A major QTL and an SSR marker associated with glycoalkaloid content in potato tubers from <Emphasis Type="Italic">Solanum tuberosum </Emphasis>× <Emphasis Type="Italic">S. sparsipilum</Emphasis> located on chromosome I

New potato (Solanum tuberosum) varieties are required to contain low levels of the toxic glycoalkaloids and a potential approach to obtain this is through marker-assisted selection (MAS). Before applying MAS it is necessary to map quantitative trait loci (QTLs) for glycoalkaloid content in potato tubers and identify markers that link tightly to this trait. In this study, tubers of a dihaploid BC(1) population, originating from a cross between 90-HAF-01 (S. tuberosum(1)) and 90-HAG-15 (S. tuberosum(2) x S. sparsipilum), were evaluated for content of alpha-solanine and alpha-chaconine (total glycoalkaloid, TGA) after field trials. In addition, tubers were assayed for TGA content after exposure to light. A detailed analysis of segregation patterns indicated that a major QTL is responsible for the TGA content in tubers of this potato population. One highly significant QTL was mapped to chromosome I of the HAG and the HAF parent. Quantitative trait loci for glycoalkaloid production in foliage of different Solanum species have previously been mapped to this chromosome. In the present research, QTLs for alpha-solanine and alpha-chaconine content were mapped to the same location as for TGA content. Similar results were observed for tubers exposed to light. The simple sequence repeat marker STM5136 was closely linked to the identified QTL.     

Metabolic engineering of high carotenoid potato tubers containing enhanced levels of beta-carotene and lutein

In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L. cultivar Desiree which normally produces tubers containing c. 5.6 microg carotenoid g(-1) DW and also in Solanum phureja L. cv. Mayan Gold which has a tuber carotenoid content of typically 20 microg carotenoid g(-1) DW. In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 microg carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c. 11 microg g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls. The crtB gene was also transformed into S. phureja (cv. Mayan Gold), again resulting in an increase in total carotenoid content to 78 microg carotenoid g(-1) DW in the most affected transgenic line. In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene. No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation. Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls. The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.     

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.     

The influence of temperature on light enhanced glycoalkaloid synthesis in potato

The influence of temperature on total glycoalkaloid (TGA) synthesis in tubers exposed to light (250 jumol m“² s”² PAR, Photosynthetically Active Radiation) or dark environments for 96 h was examined in three potato cultivars. Following 96 h light or dark the tubers were stored without light at 5°C or 24°C and TGA concentrations monitored over the subsequent 30 and 90 days. Exposure to light and cultivar were found to be major factors influencing TGA concentrations; temperature had no significant effect. TGA content in illuminated tubers of cvs ‘Pentland Hawk’ and ‘Kerrs Pink’ were significantly higher (P < 0.01) compared with tubers placed in the dark. TGA concentrations in cv. ‘Desiree’ increased significantly only following exposure to light at low temperatures (P < 0.05). Removal of tubers from storage at 5°C and immediate illumination at 24°C altered the ratio of glycoalkaloids in cvs ‘Pentland Hawk’ and ‘Kerrs Pink’. Regardless of cultivar and storage temperature TGA concentrations were higher at the end of the storage period compared with initial TGA concentrations. During storage TGA concentrations fluctuated widely and gradual accumulation of glycoalkaloids with time was rarely demonstrated except in cv. ‘Desiree’. Tubers stored at 24°C accumulated higher TGA concentrations than those stored at 5°C in cv. ‘Kerrs Pink’ but not in cvs ‘Pentland Hawk’ and ‘Desiree’. Tubers of cv. ‘Kerrs Pink’ exposed to light prior to storage accumulated glycoalkaloids more rapidly than unexposed tubers during storage at 24°C and occasionally at 5°C. Light enhanced glycoalkaloids are not degraded over time.     

α and β-solamarine in kennebec Solanum tuberosum leaves and aged tuber slices

Two major steroid glycoalkaloids, in addition to α-solanine and α-chaconine, were isolated from leaves and aged tuber slices of potato, Solanum tuberosum L. var Kennebec. They are glycosides of tomatidenol and have been identified as α- and β-solamarine. The compounds were not found in tuber peel or freshly sliced Kennebec tubers or in 20 other cultivars.     

