Colorado potato beetle resistant somatic hybrid potato plants produced via protoplast electrofusion

Leptines are natural glycoalkaloids found only in certain selections of the wild potato speciesSolanum chacoense. These rare glycoalkaloids have been identified to be phytochemical defensive agents against insect herbivores such as the Colorado potato beetle (CPB). In an attempt to introduce this CPB resistance into the cultivated potatoS. tuberosum, interspecific somatic hybrid plants were developed between a dihaploid ofS. tuberosum and a high leptine-producing germplasm selection ofS. chacoense. The somatic hybrid was fused using protoplast electrofusion and regeneration techniques. Selection of interspecies fusion cell lines was based on hybrid vigor in protoplast-callus (p-callus) growth, on shoot regeneration from p-calli, and on characteristic appearance of anthocyanin pigment. This selection system was highly efficient and 12 of 13 fully regenerated plants were identified as somatic interspecies hybrids, as determined by the analyses of morphologic biochemical, and isozyme markers. In vitro insect bioassays demonstrated that the hybrids averaged a threefold reduction in leaf consumption by the CPB when compared to cultivated potatoes.     

Molecular markers associated with leptinine production are located on chromosome 1 in Solanum chacoense

Leptines of Solanum chacoense are effective natural deterrents against the Colorado potato beetle. Leptines are the acetylated forms of the glycoalkaloids solanine and chaconine and are supposed to be synthesised via hydroxylated derivatives, called leptinines. Inheritance of leptinine production was studied in crosses of closely related S. chacoense genotypes. The segregation data supported a single-gene model for the inheritance of leptinine production. In the segregating F₁ population of a S. chacoense cross, AFLP, RFLP and RAPD markers segregating with the leptinine production have been identified. The locus involved in leptinine synthesis was localised to the short arm of chromosome 1 of the potato where a major QTL for solanidine production, and markers with tight linkage to leptine production, have been mapped before. Our data further support the previous finding that the short arm of chromosome 1 is involved in steroid alkaloid synthesis in potato, and suggest that the genes involved in leptinine and leptine production are tightly linked in S. chacoense. Received: 27 June 2000 / Accepted: 4 August 2000     

Identification of molecular markers associated with leptine production in a population of Solanum chacoense Bitter

  Solanum chacoense Bitter, a wild relative of the cultivated potato, produces several glycoalkaloids, including solanine, chaconine, and the leptines. The foliar-specific leptine glycoalkaloids are believed to confer resistance to the Colorado Potato Beetle (CPB). Using two bulked DNA samples composed of high- and low-percent leptine individuals from a segregating F₁ population of S. chacoense, we have identified two molecular markers that are closely linked to high percent solanine+chaconine and, conversely, to nil/low percent leptine. One of these, a 1,500-bp RAPD product (UBC370-1500), had a recombination value of 3% in the F₁ progeny, indicating tight linkage. UBC370-1500 mapped to the end of the short arm of potato chromosome 1, in the region of a previously mapped major QTL for solanidine, from a S. tuberosum (solanidine)×S. berthaultii (solasodine) cross. Taken together, these results suggest that either (1) a major locus determining solanidine accumulation in Solanum spp. is on chromosome 1 in the region defined by the RFLP markers TG24, CT197, and CT233, or (2) this region of chromosome 1 may harbor two or more important genes which determine accumulation of steroidal aglycones. These findings are important for the genetics of leptine (as well as other glycoalkaloid) accumulation and for the development of CPB-resistant potato varieties. Received: 5 March 1998 / Accepted: 28 July 1998     

Genetic analysis of leaf explant regenerability inSolanum chacoense

An F₁ population consisting of 51 genotypes, derived from two unresponsive parental lines ofSolanum chacoense Bitt., was examined for shoot regeneration from leaf explants. Fourteen genotypes failed to respond whereas, among the responsive genotypes, four produced multiple shoots on over 90% of the explants. Estimates of broad-sense heritability were high for both frequency of responsive explants (83%) and the number of shoots per responsive explant (82%). The segregation of the F₁ hybrid progeny was in agreement with the theoretical ratios of a genetic model for tissue culture responsiveness involving three unlinked genes. This study confirms earlier findings concerning the genetic control ofin vitro shoot regeneration from leaf explants inS. chacoense.     

