Transgenic Potato with PVY Coat Protein Gene and Its Small-Scale Field Test

Potato virus Y (PVY) infection may cause a severe yield depression up to 80%. To develop the potato (Solanum tuberosum L. ) cultivars that resist PVY infection is very crucial in potato production. The authors have been cloned the coat protein gene of PVY from its Chinese isolate. A chimaeric gene containing the cauliflower mosaic virus 35S promoter and PVY coat protein coding region was introduced into the potato cultivars “Favorita”, “Tiger head” and “K4” via Agrobacterium tumefaciens. Results from PCR and Southern blot analysis confirmed that the foreign gene has integrated into the potato chromosomes. These transgenic potato plants were mechanically inoculated with PVY virus (20 mg/L). The presence of the virus in the potato plants was determined by ELISA and method of back inoculation into tobacco. The authors observed a drastic reduction in the accumulation of virus in some transgenic potato lines. Furthermore, some transgenic potato lines produced more tubers per plant than the untransformed potato did, and the average weight of these transgenic plant tubers was also increased. In the field test, the morphology and development of these transgenic potato plants were normal, 3 transgenic lines of “Favorita” exhibited a higher yield than the untrasformed virus-free potato with an increase ranged from 20% to 30%. From these transgenic lines, it will be very hopeful to develop a potato cultivar which not only has a significant resistance to PVY infection, but also a good harvest in potato production.     

Introduction of the carrot HSP17.7 into potato (Solanum tuberosum L.) enhances cellular membrane stability and tuberization in vitro

We have examined the ability of a carrot (Daucus carota L.) heat shock protein gene encoding HSP17.7 (DcHSP17.7) to confer enhanced heat tolerance to potato (Solanum tuberosum L.), a cool-season crop. The DcHSP17.7 gene was fused to a 6XHistidine (His) tag to distinguish the engineered protein from endogenous potato proteins and was introduced into the potato cultivar 'Désirée' under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Western analysis showed that engineered DcHSP17.7 was constitutively, but not abundantly, expressed in transgenic potato lines before heat stress. Leaves from multiple regenerated potato lines that contain the transgene exhibited significantly improved cellular membrane stability at high temperatures, compared with wild-type and vector control plants. Transgenic potato lines also exhibited enhanced tuberization in vitro: under a condition of constant heat stress, at 29 degrees C, nodal sections of the transgenic lines produced larger and heavier microtubers at higher rates, compared to the wild type and vector controls. The dry weight and percentages of microtubers that were longer than 5 mm were up to three times higher in the transgenic lines. Our results suggest that constitutive expression of carrot HSP17.7 can enhance thermotolerance in transgenic potato plants. To our knowledge, this is the first study that shows that the thermotolerance of potato can be enhanced through gene transfer.     

Differential expression of invertase genes in internal and external phloem tissues of potato (Solanum tuberosum L.)

The cloning of promoter sequences of two invertase genes from potato (Solanum tuberosum L.) is described. Histochemical analysis of series of reporter transgenic lines reveals phloem-specific expression from both promoters, with one expressed preferentially in internal phloem and the other in external phloem of stem vascular bundles.     

Overexpression of the wild potato eIF4E-1 variant Eva1 elicits Potato virus Y resistance in plants silenced for native eIF4E-1

Potato virus Y (PVY) is the most important viral pathogen of cultivated potato (Solanum tuberosum) from a commercial perspective, causing severe losses in both tuber quality and yield worldwide. Specific accessions of wild potato species exhibit resistance against PVY but efforts to transfer the trait to cultivated material have not yielded widely adopted varieties. Because amino acid substitutions at specific domains of host factor eIF4E-1 often confer resistance to various crops, we sequenced the associated genes expressed in wild potato plants. A novel eIF4E-1 variant, designated here as Eva1, was identified in S. chacoense, S. demissum, and S. etuberosum. The protein contains amino acid substitutions at ten different positions when compared to its cultivated potato (S. tuberosum) homolog. In the yeast two-hybrid system, Eva1 failed to bind VPg, a viral protein required for infectivity. Overexpression of the associated cDNA conferred PVY resistance to transgenic potato plants silenced for the native eIF4E-1 gene. Because the gene sources of Eva1 are sexually compatible with potato, the molecular strategies described can be employed to develop 'intragenic' potato cultivars.     

