Transgenic Potato Lines Research Articles

Untersuchungen zur CO2-Gas-Austauschrate bei transgenen Fructan bildenden Kartoffellinien ( Solanum tuberosum L.) unter Einsatz eines modularen Großküvettensystems: (Investigations to the CO2 gas exchange rate in transgenic fructans generating potato lines ( Solanum tuberosum L.) by implementation of a modular cuvette system)

This survey was aimed at comparing the CO2 gas exchange rate of transgenic potato lines and of the isogenic line Désirée in order to establish if genetic modification has resulted in ecologically relevant changes. In examining two transgenic potato lines and one isogenic line, a modular whole-plant cuvette system was implemented for the first time. Furthermore, suitability of such a cuvette system for comparative examination was to be tested in the survey at hand. The lowest CO2 exchange rate was observed in the SST/FFT line. The SST line achieved the highest CO2 exchange rate of all genotypes tested. Measurement of CO2 exchange rate, and additionally collected data on morphologic and characteristics of yield physiology, resulted in distinctly correlating data. The phenotypical changes observed could be classified within the growth and yield profile specified by the German Federal Office of Plant Varieties (Bundessortenamt) for potato varieties permitted for cultivation in Germany. A changed growth rate of crop in the transgenic lines could not be established. A modular whole-plant cuvette system can be implemented in comparative gas exchange measurements in a partly air-conditioned cell.

Toxic Secondary Metabolite Production in Genetically Modified Potatoes in Response to Stress

Potatoes produce a number of toxic secondary metabolites, which are divided into two groups: the sesquiterpenes and the glycoalkaloids (PGAs): whereas PGAs are largely preformed and present in toxic quantities in both the foliage and "green" potatoes, it is well documented that the levels of PGAs and sesquiterpenes are effected by many biotic an abiotic stresses. The development of genetically modified potato varieties has made it prudent to ascertain whether there may be changes in the amounts or types of these secondary metabolites either as a direct effect of the transgene or due to its interactions with environmental variables. Transgenic potato lines were exposed, along with nontransgenic lines, to a range of biotic and abiotic stresses and a range of environmental conditions in the field and store. Following stressing, a comparison was made of levels of potato glycoalkaloid and sesquiterpene levels between the two groups. Significant differences were observed in the levels of both glycoalkaloid and sesquiterpene levels between transgenic and control material and between infected and noninfected material.

Effect of water stress on proline accumulation of genetically modified potatoes ( Solanum tuberosum L.) generating fructans

This study aimed to investigate the effect of water stress on the accumulation of proline in the leaves of transgenic potato lines generating fructans. The results of the series of bifactorial experiments in 2002 and 2003 indicated an increase of the proline level in leaves of all potato lines examined under water deficit. In addition, an increase in proline concentration during plant development was observed. The proline content was related to leaf water potential and relative water content (RWC), which indicates that proline could be involved in osmoregulation of potato plants under the experimental conditions. Surprisingly, under water deficit, the proline level was lowest in most of the transgenic SST/FFT-lines, which generate fructan molecules with a high degree of polymerization. Therefore, a pleiotropic effect can not be excluded as the reason for the divergence in behavior of these transgenic lines. The present results suggest that the modification of carbohydrate metabolism, especially the high content of soluble carbohydrates, may affect water stress-induced proline accumulation.

Age‐specific bioassays and fecundity of Phthorimaea operculella (Lepidoptera: Gelechiidae) reared on cry1Ac ‐transgenic potato plants

