Production of aposporous gametophytes and calli from Pteris vittata L. pinnae strips cultured in vitro

Murashige and Skoog's modified medium in 1% Difco Bacto-agar supplemented with sugar alcohols (sorbsitol, mannitol), growth regulators (1-naphthalenacetic acid, 2,4-dichlorophenoxyacetic acid, benzyladenine, kinetin) and sugars (fructose, glucose, sucrose) induced aposporous gametophytes from pinnae of Pteris vittata cultured in vitro at lower concentrations of all the mentioned components. Aposporous gametophytes and vegetative calli were produced at higher concentrations. The calli regenerated sporophytes when cultured on MS medium without growth regulators. The gametophytes grew vegetatively on MS medium but produced sporophytes when transferred into 0.1 strength MS medium. This is the first report of simultaneous production of calli and gametophytes from fern explants.     

Crown gall transformation of lentil (Lens culinaris Medik.) with virulent strains of Agrobacterium tumefaciens

Summary Four diverse strains of Agrobacterium tumefaciens (C58, Ach5, GV3111, and A281) were capable of inducing tumors at a high frequency on inoculated stems of lentil (Lens culinaris Medik. cultivar Laird) in vivo, and on excised shoot apices in vitro. GV3111 and Ach5 produced the largest and heaviest tumors in vivo, while A281 produced the heaviest tumors in vitro. Tumor formation and opine production are indicative of plant cell transformation and tumors produced appropriate opines: nopaline (C58), octopine (Ach5 and GV3111), and agropine and mannopine (A281). Southern analysis of DNA from a tumor line produced by strain C58 showed that a T-DNA fragment had been transferred into the lentil genome.     

Gene transfer in plants of Brassica juncea using Agrobacterium tumefaciens-mediated transformation

An efficient system for gene transfer into plants of Brassica juncea var. India Mustard, mediated by Agrobacterium tumefaciens. was developed through the manipulation of the culture medium and the use of the appropriate Agrobacterium strain. High frequency shoot regeneration (90–100%) was obtained from hypocotyl explants grown on medium containing 0.9% agarose, 3.3 mg/L AgNO₃ and 0.5–2 mg/L BA in combination with 0.01–0.05 mg/L 2,4-D or 0.1–1 mg/L NAA. Of all the Agrobacterium strains tested, A. tumefaciens A208-SE, carrying the disarmed Ti plasmid and a binary vector pROA93, was the most effective for B. juncea transformation. pROA93 carries the coding sequences of the NPTII and the GUS genes, both driven by a common CaMV 35S promoter in two divergent directions. Inoculated explants grown on the selection medium in the presence of 0.5 mg/L BA and 0.1 mg/L NAA gave rise to transgenic shoots at the highest frequency (9%). All R_o transgenic plants were phenotypically normal, but variation in expression patterns of the GUS gene occurred among the transgenic plants in an organ- and tissue-specific manner. Both the NPTII and the GUS genes were transmitted to the R₁ seed progeny and showed co-segregation.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid - NPTII neomycin phosphotransferase type II - GUS -glucuronidase - CaMV cauliflower mosaic virus - MS Murashige and Skoog - X-Gluc 5-bromo-4-chloro-3-indolyl-D--glucuronic acid - IBA indolebutyric acid - SDS sodium dodecyl sulfate     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Energy distribution in the photochemical apparatus of Porphyridium cruentum: Picosecond fluorescence spectroscopy of cells in state 1 and state 2 at 77 K

Excitation energy distribution in Porphyridium cruentum in state 1 and state 2 was investigated by time resolved 77 K fluorescence emission spectroscopy. The fluorescence rise times of phycoerythrin, phycocyanin and allophycocyanin (in cells in state 1 and state 2) were very similar in contrast to the emission from chlorophyll a (Chl a) associated with the two photosystems. In state 2 photosystem II (PSII) Chl a fluorescence emission rose faster than the PSI Chl a emission and decayed more rapidly, and the converse was observed in state 1. These kinetic data support the concept of increased energy transfer from PSII Chl a to PSI Chl a in state 2 in P. cruentum.Abbreviations APC allophycocyanin - Chl a chlorophyll a - PSII photosystem II - PC phycocyanin - PE phycoerythrin     

Agrobacterium tumefaciens-mediated beta-glucuronidase (GUS) gene expression in lentil (Lens culinaris Medik.) tissues

Summary Lentil (Lens culinaris Medik.) shoot apex, epicotyl, and root expiants were capable of expressing an intron-containing beta-glucuronidase (GUS) gene after inoculation with the disarmed Agrobacterium strain GV2260:p35SGUSINT. Expression occurred at all wound sites on these expiants except at the end of the root expiants proximal to the cotyledonary node. GUS expression was detected using both histochemical and fluorescence assays and was stable for at least nine days after inoculation for epicotyl and root expiants, and for at least seventeen days for shoot apices. Non-inoculated controls, or controls inoculated with an Agrobacterium strain lacking the GUS gene, did not produce any background blue staining in the histochemical assay. Expression levels for all lentil expiants were substantially lower than for comparable flax (Linum usitatissimum L.) expiants which served as a positive control.     