Effects of glycoalkaloids from Solanum plants on cucumber root growth

The phytotoxic effect of four glycoalkaloids and two 6-O-sulfated glycoalkaloid derivatives were evaluated by testing their inhibition of cucumber root growth. The bioassays were performed using both compounds singly and in equimolar mixtures, respectively. Cucumber root growth was reduced by chaconine (C), solanine (S), solamargine (SM) and solasonine (SS) with IC₅₀ values of 260 (C), 380 (S), 530 (SM), and 610 μM (SS). The inhibitory effect was concentration-dependent. 6-O-sulfated chaconine and 6-O-sulfated solamargine had no inhibitory effects, which indicated that the carbohydrate moieties play an important role in inhibiting cucumber root growth. The equimolar mixtures of paired glycoalkaloids, both chaconine/solanine and solamargine/solasonine, produced synergistic effects on inhibition of cucumber root growth. By contrast, mixtures of unpaired glycoalkaloids from different plants had no obviously synergistic effects. The growth inhibited plant roots lacked hairs, which implied that inhibition was perhaps at the level of root hair growth.     

Chloroplast DNA variation between the common cultivated potato (Solanum tuberosum ssp. tuberosum) and several South American relatives

Summary Chloroplast DNA (ctDNA) from the tuberbearing Solanum species tuberosum, vernei, phureja, and chacoense has been compared by restriction endonuclease analysis. Digestion by Hind III or Xba I reveal no differences, but digestion with Bam HI and Eco RI reveals minor differences in the ctDNA among these species. The ctDNA restriction patterns of the tetraploid common cultivated potato of North America and Europe, S. tuberosum ssp. tuberosum and the South American tetraploid, S. tuberosum ssp. andigena are identical for all four restriction endonucleases. These data suggest that ssp. tuberosum and ssp. andigena contain similar ctDNA and therefore may share a common ancestor, or direct lineage. The ctDNA restriction patterns of S. vernei and S. chacoense are identical for all four restriction endonucleases, and S. phureja ctDNA, can be distinguished from the other diploid ctDNAs by digestion with Bam HI. None of the diploids analyzed contain ctDNA identical to the tetraploids and therefore either did not contribute their chloroplast genomes to the evolution of the tetraploids, or the ctDNA has diverged since this evolutionary event. The ctDNAs studied did not contain restriction polymorphisms which could be correlated to cytoplasmic male sterility in Solanum. This is the first demonstration of ctDNA diversity in the tuber-bearing Solanum species.     

Mapping of genes associated with leptine content of tetraploid potato

High content of leptine glycoalkaloids present in Solanum chacoense has been associated with genetic resistance to Colorado potato beetle (Leptinotarsa decemlineata [Say]). From an unrecorded accession of S. chacoense, the North Dakota State University breeding program has developed a tetraploid genotype, ND4382-19, that contains foliar leptines. In this study, using a segregating population, ND5873 (ND4382-19 × Chipeta), and GC-MS to analyze foliar content of alkaloids, two loci, involved in the synthesis of leptines were identified. They segregated as two complementary epistatic genes that allowed the synthesis of leptinidine (Lep) and acetyl-leptinidine (AL), respectively. Partial AFLP maps for both parents were developed using 97 individuals from population ND5873. The total lengths mapped for ND4382-19 and Chipeta were 1,883 and 1,021 cM, respectively. The marker for Lep was located at the distal end of simplex-coupling linkage group R37. Expansion of the initial mapping population and analysis of Lep-containing individuals allowed us to identify the linkage group (R35) that enabled synthesis of AL. By the use of simple sequence repeat markers, linkage group R37 (Lep) and linkage group R35 (AL) have been identified as homologs of chromosomes II and VIII, respectively.H. Casper (deceased)     

Nuclear and cytoplasmic genome components of Solanum tuberosum + S. chacoense somatic hybrids and three SSR alleles related to bacterial wilt resistance

