Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Seasonal and physiological aspects of the isolation of tobacco protoplasts

The viability of tobacco ( N. tabacum – L. cv. Xanthi-nc) protoplasts isolated during the winter months is affected by membrane calcium, total calcium, plant age and supplementary lighting. Feeding calcium nitrate and calcium chloride helps to increase protoplast stability. Feeding is important because it increases membrane calcium, but it also has other beneficial effects which may be connected with increased nitrate and chloride (anion) uptake and cation/anion interaction. Whatever optimal feeding regime is chosen, it must be used in conjunction with the correct harvesting practice, which depends on whether or not supplementary light is given. Plants grown without supplementary light are best harvested between 8–9 weeks. If supplementary light is given, older plants (with and without supplementary calcium feeding) of approx. ten weeks are better for protoplast isolation. Plants grown under supplementary light have low leaf shape index (LSI) values (i.e. wider leaves) compared to plants in low light.
Spring and summer calcium feeding treatments had no significant effect on increasing total or membrane calcium. Likewise, there was no significant correlation between total/membrane calcium and percentage protoplast viability from days 0–5; and protoplasts isolated from plants grown in shade and fed with calcium salts, were no more stable than protoplasts from control plants. Irrespective of the feed treatments given, spring and summer plants older than about fifty days and grown under shaded and normal light conditions, produced poor protoplasts despite the fact that there was more calcium present in the membranes compared to protoplasts of younger plants. This decrease in percentage protoplast viability appears to be associated with changes in leaf shape. As plants age from 49–63 days, the leaf shape index (LSI) values increase (i.e. leaves are becoming narrower) and % protoplast viability decreases.     

Plant regeneration from leaf protoplasts of Brassica oleracea var. italica CV Green Comet broccoli

A procedure is described for regeneration of plants from leaf protoplasts of the hybrid broccoli cultivar, Green Comet (Brassica oleracea var italica). The totipotency of protoplasts isolated from plants regenerated from hypocotyl explants (GCR) was greater than that of protoplasts from plants grown directly from seed (GC). Using medium B developed by Pelletier et al (1983), division efficiencies greater than 70% were obtained in leaf protoplasts isolated from GCR. Approximately 1% of these protoplasts formed calli on solidified medium; 77% of the calli regenerated shoots. In contrast, protoplasts from seed-grown material showed a lower division efficiency (15–22%) and fewer protoplast-derived calli produced shoots. Some of the 178 protoplast-derived plants grown to maturity had variant phenotypes.Abbreviations NAA napthalene acetic acid - BA 6-benzylaminopurine - MES 1-morpholino-ethane sulfonate This work has been submitted by D. R. in partial fulfillment of the requirement for the Ph.D. degree     

A method for isolating a high yield of Arabidopsis chloroplasts capable of efficient import of precursor proteins

Chloroplasts were isolated from Arabidopsis plants grown under different conditions, and using different protocols, to determine a method that would yield chloroplasts capable of binding and importing precursor proteins. Chloroplasts isolated from protoplasts and purified on a Percoll gradient were highly import-competent, with little non-specific binding of the precursor, and a high yield of intact chloroplasts (0.1 mg chlorophyll/g FW). Chloroplasts from plants grown on agar plates had a much higher rate of import than those from plants grown on soil. Protein import remained high at all of the ages tested for chloroplasts from plate-grown plants, whereas it declined during the development of soil-grown plants. Arabidopsis chloroplasts imported a range of precursor proteins and had nucleotide requirements for binding and import similar to those reported for pea chloroplasts.     

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5–12 months old cell suspensions showing visible colony formation in the range of 180–1050 colonies/10⁶ protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10⁶ protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - NAA naphthaleneacetic acid     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Protoplast Cultures from Cell Suspensions of Daucus carota

Protoplasts, enzymatically isolated from cell suspension cultures of Daucus carota, have been grown in small Petri-dishes. After enzyme treatment and washing the protoplasts were plated in agar media. Growth and divisions were viewed through the bottom of the Petri-dishes with a light microscope. Different osmotic stabilizers were tested with respect to their ability to promote wall formation and growth of the protoplasts. Combinations of sucrose, sorbitol and “Modopeg” gave the best results. Electron micrographs of cultured protoplasts revealed normal as well as abnormal nuclear conditions.     

Culture of soybean mesophyll protoplasts in alginate beads

Mesophyll protoplasts were isolated from leaves of 10 day old aseptically grown soybean seedlings, or from surface disinfested leaves of 3 week old plants grown in environmental chambers. The protoplasts were encapsulated in 2mm diameter Ca alginate beads. Immobilized protoplasts were induced to divide by culturing in shaker flasks containing an actively growing soybean cell suspension. The feeder cell suspension supported the division of protoplasts independent of the protoplast density in the Ca alginate beads. At day 18 after encapsulation, the alginate matrix was dissolved, releasing viable callus colonies. The feeder cell suspension obviated plating of protoplasts at high density which is usually required for subsequent cell division and colony development. Since the protoplasts were embedded at low density, the cell colonies were derived from single cells.     

