A LOCAL-LESION TECHNIQUE FOR MEASURING THE INFECTIVITY OF CONIDIA OF BOTRYTIS FABAE SARDIÑA

Discrete lesions developed when conidial suspensions of Botrytis fabae Sardiña were rubbed with the forefinger on leaves of broad bean plants ( Vicia faba L.) subsequently kept in a water-saturated atmosphere. The numbers of lesions which developed were directly proportional to the concentration of inoculum. At equal concentrations, conidia from young cultures produced more lesions than conidia from old cultures.
Variation in lesion numbers between plants within a pot always considerably exceeded the variation between opposite half-leaflets of a leaf, as did variation between pots and between leaves of old, but not of young plants.
As the standard error of the number of lesions developing per half-leaflet ( x ) increases as the mean increases, the values of x need to be transformed before being statistically analysed. A suitable transformation is
z = log₁₀ 1/2{x + c +√(x²+ 2cx)}, where c = 20.     

Needs and benefits of empirical power transformations for production and quality traits in forest tree breeding

Non-normality in the distribution of individual observations of production and quality traits in forest tree breeding may cause inaccurate selection and overestimation of predicted selection gain. The distribution of individual observations of traits such as height, diameter, branch diameter, branch angle and number of branches per whorl is not always normal. We investigated how the observations were distributed and to what degree it is possible to improve normality, homogeneity of error variance and additivity by using empirical power transformations. Computer simulations showed that a seriously skewed distribution impairs selection efficiency and exaggerates selection gain expectations. If the distribution is heavily skewed, transformation might be worthwhile. It does not seem possible to offer any general advice about which varities should be transformed, but in most cases there seems to be no need of any transformation.     

Wheat regenerated from scutellum callus as a source of material for transformation

Reports of wheat transformation efficiencies vary from less than 1% to more than 5% for individual experiments. Rarely are negative experiments reported though we estimate that between one in two and one in three of all experiments fail to produce transformed plants. Consequently if transformation efficiencies were calculated from the total number of scutellum bombarded rather than from only those experiments which produced transformed plants there would be a significant fall in reported efficiencies. The use of scutellum-derived material from plants regenerated from scutellum callus and grown in a controlled environment room significantly reduced the number of experiments failing to produce plants. Though there is a small but significant increase in transformation efficiencies for individual experiments, the recovery of plants, as a direct consequence of the reduction in the number of failed experiments, increases nearly 350%, from 4.8 plants/1000 scutella from seed gown plants bombarded to 17 plants/1000 embryos bombarded from tissue culture regenerated plants. There appears to be no additional gains to be made from plants which are cycled through tissue culture more than one time. This revised version was published online in June 2006 with corrections to the Cover Date.     

扶桑绵粉蚧雌成虫在大花马齿苋上的空间格局

本文对外来入侵害虫扶桑绵粉蚧Phenacoccus solenopsisTinsley雌成虫在大花马齿苋(Portulaca grandiflora Hook)上的空间分布特征进行了研究。结果表明,在水平上的空间分布表现为聚集分布,个体间相互吸引,分布的基本成分为个体群;雌成虫密度m和平均拥挤度m*间的回归方程为:m*=12.4595+1.2649m;雌成虫在大花马齿苋枝条上、中层虫口数量均显著多于下层。确定了不同虫口密度及不同允许误差下的最适抽样数。     

Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation

AIMS: To optimize the conditions for electroporating foreign plasmid DNA into Lactobacillus acidophilus ATCC 43121. METHODS AND RESULTS: The conditions of electroporation were optimized to improve the transformation efficiency. Plasmid pNZ123 containing multicloning site and chloramphenicol resistance was employed to construct a cloning vector. The optimum electroporation conditions for the maximum transformation efficiency were a pulse strength of 12.5 kV cm(-1), a pulse number of 10, a pulse interval of 500 ms, and pNZ123 plasmid DNA concentration of 25 ng microl(-1). Under the optimum conditions the transformation efficiency of L. acidophilus ATCC 43121 was 1.84 +/- 0.13 x 10(4) (+/- standard error of measurements) CFU per mug of plasmid DNA. Other strains of L. acidophilus showed transformation efficiencies ranging from 1.38 +/- 0.02 x 10(4) to 9.32 +/- 0.54 x 10(4) under these conditions. A green fluorescent protein (GFP) was successfully expressed and detected by fluorescence microscopy when the pKU::slpA-GFP, pNZ123 containing GFP gene, was transformed in L. acidophilus ATCC 43121 under the optimum conditions. CONCLUSIONS: The results suggest that electrical parameters, antibiotic concentration, and host specificity play important roles to determine transformation efficiency of lactobacilli. The optimum conditions for the transformation of L. acidophilus ATCC 43121 may be applied to improve transformation efficiency of other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized conditions for electrotransformation may provide a mean to improve the introduction of foreign DNA into L. acidophilus to be used as a vehicle for a heterologous protein expression.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

Die Plastidenzahl als Merkmal bei der Kartoffel

Summary The average number of plastids in ten pairs of guard cells is a very useful aid for screening haploids among tetraploid plants fromS. tuberosum x S. phureja.In individual seedlings the stomatal plastid number decreases from the cotyledons to the first leaves. From the lower to the upper part of stems there exists a decreasing gradient for plastid number and an increasing gradient for the number of stomata per leaf area unit, whereas the stomatal length does not show a consistent trend.In the stomata, plastid number and stomatal length are positively correlated.Mean numbers of plastids in guard cells increase by a factor below 2 (1.8–1.9) after each doubling of the chromosome number.The stomatal plastid numbers of 48-chromosome breeding lines and of their haploid progenies show a clear positive correlation. The mean plastid numbers in various haploid families from different mother plants display significant differences.The origin of the cytoplasm (plasmone + plastome) fromS. demissum, S. stoloniferum, S. tuberosum, andigena forms ofS. tuberosum orS. spegazzinii in which the genome of the haploid is incorporated definitely influences the number of plastids in the guard cells.The stomatal numbers of plastids in 72 trisomic haploids show no significant deviation from the normal distribution of plastid numbers in haploids.Plastid number and stomatal length show a positive correlation in a population of 48 haploid lines and also in the same lines after doubling and quadrupling the chromosome number, the coefficient of regression decreasing with increasing ploidy level. In the above mentioned material there exists a strong positive correlation between the haploid and the corresponding homodiploid plants concerning stomatal length and the number of plastids.A comparison of the correlation stomatal length/plastid number at the 24-, 48- and 96-chromosome levels in three different genotypes reveals that some individuals are more sensitive to a rise in ploidy level than others and that the stomatal plastid number is a more reliable indicator of ploidy level than the stomatal length.We found some scattered polysomatic doubled stomata in leaf epidermissystems, stolons and tuber primordia of potatoes from various ploidy levels. The stomata at the border of the lamina are regularly endomitotically doubled, regardless of the original ploidy level.The hypothesis of a specific basic number of plastids for a given species, multiples of which should give origin to tissue-specific numbers, is criticized.High plastid numbers were negatively correlated with vitality. potatoes the possible correlation between plastid number in haploids and the direction of their original phyllotacticAs both the plastid number and the direction of the phyllotactic leaf spiral show some correlation with vitality in potatoes the possible correlation between plastid number in haploids and the direction of their original phyllotactic spiral was tried. The two groups with low and normal stomatal plastid numbers displayed almost equal distribution between left- and right-directed spirals; in the category with high plastid number, however, this relation was significantly displaced in favor of right spirals. It is tentatively proposed that both vitality and the direction of the phyllotactic spiral are governed by a common hormonal principle (pissibly auxins) which also influences the number of plastids.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.National Research Council of Canada Publication No. 38003     

