Cryptic Plutella species show deep divergence despite the capacity to hybridize.

BACKGROUND: Understanding genomic and phenotypic diversity among cryptic pest taxa has important implications for the management of pests and diseases. The diamondback moth, Plutella xylostella L., has been intensively studied due to its ability to evolve insecticide resistance and status as the world's most destructive pest of brassicaceous crops. The surprise discovery of a cryptic species endemic to Australia, Plutella australiana Landry & Hebert, raised questions regarding the distribution, ecological traits and pest status of the two species, the capacity for gene flow and whether specific management was required. Here, we collected Plutella from wild and cultivated brassicaceous plants from 75 locations throughout Australia and screened 1447 individuals to identify mtDNA lineages and Wolbachia infections. We genotyped genome-wide SNP markers using RADseq in coexisting populations of each species. In addition, we assessed reproductive compatibility in crossing experiments and insecticide susceptibility phenotypes using bioassays. RESULTS: The two Plutella species coexisted on wild brassicas and canola crops, but only 10% of Plutella individuals were P. australiana. This species was not found on commercial Brassica vegetable crops, which are routinely sprayed with insecticides. Bioassays found that P. australiana was 19-306 fold more susceptible to four commonly-used insecticides than P. xylostella. Laboratory crosses revealed that reproductive isolation was incomplete but directionally asymmetric between the species. However, genome-wide nuclear SNPs revealed striking differences in genetic diversity and strong population structure between coexisting wild populations of each species. Nuclear diversity was 1.5-fold higher in P. australiana, yet both species showed limited variation in mtDNA. Infection with a single Wolbachia subgroup B strain was fixed in P. australiana, suggesting that a selective sweep contributed to low mtDNA diversity, while a subgroup A strain infected just 1.5% of P. xylostella. CONCLUSIONS: Despite sympatric distributions and the capacity to hybridize, strong genomic and phenotypic divergence exists between these Plutella species that is consistent with contrasting colonization histories and reproductive isolation after secondary contact. Although P. australiana is a potential pest of brassicaceous crops, it is of secondary importance to P. xylostella.

The implementation of long-lasting insecticidal bed nets has differential effects on the genetic structure of the African malaria vectors in the Anopheles gambiae complex in Dielmo, Senegal.

BACKGROUND: Mosquitoes belonging to the Anopheles gambiae complex are the main vectors of malaria in sub-Saharan Africa. Among these, An. gambiae, Anopheles coluzzii and Anopheles arabiensis are the most efficient vectors and are largely distributed in sympatric locations. However, these species present ecological and behavioural differences that impact their vectorial capacity and complicate vector-control efforts, mainly based on long-lasting insecticidal bed nets (LLINs) and indoor residual spraying (IRS). In this study, the genetic structure of these three species in a Senegalese village (Dielmo) was investigated using microsatellite data in samples collected in 2006 before implementation of LLINs, in 2008, when they were introduced, and in 2010, 2 years after the use of LLINs. RESULTS: In this study 611 individuals were included, namely 136 An. coluzzii, 101 An. gambiae, 6 An. coluzzii/An. gambiae hybrids and 368 An. arabiensis. According to the species, the effect of the implementation of LLINs in Dielmo is differentiated. Populations of the sister species An. coluzzii and An. gambiae regularly experienced bottleneck events, but without significant inbreeding. The Fst values suggested in 2006 a breakdown of assortative mating resulting in hybrids, but the introduction of LLINs was followed by a decrease in the number of hybrids. This suggests a decrease in mating success of hybrids, ecological maladaptation, or a lesser probability of mating between species due to a decrease in An. coluzzii population size. By contrast, the introduction of LLINs has favoured the sibling species An. arabiensis. In this study, some spatial and temporal structuration between An. arabiensis populations were detected, especially in 2008, and the higher genetic diversity observed could result from a diversifying selection. CONCLUSIONS: This work demonstrates the complexity of the malaria context and shows the need to study the genetic structure of Anopheles populations to evaluate the effectiveness of vector-control tools and successful management of malaria vector control.

