Influence of enzyme solution on protoplast culture and transient gene expression in maize (Zea mays L.)

The influence of two enzyme solutions, differing only in the presence or absence of Macerozyme, on protoplast yield, colony formation and transient GUS (-glucuronidase) activity was studied. For all parameters tested the presence of Macerozyme during protoplast isolation had a negative influence. Using an enzyme solution without Macerozyme suspension aggregates gave up to 4.4 times higher protoplast yield and plating efficiencies were increased up to 10-fold. Further, protoplasts isolated without macerozyme showed a 5.2-fold higher GUS activity in transient gene expression. Apart from the presence of Macerozyme, longer incubation (3 compared with 1.5 h) of cell aggregates in the enzyme solution also had a negative effect on transient transformation efficiency. These data demonstrate that protoplast isolation conditions have a profound effect on transient gene expression and it is proposed that these factors will also influence stable transformation efficiency.Abbreviations CP cellulase pectolyase - CPM cellulase pectolyase Macerozyme - 2,4-d 2,4-dichlorophenoxyacetic acid     

Confocal Microscopy of Microtubule Cytoskeleton in the Pollen Protoplast of Narcissus

Pollen protoplasts were isolated from the mature pollen grains of Narcissus cyclamineus using cellulase Onozuka'R-10 and pectinase in Bs medium. The microtubule cytoskeleton in the pollen protoplasts was studied using immunofluorescence and confocal microscopy. In the cortical region there was a very complex microtubule network. The network contained numerous whirl-like arrays. The microtubule bundles in the whirl-like arrays were connected with each other by smaller bundles indicating that the arrangement of the whirl-like bundles were quite well organized and not at random. From the cortex to the centre of the protoplast another microtubule network having a structure different from the one in the cortical region was present. This network was much loosely packed than the cortical network. The arrangement of the microtubule bundles near the vegetative nucleus was again different. Numerous granules appeared outside the nuclear membrane. From these granules microtubule bundles radiated towards the cytoplasm. The arrangement of the microtubule network around the generative cell showed no specialized features. But inside the cell three types of microtubule arrays were present. 1. parallel arrays, 2. network, and 3. a mixture of the two. In the bursted pollen protoplast (as a result of osmotic shock treatment )some microtubule bundles could still be found attached to the ghost. The microtubule bundles associated with the ghost were much fragmented. But some still retained their branches and junctions. In the dry cleaved samples,a number of organelles still remained attached to the membrane and they included : microtubules, microfilaments, coated vesicles, endoplasmic reticulum and numerous honey-comb-like apparatus. The honey-comb-like apparatus was named as coated pits by Traas (1984). But we feel that it is more appropriate to call this organelle the honey-comb apparatus and we also believe that this organelle may be involved in microtubule and/or microfilament organization.     

The isolation, culture and division of protoplasts from citrus cotyledons

High yields (2.3 × 10⁵ to 1.3 × 10⁶ protoplasts/g.f.wt.) of isolated protoplasts were obtained from cotyledons of Cirus sinensis (L.) Osb. 'Valencia'. Osmotic potential of the medium and enzyme concentrations were important in obtaining high viability of preparations as indicated by FDA fluorescence. Adding malt extract to a Murashige-Tucker basal medium increased plating efficiencies somewhat, but not the rate or duration of cell division. However, modifying the NAA and kinetin concentration optimized plating efficiencies (up to 20%) of protoplasts and also the rate or duration of cell division. The highest plating efficiency and number of cells per colony were obtained on a defined medium containing NAA (15 μ M ). and kinetin (4.6 μ M ). Coincidence of percentage protoplast viability after 13 days (assessed by FDA fluorescence) with plating efficiency after 21 days indicates that FDA fluorescence is an accurate indicator of citrus protoplast viability.     

