High yields of cytoplasts from protoplasts of Lolium perenne and Beta vulgaris using gradient centrifugation

Methods for the isolation of cytoplasts from suspension culture-derived protoplasts of the monocot Lolium perenne (perennial ryegrass) and the dicot Beta vulgaris (sugarbeet) have been determined. After comparing a range of gradients it was found that a discontinuous sucrose/mannitol gradient gave the highest cytoplast yields for both species tested: of the protoplasts loaded onto the gradient, for Beta >30% and for Lolium up to 45% could be recovered as cytoplasts. Sufficient protoplasts could be loaded onto the gradient to produce suitable numbers of cytoplasts for use in asymmetric somatic hybridisation experiments. Cytoplasts could be isolated from several suspension cultures of different ages. The cytoplast fraction was recovered from the upper part of the gradient in all cases and was only slightly contaminated (2–8%) with protoplasts. Lolium cytoplasts were small, evacuolate cells with granular cytoplasm. In contrast, Beta cytoplasts were larger and predominantly vacuolate. Both contained mitochondria as determined using fluorescence staining.Abbreviations 2,4-d 2,4 dichlorophenoxyacetic acid - M mannitol - S sucrose - P Percoll - S/M sucrose/mannitol gradient     

Isolation and culture of protoplasts from embryogenic suspension cultures of red fescue (Festuca rubva L.)

Protoplasts were isolated from fast-growing embryogenic suspension cultures of red fescue cv. Dawson (Festuca rubra L.) without agitation. The enzyme isolation solution was highly efficient at releasing protoplasts of greater than 95% viability (5×10⁶–10⁷ protoplasts per ml of packed cell volume). A three step procedure was followed for washing and transferring protoplasts from a solution high in inorganic salts to a medium containing glucose and sucrose. The addition of 30 mM sodium thiosulfate to the wash and culture media was found to be helpful in reducing the number of lysed protoplasts. Isolated protoplasts began to divide within 48–72 h when protoplasts were plated in agarose squares and surrounded by nurse cells (mixed nurse plating technique). Maximum colony formation (plating efficiency) was approximately 1%. Many of the colonies continued to grow and produced embryos when transferred to a medium consisting of half-strength MS salts, 4 mg/l 2,4-D, 3 g/l casein hydrolysate and 30 g/l sucrose. Upon transfer to hormone-free medium and exposure to light 16 h/day, many of the embryos germinated to produce green leaves and roots.Abbreviations BA Benzylaminopurine - 2,4-D 2,4-dicholorophenoxyacetic acid - DMSO dimethyl sulfoxide - MES 2-(N-morpholino)-ethanesulfonicn acid - MS Murashige and Skoog medium (1962) - UGC Ultraclone Growth Chamber - KM Kao and Michayluk medium (1975) - NAA Naphthalene acetic acid     

Isolation and characterization of cytoplasts and miniprotoplasts derived from protoplasts of cultured cells

We have isolated and partially characterized subprotoplasts containing nuclei, i.e. miniprotoplasts, and enucleated subprotoplasts, i.e. cytoplasts, from freshly isolated protoplasts of cultured cells from Hyoscyamus muticus, Nicotiana tabacum and especially Zea mays . Protoplasts were fragmentated by centrifugation through discontinuous iso-osmotic density gradients containing colloidal silaca gel (Percoll), calcium chloride and mannitol. Using this method metabolically active miniprotoplasts and highly purified cytoplast fractions with less than 4% contamination with nucleated protoplasts were obtained. The cytoplasts prepared by our method are suitable for use in fusion experiments aimed at transferring nuclear and cytoplasmic genetic information separately.     