Expression of Potato Virus Y Coat Protein Gene in Transgenic Potato

Potato virus Y (PVY) N coat protein (CP) coding sequence was cloned into a plant expression vector pMON316 under the CaMV 35S promoter. Leaf discs of potato (Solanum tuberosum) were used to Agrobacterium-mediated gene transfer. A large number of regenerated putative transgenic plants were obtained based on kanamycin resistance. Using total DNA purified from transgenic plants as templates and two oligonucleotides synthesized from 5' and 3' of the PVY coat protein gene as primers, the authors carried out polymerase chain reaction (PCR) to check the presence of this gene and obtained a 0. 8 kb specific DNA fragment after 35 cycles of amplification. Southern blot indicated that the PCR product was indeed PVY CP gene which had been integrated into the potato genome. Enzyme-linked immunosorbent assay (ELISA) of our transgenic plants showed that CP gene was expressed in at least some transgenic potato plants.     

Evaluation of potato tuber moth (Lepidoptera: Gelechiidae) resistance in tubers of Bt-cry5 transgenic potato lines

The potato tuber moth, Phthorimaea operculella (Zeller), in tropical and subtropical countries, is the most destructive pest of potato, Solanum tuberosum L. The larvae attack foliage and tubers in the field and in storage. The purpose of this study was to evaluate the efficacy of a Bt-cry5 transgene to control the potato tuber moth in tuber tissues. Tuber bioassays using stored (11-12 mo old) and newly harvested tubers of Bt-cry5-Lemhi Russet and Bt-cry5-Atlantic potato lines showed up to 100% mortality of 1st instars. Mortality was lowest in the newly harvested tubers of Bt-cry5-Atlantic lines (47.1-67.6%). Potato tuber moth mortality was 100% in the Bt-cry5-Spunta lines that were transformed with Bt-cry5 gene controlled by the CaMV 35S promoter (pBIML5 vector) and in 2 of 3 lines transformed with Bt-cry5 gene controlled by the Gelvin super promoter (pBIML1 vector). The transgenic Spunta lines expressing Bt-cry5 controlled by the patatin promoter (pBMIL2 vector) showed the lowest tuber moth mortality (25.6 and 31.1%). The Bt-cry5 transgenic lines with high tuber expression of B. thuringiensis have value in an integrated pest management system to control potato tuber moth.     

Salicylic acid is important for basal defense of Solanum tuberosum against Phytophthora infestans

The importance of the signaling compound salicylic acid for basal defense of potato (Solanum tuberosum L. cv. Désirée) against Phytophthora infestans, the causal agent of late blight disease, was assessed using transgenic NahG potato plants which are unable to accumulate salicylic acid. Although the size of lesions caused by P. infestans was not significantly different in wild-type and transgenic NahG plants, real-time polymerase chain reaction analyses revealed a drastic enhancement of pathogen growth in potato plants depleted of salicylic acid. Increased susceptibility of NahG plants correlated with compromised callose formation and reduced early defense gene expression. NahG plants pretreated with the salicylic acid analog 2,6-dichloro-isonicotinic acid allowed pathogen growth to a similar extent as did wild-type plants, indicating that salicylic acid is an important compound required for basal defense of potato against P. infestans.     

Potato flour viscosity improvement is associated with the expression of a wheat LMW-glutenin gene

It has been previously shown that expression of a high-molecular-weight glutenin (HMW-GS) in transgenic wheat seeds resulted in the improvement of flour functional properties. In this study, potato flour viscosity was improved through a specific expression of a low-molecular-weight glutenin (LMW-GS-MB1) gene in tuber. The resulting construct was introduced into potato leaf explants (Solanum tuberosum cv Kennebec) through Agrobacterium tumefaciens-mediated gene transfer. Southern and Northern analysis of transgenic potato confirmed that the integration of LMW-GS-MB1 in genomic DNA was stable and its mRNA was abundant in transgenic line 16 tubers. Western blot analysis of line 16 extract shows a LMW-GS subunit accumulation in tuber. To demonstrate the capacity of transgenic lines to produce tubers with improved flour functional properties, transgenic lines 9 and 16 exhibiting, respectively, moderate and high expression of LMW-GS-MB1 mRNA and nontransgenic plants were transferred to field plots. The mean viscosity value of flour obtained from the field-grown tubers of transgenic line 16 exhibited a 3-fold increase in viscosity at 23 degrees C when compared to flour from nontransgenic tubers.     

