The use of agarose bead culture for the regeneration of single cell-derived colonies from protoplasts isolated from suspension cultures of Humulus lupulus

A cell-wall digestion medium has been devised to isolate protoplasts from suspension cultures of Humulus lupulus. Conditions have been developed for colony formation from protoplasts and the plating efficiency determined in three types of agar and by two culture methods. Viable calli were produced only when protoplasts embedded in Seaplaque agarose were incubated in a defined liquid medium. HPLC analysis showed that none of the isolated colonies accumulated -acids.     

An improved method for protoplast formation and its application in the fusion of Rhodotorula rubra with Saccharomyces cerevisiae

Protoplasts from various strains of red-pigmented yeasts were generated at high frequency using improved procedures. The use of sulphur-containing amino acids and 2-deoxyglucose in growth media led to impaired cell wall synthesis and rendered cells very susceptible to treatment with mercapto-ethanol and various lytic enzymes. Use of individual lytic enzymes separately resulted in relatively low frequencies of protoplasts from most of the red yeasts examined, whilst use of -glucoronidase, Novozyme and Zymolyase in series markedly increased stable protoplast formation. The latter effects were shown to be strain specific. The ability to generate large numbers of red yeast protoplasts prompted the attempt to examine intergeneric fusion between auxotrophs of a strain of Saccharomyces cerevisiae and Rhodotorula rubra. Putative hybrids were selected as variously-pigmented prototrophic colonies growing on minimal medium and stabilised by subculturing on the latter medium. Unusual cream, orange and yellow hybrid colonies were generated, composed of cells of varying morphologies (chains, multibudded). The majority of stable hybrids contained one nucleus, although several heterokaryons were also observed. Some hybrids possessed the phenotypes of both parents: fusant wcat41 grew as rapidly as the S. cerevisiae parent but also contained an inducible phenylalanine ammonia-lyase (PAL) which appeared to be more active than that of the Rhodotorula parent.     

Interspecific hybridization between Penicillium chrysogenum and Penicillium cyaneo-fulvum following protoplast fusion

Summary Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Isolation and culture of daylily mesophyll protoplasts

Mesophyll protoplasts were isolated from leaf tissues of a diploid daylily (HemerocallisxRed Magic) by enzymatic digestion with a solution containing 0.5% Pectolyase Y-23, 0.1% Cellulase R-10, 0.1% Driselase, 0.6 M sorbitol and half-strength MS inorganic salts. When cultured on MS medium supplemented with 0.5 mg/l NAA and 0.5 mg/l BA, the protoplasts underwent sustained division to produce multicellular colonies. The optimal plating density for cell division was 0.5 × 10⁵ protoplasts/ml. The highest plating efficiency was obtained in cultures grown in media solidified with 0.2% Gelrite. Under these conditions, formation of colonies occurred from 14% of cultured protoplasts. Calli were recovered from 9 colonies only after the cultures were treated with a conditioned medium. Intact plants were regenerated from protoplast-derived calli through organogenesis.Abbreviations BA 6-benzylaminopurine - FDA fluorescein diacetate - GA₃ gibberellic acid - MS medium Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Plant regeneration from protoplasts of embryogenic suspension cultures of orchardgrass (Dactylis glomerata L.)

An embryogenic suspension culture of orchardgrass (Dactylis glomerata L.) consisting of small, embryogenic cell clusters was obtained from callus formed on basal sections of young leaves through a process of selective enrichment. These suspensions were used as a source of protoplasts. The isolated protoplasts divided at a frequency of 0.5–10% when plated in an agarose solidified culture medium. Conditioned medium, in which embryogenic Dactylis suspension cultures had been grown, was found to increase the rate of cell colony formation. Protoplast-derived colonies grew rapidly in a bead-type culture system of floating agarose slabs in liquid medium. New suspension cultures formed as the colonies grew out of the agarose. These cultures were embryogenic and formed green plantlets when plated on a solid medium lacking auxin. The plantlets were established in soil and grown to mature plants.Abbreviations B5 medium according to Gamborg et al. (1968) - SH-x medium according to Schenk and Hildebrandt (1972) supplemented with x M dicamba - dicamba 3,6-dichloro-o-anisic acid - KM-8p medium 8p of Kao and Michayluk (1975)     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.     