Mesophyll protoplasts from Solanum melongena var depressum bailey regenerate to fertile plants

Mesophyll protoplasts have been isolated from soil grown plants of Solanum melongena var depressum Bailey and protoplast-derived colonies regenerated in liquid DPD culture medium. Protoplast-derived colonies proliferated on agar-solidified MS culture medium and regenerated shoots which grew to fertile plants in potting compost.on leave from Landzhou University, Peoples Republic of China     

The characterization of herbicide tolerant plants in Brassica napus L. after in vitro selection of microspores and protoplasts

Brassica napus L.(cv Topas) plants tolerant to chlorsulfuron (CS) were isolated after selection experiments utilizing microspores and haploid protoplasts. The first microspore-derived plant (M-37,) was CS tolerant, haploid and sterile. Normal plant morphology and fertility was restored after colchicine doubling. A CS tolerant plant was also selected from protoplasts (P-26) isolated from microspore-derived embryo tissue and grown on medium containing CS. P-26 was aneuploid, CS tolerant and had very low fertility. The two selected lines produced selfed progeny which were tolerant to from 10–100 times the CS levels of the corresponding Topas plants. Microspores and protoplasts derived from the selfed plants were also CS tolerant. The segregation pattern for CS tolerance from reciprocally crossed progeny of M-37 and Topas was consistent with a semi-dominant nuclear mode of inheritance. Biochemical analysis of the two mutants indicated that the microspore-derived mutant and F1 crosses contained an altered acetohydroxyacid synthase (AHAS) enzyme, while the AHAS activity of the protoplast mutant was similar to Topas. Selfed seed from the M-37 plants have provided tolerance to CS in both greenhouse and field tests. S1 plants from a second microspore selected mutant (M-42) have tolerated 30 g/ha of CS in greenhouse tests. The two single-celled selection systems are discussed and the microspore selection system highlighted as a new method for in vitro selection.     

A Method for the Isolation of Stable Mesophyll Protoplasts from Tomato Leaves throughout the Year under Standard Conditions

A method is described, based on growing tomato plants under low light intensities and feeding with calcium nitrate, whereby stable protoplasts can be isolated throughout the year under standard conditions. Plants are grown in the equivalent of spring and winter light intensities (i. e. incoming solar radiation 2.52 to 10.8 MJ m^?2 day^?1 15 h day) in the glasshouse on peat compost supplied with a balanced fertilizer containing nitrate and urea as nitrogen sources and given a supplementary feed of calcium nitrate. Under these conditions, high yields of protoplasts can be isolated on incubation in the dark using low enzyme concentrations at 25°C for 2 h. Protoplasts isolated in this way can support tobacco mosaic virus replication and are capable of cell division.     

Citrate-permeable channels in the plasma membrane of cluster roots from white lupin

White lupin (Lupinus albus) is well adapted to phosphorus deficiency by developing cluster roots that release large amounts of citrate into the rhizosphere to mobilize the sparingly soluble phosphorus. To determine the mechanism underlying citrate release from cluster roots, we isolated protoplasts from different types of roots of white lupin plants grown in phosphorus-replete (+P) and phosphorus-deficient (-P) conditions and used the patch-clamp technique to measure the whole-cell currents flowing across plasma membrane of these protoplasts. Two main types of anion conductance were observed in protoplasts prepared from cluster root tissue: (1) an inwardly rectifying anion conductance (IRAC) activated by membrane hyperpolarization, and (2) an outwardly rectifying anion conductance (ORAC) that became more activated with membrane depolarization. Although ORAC was an outward rectifier, it did allow substantial inward current (anion efflux) to occur. Both conductances showed citrate permeability, with IRAC being more selective for citrate3- than Cl- (PCit/PCl = 26.3), while ORAC was selective for Cl- over citrate (PCl/PCit = 3.7). Both IRAC and ORAC were sensitive to the anion channel blocker anthracene-9-carboxylic acid. These currents were also detected in protoplasts derived from noncluster roots of -P plants, as well as from normal (noncluster) roots of plants grown with 25 microm phosphorus (+P). No differences were observed in the magnitude or frequency of IRAC and ORAC currents between the cluster roots and noncluster roots of -P plants. However, the IRAC current from +P plants occurred less frequently than in the -P plants. IRAC was unaffected by external phosphate, but ORAC had reduced inward current (anion efflux) when phosphate was present in the external medium. Our data suggest that IRAC is the main pathway for citrate efflux from white lupin roots, but ORAC may also contribute to citrate efflux.     

