Cell wall synthesis and salt (saline) sensitivity in cultured colt cherry (Prunus avium × pseudocerasus) protoplasts

In order to investigate the cellular basis of salt tolerance, Colt cherry (Prunus avium ×pseudocerasus) protoplasts from mesophyll tissues and root cell suspension cultures were cultured in the presence of NaCl, KCl or Na₂SO₄, at normalities of 25, 50, 100 or 200 mN for each salt and with or without 2,6-dichlorobenzonitrile, an inhibitor of cell wall synthesis. Results showed that the acquisition of salt tolerance was concomitant with the onset of cell wall regeneration, with protoplasts exhibiting a greater salt tolerance than cells.Abbreviations DBN 2,6-dichlorobenzonitrile - FDA fluorescein diacetate     

Isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato

Optimum conditions for the isolation and culture of protoplasts from high anthocyanin-producing callus of sweet potato (Ipomoea batatas) were investigated. Growth phase of callus and the ratio of callus-enzyme solution affected the yield of protoplasts. Composition of the medium and protoplast density were examined for protoplast culture.Small colonies were regenerated from the protoplasts. Upon transfer to light a high amount of anthocyanin was accumulated in these colonies.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-morpholino)-ethanesulfonic acid     

Chemical inhibition of cell wall formation and cytokinesis,but not of nuclear division,in protoplasts of Nicotiana tabacum L. cultivated in vitro

The effect of cytochalasin B, colchicine, coumarin and 2,6-dichlorobenzonitrile on cell wall formation and cellular division was studied by light and electron microscopy with tobacco mesophyll protoplasts cultivated in vitro. 2,6-dichlorobenzonitrile was found to be the most effective and reversible inhibitor of cell wall formation. The other inhibitors caused irreversible damage and/or inhibited mitosis. In protoplasts cultivated in the presence of 2,6-dichlorobenzonitrile the total inhibition of cell wall formation had no effect on nuclear division, but cytokinesis was totally inhibited so that multinucleate protoplasts were obtained.Abbreviations DB 2,6-dichlorobenzonitrile=dichlobenil - CB cytochalasin B     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Isolation of protoplasts from female gametophytes of Torenia fournieri

Protoplasts from the cells of mature embryo sacs (ES-protoplasts) of Torenia fournieri were obtained during incubation of ovules in an enzyme solution. Four protoplasts which arose from each embryo sac were connected together after isolation, or aggregates of the egg cell protoplast and two synergide protoplasts dissociated from the protoplast of the central cell. The ES-protoplasts stayed viable for 2 weeks in culture, but they did not regenerate cell walls.Abbreviations ES embryo sac - FAA fixative (formalin : acetic acid : alcohol = 1 : 1 : 18) - FDA fluorescein diacetate - PAS periodic acid Schiff reaction - 2,4-D 2,4-dichlorophenoxyacetic acid     

Callus formation from protoplasts of a sugarbeet cell suspension culture

Protoplasts of sugarbeet (Beta vulgaris L.) were isolated from cell suspension cultures and cultured in modified PGo medium. Conditions required for the efficient division of the protoplasts were investigated.The optimal combination of phytohormones was found to be 1 mg/l NAA, 0.2 mg/l 2,4-D, 0.5 mg/l zeatin. Protoplast division was also considerably stimulated by the addition of 250 mg/l casein hydrolysate, 200 mg/l yeast extract, and 20% v/v conditioned culture medium to the protoplast culture medium. The highest division rate (up to 35% of the protoplasts) was achieved at a density of 4×10⁴- 1×10⁵ protoplasts/ml. From the colonies callus and suspension cultures were readily obtained.Abbreviations BAP 6-Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - Kin 6-Furfurylaminopurine (kinetin) - NAA -Naphtalenacetic acid - Zea Zeatin     

Role of the alfalfa mosaic virus movement protein and coat protein in virus transport