Benomyl: A broad spectrum fungicide for use in plant cell and protoplast culture

The systemic fungicide methyl-1-(butylcarbamoyl)-2-benzimidazole carbamate (benomyl), is a broad spectrum fungicide. Benomyl at concentrations up to 50 mg/l does not inhibit the growth of suspension cultures ofNicotiana tabacum, Datura innoxia, Daucus carota, Glycine canescens, andSolanum tuberosum nor growth ofN. tabacum orN. plumbaginifolia protoplasts if benomyl is dissolved by autoclaving or boiling. Addition of benomyl dissolved in dimethyl sulfoxide results in a visible toxicity. Benomyl, at 6.25–50 mg/l preventsPenicillium spp. growth in both protoplast and cell cultures and can be used to remove fungal contaminates after one to three transfers without visibly retarding plant cell growth. Due to the broad spectrum of fungicidal activity, and nontoxicity at high concentrations when dissolved by boiling or autoclaving, benomyl can be used effectively to control or prevent fungal contamination in plant cell and protoplast cultures.     

Somatic cell genetic studies inBrassica species

In vitro culture ofBrassica alba anthers on a growth medium containing inorganics of KB5 and organics, iron, sucrose and hormones of B5 resulted in a very high response of anthers (93.75%) towards callus induction. All the calli transferred to regeneration media responded favourably even after six months of callus induction. Numerous torpedo-shaped embryoids developed in clusters at many sites from each callus mass. Secondary embryogenesis and multiple shoot formation was also observed in many cases. The number of embryoids and plantlets produced by one embryogenic anther were as high as 169.8 and 17 respectively. 87% of the regenerated plants were haploids.     

Transformation of Solanum integrifolium poir via Agrobacterium tumefaciens: Plant regeneration and progeny analysis

Summary The wild species Solanum integrifolium represents a source of pest and disease resistance genes for breeding strategies of the cultivated species Solanum melongena. Somatic hybridization via protoplast fusion between the two species may provide a valuable tool for transferring polygenic traits into the cultivated species. The availability of S.integrifolium cells carrying dominant selectable markers would facilitate the heterokaryon rescue. An appropriate methodology for in vitro culture and plant regeneration from leaf explants of S.integrifolium is reported. Efficient leaf-disk transformation via co-cultivation with Agrobacterium tumefaciens led to the regeneration of transformed plants carrying the reporter genes GUS and NPT-II. Transformed individuals were obtained through selection on kanamycin-containing medium. Stable genetic transformation was assessed by histochemical and enzymatic assays for GUS and NPT-II activity, by the ability of leaf disks to initiate callus on Km-containing medium, Southern blot analyses of the regenerated plants, and genetic analysis of their progenies. Selfed-seed progeny of individual transformed plants segregated seedlings capable to root and grow in selective condition, while untransformed progeny did not. Genetic analyses of progeny behaviour showed that the reporter gene NPT-II segregated as single as well as two independent Mendelian factors. In two cases an excess of kanamycin-sensitive seedlings was obtained, not fitting into any genetic hypothesis.Abbreviations MS Murashige and Skoog (1962) medium - NOS nopaline synthase - NPT-II neomycin phosphotransferase - GUS beta-glucuronidase - LB Luria and Bertani medium - KIN 6-furfurylaminopurine - BAP 6-benzylaminopurine - 2iP N⁶-(2-isopentyl)adenine - ZEA zeatin - TDZ Thidiazuron     

Somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.)

Tissue culture methods for improvement of cotton has lagged seriously compared to other major crops. A method for regeneration of cotton which includes a morphogenetically competent cell suspension was needed to facilitate selection of stress-resistant variants and gene manipulation. Preliminary screening of eight strains of Gossypium hirsutum L. for embryogenic potential resulted in the production of somatic embryos in all strains. Coker 312 was selected for use in the development of a model regeneration system for G. hirsutum. Calli were initiated from hypocotyl tissues of 3-day-old-seedlings. Globular embryos were present after six weeks in culture. Calli were subcultured to liquid suspension in growth regulator-free medium. After three to four weeks, suspensions were sieved to collect globular and heart stage embryos. Collected embryos developed further when plated onto semi-solid medium. To induce germination and plantlet growth, mature embryos were placed on sterile vermiculite saturated with medium. Upon development of roots and two true leaves, plantlets were potted in peat and sand, and hardened. Mature plants and progeny have been obtained with this procedure. A high percentage of infertile plants was observed among the regenerants.Abbreviations NAA 1 naphthaleneacetic acid - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid - MS Murashige and Skoog - BA 6 benzylamino purine - 2i P N⁶-(²-isopentenyladenine     

