic bacteria.

Bacillus subtilis↗

PubMed1982

Diploid state of phenotypically recombinant progeny arising after protoplast fusion in Bacillus subtilis.

After fusion of Bacillus subtilis protoplasts the phenotypically recombinant clones isolated, whether immediately or as segregants of complementing diploid clones, have in common the following properties. They appear independently of the recN+ gene, most often as the result of apparently non-reciprocal recombination occurring in genetic intervals encompassing the origin and the terminus of replication. First indicated by reciprocal fusion crosses between ø105-lysogenic and ø105-sensitive strains, the diploidy of the recombinants was confirmed by studying the transforming activities of their DNA. These experiments establish heterozygosity at eight loci scattered on the chromosome map. By revealing the presence of the trpF+ allele in trpF7 recombinants, the results also strongly suggest that stable phenotypic recombinants may arise by genetic inactivation. Two possible genetic structures for these recombinants are discussed, one implying total inactivation of one recombinant chromosome, the other a segmentary inactivation of one unrecombined chromosome. Whatever the structure, genetic stability is not a reliable sign of haploidy in bacterial clones produced after protoplast fusion.

Bacillus subtilis↗

PubMed1998

Hormonal Control of Gene Expression During Reactivation of the Cell Cycle in Tobacco Mesophyll Protoplasts.

The roles of auxin and cytokinin in cell cycle reactivation were studied during the first 48 h of culture of mesophyll protoplasts of Nicotiana tabacum. Using hormone delay and withdrawal studies we found that auxin was required by 0-4 h of culture, whereas cytokinin was not required until hour 10-12, which is 6-10 h before S phase. Cycloheximide blocks division, indicating that protein synthesis is required. In an effort to detect a molecular response to either hormone, we examined the expression of the cell cycle marker, cdc2. Cdc2 expression was detected by 12 h of culture, coincident with the timing of the cytokinin requirement and well before the entry into S. However, cdc2 was partially induced by either auxin or cytokinin alone, suggesting that cdc2 expression is not the primary target of either hormone. Our hormone delay experiments suggest that there are separate signal transduction pathways leading from auxin and from cytokinin to reactivation of the cell cycle and that these pathways converge before S. The underlying mechanisms for these distinct pathways remain to be elucidated. Key Words. Auxin-Cytokinin-Tobacco-Protoplast-Development-cdc2

Journal Article↗

PubMed1996

External protons enhance the activity of the hyperpolarization-activated K channels in guard cell protoplasts of Vicia faba.

Hyperpolarization-activated K channels (KH channels) in the plasmalemma of guard cells operate at apoplastic pH range of 5 to over 7. Using patch clamp in a whole-cell mode, we characterized the effect of varying the external pH between 4.4-8.1 on the activity of the KH channels in isolated guard cell protoplasts from Vicia faba leaves. Acidification from pH 5.5 to 4.4 increased the macroscopic conductance of the KH channels by 30-150% while alkalinization from pH 5.5 to 8.1 decreased it only by roughly 15%. The voltage-independent maximum cell conductance, increased by -60% between pH 8.1 and 4.4 with an apparent pKa of 5.3, most likely owing to the increased availability of channels. Voltage-dependent gating was affected only between pH 5.5 and 4.4. Acidification in this range shifted the voltage-dependent open probability by over 10 mV. We interpret this shift as an increase of the electrical field sensed by the gating subunits caused by the protonation of external negative surface charges. Within the framework of a surface charge model the mean spacing of these charges was approximately 30 A and their apparent dissociation constant was 10(-4.6). The overall voltage sensitivity of gating was not altered by pH changes. In a subgroup of protoplasts analyzed within the framework of a Closed-Closed-Open model, the effect of protons on gating was limited to shifting of the voltage-dependence of all four transition rate constants.

Fabaceae↗

PubMed1985

Subcellular fractionation of wheat leaf protoplasts by centrifugal elutriation.