SummaryVariation in the susceptibility of lepidopterous pest larvae of different ages to transgenic crops and the potential for survivors to reproduce could have important consequences for the development of resistance in such pests. Experiments were undertaken in the laboratory to determine if larvae of the potato tuber moth, Phthorimaea operculella, of different ages (0 (< 1 day old), 3, 5, 7 days) varied in their susceptibility to cry1Ac9–transgenic potato (Solanum tuberosum) foliage grown in the glasshouse or field. The survival and fecundity of larvae reared on transgenic tubers was also determined in the laboratory. There were no apparent differences in susceptibility of larvae of different ages to transgenic foliage. Larvae fed glasshouse or field‐grown non‐transgenic foliage had significantly larger relative growth indices and more larvae pupated, than those fed transgenic foliage, regardless of larval age. Eggs from a laboratory colony were placed on transgenic or non‐transgenic tubers to measure survival and fecundity. Between 6% and 15% of eggs placed on transgenic tubers developed into pupae for three of the four transgenic potato lines tested. On one transgenic line, only six adults emerged from 1300 eggs. In contrast, between 71% and 97% of the eggs placed on non‐transgenic tubers developed into pupae. Male and female pupae from transgenic lines weighed less than those from non‐transgenic lines. The fecundity of females from two of four transgenic lines was lower than from the non‐transgenic parent cultivar. Although larvae of different ages did not exhibit any overall age‐dependent pattern of increasing or decreasing susceptibility to transgenic foliage of glasshouse or field‐grown plants, the ability of larvae to survive and reproduce on transgenic tubers suggests this pest has the ability to evolve resistance to the transgenic plants used in the present study.

Unexpected effects of chitinases on the peach-potato aphid (Myzus persicae Sulzer) when delivered via transgenic potato plants (Solanum tuberosum Linn�) and in vitro

With the aim of producing insect-resistant potato plants, internode explants of Solanum tuberosum L. cv. Désirée were transformed with an Agrobacterium strain C58pMP90 containing an insect (Phaedon cochleariae: Coleoptera, Chrysomelidae) chitinase gene and the neomycin phosphotransferase (nptII) gene as selectable marker, both under the control of the viral CaMV 35S promoter. Three transformed potato lines (CH3, CH5 and CH25) exhibiting the highest chitinolytic activities were selected for feeding experiments with the peach-potato aphid, Myzus persicae (Sulzer), under controlled photoperiod and temperature conditions. Aphids fed on transgenic potato plants showed a reduced pre-reproductive period and an enhanced daily fecundity. Transgenic potato lines did not affect nymphal mortality, but improved several biological parameters related to aphid population's growth. Artificial diets were used to provide active (1, 10, 100 and 500 microg ml(-1)) and inactive (500 microg ml(-1)) bacterial (Serratia marcescens) chitinase to M. persicae. These compounds increased nymph survival at all active chitinase doses when compared to the control diet, while inactive chitinase did not. Although the pre-reproductive period was slightly shortened and the daily fecundity slightly higher, active and inactive chitinase provided as food led a reduction from 1 to 1.5 day population's doubling time. Therefore chitinase activity was responsible for the probiotic effects on aphids. Our results question the relevance of a chitinase-based strategy in the context of potato culture protection.

Microbial communities of Solanum tuberosum and magainin-producing transgenic lines

Antimicrobial peptide magainin II, isolated from the skin of the African clawed toad, has shown activity in vitro against a range of micro-organisms. Transgenic potato lines expressing a synthetic magainin gene show improved resistance to the bacterial plant pathogen, Erwinia carotovora. Culturable bacterial and fungal communities associated with magainin-producing potato plants were compared with those communities from the non-transgenic parental control and with another potato cultivar. Total numbers of aerobic bacteria recovered from the leaves of the magainin-producing line, its non-transgenic parent line and an unrelated cultivar did not differ significantly. There were no detectable differences in the numbers of Gram-positive and Gram-negative bacteria, pseudomonad populations or fungi recovered from foliage from the three plant lines. Bacterial populations recovered from the roots of a magainin-expressing plant line did not differ significantly from populations recovered from the unmodified parental line. Tubers from the magainin-expressing transgenic potatoes, however, had significantly lower total numbers of bacteria than tubers produced by unmodified plants. In vitro testing of rhizosphere isolates against magainin analogues found that bacterial isolates varied in their susceptibility to the peptides. There were no significant differences in the total numbers of fungi and yeasts recovered from the various plant lines, with one exception: higher numbers of fungi were recovered from roots of magainin-expressing plants than the unmodified control plants.