Influence of primary callus induction conditions on the establishment of barley cell suspensions yielding regenerable protoplasts

Summary With the aim of the development of a culture method for efficient plant regeneration from barley (Hordeum vulgare L.) protoplasts, we examined several culture conditions for primary calli from immature embryos of cvs. Dissa and Igri, which were used for initiation of cell suspensions. Among the primary callus culture conditions tested, growth condition of donor plants had a great impact on these efficiencies; Igri protoplasts derived from embryos of plants grown in a greenhouse gave rise to albino plants and few green shoots while several cell lines originating from embryos of plants grown in a growth chamber (16h light, 12°C) yielded protoplasts developing into green plants. In contrast, cell suspensions were produced at higher frequencies from calli derived from embryos of greenhouse-grown Dissa plants. In Igri, increased levels of 2,4-dichlorophenoxyaceticacid (2,4-D) significantly reduced the efficiency of cell suspension establishment and plant regeneration from protoplasts was achieved only with suspension cells derived from calli induced at the lowest level (2.5 mg/l), while the effect of the 2,4-D concentration was not clear in Dissa. The developmental stage of immature embryos also affected the efficiency of cell suspension establishment, and the optimal embryo size was determined to be approximately 1mm in diameter. These results demonstrate the importance of callus induction conditions for successful barley protoplast culture.     

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     

Potato Cell Plasma Membrane Receptors to Ring Rot Pathogen Extracellular Polysaccharides

The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome‐labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Cms are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.     

New evidence about the relationship between water channel activity and calcium in salinity-stressed pepper plants

This study, of how Ca2+ availability (intracellular, extracellular or linked to the membrane) influences the functionality of aquaporins of pepper (Capsicum annuum L.) plants grown under salinity stress, was carried out in plants treated with NaCl (50 mM), CaCl2 (10 mM), and CaCl2 (10 mM) + NaCl (50 mM). For this, water transport through the plasma membrane of isolated protoplasts, and the involvement of aquaporins and calcium (extracellular, intracellular and linked to the membrane) has been determined. After these treatments, it could be seen that the calcium concentration was reduced in the apoplast, in the cells and on the plasma membrane of roots of pepper plants grown under saline conditions; these concentrations were increased or restored when extra calcium was added to the nutrient solution. Protoplasts extracted from plants grown under Ca2+ starvation showed no aquaporin functionality. However, for the protoplasts to which calcium was added, an increase of aquaporin functionality of the plasma membrane was observed [osmotic water permeability (Pf) inhibition after Hg addition]. Interestingly, when verapamil (a Ca2+ channel blocker) was added, no functionality was observed, even when Ca2+ was added with verapamil. Therefore, calcium seems to be involved in plasma membrane aquaporin regulation via a chain of processes within the cell but not by alteration of the stability of the plasma membrane.     

Plant regeneration from callus and protoplast cultures of Helianthus giganteus L.

Summary Methods of plant regeneration from callus and protoplasts of Helianthus giganteus L. are described. Embryogenic callus was obtained from leaf explants and plants were regenerated from these calli on MS media with different combinations of benzyladenine and naphtaleneacetic acid. Leaf protoplasts isolated from in vitro grown plants formed somatic embryos when cultured in agarose solidified droplets of V-KM medium containing benzyladenine and naphtaleneacetic acid. Embryos developed into plantlets on media with reduced auxin contents. Regenerated plants were successfully planted in soil.Abbreviations BA benzyladenine - IAA indoleacetic acid - MS Murashige and Skoog medium - NAA naphtaleneacetic acid - V-KM protoplast culture medium of Binding and Nehls     

Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation

Organic-acid secretion from higher plant roots into the rhizosphere plays an important role in nutrient acquisition and metal detoxification. In this study we report the electrophysiological characterization of anion channels in Arabidopsis (Arabidopsis thaliana) root epidermal cells and show that anion channels represent a pathway for citrate efflux to the soil solution. Plants were grown in nutrient-replete conditions and the patch clamp technique was applied to protoplasts isolated from the root epidermal cells of the elongation zone and young root hairs. Using SO4(2-) as the dominant anion in the pipette, voltage-dependent whole-cell inward currents were activated at membrane potentials positive of -180 mV exhibiting a maximum peak inward current (I(peak)) at approximately -130 mV. These currents reversed at potentials close to the equilibrium potential for SO4(2-), indicating that the inward currents represented SO4(2-) efflux. Replacing intracellular SO4(2-) with Cl- or NO3(-) resulted in inward currents exhibiting similar properties to the SO4(2-) efflux currents, suggesting that these channels were also permeable to a range of inorganic anions; however when intracellular SO4(2-) was replaced with citrate or malate, no inward currents were ever observed. Outside-out patches were used to characterize a 12.4-picoSiemens channel responsible for these whole-cell currents. Citrate efflux from Arabidopsis roots is induced by phosphate starvation. Thus, we investigated anion channel activity from root epidermal protoplasts isolated from Arabidopsis plants deprived of phosphate for up to 7 d after being grown for 10 d on phosphate-replete media (1.25 mm). In contrast to phosphate-replete plants, protoplasts from phosphate-starved roots exhibited depolarization-activated voltage-dependent citrate and malate efflux currents. Furthermore, phosphate starvation did not regulate inorganic anion efflux, suggesting that citrate efflux is probably mediated by novel anion channel activity, which could have a role in phosphate acquisition.     

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.