Endogenous ABA concentration and cytoplasmic membrane fluidity in microspores of oilseed rape (Brassica napus L.) genotypes differing in responsiveness to androgenesis induction

Key message

A better understanding of androgenesis with a focus on the changes in plasma membrane fluidity and endogenous ABA content affecting embryogenesis induction in microspore suspension of B. napus.

Abstract

Changes in plasma membrane fluidity (MF) and ABA content associated with androgenesis induction were under the study. Both parameters were monitored in microspores of two Brassica napus L. genotypes differing in their response to androgenic induction under heat (1 day at 32 °C). MF was assessed by DPH method. ABA content was evaluated by ELISA. Heat caused microspores’ plasma membrane to become more rigid. Lower MF in microspores of ‘DH 4079’ (of high androgenic potential) seems to maintain proper cell protection and leads to efficient embryogenesis induction. Plasma membrane remodelling coincided with changes of ABA content in microspores and in the culture medium in both genotypes. ABA concentration (μM) and ABA content (fmol per 10⁴ microspores or pmol g^?1 FW) were for the first time measured in microspores. ABA concentration (μM) in microspores and in the culture medium (nM) differed significantly for the genotype and the treatment. The interaction between both variables was also significant. In general, ABA content ranged from <3.5 to 87.1 fmol per 10⁴ microspores. The highest content of ABA was detected in ‘DH 4079’ microspores at 32 °C. Assuming a mean microspores’ radius of 10 μm, it corresponds to ABA concentration of 2.1 μM. Heat shock resulted in quantum of medium pH reduction (0.1–0.2) and increased levels of ABA in microspores and in the medium of both tested genotypes. However, heat induced increase of ABA content in microspores of non-responsive ‘Campino’ had no clear-cut impact, on androgenesis induction efficiency, which suggests a more complex mechanism of process initiation.     

The effect of Lr19-translocation on in vitro androgenesis and inheritance of leaf-rust resistance in DH3 lines and F2 hybrids of common wheat

Leaf-rust resistance and androgenesis were studied in the anther cultures of Triticum aestivum L., which included Saratovskaya 29 cultivar, the isogenic line Ps29, and three F1 hybrids (L503/S55, L504/S58, ATS7/L1063) with 7DS-7DL-7Ae#1L translocation of Lr19 gene (Lr19 translocation) from Agropyron elongatum (Host) P.B. The Lr19 translocation was shown to affect the induction of embryogenesis and green plant regeneration. The frequencies of Lr19 translocation differed in F2 hybrids obtained by traditional hybridization and in sets of DH lines obtained in F1 anther cultures derived from the same combinations of T. aestivum parental forms. The number of leaf-rust resistant genotypes tended to decrease. The frequency of Lr19 translocation in the set of DH3 lines derived from F1 L504/S58 was significantly lower than in other sets of DH3 lines and F2 hybrid populations.     

Confirmation of QTL controlling seed yield in spring canola (Brassica napus L.) hybrids

Allelic effects observed in QTL discovery experiments must be confirmed to be useful in subsequent breeding efforts. Two QTL affecting seed yield of spring hybrid canola (Brassica napus L.) were previously identified in two populations of inbred backcross lines (IBLs) containing germplasm introgressed from a winter cultivar. The effects of favorable alleles at these QTL were retested by crossing two selected IBLs (M5 and M31) to three spring canola lines having different genetic backgrounds. Doubled haploid (DH) lines derived from each F₁ were genotyped with RFLP markers flanking the QTL and grouped into the four possible QTL genotypes. For the first field experiment, DH lines derived by crossing the M5 line to one spring line were crossed to two female testers and evaluated as individual testcross progenies in one environment. QTL genotypes had large variances and were not significantly different. A second field experiment was conducted using the DH lines from the first experiment and two other sets of DH lines derived from the M31 line crossed to two different spring canola lines. Individual lines within each QTL genotype of each set were bulked and crossed to the same testers used in Experiment 1. Bulked hybrid seeds of each QTL genotype were planted in a split-split plot randomized block design and 12 replicates. QTL genotypes had smaller variances in this experiment, and the effects of one QTL were confirmed in some genetic backgrounds. These results suggest that bulking of QTL genotypes and use of an appropriate experimental design with many replicates are needed to detect small differences between QTL genotypes.     