The movement protein (MP) and coat protein (CP) encoded by Alfalfa mosaic virus (AMV) RNA 3 are both required for virus transport. RNA 3 vectors that expressed nonfused green fluorescent protein (GFP), MP:GPF fusions, or GFP:CP fusions were used to study the functioning of mutant MP and CP in protoplasts and plants. C-terminal deletions of up to 21 amino acids did not interfere with the function of the CP in cell-to-cell movement, although some of these mutations interfered with virion assembly. Deletion of the N-terminal 11 or C-terminal 45 amino acids did not interfere with the ability of MP to assemble into tubular structures on the protoplast surface. Additionally, N- or C-terminal deletions disrupted tubule formation. A GFP:CP fusion was targeted specifically into tubules consisting of a wild-type MP. All MP deletion mutants that showed cell-to-cell and systemic movement in plants were able to form tubular structures on the surface of protoplasts. Brome mosaic virus (BMV) MP did not support AMV transport. When the C-terminal 48 amino acids were replaced by the C-terminal 44 amino acids of the AMV MP, however, the BMV/AMV chimeric protein permitted wild-type levels of AMV transport. Apparently, the C terminus of the AMV MP, although dispensable for cell-to-cell movement, confers specificity to the transport process.     

Techniques for enhanced release of leaf protoplasts in Populus

Microscopic examination of Populus leaf tissue following enzyme treatment revealed two factors contributing to low protoplast yields: (1) poor penetration of the enzymes into the tissue, and (2) entrapment of protoplasts in leaf debris during protoplast purification procedures. A simple combination of rapid grinding of the tissue in an Omni-mixer prior to enzyme treatment and forceful washing of leaf-debris after digestion provided high exposure of the cells, uniform digestion, and high yields of protoplasts of two Populus clones. Protoplasts exhibited cell wall regeneration and long-term viability in culture. The relative yield advantages of the techniques varied with the inherent digestibility of each clone but could produce up to 600 percent greater protoplast yields in a woody plant species in which protoplast isolation was previously limited. The techniques were also applicable to an herbaceous species, Solanum etuberosum.Abbreviations BA benzyladenine - NAA naphthalene acetic acid - WPM Woody Plant Medium, Lloyd and McCown (1980) - MS Murashige and Skoog Medium (1962) - (NC-XXXX) North Central Forest Experiment Station accession number assigned to Populus hybrid clones     

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.Abbreviations BA N⁶-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA naphthaleneacetic acid     

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)     

Plant regeneration from protoplast culture of sweet potato (Ipomoea batatas Lam.)

This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1^–1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1^–1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.Abbreviations MS Murashige and Skoog basal medium - IAA Indol-3-acetic acid - NAA naphthaleneacetic acid - 2,4-D dichlorophenoxyacetic acid - Mes 2-(N-morpholino)-ethanesulfonic acid - Cpw cell and protoplast washing solution     

Plant regeneration from protoplasts of embryogenic cell suspensions of Coffea arabica L. cv. caturra

Coffee plants were regenerated from protoplasts isolated from embryogenic cell suspension cultures derived from somatic embryos of Coffea arabica L. cv. caturra. Yields of viable protoplasts ranged from 1×10⁵ to 6×10⁵ protoplast/g fresh weight. Protoplast preparations usually contained no contaminating cells, and when present, the number of cells never exceeded 0.1% of the total. Plating efficiencies of protoplast ranged from 1 to 10%. Embryogenic protocolonies obtained after several subcultures in a medium supplemented with 0.5 mg/l each of benzylaminopurine, 2,4-dichlorophenoxyacetic acid and naphtaleneacetic acid, were transferred to a medium lacking plant growth regulators. Well differentiated embryos were formed in selected protocolonies that contained many embryos-like structures. Approximately 70% of the somatic embryos developed into green rooted plantlets which were succesfully transferred to vessels containing sterilized scoria. Plants grown for two months in scoria were finally transferred to greenhouse.Abbreviations B5 medium according to Gamborg et. al.(1968) - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphtaleneacetic acid     

Regeneration of isolated mesophyll and cell suspension protoplasts to plants in Stylosanthes guianensis. A tropical forage legume

