A Comparison of Methods for Plasmid Delivery into Plant Protoplasts

Three methods of plasmid delivery to mesophyll protoplasts ofNicotiana tabacum cv. Xanthi have been evaluated. Specifically;a) chemically stimulated uptake of isolated plasmid, b) deliveryof plasmid encapsulated in liposomes, and c) fusion of plasmid-containingspheroplasts, were combined with divalent cation (Ca²⁺ and Mg²⁺)or polyalcohol [polyethylene glycol (PEG) and polyvinyl alcohol(PVA)] treatments. The quantity and quality of plasmid associatedwith intact protoplasts, was assessed by DNA-DNA blot hybridisationanalysis, following stringent washing to separate intact protoplastsfrom non-viable protoplasts and debris. Treatments which increasedassociation of plasmid with protoplasts decreased protoplastviability. Optimum association of plasmid with protoplasts,in the context of acceptable loss of viability, was achievedwhen protoplasts were interacted with either naked plasmid orliposomeencapsulated DNA in the presence of 15% w/v PEG 6000,or with Escherichia coli spheroplasts containing chloramphenicol-amplifiedplasmid in the presence of 25% w/v PEG 6000. Divalent cationsdid not stimulate significant plasmid delivery without unacceptableloss of protoplast viability. Strategies to further increasethe efficiency of plasmid delivery are discussed. (Received June 21, 1984; Accepted August 20, 1984)     

Electroporation Stimulates Plant Regeneration from Protoplasts of the Woody Medicinal Species Solarium dulcamara L.

Exposure of cell suspension protoplasts of the woody medicinalplant Solatium dulcamara L. to voltages of 250 to 1250 V cm^–1for three successive pulses, each of 10–50 us duration,stimulated growth of protoplast-derived tissues. Such tissuesexhibited increased morphogenesis and required a shorter periodin culture to exhibit this effect than tissues from untreatedprotoplasts. Regenerated shoots also rooted more readily anddeveloped more prolific root systems than shoots from untreatedprotoplasts. These observations have important implicationsfor plant genetic manipulation and may have application in therecovery and rooting of shoots from tissues of woody species,normally considered recalcitrant in culture. Key words: Electroporation, protoplasts, shoot regeneration, Solanum dulcamara (woody nightshade, bittersweet)     

Stages in the Initiation of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Reciprocal transfers of Nicotiana tabacum cv. Xanthi nc. leafexplants were made daily between root inducing medium (RIM)and shoot inducing medium (SIM), SIM and a basal medium containingno growth regulators (BM), and RIM and BM. It was found thatthe explants became determined for shoot production after 6d, while roots were produced after only 1 d on RIM before transferto BM. The competence of the explant to produce roots was greatlyreduced by culture on BM prior to culture on RIM. There wasfar less reduction in shoot numbers with preculture on BM. Explantswere found to be only weakly canalized for both caulogenesisand rhizogenesis for the first 2 d after determination. Thereafterthey became strongly canalized. Transfers were also made fromBM to SIM and back to BM, which revealed that the explants becamecompetent for caulogenesis in the absence of cytokinins priorto determination. The period for which SIM is required can bereduced to only 1 d. Key words: Nicotiana tabacum, in vitro, organogenesis, competence, determination     

Enzymatic Isolation of Protoplasts from Fern Protonemal Cells Stainable with a Fluorescent Brightener

Protoplasts were successfully isolated for the first time fromthe filamentous protonemal cells of ferns after the cells werecultured in contact with both air and medium. Sterilized sporesof Adiantum capillus-veneris and Pteris vittata were inoculatedon a piece of nylon mesh (40- {diaeresis}m mesh) placed on amat of polyester fibers which was soaked in liquid culture medium,and the spores were illuminated from above with continuous redlight. Protonemal cells, exposed to the air during this procedure,could be stained with Calcofluor White, a dye that binds tocell walls. Protoplasts were easily isolated from these protonemalcells by digestion of the cell wall with cellulase and pectinase.A total of 0.8–1.9 x 10⁴ and 0.6–2.0 x 10⁴ protoplastswere obtained from protonemata that originated from 10 mg ofdry spores of Adiantum and of Pteris respectively. Viability,as judged by staining with fluorescein diacetate was more than90% for both species. Staining with 4'6-diamidino-2-phenylindole(DAPI) revealed that about half of the protoplasts of both speciescontained a nucleus. (Received May 22, 1989; Accepted September 5, 1989)     

