In vitro screening of rice germplasm for resistance to brown spot disease using phytotoxin

Summary Two R₁ IR₈ plants derived from somatic cells cultured in 25% Helminthosporium oryzae toxin-medium, and one IR₅₄ plant derived from a control (toxin free medium) were found to have mutated resistance to brown spot disease of rice. In the second generation (R₂, the offspring of the R₁ generation) of the three resistant populations, the segregation of resistance and susceptibility to the disease was observed. This is the first report about a disease resistant mutation obtained successfully in rice by tissue culture and in vitro screening with phytotoxin.This research was conducted at the IRRI, Manila     

Cloning and characterization of a root sunflower peroxidase gene putatively involved in cell elongation

A cDNA-encoding a peroxidase (Helianthus annuus POX (HaPOX)1) was isolated and characterized from the roots of sunflower seedlings. This gene exhibited homology with other peroxidases from several higher-plant species, and its expression in the root growth was particularly abundant during cell expansion. To elucidate the precise functions of HaPOX1 in sunflower root, we examined its expression pattern in response to several plant growth regulators. Expression of HaPOX1 is down-regulated by abscisic acid (ABA), whereas indole-3-acetic acid (IAA) induced its expression. These results suggest that HaPOX1 expression is differentially regulated by phytohormonal components of signaling cascades. Since IAA appears to participate in the regulation of HaPOX1 expression, we postulate that the peroxidase encoded by HaPOX1 may be involved in the reactions that promote cell elongation during the early stage of root growth.     

AFLP mapping of QTLs for in vitro organogenesis traits using recombinant inbred lines in sunflower (Helianthus annuus L.)

Genetic control for two in vitro organogenesis traits, the number of shoots per explant plated (S/E) and the number of shoots per regenerating explant (S/RE), was investigated in 75 recombinant inbred lines (RILs) of sunflower and their two parents (PAC-2 and RHA-266). Genetic variability was observed among the 75 RILs for the organogenesis traits studied. Some RILs presented significant differences when compared with the best parental line (RHA-266). Genetic gain, in terms of the percentage of the best parent, for 32% of the selected RILs was significant. A set of 99RILs from the same cross including the 75 mentioned above was screened with 333 AFLP markers and a linkage map was constructed based on 264 linked loci. Six putative QTLs for the S/RE (tentatively named osr) and seven QTLs for the S/E (ose) trait were detected using composite interval mapping. For each trait, the QTLs explained 52% (ose) and 67% (osr) of the total phenotypic variance. These results suggested that additive gene effects predominate in explaining a large proportion of the observed genetic variation associated with regeneration ability. The coincidental location of QTLs for S/E and S/RE is discussed. Received: 20 September 1999 / Accepted: 16 May 2000     

Screening of sunflower cultivars for metal phytoextraction in a contaminated field prior to mutagenesis

Sunflower can be used for the remediation of metal-contaminated soils. Its high biomass production makes this plant species interestingfor phytoextraction and using sunflower oil for a technical purpose may improve the economic balance of phytoremediation. The aim of the present field study was to screen 15 commercial cultivars of Helianthus annuus L. grown on metal-contaminated soil, to find out the variety with the highest metal extraction, which can be further improved by mutation or in vitro breeding procedures. Two different fertilizers (ammonium sulphate and ammonium nitrate) were also used to enhance the bioavailability of metals in soil Highly significant differences were observed within tested varieties for metal accumulation and extraction efficiency. Furthermore, ammonium nitrate increased cadmium extraction, whereas ammonium sulphate enhanced zinc and lead uptake in most tested cultivars. In this field-based sunflower screening, we found enhanced cumulative Cd, Zn, and Pb extraction efficiency by a factor 4.4 for Salut cultivar. We therefore emphasize that prior to any classical breeding or genetic engineering enhancing metal uptake potential, a careful screening of various genotypes should be done to select the cultivar with the naturally highest metal uptake and to start the genetic improvement with the best available plant material.     

