A simple, accurate method for determining wet and dry weight concentrations of plant cell suspension cultures using microcentrifuge tubes

Summary A simple method using microcentrifuge tubes for determining fresh and dry weights, and collecting cell-free supernatant from plant suspension cultures is described. This method offers improvements in accuracy, precision, and time efficiency over traditional filtration methods. Using 4-day-old Nicotinia tabacum cultures, the centrifuge method was shown to remove 25% more of the interstitial water from cell aggregates compared to a suction filtration method, with significantly less variation in fresh weight data.     

Influence of BA and sucrose on the competence and determination of pepper (Capsicum annuum L. var. Sweet Banana) hypocotyl cultures during shoot formation

Summary The simultaneous presence of 6-benzyladenine (BA) and sucrose in a Murashige and Skoog medium (SIM) during the initial stages of shoot initiation have been found to be obligatory for high-frequency shoot formation in the Capsicum annuum L. var. Sweet Banana upper hypocotyl explants. The explants are determined for shoot formation following a minimum of 8 days of culture on SIM. Deprivation of exogenous sucrose from day 6 to day 20 of culture had no effect on the shoot forming response of the explants. BA and sucrose appear to act independently on different aspects of the competence of explants to respond to SIM during shoot initiation.Abbreviations BA N⁶-benzyladenine - MS Murashige and Skoog medium - SIM shoot induction medium - HFM hormone free medium - SUC sucrose minus medium     

Plant regeneration from somatic embryos ofTaxus brevifolia

Summary Taxusbrevifolia is the source of paclitaxel (Taxol®), an anticancer drug. A method for regeneration ofTaxus brevifolia from immature zygotic embryos via somatic embryogenesis is described. Embryogenic callus tissues were obtained by culturing immature zygotic embryos on Lloyd and McCown medium (MCM) supplemented with 160 M 2,4-dichlorophenoxyacetic acid (2,4-D) + 5 M benzylaminopurine (BA) + 5 M naphthaleneacetic acid (NAA) for 4 weeks. Putative embryoids were obtained following transfer of cultures to MCM medium supplemented with 4 M BA + 5 M kinetin + 1 M NAA for 6 to 8 weeks. Conversion of embryos was obtained on MCM medium supplemented with 40 M abscisic acid (ABA) + 1% activated charcoal. Development of bipolar structures with recognizable shoot and root apices was observed in somatic embryos. Five percent of somatic embryos were regenerated into plantlets on half-strength growth regulator-free MCM medium.     

48例原发性闭经患者的细胞遗传学分析

本文报告对48例原发闭经患者的临床和细胞遣传学分析,共发现染色体异常17例,占35.4%,其中包括45,X,7例;45,X/46,XX,2例;X染色体结构异常5例;核型中有Y染色体3例。讨论了原发闭经的细胞遗传学病因及异常核型与表型的关系。     

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a -1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.Abbreviations AGL Aplysia gonad lectin - PGPR plant growth-promoting rhizobacteria The authors wish to thank Sylvain Noël for excellent technical assistance. This study was supported by grants from the Fonds Québécois pour la formation de chercheurs et l'Aide à la Recherche (FCAR), the Natural Sciences and Engineering Council of Canada (NSERC) and the Ministère de l'Industrie, du Commerce, de la Science et de la Technologie (SYNERGIE).     

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.     

The effects of mating design on introgression between chromosomally divergent sunflower species

Population genetic theory suggests that mating designs employing one or more generations of sib-crossing or selfing prior to backcrossing are more effective than backcrossing alone for moving alleles across linkage groups where effective recombination rates are low (e.g., chromosomally divergent linkages). To test this hypothesis, we analyzed the effects of chromosomal structural differences and mating designs on the frequency and genomic distribution of introgressed markers using the domesticated sunflower, Helianthus annuus, and one of its wild relatives, H. petiolaris, as the experimental system. We surveyed 170 progeny, representing the end products of three different mating designs (design I, P-F₁-BC₁-BC₂-F₂-F₃; design II, P-F₁-F₂-BC₁-BC₂-F₃; and design III, P-F₁-F₂-F₃-BC₁-BC₂), for 197 parental RAPD markers of known genomic location. Comparison of observed patterns of introgression with expectations based on simulations of unrestricted introgression revealed that much of the genome was protected from introgression regardless of mating design or chromosomal structural differences. Although the simulations indicated that all markers should introgress into multiple individuals in each of the three mating designs, 20 of 58 (34%) markers from collinear linkage groups, and 112 of 139 (81%) markers from rearranged linkage groups did not introgress. In addition, the average size of introgressed fragments (12.2 cM) was less than half that predicted by theoretical models (26–33 cM). Both of these observations are consistent with strong selection against introgressed linkage blocks, particularly in chromosomally divergent linkages. Nonetheless, mating designs II and III, which employed one and two generations of sib-mating, respectively, prior to backcrossing, were significantly more effective at moving alleles across both collinear and rearranged linkages than mating design I, in which the backcross generations preceded sib-mating. Thus, breeding strategies that include sib-crossing, in combination with backcrossing, should significantly increase the effectiveness of gene transfer across complex genic or chromosomal sterility barriers.     

Inheritance of resistance to potyviruses in Phaseolus vulgaris L. IV. Inheritance, linkage relations, and environmental effects on systemic resistance to four potyviruses

We have examined the genetics of systemic resistance in Phaseolus vulgaris to azuki bean mosaic virus (AzMV) and cowpea aphid-borne mosaic virus (CABMV) and the relationship of this resistance to a phenotypically similar resistance to watermelon mosaic virus (WMV) and soybean mosaic virus (SMV). In P. vulgaris cv Great Northern 1140 (GN1140), resistance to SMV and WMV has been attributed to the genes Smv and Wmv, respectively, which have been shown to segregate as a unit. Systemic resistance to AzMV is conferred by two incompletely dominant alleles, Azm1 and Azm2, at unlinked loci. At least three resistance alleles must be present at these two loci for systemic resistance to be expressed in the plant. Systemic resistance to CABMV in GN 1140 is conditioned by a dominant allele that has been designated Cam2. Under some environmental conditions, a recessive allele at an unlinked locus, cam3, also controls a resistant response to CABMV. Resistance to AzMV and CABMV does not assort independently from Wmv/Smv, but also does not consistently cosegregate, suggesting that perhaps in each case one of the factors involved in resistance is associated with Smv/Wmv.     

Studies on the plant cytoskeleton using miniprotoplasts of tobacco BY-2 cells

Vacuoles in plant cells can be eliminated by centrifugation of protoplasts through a density gradient. In this review, properties of evacuolated protoplasts, named ‘miniprotoplasts’, and the significant roles in plant cytoskeleton studies are described. Miniprotoplasts, prepared from tobacco BY-2 cells whose cell-cycle had been synchronized at late anaphase, continued to divide to form two daughter cells. In the presence of cytochalasin B cytokinetic cleavage was enhanced, suggesting a role of actin filaments in plant cytokinesis. In the cytoplasmic extract of miniprotoplasts both tubulin and actin could be polymerized to form microtubules (MTs) and actin filaments (AFs), respectively. A purification method for tubulin, actin and related proteins was developed using the extract. To investigate the interaction between cortical microtubules and the plasma membrane, an experimental system in which MTs were reconstructed on membrane ghosts was developed by combination of membrane ghosts and the extract.     

Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.