Improved protoplast yield and cell wall regeneration in Gracilaria verrucosa (Huds.) Papenfuss (Gracilariales, Rhodophyta)

Protoplasts were isolated enzymatically from the red alga Gracilariaverrucosa using only two enzymes: agarase prepared from marinebacteria and commercial cellulase. Yields of protoplasts weredependent on the donor material and by choosing young bladesor algae in a state of higher growth rate, the production ofprotoplasts reached a maximum of 10⁷ protoplasts per gram offresh tissue. Cell viability was better with NaCI used as osmoticumthan with sorbitol in the culture medium and on reducing culturemedia to normal osmolarity in 4 d. 25% of the cultured protoplastswere able to regenerate a cell wall (i.e. cellulose) within7 d as confirmed by staining with calcofluor white althoughonly a few protoplasts were able to divide. During the first24 h of culture, the synthesized agar contained higher amountsof L-galactose-6-sulphate than the cell wall of thalli. Theamount of agar in the protoplasts, however, did not increase,indicating that the protoplasts synthesize a qualitatively differentcellwall. Key words: Agarase, agar, cell wall regeneration, Gracilaria verrucosa, protoplasts     

Regeneration of Plants from Protoplasts of Arabidopsis thaliana L. cv. Columbia (C24), Via Direct Embryogenesis

This report describes a protocol for the regeneration of fertileplants from mesophyll protoplasts of Arabidopsis thaliana raceColumbia (C24). Regeneration was rapid and reproducible. Theprotocol is especially novel in that a large proportion of regeneratingprotoplasts regenerated via direct somatic embryogenesis. Protoplastsisolated from in vitro-grown plants entered sustained divisionafter 3–5 d in culture medium and over a period of severaldays 6–22% of protoplasts underwent at least one celldivision. Approximately 2–16% of these protoplasts continuedto divide and after 3 weeks in culture had formed macroscopiccolonies, of which 70–80% were regular embryo-like structures.Four weeksafter release from the alginate culture matrix andtransfer to solid medium in the light, 68–88% of thesestructures had produced well-developed shoots. Shoots couldbe maintained in culture or established in peat blocks. Theregenerated plants were fertile. Key words: Arabidopsis thaliana, protoplast, regeneration, embryogenesis, dicamba     

A method for the microinjection and culture of protoplasts at very low densities

A method has been developed which allows the recovery of calli from a high proportion of individual, injected, mesophyll protoplasts of Nicotiana tabacum c.v. Xanthi. A small drop of low melting point agarose is used both to hold protoplasts during microinjection and for their subsequent culture in feeder dishes. The feeder dishes consist of "beads" of protoplasts at a high density set in agarose to "feed" the infected protoplasts across a liquid medium.The method has been used successfully both with normal protoplasts and protoplasts from which the vacuole has been removed.Abbreviations NT medium Nagata-Takebe medium (Nagata and Takebe, 1971) - MS medium Murashige-Skoog medium (Murashige and Skoog, 1962) - NAA 1-Naphthaleneacetic acid - BAP 6-Benzylaminopurine - LMP agarose low melting point agarose     

Metabolism of Pyrimidines in Protoplasts from Cultured Catharanthus roseus Cells

The metabolism of [2-¹⁴C]thymine, [2-¹⁴C]thymidine, [2-¹⁴C]uraciland [¹⁴C]uridine was investigated in protoplasts obtained fromsuspension cultures of Catharanthus roseus. Most of the exogenouslysupplied thymine, thymidine and uracil was degraded, and salvageof these pyrimidines accounted for 5–36 per cent of thetotal amount of ¹⁴C-labelled precursors which was metabolized.However, more than 80 per cent of the labelled uridine was utilizedfor the biosynthesis of nucleotides and nucleic acids, and therest was degraded. In contrast to the results from protoplastsof sugar cane cells in suspension culture, the data indicatethat protoplasts possess a pathway for the degradation of pyrimidines,and that the overall metabolism of these pyrimidines in protoplastsis very similar to the metabolism in the intact cells. Catharanthus roseus, madagascar periwinkle, protoplasts, pyrimidine metabolism     

Distribution of {beta}-Thioglucosidase Activity in Intact Plants, Cell and Tissue 6Brassica napus L.

