Comparative Phloem mobility of nickel in nonsenescent plants

⁶³Ni was applied to nonsenescent source leaves and found to be transported to sink tissues in pea (Pisum sativum L.) and geranium plants (Pelargonium zonale L.). The comparative mobilities (percent tracer transported out of source leaf ÷% ⁸⁶Rb transported) for ⁶³Ni in peas was 2.12 and in geranium 0.25. The value for the phloem mobile ⁸⁶Rb was 1.00. By contrast, the comparative mobility of ⁴⁵Ca, which is relatively immobile in the phloem, was low (0.05 in peas, 0.00 in geranium). Interruption of the phloem pathway between source and sink leaves by steam girdling almost completely inhibited ⁶³Ni accumulation in the sink leaves of both species. We conclude that Ni is transported from nonsenescent source leaves to sink tissues via the phloem of leguminous and nonleguminous plants.     

Over‐expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose‐scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis

Rose‐scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2‐C‐methyl‐d ‐erythritol‐4‐phosphate (MEP) pathway, 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose‐scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over‐expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose‐scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose‐scented geranium as well as functional characterization study of any gene from rose‐scented geranium through a genetic transformation system.     

Development of a mite bioassay to evaluate plant resistance and its use in determining regeneration of spider mite resistance

The zonal geranium (Pelargonium xhortorum) possesses tall glandular trichomes that secrete anacardic acids, a viscous, sticky exudate which has been suggested as the primary mechanism in two-spotted spider mite (Tetranychus urticae Koch) resistance. A new bioassay was developed using small Plexiglas^® cylinders as chambers for evaluating the resistance of geranium leaves to the two-spotted spider mite. This bioassay was easy to prepare, required only 24 hours to conduct, exhibited no problems with desiccation, condensation, or mite accountability, and yielded reproducible results. This bioassay was then used to study the regeneration of resistance of attached geranium leaves after they were made mite-susceptible by removing the excreted anacardic acids with water. Washed leaves regained full resistance after 14 days.     

Detection of Puccinia pelargonii‐zonalis‐infected Geranium Tissues and Urediniospores

The occurrence of geranium rust (caused by Puccinia pelargonii‐zonalis) in commercial greenhouses can result in unmarketable plants and significant economic losses. Currently, detection of geranium rust relies solely on scouting for symptoms and signs of the disease. The purpose of this research was to develop a rapid detection assay for P. pelargonii‐zonalis‐infected tissues or urediniospores on greenhouse‐grown geraniums. Two oligonucleotide primers were designed based on internal transcribed spacer sequence data from three isolates of P. pelargonii‐zonalis. The primers amplified a 131‐bp product from genomic DNA from each isolate of P. pelargonii‐zonalis but did not amplify a product from genomic DNA from twelve other rust fungi or four other plant pathogenic fungi. A PCR product was amplified consistently from solutions that contained 1 ng or 100 pg/ml of purified P. pelargonii‐zonalis DNA in conventional PCR and at 1 pg/ml using real‐time PCR. The detection threshold was 10² urediniospores/ml for real‐time PCR and 10⁴ urediniospores/ml for conventional PCR using urediniospores collected by vacuum from sporulating lesions. Puccinia pelargonii‐zonalis DNA was amplified by real‐time PCR from urediniospores washed from a single inoculated leaf, but recovered urediniospores were below detection threshold from one inoculated leaf with 5, 10, 25 and 50 non‐inoculated leaves. Conventional and real‐time PCR did not detect P. pelargonii‐zonalis in infected leaf tissues, presumably due to PCR inhibitors in the geranium leaf tissue. The inhibition of both conventional and real‐time PCR by geranium tissues suggests that a detection assay focusing on urediniospore recovery and microscopic examination with subsequent species verification by PCR may be the most efficient method for assessing the presence of geranium rust in greenhouses.     