Plant regeneration from hypocotyl-derived protoplasts of Brassica juncea (L.) Czern and Coss

Protoplasts isolated from etiolated hypocotyls of 6-day-old seedlings of Brassica juncea cv RLM 198 were cultured in a modified V₄₇ medium containing 7% mannitol, 2% sucrose, 1.0 mg/l 2,4-D, 0.1 mg/l NAA and 0.4 mg/l BAP, at a density of 5×10⁴ protoplasts per ml of medium. Cultures were incubated in the dark at 25+1°C. After 7 d of culture, cell colonies were diluted with 8_p medium containing 5% mannitol and a similar hormone combination as described earlier. After 14 d, cell colonies were embedded in 8_p medium containing agarose and 3.5% mannitol. Immediately upon gelling, liquid 8_p medium was added to each Petri dish as an overlayer, and cultures were incubated in the light. After a total of 3 to 4 weeks in culture, microcalli were obtained. A modified MS medium with 2% sucrose, 1.0 mg/l 2,4-D and 0.1 mg/l kinetin solidified with 0.5% agarose was used for growing microcalli into callus lines. On MS medium containing 2% sucrose, 0.1 mg/l IAA, 2.0 mg/l zeatin riboside and 2.0 mg/l BAP, solidified with 0.5% agarose, about 35% of the calli regenerated multiple shoots. The time required from culture of protoplasts to multiple shoot regeneration was about 10 weeks. Regenerated shoots were rooted and plants were re-established in a growth chamber at high frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - IBA Indole-3-butyric acid     

Callus formation and plantlet regeneration from protoplasts derived from suspension cultures of wheat (Triticum aestivum L.)

Protoplasts were isolated from anther-derived suspension cultures of commercial wheat (Triticum aestivum L. cv. Chris). The protoplasts were released enzymatically and isolated by centrifugation on a sucrose cushion. The isolated protoplasts were initially cultured in a liquid medium in the dark. Numerous microcalli were produced under these conditions, some of which differentiated into globular embryos. Upon transfer to a solid medium and exposure to 16h/8h light/dark cycle, the protocalli proliferated and many of the somatic embryos matured. Complete plantlets were obtained and maintained in sterile culture.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - MES 2-[N-morpholino] ethanesulfonic acid     

Plant regeneration from protoplasts of common buckwheat (fagopyrum esculentum)

Protoplasts were isolated from hypocotyls of etiolated seedlings from a diploid and the corresponding autotetraploid variety of common buckwheat (Fagopyrum esculentum). The isolated protoplasts started to divide after 4 days in culture in a modified MS medium. Maximum plating efficiency was approximately 1%. Regenerated calli derived from the tetraploid genotype developed roots easily but were recalcitrant to form shoots. Eighteen months following the initiation of cultures, tetraploid embryoids and shoots emerged after 3 weeks on an MS medium containing 0.1 mg/l gibberellic acid.Abbreviations 2,4-D 2,4 — dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - GA gibberellic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthaleneacetic acid     

Somatic embryogenesis and plant regeneration from cell suspension and tissue cultures of mature himalayan poplar (Populus ciliata)

Somatic embryogenesis and plantlet formation were obtained from callus and cell suspension cultures of 40-year- old Himalayan Poplar (Populus ciliata Wall ex Royle). Callus and cell suspensions were obtained by transfer of inoculum of semiorganized leaf cultures, which were maintained on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BAP), to MS with 2,4-dichlorophenoxyacetic acid (2,4-D). Reduction of 2,4-D concentration during subsequent subculture of cell suspensions resulted in the formation of embryoids. These embryoids developed further only after being transferred to agar-based MS medium supplemented with BAP and naphthalene acetic acid. Loss of embryogenic potential was observed in cell suspensions after 6 subcultures. However, callus cultures retained the embryogenic potential even after repeated subcultures for more than a year. Plantlets could be successfully hardened and grown in natural outdoor conditions.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthalene acetic acid - MS Murashige and Skoog (1962) medium     

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     

In vitro propagation and low temperature storage ofSaussurea lappa C.B. Clarke — An endangered,medicinal plant