Dedifferentiation and microtubule reorganization in the apical cell protoplast ofSphacelaria (Phaeophyceae)

Summary The apical cell ofSphacelaria, a tip-growing filamentous brown alga, and its protoplast constitute a model for the investigation of the consequences of cell wall removal on microtubular cytoskeletal organization and cell polarity. In the apical cell, the microtubular cytoskeleton is strongly polarized and, in most cases, extends from two centrosomes to the cortex where it constitutes a fine meshwork. Observations of microtubule dynamics throughout the cell cycle emphasize the coincidence between orientation of the mitotic axis and cell polarity. Just after protoplast isolation, dramatic alterations of initial polarity are observed, whatever the mitotic stage. In particular, the coincidence between cytoplasmic polarity and polarity of the system nucleus-centrosomes is lost in most cases. 12–24 h after protoplast isolation, the cell shows a more symmetrical organization while a dense cortical microtubular network spreads out concomitantly with wall reformation. Our discussion emphasizes the possible relationship between cell polarity and cell totipotency, and the relevance of such a model for higher plant studies.     

辣椒子叶原生质体分离条件的研究

以不同基因型的辣椒子叶为供体组织进行辣椒原生质体分离条件的研究,结果表明：幼龄子叶的原生质体产量与活力均高于老龄子叶;酶解过程中酶液渗透压、酶液浓度、酶解时间均对原生质体分离效果产生重要影响。对于辣椒子叶原生质体,最佳分离条件为酶液甘露醇浓度０．５ｍｏｌ／Ｌ,纤维素酶Ｃｅｌｌｕｌａｓｅ Ｏｎｚｕｋａ Ｒ－１０ １．５,果胶酶Ｍａｃｅｒｏｚｙｍｅ Ｒ－１０ ０．６％,酶解时间８－１０ｈ。不同基因型辣     

Gibberellin enhancement of the enzymic release of Pisum root cell protoplasts

Cultivar differences have been reported in the protoplast yields from Pisum sativum root cortical explants treated with preparations of commercial cellulase and pectinase. The presence of intracellular starch significantly influenced these protoplast yields. The application of gibberellin before or during the enzymic wall-degradation increased the protoplast yields from two of the five cultivars tested. For tissues of'Little Marvel' pea roots, 10 mg 1⁻¹ of gibberellin most effectively increased the release of protoplasts when the hormone preceded the enzyme incubation. One mg 1⁻¹ of gibberellin was most effective at increasing the protoplast release when the tissues were treated with the hormone simultaneously with the wall-degrading enzymes. Mitotic activity was significantly reduced in protoplasts derived from gibberellin-treated tissues.     

Colony formation and plant regeneration from mesophyll protoplasts of Solanum etuberosum

A procedure for the culture of Solanum etuberosum mesophyll protoplasts with subsequent shoot regeneration is described. Several factors affected protoplast yield, colony formation, and shoot regeneration from in vitro plants. A protoplast isolation medium with 0.6 M sucrose produced twice the yield as one with 0.3 M sucrose. uowever, a higher concentration of osmoticum was inhibitory to colony development unless it was diluted into a lower osmoticum medium in a bilayer system. A 16 hour light/8 hour dark photoperiod for stock plants allowed twice the protoplast yield compared to plants grown under continuous light but no effect was found on subsequent colony formation or shoot regeneration. The concentrations of four major salts in the protoplast plating medium were critical for a high frequency of colony formation from protoplasts. Levels of 0.25 × or 1 × were considerably better than 4 ×. Fast colony formation, but at a lower efficiency, was obtained with a monolayer plating method. A bilayer plating system allowed a higher efficiency but colonies developed more slowly. For the best treatments, the frequency of colony formation from protoplasts ranged from 2.4 to 3.6 × 10^-3 with 37% to 66% of the colonies producing shoots ten weeks after protoplast isolation.Cooperative investigation of the USDA-ARS and the Wisconsin Agric. Exp. Stn.     

