Somatic embryogenesis in protoplast derived calli of cultivated jute,Corchorus capsularis L.

Protoplasts were isolated from cotyledon, hypocotyl and mesophyll cells of Corchorus capsularis L., a major fibre crop, by one step enzyme digestion method. They were further cultured successfully on modified KM-8p (Kao and Michayluk, 1975) medium to form microcalli. The required cultural conditions could be used to achieve 34% to 78% plating efficiency, depending upon the source of protoplasts. Hypocotyl protoplasts gave the highest plating efficiency. On transfer to regeneration medium, somatic embryos developed at high frequency. The present success is a significant step forward in the development of meaningful plant cell culture methods for application in jute.Abbreviations B₅ Gamborg et al., 1968 - MS Murashige and Skoog, 1962 - SH Schenk and Hildebrandt, 1972 - Ad-SO₄ Adenine sulphate - BAP 6-benzylaminopurine - NAA 1-naphthalene acetic acid - GA₃ Gibberellic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - Kn Kinetin - IBA Indole-3-butyric acid     

Phytosulfokine stimulates cell divisions in sugar beet (Beta vulgaris L.) mesophyll protoplast cultures

The aim of this work was to improve plating efficiency of sugar beet mesophyll protoplast cultures. Preliminary experiments showed that cultures of good quality, viable protoplasts were obtained in rich media based on the Kao and Michayluk formulation and with the calcium alginate as an embedding matrix. Nevertheless, in these cultures cell divisions were either not observed or very seldom confirming earlier reported recalcitrance of sugar beet protoplasts. The recalcitrant status of these cultures was reversed upon application of exogenous phytosulfokine (PSK)—a peptidyl plant growth factor. The highest effectiveness of PSK was observed at 100 nM concentration. Plating efficiencies obtained in the presence of PSK reached approximately 20% of the total cultured cells. The stimulatory effect of phytosulfokine was observed for all tested breeding stocks of sugar beet. Our data indicate that PSK is a powerful agent able to overcome recalcitrance of plant protoplast cultures.     

Direct gene transfer to plant protoplasts by mild sonication

Summary A novel procedure employing mild sonication for transformation of plant protoplasts is described. Transient expression of a chloramphenicol acetyltransferase (CAT) gene in protoplasts of sugar beet (Beta vulgaris L.) and tobacco (Nicotiana tabacum L.) was obtained by a brief exposure of the protoplasts to 20 kHz ultrasound in the presence of plasmid DNA. Maximum levels of CAT activity were achieved by sonication for 500–900 ms at 30–70 W electric power (0.65–1.6 W/cm2 acoustic power). This reduced the viability to 15–20 % and 60 % for sugar beet and tobacco protoplasts, respectively. Up to 12 % (sugar beet) and 81 % (tobacco) of maximum transient expression could be achieved with no significant loss of viability. Protoplasts surviving exposure to ultrasound were found to have a similar long-term viability and to regenerate to micro-calli as untreated protoplasts. Plasmid DNA concentrations of 80–110 g/ml and sucrose concentrations of 21–28 % in the sonication medium were found to be optimal for transient expression.Abbreviations CAT chloramphenicol acetyltransferase     

Interspecific hybridization between Brassica juncea and B. spinescens through protoplast fusion

Hypocotyl derived protoplasts of B. juncea cv. RLM-198 were fused with mesophyll protoplasts of B. spinescens using polyethylene glycol to produce interspecific hybrids. Fusion products could be microscopically identified by characteristics of the protoplasts of both parents in the hybrid cells; they are colourless and vacuolated like the hypocotyl protoplasts and possess chloroplasts of the mesophyll protoplasts. The heterokaryotic fusion frequency was around 5%. However, the frequency of calli regenerating hybrid shoots was more than 10% of the regenerating calli. Putative somatic hybrids had morphological features characteristic of both the parents. Twelve plants analysed cytologically, possessed 52 chromosomes (26_II) at meiosis representing the complete genomes of B. juncea (18_II) and B. spinescens (8_II). For esterase isozymes, the hybrids had bands of Doth the parents. Hybrid nature of some of the plants was confirmed by their close resemblance to B. juncea, chromosome number and isozyme bands of B. spinescens as in Rsp-19. Somatic hybrids had rudimentary, non-dehiscent anthers and completely sterile pollen. However, on back crossing with B. juncea, 10 out of 12 plants produced seeds and about 100 plants were realized.Abbreviations PEG Polyethylene glycol     

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     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plant regeneration from mesophyll protoplasts of Diplotaxis muralis,a wild crucifer

