小麦原生质体分离过程中生理状态的变化

酶解处理使小麦对肉原生质体膜流动性降低,膜脂过氧化产物丙二醛（ＭＤＡ）积累,说明脱璧过程对细胞有伤害作用,损伤位点可能发生在膜上。胚性愈伤组织的具有分裂能力的原生质体,不表现上述变化。酶解脱壁还使超氧化物歧化酶（ＳＯＤ）和过氧化氢酶（ＣＡＴ）活性上升;过氧化物酶（ＰＯＸ）在叶肉原生质体中活性下降,在胚性愈伤组织来源的原生质体中活性上升。以上结果表明：在原生质体分离过程中,细胞的生理特性发生了变化;膜损伤的发生可能与原生质体能否进入正常分裂状态有关。     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Isolation of Intact and Functional Chloroplasts from Mesophyll and Bundle Sheath Protoplasts of the C(4) Plant Panicum miliaceum

A procedure is described for isolating and purifying mesophyll protoplasts and bundle sheath protoplasts of the C₄ plant Panicum miliaceum. Following enzymic digestion of leaf tissue, mesophyll protoplasts and bundle sheath protoplasts are released and purified by density centrifugation. The lower density of mesophyll protoplasts allowed rapid separation of the two protoplast types. Evidence for separation of mesophyll protoplasts and bundle sheath protoplasts (up to 95% purity) is provided from light microscopy (based on size difference in both chloroplasts and protoplasts), levels of marker enzymes in the preparations (i.e. pyruvate, Pi dikinase and phosphoenolpyruvate carboxylase for mesophyll and ribulose-1,5-bisphosphate carboxylase for bundle sheath), and differences in substrate-dependent O₂ evolution by chloroplasts isolated from protoplasts.     

Isolation of protoplasts from somatic embryos of carrot

Protoplasts were isolated enzymatically from synchronously induced globular somatic embryos from a carrot suspension culture. Among the macerating enzymes tested, Driselase was the most effective for release of protoplasts from embryos. A higher medium osmolarity was required for the isolation of protoplasts from embryos than from undifferentiated cells. Protoplasts from embryos were smaller than protoplasts from undifferentiated cells. On step gradients of Ficoll, protoplasts from embryos gave one major band. Protoplasts from undifferentiated cells gave two major bands, one lighter and the other heavier than the protoplasts from embryos.     

1-Aminocyclopropane-1-carboxylic-acid-dependent ethylene production during re-formation of vacuoles in evacuolated protoplasts of Petunia hybrida

Summary Ethylene formation from 1-aminocycloprane-1-carboxylic acid (ACC) was studied in whole protoplasts, evaluolated protoplasts and isolated vacuoles from mesophyll cells of Petunia hybrida L. cv. Pink Magic. The re-formation of the large, central vacuole in evacuolated protoplasts and morphological characteristics of both types of protoplasts were examined by electron microscopy. Both the normal, whole protoplasts and vacuoles isolated from them produced ethylene from ACC at similar rates. Freshly-prepared evacuolated protoplasts had lost the capacity to produce ethylene. Re-formation of the central vacuole in these evacuolated protoplasts occurred between 14 to 17 h of incubation in the recovery medium and was followed by the development of ethyleneforming activity. Both these processes were inhibited by cycloheximide, indicating a requirement for new protein synthesis. Light stimulated the conversion of ACC to ethylene in both the regenerating, whole protoplasts and the evacuolated protoplasts that had re-formed the central vacuole.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CHI cycloheximide     

Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

A method for isolation of protoplasts from dermatophytes

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.     

Electrofusion of protoplasts from celery (Apium graveolens L.) with protoplasts from the filamentous fungus Aspergillus nidulans

A method was developed for electrofusion of higher-plant protoplasts from celery and protoplasts from the filamentous fungus Aspergillus nidulans. Initially, methods for the fusion of protoplasts from ecch species were determined individually and, subsequently, electrical parameters for fusion between the species were determined. Pronase-E treatment and the presence of calcium ions markedly increased celery protoplast stability under the electrical conditions required and increased fusion frequency with A. nidulans protoplasts. A reduction in protoplast viability was observed after electrofusion but the majority of the protoplasts remained viable over a 24-h incubation period. A small decline in protoplast respiration rate occurred during incubation but those celery protoplasts fused with A. nidulans protoplasts showed elevated respiration rates for 3 h after electrofusion.Abbreviations AC alternating current - DC direct current     

Studies on the behavior of meiotic protoplasts IV. Protoplasts isolated from microsporocytes of liliaceous plants

Enzymatic isolation of protoplasts from microsporocytes of various species of liliaceous plants is described along with some of the features of the isolated meiotic protoplasts. Protoplasts are produced with a high viability from coherent filaments of cells preculture for 24 hr during premeiosis and early meiotic prophase, but with low survival rates from free cells at late prophase. The suspensions of protoplasts contain multinucleate cells produced by spontaneous fusion at various frequencies up to 40%. In the enzyme solution meiotic protoplasts adhere to one another. When isolated at meiotic prophase, protoplasts may be cultured through the meiotic cycle.     

Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle

Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT₅₀) (−5°C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT₅₀, the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT₅₀ (−25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI₅₀) of NA protoplasts osmotically expanded from isotonic solutions was 1122 ± 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI₅₀ values obtained from osmotic manipulation. The TSAI₅₀ of ACC protoplasts expanded from isotonic solutions (2145 ± 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area.     

甘蔗和烟草叶原生质体分离期间的膜损伤及有关酶活性变化

甘蔗和烟草叶原生质体分离期间的膜损伤及有关酶活性变化何若天,覃伟,李任强（广西农业大学实验中心,南宁５３０００５）关键词：原生质体,超氧阴离子自由基（Ｏ_２~－）,膜损伤,甘蔗,烟草植物原生质体分离期间,所用细胞壁降解酶和高渗介质等对细胞生理有深刻影响...     

Division frequency of pea protoplasts in relation to starch accumulation

Division frequency of alginate-embedded pea (Pisum sativum var. Belman) protoplasts derived from embryonic shoot tips was studied quantitatively by image analysis in relation to starch accumulation and protoplast size. Protoplast divisions were observed from day 4 on and the number of protoplasts undergoing division increased in a stepwise manner to 70% the following days. The starch content increased rapidly during the first 3 days of culture prior to the onset of division and resulted a 4.2-fold increase in the intracellular starch area and a 3.0-fold increase (from 27% to 80%) in the number of protoplasts containing starch. Subsequent periods with rapid increases the number of dividing protoplasts were preceded by further starch accumulation. Dividing protoplasts were 33–60% smaller and contained 8–42% less starch than non-dividing protoplasts. However, calculations showed that, in the dividing protoplasts, the relative area covered by starch was 6–12% higher than in non-dividing protoplasts. These data suggest that starch accumulation precedes division of pea protoplasts.     

Selectivity to K+ and Na+ of protoplast fractions isolated from different regions of Aspergillus nidulans hyphae

The selectivity to K+ and Na+ of protoplast samples representing cytoplasm isolated from different regions of the hyphal filament of Aspergillus nidulans was investigated. Concentrations of both ions contained in successive protoplast fractions were measured. During lytic digestion, protoplasts were released first from apical regions and subsequently from progressively older regions of hyphae. A low K+/Na+ ratio was found in protoplasts containing primarily apical cytoplasm and a high K+/Na+ ratio was found in protoplasts originating from older regions of hyphae. The ratios were the same whether MgSO4 or mannitol was used as stabilizer. Absolute concentrations of both ions were higher in protoplasts of apical origin. Protoplasts stabilized in mannitol lost more ions than those stabilized in MgSO4 over an 8 h incubation period. Na+ losses were higher from apical protoplasts whereas K+ losses were higher from protoplasts liberated from older regions of hyphae. The addition of divalent metal cations (1.5 mM-Mn2+ or Mg2+) reduced losses of Na+ from protoplasts but did not affect loss of K+. Data obtained using protoplast samples were related to those obtained for intact mycelium. Absolute losses of both ions from mycelium were lower than for protoplasts but when compared on a protein basis the data suggested that protoplasts possess properties similar to those of intact mycelium in terms of K+ and Na+ selectivity.     

Co-electroporation of rice protoplasts with RNAs of cucumber mosaic and tobacco mosaic viruses

Cucumber mosaic virus (CMV) RNA was used to study electroporation conditions suitable for protoplasts from rice suspension cultures. Rice protoplasts required a stronger and shorter electric pulse than tobacco protoplasts for introduction of viral RNA. Under optimized conditions, CMV infection was established in 65 % of electroporated protoplasts. In contrast, electroporation with tobacco mosaic virus (TMV) RNA did not result in infection of rice protoplasts. However, when TMV RNA was electroporated into rice protoplasts together with CMV RNA, TMV production was demonstrated in 15 % of protoplasts. Differential staining with fluorescent antibodies against the two viruses showed that the protoplasts producing TMV were without exception also infected by CMV. The results show that CMV replicates in rice protoplasts by itself, whereas TMV does so only with the aid of CMV.Abbreviations CMV cucumber mosaiv virus - PBS phosphate buffered saline - TMV tobacco mosaic virus.     