Determination of potato glycoalkaloids using high-pressure liquid chromatography-electrospray ionisation/mass spectrometry

A method for quantifying two toxic glycoalkaloids, alpha-solanine and alpha-chaconine, in potato (Solanum tuberosum) tuber tissue was developed using HPLC-electrospray ionisation (ESI)/MS. Potato samples were extracted with 5% aqueous acetic acid, and the extracts were subjected directly to HPLC-ESI/MS after filtration. By determining the intensities of the protonated molecules of alpha-solanine (m/z 868) and alpha-chaconine (m/z 852) using selected ion monitoring (positive ion mode), a sensitive assay was attained with detection limits of 38 and 14 ppb for the two glycoalkaloids, respectively. The high sensitivity and selectivity of MS detection effectively reduced the time of analysis thus enabling a high throughput assay of glycoalkaloids in potato tubers.     

Difference between light- and wound-induced biosynthesis of α-solanine and α-chaconine in potato tubers

Potato tuber tissues can incorporate mevalonic acid-2-14C into glycoalkaloids, namely α-chaconine and α-solanine. The percent incorporation of this labeled precursor into α-chaconine in light exposed tubers is more than that of mechanically injured tubers.     

Effects of steroidal glycoalkaloids from potatoes (Solanum tuberosum) on in vitro bovine embryo development

alpha-Solanine and alpha-chaconine are two naturally occurring steroidal glycoalkaloids in potatoes (Solanum tuberosum), and solanidine-N-oxide is a corresponding steroidal aglycone. The objective of this research was to screen potential cyto-toxicity of these potato glycoalkaloids using bovine oocyte maturation, in vitro fertilization techniques and subsequent embryonic development as the in vitro model. A randomized complete block design with four in vitro oocyte maturation (IVM) treatments (Experiment 1) and four in vitro embryo culture (IVC) treatments (Experiment 2) was used. In Experiment 1, bovine oocytes (n=2506) were matured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVM medium only. The in vitro matured oocytes were then subject to routine IVF and IVC procedures. Results indicated that exposure of bovine oocytes to the steroidal glycoalkaloids during in vitro maturation inhibited subsequent pre-implantation embryo development. Potency of the embryo-toxicity varied between these steroidal glycoalkaloids. In Experiment 2, IVM/IVF derived bovine embryos (n=2370) were cultured in vitro in medium supplemented with 6 microM of alpha-solanine, alpha-chaconine, solanidine-N-oxide or IVC medium only. The results showed that the pre-implantation embryo development is inhibited by exposure to these glycoalkaloids. This effect is significant during the later pre-implantation embryo development period as indicated by fewer numbers of expanded and hatched blastocysts produced in the media containing these alkaloids. Therefore, we conclude that in vitro exposure of oocytes and fertilized ova to the steroidal glycoalkaloids from potatoes inhibits pre-implantation embryo development. Furthermore, we suggest that ingestion of Solanum species containing toxic amounts of glycoalkaloids may have negative effects on pre-implantation embryonic survival.     

Comparative compositional analysis of transgenic potato resistant to potato tuber moth (PTM) and its non-transformed counterpart

In this study, the compositions of transgenic potatoes (TPs) resistant to potato tuber moth (Phthorimaea operculella) were compared with those of its non-transgenic (NTP) counterparts. The light inducible promoter, phosphoenolpyruvate carboxylase led to the expression of Cry1Ab only in the leaves and light-treated tubers of the TPs. No significant differences were found in the moisture, ash, dry weight, total soluble protein, carbohydrate, starch, fiber, ascorbate, cations, anions, fatty acids, and glycoalkaloids contents of TP and NTP. Moreover, light treatment significantly affected the contents of ascorbate, acetate and nitrite anions, palmitic, stearic and linolenic fatty acids, α-haconine and α-solanine glycoalkaloids in TP and NTP tubers. While, significant differences were observed in the amino acid contents in light-treated tubers of TPs than the NTP ones. Although, light treatment in potato tubers resulted in marked metabolic changes, all the variations observed in the metabolites compositions were found to be within the desired reference ranges for potato plants. In conclusion, the results indicated that the TPs were substantially and nutritionally equivalent to the NTP counterparts.     