Effects of using phenotypic means and genotypic values in GGE biplot analyses on genotype by environment studies on tropical maize (Zea mays).

The objective of this study was to examine the effects of the type and intensity of nutritional stress, and of the statistical treatment of the data, on the genotype x environment (G x E) interaction for tropical maize (Zea mays). For this purpose, 39 hybrid combinations were evaluated under low- and high-nitrogen and -phosphorus availability. The plants were harvested at the V6 stage, and the shoot dry mass was estimated. The variance components and genetic values were assessed using the restricted maximum likelihood/best linear unbiased prediction method, and subsequently analyzed using the GGE biplot method. We observed differences in the performances of the hybrids depending on both the type and intensity of nutritional stress. The results of relationship between environments depended on whether genotypic values or phenotypic means were used. The selection of tropical maize genotypes against nutritional stress should be performed for each nutrient availability level within each type of nutritional stress. The use of phenotypic means for this purpose provides greater reliability than do genotypic values for the analysis of the G x E interaction using GGE biplot.

Use of multicopy transposons bearing unfitness genes in weed control: four example scenarios.

We speculate that multicopy transposons, carrying both fitness and unfitness genes, can provide new positive and negative selection options to intractable weed problems. Multicopy transposons rapidly disseminate through populations, appearing in approximately 100% of progeny, unlike nuclear transgenes, which appear in a proportion of segregating populations. Different unfitness transgenes and modes of propagation will be appropriate for different cases: (1) outcrossing Amaranthus spp. (that evolved resistances to major herbicides); (2) Lolium spp., important pasture grasses, yet herbicide-resistant weeds in crops; (3) rice (Oryza sativa), often infested with feral weedy rice, which interbreeds with the crop; and (4) self-compatible sorghum (Sorghum bicolor), which readily crosses with conspecific shattercane and with allotetraploid johnsongrass (Sorghum halepense). The speculated outcome of these scenarios is to generate weed populations that contain the unfitness gene and thus are easily controllable. Unfitness genes can be under chemically or environmentally inducible promoters, activated after gene dissemination, or under constitutive promoters where the gene function is utilized only at special times (e.g. sensitivity to an herbicide). The transposons can be vectored to the weeds by introgression from the crop (in rice, sorghum, and Lolium spp.) or from planted engineered weed (Amaranthus spp.) using a gene conferring the degradation of a no longer widely used herbicide, especially in tandem with an herbicide-resistant gene that kills all nonhybrids, facilitating the rapid dissemination of the multicopy transposons in a weedy population.

Plantation forestry under global warming: hybrid poplars with improved thermotolerance provide new insights on the in vivo function of small heat shock protein chaperones.

Climate-driven heat stress is a key factor affecting forest plantation yields. While its effects are expected to worsen during this century, breeding more tolerant genotypes has proven elusive. We report here a substantial and durable increase in the thermotolerance of hybrid poplar (Populus tremula×Populus alba) through overexpression of a major small heat shock protein (sHSP) with convenient features. Experimental evidence was obtained linking protective effects in the transgenic events with the unique chaperone activity of sHSPs. In addition, significant positive correlations were observed between phenotype strength and heterologous sHSP accumulation. The remarkable baseline levels of transgene product (up to 1.8% of total leaf protein) have not been reported in analogous studies with herbaceous species. As judged by protein analyses, such an accumulation is not matched either by endogenous sHSPs in both heat-stressed poplar plants and field-grown adult trees. Quantitative real time-polymerase chain reaction analyses supported these observations and allowed us to identify the poplar members most responsive to heat stress. Interestingly, sHSP overaccumulation was not associated with pleiotropic effects that might decrease yields. The poplar lines developed here also outperformed controls under in vitro and ex vitro culture conditions (callus biomass, shoot production, and ex vitro survival), even in the absence of thermal stress. These results reinforce the feasibility of improving valuable genotypes for plantation forestry, a field where in vitro recalcitrance, long breeding cycles, and other practical factors constrain conventional genetic approaches. They also provide new insights into the biological functions of the least understood family of heat shock protein chaperones.