Distribution of myrosinase activity in extracts from seeds,intact plants, cell cultures and regenerated callus and plantsof Brassica napus L. was determined by the rate of glucose formationfrom glucosinolate hydrolysis. Calli with shoots and regeneratedplants were obtained from protoplasts or from explants. Of the seedling organs from Brassica napus L. cv. Niklas, hypocotylsshowed the highest myrosinase activity. In cotyledons a nearlyconstant enzyme activity was determined over the first 6 d,followed by a gradual decline. Roots showed a fast decline inenzyme activity over the investigated period. Freshly-isolated protoplasts contained less myrosinase activitythan the original intact tissue. The enzyme activity in developingcalli generally decreased during the first culture periods.After the initial decline a low activity was found which wasstable for a period of more than 2 years. The enzyme activityshowed fluctuations when measured at different times after mediumchange. Protoplast calli with regenerated shoots showed a considerablyhigher myrosinase activity than calli without shoots. Myrosinaseactivity was also found in explant calli including explant callifrom cotyledons and hypocotyls after induction of shoots. Myrosinase activity in seeds from 21 cultivars of Brassica napus,Brassica campestris, Sinapis alba and Raphanus sativus was testedand the highest myrosinase activity was found in seeds fromthe Sinapis alba cultivar Trico while the lowest activity wasfound in the Brassica campestris cultivar Rapido III. Leaf, stem and inflorescence from flowering regenerated or seed-grownplants contained a low but significant myrosinase activity.In contrast, roots showed a high myrosinase activity. The resultsobtained from regenerated plants indicate that the myrosinasesystem is stable in vitro culture, and that the glucosinolate-myrosinasesystem is active in calli tissue. Key words: Myrosinase (thioglucoside glucohydrolase, E.C. 3.2.3.1), in vitro cultures, intact plants     

Surface Charge Density of Hetero-Fused Plant Protoplasts: an Electrophoretic Study

Electrophoretic mobilities of hetero-fused plant protoplasts,which were obtained by electrofusion of barley mesophyll cellprotoplasts and Rauwolfia serpentina cultured cell protoplasts,and those of the unfused parent protoplasts were measured invarious media of different pH values. At pH 5.2, the zeta potentialof the fused protoplasts was intermediate between those of thebarley and R. serpentina protoplasts and the average surfacecharge density of the fused protoplasts was closer to that ofR. serpentina than to that of barley. The distribution of thesurface charge density of fused protoplast obtained at pH 5.2is discussed in terms of the surface charge densities and thesizes of parent protoplasts. These results revealed that thesurface charge density of fused protoplasts was determined bythe surface charge densities and the ratio of the surface areasof the respective parent protoplasts. (Received December 28, 1989; Accepted August 10, 1990)     

Inhibition of Photosynthesis by Sulfite in Mesophyll Protoplasts Isolated from Vicia faba L. in Relation to Intracellular Sulfite Accumulation

Inhibition of photosynthesis by Na₂SO₃ in mesophyll protoplastsisolated from Vicia faba leaves and uptake of sulfite by theprotoplasts were examined at various pH values of the incubationmedium containing Na₂SO₃. As the pH of the incubation mediumlowered, the rate of photosynthesis in the protoplasts decreasedand the amount of sulfite taken up by the protoplasts increased.Most of sulfite accumulated in the protoplasts was not metabolizedduring the dark incubation, as measured with an ion chromatograph.Photosynthetic O₂ evolution by the chloroplasts isolated fromVicia mesophyll protoplasts was inhibited by exogenously-appliedNa₂SO₃ over pH region examined (7.4–9.0). The sulfiteconcentration required for a half inhibition of photosynthesisby the isolated chloroplasts was similar to the intracellularsulfite level required for that by the protoplasts. These resultsindicate that the intracellular sulfite accumulated in the protoplastsin an unmetabolized state is responsible for the inhibitionof protoplast photosynthesis. (Received January 24, 1985; Accepted May 29, 1985)     

SHORT COMMUNICATION Factors Affecting Protoplast Culture ofCucumis melo'Green Delica'