Genetic control of early events in protoplast division and regeneration pathways in sunflower

Experiments were conducted to identify the genetic factors controlling protoplast division and to determine eventual relations between genetic factors involving organogenesis, somatic embryogenesis and protoplast division in sunflower. The present study involved protoplast culture and two traits: total division per 100 protoplasts (TOTD) and asymmetric division per 100 protoplasts (ASYD) were scored in 52 recombinant inbred lines (RILs) from a cross between PAC-2 and RHA-266. Asymmetric division is an early event in the formation of embryoids from protoplasts. Analysis of variance indicated the existence of highly significant differences among parental genotypes and their RILs. Heritability for the two protoplast division parameters (TOTD and ASYD) was high (0.87 and 0.89, respectively) and genetic gain expressed as percentage of the best parent for 10% of the selected RILs was significant. Twelve putative loci associated with total division per 100 protoplasts were identified. Eleven QTLs were also detected for asymmetric division per 100 protoplasts. The QTLs present high significant LOD scores and sum to a high percentage of phenotypic variance. The percentage of phenotypic variation explained by each QTL ranged from 2% to 24%. Some segments of the linkage groups I, XV and XVII are likely to contain genes important for organogenesis, somatic embryogenesis and protoplast division, as clustering of QTLs for these characters were described. The QTLs identified in these three linkage groups should be involved in cell division and in early events associated with cell differenciation. Received: 15 December 1999 / Accepted: 30 December 1999     

Identification of quantitative trait loci controlling resistance to gray leaf spot disease in maize

Breeding maize for gray leaf spot (GLS) resistance has been hindered by the quantitative nature of the inheritance of GLS resistance and by the limitations of selection under less than optimumal disease pressure. In order to identify the quantitative trait loci (QTLs) controlling GLS resistance, a cross was made between B73 (susceptible) and Va14 (resistant) to generate a large F₂ population. Six GLS disease assessments were made throughout the disease season for over 1000 F₂ plants in 1989, and for 600 F₂-derived F₃ lines replicated in two blocks in 1990. RFLP analysis for78 marker loci representing all ten maize chromosomes was conducted in 239 F₂ individuals including those with the extreme GLS disease phenotypes. The GLS disease scores of the three field evaluations, each averaged over six ratings, were separately used for the interval mapping in order to determine the consistency of the QTL effects. The heavy GLS disease pressure, meticulous disease ratings, and large population size of this study afforded us the sensitivity for detecting QTL effects. QTLs located on three chromosomes (1, 4, and 8) had large effects on GLS resistance, each explaining 35.0–56.0%, 8.8–14.3%, and 7.7–11.0% of the variance, respectively. These three QTL effects were remarkably consistent across three disease evaluations over 2 years and two generations. Smaller QTL effects were also found on chromosomes 2 and 5, but the chromosome-5 effect might be a false positive because it was not repeatable even in the same location. The chromosome-1 QTLs had the largest effect or highest R² reported for any quantitative trait to-date. Except for the chromosome-4 gene, which was from the susceptible parent B73, the resistance alleles at all QTL were derived from Va14. The resistance QTLs on chromosomes 1 and 2 appear to have additive effects, but those on chromosomes 4 and 8 are dominant and recessive, respectively. Significant interaction between the QTLs on chromosomes 1 and 4 was detected in all three evaluations. Cumulatively, the four QTLs identified in this study explained 44, 60, and 68% of the variance in F₂, and in F₃ replications 1 and 2, respectively.     

