Regeneration of plantlets from mesophyll protoplasts of Brassica juncea (L.) Czern

Isolated mesophyll protoplasts of Brassica juncea (L.) Czern., cv. RLM 514 upon culture in suitable growth medium, regenerated cell wall, underwent cell division and formed cellular colonies. Subsequent induction of embryoid (embryogenesis) and shoot bud (organogenesis) formations in such cell masses resulted in regeneration of 186 and 42 plantlets respectively.Abbreviations NT Nagata and Takebe, 1971 - B5 Gamborg et al. 1968 - KM Kao and Michayluk, 1975 - GK₂ Schenck and Hoffmann, 1978 - MS Murashige and Skoog, 1962 - BAP 6-benzyladenine - 2, 4-D 2, 4-dichlorophenoxyacetic acid - NAA napthaleneacetic acid - GA₃ gibberellic acid     

Regeneration of plants from mesophyll protoplasts of ground-cherry (Physalis minimal L.)

High yields of protoplasts were obtained by enzymatic digestion of leaf tissue derived from exenic shoot cultures of Physalis minima L. Isolated protoplasts underwent sustained mitotic divisions and produced calli. Shoot and root morphogenesis were achieved from these calli. The regenerated plants were established in soil.     

Direct embryogenesis from single mesophyll protoplasts in alfalfa (Medicago sativa L.)

Summary We used a tetraploid clone derived from an anther culture operation of Ladak alfalfa to study the pathway of direct embryogenesis from leaf-mesophyll protoplasts. About 72% of the protoplasts divided, and 7% of those produced proembryos. Approximately 38% of the proembryos developed into green embryos, and 33% initiated calluses. Other proembryos dedifferentiated into calluses which later redifferentiated embryos. Sixteen percent of the embryos developed directly into plants, whereas 81% produced plants indirectly via secondary embryos. The remaining 3% of the primary embryos failed to develop into plants. The lowest plating efficiency for direct embryogenesis was 0.3%. The high percentage of direct embryogenesis observed was related to the genetic nature of the clone, low density of liquid medium, low protoplast culture density, and the composition of culture media.Contribution no. 90-61-J from the Kansas Agricultural Experiment Station.     

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     

Isolation and culture of sunflower protoplasts (Helianthus annuus L.): Factors influencing the viability of cell colonies derived from protoplasts

Sunflower protoplasts from various sources (mesophyll, stems, cotyledons anhypocotyls) have been tested for their capacity to divide in culture. Only hypocotyl protoplasts divided in our media at a high and repeatable percentage (60%). Culture at low density in a medium containing glutamine or ammonium succinate as sole sources of nitrogen and a reduced amount of naphthalene acetic acid (NAA) (0.1 mg/l) were the most important conditions for obtaining calli from sunflower protoplasts. On such media, 6% of the initially plated protoplasts reached the stage of calli.     

Isolation,culture and plantlet regeneration from cotyledon and mesophyll protoplasts of two pickling cucumber (Cucumis sativus L.) genotypes

Optimal protoplast yields from cotyledons (2.0×10⁶ protoplasts/ 0.5 g tissue) and from true leaves (5.0×10⁶ protoplasts/g tissue) of two Cucumis sativus genotypes were obtained following a 16 h digestion with, respectively, 1.25% pectinase+0.5% Cellulysin and 0.5 % pectinase+ 1.0% Cellulysin. Enzyme solutions were prepared in modified MS medium containing half-strength major salts, full complement of minor salts and vitamins, 2% sucrose and 0.25 M mannitol. A plating density of 3.5–4.0× 10⁴ protoplasts/ml or higher was required for sustained division, with first division occurring in 6–7 days, second-third division in 8–9 days, and minicalli formation by day 13. Embedding in 0.4% agarose provided the highest plating efficiency (proportion that formed minicalli) of mesophyll protoplasts, which was 28.3% for genotype 3672 and 15% for genotype 3676. By comparison, liquid culture and droplet culture gave lower plating efficiencies (10–19%). Cotyledon and mesophyll protoplasts of one genotype formed minicalli on MS medium containing 2,4-D/BA at 1.0/2.5 M and 5.0/5.0 M, respectively, within 21 days, while mesophyll protoplasts of the second genotype formed minicalli on MS medium containing NAA/BA at 5.0/5.0 M within 12 days. Shoot buds or somatic embryos were obtained upon subculture of calli to MS medium containing lower concentrations (0.05–0.01 M) of 2,4-D/BA or NAA/BA and a few plantlets, ca.18, were recovered on hormone-free medium.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog medium - NAA naphthaleneacetic acid     

Isolation and culture of protoplasts from mesophyll tissue of the legume Cyamopsis tetragonoloba L.