The somatic hybrids were derived previously from protoplast fusion between Solanum tuberosum and S. chacoense to gain the bacterial wilt resistance from the wild species. The genome components analysis in the present research was to clarify the nuclear and cytoplasmic composition of the hybrids, to explore the molecular markers associated with the resistance, and provide information for better use of these hybrids in potato breeding. One hundred and eight nuclear SSR markers and five cytoplasmic specific primers polymorphic between the fusion parents were used to detect the genome components of 44 somatic hybrids. The bacterial wilt resistance was assessed thrice by inoculating the in vitro plants with a bacterial suspension of race 1. The disease index, relative disease index, and resistance level were assigned to each hybrid, which were further analyzed in relation to the molecular markers for elucidating the potential genetic base of the resistance. All of the 317 parental unique nuclear SSR alleles appeared in the somatic hybrids with some variations in the number of bands detected. Nearly 80 % of the hybrids randomly showed the chloroplast pattern of one parent, and most of the hybrids exhibited a fused mitochondrial DNA pattern. One hundred and nine specific SSR alleles of S. chacoense were analyzed for their relationship with the disease index of the hybrids, and three alleles were identified to be significantly associated with the resistance. Selection for the resistant SSR alleles of S. chacoense may increase the possibility of producing resistant pedigrees.     

The effect of genetic transformations for pest resistance on foliar solanidine-based glycoalkaloids of potato (Solatium tuberosuni)

Foliage of potato cv. Desiree was harvested from glasshouse‐cultivated plants of five experimental transgenic lines expressing three different insecticidal proteins (snowdrop lectin, Galanthus nivalis agglutinin (GNA); jackbean lectin, Concanavalin A (Con A), cowpea trypsin inhibitor; (CpTi)), tissue‐cultured control plants and standard control (non‐tissue cultured) plants. The foliage was subdivided into stems, upper, middle and lower leaves and analysed separately by HPLC for the solanidine‐based glycoalkaloids a‐solanine and a‐chaconine. The results demonstrate that one or more stages in the plant transformation process (i.e. insecticidal‐ and marker‐gene insertions, gene expression and tissue culture) resulted in a lower level of leaf glycoalkaloids than that found in either the tissue‐cultured controls or standard controls, based on the selected potato lines transformed for insecticidal protein expression. However, the distribution of glycoalkaloids throughout the plant foliage was unaffected by genetic transformation and tissue culture, with the highest glycoalkaloid levels being observed in the top third of the plant. The importance of investigating unexpected effects of genetic engineering on plant secondary metabolism is discussed from an ecological viewpoint.     

REPRODUCTIVE ISOLATION BARRIERS TO GENE EXCHANGE BETWEEN SOLANUM CHACOENSE AND S. COMMERSONII (SOLANACEAE)

In contrast to the general intercompatibility of diploid species of tuber-bearing potatoes of South America, Solanum chacoense Bitter and S. commersonii Dunal have well-established internal barriers to gene exchange. Pollen tubes of S. commersonii are inhibited in growth down the style of S. chacoense. While S. chacoense pollen tubes are not inhibited in S. commersonii stylar tissue, seed development is abnormal, resulting in production of large numbers of seeds that are not filled out. Even the apparently normal F₁ seed germinated poorly; approximately 0.1 germinable seeds were set per cross as compared with over 30 seeds per cross following intraspecific crosses. Lowland S. chacoense, including populations sympatric with S. commersonii, were not more effectively isolated from S. commersonii than were highland S. chacoense populations, over 500 km from the nearest S. commersonii. The F₁ hybrids produced were vigorous and highly fertile. Crosses between unrelated S. commersonii-chacoense F₁ hybrids produced only 1.3 germinable seeds per cross. This reduction resulted from low numbers of berries set, few seeds per berry, and low seed germination. The F₂ progeny were vigorous, but while many were both male and female fertile there were indications of an increase in sterility over that of the parents and F₁'s. The data were interpreted as indicating that the isolation barriers result from polygenic differences between the parent species and are composed principally of genes that function in several of the processes of seed setting. The accumulation of genes that keep these species isolated was interpreted as not having developed as a result of selection for such isolation in the region of overlap (Wallace effect), but rather as a byproduct of divergence that occurred during their allopatric evolution.     