向日葵茎芯多糖的提取工艺优化及其体外抗肿瘤活性研究

研究了向日葵茎芯中主要活性物质多糖的提取工艺,并对此工艺进行了优化,选取的提取方法为水提醇沉法,以多糖含量作为指标,采用单因素试验研究了提取次数、原料颗粒的大小（目数）、料液比、提取时间、提取温度对向日葵茎芯多糖含量的影响。用苯酚-硫酸法测定提取液中多糖的含量,得出向日葵茎芯中多糖的最佳提取工艺条件为：提取次数2次,原料颗粒的大小（目数）60~80目,料液比（g·mL^-1）1：50,提取时间3.0 h,提取温度90℃,在最优提取条件下,多糖的提取得率为6.56%,多糖的含量为266.03 mg·g^-1。本文也对多糖的体外抗肿瘤活性进行了研究,结果表明向日葵茎芯多糖的体外抗肿瘤活性较弱。这些条件的确定为向日葵茎芯的深入研究奠定了基础。     

银杏幼嫩茎段培养诱导愈伤组织及其细胞学研究

将银杏幼嫩茎段培养于含NAA和BA的MS和改良MS培养基中获得球型胚。培养基类型对愈伤组织的诱导和分化有明显影响,MS培养基优于DCR培养基。所有的MS和改良MS培养基上有胚性细胞的分化。在含有4mg／L的NAA的MS和改良MS培养基中,有三细胞和四细胞的原胚出现,并在MS 2mg／L NAA 0．1mg／L BA的培养基中观察到球形胚的分化。     

向日葵(Helianthus annuus L.)对133Cs、88Sr的吸收和分布

通过盆栽试验研究了向日葵(Helianthus annuus L.)对土壤中不同处理浓度¹³³Cs和⁸⁸Sr的吸收,以及¹³³Cs和⁸⁸Sr在向日葵不同部位的分布。结果表明：随着处理浓度的增加,植物中¹³³Cs或⁸⁸Sr的含量增加。同一处理浓度下,⁸⁸Sr含量约比¹³³Cs含量高一个数量级。¹³³Cs和⁸⁸Sr在植物不同部位分布不同。根部中¹³³Cs含量高于植物的其他部位（茎、叶、花）。不同于¹³³Cs在植物中的分布,⁸⁸Sr除在根中的分布外,主要转运到了叶片。¹³³Cs和⁸⁸Sr在向日葵体内的分布与目前对放射性¹³⁷Cs和⁹⁰Sr的研究结果相似,所以¹³³Cs和⁸⁸Sr可分别预测¹³⁷Cs和⁹⁰Sr的运转。向日葵是治理大面积低放核素污染土壤的较佳植物种类。     

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers

Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linked genes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes. Received: 9 April 2001 / Accepted: 10 August 2001     

Metabolic profiles of sunflower genotypes with contrasting response to Sclerotinia sclerotiorum infection

We report a comprehensive primary metabolite profiling of sunflower (Helianthus annuus) genotypes displaying contrasting behavior to Sclerotinia sclerotiorum infection. Applying a GC-MS-based metabolite profiling approach, we were able to identify differential patterns involving a total of 63 metabolites including major and minor sugars and sugar alcohols, organic acids, amino acids, fatty acids and few soluble secondary metabolites in the sunflower capitulum, the main target organ of pathogen attack. Metabolic changes and disease incidence of the two contrasting genotypes were determined throughout the main infection period (R5.2-R6). Both point-by-point and non-parametric statistical analyses showed metabolic differences between genotypes as well as interaction effects between genotype and time after inoculation. Network correlation analyses suggested that these metabolic changes were synchronized in a time-dependent manner in response to the pathogen. Concerted differential metabolic changes were detected to a higher extent in the susceptible, rather than the resistant genotype, thereby allowing differentiation of modules composed by intermediates of the same pathway which are highly interconnected in the susceptible line but not in the resistant one. Evaluation of these data also demonstrated a genotype specific regulation of distinct metabolic pathways, suggesting the importance of detection of metabolic patterns rather than specific metabolite changes when looking for metabolic markers differentially responding to pathogen infection. In summary, the GC-MS strategy developed in this study was suitable for detection of differences in carbon primary metabolism in sunflower capitulum, a tissue which is the main entry point for this and other pathogens which cause great detrimental impact on crop yield.     