A procedure forin vitro multiplication ofSaussurea lappa (Asteraceae) is described. On Murashige and Skoog's medium (MS) containing benzylaminopurine and gibberellin 3.5-fold shoot multiplication occurred every three weeks. Shoots rooted on MS containing 0.5 M naphthaleneacetic acid with 90% efficiency. The shoot cultures stored at 5°C in the dark for 12 months without an intervening subculture survived with 100% viability. The shoots cold stored for 6 months or more showed higher rates of multiplication under culture room conditions than the untreated shoots.Abbreviations MS Murashige and Skoog 1962 - BAP Benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA Indole-3-acetic acid - IBA Indolebutyric acid - NAA Naphthaleneacetic acid - GA₃ Gibberellin     

High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander (Coriandrum sativum L.)

Hypocotyl segments and zygotic embryos of coriander formed embryogenic calli at frequencies of up to 75% when cultured on MS medium supplemented with 1 mgl^–1 2,4-D. Calli were transferred to MS liquid medium with 1 mgl^–1 2,4-D to initiate cell suspension cultures. Embryogenic cells became finely dispersible in the medium as the subculture proceeded. Cultures were transferred to a nitrogen compound enriched liquid MS medium containing 2% sucrose and 0.1 mgl^–1 2,4-D, and cultured two weeks before plating on MS basal medium. Approximately 75% of cell aggregates (1 to two mm in diameter) underwent development into globular to cotyledonary somatic embryos after two weeks of plating. Most of the embryos were subsequently regenerated into plantlets. Regenerants were successfully transplanted to potting soil and grown to maturity in a phytotron.Abbreviations MS Murashige and Skoog - MS1D MS medium + 1 mgl^–1 2,4-D     

Plant regeneration from leaf protoplasts of Solanum torvum

A protocol to obtain regenerated plants from protoplasts of Solanum torvum Sw a wild species of eggplant resistant to Verticillium wilt is reported. Leaf protoplasts were enzymatically isolated from six-week old seedlings grown in a controlled environment chamber. Protoplasts were plated on modified KM medium (0.4 M glucose)+(mg/l): 1.0 p-chlorophenoxyacetic acid (CPA)+1.0 naphthaleneacetic acid (NAA)+0.5 6-benzylaminopurine (BAP) and 0.02 abscisic acid (ABA). The protoplast density was 5×10⁴ per ml with 5 ml placed in each of two quadrants in X-dishes (100×15 mm). The reservoir medium was modified KM+(mg/l): 0.1 NAA+0.5 BAP+0.1 M sucrose+0.1 M mannitol+0.6% washed agar+1% activated charcoal. Dishes were initially placed in the dark at 27°C. Protoplast division was initiated in 1–2 weeks and 4 weeks later p-calli were 1–3 mm. Plating efficiency was 11% when measured at 3 weeks. Six-week old p-calli were transferred individually onto Whatman No. 1 filter paper layered on modified KM (0.15 M sucrose)+mg/l: 2.0 indoleacetic acid (IAA)+2.0 zeatin+0.5% washed agar for 2 weeks. Subsequently, shoots occurred within 4 weeks at 70% efficiency on MS+30 g/l sucrose+2 mg/l zeatin. Shoots were rooted on half strength MS+10 g/l sucrose.Abbreviations ABA abscisic acid - BAP 6-benzylaminopurine - CPA p-chlorophenoxyacetic acid - IAA indoleacetic acid - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthaleneacetic acid - 2ip 6-dimethylallyamino purine Michigan Agricultural Experiment Station Journal Article No. 12167     

Plant regeneration from protoplasts of long-term callus cultures of Actinidia deliciosa var. deliciosa cv. Hayword (Kiwifruit)

