Differential callus type formation by auxins and cytokinin in in vitro cultures of pepper (Capsicum annuum L.)

ABSTRACT

Two types of callus were produced by pepper explants cultured in various media containing auxins, the cytokinin 6-benzylaminopurine (BAP) and the auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA). Callus produced on media containing auxins alone was friable, grey-green or green-orange in colour and more compact, whereas when BAP was added to culture media with a low concentration of auxin or when the medium contained TIBA alone, the callus produced was white and very hard. This type of callus was also produced in cultures of older tissues and of young tissues cultured on hormonefree medium. Results are discussed in relation to the role of cytokinins in wounding, phenylpropanoid metabolism and lignin biosynthesis.     

Brain-derived neurotrophic factor increases survival and differentiated functions of rat septal cholinergic neurons in culture

Brain-derived neurotrophic factor (BDNF) was found to promote the survival of E17 rat embryo septal cholinergic neurons in culture, as assessed by a histochemical stain for acetylcholinesterase (AChE). A 2.4-fold increase in neuronal survival was achieved with 10 ng/ml BDNF. After initial deprivation of growth factor for 7 days, BDNF failed to bring about this increase, strongly suggesting that BDNF promotes cell survival and not just induction of AChE. BDNF was also found to increase the levels of cholinergic enzymes; choline acetyltransferase (ChAT) and AChE activities were increased by approximately 2-fold in the presence of 50 ng/ml BDNF. BDNF produced a 3-fold increase in the number of cells bearing the NGF receptor, as detected by the monoclonal antibody IgG-192. Although NGF had no additive effect with BDNF in terms of neuronal survival, suggesting that both act on a similar neuronal population, the combination of both produced an additive response, approximately a 6-fold increase, in ChAT activity.     

The control of shoot tip necrosis in Pistacia vera L. in vitro

The effect of increased concentrations of calcium (Ca) (3–24 mM) and boron (B) (100–800 M) in the medium was studied on the occurrence of shoot tip necrosis (STN) in cultures of Pistacia vera L. STN was significantly reduced by application of Ca or B, however media with more than 200 M boron had reduced shoot multiplication. Ca (12–24 mM) supplied as calcium chloride reduced STN without any adverse effect on shoot multiplication or elongation, whereas calcium acetate reduced elongation. It is concluded that STN is a physiological mineral disorder associated with Ca and/or B deficiency in the meristematic regions of actively growing shoots. Application of Ca (up to 24 mM) as calcium chloride to the medium was the best treatment for the control of STN. Reduction of humidity or increased aeration in the culture jars did not have any significant effect on the occurrence of STN.Abbreviations MS Murashige and Skoog medium - STN Shoot tip necrosis     

Promotion of root emergence in vitro from rhizome buds of Lapageria rosea cv. Nashcourt after proliferation in the presence of paclobutrazol

Medium components that may influence the emergence of adventitious root primordia in rhizome buds of Lapageria rosea (Ruiz et Pav.) cv. Nashcourt in vitro were examined. Sucrose was the only medium component tested that affected root emergence. Increasing the sucrose concentration in the medium from 10 to 30 g 1^-1 stimulated adventitious root emergence from rhizome buds that had been proliferated on a medium containing the gibberellin biosynthesis inhibitor paclobutrazol (5 M). Successful clonal propagation of L. rosea cv. Nashcourt can now be achieved by a rhizome bud proliferation stage(s) in the presence of paclobutrazol followed by an adventitious root emergence stage in the absence of paclobutrazol.     

