Replicase mediated resistance against potato leafroll virus in potato Desirée plants

Potato leafroll virus (PLRV) is a major menace for the potato production all over the world. PLRV is transmitted by aphids, and until now, the only strategy available to control this pest has been to use large amounts of insecticides. Transgenic approaches involving the expression of viral replicases are being developed to provide protection for plants against viral diseases. The purpose of this study was to compare the protection afforded by the differential expression of PLRV replicate transgene in potato plants cv. Desirée, Plants were genetically modified to express the complete sense PLRV replicase gene. Two constructions were used, one containing the constitutive 35SCaMV promoter and the other the phloem-specific RolA promoter from Agrobacterium rhizogenes. Transgenic plants were infected with PLRV in vitro, using infested aphids. In plants in which 35SCaMV controlled the expression of the PLRV replicase gene, signs of infection were initially detected, although most plants later developed a recovery phenotype showing undetectable virus levels 40 days after infection. In turn, those plants with the RolA promoter displayed an initial resistance that was later overcome. Different molecular mechanisms are likely to participate in the response to PLRV infection of these two types of transgenic plants.     

Direct somatic embryogenesis of potato [<Emphasis Type="Italic">Solanum tuberosum</Emphasis> (L.)] cultivar ‘Kufri Chipsona 2’

Direct somatic embryo induction was achieved from leaf and internodal explants of Solanum tuberosum (L.) cultivar ‘Kufri Chipsona 2’ on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium containing 10.0 µM silver nitrate (MS1 medium) supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 2.5 µM) and 6-benzyladenine (BA; 1.0 µΜ). It was observed that in absence of AgNO₃, friable callus was induced from cut ends of the explants, which does not develop into any kind of organised structure; thus highlighting the requirement of AgNO₃ for somatic embryogenesis in potato. Furthermore, the effect of medium strength, sucrose concentration and heat shock treatment on somatic embryogenic potential of explants was also investigated. When the strength of basal medium was reduced to half, the frequency of internodal segments differentiating somatic embryos was almost double in comparison to full strength MS medium. Sucrose concentration and heat shock treatment were found to have interactive effect on somatic embryo induction. Explants subcultured on medium containing 174 mM sucrose and subjected to heat shock (1 h; 50 °C) showed maximum somatic embryo differentiation. Although, the percent explants differentiating somatic embryos decreased sharply with increase in sucrose concentration (>?174 mM), yet the number of somatic embryos differentiated per explant were found to increase with further increase in sucrose concentration. Histological observations revealed that somatic embryos directly developed from epidermis of leaf explant and cut ends of internodal segments progressed from globular to cotyledonary stage after passing through intermediate embryogenic stages (heart shaped and torpedo shaped). Conversion of somatic embryos into plantlets (92%) was achieved on MS1 medium supplemented with BA (10.0 µM) and gibberellic acid (15.0 µM) and all regenerated plants were found to be phenotypically alike.     

Transient expression of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> ascorbate peroxidase 3 in<Emphasis Type="Italic"> Nicotiana benthamiana</Emphasis> plants infected with recombinant potato virus X

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.     

Stress-induced expression of cucumber chitinase and <Emphasis Type="Italic">Nicotiana plumbaginifolia</Emphasis><Emphasis Type="Italic">β</Emphasis>-1,3-glucanase genes in transgenic potato plants

The genes encoding for a cucumber class III chitinase and Nicotiana plumbaginifolia class I glucanase were co-introduced into Slovak potato (Solanum tuberosum L.) cultivar ETA using Agrobacterium tumefaciens. Expression of both genes was driven by wound-inducible polyubiquitin promoter isolated from Slovak potato breeding line 116/86. Analyses showed inducible, peel-specific expression of both transgenes under stress conditions. The effect of transgene expression on fungal susceptibility of transformants was evaluated in vitro and in vivo. Experiments with crude protein extracts isolated from transgenic microtubers showed growth inhibition of Rhizoctonia solani hyphae in the range from 7.3 to 14.2%. In contrast, experiments performed in growth chamber conditions revealed that the polyubiquitin promoter driven transgene expression did not ensure any obvious increase of transgenic potato resistance against Rhizoctonia solani.     

Shall I compare thee to a GM potato?

A fundamental issue in the safety assessment of genetically modified crops is the question of whether unintentional changes have occurred in the crop plant as a consequence of the genetic modification. This question was addressed recently by using a powerful metabolite fingerprinting and metabolite profiling method to assess whether genetically modified potatoes are substantially similar to their corresponding conventional cultivars.     

Osmotic regulation of starch synthesis in potato tubers?