Plant regeneration of Actinidia deliciosa var. deliciosa cv. Hayword was obtained from protoplasts isolated from petiole derived long-term callus cultures. Protoplasts were cultured in liquid medium over agarose gelled medium. Regenerated green callus, plated on solid medium, could develop shoots that rooted spontaneously in hormone-less medium. The plants obtained are growing fast in soil and present a normal phenotype.Abbreviations BAP benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - DTT dithiotreitol - IAA indole-3-acetic acid - IBA indole-3-butyric acid - Kin kinetin - MES 2-(N-morpholino) ethanesulphonic acid - MS Murashige and Skoog (1962) medium - NAA naphthalene-1-acetic acid - SH Schenk and Hildebrandt (1972) medium This Research was supported by JNICT and INIC     

Protoplast isolation,culture and regeneration from Egyptian cultures of Pea and Beans

Abstract

A protocol of protoplast isolation from Egyptian varieties of pea and bean is reported. Protoplast cultures were established from apical shoots of pea (Pisum sativum) and suspension cultures of bean (Phaseolus vulgaris). To isolate protoplasts of pea, apical shoot tissues were digested for 10 h using enzyme solution containing 1% pectinase, 0.5% cellulase, 0.5% hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. For protoplast isolation from suspension culture of bean, collected cells were incubated for 6 h in digestion solution containing 0.5% pectinase, 0.25% of each of cellulase and hemicellulase, 10% mannitol and 0.1% CaCl₂-2H₂O. Purified protoplasts were cultured in liquid culture medium. Microcalli were obtained after 30 days of culture. Calli colonies with a diameter of about 5 mm were developed after one month of culturing on solid B5 medium containing 2% sucrose, 2 g/l casein hydrolysate, 0.7% agar and supplemented with either 1 mg/l of each 2,4-D and kin in case of pea or 2 mg/l 2,4-D+0.5 mg/l kin in case of bean. Protoplast derived callus of pea was successfully differentiated into shoot and root, and highest frequency of shoot organogenesis was recorded on medium containing 0.5 mg/l NAA+2 mg/l BA. Protoplast derived callus of bean, on the other hand, gave rise to a high frequency of root formation when cultured on medium containing 1 mg/l NAA, but attempts to regenerate shoots from this callus was unsuccessfull.     

Production of chrysanthemic acid and pyrethrins by tissue cultures ofChrysanthemum cinerariaefolium

Tissue cultures ofChrysanthemum cinerariaefolium were established, and then used to study the production of pyrethrin insecticides, and their precursor chrysanthemic acid. Callus cultures and root-differentiated cultures did not contain pyrethrins whereas shoot differentiated callus was found to produce the pyrethrins. Chrysanthemic acid was isolated by extraction from callus cultures, and feeding¹⁴C-labelled chrysanthemic acid to a cell suspension ofC. cinerariaefolium established that the acid accumulates largely as a glucoside ester.Abbreviations MS Murashige and Skoog (1962) medium - 2,4-D 2,4-dichlorophenoxyacetic acid - 1AA Indoleacetic acid - BAP 6-Benzylaminopurine - GC-MS Gas chromatography - mass spectrometry     

Establishment of anthocyanin-producing cell suspension cultures of Cleome rosea Vahl ex DC. (Capparaceae)

The effects of different levels of Murashige and Skoog (MS) basal medium, 2,4-dichlorophenoxyacetic acid (2,4-D), and sucrose on anthocyanin production and biomass accumulation of cell suspension cultures of Cleome rosea were investigated. Cultures were established in liquid MS medium containing 30 g l⁻¹ sucrose and supplemented with 0.90 μM 2,4-D. Proliferating cell suspension cultures achieved the highest growth capacity, a fourfold increase in biomass accumulation, following subculture at the exponential growth phase, 14–18 days of culture. Moreover, the presence of 2,4-D was essential for anthocyanin production and biomass accumulation. On the other hand, increasing levels of sucrose above 30 g l⁻¹ resulted in a drastic reduction in biomass accumulation. Anthocyanin production was highest in cell suspension cultures grown on half-strength MS medium (1/2 MS), 30 g l⁻¹ sucrose, and 0.45 μM 2,4-D. These cell suspension cultures were mainly composed of small aggregates of spherical cells with similar morphology observed in anthocyanin-producing and non-producing cultures. Moreover, microscopic analysis of anthocyanin-producing cultures showed the presence of mixtures of non-pigmented, low-pigmented, and high-pigmented cells.     