Pappus part number in annual species ofMicroseris (Compositae,Cichoriaceae)

All North American annual species of the genusMicroseris have a five-part pappus, the one South American annual,M. pygmaea, has ten pappus parts. The pappus develops over a constant number of ten provascular bundles with or without inhibition between alternate sites of pappus development. Each natural population contains a predictable proportion of achenes with aberrant pappus part numbers. Hybridization betweenM. bigelovii (5 parts) andM. pygmaea results in F 1 and F 2 plants with many aberrant achenes. In each plant either five or ten can be shown to be the basic number with aberrant numbers following a Poisson distribution for numbers added to 5 or deleted from 10. Occasional plants show no basic number but have a random distribution of numbers about an intermediate mean. The evolutionary genetics of this character is discussed.     

Rapid detection of aneuploidy in <Emphasis Type="Italic">Musa</Emphasis> using flow cytometry

We report a procedure for the rapid and convenient detection of aneuploidy in triploid Musa using DNA flow cytometry. From a population of plants derived from gamma-irradiated shoot tips, plants were selected based on aberrant morphology and their chromosome numbers were counted. Aneuploids plants with chromosome numbers 2n=31 or 32 were found as well as the expected triploid plants (2n=3x=33). At the same time, the nuclear DNA content of all plants was measured using flow cytometry. The flow cytometric assay involved the use of nuclei isolated from chicken red blood cells (CRBC), which served as an internal reference standard. The relative DNA content of individual plants was expressed as a ratio of DNA content of CRBC and Musa (DNA index). In order to estimate the chromosome number using flow cytometry, the relative DNA content of plants with unknown ploidy was expressed as a percentage of the DNA content of triploid plants. The classification based on flow cytometry fully agreed with the results obtained by chromosome counting. The results indicated that flow cytometry is a convenient and rapid method for the detection of aneuploidy in Musa.     

Estimation of Allele Frequencies at Isoloci

In some polyploid animals and plants, pairs of duplicated loci occur that share alleles encoding proteins with identical electrophoretic mobilities. Except in cases where these ``isoloci' are known to be inherited tetrasomically, individual genotypes cannot be determined unambiguously, and there is no direct way to assign observed variation to a particular locus of the pair. For a pair of diallelic isoloci, nine genotypes are possible but only five phenotypes can be identified, corresponding to individuals with 0-4 doses of the variant allele. A maximum likelihood (ML) approach is used here to identify the set of allele frequencies (p, q) at the individual gene loci with the highest probability of producing the observed phenotypic distribution. A likelihood ratio test is used to generate the asymmetrical confidence intervals around ML estimates. Simulations indicate that the standard error of p is typically about twice the binomial sampling error associated with single locus allele frequency estimates. ML estimates can be used in standard indices of genetic diversity and differentiation and in goodness-of-fit tests of genetic hypotheses. The noncentral χ(2) distribution is used to evaluate the power of a test of apparent heterozygote deficiency that results from attributing all variation to one locus when both loci are polymorphic.     

A simple transformation for sets of range sizes

Transformation of data to normality may be illuminating and useful statistically. There are two standard families of transformations, power transformations for positive numbers. bounded at the left, and folded transformations for proportions, bounded both at the left and the right. It has been shown that there is no one satisfactory power transformation for range size data. However, such measures are limited to the right as well as the left, and we consider applying folded transformations to them. Seven data sets of range sizes recorded by 10 km squares are studied. Six are British (native and introduced plants, mammals, dragonflies and two breeding bird surveys) the seventh is of Swiss breeding birds. Using these we show that the right hand limit of the distribution can be estimated and the best folded transformation found. In all cases the right hand limit is larger than the range size of the most widespread species and smaller than the notional scope of the survey. In all cases the logit or Hog, the logarithmic folded transformation, is satisfactory: in five cases it is the best. It is well known that abundance is approximately (though not exactly) log-normally distributed. The relationship of that to our discovery that range size data are approximately logit-normal is discussed. There is no fully satisfactory explanation for either observation at present.     

