A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Protoplast isolation and regeneration in Streptomyces clavuligerus

The regeneration of streptomycete protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. Reports of studies on the regeneration of protoplasts from Streptomyces clavuligerus are limited and for this reason the experiments described in this paper were carried out. An investigation of protoplast formation and cytology was made to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency were isolated from mycelium, grown in a two-stage culture system (without glycine), using lysozyme dissolved in a sucrose osmoticum containing 1% bovine serum albumin. The latter promoted improved protoplast viability. A systematic survey was made of the components of regeneration medium R5, previously used for S. clavuligerus, and other potentially advantageous components and conditions, in an attempt to raise the regeneration frequency of the protoplasts. An improved regeneration medium (R6) and protocol which supported higher and more consistent levels of regeneration of S. clavuligerus protoplasts resulted from these experiments. These improved procedures for protoplast isolation and regeneration proved to be suitable for other streptomycete species.     

Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

花脸香蘑原生质体的制备及再生条件

以珍稀食用菌花脸香蘑菌丝为原材料,对原生质体制备与再生条件进行系统研究,并通过响应面法优化酶解液种类、酶解温度、酶解时间和稳渗剂等影响因素。结果表明以0.6mol/mL甘露醇作稳渗剂,在以1%溶菌酶+1%蜗牛酶+1%纤维素酶为复合酶解液,酶解温度为30℃,60-70r/min摇床振荡培养条件下,酶解4h,原生质体产量达到2.31×10⁷CFU/mL,在以蔗糖为稳渗剂的液体培养基上再生率达到25%。研究结果可为花脸香蘑后期研究提供依据。     

Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Plant regeneration from cell suspension-derived protoplasts of Nicotiana africana

Plants have been regenerated from Nicotiana africana Merxm. protoplasts isolated from cell suspensions. Two different sequences of media were assayed, one usually used to regenerate tobacco mesophyll protoplasts (K3,RMO) the other previously recommended for potato mesophyll protoplast regeneration (W-S-S, ST-1, ST-2, S-3). Only the media for potato protoplasts were efficient for African tobacco plant regeneration. The regeneration efficiency was 6.3 plants per 1000 plated protoplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

Isolation,Culture and Plant Regeneration of Protoplasts from an Embryogenic Callus Tissue of Gossypium hirsutum

Protoplasts were isolated and cultured from hypocotyl embryogenic callus tissue of Gossypium hirsutum L. cv. "Lumian 6". The highest yields of viable protoplasts were obtained from a vigorous embryogenic callus 7 to 9 d old subcultured on MS medium supplemented with 2 mg/L IAA and 1 mg/L KT using a solution of 1% cellulase Onozuka R-10, 1% pectinase, 0.7 mmol/L KH2PO4, 2.5 mmol/L Ca2+ , and 0.5 mol/L osmoticum (mannitol), at pH 5.8 and at a temperature of 30 ℃. After separation and purification (in 21% sucrose floatation medium), the protoplasts were laid up in a quiet liquid protoplast culture medium containing K3 salts, NT vitamins with 0.1 mg/L 2,4-D, 0.2 mg/L KT and 0.45 mol/L glucose for 10 to 15 min. The protoplasts were fractioned into an upper and a lower layer in the centrifugal tube. Most of the protoplasts in the lower layer were smaller, round and rich in cytoplasts in which contain many granular substances. When this kind of protoplasts were cultured in the thin liquid protoplast culture medium with a density of 1 x l0s to 5 x los protoplasts/mL, the division and the callus formation of the regenerated cells were easily observed. The first divisions occurred in 3 days and small cell clusters could be seen after 2 to 3 weeks in the culture. At this moment, the addition of the protoplast culture medium with decreased osmoticum once or twice is needed for the continuous protoplasts division to form calli. Regenerated calli, 3 to 5 mm in diameter, were transferred in succession on MS medium with 2 mg/L IAA and 1 mg/L KT for the initiation of embryogenesis. The embryoids germinated on the hormonefree MS medium and a number of plantlets were obtained. It seems that using vigorous embryogenic callus and decreasing osmoticum are the two critical factors for plant regeneration of cotton protoplasts.     

Isolation and characterization of four cell wall purple acid phosphatase genes from tobacco cells

Four full-length cDNAs were isolated from a cDNA library prepared from tobacco cultured cells and designated NtPAP4, NtPAP12, NtPAP19 and NtPAP21, which could correspond to purple acid phosphatase (PAP). Levels of both NtPAP12 and NtPAP21 mRNA in the protoplasts immediately increased after the protoplasts were transferred to a medium for cell wall regeneration, and the accumulation of the mRNA was correlated with cell wall regeneration for 3 h. It is likely that the NtPAP12 and NtPAP21 gene products are wall-bound PAPs at the early stage of regenerating walls in tobacco protoplasts.     

Rapid plant regeneration from Nicotiana mesophyll protoplasts

A procedure is described for rapid plant regeneration from tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Six to seven days after protoplast isolation, colonies are placed on double filter feeder plates that consist of a strong regeneration medium containing 7.5 mg/l 6-(γ,γ-dimethylallylamino)-purine (2iP) and 0.1 mg/l p-chlorophenoxyacetic acid (pCPA). Complete plants are regenerated in about 5 weeks after transfer to a rooting medium (hormone-free Murashige and Skoog (MS) medium). However, upon remaining on shoot regeneration medium, 50–75 shoots are regenerated from single colonies derived from individual protoplasts. This procedure may reduce the amount of somaclonal variation (as measured by ploidy level) which is usually expressed in plants obtained by conventional regeneration techniques.     