Selection of maize hybrids for tolerance to aluminum in minimal solution.

The aim of this study was to determine the Al concentration and the period of exposure of the roots of maize hybrids in minimal solution for efficient selection of genotypes that are Al-tolerant. Two experiments were performed (48 and 96 h of exposure) with increasing doses of Al in minimal solution; the block design was completely randomized in a split-plot design with 3 replications. By assessing differences in root growth (cm) and the percentage of inhibition of the growth of the main root (%), a marked decrease was observed in maize root growth with increasing Al concentration in the solution. Exposure of the roots to 2 mg/L Al for 48 h in minimal solution was the most efficient for selecting sources of tolerance, particularly for the hybrids H 44 and H 38.

The expression of IL6 and 21 in crossbred calves upregulated by inactivated trivalent FMD vaccine.

Foot and mouth disease (FMD) is an economically important disease and a whole-virus inactivated trivalent virus vaccine is the mainstay for controlling the disease in India. The protective humoral immune response to FMD vaccination is a complex, but, tightly regulated process mediated by the interplay of interleukins (IL). Based on the specific role of IL6 and 21 in adaptive immune response, we hypothesized that inactivated trivalent FMD vaccine would stimulate IL6 and 21 expression in the circulating lymphocytes. The expressions of IL6 and 21 were assayed on 0, 28, 60, 90, and 120 d post-vaccination (DPV) by quantitative PCR (qPCR) with simultaneous assessment of FMDV antibody titer by liquid phase blocking ELISA. The results revealed that the peak expression of IL6 and 21 was on DPV 28 which correlated well with the FMDV antibody titer and plummeted to the prevaccination titer level by 60 DPV. As IL21 is the final effector of antibody production as compared to IL6, we investigated the expression of IL21 in calves that had protective titer (>1.8) with the unprotected group (<1.8). Expression of IL21 on 28 DPV was numerically higher in the protected than that of the unprotected group of calves.

Influence of hybrid giant Napier grass on salt and nutrient distributions with depth in a saline soil.

Cultivation of the biofuel plant, hybrid giant Napier grass (HGN), in saline soil was investigated in a greenhouse study. The results show that HGN is a salt tolerant plant which can flourish in saline soil and product a large amount of biomass. The extensively developed fibrous root system of HGN plays a significant role in the uptake of sodium from saline soil so that both soil salinity and pH are reduced. Fibrous roots of HGN are well distributed in the soil below the surface, where the metabolism of the root system produces a gradient at the depth between 10 and 20 cm in soil salinity, pH and organic content. The degradation of the HGN by the biota within the soil results in an increase in nutrients and improved soil quality. The experimental results suggest that HGN adapts to saline soil, which is promising for phytoremediation of such soils. Additional advantages of HGN include the large biomass produced which can be used for renewable energy generation.

Molecular cloning and functional characterization of a mouse ccl6 analog gene in the rat.

Suppression subtractive hybridization was used to analyze differential expression of genes in rat peritoneal macrophages after granulocyte macrophage colony-stimulating factor treatment. We identified and cloned the mouse C10 analog gene in the rat, and named it as ccl6. The full-length cDNA of rat ccl6 was 467 bp, which contains a single-open reading frame and encodes 116 amino acid residues. Compared with other C-C chemokines, the rat ccl6 gene had an unusual four-exon genome structure instead of the typical three exons, it had the highest homology with murine ccl6. The rat ccl6 gene was localized on chromosome 10, where most of the C-C chemokine superfamily members are located. The recombinant rat C-C chemokine ligand 6 (CCL6) protein was expressed by the pGEX4T-1 plasmid in Escherichia coli BL21. The purified recombinant protein had bioactivity similar to that of mouse CCL6, which is a chemoattractant for macrophages and lymphocytes, but not for neutrophils.