Evaluation of genetic diversity and uniformity of head cabbage DH lines by the use of RAPD markers

DH lines derived from cabbage cvs. Kamienna G?owa, S?awa z Enkhuizen and Langendijker, representing R1 generation, were analysed by the use of RAPD markers for their diversity and uniformity. For the evaluation of genetic diversity, eight primers yielding informative bands were used. Of the total of 83 RAPD bands scored in this study, 16.9% were polymorphic between a set of 13 DH lines. The similarity of the DH lines, estimated by Jaccard's coefficient, was depicted in the UPGMA dendrogram. Fourteen generated informative RAPD bands allowed the identification of DH lines developed from each cultivar. The evaluation of the uniformity for six closely related DH lines was possible by the use of three primers which generate one or two polymorphic bands. The lack of differences among ten plants of the five investigated DH lines manifested their uniformity. One line showed intraline polymorphism with two RAPD primers. The occurrence of the differences at the molecular level among ten plants indicated that their parental R0 plant was probably obtained from somatic cells, not by androgenesis.     

Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.)

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F₁ hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F₂ progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F₂ progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN. Received: 2 July 1998 / Accepted: 11 March 1999     

Genotypic variation of quantitative trait loci controlling in vitro androgenesis in maize.

A quantitative trait loci (QTL) analysis for androgenetic capability has been conducted on three different crosses in maize, including very high and nonresponding lines for androgenesis. The doubled haploid lines derived by anther culture from the crosses DH5 x DH7, A188 x DH7, and R6 x DH99 showed a range of 0-70%, 0-40%, and 0-50% androgenetic responding anthers, respectively. The genotypic heritability of means for this trait is close to 0.90 for A188 x DH7 and 0.78 for R6 x DH99. The QTL analysis involved in each population the mapping of more than 100 loci covering a large part of the genome with reasonably spaced markers averaging 12 cM. Different measurements describing the androgenetic process were studied: AC, percentage of responding anthers; ELS, number of androgenetic embryos produced per 100 plated anthers; PLE, number of plantlets regenerated per 100 embryos; PLA, number of plantlets per 100 plated anthers. In each cross, three to four QTLs were found for AC, explaining 30-40% of the phenotypic variation. The QTL detected for PLA was also strong QTL for AC or ELS. This agrees with the observation that these last two traits are good predictors for final plantlet yield. The QTLs found were specific, although the same line DH7 was used in two crosses and DH99 derived from DH5 and DH7 in the third cross. These results suggest that the transfer of the androgenetic capabilities in elite germplasm will still involve a phenotypic evaluation of the androgenetic performances. A backcross-assisted selection based only on the genotype at the QTL is probably possible but only within the crosses used for this QTL analysis.     

Increased frequency of dividing microspores and improved maintenance of multicellular microspores of Coffea arabica in medium with coconut milk

The number of dividing microspores of Coffea arabica L. cv. Catuai and Catimor could be drastically increased in microspore media containing 16% (w/v) coconut milk, allowing cell divisions to continue in the microspore and multicellular microspores to survive until day 60. After a cold treatment, the microspores were mechanically isolated prior to cultivation in Murashige and Skoog medium supplemented with sucrose and maltose and (mg l^-1) 2,4-d: 2, BAP: 1 or a combination of kinetin: .5, 2,4-d: .5 and NAA: .5 as stationary suspension at a density of 1,200/ml. The crucial stage during microsporogenesis suitable for in vitro androgenesis proved to be mid uninucleate till early binucleate in flowerbuds with the size of 13–15 mm two to three days before anthesis. The initial steps of androgenesis were determined.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxy-acetic acid - NAA 1-naphthaleneacetic acid     

Differential mRNA display analysis of two related but functionally distinct rat insulinoma (RIN) cell lines: identification of CD24 and its expression in the developing pancreas