Evaluation of parameters in the isolation of viable protoplasts from cultured tobacco cells

A systematic evaluation disclosed the following conditions to be optimum for the isolation of viable protoplasts from cultured cells of Nicotiana tabacum L. `Bright Yellow' grown in liquid suspensions: (a) the cell culture in the early phase of cell number increase, (b) an enzyme mixture of 1% cellulase “Onozuka” and 0.2% Macerozyme, (c) an enzyme solution pH of 4.7 or 5.7, (d) a 2- to 3-hr incubation period, (e) 5 ml of enzyme solution per 500 mg cells and contained in a 50-ml Delong flask, (f) agitation on a gyrotory shaker at 50 rpm, and (g) 0.3 to 0.8 m mannitol as osmoticum in the cell enzyme mixture. The incubation temperature may be varied from 22 to 37 C. The procedure enabled 30% of the tobacco cells to form protoplasts, 80% of which regenerated cell walls in 4 days and 40% resumed cell division activity when returned to cell culture medium.     

PROTOPLAST ISOLATION AND CELL DIVISION IN THE AGAR-PRODUCING SEAWEED GRACILARIA (RHODOPHYTA)1

Methods were developed for the isolation of large numbers of healthy protoplasts from two species of the agarophyte Gracilaria; G. tikvahiae McLachlan and G. lemaneiformis (Bory) Weber-van Bosse. This is the first report of protoplast isolation and cell division in a commercially important, phycocolloid-producing red seaweed, as well as for a member of the Florideophycidae. The optimal enzyme composition for cell wall digestion and protoplast viability consisted of 3% Onozuka R-10, 3% Macerozyme R-10, 1% agarase and 0.5% Pectolyase Y- 23 dissolved in a 60% seawater osmoticum containing 1.0 M mannitol. The complete removal of the cell wall was confirmed by several different methods, including electron microscopic examination, and the absence of Calcofluor White (for cellulose) and TBO (for sulfated polysaccharide) staining. Spontaneous protoplast fusion was observed on several occasions. Protoplast viability was dependent upon the strain and age of the parent material, as well as the mannitol concentration of the enzyme osmoticum. Cell wall regeneration generally occurred in 2-6 days; cell division in 5-10 days. Protoplast-produced cell masses up to the 16-32 cell stage have been grown in culture. However, efforts to regenerate whole plants have been unsuccessful to date.     

Protoplast liberation and regeneration in the ascomycete Hypomyces ochraceus (Pers.) Tul

A rapid and convenient method for producing protoplasts from 3 d old mycelium of the ascomycete Hypomyces ochraceus is described. The procedure involves a Helix pomatia enzyme preparation and sucrose (20%) for stabilization. Pretreatment with disulfide bond reducing agents reduced the amount of viable protoplasts. Formation of protoplasts and different stages of regeneration were observed by phase contrast microscopy. There was only one type of true regeneration from protoplasts to hyphae in 15-30% gelatine medium by direct forming a germ tube from the original protoplast. Cytological events and physiological conditions are discussed.     

甘蔗和烟草幼叶原生质体的核酸含量与核酸酶活性及其影响因素

与幼叶组织相比,酶法新鲜分离的甘蔗和烟草幼叶原生质体内的ＲＮＡ、ＤＮＡ及总核酸含量均降低。其原因可能是刚游离的原生质体内酸性和碱性ＲＮＡ酶与ＤＮＡ酶等活性提高所致。甘蔗叶原生质体内的核酸降低量和ＲＮＡ酶与ＤＮＡ酶活性的增加程度均高于烟草。随用作渗透压稳定剂的甘露醇浓度增加,甘蔗和烟草叶原生质体的ＲＮＡ酶和ＤＮＡ酶活性均相应提高。其中以甘蔗叶原生质体的核酸酶活性增加水平较明显。在细胞壁降解产物的作用下,除了甘蔗原生质体内的ＲＮＡ酶活性略被促进外,其ＤＮＡ酶和烟草叶原生质体内的核酸酶均不受影响     

Optimal Cultural and Physiological Conditions for Handling Streptomyces rimosus Protoplasts

A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no alpha, epsilon-ll-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.     

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10⁶ / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl₂ and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.Abbreviations BAP benzylaminopurine - NAA naphthaleneacetic acid - PCR Polymerase Chain Reaction - fw fresh weight     

Wirkung von Virushemmstoffen auf die Infektion von Tabakprotoplasten mit dem Tabakmosaik-Virus

Effects of virus inhibitors on the infection of tobacco protoplasts with tobacco mosaic virus Yeast extract inhibits the infection of Nicotiana glutinosa plants with tobacco mosaic virus (TMV), whereas in N. sandérae yeast extract is not effective. This phenomena was compared with the effect of yeast extract on protoplasts, and on the infection of protoplasts of both tobacco species with TMV. Additionally, skim milk and ribonuclease were included in the experiments as further inhibitors of early stages of virus infection. It was examined whether these inhibitors damage non-inoculated protoplasts (a), and whether they affect virus infections in protoplasts as they do in cells of intact plants (b). To investigate protoplast damage by the inhibitors, conductivity measurements of protoplast suspensions containing inhibitors, and the ability of protoplasts for cell wall regeneration after treatment with the inhibitors, were used. Inhibitor concentrations which prevent virus infections in plants did not damage the protoplasts. The inhibitor effect on the course of infection was investigated by protoplast treatments before, during and after inoculation with TMV, and by addition of the substances to the culture medium. Measurements of virus content in protoplasts after cultivation revealed different results for the three inhibitors, however, there was no difference in the response of protoplasts from the two tobacco species to yeast extract. It is concluded that there are principal differences between the inhibition of plant and protoplast infections. Therefore, it is unlikely that protoplasts are a useful system for the mode of action studies on inhibitors of early stages of virus infection in plants.     

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.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.