Plant regeneration from protoplasts isolated from callus ofGentiana crassicaulis

Fast growing calli induced from hypocotyl segments ofGentiana crassicaulis were used for preparation of protoplasts. High yields of viable protoplasts were produced in an enzyme solution containing 1–2% cellulase, I% pecfinase, and 0.5% Hemicellulase. Protoplasts were cultured in KM8P medium containing 1 mg/l 2,4-D, 0.5 mg/l 6BA, 500 mg/l LH, 0.5 M glucose and 0.1 M mannitol by the solid-liquid dual layer culture method. First division occurred within 4–5 days of culture at a frequency of 17.8%. Sustained divisions led to callus formation. Periodically diluting the cultures with freshly prepared liquid medium containing 1% glucose was critical for colony formation. Protocolonies about 2 mm in size were transferred onto MS medium supplemented with 3 mg/l ZT, 2 mg/l 6BA, 1 mg/l GA₃, 1 mg/l NAA and 6% sucrose to obtain embryogenic calli. Plantlets were regenerated via somatic embryogenesis at high frequency on hormone-free MS Medium.Abbreviations 6BA 6-benzylaminopurine - NAA naphthaleneacetic acid - 2,4-D 2,4 - dichlorophenoxyacetic acid - ZT zeatin - GA₃ gibberellic acid - LH lactalbumin hydrolysate - MES 2-(N-morpholino)-ethane sulfonic acid - MS Murashige & Skoog's medium(1962)     

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     

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     

生姜离体叶片愈伤组织悬浮细胞系建立与植株再生

以‘莱芜大姜’为试材,研究了生姜离体叶片愈伤组织的诱导以及细胞悬浮系建立与植株再生。结果表明,以生姜试管苗叶片为外植体,接种到MS+1.0 mg/L 2,4-D+0.5 mg/L 6-BA+30 g/L蔗糖的培养基上,可有效诱导出生长迅速、质地疏松的愈伤组织。将获得的愈伤组织接种到MS+0.15 mg/L 2,4-D+6.0 mg/L 6-BA+30 g/L蔗糖的液体培养基上,25℃黑暗条件下震荡培养25-30 d,可建立分散性好、生长迅速的悬浮细胞系,细胞悬浮系培养的适宜参数为:初始接种量为1.0-1.5 g,继代培养的适宜间隔期为15 d,继代培养液体培养基更新比例为3/4。将悬浮细胞接种到固体培养基MS+0.2 mg/L NAA+10.0 mg/L 6-BA+30 g/L蔗糖上可获得再生植株。     

Plant regeneration from mesophyll protoplasts of Centaurea cyanus,Senecio x hybridus and Callistephus chinensis

Protoplasts were isolated from leaves of glasshouse-grown plants of Centaurea cyanus and axenic shoot cultures of Senecio x hybridus. Upon culture, using modified MS-based media, protoplasts of both systems entered division to produce callus, followed by plant regeneration. Leaf protoplasts of Callistephus chinensis entered sustained division only following the preconditioning for 24h of peeled leaf tissues on agar-solidified MS-based medium. Protoplasts were also isolated from cell suspensions of C. chinensis and divided in MS-based or KM media. However, only leaf mesophyll protoplasts of Callistephus produced callus, which developed shoots.The establishment of protoplast-to-plant protocols for these ornamental species has provided a basis for broadening their gene pools through somatic hybridisation.Abbreviations BAP 6-benzylaminopurine - NAA -naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) - KM Kao and Michayluk (1975) - g.f.wt. gram fresh weight