Regeneration of plants from leaf explants of micropropagated clonal Eucalyptus grandis

Summary Callus production along with caulogenesis was obtained from leaf explants of micropropagated clonal Eucalyptus grandis after six to twelve weeks of culture. Out of eight clones tested, six were amenable to shoot production using simple media containing naphthaleneacetic acid and either 6-benzyladenine or zeatin. Differences in growth regulator requirements for organogenesis were observed between different clones. These shoots were then elongated on a medium containing gibberellic acid and rooted using media derived from the micropropagation medium. Light conditions were also found to be important for regeneration. This protocol is the first published on regeneration from nonseedling material and it will facilitate the Agrobacterium tumefaciens -mediated transformation of selected clonal Eucalyptus grandis.Abbreviations BA 6-benzyladenine - IBA indole-3-butyric acid - MS Murashige and Skoog (1962) medium - NAA naphthaleneacetic acid - PAR photosynthetically active radiation - PVP polyvinyl pyrrolidone - SH Schenk and Hildebrandt (1972) medium     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

A method to determine tannin concentration by the measurement and quantification of protein — tannin interactions

Summary Protein determinations on protein-tannin complexes after protein isolation (gel filtration and trichloroacetic acid [TCA] precipitation) or phenolic extraction (polyvinyl pyrrolidone [PVP] and organic solvent precipitation) were unsuccessuful. Kjeldahl determinations of the amount of unprecipitated protein bovine serum albumin [BSA] showed a sigmoid relationship with increasing concentrations of tannins. A similar relationship was found for the reduced viscosity of BSA and plant protein, and the concentration of tannin. Non-linear regression and curve normalization allowed three variable (k ₁, k ₂ and T _1/2) to be defined for the quantification of the protein-tannin interaction/s. Such a treatment may be useful in studies of the role of tannins in plant-herbivore interactions.     

Adaptation of the Spodoptera exigua Se301 insect cell line to grow in serum-free suspended culture. Comparison of SeMNPV productivity in serum-free and serum-containing media

Spodoptera exigua Se301 cells have been successfully adapted to two different commercial serum-free media (SFM; Ex-Cell 420 and Serum-Free Insect Medium-1) by gradually reducing the 10 %-added serum-containing medium content from 100 % to 0 % (v/v) in suspended cultures. Both direct adaptation to a serum-free medium and cell growth in the absence of protective additives against fluid dynamic stress [polyvinyl pyrrolidone and polyvinyl alcohol] and disaggregation [dextran sulfate] proved impossible. Cells grew reproducibly in both SFMs once the serum had been completely removed, although the use of Ex-Cell 420 resulted in higher growth rates and cell densities. Turbulence was sufficiently high to reduce growth rates and final cell densities at the highest Reynolds number investigated, although no clear influence of agitation was observed on virus productivity. Both attached and suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Cells adapted to different conditions (attached or suspended culture, serum-containing or serum-free medium) showed different occlusion bodies productivities at the two multiplicities of infection assayed (0.1 and 0.5).     

Studies of cotyledon protoplast cultures from B napus,B. campestris and B. oleracea. II: Callus formation and plant regeneration

Cotyledons from twelve cultivars of Brassica; B. napus (Westar, Eureka, Global, Pivot and Narc 82); B. campestris: (Arlo, Sonja, Bunyip and Wonk Bok) and B. oleracea (Phenomenal Early, Sugar Loaf and Earliball) were used for protoplast isolation and culture in a comparative study of cell colony and callus formation, and plant regeneration. The formation of cell colonies and callus from protoplast cultures were significantly influenced by the light conditions of seed germination. All twelve cultivars showed callus formation from protoplast cultures derived from cotyledons of seedlings grown in dark for 3 days followed by 1 day dim light (dark/dim light-grown). Callus was obtained in all five liquid media used: modified K8P(1), modified K8P(2), modified MS, modified B and modified NN. In contrast, only six cultivars exhibited callus formation from the protoplasts isolated from cotyledons of seedlings germinated under light conditions for 7 days (light-grown) and in only three media: modified K8P(1), modified MS, modified B.Callus, derived from protoplast cultures isolated from dark/dim light-grown cotyledons and grown on K3 or MS series solid media for about 1 month, could develop shoots when further transferred onto MS series regeneration media. All five cultivars of B. napus, three of the four cultivars of B. campestris (Arlo, Sonja and Bunyip) and one of the three cultivars of B. oleracea (Sugar Loaf) exhibited shoot regeneration from protoplast cultures within 2–3 months after protoplast isolation. The frequency of shoot regeneration ranged among 1–22.5%. A high degree of reproducibility was observed in cultivars Westar, Eureka, Global, Arlo, Bunyip and Sugar Loaf. In contrast, among the six cultivars that formed callus in protoplast culture derived from light-grown cotyledons, only three cultivars from B. napus (Westar, Eureka, Global) exhibited shoot regeneration 5.5 months after protoplast isolation. Regenerated shoots from cultivars Westar, Eureka and Bunyip and Sugar Loaf, which derived from protoplasts of dark/dim light germinated seedling and were induced to root on rooting media, survived in soil and grew to produce silique and set seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - BA benzylaminopurine - EDTA ethylenediaminetetraacetic acid - IAA indole-3-acetic acid - IBA indole-3-butyric acid - KT kinetin - GA₃ gibberellic acid - MS Murashige and Skoog medium - NAA -naphthaleneacetic acid - PAR photosynthetically active radiation     