Characterization of Geranium (Pelargonium graveolens) Chloroplast EF-Tu cDNA

A geranium (Pelargonium graveolens) chloroplast translational elongation factor EF-Tu (tufA) cDNA was isolated. The geranium tufA cDNA is 1,584 bp long with 20 bp of 5 untranslated region (UTR) and 139 bp of 3 UTR. It encodes 474 amino acids including a putative chloroplast transit peptide of 65 amino acids. The deduced polypeptides of the geranium tufA cDNA contains four GTP binding sequences in its N-terminal region and two chloroplast EF-Tu signature regions in the C-terminal region. The predicted molecular weight of the mature geranium chloroplast EF-Tu protein was about 45,000 and its amino acid sequence identity with the chloroplast EF-Tu proteins of tobacco, pea, Arabidopsis, rice, and soybean ranges from 85% to 91%. The geranium tufA appears to exist as a single copy gene like Arabidopsis and rice, whereas other known dicot plants have more than one copy in their nuclear genomes.     

A reliable and efficient method for total rna isolation from various members of spurge family (Euphorbiaceae)

Introduction – It is prerequisite and crucial to extract RNA with high quality and integrity in order to carry out molecular biology studies in any plant species of a family. Euphorbiaceae members are known for high levels of their waxes, oils with polysaccharides, polyphenolics and secondary metabolites. These conditions are recognised to interfere unfavourably with various methodologies of RNA isolation. Objective – To develop a simple, rapid and reproducible cetyltrimethylamonium bromide (CTAB)‐based protocol, to reduce the time and cost of extraction without reducing quality and yield of RNA extracted from various recalcitrant Euphorbiaceae member plant tissues such as from tree leaves (Hevea brasilensis), woody shrubs leaves (Ricinus communis, Jatropha curcas, Manihot esculenta) and storage root tissue (M. esculenta). Methodology – Simple modifications and fast steps were introduced to the original CTAB protocol. All centrifugation steps were carried out at 4°C at 12000 rpm for 10 min, the sample weight was decreased and usage of spermidine and LiCl was omitted, reducing incubation time prior to RNA precipitation. This rapid CTAB protocol was compared with various RNA isolation methods intended for use with plants rich in polysaccharides and secondary metabolites. Results – The procedure can be completed within 2 h and many samples can be processed at the same time. RNA of high quality could be isolated from all the tissues of species that we tried. The isolated RNA from different species served as a robust template for RT‐PCR analysis. Conclusion – The study has shown that the improvement of a CTAB‐based protocol allows the rapid isolation of high‐quality RNA from various recalcitrant Euphorbiaceae members. Copyright © 2010 John Wiley & Sons, Ltd.     

Molecular cloning of a cytosolic ascorbate peroxidase cDNA from cell cultures of sweetpotato and its expression in response to stress

A cDNA encoding a cytosolic ascorbate peroxidase (APX), swAPX1 , was isolated from cell cultures of sweetpotato (Ipomoea batatas) by cDNA library screening, and its expression in the context of various environmental stresses was investigated. swAPX1 contains an ORF of 250 amino acids (27.5 kDa) encoding a protein with a pI value of 5.32. The swAPX1 ORF does not code for a transit peptide, suggesting that the product is a cytosolic isoform. RNA blot analysis showed that swAPX1 gene is expressed in cultured cells and mature leaves, but not in stems, non-storage or storage roots of sweetpotato. The level of swAPX1 RNA progressively increased during cell growth in suspension cultures. In leaf tissues, the gene responded differentially to various abiotic stresses, as revealed by RT-PCR analysis. swAPX1 was highly induced in leaves by wounding, and treatment with methyl viologen (50 M), hydrogen peroxide (440 mM), abscisic acid (ABA; 100 M) or exposure to high temperature (37°C). In addition, the gene was strongly induced in the leaves following inoculation with a bacterial pathogen (Pectobacterium chrysanthemi). These results indicate that swAPX1 may be involved in hydrogen peroxide-detoxification and thus help to overcome the oxidative stress induced by abiotic and biotic stresses.Communicated by G. Jürgens     