Protoplasts were isolated from leaf mesophyll and cell suspensions of two accessions of Stylosanthes guianensis (Aubl.), a tropical forage legume. When cultured in VKM liquid culture medium, both types of protoplasts divided at a rate of 4–8%, and subsequently formed cell colonies. Protoplast-derived calluses produced numerous shoots when transferred to regeneration medium. Regenerated shoots could be easily rooted, and plantlets were transferred to soil. The effects of several factors on the efficiency of this protoplast system have been investigated.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - NAA Naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Zea Zeatin     

Plant regeneration from protoplasts of Panax ginseng (C.A. Meyer) through somatic embryogenesis

Summary Protoplasts of Panax ginseng were isolated from embryos obtained from the 4-year old embryogenic cell line KCTC PCL 49031 which was derived from a zygotic embryo. High protoplast yields of 22–25 × 10⁶ protoplast / g tissue were obtained following 5–6 h digestion with 2% Cellulysin, 1% Pectinase and 1% Macerasae in half strength Murashige and Skoog's medium containing 12% mannitol. A plating density of 1×10⁵ protoplasts /ml was found optimal for protoplast culture. An initial division frequency of 10% was obtained in an agarosegelled defined medium. Myo-inositol (6%) was found to be the most suitable osmoticum. Somatic embryos were formed from protoplast derived embryogenic callus, which regenerated into plantlets.Abbreviations NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kn kinetin - BA benzyladenine - GA₃ gibberellic acid - MS Murashige and Skoog medium     

Intracellular localization and movement phenotypes of alfalfa mosaic virus movement protein mutants

Thirteen mutations were introduced in the movement protein (MP) gene of Alfalfa mosaic virus (AMV) fused to the green fluorescent protein (GFP) gene and the mutant MP-GFP fusions were expressed transiently in tobacco protoplasts, tobacco suspension cells, and epidermal cells of tobacco leaves. In addition, the mutations were introduced in the MP gene of AMV RNA 3 and the mutant RNAs were used to infect tobacco plants. Ten mutants were affected in one or more of the following functions of MP: the formation of tubular structures on the surface of protoplasts, association with the endoplasmic reticulum (ER) of suspension cells and epidermal cells, targeting to punctate structures in the cell wall of epidermis cells, movement from transfected cells to adjacent cells in epidermis tissue, cell-to-cell movement, or long-distance movement in plants. The mutations point to functional domains of the MP and support the proposed order of events in AMV transport. Studies with several inhibitors indicate that actin or microtubule components of the cytoskeleton are not involved in tubule formation by AMV MP. Evidence was obtained that tubular structures on the surface of transfected protoplasts contain ER- or plasmalemma-derived material.     

The isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca (moench) voss

Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl₂.2H₂O produced an average of 4.5 × 10⁶ protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 10⁵ protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxy-acetic acid - FDA fluorescein diacetate NRCC No. 27937     

Intergeneric protoplast fusion between Brassica carinata and Camelina sativa

Camelina sativa is a wild crucifer that is reported to be resistant to Alternaria blight. Polyethylene glycol mediated fusion was attempted between protoplasts from etiolated hypocotyls of Brassica carinata and mesophyll protoplasts of Camelina sativa. The mean frequency of heterokaryons was 6.8%. Three hybrid shoots were regenerated, each from a single fusionderived callus. These shoots failed to produce roots capable of withstanding transplantation. Confirmation of hybridity was obtained from the morphology of in vitro produced leaves, somatic chromosome number in leaf tips, and restriction fragment length polymorphism for a nuclear rDNA probe. Analysis for organelle constitution using RFLPs indicated that the hybrid contained chrloroplasts derived from the wild species and mitochondria from the cultivated Brassica species.Abbreviations 2,4-D 2,4-dichlorophenoxy-acetic acid - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid - IBA Indole-3-butyric acid - GA₃ gibberellic acid - BAP 6-Benzylaminopurine - MS Murashige and Skoog (1962) basal medium     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Induction of efficient cell division in alfalfa protoplasts

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2–10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.Contribution No. 774 Ottawa Research Station