Potato gene Y-1 is an N gene homolog that confers cell death upon infection with potato virus Y

ADG2 is a DNA sequence mapped to a resistance (R) gene-rich region at the distal end of chromosome XI in potato (Solanum tuberosum subsp. andigena). The gene, in which ADG2 represents the predicted nucleotide-binding domain (NBS), was cloned and characterized. The coding region of the gene (designated as Y-1) is 6,187 bp long and structurally similar to gene N that confers hypersensitive resistance to Tobacco mosaic virus in Nicotiana spp. Both belong to the TIR-NBS-LRR class of genes and show 57% identity at the amino acid sequence level. The introns of Y-1 were spliced as predicted from the sequence. Y-1 cosegregated with Ry(adg), a gene for extreme resistance to Potato virus Y (PVY) on chromosome XI, as tested in a potato-mapping population and with independent potato cultivars. Leaves of the transgenic potato plants expressing Y-1 under the control of Cauliflower mosaic virus 35S promoter developed necrotic lesions upon infection with PVY, but no significant resistance was observed, and plants were systemically infected with PVY.     

Glycoalkaloid aglycone accumulations associated with infection by Clavibacter michiganensis ssp. sepedonicus in potato species Solanum acaule and Solanum tuberosum and their interspecific somatic hybrids

Solanum acaule Bitt., a wild potato species, is closely related to cultivated potato (Solanum. tuberosum L.). Incorporation of desirable traits from allotetraploid [2n=4x=48, 2 endosperm balance number (EBN)] S. acaule (acl) into autotetraploid (2n=4x=48, 4EBN) S. tuberosum (tbr) is difficult due to incongruity boundaries. In this study, three hybrid combinations, each with a specific genome constitution, were produced through protoplast fusion: (1) hexaploid 2x acl (+) 4x tbr, (2) tetraploid 2x acl (+) 2x tbr, and (3) hexaploid 4x acl (+) 2x tbr hybrids. In terms of glycoalkaloid aglycones, the hybrids produced demissidine, tomatidine and solanidine, similarly to the S. acaule parental species, but S. tuberosum synthesised only solanidine. Inoculations with Clavibacter michiganensis ssp. sepedonicus (Cms), which is the causal agent of bacterial ring rot in potato, yielded significantly lower total glycoalkaloid aglycone accumulation both in S. acaule plants and in interspecific hybrids in comparison with the corresponding mock-inoculated plants. However, in S. tuberosum the aglycone levels were either higher or unchanged as a result of infection by Cms. To incorporate the desirable traits of the interspecific somatic hybrids into 4EBN S. tuberosum, sexual backcrosses were carried out. The hexaploid 4x acl (+) 2x tbr hybrids with the hypothetical 4EBN showed the greatest capacity to undergo backcrosses with S. tuberosum.     

Novel somatic hybrids (Solanum tuberosum L. + Solanum tarnii) and their fertile BC1 progenies express extreme resistance to potato virus Y and late blight

Solanum tarnii, a wild diploid, tuber-bearing Mexican species belonging to the series Pinnatisecta is highly resistant to Potato virus Y (PVY) and Colorado potato beetle and shows a strong hypersensitive reaction to Phytophthora infestans. Therefore, it could be a potential source of resistance to pathogens for potato breeders. S. tarnii (2n = 2x = 24) is reproductively isolated from tetraploid Solanum tuberosum and hence difficult to include in potato breeding programmes. In this study, interspecific somatic hybrids were produced for the first time by protoplast electrofusion of the cells of potato cv. Delikat (Solanum tuberosum L.) and Solanum tarnii. The hybrid nature of the regenerants was confirmed by simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers and by morphological analysis and flow cytometry. Selected somatic hybrids were successfully backcrossed with cv. Delikat. Parental lines, primary somatic hybrids and BC₁ progeny were assessed for resistance to PVY by mechanical inoculation, grafting and exposure to viruliferous aphid vectors in the field, and resistance to late blight (P. infestans) by detached leaflet and whole tuber tests. The somatic hybrids showed no symptoms of viral infection and most of them displayed high levels of resistance to foliage blight. The BC₁ progenies were highly resistant to PVY and a few were resistant to foliage blight. Selected hybrids and BC₁ clones were evaluated in the field for tuber quality and tuber yield. Some BC₁ clones produced yields of good quality tubers. The results confirm that both the resistance to PVY and to late blight of S. tarnii is expressed in somatic hybrids, and PVY resistance is transferred to BC₁ progeny, whereas blight resistance is harder to transfer. Somatic hybridization again proved to be a valuable tool for producing pre-breeding material with increased genetic diversity.     