Mesophyll protoplasts from leaves of aseptically grown shoot tips of Diplotaxis muralis were isolated (6.2–7.1×10⁵ protoplasts/g fresh weight of tissue) using one step enzyme digestion. The protoplasts (71% viability) underwent divisions (4.2+0.1%) on plating in M8PS₂ medium and ultimately formed calli with 0.45+0.03% plating efficiency. Plant regeneration could be achieved both through embryogenesis and organogenesis. The efficiency of plant regeneration through organogenesis was 9 times higher than embryogenesis. Forty eight out of 52 plants regenerated so far from 3 independent experiments were normal with respect to fertility and meiotic chromosomal behavior.Abbreviations BAP 6-benzylaminopurine - GA₃ Gibberellic acid - A Kao and Michayluk, 1981 - KM Kao and Michayluk, 1975 - MK₃ Modified K₃ - M8P Modified 8P - MS Murashige and Skoog, 1962 - NAA 1-naphthalene acetic acid - PE Plating efficiency     

High frequency of fusion induced in freely suspended protoplast mixtures by polyethylene glycol and dimethylsulfoxide at high pH

Fusion of freely suspended protoplast mixtures (hypocotyl protoplasts of Brassica napus mixed with mesophyll protoplasts of either B. campestris or Nicotiana plumbaginifolia) was induced by a solution containing 10% polyethylene glycol, 10% dimethyl-sulfoxide and 0.1M glycine-NaOH buffer (pH 10.0). The fusion products represented 15 to 17 percent of the surviving cells. More than 50% of the fusion products divided within two days after fusion, indicating that the fusion procedure did not significantly affect the viability of fused cells. The fusion products were not bound to the surface of the fusion vessel, so they could be isolated with a micropipette immediately after fusion.Abbreviations PEG polyethylene glycol - DMSO dimethylsulfoxide     

Isolation of protoplasts and regeneration of callus from suspension cultures of cultivated beets

Conditions necessary for the isolation and culture of protoplasts from suspension cultures of sugar, fodder and garden beets were investigated. Good yields of protoplasts were obtained by treating cells with a mixture of cellulase, Macerozyme and Driselase enzymes. Nutritional requirements of beet protoplasts were found to be quite simple: protoplasts could be cultured in MS, B₅ or PGo based media with 0.4 M glucose with the optimum result being produced on KM8p medium. Plating efficiency (P.E) was genotype-dependent with the sugar beet giving better P.E. than the fodder or garden beets used, and higher values being achieved with the use of desalted Driselase for isolation followed by culture on KMBp medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP N⁶ benzylaminopurine - NAA naphthaleneacetic acid - P.E. plating efficiency - * University of Birmingham beet germplasm accession number     

Organogenesis of stem and leaf protoplasts of a haploid golden delicious apple clone (Malus Xdomestica Borkh.)

Summary Highly viable protoplasts were isolated in large numbers from in vitro-grown leaf and stem tissues of a haploid clone of the apple scion cultivar Golden Delicious (Malus Xdomestica Borkh.). Protoplasts from both sources divided rapidly to give microcallus, when cultured in a modified Kao and Michayluk-based medium. Following two successive subcultures for callusing, shoot buds were regenerated from such calli, on half-strength Murashige and Skoog medium with an increased concentration of group B vitamins and containing 5.0 mg.l^-1 6-benzyl-aminopurine and 0.1 mg.l^-1 l-naphthaleneacetic acid (for the leaf protoplast-derived calli) or 4-indole-3yl-butyric acid (for stem protoplast-derived calli). The mesophyll protoplast-derived shoots were enfeebled and vitrified, in time with their ultimate death. Conversely, for those shoots deriving from the stem protoplasts, in vitro propagation was successfully achieved. This is the first report on the successful isolation, culture and organogenesis from stem protoplasts of a woody plant genotype.Abbreviations BAP 6-benzylaminopurine - FPE final plating efficiency - IBA 4-indole-3yl-butyric acid - IPE initial plating efficiency - f wt fresh weight - KM Kao and Michayluk (1975) - MES 2-N-morpholino ethane sulfonic acid - MPE intermediate plating efficiency - MS Murashige and Skoog (1962) - NAA l-naphthaleneacetic acid - PVP-10 polyvinylpyrrolidone (Av MW 10,000)     

Improved efficiency of plant regeneration from protoplasts of eggplant Solanum melongena L.

Eggplant (Solanum melongena L.) mesophyll protoplasts were obtained from in vitro growing plants of line 410 and cv. Classic. Relatively high (15%) plating efficiency was achieved using petri dishes with alternate quadrants containing reservoir medium (R medium + 1% activated charcoal) and culture medium. Shoot regeneration occurred within 6 weeks following initiation of protoplast culture.Contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan Israel, No. 1164-E, 1984 Series.     