Evaluation of methods of screening strawberry cultivars for resistance to crown rot caused by Phytophthora cactorum

Four methods were evaluated in measuring resistance of strawberry cultivars to crown rot caused by Phytophthora cactorum. Meristem propagated plants grown in vitro were inoculated with mycelial discs. Four to five days after inoculation, it was possible to distinguish between cultivars with large differences in susceptibility to the disease. Ten days later, all plants were totally necrotic making it impossible to distinguish between cultivars. When detached leaves were inoculated by inoculating a plug of mycelium into the petiole, disease symptoms developed more slowly in resistant cultivars, but leaf age greatly affected the rate of symptom development. When plug plants (not cold stored) were lightly wounded in the rhizome with a scalpel and inoculated with either zoospores or mycelium, differences in disease development between cultivars were mainly as would be expected from previous information on susceptibility, but both age and size of plants influenced the rate of disease development. Unwounded, inoculated plants did not develop symptoms. When cold‐stored plug plants were either unwounded or lightly wounded with a scalpel in the rhizome and inoculated with zoospores, the relative rates of disease development consistently reflected the susceptibility to crown rot. At the time of final assessment, disease was much more severe in wounded plants, but the relative susceptibility of cultivars was not affected by the wounding.     

Primary culture of germ cells that portray stem cell characteristics and recipient preparation for autologous transplantation in the rhesus monkey

Fertility preservation for prepubertal cancer patients prior to oncologic treatment is an emerging issue, and non‐human primates are considered to constitute suitable models due to the limited availability of human testicular tissues. However, the feasibility of spermatogonial stem cell (SSC) propagation in vitro and autologous testicular germ cell transplantation in vivo requires further exploration in monkeys. Herein, we characterized germ cells in macaque testes at 6 months (M), 18 M and 60 M of age, and effectively isolated the spermatogenic cells (including the spermatogonia) from macaque testes with high purity (over 80%) using combined approaches of STA‐PUT separation, Percoll gradients and differential plating. We also generated recipient monkey testes with ablated endogenous spermatogenesis using the alkylating agent busulfan in six macaques, and successfully mimicked autologous cell transplantation in the testes under ultrasonographic guidance. The use of trypan blue led to successful intratubular injection in 4 of 4 testes. Although SSCs in culture showed no significant propagation, we were able to maintain monkey testicular germ cells with stem cell characteristics for up to 3 weeks. Collectively, these data provided meaningful information for future fertility preservation and SSC studies on both non‐human primates and humans.     

Characterization of embryogenic mango cultures selected for resistance to Colletotrichum gloeosporioides culture filtrate and phytotoxin

 Embryogenic nucellar cultures of two polyembryonic mango cultivars, ‘Hindi’ and ‘Carabao’, were selected for resistance to the culture filtrate and phytotoxin of a virulent strain of Colletotrichum gloeosporioides Penz. that was isolated from mango leaves. The cultures were recurrently selected either with progressively increasing concentrations of culture filtrate or by continuous challenge with the same concentration of either culture filtrate phytotoxin. Mycelium growth was inhibited when the pathogen was cocultured with the selected, resistant embryogenic cultures. Conditioned plant growth medium containing macerated resistant embryogenic cultures did not inhibit mycelium growth, confirming that extracellular antifungal compounds were involved in the defense response. Enhanced secretion of chitinase and glucanase was observed in the plant growth medium in which resistant embryogenic cultures and regenerated somatic embryos were grown in comparison with the controls. Received: 6 February 1997 / Accepted: 4 November 1997     

Use of random amplified polymorphic DNA analysis to detect genetic variation inPyrus species