Using potato (Solanum tuberosum L.) tuber discs incubated in a range of mannitol concentrations it has been demonstrated that both sucrose uptake and the conversion of sucrose to starch are sensitive to the osmotic environment of the storage cells. Starch synthesis was optimised at 300 mM but declined sharply at both lower and higher osmotic concentrations. The decline in starch synthesis on either side of optimum was not proportional to the change in mannitol concentration, indicating different inhibitory mechanisms under low and high osmotica. The fraction of the total sucrose converted to starch i.e. the partitioning between sucrose and starch, was also influenced by osmotic environment. The amount of soluble material taken up by the storage cells, but not converted to starch, was maintained under mannitol concentrations (300–400 mM) which inhibited starch synthesis, indicating that sucrose uptake continued during declining starch synthesis. At mannitol concentrations above 400 mM, sucrose uptake was greatly enhanced but no significant change in starch synthesis occurred.     

Characterization of tryptophan-overproducing potato transgenic for a mutant rice anthranilate synthase α-subunit gene (<Emphasis Type="Italic">OASA1D</Emphasis>)

Potato plants (Solanum tuberosum cv. May Queen) transgenic for OASA1D, which encodes a point mutant of an -subunit of rice (Oryza sativa) anthranilate synthase (AS, EC 4.1.3.27), were generated in order to determine the effects of the mutant gene on levels of free tryptophan (Trp) and AS activity in this important crop. Expression of OASA1D in potato induced a 2- to 20-fold increase in the amount of free Trp. This increase was likely due to a reduction in the sensitivity of AS containing the mutant -subunit to feedback inhibition by Trp. Nontargeted metabolite profiling by high-performance liquid chromatography coupled with ultraviolet photodiode array detection as well as targeted profiling by liquid chromatography coupled with mass spectrometry revealed no marked changes in the levels of other metabolites, with the exception of indole-3-acetic acid (IAA), in the transgenic plants. The level of IAA in the upper part of the shoot was increased by a factor of 8.3–39, depending on the transgenic lines, with no detectable effect on plant growth or development. The effects of transformation thus appeared limited to the biosynthesis of Trp and IAA, with the overall metabolic network in potato being virtually unaffected. These results suggest that transformation with OASA1D may prove effective for the breeding of crops with an increased level of free Trp.     

Somatic hybrids between potato <Emphasis Type="Italic">Solanum tuberosum</Emphasis> and wild species <Emphasis Type="Italic">Solanum verneï</Emphasis> exhibit a recombination in the plastome

In the present survey, interspecific somatic protoplast fusion between a dihaploid cultivated potato Solanum L. tuberosum (BF15) and a wild species Solanum verneï (V3) has been performed using the PEG method. Five putative hybrids were first selected. Among them, only two were retained. DNA analysis, using flow cytometry, showed that the first hybrid (VB2) was dihaploid (2n=2x=24 chromosomes), whereas the second (VB1) was hexaploid (2n=6x=72 chromosomes). In the greenhouse, these putative hybrids showed that they were able of raising and producing large tubers but with a modified shape compared to parental ones. Furthermore, they harbored an intermediate leaf morphology compared to their parents. The hybrid nature of these plants was first confirmed according to their esterase and peroxidase isoenzyme patterns. The RAPD analysis of genomic DNA and microsatellite based amplification (I-SSR) showed that both clones VB1 and VB2 are asymmetric somatic hybrids. They seem to have eliminated the major part of V3 parental nucleus but not the totality. The analysis of chloroplast DNA suggests that both hybrid plastomes were the result of a recombination between parental ones.     

Impact of Bacillus thuringiensis – insecticides on population dynamics and egg predation of the Colorado potato beetle in North Carolina potato plantings

Field studies to assess the impact of Bacillus thuringiensis var. tenebrionis (Btt)-insecticides on Colorado potato beetle populations, egg survivorship and levels of predation on egg masses were conducted in replicated field research plots during two years. Stage-specific abundance of the Colorado potato beetle and predation on egg masses were monitored in Btt-treated and untreated potato plots in both years. The Btt-treatments significantly reduced densities of large (third and fourth instar) Colorado potato beetle larvae. The densities of large larvae remained below 0.5 and 3 per plant in the Btt-treatment while peak densities of 4.5 and 21 large larvae per plant occurred in the untreated control in 1992 and 1993, respectively. Regular sampling of egg masses indicated that predation rates in Btt-treated and untreated plots did not differ significantly although, in 1993, predation rates of up to 100% were recorded, only in Btt-treated plots. In a predator exclusion study carried out in 1992, survivorship of protected eggs was consistently higher than of eggs exposed to predation. Seasonal survivorship of exposed eggs was significantly lower in the Btt-treated than in untreated plots. Btt insecticides for control of Colorado potato beetles provided direct protection of the crop and were compatible with naturally-occurring biological control of Colorado potato beetle eggs due to predation.     