Protoplasts of Cyamopsis tetragonoloba were isolated from leaves of in vitro grown plants. The yield of the protoplasts, their viability and subsequent divisions were greatly influenced by the pH of the media used for isolation and culture of protoplasts. Sustained divisions of the cultured protoplasts were best supported by a modified Kao and Michayluk (1975) nutrient medium containing glucose (0.4 M), NAA (4 mgl^–1), 2,4-D (1 mgl^–1) and KIN (2 mgl^–1 ). The protoplast derived cells developed calli on transfer to Murashige and Skoog (1962) medium supplemented with 1 mgl^–1 each of 2,4-D, NAA and KIN.     

Plant regeneration from mesophyll protoplasts of Matthiola incana (L.) R. Br.

Summary A protocol for obtaining regenerated fertile plants from mesophyll protoplasts of three lines of Matthiola incana is described. Protoplasts were isolated from leaves of 21–28 days old Matthiola plants grown in controlled environment. Sustained divisions were achieved when protoplasts were embedded in sodium alginate. Up to 2.0 % of the protoplasts developed into colonies which could be transferred to shoot regeneration media. More than 25 % of the obtained calluses regenerated shoots. About 4 % of these shoots could be rooted and after transfer to soil phenotypically normal plants have been obtained.Abbreviations 2,4-D 2,4-dichlorphenoxyacetic acid - NAA naphthalene acetic acid - IAA indole-3-acetic acid - BAP 6-banzylaminopurine - IPA isopentenyladenine - IPAR isopentenyladenosine - MES (2-[N-morpholino]) ethanesulfonic acid     

Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Secondary phenolic products in isolated guard cell,epidermal cell and mesophyll cell protoplasts from pea (Pisum sativum L.) leaves: Distribution and determination

Summary Guard cells and epidermal cells of the abaxial (lower) and adaxial (upper) epidermis ofPisum sativum L., mutant Argenteum, are the predominant sites of flavonoid accumulation within the leaf. This was demonstrated by the use of a new method of simultaneous isolation and separation of intact, highly-purified guard cell and epidermal cell protoplasts from both epidermal layers and of protoplasts from the mesophyll. Isolated guard and epidermal protoplasts retained flavonoid patterns of the parent epidermal tissue; quercetin 3-triglucoside and its p-coumaric acid ester as major constituents, kaempferol 3-triglucoside and its p-coumaric acid ester as minor compounds. Total flavonoid content in the lower epidermis was estimated to be ca. 80 fmol per guard cell protoplast and 500 fmol per epidermal cell protoplast. Protoplasts isolated from the upper epidermis had about 20–30% as much of these flavonoids. Mesophyll protoplasts retained only about 25 fmol total flavonoid per protoplast.By fluorescence microscopy, using the alkaline-induced yellow-green fluorescence characteristics of flavonols, we suggest that these flavonol glycosides are present in cell vacuoles. There was no indication for the presence of flavine-like compounds.Abbreviations uE adaxial (upper) epidermis - IE abaxial (lower) epidermis - GCP guard cell protoplasts - ECP epidermal cell protoplasts - MCP mesophyll cell protoplasts - PP protoplasts - HPLC high performance liquid chromatography - TLC thin layer chromatography - CC column chromatography - HOAc acetic acid     

Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Avena sativa L.) leaves

The uptake of [1-³H]geranylgeranyl diphosphate (GGPP) into protoplasts and intact etioplasts and the metabolic interconversion therein was studied after a 2 min pulse of white light. The chlorophyll synthetase reaction, Chlide+GGPPChl_GG, was taken as a natural probe for the etioplast compartment. This reaction yields labeled ChL_GG and, by hydrogenation, labeled Chl_P, when [1-³H]GGPP receives access to the etioplast stroma. It was found that penetration across the plastid envelope was rapid and that penetration across the plasma membrane of protoplasts, however, was slow. A cellular pool of soluble GGPP was detected. This pool was lost, in part, during preparation of the protoplasts and almost completely during preparation of the etioplasts. The membrane-bound phytol pool of etioplasts could not be replaced by exogenous [³H]GG. The endogenous GG and phytol pools of protoplasts, which were larger than those of etioplasts, could be replaced in part by exogenous [³H]GGPP. That part of this pool exists as soluble GGPP or as a direct precursor in the cytoplasm is discussed.Abbreviations GGPP geranylgeranyldiphosphate - Chl_GG geranylgeranyl chlorophyllide a - Chl_P phytyl chlorophyllide a - IPP isopentenyl diphosphate - Chlide chlorophyllide a     

