Response to chilling of tomato mesophyll protoplasts

Freshly isolated protoplasts from tomato leaves show two completely different responses to a chilling treatment of 12 h at 7° C prior to culture at 29° C, depending on the presence or absence of glucose in the medium. In the culture medium with glucose as osmoticum, where the rate of cell divisions under optimal culture conditions is relatively high (about 20% plating efficiency), protoplasts were drastically injured by the chilling procedure and died. In the medium with mannitol as the osmoticum instead of glucose, where the plating efficiency even under optimal conditions is rather low (about 8%), protoplasts withstand the chilling procedure. More-over, after the chilling treatment when the protoplasts were transferred to the optimal culture temperature of 29° C, the plating efficiency was raised to about 20%, which is the same level as in the glucose-containing medium without chilling. This effect was not observed when the medium in which the protoplasts were suspended during the chilling period was replaced with fresh medium. This suggests that under these conditions tomato protoplasts produce and excrete a factor in the cold that improves the vitality of the cells or stimulates cell division. The possible relationship between chilling sensitivity of tomato protoplasts and their ability to divide will be discussed.     

Cell wall regeneration and cell division in isolated tobacco mesophyll protoplasts

Summary Protoplasts were isolated from palisade tissue of tobacco leaves by treatment with pectinase and cellulase under aseptic conditions, and were cultured in a synthetic liquid medium. Calcofluor, a fluorescent brightener, was found to be an excellent stain for plant cell walls and was used to demonstrate regeneration of cell walls in these protoplasts. The cultured protoplasts regenerated cell walls by the 3rd day of culture, giving rise to spherical cells. The majority of the protoplasts regenerating cell walls underwent mitosis and cell division. The cycle of mitosis and cell division was repeated 2–3 times during 2 weeks of culture. Some of the nutritional conditions affecting division in the cultured protoplasts were studied.     

The effect of 8 days of microgravity on regeneration of intact plants from protoplasts

The growth and development of protoplasts of rapeseed (Brassica napus L. cv Line) and carrot (Daucus carota L. cv. Navona) were studied onboard the Space Shuttle‘Discovery’during an 8-day International Microgravity Laboratory [IML-l) mission in January 1992. The Flight experiments were carried out in‘Biorack'. a fully controlled cell biological experimental facility. under microgravity conditions and in a l-g centrifuge. Parallel experiments were performed in a‘Biorack’module on the ground. After retrieval, some samples were subcultured on appropriate media and analysed for callus growth and regeneration to intact plants. The remainder were used for biochemical analysis. Samples fixed on board the Space Shuttle were kept in l% glutaraldehyde fixative at 4°C for 3–7 days for microscopy analysis after retrieval. Protoplasts exposed to microgravity conditions showed a delay in cell wall synthesis. Cells were swollen in appearance and formed cell aggregates with only few cells. Callus were obtained from protoplasts cultured under microgravity (Fogl). on the l-g centrifuge on board the shuttle (Flg), under normal l-g conditions on the ground (G1g) and on a centrifuge on the ground giving 1.4 g (Gl.4g). Regeneration of intact rapeseed plants was obtained from Flg. Glg and G1.4g. However, no plants were regenerated from protoplasts exposed to microgravity (Fog). Biochemical analysis indicated that the microgravity samples (Fog displayed a reduced packed cell volume, an increased concentration of soluble proteins per cell, and a reduced specific activity of peroxidase in the cytoplasm. Morphometric analysis of fixed samples demonstrated that 3-day old protoplasts under microgravity conditions were significantly larger than protoplasts kept on the l-g centrifuge in space. UItrastructural analysis by transmission electron microscopy showed that protoplasts exposed to microgravity conditions for 3 days had larger vacuoles and a slightly reduced starch content compared to Flg cells. Cell aggregates formed under microgravity conditions (Fog) had an average of 2–I cells per aggregate while aggregates formed under Flg had 8–12 cells.     