Engineering ketocarotenoid biosynthesis in potato tubers

Consumption of astaxanthin is increasingly associated with a range of health benefits. Attempts to engineer ketocarotenoid biosynthesis in plants have been successful although there are no reports of nutritionally significant levels of astaxanthin in plant storage organs. Thus, in this study, ketocarotenoid biosynthesis was engineered in potato tubers. Both Solanum tuberosum and Solanum phureja transgenic lines were produced that expressed an algal bkt1 gene, encoding a beta-ketolase, and accumulated ketocarotenoids. Two major ketocarotenoids were detected, ketolutein and astaxanthin. The level of unesterified astaxanthin reached ca. 14 microg g(-1) DW in some bkt1 expressing lines of S. phureja but was much lower in the S. tuberosum background. Co-transformation of S. tuberosum with crtB, encoding phytoene synthase, and the bkt1 gene was achieved in order to determine whether this would enhance the levels of S. tuberosum ketocarotenoid.     

Genomic instability in Solanum tuberosum x Solanum kurtzianum interspecific hybrids.

The use of interspecific crosses in breeding is an important strategy in improving the genetic base of the modern cultivated potato, Solanum tuberosum L. Until now, it has normally been interspecific Solanum hybrids that have been morphologically and cytologically characterized. However, little is known about the genomic changes that may occur in the hybrid nucleus owing to the combination of genomes of different origin. We have observed novel AFLP bands in Solanum tuberosum x Solanum kurtzianum diploid hybrids; 40 novel fragments were detected out of 138 AFLP fragments analyzed. No cytological abnormalities were observed in the hybrids; however, we found DNA methylation changes that could be the cause of the observed genomic instabilities. Of 277 MSAP fragments analyzed, 14% showed methylation patterns that differed between the parental species and the hybrids. We also observed frequent methylation changes in the BC1 progeny. Variation patterns among F1 and BC1 plants suggest that some methylation changes occurred at random. The changes observed may have implications for potato breeding as an additional source of variability.     

Development of SCAR markers to the PVY resistance gene Ryadg based on a common feature of plant disease resistance genes.

Sequence-characterized amplified regions (SCARs) were developed, based on nucleotide differences within resistance gene-like fragments isolated from a potato plant carrying the Ryadg gene, which confers extreme resistance to potato Y potyvirus (PVY). It originates from Solanum tuberosum subsp. andigena, and a susceptible potato plant. SCARs were tested using 103 potato breeding lines and cultivars with diverse genetic backgrounds derived from Europe, North America, and Japan. Two markers showed high accuracy for detection of the Ryadg gene. The SCAR marker RYSC3 was generated only in genotypes carrying Ryadg. The SCAR marker RYSC4 was detected in all genotypes carrying Ryadg but also in four PVY-susceptible genotypes. Neither marker was detected in genotypes carrying other Ry genes originating from different species than S. tuberosum subsp. andigena. Therefore, these SCAR markers should be powerful tools in marker-assisted selection for Ryadg in potato breeding programs, and should also be useful for cloning of the Ryadg gene.     

Nitrogen Source Regulation of Growth and Photosynthesis in Beta vulgaris L

A chimeric gene containing the patatin promoter and the transit-peptide region of the small-subunit carboxylase gene was utilized to direct expression of Escherichia coli glycogen synthase (glgA) to potato (Solanum tuberosum) tuber amyloplasts. Expression of the glgA gene product in tuber amyloplasts was between 0.007 and 0.028% of total protein in independent potato lines as determined by immunoblot analysis. Tubers from four transgenic potato lines were found to have a lowered specific gravity, a 30 to 50% reduction in the percentage of starch, and a decreased amylose/amylopectin ratio. Total soluble sugar content in these selected lines was increased by approximately 80%. Analysis of the starch from these potato lines also indicated a reduced phosphorous content. A very high degree of branching of the amylopectin fraction was detected by comparison of high and low molecular weight carbohydrate chains after debranching with isoamylase and corresponding high-performance liquid chromatography analysis of the products. Brabender viscoamylograph analysis and differential scanning calorimetry of the starches obtained from these transgenic potato lines also indicate a composition and structure much different from typical potato starch. Brabender analysis yielded very low stable paste viscosity values (about 30% of control values), whereas differential scanning calorimetry values indicated reduced enthalpy and gelatinization properties. The above parameters indicate a novel potato starch based on expression of the glgA E. coli gene product in transgenic potato.     