The sweet potato—Its origin and primitive storage practices

In pre-Columbian times sweet potatoes provided food for the widely separated Mayans of Central America, the Incas of Peru and the Maoris of New Zealand, while two other species of the genus were occasionally used by Indians of western North America.     

Double hydroperoxidation of α-linolenic acid by potato tuber lipoxygenase

The potato tuber lipoxygenase preparations convert α-linolenic acid not only to 9(S)-HPOTE, but also to some more polar metabolites. Two of these polar products, I and II, with ultraviolet absorbance maxima at 267 nm were purified by HPLC. It was found that metabolites I and II have, respectively, one and two hydroperoxy groups. Products of NaBH₄ reduction of both I and II were identified by their chemical ionization and electron impact mass spectra and by ¹H-NMR spectra as 9,16-dihydroxy-10(E), 12(Z), 14(E)-octadecatrienoic acid. The obtained results suggest that compound II is 9,16-dihydroperoxy-10(E), 12(Z), 14(E)-octadecatrienoic acid and product I is a mixture of two positional isomers, 9-hydroxy-16-hydroperoxy-10(E),12(Z),14(E)-octadecatrienoic and 9-hydroperoxy-16-hydroxy-10(E),12(Z), 14(E)-octadecatrienoic acids. Lipoxygenase converts efficiently [¹⁴C]9-HOTE into product I. Also, both metabolites I and II are the products of double dioxygenation. The second oxygenation at C-16 position as well as the first one at C-9 is controlled by lipoxygenase.     

Effect of the <Emphasis Type="Italic">desA</Emphasis> gene encoding Δ12 acyl-lipid desaturase on the chloroplast structure and tolerance to hypothermia of potato plants

Effects of the desA gene from the cyanobacterium Synechocystis sp. encoding Δ12 acyl-lipid desaturase and increasing the level of unsaturated fatty acids (linoleic acid (18:2) primarily) in membrane lipids, which was inserted into potato (Solanum tuberosum L., cv. Desnitsa) plants, on chloroplast ultrastructure and plant tolerance to low temperatures were studied. The main attention was focused on modifications in the chloroplast structure and their possible relation to potato plant tolerance to oxidative and low-temperature stresses under the influence to their transformation with the Δ12 acyl-lipid desaturase gene from cyanobacterium (desA-licBM3-plants). Morphometric analysis showed that, in comparison with wild-type (WT) plants, in desA-licBM3-plants the number of grana in chloroplasts increased substantially. The total number of thylakoids in transformant chloroplasts was almost twice higher than in WT plants. The number of plastoglobules per chloroplast of transformed plants increased by 25%. A marked increase in the number of grana, total number of thylakoids, and the number of plastoglobules in chloroplasts of desA-licBM3-plants indicates their more intense lipid metabolism, as compared with WT plants, and this resulted in the conservation of some part of lipids in plastoglobules. In addition, the expression of heterological desA gene encoding Δ12 acyl-lipid desaturase positively influenced stabilization of not only structure but also functioning of chloroplast membranes, thus preventing a transfer of electrons from the ETR to oxygen and subsequent ROS generation at hypothermia. This was confirmed by the analysis of the rate of superoxide anion generation in tested genotypes.     

Genetic diversity and antimicrobial activity of lactic acid bacteria in the preparation of traditional fermented potato product ‘<Emphasis Type="Italic">tunta’</Emphasis>

Fermentation microorganisms, lactic acid bacteria (LAB) and yeast from 12 samples of tunta production chain were quantified, from the native potatoes used by the process fermentation of potatoes in the river up to the final product. During fermentation, the LAB population steadily increased from 3 to 4 to 8 log CFU/g during the first 8 days in the river and the yeast population increased from 2 to 3 to 3–4 log CFU/g. Overall, 115 LAB strains were isolated using a culture-dependent method. Molecular techniques and 16S rRNA gene sequencing enabled the identification of native species. In LAB isolates, members of the Lactobacillaceae (64%), Leuconostocaceae (9%) and Enterococcaceae (2%) families were identified. The most prevalent LAB species in the tunta production chain was Lactobacillus curvatus, followed by Leuconostoc mesenteroides and Lactobacillus sakei, Lactobacillus brevis and Enterococcus mundtii were also present. Only 13 LAB strains showed anti-listerial activity, and one of them, identified as En. mundtii DSM 4838^T [MG031213], produced antimicrobial compounds that were determined to be proteins after treatment with proteolytic enzymes. Based on these results, we suggest that traditional fermented product-derived LAB strains from specific environments could be selected and used for technological application to control pathogenic bacteria and naturally protect food from post-harvest deleterious microbiota.     

