An efficient protocol for plant regeneration from protoplasts of the moss <Emphasis Type="Italic">Atrichum undulatum P. Beauv in vitro</Emphasis>

A system for plant regeneration from protoplasts of the moss, Atrichum undulatum (Hedw.) P. Beauv. in vitro, is first reported. Viable protoplasts were isolated at about 9 × 10⁵ protoplasts g⁻¹ fresh weight from 10 to 18 days protonemata. For regeneration of protoplasts, viable protoplasts were cultured in liquid–solid medium containing surface liquid medium MS (0.4 M mannitol) and subnatant solid medium Benecke (0.3 M mannitol) at 20 °C under a 16-h photoperiod white light after 12 h preculture in darkness at 20 °C. The great majority of protoplasts follow a regenerative sequence: formation of asymmetric cells in 2–3 days; division of the asymmetric cells to 2–3 cells in 4–5 days, and further develop to produce a new chloronemal filament in 15 days. Juvenile gametophyte can be visible in 20 days. The plating ratio of cell cluster regenerated from protoplasts reaches up to 45%. Transient expression experiments indicate the electroporation uptake of DNA is possible.     

THE GROWTH OF DUCKWEEDS IN MINERAL NUTRIENT SOLUTIONS WITH AND WITHOUT ORGANIC EXTRACTS

1. Detmer''s solution and a modified Knop''s solution are unfavorable culture media for the growth of Spirodela polyrhiza. 2. When the modified Knop''s solution was diluted to 10 times its volume, Spirodela polyrhiza and Lemna valdiviana grew and reproduced for periods of 26 months and 21 months, respectively. 3. Growth in the dilute Knop''s solution, which alone can support the growth of Spirodela indefinitely, was considerably stimulated over a period of 23 days by adding to every liter the water-soluble material of 0.4 gm. autolyzed yeast, or the material of 2.5 gm. peat soluble in a 1 per cent solution of NaHCO₃. 4. The nature of the stimulus or of the protection afforded by the organic material is as yet unknown. 5. The necessity of organic accessory foods (auximones) in the nutrition of green plants cannot be accepted as an established fact.     

Isolation of protoplasts from fern prothallia and their regeneration to gametophytes

Protoplasts were isolated enzymatically from prothallia ofLygodium japonicum. The protoplasts grown in a culture medium containing 0.6 M mannitol and 0.05 M sucrose began to divide within 8 days of culture, and after 30 days 10-cell clusters were present. When the cell-clusters were transferred into fresh media followed by sequential reduction of mannitol concentration, they developed rhizoids and protonemata. The reduction of mannitol concentration to 0.3 M resulted in the regeneration of a common gametophyte within 50 days of culture, and subsequently the regenerated gametophytes produced sporophytic leaves and roots.     

Development of gametophores from isolated protoplasts of the mossAnoectangium thomsvnii Mitt.

Summary Protoplasts of the mossAnoectangium thomsonii Mitt. were isolated from preplasmolyzed protonemal filaments, grown in suspension cultures, after the digestion of the cell wall by the enzymes cellulase and macerozyme or driselase. Driselase was more effective than cellulase and macerozyme. After purification these protoplasts were plated in the form of small agar drops in modified Kofler's medium without hormones and incubated in the dark at 26 ± 2 °C. Cell walls regenerated within three days and cell divisions started seven days after the initiation of the cultures. When the regenerants were transferred to normal protonemal culture medium and illuminated by 3,000 lux continuous light, a multi-cellular protonema developed which formed leafy gametophores on salicylic acid supplemented medium.     

STUDIES ON STRIGA SENEGALENSIS III. IN VITRO CULTURE OF SEEDLINGS. ESTABLISHMENT OF CULTURES

Striga senegalensis seeds were cultured on Knop's and modified Murashige-Skoog's medium. An aqueous root exudate/extract of the host (Sorghum vulgare) was found essential for the germination of seeds in vitro. The seedlings grew better on a medium containing sucrose than on mineral salts alone. Only limited growth was achieved on Knop's medium. Better growth was supported by Murashige-Skoog's medium. By transfer to larger culture vessels seedlings were kept in the latter for several months. The seedlings developed chlorophyllous shoots in light and flowered after 4–4½ months. The successful culture of these seedlings in a simple medium of only mineral salts and sugar indicates that the parasitic seedling depends on the host for only water, mineral salts and sugar and not for more elaborate substances.     