To identify genes that might play a role in growth and differentiation of pancreatic beta-cells, we have applied the technique of differential mRNA display to the lineage-related, but functionally distinct rat insulinoma (RIN) cell lines RIN-5AH and RIN-A12. Direct comparison of PCR-generated RIN-5AH and RIN-A12 cDNAs on DNA sequencing gels revealed 31 differentially expressed bands. By Northern blot hybridization, authentic differential expression was confirmed for three cDNAs derived from RIN-5AH cells and four cDNAs from RIN-A12 cells. Nucleotide sequences were determined for these cDNAs and database searches identified one known gene that encoded heat stable antigen CD24. Of the remaining six genes, three matched with established sequence tags from fetal tissue, and three were potentially novel. By RT-PCR analysis, five of the seven genes were expressed in normal fetal and/or adult pancreas. In a detailed survey of CD24 protein expression in the pancreas using the CD24-specific monoclonal antibody J11d, CD24 was predominantly expressed in ductal epithelial cells (E13.5-15.5), developing endocrine (alpha, beta and delta) and exocrine cells (E15.5-20.5) and mature exocrine and peripheral islet delta-cells post E20.5. The retention of CD24 expression in a large proportion of delta-cells but only in a minority of alpha- and beta-cells leads us to hypothesize that CD24 may mark a pool of precursor endocrine cells within adult islets.     

The Development of Male and Female Regenerants by In Vitro Androgenesis in Dioecious Plant Melandrium album

We examined induced androgenesis in vitro in the dioecious plantMelandrium album and aimed to produce complete plants from culturedimmature microspores. Flow cytometric analysis of nuclear DNAcontent was used to screen ploidy levels in regenerated plantsand to estimate the nuclear genome size in plants differingin sex. Haploid and spontaneous dihaploid (polyhaploid) femalesdominated among androgenic regenerants. Androgenic males occurredsporadically. They were exclusively dihaploid and geneticallysupermales (AAYY). The progenies obtained as a result of thecrosses between supermales and standard females contained onlymales. This is the first report on complete androgenesis inM. album from the microspores carrying the Y chromosome.Copyright1994, 1999 Academic Press Melandrium album (Miller) Garcke, pollen androgenesis, sex, female, male, supermale, flow-cytometry, nuclear genome size     

Segregation distortion at marker loci: variation during microspore embryogenesis in maize

In the present study, we analyzed the segregation distortions of markers during in vitro androgenesis in maize. This was based on four segregating populations derived from the A188×DH7 one-way-cross. These populations consisted of very young androgenetic embryos, well-developed calluses, haploid regenerated plantlets and spontaneous diploid plantlets. These structures all represented different developmental stages, from that of microspores to the regenerated plantlets. This study complemented a previous one by Murigneux et al. 1994, where distorted segregations of RFLP markers were detected in a single-seed-descent population and in a doubled-haploid population derived from the same cross. The weakly biased SSD maize genetic map was used as a reference to locate 145 AFLP loci whose allelic segregations were also analyzed in the androgenetic segregating populations. Segregation distortions were determined based on chi-square analysis (P<0.01 and P<0.001). Regions on chromosomes 2 and 8 showed distortions from the beginning of embryo formation, with large effects throughout the process. Regions on chromosomes 3, 4, 6 and 10 could control callus formation from microspores. Other deviations of marker genotypes on chromosomes 1, 4, 6 and 10 could be associated with the regeneration phase. Moreover, the statistical method of Cheng et al. for mapping a lethal factor locus inside segments of linked distorted markers was used to estimate the position of seven partial lethal androgenetic factors on chromosomes 1, 2, 8 and 10. These factors could represent selective genes actively involved in maize androgenesis. Received: 31 July 2000 / Accepted: 2 January 2001     

The agronomic performance of wheat doubled haploid lines derived from wheat x maize crosses

Summary The agronomic performance of 9 doubled haploid (DH) lines of Chinese Spring, 6 DH lines of Hope, 14 DH lines of the single chromosome substitution line Chinese Spring (Hope 5 A) and their respective parents was analyzed under field conditions. Seventeen Chinese Spring DH lines derived from wheat x Hordeum bulbosum crosses were also included for comparison. No significant variation was detected in either population of Chinese Spring DH lines and neither DH population differed from its parent. The Hope DH lines differed significantly for tiller biomass, spikelet number per ear, ear grain weight and 50-grain weight. However, all the variation could be attributed to the poor performance of only one line. Chinese Spring (Hope 5 A) DH lines showed significant variation for ear emergence time, but this was probably due to genetic heterogeneity in the parental stock. Overall, the results suggest that most DH lines produced by the wheat x maize method resemble their wheat parent, and that the variation induced in DH production is likely to be similar to that found in DHs from wheat x Hordeum bulbosum crosses.     