Effect of atmospheric pressure on sunflower (Helianthus annuus L.) protoplast division

Summary Sunflower protoplasts were cultured in liquid medium under high atmospheric pressure (0.2 to 0.6 MPa) and the plating efficiency, cell wall synthesis and microtubule organization were assessed. In 7-day-old cultures under a pressure of 0.4 MPa and above, the division rate was strongly reduced by more than 60% as compared to the control. Although most of the protoplasts had begun to regenerate a new cell wall they were unable to complete this process. Pressure also had an inhibitory effect on microtubule synthesis. The percentage of protoplasts showing a disassembled cortical network of microtubules was significantly increased up to 60% of the population. These effects were reversible: when protoplasts were transferred to normal pressure most of them rapidly recovered their capacity to divide and afterwards developed normally. Culturing protoplasts under a pressurized atmosphere revealed to be a good model system for studying cortical microtubule dynamics.Abbreviations BSA bovine serum albumin - PBS phosphate buffered saline - TBS tris buffer saline - MT(s) microtubule(s)     

Reorganization of interphase microtubules in root cells of <Emphasis Type="Italic">Medicago sativa</Emphasis> L. during acclimation to osmotic and salt stress

We examined the organization of microtubule system of interphase cells in roots of Medicago sativa L. during acclimation to salt and osmotic stress at different concentrations of NaCl, Na₂SO₄, and mannitol. We identified morphological changes of tubulin cytoskeleton in different root tissues during the acclimation to salt and osmotic stress: (1) decreased density of the cortical microtubule network, (2) random orientation of cortical microtubule bundles, (4) thickening of the bundles, (3) nonuniform density of the bundles, (4) fragmentation of the bundles, and (5) formation of microtubule converging centers. Network thinning and thickening of the bundles were observed both under osmotic and salt stress. Random orientation of cortical microtubules was visualized under osmotic stress but not during salt stress. Fragmentation of microtubule bundles took place under salt stress with a high concentration of mannitol. Formation of microtubule converging centers was common under prolonged action of sodium sulfate, less evident under sodium chloride, and not found after mannitol treatment. Our data show that, in alfalfa root cells, cortical microtubules rearrange not only in response to different ions, but also to osmotic pressure. Thus, the signaling pathways and molecular mechanisms inducing reorganization of the microtubule system may be triggered by sodium cations, as well as by sulfate and chloride anions at concentrations that do not cause irreversible cell damage.     

玉米、小麦、水稻原生质体制备条件优化

玉米Zea mays L.、小麦Triticum aestivum L.、水稻Oryza sativaL.是三大重要粮食作物,对其原生质体制备条件的优化具有重要意义.以玉米(综3)、小麦(中国春)、水稻(日本晴)10日龄幼苗为材料,研究了叶肉细胞原生质体分离过程中的酶浓度、酶解时间和离心力大小等因素对产量和活力的影响.结果表明:酶浓度和酶解时间对原生质体产量影响显著,随着酶解液浓度和酶解时间的提高,原生质体产量增加,但细胞碎片同时增多.水稻经真空处理后,原生质体产量大幅度提高.通过正交实验设计得出如下结果:玉米叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解7h,100×g离心2 min收集,原生质体产量为7×106/g FW;小麦叶肉细胞原生质体分离的最佳条件为:纤维素酶1.5％,离析酶0.5％,50 r/min酶解5h,100×g离心2 min收集,原生质体产量为6×106/g FW;水稻叶肉细胞原生质体分离的最佳条件为:纤维素酶2.0％,离析酶0.7％,50 r/min酶解7h,1 000×g离心2 min收集,得到的原生质体产量为6×106/g FW.通过二乙酸荧光素染色发现原生质体活力均在90％以上.用PEG-Ca2+介导法将含有绿色荧光蛋白的质粒转化入原生质体,转化率可达50％ ～80％.     