An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

An improved method for electroporation in plant protoplasts: infection of tobacco protoplasts by tobacco mosaic virus particles

Conditions of electroporation were optimized for introduction of tobacco mosaic virus (TMV) particles into tobacco mesophyll protoplasts (Nicotiana tabacum L. cv. Petit Havana SR1). Compared with conditions for TMV-RNA uptake, a longer electric pulse was necessary at the same voltage to induce TMV particle entry. Up to 80–90% of the protoplasts were infected with TMV particles after exposure to a 10 msec pulse at 200 V (0.67 KV/cm) in a 0.5 M mannitol solution. Protoplast viability was slightly lower than for controls which did not undergo electroporation. The presence of buffer in the mannitol solution reduced the net voltage in the solution which resulted in a significant decrease of the level of infection. These results suggest that the membrane pores resulting from an electrical pulse were wide enough for TMV particles (300 × 18 nm) to enter protoplasts.     

The culture response of Arabidopsis thaliana protoplasts is determined by the growth conditions of donor plants

Procedures previously established for plant regeneration from mesophyll protoplasts of Arabidopsis thaliana have been optimized. The protoplast plating efficiency (number of microcalli per number of plated protoplasts) is strongly dependent on the growth conditions of the donor plant, especially medium composition and illumination conditions. The yield of microcalli is markedly influenced by the nitrogen source in the protoplast culture medium. The developed procedure gives reproducible plating efficiencies of 7–10%, i.e. 15–20 times higher than previously published (0.5%) for A. thaliana race C24.     

A method for production of interspecific hybrids within Brassiceae via somatic hybridization,using resynthesis of Brassica napus as a model

A general method for the production of somatic hybrids within Brassiceae has been developed using mesophyll protoplasts from one parent and hypocotyl protoplasts from the other. Fusion products were easily identified by their intermediate phenotype between the parental protoplasts. They could be isolated 24 h after fusion with micropipettes using a micromanipulator. At that time their frequency was about 15%. They were cultured in small volumes, 10 μl, and a plating efficiency of 14% was obtained. Hybrid calli were obtained from the fusion products, which was confirmed by isozyme analysis. Ploidy level of one hybrid shoot was determined by flow cytometric DNA analysis.     

Iron deficiency decreases the Fe(III)-chelate reducing activity of leaf protoplasts

The ferric-chelate reductase (FC-R) activity of mesophyll protoplasts isolated from Fe-sufficient (control) and Fe-deficient sugar beet (Beta vulgaris L.) leaves has been characterized. Measurements were made in an ionic environment similar to that in the apoplastic space of the sugar beet mesophyll cells. The FC-R activity of Fe-sufficient and Fe-deficient protoplasts was dependent on light. Fe deficiency decreased markedly the FC-R activity per protoplast surface unit. The optimal pH for the activity of the FC-R in mesophyll protoplasts was in the range 5.5 to 6.0, typical of the apoplastic space. Beyond pH 6.0, the activity of the FC-R in mesophyll protoplasts decreased markedly in both Fe-sufficient and Fe-deficient protoplasts. These data suggest that both the intrinsic decrease in FC-R activity per protoplast surface and a possible shift in the pH of the apoplastic space could lead to the accumulation of physiologically inactive Fe pools in chlorotic leaves.     

Immobilization of plant protoplasts: Viability studies

Protoplasts of Daucus carota Ca68 and Catharanthus roseus have been immobilized by entrapment in gelforming polysaccharides (kappa-carrageenan, agarose and alginate). Uniform spherical beads of carrageenan and agarose containing the protoplasts have been prepared by utilizing an inert hydrophobic phase (vegetable oil). The entrapped protoplasts are viable and stabilized towards osmotic shock by the polymeric backbone. Standard methods have been used to study the viability and integrity of the entrapped protoplasts. Upon incubation in a relatively simple medium the immobilized protoplasts show a much higher viability after 14 days as compared to free protoplasts under the same conditions. The viability of D. carota protoplasts has also been monitored by an enzyme activity present in the cells (digitoxigenin 58-hydroxylase).     

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     

Examination of genome stability in cultured Lycopersicon

The genetic stability of plants regenerated from either mesophyll protoplasts or leaf slices of the F1 hybrid between Lycopersicon esculentum and L. pennellii was assayed by comparing the ploidy level, leaf morphology and isozyme patterns of the regenerants with their somatic parents. Regenerants from protoplasts were predominantly tetraploid, regenerants from leaf slices were predominantly diploid; both classes of regenerants had isozyme patterns identical to those of the parent plant. Callus was analyzed that grew up from cultures containing fused protoplasts from either irradiated or untreated protoplasts of L. esculentum and L. pennellii. The L. pennellii cell line used was 18 months old and could no longer regenerate. Out of 75 calli scored at 3 isozyme loci, 51 were heterozygous at only one or two of the loci. Irradiation of the two parental lines was not necessary to produce fusion products exhibiting asymmetric expression of parental genes.Abbreviations Got-2 glutamate oxaloacetate transaminase-2 - Pgi-1 phosphoglucoisomerase-1 - Pgm-2 phosphoglucomutase-2     

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