D-glufosinate as a male sterility agent for hybrid seed production.

A chemical male sterility system based on anther-localized conversion of the inactive D-enantiomer of the herbicide, glufosinate (2-amino-4-(methylphosphinyl)-butanoate) to the phytotoxic L is described. Highly pure D-glufosinate was isolated in >98% enantiomeric excess from the racemate via fermentation with a strain of Escherichia coli expressing the PAT (L-glufosinate N-acetyl transferase) gene and purification of the unreacted D-enantiomer from the broth by ion exchange. A modified (F58K, M213S) form of the D-amino acid oxidase (DAAO) (EC 1.4.3.3) from Rhodosporidium toruloides was designed, tested in vitro and found to efficiently oxidize D-glufosinate to its 2-oxo derivative [2-oxo-4-(methylphosphinyl)-butanoic acid]. Tobacco (Nicotiana tabacum) plants were transformed to express this modified oxidase under control of the TAP1 tapetum-specific promoter. A number of the resultant transgenic lines exhibited complete male sterility that persisted for two or more weeks immediately following foliar treatment with 75 or 200 g/ha of D-glufosinate without exhibiting obvious phytotoxic symptoms or any measurable decline in female fertility. Similarly, plants containing the same construct and, additionally, a PAT gene expressed from a plastocyanin promoter exhibited significantly reduced male fertility and no reduction in female fertility following foliar application of racemic glufosinate. Thus, foliar application of d-glufosinate either purified or as the commercial herbicide, combined with anther expression of a modified DAAO promises to provide a cost-effective conditional chemical male sterility system with the characteristics necessary for practical F1 hybrid seed production.

Transfer of the barrier to radial oxygen loss in roots of Hordeum marinum to wheat (Triticum aestivum): evaluation of four H. marinum-wheat amphiploids.

• Wide hybridization of waterlogging-tolerant Hordeum marinum with wheat (Triticum aestivum) to produce an amphiploid might be one approach to improve waterlogging tolerance in wheat. • Growth, root aerenchyma and porosity, and radial oxygen loss (ROL) along roots were measured in four H. marinum-wheat amphiploids and their parents (four accessions of H. marinum and Chinese Spring wheat) in aerated or stagnant nutrient solution. A soil experiment was also conducted. • Hordeum marinum maintained shoot dry mass in stagnant nutrient solution, whereas the growth of wheat was markedly reduced (40% of aerated control). Two of the four amphiploids were more tolerant than wheat (shoot dry masses of 59-72% of aerated controls). The porosity of adventitious roots when in stagnant solution was higher in H. marinum (19-25%) and the four amphiploids (20-24%) than in wheat (16%). In stagnant solution, adventitious roots of H. marinum formed a strong ROL barrier in basal zones, whereas, in wheat, the barrier was weak. Two amphiploids formed a strong ROL barrier and two formed a moderate barrier when in stagnant solution. • This study demonstrates the transfer of higher root porosity and a barrier to ROL from H. marinum to wheat through wide hybridization and the production of H. marinum-wheat amphiploids.

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora.

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0-32.6% of cultured ovary pieces and 0-18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.

Transcriptomic changes following recent natural hybridization and allopolyploidy in the salt marsh species Spartina x townsendii and Spartina anglica (Poaceae).