Engineering resistance to PVY in different potato cultivars in a marker-free transformation system using a ‘shooter mutant’ <Emphasis Type="Italic">A. tumefaciens</Emphasis>

In this work, Potato virus Y (PVY) resistant potatoes were generated using an environmentally safe construct. For this purpose, a ‘shooter’ mutant Agrobacterium-based transformation system was used. The isopentenyl transferase gene (ipt) present on the Ti plasmid of ‘shooter’ strains enhances shoot regeneration and can be used as a phenotypic selection marker. The introduced marker-free binary vector carried a hairpin construct derived from the coat protein gene of PVY-NTN strain in order to induce gene silencing. Transformation resulted in high regeneration rates (1.4–5.7 shoots per explant). With pre-selection for the ipt ⁺ phenotype the transformation frequency was 24–53%, while without selection 12–28% of the shoots were PCR positive. The presence of the transgene was verified by Southern hybridization. In 16 of 31 challenged transformant lines PVY could be detected neither by RT-PCR nor by back inoculation. A 62.5% of these resistant lines proved to be also ipt-free. This transformation system was reproducible in four potato cultivars, suggesting that it could easily be adapted for other species.     

A putative β-glucanase pseudogene behind the potato GBSS gene

We identified an open reading frame (ORF) which is located closely behind the gene encoding granulebound starch synthase (GBSS) of potato (Solanum tuberosum L.). The ORF ends with a perfect 43 bp direct repeat, which carries the stop triplet precisely at the beginning of the second repeat. The deduced protein shows homology with all known isoforms of plant -1,3-glucanases and -1,3-1,4-glucanases. Although the DNA sequence is unique in potato and tomato (Lycopersicon esculentum L.), no expression of the gene was found in these species. Taken together with the unusual codon usage and length of the predicted protein, this sequence could be a pseudogene.     

Transgenic <Emphasis Type="Italic">indica</Emphasis> rice cultivar ‘Swarna’ expressing a potato chymotrypsin inhibitor <Emphasis Type="Italic">pin2</Emphasis> gene show enhanced levels of resistance to yellow stem borer

Transgenic rice was developed from ‘Swarna’, the most popular indica rice cultivar (Oryza sativa L.) in South East Asia, with a potato chymotrypsin inhibitor gene (pin2) through Agrobacterium-mediated transformation. Four out of nine primary transgenic plants had a single-copy T-DNA insertion while other five plants had two copies. Mendelian pattern of inheritance of the transgene (pin2) was observed in the T₁ generation progeny plants. Whole plant bioassays conducted at both vegetative and reproductive stages and cut stem assays showed enhanced levels of resistance of transgenic rice against yellow stem borer. The transgenic rice lines with plant derived proteinase inhibitor genes would develop into resistant cultivars to fit into resistance breeding strategies as an important component of integrated pest management in rice.     

Monte Carlo simulation of the α-amylolysis of amylopectin potato starch

The branched structure of potato amylopectin (degree of polymerization ~200,000) was modeled in a computer matrix. The chain-length distribution and the length and width of a cluster of the amylopectin molecule were used as input variables in the model. Independent literature values related to the structure of amylopectin (percentage #-hydrolysis and ratio of A- to B-chains) were used for evaluation of the branching characteristics (length of branch area and chance of branching) of the modeled amylopectin. The structural parameters predicted by the model agreed very well with data from the literature. The chain-length distribution and values for the percentage of #-hydrolysis were the two most important parameters required to model the structure of amylopectin. This computer-generated model of potato amylopectin in solution can be used to simulate various enzymatic (i.e., !-amylase, #-amylase, glucoamylase, pullunanase) or chemical reactions (i.e., acid hydrolysis, hypochlorite oxidation). The modeling approach described in this paper is also suitable for starches from other botanical sources (i.e., corn, wheat, tapioca).     

Enhanced operation of 4-aminobutyrate shunt in γ-irradiated potato tubers

γ-Radiation at a sprout inhibiting dose of 100 Gy enhanced the operation of the 4-aminobutyrate shunt by about 3-fold 48 hr after irradiation, but there was no accumulation of the amino acid in irradiated potatoes during storage up to 6 months. γ-Radiation increased the activity of the first enzyme of the 4-aminobutyrate shunt, glutamate decarboxylase and the maximum increase of 2.6-fold was found on the fourth day after irradiation. γ-Radiation had no pronounced effect on the activity of the other two enzymes of the pathway, 4-aminobutyrate transaminase and succinate semialdehyde dehydrogenase. Based on the results obtained, a possible role for the 4-aminobutyrate shunt in potato tuber physiology was suggested.