Changing desiccation tolerance of pea embryo protoplasts during germination

Protoplasts were isolated from pea (Pisum sativum L. cv. Alaska) embryonic axes during and after germination to determine whether the loss of desiccation tolerance in the embryos also occurs in the protoplasts. At all times studied, protoplast survival decreased as water content decreased; however, the sensitivity to dehydration was less when the protoplasts were isolated from embryos that were still desiccation-tolerant (12 h and 18 h of imbibition) than when protoplasts were derived from axes that were sensitive (24 h and 36 h of imbibition). The water content at which 50% of the population was killed (WC50) increased throughout germination and early seedling growth for both the intact tissue and the protoplasts derived from them. Prior to radicle emergence, protoplasts were less desiccation-tolerant than the intact axes; however, protoplasts isolated from radicles shortly after emergence had lower WC50s than the intact radicles. A comparison of protoplast survival after isolation and dehydration in either 500 mM sucrose/raffinose or 700 mM sucrose revealed no difference in tolerance except at 24 h of imbibition, when protoplasts treated in the more concentrated solution had improved tolerance of dehydration. Although intact epicotyls are generally more desiccation-tolerant than radicles, protoplasts isolated separately from epicotyls and radicles did not differ in tolerance. Collectively, these data suggest that protoplasts gradually lose desiccation tolerance during germination, as do the orthodox embryos from which they were derived. However, even prior to radicle emergence, protoplasts display a sensitivity to progressive dehydration that is similar to that shown by recalcitrant and ageing embryos.     

Structural and ultrastructural analysis of Solanum lycopersicoides protoplasts during diploid plant regeneration

Light, fluorescence and electron microscopy were used to analyse the structural properties of protoplasts obtained from established suspension culture of Solanum lycopersicoides Dun, composed of meristematic cell aggregates. Four types of protoplasts were distinguished immediately after isolation: (1) mononuclear; (2) polynuclear, (3) anuclear and (4) homogeneous protoplasts. Only mononuclear protoplasts were capable of complete cell wall regeneration and mitotic division. Other types of protoplasts were eliminated during culture. Three phases were distinguished in the developing protoplast culture: (1) the elimination phase during which protoplasts damaged during isolation underwent complete degradation; (2) a phase of intense division during which both mitotic cell division and amitotic nuclear division took place; and (3) a stabilization phase leading to the formation of suspension culture. The cell suspension culture obtained from protoplasts was capable of regenerating diploid plants.     

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.     

Microviscosity of plasmalemmas in rose petals as affected by age and environmental factors

The microviscosity of the plasmalemma of protoplasts isolated from rose (Rosa hyb. cv. Golden Wave) petals was measured by fluorescence depolarization. The plasmalemma's microviscosity was found to increase in petals which were allowed to age on cut flowers or after isolation as well as in isolated protoplasts aged in an aqueous medium. Increasing the temperature of the cut flowers or the isolated protoplasts enhanced the increase of the microviscosity of the protoplast plasmalemma. The mole ratio of free sterol to phospholipid was greater in protoplasts isolated from old flowers or in protoplasts aged after isolation than in protoplasts isolated from younger flowers. Microviscosity was greatest when protoplasts were aged at pH 4.4 and in the presence of Ca²⁺. Artificial alterations of the sterol to phospholipid ratio in the protoplasts, induced by treatment with liposomes, caused similar changes in their measured microviscosity.

These findings strongly suggest that the increase in the petal plasmalemma microviscosity with age is associated with an increase in the sterol to phospholipid ratio which results, at least partially, from the activity of endogenous phospholipases.

     

Seawater-resistant, non-spherical protoplasts from seagrass leaves

Two distinct types occurred among enzymatically isolated protoplasts from leaves of eelgrasses ( Zostera marina L., Z. japonica Ascherson and Phyllospadix iwatensis Makino). Spherical protoplasts with a smooth cell membrane were obtained only from young leaf tissues at the basal portions of blades protected from seawater by tightly enclosing sheaths. Non-spherical protoplasts had a highly invaginated cell membrane and were obtained from mature leaf blades, where the cells also in situ have this type of membrane. The protoplasts from mature leaves were rather rigid in shape and resistant to wide ranges of osmotic potential and salinity without change in their non-spherical shape, while the spherical protoplasts were rapidly destroyed in seawater. Detergents lysed the spherical protoplasts but not the non-spherical ones, suggesting that the highly invaginated enclosing structures of the non-spherical protoplasts contained detergent-resistant materials. Thus, the seagrass leaf cells develop seawater resistance, and this change alters the nature of the enclosing structures during the growth of the leaf blades. The non-membranous enclosing structures and their characteristic materials in the mature leaf cells remain to be defined.