Influence of explant source, plant growth regulators and culture environment on culture initiation and establishment of Quercus robur L. in vitro

Suitable cytokinin supplements and culture environments havebeen determined for the initiation and establishment of shootcultures of Quercus robur seedling tissue. Initiation of axillaryshoot development from nodal explants required culture mediumsupplemented with BA (6-benzylamminopurine). The greatest numbersof stem segments for culture proliferation were obtained using1.0 mg I^-1 BA after 56 d culture. The frequency of shoot developmentand subsequent formation of multiple shoots at initiation wasinfluenced by the position of the nodal explant in the seedlingshoot, incubation temperature and daylength. Explants from basaland apical regions, which contained multiple axillary buds,produced the lowest frequencies of axillary shoot developmentand multiple shoot formation, many remained quiescent. Axillaryshoot development was greatest in single nodal explants excisedfrom the midstem positions, elongated regions of the shoot wherenodes were formerly associated with a leaf. Higher temperaturesstimulated shoot formation with greater numbers of stem segmentsfor culture multiplication being obtained from nodal explantsincubated at 25C. Axillary shoot development was promoted innodal explants maintained under daylengths of 16 h or more.Stem segments cut from axillary shoots which developed fromnodal explants were used to establish shoot multiplication cultureson medium supplemented with 0.4 mg I^-1 BA. Shoot formation fromstem segments was greater at higher incubation temperaturesof 25C and 30C. Multiplication coefficients for stem segmentsincreased after one subculture. Key words: Quercus robur, oak, micropropagation, cytokinin, temperature, daylength, rest, quiescence     

Lectin-induced cell agglutination and membrane cholesterol levels

The membranes of human and guinea pig erythrocytes were enriched with, or depleted of cholesterol. Ehrlich ascites carcinoma cells were also enriched with cholesterol and the extra slerol shown to be present in the plasma membrane. Enrichment of the cells with sterol made them less susceptible to agglutination by concanavalin A (ConA) or phytohemagglutinin (PHA), while removal of sterol from the erythrocytes increased their susceptibilily to agglutination. It is suggested that following changes in surface membrane sterol levels there are changes both in short-range movement of individual receptor molecules and in cell shape and deformability which control the agglutinability of the cells.     

Reporter system for the detection of in vivo gene conversion

We have devised a system for the study of in vivo gene correction based on the detection of color variants of the green fluorescent protein (GFP) from the jellyfish Aequorea victoria. The intensity and spectra of the fluorescence emitted by the blue (BFP) and red-shifted (EGFP) variants of GFP differ from each other. We modified one nucleotide from an EGFP expression vector that we predicted would yield a blue variant (TAC-CAC, Tyr⁶⁶-His⁶⁶). Cells that were either transiently or stably transfected with the reporter system were used to test the functionality and feasibility of the detection of in vivo gene correction. A thio-protected single-stranded oligonucleotide designed to convert the genotype of the blue variant to that of the EGFP variant by the correction of a single base pair was delivered to the reported cells using a variety of methodologies and strategies. Conversion events were easily observed using fluorescent microscopy because of the enhanced emission intensity and different spectra of the EGFP variant.     

Rapid determination of vapA/vapB genotype in Rhodococcus equi using a differential polymerase chain reaction method

Rhodococcus equi is a facultative pathogen of foals. Infection causes an often fatal pulmonary pneumonia. The organism has also been isolated from pigs, cattle, humans and the environment. Equine virulence has a high positive correlation with the expression of a 17.4 kD polypeptide of unknown function, VapA, the product of the plasmid-encoded vapA gene. More recently an isogene of vapA, referred to as vapB and encoding an 18.2 kDa polypeptide, has been identified among pig and human isolates. The two genes share > 80% sequence identity, yet their host strains apparently exhibit different pathogenicity profiles (for example by reference to virulence in mouse model system and host specificity). In this study, a polymerase chain reaction (PCR) technique was developed that permits the selective amplification of vapA and vapB. Using this technique the distribution of the two genes among 35 randomly selected isolates of Rhodococcus equi from various animal and environmental sources was determined. Using this technique the genotype of each isolate could be unambiguously assigned as vapA+, vapB+ or vap- (i.e., scoring negative for both vapA and vapB). No isolate scored positive for both vapA and vapB. 100% of equine isolates scored vapA+, confirming the status of vapA as a reliable marker of equine virulence. All three genotypes were found among human isolates; porcine isolates scored either vapB+ or vap- and no vapA+ isolates were present in this sample. Rigorous statistical analysis using the Fisher Exact test confirmed that the high frequency of vapA+ among equine isolates is significant; however the sample size was too small to draw statistically significant conclusions regarding the distribution of genotypes among within other animal groups.