Hypocotyls, cotyledons and etiolated half-expanded leaves ofCucumismelo‘Green Delica’ were used as explants for protoplastisolation and culture. Protoplasts isolated from cotyledonsand etiolated half-expanded leaves cultured in Durand, Potrykusand Donn (DPD) medium supplemented with 0.9 µMbenzylaminopurine(BAP), 3.6 µM2,4-dichlorophenoxyacetic acid (2,4-D) and1% sucrose, using the agarose bead culture method, were ableto form cell walls and subsequently go through cell division.Pretreatment of half-expanded leaf explants in the dark for14 d provided the best material for protoplast isolation andcell division. Approximately one third of protoplasts from etiolatedhalf-expanded leaves formed microcolonies. For hypocotyl protoplasts,none of the treatments used were suitable to induce cell division.There was no significant difference between sucrose, glucose,and sucrose plus glucose, in culture media on the plating efficiencyof leaf protoplasts ofC. melo‘Green Delica’; however,bigger colonies were formed in media supplemented with 1% sucrose.No shoot or whole plant regeneration was achieved. However,the methods reported here provide further information onC. meloprotoplastculture.Copyright 1998 Annals of Botany Company Cucumis melo,protoplast culture, 2,4-D, BAP, yeast extract, casein hydrolysate.     

Carbon Dioxide Fixation by Guard Cell Protoplasts of Commelina communis

Biochemical studies of epidermal tissue may not reflect metabolismof the guard cells which represent less than 5% of the tissuevolume. Pure samples of guard cell protoplasts of Commelinacommunis were therefore used to investigate CO₂ fixation ratesand ¹⁴C-labelling patterns of metabolites in the light and thedark. Qualitatively, results were similar in most respects tothose obtained in a previous study (Schnabl, 1980) for guardcell protoplasts of Vicia faba. CO₂ fixation rates by guardcell protoplasts of C. communis were the same in the light andthe dark but about 50 times lower than the values Schnabl obtainedfor V.faba. The ¹⁴C-labelling pattern of metabolites in C. communiswas also similar in the light and the dark: over 60% of thetotal fixed was in malate with only 1% in sugar phosphates.Label was also detected in starch, aspartate, glutamate andcitrate but not in glycollate as previously recorded in V. fabaguard cell protoplasts. The results confirm the view that the reductive pentose phosphatepathway does not occur in guard cells of C. communis. Key words: CO₂ fixation, Guard cell protoplasts, Stomata     

Isolation and Biochemical Characteristics of Protoplasts of Jojoba (Simmondsia chinensis (Link) Schneider)

Two types of protoplasts were isolated from leaves of shootsor callus of subcultures of jojoba (Simmondsia chinensis (Link)Schneider). Protoplasts from leaves were rich in chloro-plastsand were about half the volume of protoplasts from callus. Theviability of preparations as determined by the Evans blue techniquewas 80%. From cell cycle analysis by flow cytometry of nuclei,leaf protoplasts were uniformly in a non-proliferating phase(G0-G1), while callus protoplasts presented many phases of thecell cycle. Protoplasts from calli had only half the Chi ofthose from leaves. Yet Chi a/b ratio, as well as protein andtotal lipid content per cell, were similar in both types ofprotoplasts. A major drop in polar lipids, chiefly in mono-and digalactosyldiacylglycerol, and a parallel increase in neutrallipids occurred during protoplast isolation. The 18:2/18:3 ratiodecreased in neutral lipids concomitant with an increase intriglycerides rich in linolenic acid. Our results suggest atriggering of lipolytic acylhydrolases during the protoplastisolation, as reported for other species. Plasmolysis of thecells with high osmolarity medium and long incubation timeswere required to get a good yield of jojoba protoplasts. Inthe course of this procedure water-deficit stress takes place.A parallel with lipid changes occurring under this type of stressis discussed. (Received April 22, 1991; Accepted July 3, 1991)     

Developmental Pattern of Root and Shoot Organogenesis in Cultured Leaf Explants of Nicotiana tabacum cv. Xanthi nc.

