Interaction of purified DNA with plant protoplasts of different cell cycle stage: The concept of a competent phase for plant cell transformation

Summary The polycation mediated attachment of purified tritiated DNA to plant protoplasts has been measured by quantitative microautoradiography. The automated grain counting technique used, also provides information on the cell cycle stage of individual protoplasts, which circumvents the need to synchronize the plant cell population before preparation of protoplasts. With protoplasts from asynchronously dividing suspension cultures of Nicotiana syhestris (NS-1), S-phase protoplasts appear to be inefficient binders of ³H-DNA, as compared with G₁ or G₂ protoplasts. Protoplasts derived from a tumour line of Crepis capillaris (CAPT) exhibit ³H-DNA binding at all cell cycle phases, but Sphase protoplasts appear to be preferential binders. These differences are discussed with reference to cell cycle kinetics, membrane charge variation and the possibility of increasing the efficiency of genetic transformation of higher plant cells in culture.     

Co-expression and inheritance of foreign genes in transformants obtained by direct DNA transformation of tobacco protoplasts

Summary A plasmid containing two marker genes for expression in plants was constructed. This 16 kb vector, pCT1T3, contains an intact nopaline synthase gene and a chimaeric gene consisting of the promoter and terminator regions from cauliflower mosaic virus gene VI and a structural gene, aminoglycoside phosphotransferase (APH(3′)II), from the bacterial transposon Tn5. After transformation of tobacco mesophyll protoplasts with this plasmid, several kanamycin-resistant transformants were obtained. Intensive studies on the drug tolerance of growth and differentiation of the transformants showed that the chimaeric gene was stably expressed. Of 17 independent transformants, 3 (about 18%) expressed the two marker genes, regardless of the state of differentiation, as did individual plants regenerated from the same callus. Multiple copies of the inserted DNA were found in some transformants. Viable seeds were produced by 12 out of 15 independent transformants. Seeds obtained by self-pollination were germinated on medium containing kanamycin sulphate. With the exception of one clone, resistant seedlings with green leaves and sensitive seedlings with white leaves were found to segregate in a 3:1 ratio. This suggests that the inheritance of the inserted gene is Mendelian. A reciprocal cross between the transformants and wild-type tobacco also showed nuclear transmission of the APH(3′)II gene. This was consistently maintained in a subclone of the same transformant derived from the same callus line. Stable inheritance of the single dominant character was also seen among seeds formed in several different flower pods of the same individual plants. Two clones were also found to synthesize nopaline in addition to expressing APH(3′)II. Analysis of the progeny obtained by self-crosses of such transformants revealed the simultaneous expression of these two enzymes, indicating that the two marker genes are linked on the same chromosome.     

DNA replication in soybean protoplasts and suspension-cultured cells: Comparison of exponential and fluorodeoxyuridine synchronized cultures

Cell-suspension cultures of soybean (Glycine max (L.) Merr., line SB-1) have been used to study DNA replication. Cells or protoplasts incorporate either radioactive thymidine or 5-bromodeoxyuridine (BUdR) into DNA. The DNA has been extracted as large molecules which can be visualized by autoradiography. Nuclei were isolated and lysed on slides thus avoiding degradation of DNA by a cytoplasmic endonuclease. The autoradiograms demonstrated that DNA synthesis occurs at several sites tandemly arranged on single DNA molecules separated by center to center distances ranging from 10 to 30 m. Velocity sedimentations through alkaline gradients confirm the lengths of the replicated regions seen in autoradiograms. By using velocity sedimentation it also has been possible to demonstrate that replication proceeds by the synthesis of very small (4–6S) DNA intermediates which join to form the larger, replicon-size pieces seen in autoradiograms. Both small (4–6S) and large (20–30S) intermediates are observed in synchronized and exponential cultures. However, after synchronization with fluorodeoxyuridine (FUdR) the rate of DNA synthesis is reduced. Since the size of intermediates is not reduced by FUdR treatment, it is concluded that the slower rate of replication results from a reduction in the number of tandem replication units but not in the rate at which they are elongated. After FUdR treatment, the density analogue of thymidine, BUdR, can be substituted for almost all of the thymidine residue in DNA, resulting in a buoyant density increase (in CsCl) from 1.694 to 1.747 g/cm³. Using this density analogue it is possible to estimate the amount of template DNA attached to new replication sites. When this is done, it can be shown that synchronized cells initiate replication at about 5,000 different sites at the beginning of S. (Each such site will replicate to an average length of 20 m.) Use of BUdR also substantiates that at early stages of replication, very small replicated regions (<8S) exist which are separated by unreplicated segments of DNA which replicate at a later time. Most of these conclusions agree with the pattern of DNA replication established for animal cells. However, a major difference appears to be that after prolonged inhibition of soybean cell replication with FUdR, very small, as well as replicon-size intermediates accumulate when replication is restored. This indicates that regulation of replication in these cells may be different from animal cells.Abbreviations BUdR 5-Bromodeoxyuridine - FUdR 5-Fluorodeoxyuridine     