Pyrus communis L. is the most important pear species for European production. Very few cultivars satisfy standards for fruit quality and clonal fidelity; thus, accurate verification of cultivar identity for checking propagation material and patent protection is important. We evaluated the randomly amplified polymorphic DNA (RAPD) technique for its ability to identify genetic differences among standard pear (Pyrus communis L.) cultivars, William, Passa Crassana, and Conference, and three gamma-ray induced variants. To identify genotype-specific markers, we used thirty 10-mer and two 11-mer sequences, annealing temperatures from 36–45°C, 2Taq polymerases (AmpliTaq and Stoffel fragment, both from former Perkin Elmer Cetus), and 2–4 replicate amplifications. Of the 32 primers (30 from Operon Technologies, Alameda, CA, USA), very few distinguished William from Passa Crassana, and only 1 could clearly differentiate all 3 cultivars. Two primers that did not reveal polymorphisms when used singly, generated polymorphic patterns that distinguished standard from gamma-ray-treated material when used in combination. We show that RAPD analyses can discriminate pear genotypes and suggest this technique as a reliable and inexpensive method for marker-facilitated screening of propagation material and for patent protection.     

Selection for resistance to filtrates of Fusarium spp. in embryogenic cell suspension culture of Medicago sativa L.

A highly embryogenic cell suspension of alfalfa derived from a genotype sensitive to Fusarium oxysporum was successfully used for selection in vitro for resistance to culture filtrates of F. oxysporum, F. solani and F. avenaceum. Fifty two stable resistant cell lines were obtained and 500 plants regenerated from them. Among the 167 regenerants tested under glass there were 12–20% more plants with increased resistance to pathogens than in the group of plants regenerated from a control cell line. It was also found that the cell suspension cultures derived from genotypes of alfalfa with increased resistance to Fusarium spp. better tolerated filtrates of the pathogen. The results of a comparison of virulence of individual isolates of several species of Fusarium with toxicity of their filtrates to plants in vivo and in cell cultures were not unequivocal.     

Use of culture filtrates of Ceratocystis ulmi as a bioassay to screen for disease tolerant Ulmus americana

Cullus cultures of elm (Ulmus americana L.) derived from Dutch elm disease susceptible, intermediate-resistant, and resistant genotypes were exposed to the culture filtrates of three patogenic isolates of Ceratocystis ulmi, the causal agent of Dutch elm disease. Callus fresh weights, cell viability, and reactions of stem cuttings were determined after exposure to various concentrations of the filtrates. Calli from the susceptible elm failed to increase in fresh weight and lost viability after exposure to media containing culture filtrate. Calli from the resistant and the intermediate-resistant elms exhibited growth rates and maintained viability similar to controls not exposed to culture filtrate. Stem cuttings of the susceptible elm wilted after exposure to the culture filtrate. The symptoms were similar to wilt seen with the disease. Cuttings from the resistant elm had no disease symptoms whereas, the intermediate elm had some leaf chlorosis. Callus screening may thus be useful for selection of Ulmus germplasm for Dutch elm disease resistance.     

Screening of cassava and yam cultivars for resistance to anthracnose using toxic metabolites of colletotrichum species

Collectotrichum gloeosporioides f. sp. manihotis and C. gloeosporioides, causal agents of cassava (Manihot spp.) and yam (Dioscorea spp.) anthracnose diseases, respectively, produce toxic metabolites in culture that fluoresce at 254 nm and 366 nm, producing bands with Rf of 0.65 and 7.0, respectively. Symptoms induced on yam and cassava by the extracted metabolites were similar to those induced by the pathogens. Twenty-four clones of tropical D. rotundata (TDr), D. alata (TDa), D. esculenta (TDe), and D. cayenensis (TDc) were screened by applying toxic metabolites of C. gloeosporioides to their leaves and stems. Only TDr131, TDe 179 and TDc750 were resistant. Other clones were susceptible to varying degrees. Nineteen of the 45 clones of M. esculenta were resistant to varying degrees of toxic metabolites of C. gloeosporioides f. sp. manihotis. Results from in vitro screening of’ cassava and yam clones using toxic metabolites compared favourably with field screening based on natural epidemics. Using toxic metabolites appears to be a more effective technique for screeningfor disease resistance than conventional inoculation with plant. pathogens. This revised version was published online in June 2006 with corrections to the Cover Date.