Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.     

Distribution of photorespiratory enzymes between bundle-sheath and mesophyll cells in leaves of the C3−C4 intermediate species Moricandia arvensis (L.) DC

In order to study the location of enzymes of photorespiration in leaves of the C₃–C₄ intermediate species Moricandia arvensis (L.). DC, protoplast fractions enriched in mesophyll or bundlesheath cells have been prepared by a combination of mechanical and enzymic techniques. The activities of the mitochondrial enzymes fumarase (EC 4.2.1.2) and glycine decarboxylase (EC 2.1.2.10) were enriched by 3.0- and 7.5-fold, respectively, in the bundle-sheath relative to the mesophyll fraction. Enrichment of fumarase is consistent with the larger number of mitochondria in bundle-sheath cells relative to mesophyll cells. The greater enrichment of glycine decarboxylase indicates that the activity is considerably higher on a mitochondrial basis in bundle-sheath than in mesophyll cells. Serine hydroxymethyltransferase (EC 2.1.2.1) activity was enriched by 5.3-fold and glutamate-dependent glyoxylate-aminotransferase (EC 2.6.1.4) activity by 2.6-fold in the bundle-sheath relative to the mesophyll fraction. Activities of serine- and alanine-dependent glyoxylate aminotransferase (EC 2.6.1.45 and EC 2.6.1.4), glycollate oxidase (EC 1.1.3.1), hydroxypyruvate reductase (EC 1.1.1.81), glutamine synthetase (EC 6.3.1.2) and phosphoribulokinase (EC 2.7.1.19) were not significantly different in the two fractions. These data provide further independent evidence to complement earlier immunocytochemical studies of the distribution of photorespiratory enzymes in the leaves of this species, and indicate that while mesophyll cells of M. arvensis have the capacity to synthesize glycine during photorespiration, they have only a low capacity to metabolize it. We suggest that glycine produced by photorespiratory metabolism in the mesophyll is decarboxylated predominantly by the mitochondria in the bundle sheath.Abbreviation RuBP ribulose 1,5-bisphosphate     

Improvements in the transformation of Bacillus subtilis protoplasts with plasmid DNA

Abstract The transformation system currently used for Bacillus subtilis protoplasts has been improved. Special emphasis was made on three parameters of practical importance:
(a) conditions for direct selection of transformants, (b) optimization of the transformation system for Rec⁻ mutants, and (c) conservation of protoplast suspensions for further use.
Selective regeneration was efficiently achieved for kanamycin or neomycin. Chloramphenicol, tetracycline and erythromycin were only expressed when low concentrations of the antibiotics were used to select transformants during regeneration.     

Relative performance of monohaploid potato clones and their diploid parents at plant level and after protoplast isolation and subsequent fusion

Summary Plant growth performance was studied in 118 potato monohaploids and in their diploid parents. Of these monohaploids 76 were also investigated at the protoplast level and eight of these were used in protoplast fusion experiments as well. No correlation was found between relative performance of greenhouse grown and in vitro grown plants. No or only weak correlations were found between different in vitro characteristics such as plant growth, protoplast yield per gram plant material, plating efficiency and callus growth. This indicates the unpredictability of these characters.The protoplast fusion experiments indicated that only in some genotype combinations increased callus growth rates may be found. However, it is not clear whether such calli were hybrids or not. In protoplast monocultures only diploid and tetraploid regenerants were obtained. After fusion, tetraploids but also some triploids could be regenerated. The finding of triploids indicates that monoploid protoplasts were involved in fusion. Isozyme analysis and morphological assessment of the plants pointed out that the majority of the fusion regenerants were hybrids. The implications of these results are discussed.     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion

Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.     