Production of bioflavor by regeneration from protoplasts of Ulva pertusa (Ulvales,Chlorophyta)

Protoplasts were isolated from thalli of Ulva pertusa using a mixed enzyme solution of 2.0% Cellulase Onozuka R-10, 2.0% Macerozyme R-10, and 2.0% Driselase. Isolated protoplasts regenerated cell walls, developed into thalli, and propagated in large numbers under aeration in the preparative scale-culture system. Typical bioflavor compounds produced from the regenerated plants, as well as from field-collected plants, were found to be long chain aldehydes, which gave a typical seaweed odor. The long chain aldehydes were formed enzymatically from unsaturated fatty acids and released into the culture fluid. A Percoll/mannitol discontinuous density gradient separation of the heterogeneous protoplasts led to a selection of cell lines with high production of bioflavor. The cells that regenerated from protoplasts were immobilized by polymer matrices such as alginate, -carrageenan, agarose, and agar. Living cells entrapped in alginate beads in aerated cultures survived best. However, the beads started to breakdown after two months. The immobilized cells demonstrated a higher bioflavor production than did the cultured cells.     

Immunological evidence for transfer of the barley nitrate reductase structural gene to Nicotiana tabacum by protoplast fusion

Summary A protoplast fusion experiment was designed in which the selectable marker, nitrate reductase (NR), also served as a biochemical marker to provide direct evidence for intergeneric specific gene transfer. NR-deficient tobacco (Nicotiana tabacum) mutant Nia30 protoplasts were the recipients for the attempted transfer of the NR structural gene from 50 krad -irradiated barley (Hordeum vulgare L.) protoplasts. Barley protoplasts did not form colonies and Nia30 protoplasts could not grow on nitrate medium; therefore, selection was for correction of NR deficiency allowing tobacco colonies to grow on nitrate medium. Colonies were selected from protoplast fusion treatments at an approximate frequency of 10^-5. This frequency was similar to the Nia30 reversion frequency, and thus provided little evidence for transfer of the barley NR gene to tobacco. Plants regenerated from colonies had NR activity and were analyzed by western blotting using barley NR antiserum to determine the characteristics of the NR conferring growth on nitrate. Ten plants exhibited tobacco NR indicating reversion of a Nia30 mutant NR locus. Twelve of 26 regenerated tobacco plants analyzed had NR subunits with the electrophoretic mobility and antigenic properties of barley NR. These included plants regenerated from colonies selected from 1) co-culturing a mixture of Nia30 protoplasts with irradiated barley protoplasts without a fusion treatment, 2) a protoplast fusion treatment of Nia30 and barley protoplasts, and 3) a fusion treatment of Nia30 protoplasts with irradiated barley protoplasts. No barley-like NR was detected in plants regenerated from a colony that grew on nitrate following selfed fusion of Nia30 protoplasts. Because tobacco plants expressing barley-like NR were recovered from mixture controls as well as fusion treatments, explanations for these results other than protoplast fusionmediated gene transfer are discussed.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Polyethylene-glycol induced fusion of bacterial protoplasts

Summary Direct selection for recombinants by supplemented minimal media from polyethylene-glycol (PEG)-induced fusion of protoplasts of polyauxotrophic strains of B. megaterium revealed striking physiological influences on the yield of recombinants. Cytoplasmic state of the protoplasts to be fused, rather than genetic events, determined the number of colonies obtained on the selection media. It is suggested that the physiological effects primarily influenced the ability of the fused protoplasts to revert to bacillary form.     