Isolation and culture of mesophyll protoplasts from Rosa hybrida

After 1 h plasmolysis in CPW13M solution, highly viable (>75%) protoplasts were isolated from leaves of axenic shoot cultures of Rosa hybrida L. cv. Abraham Darby using an enzyme mixture containing 1.0% (w/v) Hemicellulase, 0.1% (w/v) Macerozyme, 1.0 (w/v) Cellulase RS, 0.05% (w/v) Pectolyase Y23 and 1.0% (w/v) PVP-10 and from cv. Marie Pavié using an identical mixture but with Cellulase RS and Pectolyase Y23 at 0.7% (w/v) and 0.1% (w/v), respectively. With both cvs., sustained protoplast division was achieved after plating in agarose beads with modified KM8p medium containing 1.0% (w/v) polyvinylpyrrolidone (mol. wt. 10 000; PVP-10), 8.91 μM naphthaleneacetic acid (NAA) and 4.44 μM 6-benzyladenine (BA). Protoplast-derived callus gave rise to roots after transfer to SH medium containing 14 μM 2,4-D. This revised version was published online in June 2006 with corrections to the Cover Date.     

Modulation of photosynthesis and respiration in guard and mesophyll cell protoplasts by oxygen concentration

Stomatal movement is an energetic oxygen-requiring process. In the present study, the effect of oxygen concentration on mitochondrial respiratory activity and red-light-dependent photosynthetic oxygen evolution by Vicia faba and Brassica napus guard cell protoplasts was examined. Comparative measurements were made with mesophyll cell protoplasts isolated from the same species. At air saturated levels of dissolved oxygen in the protoplast suspension media, respiration rates by mesophyll protoplasts ranged from 6 to 10μmoles O₂ mg^?1 chl h^?1, while guard cell protoplasts respired at rates of 200–300 μmoles O₂ mg chl^?1 h^?1, depending on the species. Lowering the oxygen concentration below 50–60 mmol m^?3 resulted in a decrease in guard cell respiration rates, while rates by mesophyll cell protoplasts were reduced only at much lower concentrations of dissolved oxygen. Rates of photosynthesis in mesophyll cell protoplasts isolated from both species showed only a minor reduction in activity at low oxygen concentrations. In contrast, photosynthesis by guard cell protoplasts isolated from V. faba and B. napus decreased concomitantly with respiration. Oligomycin, an inhibitor of oxidative phos-phorylation, reduced photosynthesis in mesophyll cell protoplasts by 27–46% and in guard cell protoplasts by 51–58%. The reduction in both guard cell photosynthesis and respiration following exposure to low oxygen concentrations suggest close metabolic coupling between the two activities, possibly mediated by the availability of substrate for respiration associated with photosynthetic electron transport activity and subsequent export of redox equivalents.     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Influence of acetylcholine agonists and antagonists on the swelling of etiolated wheat (Triticum aestivum L.) mesophyll protoplasts