Effects of transgenic potatoes with an altered starch composition on the diversity of soil and rhizosphere bacteria and fungi

The aim of this study was to investigate potential effects on the composition of the bacterial and fungal diversity in rhizosphere and soil of a transgenic potato line (SIBU S1) which was modified in its starch composition by RNA anisensing, compared to the non-transgenic parental cultivar (SIBU) at the flowering stage in 2000. Furthermore a second non-transgenic cultivar (SOLANA) was included in the study. To avoid artefacts derived from cultivation depending approaches, molecular techniques based on 16S-(bacteria) and 18S-(fungi) rDNA respectively were used to describe the microbial community structure. Comparing 16S- and 18S-rDNA DGGE fingerprints from the different bulk soil samples, it could be shown that no significant differences between the two cultivars and the transgenic line were found. Similar results were obtained for the rhizosphere samples using the eubacterial, α-and β-proteobacterial and fungal specific primers with the exception of, the eubacterial DGGE patterns obtained for the rhizosphere of SOLANA. These patterns revealed that the relative abundance of one band was enhanced compared with the patterns of SIBU and SIBU S1 and the sequence of the differentiating band showed the highest similarity with Enterobacter amnigenus. When Pseudomonas specific primers were used, relevant differences were found between the rhizosphere patterns of the transgenic potato line (SIBU S1) and the parental cultivar (SIBU). However, clear effects of the cultivar SOLANA on the structure of the Pseudomonas community compared to SIBU were also detected.

Resistance of potatoes transgenic for a cry1Ac9 gene, to Phthorimaea operculella (Lepidoptera: Gelechiidae) over field seasons and between plant organs

SummaryStable performance of insect‐resistant transgenic plants across field seasons and between plant organs damaged by the insect pest is critical for management of this resistance in the field. To evaluate this, potato (Solanum tuberosum) lines transgenic for a cry1Ac9 gene with resistance to potato tuber moth (Phthorimaea operculella) were established in the field during the southern hemisphere summers of 1997/98, 1998/99 and 1999/00 as small field plots, each of 10 plants. Replicate plots of the non‐transgenic parent cultivars (at least one for every three independently derived transgenic lines) were planted randomly throughout the trials. Field‐grown foliage was challenged with larvae in the laboratory and a growth index (GI) was calculated for recovered larvae from each transgenic and non‐transgenic potato line. Larval growth on young and mature leaves, and on newly harvested or stored tubers was also measured in the laboratory. Foliage from the transgenic lines inhibited larval growth in all seasons tested. For both control and transgenic lines, larvae had slightly lower GIs when reared on mature leaves compared with young leaves, although the correlation between mean GI for young and mature transgenic leaves was high (r = 0.97). The correlation between the mean GIs of larvae on newly harvested tubers and on those stored for 5 months was also high (r = 1.0). However, the GIs of larvae on newly harvested transgenic tubers were larger than on transgenic tubers stored for 5 months. The relative growth indices (RGI = mean GI/number days before final weighing) of larvae reared on newly harvested tubers from transgenic lines were generally higher than those from young transgenic foliage, while the RGIs of larvae reared on non‐transgenic tubers were slightly lower than those fed non‐transgenic foliage. The correlation between mean RGIs of larvae fed tubers or foliage was 0.62. The transgenic potato lines exhibited stable resistance to larvae across field seasons, between affected plant organs, and between plant organs of different ages.

An agglutinating chitinase with two chitin-binding domains confers fungal protection in transgenic potato

Brassica juncea BjCHI1 is a unique chitinase with two chitin-binding domains. Here, we show that, unlike other chitinases, potato-expressed BjCHI1 shows hemagglutination ability. BjCHI1 expression in B. juncea seedlings is induced by Rhizoctonia solani infection, suggesting its protective role against this fungus. To verify this, transgenic potato (Solanum tuberosum L. cv. Desiree) plants expressing BjCHI1 generated by Agrobacterium-mediated transformation were challenged with R. solani. We also transformed potato with a cDNA encoding Hevea brasiliensis beta-1,3-glucanase, designated HbGLU, and a pBI121-derivative that contains cDNAs encoding both BjCHI1 and HbGLU. In vitro fungal bioassays using Trichoderma viride showed that extracts from transgenic potato lines co-expressing BjCHI1 and HbGLU inhibited fungal growth better than extracts from transgenic potato expressing either BjCHI1 or HbGLU, suggesting a synergistic effect. Consistently, in vivo fungal bioassays with soil-borne R. solani on young transgenic potato plants indicated that the co-expressing plants showed healthier root development than untransformed plants or those that expressed either BjCHI1 or HbGLU. Light microscopy and transmission electron microscopy revealed abundant intact R. solani hyphae and monilioid cells in untransformed roots and disintegrated fungus in the BjCHI1-expressing and the BjCHI1 and HbGLU co-expressing plants. Observations of collapsed epidermal cells in the co-expressing potato roots suggest that these proteins effectively degrade the fungal cell wall, producing elicitors that initiate other defense responses causing epidermal cell collapse that ultimately restricts further fungal penetration.