Changes in antioxidant systems in sunflower partial resistant and susceptible lines as affected by Sclerotinia sclerotiorum

Sunflower, Helianthus annuus L., is a major oil-seed crop widely cultivated throughout the globe. White mold, caused by necrotrophic pathogen Sclerotinia sclerotiorum (Lib.) de Bary, is a common and widespread disease of sunflower. Changes in various physiological activities such as production of malondialdehyde (MDA) as the main end product of lipid peroxidation, enzymatic and non-enzymatic activities and proline content, were investigated in leaves and stems of sunflower lines either resistant or susceptible to S. sclerotiorum. The results showed accumulation of high amount of free proline in the leaves of the resistant line. S. sclerotiorum invasion resulted in increase in the MDA content in both lines compared to the controls. Superoxide dismutase, ascorbate peroxidase and guaiacol peroxidase activities significantly increased in the stem and leaves of inoculated lines. In the resistant line, Sclerotinia infection significantly induced catalase (CAT) activity both in the stem and leaves of contaminated plants. The activity of CAT was significantly decreased in susceptible line. Based on our results the activity of antioxidant enzymes was much pronounced in sunflower resistant line than in susceptible one. Studied lines showed distinctive activity considering different biochemical parameters, which may point to further directions in exploring host-pathogen interaction and lead to selection and production of new lines to achieve an improvement of plant adaptation to pathogen.     

The S11 and S13 self incompatibility alleles in Solanum chacoense Bitt. are remarkably similar

A genomic clone of the S11 allele from the self-incompatibility locus (S locus) in Solanum chacoense Bitt. has been isolated by cross-hybridization to the S. chacoense S13 allele and sequenced. The sequence of the S11 allele contains all the features expected for S genes of the Solanaceae, and S11 expression, as assessed by northern blots and RNA-PCR, was similar to that of other S. chacoense S alleles. The S11 protein sequence shares 95% identity with the phenotypically distinct S13 protein of S. chacoense and is the gametophytic S allele with the highest similarity to an existing allele so far discovered. Only 10 amino acid changes differentiate the mature proteins from these two alleles, which sets a new lower limit to the number of changes that can produce an altered S allele specificity. The amino acid substitutions are not clustered, suggesting that an accumulation of random point mutations can generate S allele diversity. The S11 intron is unusual in that it could be translated in frame with the coding sequence, thus suggesting an additional mechanism for the generation of new S alleles.     

Stomatal Response of some Cultivated and Wild Tuber-bearing Potatoes in Warm Tropics as Influenced by Water Deficits

Leaf resistances of 14 cultivated potato genotypes (Solanumspp) and three tuber-bearing wild Solanum species were comparedwhen plants were grown under water stress at two tropical sitesFactors investigated were diurnal changes in leaf resistance,the effect of plant age, transient drought versus well-wateredconditions of potted and field-grown plants These measurementswere carried out in order to determine the stomatal behaviourof tuber-bearing genotypes and species Significant genotypic differences in leaf resistances were notedwithin the cultivated genotypes All genotypes had higher resistanceswhen water-stressed, but LT-7 appeared to have the lowest leafresistances Genetic differences in stomatal behaviour of tuber-bearingSolanum species were confirmed Abaxial stomatal resistancesof water-stressed plants of the species ranged between 1 74and 13 8 s cm^–1 Stomata of S chacoense were less affectedby drought (three-fold) than S tuberosum (four-fold) The greatesteffect was on S jungasense (five-fold) and on S raphanifoliumThese data show that stomata behaviour among tuber-bearing Solanumspecies is sufficiently different to warrant investigationsof drought-resistance in potato species under dry hot conditions Solanum tuberosum L., Solanum raphanifolium, Solanum chacoense, Solanum jungasense, leaf resistance     

Improve freezing tolerance in transgenic poplar by overexpressing a ω-3 fatty acid desaturase gene

Polyunsaturated fatty acids are a major component of glycerolipids in plant cellular membranes, and play important role in the maintenance of its fluidity. In this study, we isolated a novel ω-3 fatty acid desaturase gene (PtFAD3) from Populus tomentosa Carr. and transformed it into hybrid poplar (Populus alba × P. glandulossa) K84 lines by overexpression and RNAi silencing methods. Quantitative PCR analysis revealed varied expression levels of PtFAD3 among the transgenic lines. Two overexpressing lines (OE lines) and two gene down-regulated RNAi lines (DR lines) were chosen for further analysis. The level of the major trienoic fatty acid species, linolenic acid, increased by an average of 10% in the two OE lines and decreased by an average of 5% in the two DR lines. When young poplar cuttings with three leaves were exposed to a freezing temperature (?4°C) for 3 h without cold acclimation, survival rates of the two independent OE lines (70 and 77%) were far superior to the wild type (36.7%) and the two DR lines (10% for both). Damage caused by freezing was alleviated significantly in the two OE lines compared to wild-type controls. Phenotypic evaluation indicated that the variable responses to freezing were attributable to genotypic variation. Therefore, in the case of poplar, a significant increase in freezing tolerance was achieved on a small scale following the introduction of PtFAD3. This strategy can be used for freezing tolerance breeding in hybrid poplar and other economically important forest trees.     