Etiolated wheat (Triticum aestivum L.) mesophyll protoplasts swell within 30 min in darkness after a red light (R) pulse or addition of acetylcholine (ACh), if 0.5 mM CaCl₂ is present in the medium. In addition, ACh is also able to induce swelling in the presence of both 0.1 mM KCl or NaCl. Besides ACh, only carbamylcholine out of the choline derivatives tested was active in induction of swelling in the presence of K⁺ or Na⁺. The K⁺/Na⁺-dependent ACh-induced protoplast swelling was nullified by a ‘calmodulin inhibitor’, but not by Ca²⁺-channel blockers, Li⁺ or VO ₄ ^3- . The antagonists atropine (of muscarine-sensitive ACh receptors, mAChRs) andd-tubocurarine (of nicotine-sensitive ACh receptors, nAChRs) nullified the Ca²⁺ — and the K⁺/Na⁺-dependent protoplast swelling responses, respectively, while having no effect on the Ca²⁺-dependent R-induced swelling response. Moreover, muscarine and nicotine mimicked ACh in the Ca²⁺- and K⁺/Na⁺-dependent swelling responses respectively. Just as is the case in animal cells, the proposed mAChRs appear to be associated with a phosphatidylinositol-dependent pathway, whereas the proposed nAChRs are phosphatidylinositol independent. Similarity between the action of ACh via the proposed mChRs and R via phytochrome in protoplast swelling indicates they share in common signal-transduction pathway. We dedicate this paper to Hans Mohr on the occasion of his 60th birthday We thank the Department of Molecular Biology of the Agricultural University, Wageningen for the use of the photomicroscope and Dr. G. Fassina, Department of Pharmacology, University of Padua, Italy for the gift of nifedipine. These studies were supported by The Foundation for Fundamental Biological Research (BION) which is subsidized by The Netherlands Organization for the Advancement of Research (NWO). A.T. was also supported by: a Research Fellowship from the Agricultural University, Wageningen; a Visitors Fellowship from NWO, the Netherlands; RP II 12.15 from Ministry of Education, Poland.     

茶树叶肉原生质体的分离与纯化

以茶树幼苗叶片为材料,分析了甘露醇浓度、不同酶液组合、酶解时间等因素对叶肉原生质体分离及2种高密度分离液对原生质体纯化的影响。结果表明,叶片在含0.6 mol/L的甘露醇、1.5%的纤维素酶和2.5%的离析酶的酶解液中黑暗酶解16~18 h,叶肉原生质体的分离效果最好。此外,研究发现原生质体在10%的甘露醇与25%的碘克沙醇界面处聚集形成了一条带。     

Isolation and characterization of aleurone protoplasts from a malting variety of barley (Hordeum vulgare L.)

Summary A procedure has been developed to isolate protoplasts from mature aleurone layers of the malting variety Alexis and four other barley genotypes. It combines induction of endogenous cell wall degrading enzymes together with use of Onuzuka cellulase R 10 and driselase and results in better yields for two varieties than can be obtained with the huskless variety Himalaya. The viability of the freshly isolated protoplasts is greater than 90% and in spite of the presence of gibberellic acid during isolation procedures, most of the protoplasts are at an early developmental stage, as judged by ultrastructure. Gibberellic acid-induced changes in protoplast structure resemble those reported for Himalaya protoplasts. The protoplasts secrete both -amylase (EC 3.2.1.1) and (1-3, 1-4)--glucanase (EC 3.2.1.73) into the surrounding medium. Transfection studies using a low pI -amylase promoter to direct chloramphenicol acetyltransferase expression in aleurone protoplasts from Alexis and Himalaya revealed significant differences in their hormone responsiveness. In the absence of hormones, low levels of expression of the reporter enzyme were obtained in Alexis protoplasts, while high levels were characteristic for Himalaya protoplasts. An 8-fold increase in the expression of the reporter gene was induced by supplying the transfected Alexis protoplasts with gibberellin A₃, whereas expression in Himalaya protoplasts remained unchanged. When Himalaya protoplasts were isolated from aleurone layers that had not been incubated with GA₃ during the initial stages of protoplasting (the classical procedure), the hormone response of the promoter was 2.5-fold. It is thus possible to optimize the aleurone protoplast isolation procedure for different barley genotypes and mutants of interest in studies of transgenic gene expression and hormone induced secretion of proteins from this unique secretory plant tissue.Abbreviations ABA abscisic acid - APIM aleurone protoplast isolation medium - CAT chloramphenicol acetyltransferase - EDTA ethylenediamine tetraacetic acid - ER endoplasmic reticulum - GA₃ gibberellin A₃ - IgG immunoglobulin G - MES 2-(N-morpholino)-ethanesulfonic acid - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - pI isoelectric point - PIPES piperazine N,N-bis-(diethanesulfonic acid) - SDS sodium dodecyl sulfate     

Effects of a Ficoll-agarose system on early division and development of mesophyll protoplasts of winter oilseed rape (Brassica napus L.)

A method is described for regenerating callus from mesophyll protoplasts of a winter variety of Brassica napus. The method combines the use of Ficoll in an initial liquid medium, enhancing early protoplast division and cell colony formation, with a transfer to an agarose system after 10 days culture to give rapid microcalli formation. Further transfers resulted in callus regeneration and the initiation of organogenesis.