Effect of simulated and real weightlessness on early regeneration stages of Brassica napus protoplasts

Summary Results from experiments using protoplasts in space, performed on the Biokosmos 9 satellite in 1989 and on the Space Shuttle on the IML-1-mission in 1992 and S/MM-03 in 1996, are presented. This paper focuses on the observation that the regeneration capacity of protoplasts is lower under micro-g conditions than under 1 g conditions. These aspects have been difficult to interpret and raise new questions about the mechanisms behind the observed effects. In an effort to try to find a key element to the poor regeneration capacity, ground-based studies were initiated focusing on the effect of the variable organization and quantity of corticular microtubules (CMTs) as a consequence of short periods of real and simulated weightlessness. The new results demonstrated the capacity of protoplasts to enter division, confirming the findings in space that this was affected by gravity. The percentage of dividing cells significantly decreased as a result of exposure to simulated weightlessness on a 2-D clinostat. Similar observations were made when comparing the wall components, which confirmed that the reconstitution of the cell wall was retarded under both space conditions and simulated weightlessness. The peroxidase activity in protoplasts exposed to microgravity was slightly decreased in both 0 g and 1 g flight samples compared with the ground controls, whereas activity in the protoplasts exposed to simulated weightlessness was similar to activity in the 1 g control. The observation that protoplasts had randomized and more sparse corticular microtubules when exposed to various forms of simulated and real weightlessness on a free-fall machine on the ground could indicate that the low division capacity in 0 g protoplasts was correlated with an abnormal CMT array in these protoplasts. This study has increased our knowledge of the more basic biochemical and cell biological aspects of g effects. This is an important link in preparation for the new space era, when it will be possible to follow the growth of single cells and tissue cultures for generations under microgravity conditions on the new International Space Station, which will be functional on a permanent basis from the year 2003.     

Formation and Regeneration of Methanococcus voltae Protoplasts

Methanococcus voltae cells were converted into protoplasts by suspension in anaerobic 0.1 M Tris-HCl buffer containing 0.4 M sucrose and 0.05 M NaCl as osmoprotectants. Protoplast formation was monitored microscopically by observing the conversion of the typical irregularly shaped (uneven peripheries) coccoid whole cells to rounded forms with smooth peripheries. Although the procedure resulted in about 50% lysis of the initial number of cells, the remainder were converted to the rounded form. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electron microscopy of negatively stained cell preparations indicated that the treatment removed the wall layer from whole cells to yield protoplasts. Protoplast regeneration was evaluated by using optimized plating conditions and an anaerobic microplating technique. Between 50 and 63% of the initial number of protoplasts regenerated as colonies on agar medium (35°C, 7 days). The colony and cell morphologies of the regenerated protoplasts were indistinguishable from those of whole cells plated under identical conditions.     

Differential reponses to paraquat‐induced oxidative injury in a pea (Pisum sativum) protoplast system

Antioxidant responses to varying degrees of paraquat stress in freshly isolated photosynthesizing pea (Pisum sativum L.) protoplasts from cultivars Progress and Nugget were studied. Leaves of comparable maturity were used for protoplast isolation. Nugget protoplasts were more resistant to paraquat in the micromolar range under our conditions. In Nugget, a non-bleaching paraquat concentration (10 µM) inhibited CO₂-dependent O₂ evolution ca 50% during the first 40 min, remaining at that rate (“coping behavior”) for up to 100 min. In contrast, Progress protoplasts treated with the same concentration of paraquat did not exhibit coping behavior. Antioxidant enzyme activities were unaltered throughout the time course of the experiment in treated protoplasts from Nugget and in chloroplasts isolated from them. Thus, the coping behavior of Nugget protoplasts cannot be attributed to changes in activities of the three antioxidant enzymes tested. Paraquat treatment did not affect antioxidant enzyme activities in Progress protoplasts nor in chloroplasts isolated from them. When higher doses of paraquat were used (12 h, 0.1 mM paraquat), protoplasts from both cultivars were rapidly bleached and total protein decreased to ca 30% of pre-stress levels. Glutathione reductase (GR, EC 1.6.4.2) activity dropped in protoplasts from both cultivars under the severe stress conditions in concert with declines in protein levels. However, superoxide dismutase (SOD, EC 1.15.1.1) activity remained constant over the first 9 h of the time course, increasing to ca 150& of original levels by the final, 12-h time point. The activity of the plastid Cu,Zn isoform, expressed as a percentage of total SOD activity, declined over the time course of the experiment while that of mitochondrial MnSOD appeared to increase. This change in activity of MnSOD correlated with cell decline, therefore, and was not correlated with protection. These data are in agreement with some earlier reports and are compatible with the hypothesis that SOD activity levels increase in response to reactive oxygen species levels, even under conditions leading to cell death.     