Non-endocytic penetration of core histones into petunia protoplasts and cultured cells: a novel mechanism for the introduction of macromolecules into plant cells

The results of the present work demonstrate that core histones are able to penetrate the plasma membrane of plant cells. Confocal microscopy has revealed that incubation of petunia protoplasts with fluorescently labeled core histones resulted in cell penetration and nuclear import of the externally added histones. Intracellular accumulation was also confirmed by an ELISA-based quantitative method using biotin-labeled histones. Penetration into petunia protoplasts and cultured cells was found to be non-saturable, occurred at room temperature and at 4 °C and was not inhibited by Nocodazole. Furthermore, penetration of the biotinylated histone was neither blocked by the addition of an excess of free biotin molecules, nor by non-biotinylated histone molecules. All these results clearly indicate that the observed uptake is due to direct translocation through the cell plasma membrane and does not occur via endocytosis. Our results also show that the histones H2A and H4 were able to mediate penetration of covalently attached BSA molecules demonstrating the potential of the histones as carriers for the delivery of macromolecules into plant cells. To the best of our knowledge, the findings of the present paper demonstrate, for the first time, the activity of cell penetrating proteins (CPPs) in plant cells.     

Introduction of foreign DNA into walled plant cells via liposomes injected into the vacuole: a preliminary study

Plasmids containing the firefly luciferase reporter gene were introduced into tobacco and maize by electroporation. They were used to calibrate the performance characteristics of an Hamamatsu C1966 AVEC/VIM photonic camera-Leitz photomicroscope image processing system. Luciferin-dependent light emission was readily detected, on an individual cell basis, using the analytical photon counting mode of the Hamamatsu system. An efficient liposome-DNA encapsulation protocol was developed and used to introduce the firefly luciferase plasmid into walled cells of tobacco, maize, carrot and rice. This was achieved by pressure-injecting the liposomes (DNA encapsulated inside the vesicle) into the vacuole where they subsequently fused with the tonoplast, releasing the DNA into the cytoplasm. Analytical photon counting studies were conducted on injected cells to determine whether the DNA introduced in this way was expressed. To date all experiments have proved negative. Reasons for this lack of expression are discussed.     

Influence of lipophilic groups in cationic alpha-helical peptides on their abilities to bind with DNA and deliver genes into cells.

For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide.     

The interaction between exogenous DNA and sperm cells.

Epididymal sperm cells, incubated with plasmid DNA, showed a spontaneous tendency to interact with the exogenous nucleic acid. We have investigated the molecular basis of such interaction. Exogenous DNA is taken up by sperm cells over a 15- to 20-min period and is specifically localized on the nuclear area of the sperm head. DNA was reversibly bound to spermatozoa since it can be competed out by excess of cold competitor DNA or by other polyanions as heparin and dextran sulphate. By contrast, poly-L-lysine, a polycation, favours the uptake. DNA molecules of large size (7 kb) were preferentially taken up as compared to smaller ones (150-750 bp). Acidic proteins were also taken up and concentrated, as for DNA, at the nuclear level. These data strongly suggested that ionic interactions may occur between foreign molecules and a substrate located in the sperm head. On the basis of Southwestern analysis, a sperm head protein(s) of 30-35 KD is identified as potential substrate for exogenous DNA binding. Moreover, we have found that seminal plasma contains factor(s) which abolish sperm permeability, exerting a powerful inhibitor effect on DNA uptake. The presence of a specific binding protein for the DNA and of a factor inhibiting such interaction support the existence of a mechanism controlling, through specific factors, the sperm-DNA interaction.     

A simple and efficient procedure to improve plant regeneration from protoplasts isolated from long-term cell-suspension cultures of indica rice

Summary A simple and efficient method has been developed to improve plant regeneration from protoplasts isolated from >1-yr-old cell-suspension culture of Indica rice (Oryza sativa) cv. Pusa Basmati 1. A two-step regeneration procedure, involving the transfer of calluses to shoot-regeneration medium containing 1% (w/v) agarose prior to culture on a medium containing 0.4% (w/v) agarose, was found to improve plant regeneration. High concentrations of kinetin in the regeneration medium were also found to be beneficial. The two-step regeneration procedure, combined with a high concentration of kinetin (10.0 mgl⁻¹) in the medium, significantly increased plant regeneration. By this method, even though protoplasts were isolated from over 1-yr-old cell-suspension cultures, protoplast-derived plant regeneration frequency reached 16.1% compared with <4% regeneration frequency without such treatment. Use of a similar protocol might improve plant regeneration from other plant species, especially recalcitrant species.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Cloning of restriction and modification genes in E. coli: The HhaII system from Haemophilus haemolyticus