Drought response of two bedding plants

Bedding plants are an important part of the urban public space and private gardens. However, they are not always properly watered and suffer from drought stress, especially when grown in containers. In this trial a response to water stress of two commonly used species, impatiens (Impatiens walleriana Hook) and geranium (Pelargonium hortorum L. H. Bailey) were compared. The former is highly herbaceous and prone to wilting whereas the latter has hairy leaves and is better adapted to drought. Plants were grown at three levels of soil water content (SWC): 80% (control), 60% (mild stress) and 30% (severe stress). Drought was maintained during three 10 day cycles, separated by 10 day periods of normal watering. In both species roots were significantly longer in plants grown at 30% SWC as compared to 80% SWC while plant height and flower number were reduced by drought only in impatiens. The initial relative water content (RWC) was lower in geranium and decreased less in response to drought than in impatiens. Ammonium content in leaves of both species increased significantly under stress but the ranges of increase were different in both species. There was a significant increase in the free amino acids content in leaves of impatiens as compared to geranium but this rise was more time than drought dependent. The reduction in the a + b chlorophyll concentration in leaves of impatiens was significantly time and stress dependent while no reaction in geranium was observed. The above results show that changes in leaf RWC merit further attention as a possible indicator of plant response to drought stress in ornamental plants but additional studies are needed before this or other parameters can be used to evaluate new bedding plants for introduction into urban growing conditions, or as selection criteria in breeding for adaptation to demanding growing conditions.     

Induction of Trichocyst Discharge in Paramecium bursaria*

SYNOPSIS. A method is described for obtaining trichocyst discharge in Paramecium using an extract of geranium leaves. The extract is easily prepared, gives readily reproducible results, and is only slightly toxic to the cells. Using this method, large quantities of trichocysts can be obtained for a variety of analyses. Preliminary evidence suggests that the component of the extract active in causing trichocyst discharge is geranium tannic acid.     

Direct shoot regeneration from leaf segments of mature plants of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> (L.) moench

Summary This is the first communication of direct shoot regeneration from fully developed leaves of potted mature Echinacea purpurea plants. Shoot buds were induced directly on the adaxial surface of mature leaf tissues of E. purpurea 30 d after culture initiation on Woody Plant Medium (WPM) supplemented with various levels of 6-benzyladenine (BA). Maximum shoot organogenesis, with 12–20 shoots per leaf segment, was obtained with 5% coconut milk and 2.5 mg l⁻¹ (6μM) BA in 30 d. Callus was induced using 0.5 mgl⁻¹ (1μM) α-naphthaleneacetic acid and 2.5 mgl⁻¹ (6μM) BA. The regenerated shoots were rooted on WPM supplemented with 1.5 mgl⁻¹ (3μM) of indole-3-butyric acid, 3% sucrose, and 0.85% agarose. Rooted plants were successfully transferred to soil in pots and appeared morphologically normal and flowered in a growth chamber.     

IS POLYPLOIDY NECESSARY FOR TISSUE DIFFERENTIATION IN HIGHER PLANTS?

Measurements of relative DNA per nucleus of cells from various tissues show that cell differentiation can occur in the absence of polyploidy in higher plants. In Pisum polyploidy was present in roots, sepals, pods, pistils, and stamens but not in petals or leaves. In Triticum cells of leaves exhibited some polyploidy, but no polyploid cells were present in mature roots. No polyploid cells were found in any tissue of Helianthus examined (roots, cotyledons, stems, leaves, sepals, petals, pistils, and stamens). Therefore, as a general rule, polyploidy should not be considered essential in tissue or organ differentiation of higher plants. In Helianthus polyploidy is unnecessary for the completion of the life cycle.     