Allopolyploidy results from two events: the merger of divergent genomes and genome duplication. Both events have important functional consequences for the evolution and adaptation of newly formed allopolyploid species. In spite of the significant progress made in recent years, few studies have decoupled the effects of hybridization from genome duplication in the observed patterns of expression changes accompanying allopolyploidy in natural conditions. We used Agilent rice oligomicroarrays to explore gene expression changes following allopolyploidy in Spartina that includes a classic example of recent allopolyploid speciation: S. anglica formed during the 19th century following genome duplication of the hybrid S. x townsendii. Our data indicate important, but different, effects of hybridization and genome duplication in the expression patterns of the hybrid and allopolyploid. Deviation from parental additivity was most important following hybridization and was accompanied by maternal expression dominance, although transgressively expressed genes were also encountered. Maternal dominance was attenuated following genome duplication in S. anglica, but this species exhibits an increased number of transgressively overexpressed genes. These results reflect the decoupled effects of the 'genomic shock' following hybridization and genome redundancy on the genetic, epigenetic and regulatory mechanisms characterizing transcriptomic evolution in allopolyploids.

Ectopic expression of LEAFY COTYLEDON1-LIKE gene and localized auxin accumulation mark embryogenic competence in epiphyllous plants of Helianthus annuus x H. tuberosus.

BACKGROUND AND AIMS: The clone EMB-2 of the interspecific hybrid Helianthus annuus x H. tuberosus provides an interesting system to study molecular and physiological aspects of somatic embryogenesis. Namely, in addition to non-epiphyllous (NEP) leaves that expand normally, EMB-2 produces epiphyllous (EP) leaves bearing embryos on the adaxial surface. This clone was used to investigate if the ectopic expression of H. annuus LEAFY COTYLEDON1-LIKE (Ha-L1L) gene and auxin activity are correlated with the establishment of embryogenic competence. METHODS: Ha-L1L expression was evaluated by semi-quantitative RT-PCR and in situ hybridization. The endogenous level and spatial distribution of free indole-3-acetic acid (IAA) were estimated by a capillary gas chromatography-mass spectrometry-selected ion monitoring method and an immuno-cytochemical approach. KEY RESULTS: Ectopic expression of Ha-L1L was detected in specific cell domains of the adaxial epidermis of EP leaves prior to the development of ectopic embryos. Ha-L1L was expressed rapidly when NEP leaves were induced to regenerate somatic embryos by in vitro culture. Differences in auxin distribution pattern rather than in absolute level were observed between EP and A-2 leaves. More precisely, a strong IAA immuno-signal was detected in single cells or in small groups of cells along the epidermis of EP leaves and accompanied the early stages of embryo development. Changes in auxin level and distribution were observed in NEP leaves induced to regenerate by in vitro culture. Exogenous auxin treatments lightly influenced Ha-L1L transcript levels in spite of an enhancement of the regeneration frequency. CONCLUSIONS: In EP leaves, Ha-L1L activity marks the putative founder cells of ectopic embryos. Although the ectopic expression of Ha-L1L seems to be not directly mediated by auxin levels per se, it was demonstrated that localized Ha-L1L expression and IAA accumulation in leaf epidermis domains represent early events of somatic embryogenesis displayed by the epiphyllous EMB-2 clone.

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity.

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.

Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids.

Mitochondria play a critical role in the generation of metabolic energy and are crucial for eukaryotic cell survival and proliferation. In most sexual eukaryotes, mitochondrial DNA (mtDNA) is inherited from only one parent in non-Mendelian inheritance in contrast to the inheritance of nuclear DNA. The model organism Saccharomyces cerevisiae is commonly used to study mitochondrial biology. It has two mating types: MATa and MATα. Previous studies have suggested that the mtDNA inheritance patterns in hybrid diploid cells depend on the genetic background of parental strains. However, the underlying mechanisms remain unclear. To elucidate the mechanisms, we examined the effects of environmental factors on the mtDNA inheritance patterns in hybrids obtained by crossing S. cerevisiae with its close relative S. paradoxus. The results demonstrated that environmental factors can influence mtDNA transmission in hybrid diploids, and that the inheritance patterns are strain dependent. The fitness competition assay results showed that the fitness differences can explain the mtDNA inheritance patterns under specific conditions. However, in this study, we found that fitness differences cannot fully be explained by mitochondrial activity in hybrids under stress conditions.