Efficient isolation, culture and regeneration of Lotus corniculatus protoplasts

This paper reports an improved protocol for isolation, culture and regeneration of Lotus corniculatus protoplasts. A range of parameters which influence the isolation of L. corniculatus protoplasts were investigated, i.e., enzyme combination, tissue type, incubation period and osmolarity level. Of three enzyme combinations tested, the highest yield of viable protoplasts was achieved with the combination of 2% Cellulase Onozuka RS, 1% Macerozyme R-10, 0.5% Driselase and 0.2% Pectolyase. The use of etiolated cotyledon tissue as a source for protoplast isolation proved vital in obtaining substantially higher protoplast yields than previously reported. Culture of the protoplasts on a nitrocellulose membrane with a Lolium perenne feeder-layer on the sequential series of PEL medium was highly successful in the formation of micro-colonies with plating efficiencies 3–10 times greater than previous studies. Shoot regeneration and intact plants were achieved from 46% of protoplast-derived cell colonies.     

三倍体‘银中杨’叶肉原生质体制备的优化

以三倍体杨树品种‘银中杨’（Populus alba×P.berolinensis Yinzhong）无菌苗叶片为材料,对其原生质体分离及纯化条件进行研究,为进一步通过细胞融合、基因工程等进行品种改良探索新的途径。结果表明：酶的种类及浓度、渗透压、酶解时间对‘银中杨’叶肉原生质体分离效果有显著影响,适宜的分离条件为CPW+3% Cellulase RS+0.5% Macerozyme R-10+0.3% Pectinse Y-23+0.6 mol/L甘露醇+0.6 g/L MES+1 g/L BAS,酶解时间为8 h,原生质体产量和活力分别为2.13×10⁷个/g和80.18%;‘银中杨’叶肉原生质体纯化最佳方法为上浮法蔗糖等密度离心,且蔗糖浓度为40%时原生质体产量最高（1.06×10⁷个/g）,可满足进一步的原生质体培养等技术的要求。     

‘过山香’香蕉胚性悬浮细胞原生质体分离的方法研究

目的：研究不同的方法对‘过山香’胚性悬浮细胞原生质体分离的影响,筛选最适合用于‘过山香’香蕉胚性悬浮细胞原生质体分离的方案。方法：用不同浓度、不同组合的酶液对‘过山香’原生质体进行分离,并对酶液的甘露醇含量、pH值进行调节。结果：3．0％纤维素酶R-10＋0．2％果胶酶Y-23的是最佳酶组合;酶解8h、酶液中含0．41M甘露醇、酶液pH值为5．3时,获得原生质体产量最高。结论：合适的酶组合、酶解时间、酶液的渗透压和pH值对‘过山香’香蕉胚性悬浮细胞原生质体的分离有明显的促进作用。     

The impact of homologous and heterologous nurse cells on the division capability of sparsely plated cells

The growth-promoting effects of nurse cells of carrot, tomato, patato, maize, bean, carnation and two species of tobacco were studied on carrot, tomato, tobacco and potato cells plated at low densities. In an area immediately below the nurse cells the plating efficiency was very high and found to be independent of cell density. In an area outside the nurse cells, in some cases, the plating efficiency tended to be much higher in combinations with cells from a heterologous source as compared with those from a homologous source. Moreover, in the same area with some combinations the plating efficiency decreased when cell density was lowered, while with other combinations this phenomenon did not occur. This decrease was independent of the absolute value of plating efficiency. In experiments in which the concentration of conditioning factors was presumably changed, no significant difference in the plating efficiency was noticed. We therefore suggest that different plating efficiencies observed with heterologous nurse cells were not due to a higher level of conditioning factors, but rather to the production of different types of conditioning factors that are presumably degraded with different efficiency.     

Two kinesin-related proteins associated with the cold-stable cytoskeleton of carrot cells: characterization of a novel kinesin, DcKRP120-2