Uptake of nystatin by protoplasts of sensitive and resistant cells of Saccharomyces cerevisiae

The uptake of nystatin by protoplasts derived from sensitive and resistant cells of Saccharomyces cerevisiae has been studied as a function of nystatin concentration, temperature and pH. The presence or absence of glucose in the uptake experiments was also studied. Activation energies (Ea) for nystatin uptake revealed profound differences between protoplasts derived from sensitive and resistant cells. Those for the latter closely resembled their whole cell counterparts. The values of Ea for the uptake of nystatin under all the conditions studied indicate the importance of the cell wall in the uptake process.     

Auxin Conjugation by Tobacco Mesophyll Protoplasts : Correlations between Auxin Cytotoxicity under Low Density Growth Conditions and Induction of Conjugation Processes at High Density

Auxin induction of the proliferation of Nicotiana tabacum (cv Xanthi) mesophyll protoplasts and of protoplast-derived cells was studied. The growth-promoting properties and cytotoxicities at high concentrations of IAA and naphthaleneacetic acid were strongly affected by cell density. The induction of growth by 2,4-dichlorophenoxyacetic acid and picloram was not affected by cell density. The comparison of catabolism of these [¹⁴C]-labeled auxins by protoplasts showed that IAA and naphthalene-acetic acid were rapidly accumulated and conjugated unlike 2,4-dichlorophenoxyacetic acid and picloram. The major catabolite derived from naphthaleneacetic acid was identified as naphthaleneacetyl-l-aspartate. The biosynthesis of this conjugate in protoplasts was inducible by naphthaleneacetic acid concentrations found to be cytotoxic under low density growth conditions. However, although it was taken up by cells, the conjugate was not cytotoxic at concentrations as high as 0.2 mm under low density growth conditions. The relationship between conjugation processes and auxin cytotoxicity is discussed.     

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.     

Isolation and transformation of rice aleurone protoplasts

Summary Protoplasts isolated from the aleurone have been used extensively in molecular studies focusing on hormone-mediated regulation of gene expression in barley seed. To extend the use of aleurone protoplasts to other species, we have determined the conditions necessary for the isolation of protoplasts from rice aleurone layers of germinated seed. Many of the common cell wall degrading enzymes used in making protoplasts were tested for their ability to release protoplasts from rice aleurone layers. Cellulysin was found to be the most effective. Transformation of these aleurone protoplasts was accomplished using polyethylene glycol and DNA constructs containing the firefly luciferase reporter gene under the control of two different promoters were tested. Luciferase expression was 24-fold greater when the reporter gene was under the control of the CaMV 35S promoter than when the promoter from the alcohol dehydrogenase 1 gene was used. With the isolation and transformation of aleurone protoplasts from rice, it is now possible to investigate molecular events occurring in this tissue during germination.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium.

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

Protoplast formation and regeneration of dehydrodivanillin-degrading strains of Fusobacterium varium and Enterococcus faecium

Two strains of rumen anaerobes isolated from dehydrodivanillin-degrading cultures were identified as Fusobacterium varium and Enterococcus faecium. These organisms degraded dehydrodivanillin synergistically to 5-carboxymethylvanillin and vanillic acid. Specific conditions for protoplast formation and cell wall regeneration for both bacteria were determined, under strictly anaerobic conditions, to be as follows. The cell wall of each bacterium in yeast extract medium was loosened by adding penicillin G during early log-phase growth. The cell wall of F. varium was lysed by lysozyme (1 mg/ml) in glycerol (0.2 M)-phosphate buffer (0.05 M; pH 7.0). The addition of NaCl (0.08 M) with lysozyme was necessary for lysis of E. faecium in this solution. Almost all cells were converted to protoplasts after 2 h of incubation at 37 degrees C. Regeneration of both protoplasts was 20 to 30% on an agar-containing yeast extract medium.     

野大麦幼穗原生质体的分离和培养

以野大麦(Hordeum brevisubulatum)的幼穗诱导愈伤组织,建立悬浮细胞系,利用胚性悬浮细胞系在不同的酶解条件和不同的酶解液中分离原生质体并进行培养,获得了细胞分裂,形成细胞团。     

The inheritance of diastatic power and alpha-amylase contents in spring barley

Summary Nuclei were isolated from various types of donor protoplasts and were transferred into receptor protoplasts in numerous combinations. Five percent uptake was achieved under conditions which did not interfere with viability and subsequent culture of receptor protoplasts. Methodological investigations on nuclei uptake were carried out with cereal and tobacco protoplasts. To look for biological proof of integration and replication of transferred nuclear genes, two complementing, chlorophyll-deficient, light-sensitive mutants of tobacco were used as sources of nuclei and receptor protoplasts. Ca. 5.5 × 10⁷ receptor protoplasts were cultured following transplantation experiments involving these complementing mutants and about 1.8 × 10⁷ of the resulting calli were subjected to selective conditions which discriminate against the parental types. No nuclear hybrids were detected, although in control experiments somatic hybrids were obtained by protoplast fusion. Some explanations for failure of nuclear hybrid formation are discussed together with other possible approaches for selective somatic combination of plant cell genophores.     