The effect of polyvinyl alcohol and polyvinyl pyrrolidone on diffusion artifacts in lactate dehydrogenase histochemistry

Summary The effect of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), alone and in combination, on diffusion artifacts in histochemical incubations has been investigated using LDH as model enzyme. By measuring the amount of formazan in the medium at the end of the incubation it has been shown that both substances, but especially PVA, are effective in limiting diffusion. The significance of this is discussed in general as well as in relation to other procedures used to reduce diffusion artifacts.The Following Abbreviations are used in the Article NAD -Nicotinamide Adenine Dinucleotide - NADH -Nicotinamide Adenine Dinucleotide, Reduced form - PVA Polyvinyl alcohol - PVP Polyvinyl pyrrolidone - PMS Phenazine methosulfate - tris tris (hydroxymethyl)-aminomethan - Nitro-BT Nitro Blue Tetrazolium - LDH Lactic dehydrogenase     

A mitochondrial glycolate: Cytochrome C reductase in Chlamydomonas reinhardii

Summary A glycolate: cytochrome c reductase is reported in a thin-walled mutant of Chlamydomonas reinhardii. The reductase is of mitochondrial origin and is sensitive to certain mitochondrial electron transport system inhibitors.Abbreviations ETS electron transport system - DCIP 2.6 dichlorophenol indophenol - PVP polyvinyl pyrrolidone     

Efficient plant regeneration from rice protoplasts in general medium

Summary We established an efficient and reproducible procedure for protoplast propagation and fertile plant regeneration of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Selection of scutellum-derived secondary calli, the use of General medium and nurse culture were all found to be critical in the procedure. When 5 basal media (Murashige and Skoog, RY-2, modified R2, Amino Acid and General media) were compared, suspension callus growth rate, protoplast yield and plating efficiency were all about 30% higher in General medium than in the second-best R2 medium. Only one month was required to develop suspension cultures for protoplast isolation using General medium. A plating efficiency as high as 17% and a plant regeneration frequency of 67% were achieved by the improved procedure. Agronomic traits of protoplast- and seed-derived plants were found to be similar.Abbreviations AA Amino Acid medium (Muller and Grafe 1978) - MS Murashige and Skoog (1962) medium     

Reproductive capacity of bacteroids in nodules of Trifolium repens L. and Glycine max (L.) Merr.

Reproductive growth of intracellular bacteria from isolated protoplasts in nodules of clover and soybean was directly investigated using a microchamber with visual and video recording. Differentiated bacteriods from clover nodules uniformly failed to reproduce. Such growth as occurred came from undifferentiated rhizobia from within the protoplast or extracellularly in the nodule. Plating investigation gave results in agreement with this conclusion. Osmoprotective media failed to secure the reproduction of differentiated clover bacteroids. Reproductive growth of bacteroids from protoplasts and crushed nodules of soybean was regularly observed in the microchamber and determined as proportionate colony-forming ability (CFA) on laboratory media. The CFA markedly increased with age of nodule and with the addition of nodule or root extract. The promoting effect of such extracts was reduced after heating for 60 min at 100°C, and lost completely after 20 min at 121°C. High osmolarity in the suspending and culture media was detrimental to bacteroid recovery.Abbreviations BMM Bergersen's modified medium - B^+m BMM with additional mannitol - CDB Chlamydomonas dilution buffer - PDB protoplast dilution buffer - PDB PDB without mannitol or sorbitol - RMM Rhizobium minimal medium - R^+m RMM with mannitol instead of sucrose - YMA, YMB yeast mannitol agar and broth, respectively. For details, see Materials and methods     