Performance of rose scented geranium (Pelargonium graveolens) in heavy metal polluted soil vis-à-vis phytoaccumulation of metals

An investigation was carried out to evaluate the effect of heavy metal toxicity on growth, herb, oil yield and quality and metal accumulation in rose scented geranium (Pelargonium graveolens) grown in heavy metal enriched soils. Four heavy metals (Cd, Ni, Cr, and Pb) each at two levels (10 and 20 mg kg–1 soil) were tested on geranium. Results indicated that Cr concentration in soil at 20 mg kg–1 reduced leaves, stem and root yield by 70, 83, and 45%, respectively, over control. Root growth was significantly affected in Cr stressed soil. Nickel, Cr, and Cd concentration and accumulation in plant increased with higher application of these metals. Chromium, nickel and cadmium uptake was observed to be higher in leaves than in stem and roots. Essential oil constituents were generally not significantly affected by heavy metals except Pb at 10 and 20 ppm, which significantly increased the content of citronellol and Ni at 20 ppm increased the content of geraniol. Looking in to the higher accumulation of toxic metals by geranium and the minimal impact of heavy metals on quality of essential oil, geranium can be commercially cultivated in heavy metal polluted soil for production of high value essential oil.     

Molecular cloning and nucleotide sequence of cDNA for sporamin,the major soluble protein of sweet potato tuberous roots

Summary Sporamin accounts for more than 80% of the total soluble proteins of tuberous roots of sweet potato, but very little, if any, in other tissues of the same plant. In vitro translation of RNA fractions from the tuberous roots in wheat germ extract and subsequent immunoprecipitation with the antibody to sporamin indicated that this protein is synthesized by membrane-bound polysomes as a precursor 4 000 daltons larger than the mature protein. A cDNA expression library was constructed from the total poly(A)⁺ RNA from the tuberous roots by a vector-primer method, and an essentially full-length cDNA clone for the sporamin mRNA was selected by direct immunological screening of the colonies. Northern blot analysis showed that sporamin mRNA is approximately 950 nucleotides in length and is specifically present in tuberous roots and very little, if any, in leaves, petioles and non-tuberous roots. Nucleotide sequence of the cDNA predicts a 37 amino acid extension in the precursor at the amino-terminus of the mature protein.     

A simple model for nondestructive leaf area estimation in bedding plants

Measurement of leaf area is commonly used in many horticultural research experiments, but it is generally destructive, requiring leaves to be removed for measurement. Determining the individual leaf area (LA) of bedding plants like pot marigold (Calendula officinalis L.), dahlia (Dahlia pinnata), sweet William (Dianthus barbatus L.), geranium (Pelargonium × hortorum), petunia (Petunia × hybrida), and pansy (Viola wittrockiana) involves measurements of leaf parameters such as length (L) and width (W) or some combinations of these parameters. Two experiments were carried out during spring 2010 (on two pot marigold, four dahlia, three sweet William, four geranium, three petunia, and three pansy cultivars) and summer 2010 (on one cultivar per species) under greenhouse conditions to test whether a model could be developed to estimate LA of bedding plants across cultivars. Regression analysis of LA versus L and W revealed several models that could be used for estimating the area of individual bedding plants leaves. A linear model having LW as the independent variable provided the most accurate estimate (highest R ², smallest mean square error, and the smallest predicted residual error sum of squares) of LA in all bedding plants. Validation of the model having LW of leaves measured in the summer 2010 experiment coming from other cultivars of bedding plants showed that the correlation between calculated and measured bedding plants leaf areas was very high. Therefore, these allometric models could be considered simple and useful tools in many experimental comparisons without the use of any expensive instruments.     

Cadmium and zinc induced chlorosis in Indian mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern] involves preferential loss of chlorophyll <Emphasis Type="Italic">b</Emphasis>

Plants of Indian mustard (Brassica juncea) were treated with either 50 μM Cd, 250 μM Zn, or 25 μM Cd+125 μM Zn and the progression of chlorosis in the mature leaves monitored. As relative chlorophyll (Chl) contents in the mature leaves decreased to 75, 50, and 25 % relative to controls, both mature and young leaves were harvested and the Chl pools extracted. The metal treatments caused a greater loss of Chl b than Chl a. As mature leaves underwent progressive chlorosis, the young leaves displayed a characteristic over-greening, due largely to increased content of Chl b. However, as the young leaves began to experience chlorosis, a greater loss of Chl b was also observed. Thus during metal induced chlorosis, there is a preferential turnover of the Chl b pool in mature and young leaves.