Assessment of genetic variability of haploids extracted from tetraploid (2n = 4x = 48) Solanum tuberosum.

The objectives of this study were to assess the genetic variability of haploids (2n = 2x = 24) extracted from tetraploid Solanum tuberosum through 4x x 2x crosses with Solanum phureja. Molecular and phenotypic analyses were performed to fingerprint the genotypes used and to evaluate their potential use in breeding programs. AFLP analysis revealed the presence of specific bands derived from the tetraploid seed parent S. phureja, as well as ex novo originated bands. On average, 210 bands were visualized per genotype, 149 (70%) of which were common to both parental genotypes. The percentage of S. tuberosum specific bands ranged from 25.1% to 18.6%, with an average of 22%. The fraction of genome coming from S. phureja ranged from 1.9% to 6.5%, with an average value of 4%. The percentage of ex novo bands varied from 1.9% to 9.0%. The presence of S. phureja DNA is very interesting because it indicated that S. phureja pollinator is involved in the mechanism of haploid formation. The characterization for resistance to Erwinia carotovora subsp. carotovora and potato virus X (PVX) provided evidence that haploids may express traits that are lacking in the tetraploids they come from, which can be useful for both genetic studies and breeding purposes. It is noteworthy that genotypes combining resistance to both diseases and good pollen stainability were identified. Other possible breeding implications owing to the presence of S. phureja genome in the haploids analyzed are discussed.     

Optimization of Glycoalkaloid Analysis for Use in Industrial Potato Fruit Juice Downstreaming

Annually, within the European Union about 1.7 million tons of starch is produced by processing over 8 million tons of potato tubers, Solanum tuberosum. In recent years, the potato protein content has gained tremendous industrial interest, since these proteins have excellent nutritional value. As naturally occurring, secondary plant metabolites steroidal potato glycoalkaloids are formed in potatoes. The two major glycoalkaloids in potatoes are α‐solanine and α‐chaconine. Because of the significant toxicity of the glycoalkaloids for human and for animal nutrition it was essential to develop efficient extraction processes. The need for an easy, fast, sensitive and reliable glycoalkaloid assay at the very beginning of the production chain is obvious. In this study an efficient analytical assay for potato glycoalkaloids from powdery protein samples under industrially relevant conditions is described: sample extraction, analyte pre‐purification, and final HPLC analysis. An acetic acid extraction/homogenization process was used for glycoalkaloid extraction from potato protein samples. The extracts were purified by means of solid phase extraction cartridges using the different washing steps developed in this study. The final determination was performed through an HPLC method using a Reprosil‐Pur NH₂ column. The limit of detection was 5 μg/mL for α‐solanine and α‐chaconine, respectively, corresponding to concentrations of 20 ppm in potato protein powder.     

Biosensors for assay of glycoalkaloids in potato tubers

The possibility of practical application of biosensors based on pH-sensitive field-effect transistors and butyrylcholinesterase to glycoalkaloid analysis in potato tubers was studied. The main analytical features of the designed biosensors and measurement conditions were optimized. The biosensor was applied to quantitative analysis of glycoalkaloids in tubers of different potato varieties. The results proved to be in good agreement with those obtained by conventional protocols. Experiments on glucose assay were performed. An inverse correlation between the contents of glucose and glycoalkaloids in potato tubers was demonstrated.     

Biosensors for assay of glycoalkaloids in potato tubers

The possibility of commercial application of biosensors based on pH-sensitive field-effect transistors and butyrylcholinesterase to glycoalkaloid analysis in potato tubers was studied. The main analytical features of the designed biosensors and measurement conditions were optimized. The biosensor was applied to quantitative analysis of glycoalkaloids in tubers of different potato varieties. The results proved to be in good agreement with those obtained by conventional protocols. Experiments on glucose assay were performed. An inverse correlation between the contents of glucose and glycoalkaloids in potato tubers was demonstrated.     