High-level expression of apple PGIP1 is not sufficient to protect transgenic potato against Verticillium dahliae

Polygalacturonase-inhibiting proteins (PGIPs) are plant proteins believed to play a role in the defence against pathogenic fungi. Purified apple PGIP1 inhibited polygalacturonases (PGs) secreted by Verticillium dahliae grown on potato root cell walls and pectin. We therefore hypothesised that apple PGIP1 could be used to confer resistance against Verticillium-wilt, a major disease of potato caused by the fungus V. dahliae. Transgenic lines containing the apple pgip1 gene under control of the enhanced CaMV 35S (e35S) promoter were generated. Stable integration of the apple pgip1 transgene into the potato genome was shown by the polymerase chain reaction (PCR) and Southern blot. Cross hybridisation with potato pgip(s) was not observed. High-level expression of the apple PGIP1 in several independent transgenic potato events was verified by silver staining of SDS-PAGE separated proteins and Western blot. All but one of the PGIP1 extracts prepared from the transgenic potato lines were successful in inhibiting V. dahliae PGs. Active PGIP1 was expressed in the leaves as well as the roots of the transgenic plants. The apoplastic localisation of PGIP activity in the pgip-transgenic potato plants was demonstrated by a vacuum infiltration–extraction experiment. A glasshouse trial indicated that six transgenic lines (A10, B10, B13, A3, A14 and B16) had significantly reduced disease symptoms compared to the untransformed control and other lines when grown in the inoculated soil, but five of them also showed significantly slower senescence symptoms when grown in the control soil. It is proposed that an extended juvenile phase in the transgenic lines resulted in the apparent increased disease resistance, and this could not be attributed to inhibition of V. dahliae PGs, despite high-level expression of the apple PGIP1.

Heterologous pectate lyase isoenzymes are not different in their effects on soft rot resistance in transgenic potatoes

Pectate lyase (PL) isoenzymes secreted by Erwinia carotovora ( Ec) bacteria are of central interest to both basic and applied plant physiology because they depolymerise cell wall pectin into unsaturated oligogalacturonates that trigger various plant defence responses. Therefore, the effects of heterologously expressed PL1 and PL3 were examined with emphasis on plant resistance to soft rot disease caused by Ec bacteria. Agrobacterium-mediated, transformation resulted in transgenic potato lines of cv. Désirée, with activities of the recombinant PL1 ranging between 22 and 1402 mU ml −1. Compared with the non-transgenic plants, potato lines expressing PL1 provided enhanced resistance to soft rot-causing Erwinia bacteria. Thus, the degree of cell lysis caused by bacterial maceration as well as the extension of Ec-rotting was significantly diminished in PL-expressing tuber tissue. This was coincident with a strengthened formation of necrosis on the wound surface of tubers. Spreading of Ec bacteria appear to be prevented—most likely because of enhanced activities of polyphenol oxidase, phenylalanine ammonia-lyase and peroxidase in PL-transgenic tubers. However, key finding reveal that there are no significant differences in resistance to soft rot between potato lines expressing PL1 and PL3. Despite differences in their action patterns and reaction products, it is shown that both PL isoenzymes share similar molecular roles in the events leading to induction of plant defence responses.

Colorado potato beetles compensate for tomato cathepsin D inhibitor expressed in transgenic potato.