Phosphorothioate oligodeoxynucleotides--anti-sense inhibitors of gene expression?

Phosphorothioate (PS) oligodeoxynucleotides are relatively nuclease resistant, water soluble analogs of phosphodiester (PO) oligodeoxynucleotides. These molecules are chiral but still hybridize well to their RNA targets. While considered for use as in vivo anti-sense inhibitors of gene expression, their biology, especially in the anti-viral area, is dominated by non-sequence specific effects. This review discusses both the sequence and non-sequence specific biologic effects of PS oligomers, and attempts to more clearly indicate their ultimate therapeutic potential.

De Novo Assembly and Transcriptome Analysis of Wheat with Male Sterility Induced by the Chemical Hybridizing Agent SQ-1.

Wheat (Triticum aestivum L.), one of the world's most important food crops, is a strictly autogamous (self-pollinating) species with exclusively perfect flowers. Male sterility induced by chemical hybridizing agents has increasingly attracted attention as a tool for hybrid seed production in wheat; however, the molecular mechanisms of male sterility induced by the agent SQ-1 remain poorly understood due to limited whole transcriptome data. Therefore, a comparative analysis of wheat anther transcriptomes for male fertile wheat and SQ-1-induced male sterile wheat was carried out using next-generation sequencing technology. In all, 42,634,123 sequence reads were generated and were assembled into 82,356 high-quality unigenes with an average length of 724 bp. Of these, 1,088 unigenes were significantly differentially expressed in the fertile and sterile wheat anthers, including 643 up-regulated unigenes and 445 down-regulated unigenes. The differentially expressed unigenes with functional annotations were mapped onto 60 pathways using the Kyoto Encyclopedia of Genes and Genomes database. They were mainly involved in coding for the components of ribosomes, photosynthesis, respiration, purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, RNA transport and signal transduction, reactive oxygen species metabolism, mRNA surveillance pathways, protein processing in the endoplasmic reticulum, protein export, and ubiquitin-mediated proteolysis. This study is the first to provide a systematic overview comparing wheat anther transcriptomes of male fertile wheat with those of SQ-1-induced male sterile wheat and is a valuable source of data for future research in SQ-1-induced wheat male sterility.

The Use of Maleic Hydrazide for Effective Hybridization of Setaria viridis.

An efficient method for crossing green foxtail (Setaria viridis) is currently lacking. S. viridis is considered to be the new model plant for the study of C4 system in monocots and so an effective crossing protocol is urgently needed. S. viridis is a small grass with C4-NADP (ME) type of photosynthesis and has the advantage of having small genome of about 515 Mb, small plant stature, short life cycle, multiple tillers, and profuse seed set, and hence is an ideal model species for research. The objectives of this project were to develop efficient methods of emasculation and pollination, and to speed up generation advancement. We assessed the response of S. viridis flowers to hot water treatment (48°C) and to different concentrations of gibberellic acid, abscisic acid, maleic hydrazide (MH), and kinetin. We found that 500 µM of MH was effective in the emasculation of S. viridis, whilst still retaining the receptivity of the stigma to pollination. We also report effective ways to accelerate the breeding cycle of S. viridis for research through the germination of mature as well as immature seeds in optimized culture media. We believe these findings will be of great interest to researchers using Setaria.

Enhanced efficiency of cell hybridization by neuraminidase treatment.

Cell hybridization techniques have already been utilized, but the outcome is usually not very stable. In order to achieve high stability we tried to achieve closer intercellular contact. Neuraminidase treatment was used to remove sialic acid from the cell membrane, since sialic acid usually prevents close contact between B cells and myeloma cells. After neuraminidase treatment, not only mouse-mouse hybridization but also human-mouse hybridization produced significantly more clones. Our results indicate that neuraminidase treatment is a useful method for generating hybridomas efficiently.