Transformation of <Emphasis Type="Italic">Solanum tuberosum</Emphasis> plastids allows high expression levels of β-glucuronidase both in leaves and microtubers developed in vitro

Plastid genome transformation offers an attractive methodology for transgene expression in plants, but for potato, only expression of gfp transgene (besides the selective gene aadA) has been published. We report here successful expression of β-glucuronidase in transplastomic Solanum tuberosum (var. Desiree) plants, with accumulation levels for the recombinant protein of up to 41% of total soluble protein in mature leaves. To our knowledge, this is the highest expression level reported for a heterologous protein in S. tuberosum. Accumulation of the recombinant protein in soil-grown minitubers was very low, as described in previous reports. Interestingly, microtubers developed in vitro showed higher accumulation of β-glucuronidase. As light exposure during their development could be the trigger for this high accumulation, we analyzed the effect of light on β-glucuronidase accumulation in transplastomic tubers. Exposure to light for 8 days increased β-glucuronidase accumulation in soil-grown tubers, acting as a light-inducible expression system for recombinant protein accumulation in tuber plastids. In this paper we show that plastid transformation in potato allows the highest recombinant protein accumulation in foliar tissue described so far for this food crop. We also demonstrate that in tubers high accumulation is possible and depends on light exposure. Because tubers have many advantages as protein storage organs, these results could lead to new recombinant protein production schemes based on potato.     

Reduction of cholesterol and glycoalkaloid levels in transgenic potato plants by overexpression of a type 1 sterol methyltransferase cDNA

Transgenic potato (Solanum tuberosum cv Désirée) plants overexpressing a soybean (Glycine max) type 1 sterol methyltransferase (GmSMT1) cDNA were generated and used to study sterol biosynthesis in relation to the production of toxic glycoalkaloids. Transgenic plants displayed an increased total sterol level in both leaves and tubers, mainly due to increased levels of the 24-ethyl sterols isofucosterol and sitosterol. The higher total sterol level was due to increases in both free and esterified sterols. However, the level of free cholesterol, a nonalkylated sterol, was decreased. Associated with this was a decreased glycoalkaloid level in leaves and tubers, down to 41% and 63% of wild-type levels, respectively. The results show that glycoalkaloid biosynthesis can be down-regulated in transgenic potato plants by reducing the content of free nonalkylated sterols, and they support the view of cholesterol as a precursor in glycoalkaloid biosynthesis.     

The inhibition of potato sprout growth by light.

When potato seed tubers (Solanum tuberosum cv. Pentland Javelin) were stored in darkness or diffuse daylight at 9°C and transferred at intervals to conditions suitable for sprouting, their capacity for sprout growth was unaffected by the presence or absence of light during previous storage. When similar tubers were stored at 10°C, 18°C or 25°C, sprout growth commenced earliest at 25°C, but the date was unaffected by fluorescent light. It was concluded that light did not affect the length of the dormant period, but only the rate of sprout elongation after that period had ceased. When tubers with growing sprouts at 10°C or 18°C were transferred from darkness into fluorescent light, sprout growth virtually ceased. Transfer from light into darkness resulted in immediate sprout growth, at a rate comparable with tubers stored continuously in the dark. Tubers of three Peruvian cultivars, stored in farm-scale diffuse-daylight stores, grew progressively shorter sprouts with increasing daily exposure to light from 30 min to 12 h. Storage of cv. Wilja under 21 Wm^-2 (total) of white fluorescent light for 10 h per day maintained the sprouts at the same length as ten times this light intensity for 1 h per day. In a subsequent experiment with cv. Bintje the 10 h, low-intensity light regime gave slightly shorter sprouts. It appeared that the total light energy falling on the tubers was the dominant factor controlling sprout growth.     

The glycoalkaloids of Solanum demissum

Two glycoalkaloids previously unreported in Solanum demissum have been isolated and identified as commersonine and neotomatine.