A new lineage sheds light on the evolutionary history of Potato virus Y

Potato virus Y (PVY) is one of the rare plant viruses for which some biological traits (host range and symptomatology) are highly correlated with phylogeny, allowing the reconstruction of the evolutionary history of these traits. In this article, a new lineage of PVY isolates from Chile is described, showing unique genomic and biological properties. This lineage was found to be the sister group of all other PVY isolates and helped in the reconstruction of the ancestral traits and evolutionary history of PVY, suggesting that veinal necrosis in tobacco is an ancestral state and that adaptation to pepper ( Capsicum spp.) and potato ( Solanum tuberosum ) has been modified several times during PVY history.     

Crop borders reduce potato virus Y incidence in seed potato

Crop borders of soybean (Glycine max), sorghum (Sorghum bicolor), winter wheat (Triticum aestivum) and potato (Solanum tuberosum) were tested as a means of reducing potato virus Y (PVY) incidence in seed potato. Borders of fallow cultivated ground served as controls. Aphid landing rates were monitored weekly in plots using green tile traps, and PVY incidence was assessed by serologically testing tuber progeny from selected rows in each plot. Average weekly aphid landing rates in fallow-bordered and crop-bordered plots were not significantly different in 1992 (29.4 and 25.2 aphids, respectively) or 1993 (7.3 and 6.6 aphids, respectively). However, crop borders significantly reduced PVY incidence. In 1992, fallow-bordered and soybean-bordered plots averaged 47.8% and 35.0% PVY infection, respectively. In 1993, PVY infection averaged across all crop (soybean, sorghum, and wheat) bordered plots was 2.7% compared to 6.8% in fallow-bordered plots. PVY incidence in the centre rows of fallow-bordered and crop-bordered plots was statistically equivalent, while outer rows of crop-bordered plots had significantly less PVY than outer rows of fallow-bordered plots. Crop borders apparently reduced the number of viruliferous aphids landing on the edge of the plot. The choice of crop species used as a border, or treating the border with a systemic insecticide, did not affect aphid landing rates or PVY incidence. In 1995, PVY incidence in the centre 10 row block of potatoes averaged 2.1% across all crop borders (potato and soybean). PVY infection in the four row potato border averaged 5.7%. Crop borders are readily adaptable to current production practices, although the greatest benefits in reducing PVY incidence would occur in average sized, generation 0 (< 0.2 ha), elite seed potato fields.     

Field resistance of transgenic burley tobacco lines and hybrids expressing the tobacco vein mottling virus coat protein gene

Fifty transgenic lines expressing the tobacco vein mottling virus (TVMV) coat protein (CP) gene in five genetic backgrounds were evaluated under field conditions for response to mechanic inoculation with TVMV, tobacco etch virus (TEV) and potato virus Y (PVY). TVMV CP transgenic lines conferred resistance to TVMV, TEV and PVY under field conditions. Combining two strategies, coat protein-mediated resistance (CPMR) coupled with an endogenous resistance gene (Virgin A Mutant, VAM) significantly extended the range and magnitude of virus resistance and provided a potential valuable new source of protection against potyviruses. CP transgenic lines lacking the VAM gene had high resistance to TEV, medium resistance to PVY, and a recovery phenotype to TVMV. A series of hybrids involving transgenic lines were generated and tested under field conditions for response to virus inoculation. One copy of TVMV-CP gene presented in lines homozygous for the VAM gene provided effective resistance to all three potyviruses. These studies also suggested that selection of a suitable recipient genotype was critical and that field evaluation was necessary in order to select elite resistant transgenic lines. Engineering viral CP genes into genotypes possessing some level of virus resistance could be critical to achieve an effective level of resistance.     

Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0-8.0. Addition of tryptophan also decreased the Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.