Adaptation of the Se301 insect cell line to suspension culture. Effect of turbulence on growth and on production of nucleopolyhedrovius (SeMNPV)

As chemical pesticides are being banned as control agents for agricultural pests, the use of the highly specific, safe to non-target organisms baculoviruses has been proposed. These viruses can be produced either in vivo or in vitro. In vitro production requires appropriated host insect cell lines with the ability for growing as freely-suspended cells. In this work, the Spodoptera exigua Se301 cell line was used to produce the commercially available S. exigua nucleopolyhedrovirus (SeMNPV) in suspension. Se301 cells showed to be very sensitive to the hydrodynamic shear rates developed in bioreactors. A process of progressive adaptation to freely-suspended cultures using protective additives against shear stress and disaggregant was proposed. The best combinations were polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) with the disaggregant dextran sulfate (DS). Both static and freely-suspended Se301 cell cultures were successfully infected with the SeMNPV baculovirus. Production of occluded baculovirus (OB) increased with the multiplicity of infection (MOI > 0.1).     

The effect of oncotic pressure on heart muscle mitochondria

The role of oncotic pressure (i.e. pressure created by non-penetrants of high molecular weight) in structural responses of mitochondria has been studied.

It has been found that treatment of beef of rabbit heart mitochondria by a synthetic non-penetrant of high molecular weight, polyvinyl pyrrolidone, induces a decrease in the intermembrane (intracristal) space and an increase in the matrix space of mitochondria. As a result, the appearance of the in vitro mitochondria proves to be similar to that of the in situ ones. If a Waring blender is used to homogenize the tissue, only a portion of the mitochondria respond to polyvinyl pyrrolidone. If a glass-Teflon homogenizer is used instead all the mitochondria prove responsive. The addition of 0.5 mM polyvinyl pyrrolidone is found to be sufficient for the effect to be observable.

In the presence of polyvinyl pyrrolidone, energy-dependent changes in mitochondrial structure can be demonstrated. The increase in matrix space by polyvinyl pyrrolidone treatment enlarges even more when an energy source, a penetrating weak acid and a penetrating cation are added. The size of the matrix increases in the following order: (1) de-energized mitochondria without polyvinyl pyrrolidone, (2) de-energized + polyvinyl pyrrolidone, (3) energized + polyvinyl pyrrolidone, (4) as (3) + phosphate (“twisted” configuration of cristae), (5) as (3) + phosphate + Ca²⁺. Structural changes resembling those indicated in points (2)–(5) are shown for mitochondria in the tissue, when pieces of rat diaphragm muscle treated with an uncoupler, phosphate, and Ca²⁺ were studied in conditions excluding anaerobiosis.

The effect of polyvinyl pyrrolidone is suggested to be due to it balancing the oncotic pressure created by high molecular weight compounds dissolved in the intermembrane water, which are incapable of penetrating the outer mitochondrial membrane. A concept is discussed considering mitochondrial structure changes as a function of the osmotic gradient across the inner membrane and the oncotic gradient across the outer membrane of mitochondria.     

Effects of the high-molecular-weight cryoprotectant dextran on fluid secretion by Calliphora salivary glands

The rate of fluid secretion by isolated salivary glands of Calliphora was inhibited as a linear function of dextran and poly vinyl pyrrolidone concentrations in the range 15–35% w/w. This inhibition was not overcome by supramaximal concentrations of 5-hydroxytryptamine, nor was it caused by a decreased availability of K⁺ from the medium. Although the polymers caused large decreases of freezing point (and vapor pressure) of the incubation medium, the glands did not respond to this by secreting a more K ⁺-rich saliva. When dextran and polyvinyl pyrrolidone were added as powders to salt solutions, the total freezing-point depression of the mixture was equal to the sum of that exerted by the pure salt solution and that expected for the polymer concentration. The activities of K⁺ and Cl^?, as measured by ion-selective electrodes, were not increased in solutions by the addition of dextran. Dextran was demonstrated by electron microscopy to penetrate into the basal clefts and intercellular spaces of the isolated glands. These results demonstrate that addition of dextran (and probably of polyvinyl pyrrolidone) does not decrease the solvent activity of water in physiological salt solutions. The inhibition of fluid secretion by isolated salivary glands of Calliphora seems therefore due only to the altered physical characteristics of the medium.     