The genes for a Class II restriction-modification system (HhaII) from Haemophilus haemolyticus have been cloned in Escherichia coli. The vector used for cloning was plasmid pBR322 which confers resistance to tetracycline and ampicillin and contains a single endonuclease R·PstI site, (5′)C-T-G-C-A^↓-G (3′), in the ampicillin gene. The procedure developed by Bolivar et al. (1977) was used to form DNA recombinants. H. haemolyticus DNA was cleaved with PstI endonuclease and poly(dC) extensions were added to the 3′-OH termini using terminal deoxynucleotidyl transferase. Circular pBR322 DNA was cleaved to linear molecules with PstI endonuclease and poly(dG) extensions were added to the 3′-OH termini, thus regenating the PstI cleavage site sequence. Recombinant molecules, formed by annealing the two DNAs, were used to transfect a restriction and modification-deficient strain of E. coli (HB101 r^?m^?recA). Tetracycline-resistant clones were tested for acquisition of restriction phenotype (as measured by growth on plates seeded with phage λcI·O). A single phage-resistant clone was found. The recombinant plasmid, pDI10, isolated from this clone, had acquired 3 kilobases of additional DNA which could be excised with PstI endonuclease. In addition to the restriction function, cells carrying the plasmid expressed the HhaII modification function. Both activities have been partially purified by single-stranded DNA-agarose chromatography. The cloned HhaII restriction activity yields cleavage patterns identical to HinfI. A restriction map of the cloned DNA segment is presented.     

A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK

A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.     

The major DNA polymerase in cultured plant cells: Partial purification and correlation with cell multiplication

A DNA polymerase activity was isolated from cells of Oryza sativa L. grown in suspension culture. Molecular mass ( 180,000), optimal requirements for pH (neutral), Mg²⁺ (5–10 mM), Mn²⁺ (1 mM), template preference (activated DNA), lack of activity with native or denatured DNA, and sensitivity to N-ethylmaleimide and ionic strength are similar to those of the vertebrate -polymerase. Like DNA polymerase , the DNA polymerase described in this work is the most abundant in proliferating cells of Oryza sativa L., Parthenocissus tricuspidata (Siebold et Zucc.) Planchon, Acer pseudoplatanus L., and Medicago sativa L. and responds to changes in the rate of cell multiplication. We therefore postulate that this -like DNA polymerase is the replicating enzyme of plant cells.Abbreviations BSA bovine serum albumin - EDTA ethylendiamino-tetracetic acid - DTT dithiothreitol - PTSF p-toluenesulfonyl fluoride     

A simple and rapid procedure to establish embryogenic cell suspensions as a source of protoplasts for efficient plant regeneration from two Chinese commercial rice cultivars

An efficient and rapid procedure has been developed to establish embryogenic cell suspension cultures of two Japonica Chinese commercial rice cultivars, Zhonghua 8 and Eryi 105. Embryogenic cell suspensions of both varieties were established from 0.5–1.0 g fresh weight of embryogenic callus in AA medium within 2.5 months of the initiation of callus from sterilised seeds. The previously reported subculture of callus on semi-solid medium for 4–8 weeks prior to transfer into liquid medium was unnecessary and caused delay in the establishment of embryogenic cell suspensions. Protoplasts were isolated reproducibly from cell suspensions up to 18 months after their initiation, with protoplast plating efficiencies attaining 0.15–0.37%. Reproducible plant regeneration from 14–26% of the protoplast-derived tissues was achieved without the requirement for nurse cells. This revised version was published online in June 2006 with corrections to the Cover Date.     

The lipid bilayer and aquaporins: parallel pathways for water movement into plant cells

The plant plasma membrane is the the major barrier to water flow between cells and their surroundings. Water movement across roots involves pathways comprising many cells and their walls. There are three possible pathways which water can follow, (i) a trans-cellular pathway, which involves serial movement into and out from radial files of cells, (ii) a symplasmic pathway through the plasmodesmata, which creates a cytoplasmic continuum and (iii) a tortuous, extracellular pathway through the cell walls, the apoplasmic pathway. In each of these pathways water movement across cell membranes occurs at some stage. The possible role of water-channels in membranes is discussed in relation to this movement. The molecular identity of water-channel proteins in plasma membranes of plants has been confirmed but there remain a number of unresolved questions about their role in cell and tissue water relations, their interaction with the lipid components of membranes and the relationship between water movement through membranes by diffusion in the bilayer.     

T-DNA genes to study plant development: precocious tuberisation and enhanced cytokinins in A. tumefaciens transformed potato

Summary Potato Line Mb1501B is a derivative of the cultivar Maris Bard (Solanum tuberosum), transformed with T-DNA from A. tumefaciens strain LBA1501. In culture it grew as frequently branching stunted shoots with a basal callus, lacking roots. These shoots did not form tubers. When grafted, Mb1501B shoots gradually became morphologically more normal and aerial tubers formed readily. Cultured Mb1501B shoots contained 100–200-fold higher concentrations of the biologically-active cytokinins zeatin, zeatin riboside and their corresponding side-chain o-glucosides than untransformed Maris Bard shoots. Cultured Mb1501B shoots contained approximately a 3-fold lower concentration of indole acetic acid (IAA). In grafted Mb1501B plants a 3–10-fold higher concentration of the active cytokinins was found compared with untransformed plants and no difference in IAA concentration.