Transgenic disease-resistant wheat does not affect the clonal performance of the aphid Metopolophium dirhodum Walker

Ever since the introduction of transgenic crops one of the main concerns has been their potential impact on non-target organisms. In this study we looked at the impact of transgenic disease-resistant wheat on different clones of the aphid Metopolophium dirhodum. Looking at different clones allowed us to assess whether impacts depended on aphid clone and whether there were aphid clone×wheat line interactions (genotype×environment interactions). The performance of 30 aphid clones on four different transgenic wheat lines and their corresponding control lines was studied in a life-table experiment assessing the following aphid life-history parameters: development time, adult weight, daily fecundity, total offspring number and the fitness estimate F_i′. As expected, we found significant variation among aphid clones for all the measured life-history parameters. However, our experiments did not reveal any major impact of the transgenic wheat lines on aphid performance. The only significant difference was found for total offspring number which was reduced by 3.33% on the transgenic wheat lines compared with the control lines. There was no evidence for a genotype×environment interaction between aphid clones and wheat lines. In sum, our results imply that the genetically modified plants used in this assay were of similar host plant quality as the non-transformed control lines and that the introduced transgene had no major effect on the performance of individual aphid clones.     

Organization of the Drosophila melanogaster SF1 insulator and its role in transcription regulation in transgenic lines

The SF1 insulator was found to contain a polyadenylation signal, which corresponded to the functional polyadenylation signal in embryonic S2 cells and the transgenic lines of Drosophila and bi-directional promoter that functioned in S2 cells. The studies performed did not confirm the ability of the SF1 insulator to protect expression of reporter gene white from the chromosome position effect in transgenic lines.     

Allelic variation in genes contributing to glycoalkaloid biosynthesis in a diploid interspecific population of potato

Key message

Variation for allelic state within genes of both primary and secondary metabolism influences the quantity and quality of steroidal glycoalkaloids produced in potato leaves.

Abstract

Genetic factors associated with the biosynthesis and accumulation of steroidal glycoalkaloids (SGAs) in potato were addressed by a candidate gene approach and whole genome single nucleotide polymorphism (SNP) genotyping. Allelic sequences spanning coding regions of four candidate genes [3-hydroxy-3-methylglutaryl coenzyme A reductase 2 (HMG2); 2,3-squalene epoxidase; solanidine galactosyltransferase; and solanidine glucosyltransferase (SGT2)] were obtained from two potato species differing in SGA composition: Solanum chacoense (chc 80-1) and Solanum tuberosum group Phureja (phu DH). An F₂ population was genotyped and foliar SGAs quantified. The concentrations of α-solanine, α-chaconine, leptine I, leptine II and total SGAs varied broadly among F₂ individuals. F₂ plants with chc 80-1 alleles for HMG2 or SGT2 accumulated significantly greater leptines and total SGAs compared to plants with phu DH alleles. Plants with chc 80-1 alleles at both loci expressed the greatest levels of total SGAs, α-solanine and α-chaconine. A significant positive correlation was found between α-solanine and α-chaconine accumulation as well as between leptine I and leptine II. A whole genome SNP genotyping analysis of an F₂ subsample verified the importance of chc 80-1 alleles at HMG2 and SGT2 for SGA synthesis and accumulation and suggested additional candidate genes including some previously associated with SGA production. Loci on five and seven potato pseudochromosomes were associated with synthesis and accumulation of SGAs, respectively. Two loci, on pseudochromosomes 1 and 6, explained phenotypic segregation of α-solanine and α-chaconine synthesis. Knowledge of the genetic factors influencing SGA production in potato may assist breeding for pest resistance.     

New glycoxanthones and flavanone glycosides of Hoppea dichotoma

The whole plant of Hoppea dichotoma has been shown to contain eleven xanthones, two flavanones and two flavones, as major chemical entities, five of which are new naturally occurring compounds. Additionally, four known triterpenes, gluanone, gluanol, friedelin and friedelin-3β-ol, have been isolated as minor entities. The taxonomic significance of the chemical characters of H. dichotoma, which are closely similar to those of Canscora decussata, is appraised.