Quinoprotein D-glucose dehydrogenases inAcinetobacter calcoaceticus LMD 79. 41: Purification and characterization of the membrane-bound enzyme distinct from the soluble enzyme

Acinetobacter calcoaceticus is known to contain soluble and membrane-bound quinoprotein D-glucose dehydrogenases while other oxidative bacteria such asPseudomonas orGluconobacter contain only membrane-bound enzyme. The two different forms were believed to be the same enzyme or interconvertible. Present results show that the two different forms of glucose dehydrogenase are distinct from each other in their enzymatic and immunological properties as well as in their molecular size.The soluble and membrane-bound glucose dehydrogenases were separated after French press-disruption by repeated ultracentrifugation, and then purified to nearly homogeneous state. The soluble enzyme was a polypeptide of 55 Kdaltons, while the membrane-bound enzyme was a polypeptide of 83 Kdaltons which is mainly monomeric in detergent solution. Both enzymes showed different enzymatic properties including substrate specificity, optimum pH, kinetics for glucose, and reactivity for ubiquinone-homologues. Furthermore, the two enzymes could be distinguished immunochemically: the membrane-bound enzyme is cross-reactive with an antibody raised against membrane-bound enzyme purified fromPseudomonas but not with antibody elicited against the soluble enzyme, while the soluble enzyme is not cross-reactive with the antibody of membrane-bound enzyme.Data also suggest that the membrane-bound enzyme functions by linking to the respiratory chain via ubiquinone though the function of the soluble enzyme remains unclear.     

Cryopreservation of isolated mint shoot tips by vitrification

Shoot tips isolated from a mint clone, Mentha aquatica x M. spicata, were gradually exposed to a mixture containing 35% ethylene glycol, 1 M dimethylsulfoxide and 10% polyethylene glycol-8000 and then immersed into liquid nitrogen. Cooling and warming rates were approximately 4800°C/min and 9000°C/min respectively. Survival after liquid nitrogen treatment ranged from 31% to 75% among experiments. There was no obvious reason for this variation. In many cases the treated shoot tip directly developed into a shoot without any or with only slight callus formation.Abbreviations DSC differential scanning calorimetry - DMSO dimethylsulfoxide - EG ethylene glycol - PEG-8000 polyethylene glycol - MW avg. 8000 - LN liquid nitrogen - IBA indolebutyric acid - BA benzyladenine     

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus

Summary Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82–120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33–78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.     

Differential response of the two cotyledons of Vigna radiata in vitro

Summary Cotyledons of mature seeds of Vigna radiata were found to be variable in their response to N 6-benzytadenine, kinet in and zeatin. The two cotyledons of a seed were designated as CE and C; where CE referred to the cotyledon that remained closely attached to the embryonal axis, and the other more loosely attached cotyledon was referred to as C. Shoots formed from the proximal end of both explants in all nine cultivars studied. Shoot regeneration was faster and regeneration efficiency was higher in CE explants than in C explants in these cultivars. BA was found to be the most suitable cytokinin for both multiple shoot induction and regeneration.Abbreviations BA N6- benzyladenine - Cv cultivar - KIN kinetin - NAA naphthalene acetic acid - RE regeneration efficiency - ZEA zeatin     

Identification of a penicillin V acylase processing fungus

In our studies with the penicillin V acylase of Bovista plumbea strains NRRL 3501 and NRRL 3824, we wanted to receive spores of these fungi. Surprisingly the fruiting bodies obtained in our work were not identical with those characteristic for Bovista plumbea. We identified them as Pleurotus ostreatus. For this reason we have to correct the name of the fungi known as Bovista plumbea NRRL 3501 and NRRL 3824.     

Increasing the variability of Lycopersicon peruvianum Mill. by protoplast fusion with Petunia hybrida L.

Somatic hybridization of Lycopersicon peruvianum and Petunia hybrida was carried out to transfer cytoplasmic male sterility from Petunia to Lycopersicon. Cytological, morphological and biochemical analyses were performed to characterize the regenerated plants. Two regenerated plants, R₃ and R₆, were male sterile. R₃ possessed chromosomes morphologically similar to those of both parental types. Leaf morphologies of these two plants and a third plant, R₇, were intermediate between the two parents. The stability of RUBPCase was verified during parental plant development and after in vitro culture. Plant R₇ presented a new form of the large subunit of RUBPCase.     