Isolation and Regeneration of Tobacco Mesophyll Cell Protoplasts under Low Osmotic Conditions

A method is described for the isolation of large numbers of tobacco (Nicotiana tabacum L. cv. Xanthi-nc) mesophyll cell protoplasts under relatively low external osmotic conditions. The procedure utilized 0.2 m sucrose as the primary osmoticum and a mixture of 0.5% macerozyme, 4% cellulase, and 2% polyvinylpyrrolidone, pH 5.4. The viability of resultant protoplasts was confirmed through regeneration of fertile plants. Plating and regeneration studies revealed, however, that qualitative and quantitative modifications in plating and differentiation media were necessary for protoplasts prepared in this manner. Over-all, the procedure was found to be a simplified alternative to those previously described for tobacco protoplast regeneration. In addition, the system should permit studies related to the influence of differing osmoticum levels on a variety of cell functions.     

Induction of a long-lived fusogenic state in viable plant protoplasts permeabilized by electric fields

Electropermeabilized tobacco mesophyll protoplasts are shown to fuse by creating cell contact several minutes after electropulsation. Electropermeabilization was analysed by measuring calcein uptake. Experiments were performed at low temperature to avoid resealing of protoplast transient permeation structures. These results confirm that the long-lived permeabilized state induced by the electric field is associated to a fusogenic state, under viability conditions. This is indicative that as for mammalian cells, the electric field-induced membrane modifications, which give the permeable state, are such as to decrease the magnitude of the intercellular repulsive forces between plant protoplasts. Such a fusion method may be useful for somatic hybrids production with protoplasts showing morphological and physiological differences.     

介质钙离子对白芷悬浮培养细胞及其原生质体增殖的影响

本工作首先利用《复杂络合平衡体系》计算并配制了对自由钙离子浓度具有络合平衡缓冲能力的MS液体培养基,并用电极法验证了其可靠性。在精确控制Ca~(2 )浓度条件下,利用计数法和~3H-TdR标记DNA合成的方法系统研究了不同钙离子浓度对白芷悬浮细胞及原生质体细胞增殖的影响。原生质体第一次细胞分裂所需Ca~(2 )浓度(10mmol/L)比细胞增殖所需Ca~(2 )浓度(1mmol/L)为高;不同钙离子浓度对原生质体壁再生、活力及第一次细胞分裂的作用也不一样,壁再生所需最适Ca~(2 )浓度为50mmol/L,原生质体存活以及第一次细胞分裂所需最适Ca~(2 )浓度为5—10 mmol/L,当Ca~(2 )浓度小于10~(-4)mol/L时细胞及原生质体的增殖受到很大程度的抑制,细胞死亡数目较多。结果表明介质钙离子浓度与细胞及原生质的增殖密切相关。     

野大麦幼穗原生质体的分离和培养

以野大麦（Ｈｏｒｄｅｕｍｂｒｅｖｉｓｕｂｕｌａｔｕｍ）的幼穗诱导愈伤组织,建立悬浮细胞系,利用胚性悬浮细胞系在不同的酶解条件和不同的酶解液中分离原生质体并进行培养,获得了细胞分裂,形成细胞团。     

赤霉素产生菌~#85104菌株原生质体的形成再生和紫外光对原生质体诱变条件的研究

赤霉产生菌在正常条件下不产生孢子,育种材料以多核菌丝体及其碎片为主,大部分常用的诱变剂对它们诱变效应较差。本文报导有关赤霉素产生菌~#85104菌株原生质体的获得、再生和紫外光诱变原生质体的条件,甘氨酸对菌丝体生长的影响,以及采用二硫苏糖醇预处理菌丝体等方面条件的研究。在采用紫外光照射原生质体的诱变处理下,已获得数株高产的新菌株。