Protoplasts of Cosmarium as a Potential Protein Source

Cosmarium turpinii, a fast-growing desmid alga, transforms into protoplasts in 4 h when incubated in a mineral medium + 0.4 M mannitol + 0.5% Cellulysin.     

Isolation and culture of protoplasts from cotyledons of Pinus coulteri D. Don.

A protocol was developed for the isolation and culture of protoplasts from the cotyledons of seedlings of Pinus coulteri D. Don. Incubation of cotyledon pieces in a mixture consisting of cellulase Onozuka R10 2%, Pectolyase Y-23 0.1%, mannitol 10%, CaCl₂ 500 mg/l and other macro and micro-nutrients yielded viable protoplasts. After 24 hours of culture in a complex nutrient medium, the protoplasts regenerated new cell walls and the first divisions were observed within 7–10 days. Small cell colonies were formed within 15–20 days, but these started to accumulate phenolics and no further growth of the colonies was observed.     

Effects of nutrients,cell density and culture techniques on protoplast regeneration and early protonema development in a moss,Physcomitrella patens

To regenerate auxotrophic mutants of Physcomitrella patens, two media of increasing complexity were developed. The survival rate of protoplasts was around 30% higher on full medium when compared to standard minimal medium. Protoplast survival was higher in a medium containing 2.5 mmol/L ammonium tartrate compared to a medium with 5 mmol/L of this compound. Solid medium had a positive effect on protoplast survival compared to either liquid medium or solid medium overlaid with cellophane; the maximum survival rate being 31.6%. However, the number of surviving protoplasts without any cell division during the first ten days increased on solid medium. Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly. The effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.     

Isolation and regeneration of protoplasts from gills ofAgaricus bisporus

Protoplasts were isolated from fruit-bodies ofAgaricus bisporus, and highest yields were derived from basidia. When gill fragments were treated with a combination of Novozyme 234, chitinase, and cellulase Onozuka R-10, and with 0.35m KCl as the osmotic stabilizer, high yields (3–4×10⁷ protoplasts/g fresh wt gills) were obtained within 1 h of incubation. About 20–30% of protoplasts regenerated in a solid MMNC medium. Investigation of specific activities of glucose-6-phosphate dehydrogenase and mannitol dehydrogenase indicated a highly active pentose phosphate pathway and a good capacity for mannitol synthesis in protoplasts, as well as in other cells of fruit-bodies of the species. The simple and efficient procedure provides a new approach for further investigation of the mushroom, and possibly of other basidiomycetes with hemiangiocarpus, by use of protoplasts.     

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     

ROLE OF ASYMMETRIC CELL DIVISION IN PTERIDOPHYTE DIFFERENTIATION. II. EFFECT OF CA2+ ON ASYMMETRIC CELL DIVISION,RHIZOID ELONGATION,AND ANTHERIDIUM DIFFERENTIATION IN VITTARIA GEMMAE

Gametophytes of Vittaria graminifolia reproduce vegetatively by means of gemmae. Each gemma consists of a linear array of six cells: four body cells and a knob-shaped terminal cell at each end. When gemmae are shed from the gametophyte onto Knop's mineral medium, the two terminal cells do not divide, but elongate to form primary rhizoids. The body cells undergo asymmetric cell division, and the smaller daughter cells differentiate into either secondary rhizoids or prothalli. When gibberellic acid is included in the medium, antheridia are formed as a result of asymmetric cell division instead of vegetative structures. We studied the effect of Ca²⁺ on asymmetric cell division, rhizoid elongation, and antheridium formation in gemmae cultured on Knop's mineral medium and variations of Knop's medium. Ca²⁺ inhibited the onset of cell division and rhizoid elongation, but was required for differentiation of antheridia. Treatments which lowered the Ca²⁺ content of gemmae (EGTA and dilute HCl extraction, culture on verapamil-containing and Ca²⁺-deficient medium) caused an early onset of cell division and rhizoid elongation. The stimulation of growth was most pronounced when gemmae were deprived of Ca²⁺ during the first 24 hr of culture. The proportion of cell divisions which differentiated into antheridia in response to GA was greatly reduced when the Ca²⁺ status of gemmae was lowered with verapamil and Ca²⁺-EGTA buffers.     