Biochemical differences between carrot inbreds differing in plant regeneration potential

Cultures derived from domestic carrot (Daucus carota L.) inbreds were found to vary with respect to regeneration potential as measured by the production of somatic embryos in suspension cultures. A number of biochemical parameters previously reported to distinguish embryogenic from non-embryogenic cultures of other species were measured in these carrot cell lines. Ethylene production was found to be inversely related to regeneration potential. The cell line producing the greatest number of somatic embryos exhibited the lowest rate of ethylene biosynthesis, even when grown on 2, 4-D-containing maintenance medium. A specific isozyme of acid phosphatase was associated with embryogenic calli. Proteins visualized by SDS-PAGE did not discriminate between embryo-forming and proliferating calli in all inbreds.     

The Highly Charged Region of Plant β-type Phosphatidylinositol 4-kinase is Involved in Membrane Targeting and Phospholipid Binding

In Arabidopsis thaliana and Oryza sativa, two types of PI 4-kinase (PI4Ks) have been isolated and functionally characterized. The α-type PI4Ks (~220 kDa) contain a PH domain, which is lacking in β-type PI4Ks (~120 kDa). β-Type PI4Ks, exemplified by Arabidopsis AtPI4Kβ and rice OsPI4K2, contain a highly charged repetitive segment designated PPC (Plant PI4K Charged) region, which is an unique domain only found in plant β-type PI4Ks at present. The PPC region has a length of ~300 amino acids and harboring 11 (AtPI4Kβ) and 14 (OsPI4K2) repeats, respectively, of a 20-aa motif. Studies employing a modified yeast-based “Sequence of Membrane-Targeting Detection” system demonstrate that the PPC(OsPI4K2) region, as well as the former 8 and latter 6 repetitive motifs within the PPC region, are able to target fusion proteins to the plasma membrane. Further detection on the transiently expressed GFP fusion proteins in onion epidermal cells showed that the PPC(OsPI4K2) region alone, as well as the region containing repetitive motifs 1–8, was able to direct GFP to the plasma membrane, while the regions containing less repetitive motifs, i.e. 6, 4, 2 or single motif(s) led to predominantly intracellular localization. Agrobacterium-mediated transient expression of PPC-GFP fusion protein further confirms the membrane-targeting capacities of PPC region. In addition, the predominant plasma membrane localization of AtPI4Kβ was mediated by the PPC region. Recombinant PPC peptide, expressed in E. coli, strongly binds phosphatidic acid, PI and PI4P, but not phosphatidylcholine, PI5P, or PI(4,5)P₂ in vitro, providing insights into potential mechanisms for regulating sub-cellular localization and lipid binding for the plant β-type PI4Ks. The nucleotide sequences reported in this paper have been submitted to the GenBank^TM/EMBL Data Bank under accession number AY536061 (highly charged region of OsPI4K2) and AJ277791 (partial cDNA of OsPI4K2). Ying Lou and Hui Ma: These authors contributed equally     

The influence of auxins,light and cell differentiation on solasodine production bySolanum eleagnifolium Cav. calli

The effect of auxins, light and cellular production ofSolanum eleagnifolium Cav. calli were studied. 2,4-dichlorophenoxyacetic acid (4.5 M) was the plant growth regulator used for calli initiation and this produced the highest solasodine concentration. The solasodine concentration in darkness was significantly lower than that achieved under a photoperiod of 16 h. Differentiated tissue obtained by adequate hormonal balance (several ratios of 3-indolebutyric acid to 6-benzylaminopurine) produced higher yields of solasodine than non-differentiated tissue. 3-indolebutyric acid (2.5 M) and 6-benzylaminopurine (8.8 M) increased the productivity of solasodine by 100%.Abbreviations BAP 6-benzylaminopurine - KIN Kinetin - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA 3-indoleacetic acid - NAA 1-naphtaleneacetic acid - IBA 3-indolebutyric acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - DW dry weight - GI      

In vitro culture of isolated microspores and regeneration of plants in Brassica campestris

A protocol previously developed for B. napus microspore culture was modified to produce embryos from several lines of Brassica campestris. Bud size, genotype, media constituents, and incubation time and temperature were examined. Donor plants were grown in a growth cabinet at a day/night temperature of 10/5°C. Microspores were isolated from buds 2.0 – 2.9 mm in length and cultured in modified Lichter (1982) medium containing 17% sucrose, pH 6.2. After 48 h at 32°C, the incubation medium was replaced with NLN (Lichter 1982) medium containing 10% sucrose. Microspores were cultured at 24°C in darkness and embryos developed after three weeks. More than 1000 plants have thus far been regenerated. Genotypic differences were observed for microspore embryogenesis. The majority of the regenerants were haploid, however colchicine could be effectively used to achieve chromosome doubling.