Estimating data transformations in nonlinear mixed effects models

A routine practice in the analysis of repeated measurement data is to represent individual responses by a mixed effects model on some transformed scale. For example, for pharmacokinetic, growth, and other data, both the response and the regression model are typically transformed to achieve approximate within-individual normality and constant variance on the new scale; however, the choice of transformation is often made subjectively or by default, with adoption of a standard choice such as the log. We propose a mixed effects framework based on the transform-both-sides model, where the transformation is represented by a monotone parametric function and is estimated from the data. For this model, we describe a practical fitting strategy based on approximation of the marginal likelihood. Inference is complicated by the fact that estimation of the transformation requires modification of the usual standard errors for estimators of fixed effects; however, we show that, under conditions relevant to common applications, this complication is asymptotically negligible, allowing straightforward implementation via standard software.     

Promoter subfragments of the sugar beet V-type H+-ATPase subunit c isoform drive the expression of transgenes in the moss Physcomitrella patens

Based on the relative ease of performing targeted nuclear gene knockout, the moss Physcomitrella patens has recently been developed as a model system for plant functional genomics. To address the need for new promoters that could drive expression of transgenes in this moss, we tested two fragments of the promoter region of the gene for the sugar beet ( Beta vulgaris) V-type H ⁺-ATPase subunit isoform c. Four gene knockout constructs were tested in which the neomycin phosphotransferase II selection marker gene was put under the control of two distinct V-type H ⁺-ATPase promoter fragments, the NOS promoter, or the CaMV 35S promoter. In each case the selection cassettes were flanked by moss FtsZ1 cDNA sequences to facilitate chromosomal targeting. From a total of more than 440 transformed plants, the number of plants generated per construct was monitored and found to be in the range of 5 to 11 stable transgenics per transformation. Both V-type H ⁺-ATPase promoter fragments lead to NPTII expression levels that were sufficiently high to generate large numbers of stable transgenic plants. The numbers of plants obtained with the two V-type H ⁺-ATPase promoter fragments were comparable to those with constructs containing the standard NOS and 35S promoters. We propose that the higher plant V-type H ⁺-ATPase promoter can be used for the expression of transgenes in the bryophyte P. patens.     

Parentage and sibship inference from markers in polyploids

Many plants and some animal species are polyploids. Nondisomically inherited markers (e.g. microsatellites) in such species cannot be analysed directly by standard population genetics methods developed for diploid species. One solution is to transform the polyploid codominant genotypes to pseudodiploid‐dominant genotypes, which can then be analysed by standard methods for various purposes such as spatial genetic structure, individual relatedness and relationship. Although this data transformation approach has been used repeatedly in the literature, no systematic study has been conducted to investigate how efficient it is, how much marker information is lost and thus how much analysis accuracy is reduced. More specifically, it is unknown whether or not the transformed data can be used to infer parentage and sibship jointly, and how different sampling schemes (number and polymorphism of markers, number of individuals) and ploidy level affect the inference accuracy. This study analyses both simulated and empirical data to examine the effects of polyploid levels, actual pedigree structures and marker number and polymorphism on the accuracy of joint parentage and sibship assignments in polyploid species. We show that sibship, parentage and selfing rates in polyploids can be inferred accurately from a typical set of microsatellite loci. We also show that inferences can be substantially improved by allowing for a small genotyping error rate to accommodate the distortion in assumed Mendelian inheritance of the converted markers when large sibship groups are involved. The results are discussed in the context of polyploid data analysis in molecular ecology.     