A new method using centrifugal elutriation for subcellular fractionation of plant cells has been developed. This method takes advantage of the fact that particles sedimenting in a gravitational field can be eluted by flow against the field. A wheat protoplast homogenate was fed into an elutriation rotor spinning at high speed and the flow rate into the rotor was gradually increased. The smaller and less dense materials such as mitochondria, microbodies, endoplasmic reticulum, and cytoplasm were elutriated earlier than the larger and denser nuclei and chloroplasts. The intact chloroplasts, free of mitochondria, microbodies, endoplasmic reticulum, and cytoplasm, could be obtained within 40 min following the rupture of protoplasts. The chlorophyll-free mitochondria could be obtained within 80 min.

Cell Fractionation↗

PubMed1989

Electric pulse induced membrane permeabilization. Spatial orientation and kinetics of solute efflux in freely suspended and dielectrophoretically aligned plant mesophyll protoplasts.

Asymmetric breakdown (occurring in only one hemisphere of the cell) was induced in freely suspended and dielectrophoretically aligned vacuole-containing or evacuolated plant protoplasts as well as in isolated vacuoles. In suspended cells breakdown was restricted to the hemisphere facing the anode and in isolated vacuoles to the opposite hemisphere. This difference in the orientation of the asymmetric breakdown can be explained by the opposite direction of the intrinsic membrane potentials of isolated vacuoles and of cells on which the generated potential difference is superimposed. The ensuing permeabilization of the membrane was microscopically monitored by dye uptake and by release of chloroplasts and of cytoplasmic and/or vacuolar solutes. The asymmetric release of intracellular substances (organic acids and/or amino acids) was detected by accumulation of chemotactic bacteria (Pseudomonas aeruginosa) close to the permeabilised membrane area of the cells or vacuoles. Maximum bacteria accumulation required about 5 min and subsequently disappeared after a further 20 min presumably because of the restoration of the original membrane impermeability. With vacuoles retention of the accumulated bacteria was shorter indicating that the resealing process of the tonoplast membrane was faster than that of the plasmalemma. From the kinetics of bacteria accumulation and retention it is therefore possible to deduce information about the life-span and the resealing properties of electropermeabilized membrane areas on the single-cell level. Symmetric breakdown in both hemispheres of the cells could be achieved by electric field-mediated cell rotation of about 180 degrees between two pulses of the same polarity or by application of two pulses of alternating polarity. In dielectrophoretically aligned protoplasts of comparable diameter, breakdown occurred in both hemispheres, even though the breakdown was still asymmetric. It could be demonstrated by the uptake of the vital dye neutral red that the size of the membrane area which was permeabilized was much larger in that hemisphere oriented to the anode than in the other one. The relevance of these observations for further improvement of electroinjection of macromolecules and of electrofusion is discussed. In particular, it is pointed out that positioning of differently sized cells in electric field-mediated hybridisation and the polarity of the breakdown pulse is of great importance with respect to hybrid yield.

Cell Membrane Permeability↗

PubMed1985

The effects of dicyclohexylamine on polyamine biosynthesis and incorporation into turnip yellow mosaic virus in Chinese cabbage protoplasts infected in vitro.

We have reported (R. Balint and S. S. Cohen, 1985, Virology 144, 181-193) that protoplasts from plants infected with turnip yellow mosaic virus (TYMV) continue to produce virus in culture and that newly formed virus particles contained predominantly newly synthesized spermidine and spermine. Inhibition of spermidine synthesis by dicyclohexylamine (DCHA), however, led to incorporation of preexisting spermidine and increased amounts of spermine into newly formed virions. We now report similar results with healthy protoplasts infected in vitro, in which essentially all of the virus is newly formed. Again, newly synthesized spermidine and spermine were preferentially incorporated into virus. DCHA inhibited spermidine synthesis by 85%, leading in 20 hr to a 60% depletion of the cellular spermidine and a 30% reduction in the amount of spermidine per virion. Spermine synthesis increased, however, producing a 40% increase in cellular spermine and 50-100% increase in the amount of spermine per virion. Thus, in spite of spermidine depletion, the total positive charge contributed by polyamines to the virus was essentially conserved.

Brassica↗