Effects of Thermal Curing Conditions on Drug Release from Polyvinyl Acetate–Polyvinyl Pyrrolidone Matrices

This study aimed to investigate the effects of dry and humid heat curing on the physical and drug release properties of polyvinyl acetate–polyvinyl pyrrolidone matrices. Both conditions resulted in increased tablet hardness; tablets stored under humid conditions showed high plasticity and deformed during hardness testing. Release from the matrices was dependent on the filler's type and level. Release profiles showed significant changes, as a result of exposure to thermal stress, none of the fillers used stabilized matrices against these changes. Density of neat polymeric compacts increased upon exposure to heat; the effect of humid heat was more evident than dry heat. Thermograms of samples cured under dry heat did not show changes, while those of samples stored under high humidity showed significant enlargement of the dehydration endotherm masking the glass transition of polyvinyl acetate. The change of the physical and release properties of matrices could be explained by the hygroscopic nature of polyvinyl pyrrolidone causing water uptake; absorbed water then acts as a plasticizer of polyvinyl acetate promoting plastic flow, deformation, and coalescence of particles, and altering the matrices internal structure. Results suggest that humid heat is more effective as a curing environment than dry heat for polyvinyl acetate–polyvinyl pyrrolidone matrices.     

Protoplast isolation and culture for somatic hybridization of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis> and <Emphasis Type="Italic">L</Emphasis>. <Emphasis Type="Italic">subcarnosus</Emphasis>

A method for isolation and shoot regeneration from electrofused protoplasts of L. angustifolius and L. subcarnosus was developed. Viable protoplasts were isolated from leaves of in-vitro grown seedlings at an average yield of 6 × 10⁵ protoplasts g⁻¹ fresh weight. Liquid and agarose solidified B5 media were used for protoplast culture. In the liquid-culture system, all tested media, VKM, P1 and KM8p, were applicable for inducing cell division (84% of all tested petri dishes at four weeks) and colony formation. Media containing additional carbohydrates were suitable to produce compact calli with green and brown pigmentations in different combinations. Analysis of callus with molecular markers allowed to identify six somatic hybrids. However, none of the parental-protoplast derived cell colonies could develop shoots. This is the first report on protoplast fusion of L. angustifolius and L. subcarnosus with subsequent shoot regeneration.     

Plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides

A protocol was developed for the isolation, culture and plant regeneration of protoplasts isolated from suspension cultures of Solanum lycopersicoides Dun. (LA 1990). Protoplasts were isolated by an overnight enzyme digestion, further purified by washing in W5 salts solution, and plated in two modified MS protoplast culture media with and without type VII agarose. The addition of agarose to the two culture media did not enhance plating efficiencies and shoot regeneration percentages and in some cases was even inhibitory. Unlike the experience with some other solanaceous species, the deletion of ammonium from the protoplast culture medium was not found to be beneficial. Protoplasts sustained continuous division in the modified MS media and up to 70% of the protoplast-derived calli readily regenerated shoots on MS salts and vitamins medium containing zeatin and GA.     

Applying Proteolytic Enzymes on Soybean

The soybean cold-insoluble protein was hydrolyzed with pepsin and the hydrolyzate was dialyzed. The diffusate was submitted to gel permeation chromatography of Sephadex G-10 and thin layer chromatography of silica gel G. A ninhydrin-negative bitter peptide was detected by chlorine-starch-iodine test and was purified by paper electrophoresis and by rechromatography on the silica gel. Azeotropic HCl hydrolysis of the peptide gave equal molar ratio of Ala, Glu, Gly, Ile, Leu, Phe, Ser and Val. N-Terminal residue was composed of pyrrolidone carboxylic acid, which was tentatively identified in comparison with the authentic sample by paper electrophoresis and thin layer chromatography. Hydrazinolysis of the peptide, followed by the 2,4-dinitrophenylation, produced α,α,γ-tri-DNP-glutamic acid-α,γ-dihydrazide and DNP-pyrrolidone carboxylic acid hydrazide, also supporting the N-terminal structure. Hydrolysis of the peptide by carboxypeptidase A gave Leu, Val, Phe, Ile, Ala, etc. in the order of the liberation rate. As the peptide fragments remaining in this hydrolyzate were detected pyrrolidone carboxyl-Gly·OH, pyrrolidone carboxyl-Gly-Ser·OH, pyrrolidone carboxyl-Gly-Ser-Ala·OH, etc. In conclusion, the structure of this peptide was proposed as: pyrrolidone carboxyl-Gly-Ser-Ala-Ile-Phe-Val-Leu·OH. Quantitative information about the total amount of pyrrolidone carboxyl residue contained in the peptic hydrolyzate of the soybean protein and sensory study on the bitterness of the peptide as referred by standard solution of phenylthiourea, were demonstrated.     