Induction of a regulatory phenotype in human CD4+ T cells by streptococcal M protein

Regulatory T cells (Tregs) participate in the control of the immune response. In the human system, an IL-10-secreting, T regulatory type 1 cell (Tr1)-like subset of Tregs can be induced by concurrent cross-linking of the TCR and CD46 on naive CD4(+) T cells. Because many viral and bacterial pathogens, including the major human pathogen Streptococcus pyogenes, bind to CD46, we asked whether this bacterium can directly induce Tr1-like cells through the streptococcal ligand for CD46, the M protein. The M5 and M22 proteins were found to induce T cells to develop into the IL-10-producing Tr1-like phenotype. Moreover, whole M5-expressing bacteria, but not isogenic M-negative bacteria, led to proliferation and IL-10 secretion by T cells. The interaction between the M5 protein and T cells was dependent on CD46 and the conserved C repeat region of M5. Supernatants derived from T cells stimulated with M proteins or M protein-expressing bacteria suppressed bystander T cell proliferation through IL-10 secretion. In addition, activation of CD46 through streptococcal M protein induced the expression of granzyme B, providing a second means for these cells to regulate an immune response. These findings suggest that binding to CD46 and exploiting its signaling pathway may represent a strategy employed by a number of important human pathogens to induce directly an immunosuppressive/regulatory phenotype in T cells.     

Effect of IAA content Modulators on peroxidase activity and on endogenous IAA during cold pretreatment of maize anthers prior to androgenesis

A cold pretreatment is usually applied to induce maize androgenesis. Peroxidase activity, including indole-3-acetic acid (IAA) oxidase activity, and endogenous IAA concentrations were followed during a cold pretreatment (14 days, 7°C) in anthers of two maize genotypes, Seneca 60 and DH5×DH7, respectively with a low or high androgenetic response. The most prominent result was the absence of a detectable IAA oxidase activity in DH5×DH7. Adding effectors of IAA-oxidase activity or IAA transport did not affect significantly the crude peroxidase activity of DH5×DH7 anthers while inducing a clear inhibition of androgenesis at higher concentrations. No strict correlation was found between IAA level and physiological response, the low responding variety having as much IAA as DH5×DH7. However, for DH5×DH7, every treatment that lowered the IAA level after 14 days of cold resulted in a decrease in androgenetic response.     

Field evaluation of doubled haploid plants in the Apiaceae: dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.)

The Apiaceae family includes vegetables, as well as herb and spice crops. Compared to major crops, there have been few breeding or genetic improvement programs for any of the Apiaceae, especially the herb and spice species. Haploidy technology can be used to develop uniform, true-breeding lines, as well as to accelerate breeding programs. Field trials of dill (Anethum graveolens L.), caraway (Carum carvi L.), and fennel (Foeniculum vulgare Mill.) doubled haploid (DH) lines were conducted over 2–5 cropping seasons. Several of the DH dill lines had desirable agronomic characteristics such as short uniform stature along with early maturity that would be useful for crop improvement. Seed yields and the essential oil content of the seed harvested from the best performing DH dill lines were either equal to or higher than the parental line. A DH annual caraway line was identified that produced higher seed yields than the industry standard. The main constituents of the essential oil for the DH lines of both dill and caraway were similar to the parental lines. Fennel DH lines exhibited differences in height but were too late in maturity for seed production under prairie conditions. The results indicate that not only were we able to generate DH lines that could be used in a crop improvement program, but we developed DH lines that could be used directly as cultivars as these lines performed better than the industry standard (parental line).     

Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (<Emphasis Type="Italic">Brassica oleracea</Emphasis> var. <Emphasis Type="Italic">botrytis</Emphasis>)

Protoplasts were isolated from hypocotyls of 5-day-old seedlings of five doubled-haploid (DH) lines of cauliflower after enzymatic digestion in 0.1% macerozyme R-10 and 1.0% cellulose R-10. Shoot regeneration was achieved in four of the five DH lines. Protoplast yield and frequency of cell division and shoot regeneration varied among experiments and DH lines. Sequence-related amplified polymorphism (SRAP) analysis on all the regenerated plants of each DH line indicated that their genetic compositions were homologous with their mother plants, but few regenerated plants of DH lines no. 3 and 5 were detected with changes in ploidy levels.     

Differentiation and classification of parental lines and favorable genic interactions affecting F1 fertility in distant crosses of rice (Oryza sativa L.)

 This study was intended to investigate the extent of genetic differentiation in parental lines of rice hybrids and to analyze the genetic basis underlying the fertility phenomenon in distant crosses. Two subsets of rice material (111 entries in total) were used, including 81 doubled-haploid (DH) lines and 30 Indica and Japonica rice varieties or lines (as a control). The DH lines was derived from a heterotic Indica/Japonica cross (Gui630/02428) by anther culture. The materials in the control represent a broad spectrum of the Asian cultivated rice gene pool including landraces, primitive cultivars, historically important cultivars, modern elite cultivars, super rice and parents of superior hybrids. In accordance with the NC II design, 57 out of the DH lines were test-crossed to two important wide compatibility lines: photoperiod-sensitive genetic male sterile (PGMS) line N422s and thermo-sensitive genetic male sterile (TGMS) line Peiai64s. The F₁s and their parents, 182 entries in total, were examined for the performance of seven traits in a replicated field trial. All the rice materials was surveyed for polymorphisms using 92 RFLP markers selected from two published molecular marker linkage maps. Genotypes of the F₁ hybrids at the molecular-marker loci were deduced from the parental genotypes. The analysis showed that there were two types of genetic differentiation in the two subsets of rice material; that is, qualitative differentiation in the control and quantitative differentiation in the DH lines. In addition, favorable genic interactions (both intra- or inter-locus) contributed to better increase the fertility in hybrids of distant crosses through incorporation of a wide-compatibility line as the female parent. Favorable genic interactions can be applied in hybrid rice breeding programs by selecting parents with an appropriate extent of genetic differentiation. Received: 5 June 1997 / Accepted: 10 September 1997     

Hormonal regulation of murine mammary tumor virus RNA expression during mammary tumorigenesis in BALB/c mice.

The effects of glucocorticoids and prolactin on murine mammary tumor virus (MuMTV) RNA expression in preneoplastic outgrowth lines and mammary tumors in BALB/c mice were investigated. Hyperplastic alveolar nodules (HAN) and a ductal hyperplasia (DH) are induced in virgin BALB/c mice by prolonged hormonal stimulation or treatment with 7,12-dimethylbenz(a)anthracene or both. Mice bearing HAN or DH outgrowth lines and mammary tumors that arose from the outgrowth lines were treated with glucocorticoids or prolactin. MuMTV RNA was quantitated by hybridization with a representative complementary DNA probe specific for MuMTV RNA. Prolactin treatment did not increase MuMTV RNA in the BALB/c HAN or DH outgrowth lines or tumors. MuMTV RNA increased after glucocorticoid treatment in the C3, C4, and C5 HAN outgrowth lines and in tumors that arose from the D1, D2, C4, and C5 HAN and CD8 DH outgrowth lines. No increase in MuMTV RNA with glucocorticoid treatment was observed in the D1 or D2 HAN outgrowth line, in the CD8 DH outgrowth lines, and in tumors that arose from the C3 HAN outgrowth line. The ability of glucocorticoids to stimulate MuMTV expression was specific since the response was dose dependent and specific for glucocorticoid hormones. Glucocorticoid treatment did not increase the level of type C viral RNA in the majority of hormone- or 7,12-dimethylbenz(a)anthracene-induced HAN outgrowth lines or tumors. These observations suggested that glucocorticoids may influence MuMTV expression during mammary tumorigenesis in BALB/c mice.     

Structure and expression of the human gene encoding major heat shock protein HSP70.

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.