Intraspecific heterokaryon and fruit body formation in Coprinus macrorhizus by protoplast fusion of auxotrophic mutants

Summary Fusion of protoplasts of Coprinus macrorhizus mutants with different amino acid requirements resulted in the production of prototrophic clones at frequencies of 1–4% of the protoplasts surviving the fusion treatment. The frequencies were at least 200 times higher than those of the appearance of revertants. Few prototrophic colonies appeared also when the mutant protoplasts were individually subjected to fusion treatment, or when they were mixedly cultured without fusion treatment. It was thus concluded that intraspecific heterokaryons were formed by protoplast fusion.The auxotrophic mutants did not form fruit bodies when cultured singly or mixedly with each other. In contrast, the heterokaryons produced by protoplast fusion between the mutants of compatible mating types developed into fruit bodies with intermediate morphology of those of the strains from which the mutants were derived. Heterokaryons were also formed by fusion of mutant protoplasts with identical mating genotype, but they failed to form fruit bodies.Abbreviations PEG polyethyleneglycol - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid     

Monitoring trichloroethylene mineralization by Pseudomonas cepacia G4 PR1

To analyze the extent of mineralization of trichloroethylene (TCE) without disturbing an actively growing biofilm, a minimal growth medium was formulated that reduces the concentration of chloride ions to the extent that the chloride ions generated from TCE mineralization may be detected with a chloride-ion-specific electrode. By substituting chloride salts with phosphates and nitrates, a chloride-free minimal medium was produced that yields a specific growth rate for Pseudomonas cepacia G4 PR1 which was 93% of that in chloride-ion-containing minimal medium. Furthermore, TCE degradation by resting cell suspensions was similar in both media (85% of 75 M TCE degraded in 6 h), and complete mineralization of TCE was slightly superior in the chloride-free minimal medium (77% compared to 60% of 75 M TCE mineralized in 6 h). In addition, indole-containing, minimal-medium agar plates were developed to indicate the presence of the TCE-degrading enzyme toluene ortho-monooxygenase (fire-engine-red colonies) as well as to distinguish this enzyme from other TCE-degrading enzymes (toluene dioxygenase and toluene para-monooxygenase).     

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Two novel techniques improve division and colony formation from protoplasts:

1. Plating in agarose stimulates colony formation of protoplasts from a wide range of species. Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid. Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium.
2. Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (e.g. Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida).

The combination of ‘agarose plating’ and ‘bead culture’ dramatically improved plating efficiencies of protoplasts in all species tested.     

Recombination after protoplast fusion in the yeast Candida tropicalis

Candida tropicalis protoplasts obtained by snail enzyme treatment were induced to fuse by the use of polyethylene-glycol. Heterokaryons formed by two auxotrophic strains were selected by complementation on minimal medium. These heterokaryons were unstable and readily dissociated into their nuclear components. Under appropriate conditions, the parental nuclei of an heterokaryon fused. The homokaryon so obtained was unstable and segregated into various types of auxotrophic and prototrophic recombinants.List of Abbreviations Used MM minimal medium - YEA yeast extract agar (complete medium) - YPGT yeast-peptone-glucosethiol (medium for protoplast preparation) - PTP medium for cell pretreatment (used before the action of snail enzyme) - PEG polyethylene glycol - p-FPA para-fluorophenylalanine - 5-FC 5-fluorocytosine     

Isolation and Identification of Ciliatine (2-Aminoethylphosphonic Acid) from Lipids of Edible Antarctic Krill,Euphausia superba

An improved method for regenerating Bacillus subtilis protoplasts at the frequency of 92～100% on a semi-synthetic medium was found. Protoplasts were preincubated in HCP-3 medium, an isotonic semi-synthetic medium supplemented with polyvinylpyrrolidone, and then plated on HCP-1.5 agar medium by overlaying. By this method, even on the regeneration medium supplemented with minimal nutritional requirements protoplasts regenerated at a frequency of as high as 20%. The modified method was applicable to the direct-selection of prototrophic recombinants after fusion (the highest recombination yield from the input protoplasts was 1.3%) and to protoplast transformation with plasmid DNA.     