We have previously described the biochemical isolation of 65 kDa and 120 kDa microtubule-associated proteins from carrot cytoskeletons. The 65 kDa MAPs have subsequently been shown to be structural MAPs that reconstitute 30 nm cross-bridges of the kind that maintain cortical microtubules in parallel groups. By exploiting its avid binding to microtubules, we have now devised a method for isolating MAP120 from protoplast extracts, and shown that it has properties of a kinesin-related protein. MAP120 segregates with the cold stable pool of microtubules in carrot cytoskeletons, whilst the 65 kDa MAPs are also associated with the cold-sensitive microtubules. On gradient gels, MAP120 resolves as two kinesin-like bands. We report the isolation of a carrot cDNA, DcKRP120-2, corresponding to a novel kinesin of the BimC class known to move to the plus ends of microtubules. Antibodies raised against specific expressed sequences recognize the upper band, while the lower band is recognized by antibodies to the tobacco kinesin-related protein, TKRP125. We have also isolated a partial genomic carrot DNA, DcKRP120-1, homologous to the motor region of tobacco TKRP125. Immunofluorescence of the two proteins produces different staining patterns. Anti-TKRP125 labels the cortical microtubules and the pre-prophase band, but anti-DcKRP120-2 does so only weakly. Both clearly stain the spindle and the phragmoplast, but in a proportion of cells anti-DcKRP120-2 strongly decorates the phragmoplast mid-line where the plus ends of the microtubules overlap. We discuss the potential roles of these proteins during the microtubule cycle.     

红曲霉原生质体的制备、再生及其遗传转化系统

原生质体是研究和建立真菌遗传转化系统的重要工具。为了建立原生质体介导的红曲霉遗传转化系统,考察了各种细胞壁裂解酶和渗透压稳定剂等对红曲霉原生质体形成和再生的影响。将红曲霉分生孢子在铺有玻璃纸的平板上30℃培养30~40 h收获的菌丝体最有利于原生质体的形成和释放。红曲霉菌丝体形成和释放原生质体最适裂解酶和酶解时间分别为：0.3 % lysing enzyme、0.1 % cellulase和1 % snailase的酶组合,30℃作用2.5 h;最适渗透压稳定剂是：1mol /L MgSO4。最适合原生质体再生的培养基为含0.6 mol/L蔗糖的CM培养基。原生质体液涂布单层再生培养基的方法,再生率最高,菌株M34和N18分别为8.5 %和36.4 %。在PEG和CaCl2存在下,以潮霉素B为抗生素选择标记,用质粒pBC-Hygro和pNL1共转化菌株M34原生质体,每微克DNA克获得100个稳定转化子。     

Plant donor tissue and isolation procedure effect on early formation of embryoids from protoplasts of Helianthus annuus L.

The effects of factors influencing sunflower protoplast isolation yield, plating efficiency (PE) and the early differentiation into embryoids (embryogenic capacity, EC) have been studied. Only hypocotyl-derived protoplasts divided. The variations of PE and EC in the various treatments did not seem to be linked to the protoplast yields. From statistical analysis of the data, we concluded that, the sunflower genotype, the age and height of seedlings, the part of hypocotyl used, the incubation time (from 6 to 16 hours) in enzymes of explants or of protoplasts alone, influenced PE but large variations were detected for EC. A comparison of the factors effecting EC suggested an origin, inside the hypocotyl, of cells able to give rise, after induction, to embryogenic protoplasts.     

多种被子植物花粉生殖细胞大量分离技术的比较研究

从11种植物的成熟花粉粒中分离出大量生活的生殖细胞。比较了四种分离方法(一步渗透压冲击法、二步渗透压冲击法、低酶法和花粉原生质体释放法)在不同植物中的效果。归纳出三类植物适于采用三种分离方法。研究了影响分离效果的若干重要因素。对5种植物的分离生殖细胞进行了纯化。经鉴定,纯化的细胞群体中80%以上的细胞是生活的。     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

小麦叶肉细胞原生质体中微管骨架的排列格局与[Ca^2＋]cyt的关系

以小麦叶肉细胞原生质体为材料,通过免疫荧光标记和Ca^2＋荧光染料的装载并结合药物学试验,借助激光共聚焦扫描显微镜观察,探讨微管骨架和Ca^2＋之间的内在联系。试验结果表明,[Ca^2＋]cyt的升高能够诱发微管骨架的解聚;而微管骨架的解聚也会促使胞外Ca^2＋内流,进而造成[Ca^2＋]cyt的升高。