Caulogenesis and rhizogenesis were studied in cultured leafexplants of Nicotiana tabacum cv. Xanthi nc. using both lightand scanning electron microscopy. The timing of organ appearancewas also recorded. The patterns of development seen were comparedto each other and to that in explants grown on growth regulator-freemedium. Shoots first appeared after 12 d in culture and rootsafter 7 d. In caulogenesis nodules appear at the explant edgeand from these the shoots arise. The nodules are mainly derivedfrom palisade mesophyll cells, along with some spongy mesophylland bundle-sheath cells. The nodules form a continuous row alongthe edge of the explant and their initiation appears to be centredon veins. Shoots are produced indirectly. Roots are produceddirectly from bundle-sheath and vein parenchyma cells. Withoutplant growth regulators bundle-sheath cells still divide, althoughonly a few divisions were seen. Key words: Nicotiana tabacum, in vitro, caulogenesis, rhizogenesis     

Intraclonal Genetic Variation for Protoplast Regenerative Ability Within Solatium tuberosum cv. Record

The potato cv. Record is recognized as a recalcitrant cultivarin tissue culture and attempts in the past to obtain regenerationfrom protoplasts continually failed, despite media and protocolalterations. By sampling a large number of Record tubers, significantdifferences between lines were obtained for regeneration fromleaf discs. Eight such lines exhibiting a range of responseto regeneration from leaf discs were used in the present studyto examine protoplast culture response. Significant variationwas detected in protoplast plating efficiency and in the numberof regenerants produced. These results are discussed in relationto the exploitation of protoplasts in potato improvement andin terms of the role of tissue culture techniques for the maintenanceof potato cultivars. Solarium tuberosum, cv. Record, potato, protoplasts, intraclonal variation     

Direct Transfer of Plasmid DNA from Intact Yeast Spheroplasts into Plant Protoplasts

We developed a polyethylene glycol (PEG)-mediated direct DNAtransfer method from intact Saccharomyces cerevisiae spheroplastsinto Arabidopsis thaliana protoplasts. To monitor the DNA transferfrom yeast to plant cells, ß-glucuronidase (GUS) reportergene in which a plant intron was inserted was used as a reporter.This intron-GUS reporter gene on a 2µm-based plasmid vectorwas not expressed in yeast transformants, while it expressedGUS activity when the plasmid DNA was introduced into plantcells. When a mixture of 1 x 10⁸ of S. cerevisiae spheroplastsharboring the plasmid and 2 x 10⁶ of A. thaliana protoplastswas treated with PEG and high pH-high Ca²⁺ solution (0.4 M mannitol,50 mM CaCl₂, 50 mM glycine-NaOH pH 10.5), GUS activity was detectedin the extract of the plant cells after a three-day culture.The GUS activity was higher than that of a reconstitution experimentin which the mixture of 1 x 10⁸ of S. cerevisiae spheroplastswhich did not carry the reporter gene, 2 x 10⁶ of A. thalianaprotoplasts and the same amount of the reporter plasmid DNAas that contained in 1 x 10⁸ of S. cerevisiae spheroplasts,was treated with PEG and high pH-high Ca²⁺ solution. Moreover,the GUS gene expression was resistant to micrococcal nucleasetreatment before and during PEG treatment. From these results,we concluded that plasmid DNA can be directly transferred fromintact yeast spheroplasts to plant protoplasts by a nuclease-resistantprocess, possibly by the cell fusion. ²Deceased on September 15, 1992.     

Studies of the recovery of tobacco mesophyll protoplasts from an evacuolation treatment

Summary Mesophyll protoplasts ofNicotiana tabacum cv. Xanthi were subjected to centrifugation through a Percoll gradient. This resulted in the removal of the central vacuole from each protoplast, and improved mechanical and osmotic stability. Electron microscope studies showed that the remaining cell contents, including small vacuoles, were of normal morphology. Fixation of protoplasts at various times during subsequent culture showed that the central vacuole was restored after about 12 hours. Cell-wall formation was well advanced after 24 hours of culture. These results are discussed in terms of the potential use of evacuolate protoplasts and the mechanisms of vacuole formation.Abbreviations ER endoplasmic reticulum     

Recovery of Totipotent Cells and Plantlet Production from Daylily Protoplasts

The release of protoplasts by means of enzymes from a totipotentcell suspension of a diploid daylily (Hemerocallis cv. ‘AutumnBlaze’), their collection and distribution in plasticculture vessels and their subsequent regeneration is described.Attention is given to aggregation and to the different formsof growth during the initial stages of culture. The methodsfor the subsequent multiplication of the regenerated cells intomorphogenetically competent compact clusters and the manipulationof these clusters to yield embryo-plantlets are also outlined.The implications of all this in terms of potential for the geneticmodification of daylily is discussed. Hemerocallis, cloning, protoplasts