Immobilisation and mechanical support of individual protoplasts

Mesophyl cell protoplasts of Vicia faba were suspended in a solution consisting of 10% sodium alginate and 0.4 M mannitol. The protoplasts could be immobilized by cross-linking the alginate in the presence of 100 mM CaCl₂. Changes in the osmolarity of the external medium led to reversible shrinkage and swelling of the entrapped protoplasts. It was demonstrated by using the pressure probe technique that a pressure gradient (cell turgor pressure) of several 100 mbar is built up when the immobilized cells were transferred to hypotonic solution. By complexing the Ca²⁺ in the alginate matrix with sodium citrate buffer the protoplasts could be released from the matrix. No morphological change or alteration of the membrane permeability of the immobilized protoplasts was observed after a storage period of up to 14 days at 4°C in the matrix.     

In vitro Post-germination Growth and Development of Embryos of Alectra (Scrophulariaceae)

In vivo, seeds of the obligate root parasite Alectra vogelii Benth. (Scrophulariaceae) germinate only after being soaked in water for a period of time (pretreatment) followed by stimulation by certain factors exuded from a host root. Germinated seedlings do not develop beyond radicle emergence, and finally die, unless their radicles make contact with and penetrate into a host root conductive system. In vitro, germinated embryos obtained by exposing sterilized and pretreated seeds to root exudate of Vigna unguiculata were aseptically cultured on Knop's, White's and Murashige and Skoog's media. The embryos grew into seedlings with shoots and roots on a medium containing mineral salts and sucrose, but not on mineral salts alone. Seedling performance was generally not improved when the mineral salts-sucrose media were supplemented with vitamins. Shoot extension growth was better on Murashige and Skoog's mineral salts-sucrose medium than on Knop's or White's medium. However, seedling development was greatly boosted when cultivated on White's minerals salts-sucrose medium supplemented with coconut milk. Seedlings turned green on transfer to light but did not flower. The successful culture of these embryos and seedlings on a simple, chemically defined medium of mineral salts and sugar suggests that these nutrient components are the minimal external requirements for stimulation and support of normal seedling growth. These may be obtained in vivo by the parasite's tapping of the host root conductive system.     

A rapid procedure for plant regeneration from protoplasts isolated from suspension cultures and leaf mesophyll cells of wild Solanum species and Lycopersicon pennellii

A rapid procedure for protoplast isolation, culture and plant regeneration has been developed for two Solanum species (S. lycoperisicoides and S. verrucosum) and Lycopersicon pennellii. Freshly isolated protoplasts were initially cultured in liquid Solanum Culture Medium (SCM), containing 2,4-dichlorophenoxy acetic acid (2,4-D). Subsequent dilution with fresh culture medium without auxins appeared to be essential to obtain rapid regeneration medium later on. The resulting micro calli were first grown in a culture medium containing 0.5 mg/l 6-BAP and 0.05 mg/l NAA and 0.2 M mannitol and 7.3 mM sucrose to induce greening, at a lower osmolarity (300 mOsm · kg⁻¹). Then, the green micro calli were transferred to shoot induction medium, containing 2 mg/l zeatin, 0.1 mg/l IAA and 2% sucrose (150 mOsm · kg⁻¹). In this way plants could be regenerated from leaf mesophyll protoplasts and suspension cell-derived protoplasts of L. pennellii and S. lycopersicoides within 2 months. Shoot regeneration from leaf mesophyll protoplasts of the two lines of S. verrucosum could be obtained 3 months after protoplast isolation.     