We reported earlier the importance of digestive cathepsin D-like activity for initiating dietary protein hydrolysis in Colorado potato beetle, Leptinotarsa decemlineata Say [Brunelle et al. (1999) Arch. Insect Biochem. Physiol. 42:88-98]. We assessed here whether transgenic lines of potato (Solanum tuberosum L.) expressing a cathepsin D inhibitor (CDI) from tomato would show resistance to the beetle, or if the insect would compensate for the loss of cathepsin D activity after ingesting the recombinant inhibitor. Transgenic potato lines expressing tomato CDI were developed by Agrobacterium tumefaciens genetic transformation, and selected based on their relative amount of CDI. After confirming the absence of detectable visible effects of CDI on the plant's phenotype, diet assays with control and transgenic lines were carried out to assess the impact of the inhibitor on growth and development of the insect. Leaf consumption, relative growth rate, molting incidence, and digestive protease activity were monitored at 12-h intervals over 132 h for 3rd-instar larvae provided with transgenic potato foliage. Leaf consumption and relative growth rate were slightly reduced during the first 12 h for larvae fed CDI, but no significant differences were observed thereafter. In contrast, time for molting to the 4th larval stage was significantly longer for larvae fed modified plants, with developmental delays of approximately 10 h (0.5 day) compared to control larvae. Recombinant CDI also had an impact on the insect's digestive physiology, readily inducing overproduction of digestive proteases (rubiscases), followed by a gradual decrease of total and pepstatin-sensitive activity. Overall, these observations show the ability of Colorado potato beetle to compensate for the loss of cathepsin D activity by modulating its digestive protease complement in response to aspartate-type inhibitors in the diet. From a practical viewpoint, these data stress the importance of devising improved strategies for the effective inhibition of insect digestive proteinases in vivo, based on the use of hybrid inhibitors active against different protease classes.

REDUCTION OF TOTAL STEROIDAL GLYCOALKALOIDS IN POTATO TUBERS USING ANTISENSE CONSTRUCTS OF A GENE ENCODING A SOLANIDINE GLUCOSYL TRANSFERASE

REDUCTION OF TOTAL STEROIDAL GLYCOALKALOIDS IN POTATO TUBERS USING ANTISENSE CONSTRUCTS OF A GENE ENCODING A SOLANIDINE GLUCOSYL TRANSFERASE

Preferential expression of a plant cystatin at nematode feeding sites confers resistance to Meloidogyne incognita and Globodera pallida

The expression patterns of three promoters preferentially active in the roots of Arabidopsis thaliana have been investigated in transgenic potato plants in response to plant parasitic nematode infection. Promoter regions from the three genes, TUB-1, ARSK1 and RPL16A were linked to the GUS reporter gene and histochemical staining was used to localize expression in potato roots in response to infection with both the potato cyst nematode, Globodera pallida and the root-knot nematode, Meloidogyne incognita. All three promoters directed GUS expression chiefly in root tissue and were strongly up-regulated in the galls induced by feeding M. incognita. Less activity was associated with the syncytial feeding cells of the cyst nematode, although the ARSK1 promoter was highly active in the syncytia of G. pallida infecting soil grown plants. Transgenic potato lines that expressed the cystatin OcIDeltaD86 under the control of the three promoters were evaluated for resistance against Globodera sp. in a field trial and against M. incognita in containment. Resistance to Globodera of 70 +/- 4% was achieved with the best line using the ARSK1 promoter with no associated yield penalty. The highest level of partial resistance achieved against M. incognita was 67 +/- 9% using the TUB-1 promoter. In both cases this was comparable to the level of resistance achieved using the constitutive cauliflower mosaic virus 35S (CaMV35S) promoter. The results establish the potential for limiting transgene expression in crop plants whilst maintaining efficacy of the nematode defence.