Cytoplasmic-nuclear interaction and medium effect for protoplast culture in sunflower

Protoplasts of 6 alloplasmic and 2 euplasmic sunflower inbred lines were isolated from dark grown seedling hypocotyls with a density of 2×10⁴ protoplasts/ml. The protoplast suspension was mixed with a solution of 0.5% agarose (sigma – type 1), then pipetted in droplets of about 1000 protoplasts. Droplets were surrounded by two different liquid media. After 30 days droplets from both media were transferred to solid differentiation medium. Protoplast division, microcolony frequency and the number of calluses produced were strongly dependent on medium composition and genotype. The number of calluses per 1000 protoplasts plated range from 0.3 to 5.0 according to the genotype and the method used. The alloplasmic line RHA274-PEF1, was the best responding genotype for calluses produced in both media used. In all cases, the percentage of calluses for alloplasmic lines were significantly higher when compared with the nucleus donor genotype. H. petiolaris fallax cytoplasm increased both the number of calluses produced and the percentage of microcolonies. The complex interaction among genotypes tested indicates that protoplast culture responses are affected independently by nuclear-cytoplasm interactions. Some nucleus-cytoplasm combinations can improve the protoplast culture responses in sunflower. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of entrapment on nucleic acid content,cell morphology,cell surface property,and stress of pure cultures commonly found in biological wastewater treatment

The effects of cell entrapment on nucleic acid content, cell morphology, cell surface property, and stress of major groups of bacteria (betaproteobacteria and gammaproteobacteria) in biological municipal wastewater treatment were investigated. Three different entrapment media (alginate, carrageenan, and polyvinyl alcohol) were examined. Results indicated that the entrapment and type of entrapment media affected nucleic acid content, cell morphology, cell surface property, and stress of the three representative species (Alcaligenes faecalis, Comamonas testosteroni, and Pseudomonas putida) studied. The highest deoxyribonucleic acid and ribonucleic acid increases were observed with the alginate and polyvinyl alcohol (PVA) entrapment, respectively. A cell morphological change from bacilli to coccoidal was observed in the case of alginate entrapment while the PVA-entrapped cells had a slim morphology when compared to non-entrapped cells and formed putative nanowires. The entrapment increased or decreased the surface roughness of cells depending on the type of entrapment media. Expression of a nitrosative stress gene, which is linked to oxygen deprivation, was observed more in the alginate-entrapped cells. These research findings advance the fundamental understanding of the entrapped cell physiology which can lead to more efficient entrapped cell-based wastewater treatment.     

Enhanced protoplast division by encapsulation in droplets: An advance towards somatic hybridisation in recalcitrant white lupin

Lupins are highly nutritious fodder and pulse crops but the greatest challenge in their genetic enhancement is the difficulty in obtaining hybrids through conventional sexual approaches. To bypass this, a procedure for the culture of hitherto recalcitrant lupin protoplasts is now being developed so that the somatic hybrids can be regenerated. This study provides a basis for a regime to culture lupin protoplasts. Cotyledonary protoplasts of white lupin (Lupinus albus) were plated in two diverse media for the evaluation of various plating regimes. The protoplasts divided in agarose as well as in Gelrite? but embedding in agarose at 6 g L^?1 concentration resulted in a higher rate of mitosis. Sodium alginate embedding inhibited protoplast division. Protoplast plating in the form of liquid suspension was significantly inferior to embedding. A filter paper substratum was clearly noxious to protoplast division. Vis‐à‐vis other designs of plating, a 400% improvement in protoplast elongation and division was achieved by plating in the form of 25 μL droplets at the base of 60 mm × 15 mm Nunclon? dish and overlaying with liquid medium. Better results in terms of protoplast elongation and division were obtained with K8p medium as compared to the AS medium. This report on lupin protoplast culture represents a significant breakthrough in the genus in which morphogenesis has not been described to date.     

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 10³ per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H² 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 10⁴ cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.