Callus regeneration from Alnus incana protoplasts isolated from cell suspensions

Nagata and Takebe's (NT) medium, supllementedte with 2.5 μm 2,4-dichlorphenoxyacetic acid (2,4-D), induced development of friable calluses from leaves of axenic shoot cultures of Alnus incana. Fast-growing cell suspensions were established in the same medium without agar. Suspensions gave high yields of viable protoplasts after an overnight incubation in an enzyme mixture consisting of 1% (w/v) Onozuka R-10, 0.5% (w/v) Rhozyme HP-150, 0.03% (w/v) Macerase, CPW salts, and 13% (w/v) mannitol (pH 5.8). Protoplasts cultured on K8p medium underwent cell wall regeneration within 24 h. The optimum protoplast-derived colony formation and growth was obtained on the NT medium supplemented, as was the K8p medium, with glucose as the osmoticum, growth regulators, coconut milk and casein hydrolysate. Compared with other culture techniques, the agarose bead technique of Shillito et al. (Plant Cell Reports, 2 (1983) 244) improved cell division and colony formation frequency. Protoplast-derived macrocalluses grew under the same conditions as those used for leaf calluses.     

仙鹤藓的孢子萌发及愈伤组织诱导

通过对采自河北雾灵山海拔1500m的仙鹤藓（Atrichum undulatum）的孢子萌发以及原丝体发育的观察,发现仙鹤藓孢子无休眠现象,孢子接种3天左右萌发：其原丝体发育分为绿丝体和轴丝体两个阶段。扩大培养实验结果表明。仙鹤藓茎叶体在添加2％葡萄糖的MS培养基上,置于25℃／20℃、14小时光照/10小时黑暗、36μmol·m^-2·s^-1条件下培养．产生新生茎叶体最多,且茎叶体长势最好,可以获得大量无菌材料。仙鹤藓愈伤组织诱导实验显示,形成愈伤组织的最佳培养基为添加2％葡萄糖和1．0mg·L^-16-BA的MS培养基。     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Plant Regeneration from Callus Protoplasts of Codonopsis pilosula

Embryogenic cell line was established from hypocotyl segments of Codonopsis pilosula (Franch.)Nannf. 4--8 day old embryogenic callus was used to isolate protoplasts in an enzyme solution containing 1.5 % cellulase Onozuka R-10 and 3 % pectinase. Protoplasts were cultured in MS,C81V,DPD and KMSp basal medium supplemented with 1.2 mg/L 2,4-D, 0.2 mg/L NAA, 0. 2 mg/L BAP, 0. 1 mg/L ZT,and different combinations of glucose and mannitol . Protoplast-derived cells underwent sustained divisions in KM8p medium. As an osmoticum, glucose was more beneficial to protoplast division. A combination of 0. 30 mol/L glucose with 0.10 mol/L mannitol gave the best result. Under proper conditions , protoplasts underwent the first division on the 3rd day of culture,formed colonies within 30 days , and developed into microcalli in 6 weeks. Plantlets were regenerated from protoplast-derived calli through somatic embryogenesis. 0.2 % activated charcoal promoted embryoid formation and root development.     

Mesophyll protoplast culture of sweet potato (Ipomoea batatas L.)

Mesophyll protoplasts of sweet potato (Ipomoea batatas L.) were readilyisolated by soaking chopped leaf tissue in distilled water for 16 h prior to enzymatic digestion. Isolated mesophyll protoplasts began to divide three days after start of culture in liquid modified N₆ medium and and formed colonies after 30 days of culture. The colonies transferred to solid medium grew rapidly and differentiated into calli. Some of the calli transplanted onto regeneration medium produced roots.     

Modulation and direction of the electrofusion response in plant protoplasts

In experiments on electrofusion of protoplasts (from Solanum brevidens, S. tuberosum, Nicotiana plumbaginifolia) the presence of divalent cations (Ca²⁺) at 1 mM in the fusion medium was found to increase the yield of hybrids observed directly after fusion and decrease the duration of pulse needed for fusion. Pretreatment of protoplasts with the polyamine spermine also enhanced fusion yield, and when combined with 1 mM Ca²⁺ the effects were additive. The improvement in fusion yield (2–4 fold) was most marked for protoplast populations (e.g. from suspension cultured cells) that were least responsive to electrofusion in mannitol alone. Short term viability, judged from FDA fluorescence was found to be high at these increased fusion levels. Optimum fusion parameters for electrofusion thus may be determined from short term experiments. Attempts to direct to fusion response between populations of protoplasts of identical properties by pretreatment of one fusion partner with spermine were inconclusive.