Callus proliferation from the protoplasts of embryogenic cells of Quercus serrata

Embryogenic culture was induced from the immature embryos of Quercus serrata using Marashige and Skoog's medium (MS) containing 0.1 M each of 2,4-d and BAP, and subcultured for seven months before isolation of protoplasts by using 1% Cellulase RS in 0.6 M mannitol solution. Efficient colony formation was obtained when protoplasts were cultured in a liquid MS medium containing 0.6 M mannitol, 3% sucrose and combination of 0.1 M or 1 M each of 2,4-d and BAP. Excluding ammonium nitrate from the MS medium resulted in the decrease of the percentage of colony formation. From colonies, both agar culture and liquid culture were sustained in the MS media without mannitol containing no plant growth regulator, or containing 0.1 M of BAP in combination with 0.1 M or 1 M of 2,4-d.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxyacetic acid - MS medium after Murashige & Skoog (1962).     

In vitro shoot regeneration from leaf mesophyll protoplasts of hybrid poplar (Populus nigra x P. maximowiczii)

Summary Protoplasts were isolated from leaf mesophyll of hybrid poplar (Populus nigra X P. maximowiczii) with a mean yield of 10.4 x 10⁶ protoplasts per g fresh weight using 2.0% Cellulase Onozuka R-10, 0.8% Macerozyme R-10, 1.2% Hemicellulase, 2.0% Driselase, and 0.05% Pectolyase Y-23 with CPW salts solution containing 0.6 M mannitol, 0.002 M DTT, 3 mM MES at pH 5.6. A liquid plating method produced the highest frequency of dividing protoplasts (48.6%) using an MS medium without NH₄NO₃. The highest percent of colony formation was 22.8%, produced with fabric supported semi-solid (0.5% w/v) agar plating method using the same culture medium. Growing cell colonies and/or micro-calli were transferred to a fresh semisolid agar medium containing 0.44 M BAP and 9.0 M 2,4-D. Multiple shoots were produced from protoplast-derived callus after culture on MS medium containing 6.8 M zeatin. After root induction on half-strength MS medium that lacked growth regulators, shoots were transferred to pots containing artificial soil mix.Abbreviations CPW Cell and Potoplast Wash solution - LPM Liquid Plating Method - LDM Liquid Drop Method - HDM Hanging Drop Method - FSPM Fabric supported Semi-solid agar Plating Method - DTT Dithiothreitol - MES 2-(N-morpholino) ethane sulfonic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - MS Murashige and Skoog (1962)     

Plating of isolated tobacco mesophyll protoplasts on agar medium

Summary A technique was developed to derive cell and plant clones from isolated mesophyll protoplasts of tobacco. The protoplasts, plated on a fully defined agar medium, divided and grew actively forming visible colonies after one month of culture. Efficiency of colony formation depended on cell density and light condition during incubation. Under standard conditions, 60% of plated protoplasts formed colonies. Upon transfer onto suitable media, these colonies differentiated shoots and roots, and eventually regenerated whole plants. Advantages of mesophyll protoplasts as the source of clones as well as implication of the plating technique for genetical studies are discussed.     

Transformation of wheat (Triticum aestivum L.) through electroporation of protoplasts

Protoplasts isolated from embryogenic suspension cultures of wheat (Triticum aestivum cv. Hartog) were electroporated in the presence of plasmid pEmuGN and/or pEmuPAT, which contained the reporter gene gus and selectable marker gene bar, respectively. Under optimised electroporation conditions, up to 0.9% of viable protoplasts displayed gus activity two days after electroporation. To select for phosphinothricin (PPT) resistant colonies, electroporated protoplasts were incubated for six weeks in a medium containing 10 g/ml PPT. The cells surviving the selection were maintained as individual colonies on solid medium or as suspension cultures. More than 60% of these colonies exhibited tolerance to 40 g/ml PPT when tested 10 months after initial selection. To date, 57 green plants have been regenerated from these colonies and 24 have been transferred to soil. Southern blot analyses of colonies and plants, using the bar gene sequence as the probe, confirmed transformation of the cells. Positive PAT assays of both regenerated colonies and plants indicated the presence of the bar gene product. These results provide a basis for the establishment of routine procedures for transformation of wheat by direct gene transfer into protoplasts.Abbreviations gus -glucuronidase - PAT phosphinothricin N-acetyltransferase - PPT phosphinothricin - MS Murashige and Skoog medium