A protoplast to plant system for the chrysanthemum Dendranthema zawadskii x D. grandiflora

Summary Plantlets were regenerated from protoplasts of in vitro shoot cultures and leaf-derived de novo shoots of the chrysanthemum Dendranthema zawadskii x D. grandiflora. Isolated protoplasts reformed cell walls and then began to divide within 24 hours of culture in streaky plate agarose lenses surrounded by liquid V-KM medium. Twenty one days after isolation, 1 mm diameter callus clumps were transferred to shoot regeneration medium. After a further 33 days leaves became visible. Elongated shoots were rooted on half strength hormone-free MS medium.Abbreviations BAP 6-benzylaminopurine - IAA 3-indoleacetic acid - MS Murashige and Skoog (1962) - NAA 1-naphthylacetic acid - Pfr Photon fluence rate - V-KM Binding and Nehls (1977)     

Cortical microtubular lattices: Absent from mature mesophyll and necessary for cell division?

Using immunofluorescence microscopy, the cortical microtubular net which is regularly present in cells of young, growing tissue is shown to be absent, or largely reduced, in mature mesophyll cells of Nicotiana plumbaginifolia Viv., N. tabacum L., Petunia hybrida Hort. and Brassica napus L. The onset of division in protoplasts isolated from these fully differentiated tissues is preceded by a period of dedifferentiation. One of the early events during dedifferentiation, as shown for N. plumbaginifolia, is the re-establishment of a net of cortical microtubules, prior to spindle formation. These findings indicate that the presence of the cortical microtubular lattice is a prerequisite for protoplast division. Cell-wall regeneration, which also must precede division, occurs simultaneously with the formation of the lattice. However, the cortical microtubules seem to not exert any influence on the orientation of the microfibrils.     

Culture of plant somatic hybrids following electrical fusion

Summary Electrically-induced protoplast fusion has been used to produce somatic hybrids between Nicotiana plumbaginifolia and Nicotiana tabacum. Following fusion of suspension culture protoplasts (N. plumbaginifolia) with mesophyll protoplasts (N. tabacum) heterokaryons were identified visually and their development was followed in culture. Because electrical fusion is a microtechnique, procedures were developed for culturing the heterokaryons in small numbers and at low density. The fusion and culture procedures described are rapid, uncomplicated and repeatable. Good cell viabilities indicate that the fusion procedure is not cytotoxic. Fused material was cultured 1–2 days at high density in modified K3 medium (Nagy and Maliga 1976). The heterokaryons were isolated manually and grown, at low density in conditioned media. Calli have been regenerated. Esterase isozyme patterns confirm the hybrid character of calli and clonally-derived plantlets recovered from these fusions.     

金针菇褐化病毒(FvBV)脱毒方法

【目的】评价5种不同脱毒方法对金针菇(Flammulina velutipes)菌株的脱毒效果,筛选出脱毒率高和脱毒后金针菇菌株菌丝生长速度、生物量、漆酶活力等性状改善明显的脱毒方法。【方法】以栽培金针菇菌株F-4889为研究材料,从菌丝体中提取大小约2.0 kb的病毒dsRNA,经RT-PCR鉴定该病毒为金针菇褐化病毒(FvBV)。采用菌丝尖端分离、原基组织分离、原生质体单核化、有性生殖和核迁移5种脱毒方法对金针菇菌株进行脱毒处理,利用dsRNA技术和RT-PCR检测脱毒效果。【结果】菌丝尖端分离脱毒后得到1株脱毒菌株;原基组织分离法未能脱毒;原生质体单核化脱毒法得到3株脱毒单核菌株和2株原单杂交脱毒菌株;有性生殖脱毒法获得脱毒孢子单核菌株23株和单孢杂交脱毒菌株8株;核迁移脱毒后得到5株核迁移脱毒菌株。脱毒率依次为25.0%、0、7.5%、57.5%和100%。脱毒菌株的菌丝生长速度、生物量、漆酶活力等均优于出发菌株、菌丝尖端和原基组织分离菌株。【结论】这5种方法中原生质体单核化、有性生殖和核迁移脱毒法脱毒效果较佳,均能有效脱除FvBV,脱毒率高,脱毒后菌株菌丝生长速度、生物量、漆酶活力等均明显提高。