Inheritance of a pectate lyase mediated soft rot resistance in crosses between transgenic potato lines of cv. désirée andS. tuberosum cultivars

Expression of the pectate lyase (PL) isoenzyme 3 in transgenic potato lines of cv. Desiree mediates an enhanced resistance of tuber tissue toErwinia carotovora (Ec) soft rot due to a pre-activation of plant defence mechanisms. Therefore, theSolanum tuberosum cvs Agave and Adretta with a moderate level of soft rot resistance were crossed with such PL-expressing potato lines. The resulting progenies were assessed with respect to plant/tuber characteristics over a period of four years and then tested for PL3-expression as well as for soft rot resistance. 71% of the selected progeny lines exhibited a stable production of the PL3 enzyme. Statistical analysis revealed differences between the transgenic and the non-transgenic progeny concerning the soft rot resistance of tuber tissue. Compared with the PL-inactive progeny, extension of Ec-rotting on the wound surface of PL-transgenic potatoes was diminished on average by 51.8%. Similarly, the degree of cell lysis caused by bacterial maceration was significantly (P<0.05) reduced in tuber tissue of PL-expressing progeny lines. The latter also revealed an enhanced PPO and PAL activity in their tuber tissue indicating an active plant defence. It is concluded therefore that the PL-mediated soft rot resistance introduced into potatoes by means of molecular techniques is heritable.

Enhancement of the primary flavor compound methional in potato by increasing the level of soluble methionine.

The primary flavor compound in potato, methional, is synthesized from methionine by the Strecker degradation reaction. A major problem associated with potato processing is the loss of methional. Methional or its precursor, methionine, is not added back during potato processing due to high costs of production. A novel approach to enhance the methional level in processed potato would be to increase the production of its precursor, soluble methionine (Met). Cystathionine gamma-synthase (CGS) is a key enzyme regulating methionine biosynthesis in plants. To increase the level of soluble methionine in potato, Arabidopsis thaliana CGS cDNA was introduced under transcriptional control of the cauliflower mosaic virus 35S promoter into Russet Burbank potato by Agrobacterium-mediated transformation. Ten different transgenic potato lines (CGS1-10) were analyzed. Immunoblot analysis demonstrated that Arabidopsis CGS is expressed in the leaves, tubers, and roots of transgenic potato plants. CGS enzymatic activity was higher in the leaves and roots of the transgenic potato lines compared to the wild-type potato. Methionine levels in the leaves, roots and tubers of transgenic potato lines were enhanced as high as 6-fold compared to those in wild type potato plants. The methional level in baked tubers of field-grown transgenic potato lines was increased between 2.4- and 4.4-fold in lines CGS1, CGS2, and CGS4. The increase observed in methional levels correlated with the soluble methionine level in the tubers from the same lines measured before processing. These results provide the first evidence that the methional level can be enhanced in processed potatoes by increasing the production of its precursor, methionine.

Evidence of medium-chain-length polyhydroxyoctanoate accumulation in transgenic potato lines expressing the Pseudomonas oleovorans Pha-C1 polymerase in the cytoplasm

The phaC1 gene from Pseudomonas oleovorans, coding for the Pha-C1 polymerase, was introduced into the potato genome. Transgenic callus and plant lines which transcribed and translated the transgene were selected and cell suspension cultures from the wild type and transgenic lines were established. The substrate for the Pha-C1 polymerase, 3-( R)-hydroxyoctanoate, was provided to the growth medium. In the transgenic lines, but not in the wild type or in transgenic cell suspension cultures without Pha-C1 expression, evidence of medium-chain-length polyhydroxyalkanoate accumulation ranging from 0.02 to 9.7 mg of polymer per gram of dry weight was observed after feeding in the growth medium the substrate.

Field Evaluation of Natural, Engineered, and Combined Resistance Mechanisms in Potato for Control of Colorado Potato Beetle