马尾松林向香樟林改造林下植物功能多样性研究

在生物多样性研究中,功能多样性比物种多样性可以更直接地反映生态系统的结构和功能,从而在群落生态学研究中受到越来越多的推崇。马尾松次生纯林是一种亟待改造的森林类型,而在亚热带地区香樟是马尾松林改造的理想树种之一。为了完善马尾松林向香樟林改造的研究,以更好地指导马尾松林改造的实践,本文用空间代替时间的方法,选取马尾松林向香樟林改造过程中4种不同阶段群落为研究对象,对林下灌木层和草本层植物的功能性状及其多样性展开了研究。结果表明:(1)随着森林改造的进行,林下植物的物种数目、功能性状多样性均表现为先上升后下降的变化趋势,其中功能丰富度和功能均匀度在改造前期达到峰值,而物种数目和功能离散度则在改造中期表现最高。(2)比较森林改造的4个时期,林下植物功能多样性的综合表现大致为:改造前期改造中期改造后期未改造时期。(3)森林改造过程中,林下植物"光响应性状"和"繁殖性状"的功能多样性变化趋势相似,但前者比后者对改造的响应更敏感。(4)在森林改造过程中,林下植物的生长、发育和扩散受到林下光照资源的影响,并在长时间的生物竞争中逐渐表现为物种种类、多度和空间分布格局的变化,最终影响生态系统功能。(5)马尾松林向香樟林的改造加速了群落的演替进程,提升了群落的功能,但仍存在林下植物功能多样性下降等问题。在对马尾松次生纯林改造过程中,应选择合适的造林密度和混交林的改造模式。     

SOME EFFECTS OF HOST NUTRITION ON THE SUSCEPTIBILITY OF PLANTS TO INFECTION BY CERTAIN VIRUSES

Fertilizer treatments that greatly influenced the growth of tobacco and potato plants in pots had little effect on the number that became infected with potato virus Y when the plants were colonized by equal numbers of infective aphids, though the number was slightly decreased by nitrogen and increased by phosphorus.
The number of local lesions produced on leaves of tobacco and Nicotiana glutinosa by tomato aucuba mosaic and tobacco mosaic viruses was increased by additions of both nitrogen and phosphorus, provided that these also increased growth. The predominant effect of both nutrients in increasing susceptibility was indirect by increasing plant size, but over certain critical ranges both elements also increased the numbers of lesions produced per unit leaf area. Conditions of maximum susceptibility approximated closely to those producing optimal growth, and susceptibility, whether measured by lesions per half-leaf or per unit area, was decreased by a deficiency or excess of either element. Sometimes the addition of nitrogen reduced susceptibility when still increasing plant growth.     

Investigations of copy number of transgene,fertility and expression level of an introduced GUS gene in transgenic NERICA produced by <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated methods

In the use of genetic transformation in breeding, there are several possible problems including multiple copy insertion of transgene, sterility caused by somaclonal variation and gene silencing. In this study, we characterized transgenic New Rice for Africa (NERICA) produced by Agrobacterium-mediated methods with respect to copy number of transgene, fertility, and expression level of an introduced GUS gene. Southern blot analysis of primary transformants demonstrated that about half of the events carried a single copy of the transgene regardless of the cell density of Agrobacerium for inoculation. We examined ten procedures, consisting of different time periods and times of subculture for callus formation and the starting times of hygromycin-based selection of transformed cells, for transformation of NERICA cultivars to produce transformants within a short culture period at high frequency. A new culture method developed in this study required only about 1.5 mo from the beginning of tissue culture to transformants, whereas a standard protocol we developed previously needed about 2 mo of culture; however, it did not significantly reduce percentages of sterile plants. Fertile T₀ plants produced fertile T₁ plants at higher frequency. However, fertility was not inherited in a simple fashion: both fertile and partially sterile T₀ plants produced fertile, partially sterile and sterile T₁ plants. Expression assay of an introduced GUS gene revealed position effects in seven independent homozygous transformed lines carrying one copy of the transgene. Points to pay attention to in the use of genetic transformation in breeding are discussed.     

根癌农杆菌介导的高效大豆遗传转化体系的建立

利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。