The colorado potato beetle, Leptinotarsa decemlineata Say (Coleoptera: Chrysomelidae), is the leading insect pest of potato (Solanum tuberosum L.) in northern latitudes. Host plant resistance has the potential use in an integrated pest management program for control of colorado potato beetle. During the 1998 and 1999 seasons, field studies were conducted to compare natural (leptine glycoalkaloids and glandular trichomes), engineered (Bt-cry3A and Bt-cry5 transgenic potato lines), and combined (Bt-cry5+glandular trichomes) plant resistance mechanisms of potato for control of colorado potato beetle. Nine different potato clones representing five different host plant resistance mechanisms were evaluated under natural colorado potato beetle infestation at the Montcalm Research Farm in Entrican, Michigan. The Bt-cry3A transgenic lines, the high leptine line (USDA8380-1), and the high foliar glycoalkaloid line (ND5873-15) were most effective for controlling defoliation by colorado potato beetle adults and larvae. The Bt-cry5 line (SPc5-G2) was not as effective as the Bt-cry3A transgenic lines ('Russet Burbank Newleaf,' RBN15, and YGc3.1). The glandular trichome (NYL235-4) and Bt-cry5+glandular trichome lines proved to be ineffective. Significant rank correlations for the potato lines between the two years were observed for egg masses, second and third instar, and fourth instar seasonal cumulative mean number of individuals per plant, and defoliation. Egg mass and first instar seasonal cumulative mean number of individuals per plant were not strong indicators of host plant resistance in contrast to second and third instars or adults. Based on these results, the Bt-cry3A transgenic lines, the high leptine line, and the high total glycoalkaloid line are effective host plant resistance mechanisms for control of colorado potato beetle.

Development and Evaluation of Potatoes Transgenic for a cry1Ac9 Gene Conferring Resistance to Potato Tuber Moth

Transgenic potato (Solanum tuberosum L.) lines of two cultivars, Ilam Hardy and Iwa, were developed using Agrobacterium-mediated transformation to transfer a cry1Ac9 gene under the transcriptional control of the CaMV 35S promoter. PCR confirmed the presence of the nptII selectable marker gene in all recovered lines. All ten lines of Ilam Hardy and 14 of 15 Iwa lines were PCR-positive for the cry gene. In greenhouse trials, all Ilam Hardy transgenic lines produced phenotypically normal plants and significantly inhibited larval growth of potato tuber moth (Phthorimaea operculella Zeller). In contrast, only 60% of the Iwa transgenic lines produced phenotypically normal plants, but all lines positive for the cry gene significantly inhibited larval growth. All transgenic lines with a greenhouse appearance equivalent to the nontransgenic controls and improved resistance to potato tuber moth larvae were planted in the field. Three of the ten Ilam Hardy lines and two of the eight Iwa lines retained phenotypically normal appearance in the field and produced tuber yields equivalent to the nontransgenic controls. All five of these transgenic lines significantly inhibited larval growth of potato tuber moth on excised field-grown leaves. A high correlation was established between larval growth indices from the greenhouse and the field. A transgenic line from each cultivar inhibited larval growth by over 40%, and the line derived from Ilam Hardy prevented pupation of all larvae. Southern analysis on these five elite lines revealed that they contained either one or two copies of the cry1Ac9 gene. The amount of Cry protein in all transgenic lines tested was less than 60 ng·g-1 of fresh leaf tissue. A transgenic line from each cultivar was identified with comparable phenotypic appearance and yield to their parent cultivars coupled with high resistance to PTM.

Effects of T4 lysozyme release from transgenic potato roots on bacterial rhizosphere communities are negligible relative to natural factors.

Rhizosphere bacterial communities of two transgenic potato lines which produce T4 lysozyme for protection against bacterial infections were analyzed in comparison to communities of wild-type plants and transgenic controls not harboring the lysozyme gene. Rhizosphere samples were taken from young, flowering, and senescent plants at two field sites in three consecutive years. The communities were characterized in a polyphasic approach. Cultivation-dependent methods included heterotrophic plate counts, determination of species composition and diversity based on fatty acid analysis of isolates, and community level catabolic profiling. Cultivation-independent analyses were based on denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments amplified from rhizosphere DNA using primers specific for Bacteria, Actinomycetales, or alpha- or beta-Proteobacteria. Several bands of the DGGE patterns were further characterized by sequence analysis. All methods revealed that environmental factors related to season, field site, or year but not to the T4 lysozyme expression of the transgenic plants influenced the rhizosphere communities. For one of the T4 lysozyme-producing cultivars, no deviation in the rhizosphere communities compared to the control lines was observed. For the other, differences were detected at some of the samplings between the rhizosphere community structure and those of one or all other cultivars which were not attributable to T4 lysozyme production but most likely to differences observed in the growth characteristics of this cultivar.