Haploids of tetraploid (2n=4x=48) Mexican potato species—Their extraction,cytology and crossability

Successful isolation of haploids (2n=2x=24) from 4x(2EBN) Mexican species was achieved by using Modified Wenzel (MW) and Nitsch and Nitsch (NN) media for anther culture. Both media, MW and NN, gave consistent regeneration of plantlets from 4x(2EBN) Mexican species, and responses to the media were both species and accession (PI) specific. Plantlets regenerated directly from microspores, by-passing the callus cycle, were mostly haploid (2n=2x=24). Haploid plants appeared smaller and weaker than their parents with a drastic reduction in both male and female fertility. Abnormalities observed during meiosis and the lack of success in crossing confirmed the effect of the meiotic irregularities observed in the haploids. A total of 290 seeds was obtained from the 4x × 2x crosses of 4x Mexican species with irradiated pollen of 2xS. cardiophyllum and 2xS. chancayense; however, no haploid (2n=2x=24) plants resulted. Extraction of haploids from colchicinedoubled 4x (2EBN) or natural 6x(4EBN) Mexican species using a Phureja pollinator was examined to provide clues to the potential of using 2x (1EBN) pollinators for haploid induction in 4x(2EBN) × 2x(1EBN) crosses.     

Hybridization between Gp. Tuberosum Haploids and 1EBN wild potato species

Diploid wild potato species, classified as 1EBN (Endosperm Balance Number), do not cross with tetraploid or diploid forms ofS. tuberosum Gp. Tuberosum. The crossing of 2n pollen producing 1EBN clones as well as chromosome-doubled 1EBN clones with Gp. Tuberosum haploids was initiated to overcome this hybridization barrier. The screening of eleven 1EBN species,S. brachistotrichum, S. bulbocastanum, S. cardiophyllum, S. chancayense, S. commersonii, S. etuberosum, S. fernandezianum, S. jamesii, S. mochicense, S. pinnatisectum andS. trifidum, resulted in the identification of 2n pollen producers in eight of the species examined. Direct hybridization with Gp. Tuberosum haploids utilizing the above-mentioned crossing scheme was successful withS. chancayense andS. commersonii. The hybrids obtained, however, were male and female sterile. Abnormal microsporogenesis in the hybrids was postulated to be the result of an interaction between Gp. Tuberosum cytoplasm and nuclear genes contributed by the 1EBN male parent. Analyses of the growth of 1EBN species’ pollen tubes in the stigma/style of Gp. Tuberosum haploids were also conducted. Interspecific incompatibilities were observed with the most severe forms found with the use of species in the Series Etuberosa. The use of the Endosperm Balance Number theory can aid in designing crosses which overcome barriers to successful endosperm development. Sterilities in the hybrids obtained as well as interspecific incompatibilities will also need to be addressed, however, before 1EBN species germplasm can be successfully utilized for the improvement of the cultivated potato.     

Interspecific crossability and cytogenetic analysis of sexual progenies of Mexican wild diploid 1EBN speciesSolanum pinnatisectum andS. cardiophyllum

Mexican wild diploid species,Solanum pinnatisectum (S. pnt) (2n=2x=24, 1EBN: endosperm balance number) is a useful germplasm source of late blight and Colorado potato beetle (CPB) resistance in potato improvement. However, it is very difficult to cross this species with other 1EBNSolanum species. Sexual hybrids among three accessions ofS. pnt and two accessions ofS. cardiophyllum (S. cph) (2n=2x=24, 1EBN) were studied. There were large differences in the cross-compatibility among the genotypes and accessions from these two 2x-1EBN species. Interspecific incompatibility existed in the crosses betweenS. pnt andS. cph, in whichS. cph functioned only as the male parent. The crosses with accessions PI 275236 ofS. pnt produced 35 hybrids following the conventional propagation procedures. Seven hybrids were obtained with theS. pnt accession PI 275233 with the aid of embryo rescue, while hybridization involving accession PI 253214 resulted in no fruit. The cytogenetic analysis indicated that all of the hybrids derived from crosses of accession PI 275233 ofS. pnt withS. cph had 2n = 24 chromosomes and were pollen fertile. Crosses involving accession PI 275236 ofS. pnt produced both 2x (2n=24) and 3x (2n=36) hybrids. Meiotic analysis at metaphase I of pollen mother cells (PMCs) showed a fairly high rate of chromosome pairing that averaged between 10.10 and 11.10 bivalents per cell in most of the 2x hybrids, indicatingS. pnt andS. cph have the similar genome. A high frequency of trivalents and quadrivalents were observed in 3x hybrids derived fromS. pnt 2 ×S. cph 1, indicating homology exists among these chromosomes. These results suggest that specific gene(s) and not either differences between genomes or EBN controlled interspecific crossability and embryo development. The disease and insect tests on the 2x and 3x hybrids revealed that all have high levels of resistance to both late blight and CPB. Thus it could appear that selection of genotypes is a key for successful interspecific hybridization when using Mexican wild diploid species as a source of economic important traits.     

UseoftheEndospermBalanceNumber(EBN)inPotatoGeneticsandBreeding

在马铃薯初花期喷施植物生长调节剂多效唑,试验结果表明:多效唑显著抑制植株的高度,单株结薯数下降,大薯率上升,当每667m2喷施多效唑75g,浓度在2.5g.L-1时是本试验中提高产量的最佳浓度,干物质和淀粉均有明显的提高。说明喷施多效唑具有控上促下的作用,从而有利于提高产量和品质。     

多效唑对晋薯7号马铃薯的调控作用

在马铃薯初花期喷施植物生长调节剂多效唑,试验结果表明:多效唑显著抑制植株的高度,单株结薯数下降,大薯率上升,当每667m2喷施多效唑75g,浓度在2.5g.L-1时是本试验中提高产量的最佳浓度,干物质和淀粉均有明显的提高。说明喷施多效唑具有控上促下的作用,从而有利于提高产量和品质。     

Seed set in 4x x 2x crosses as related to 2n pollen frequency

Seeds per fruit following 4x × 2x crosses in potato are roughly related to the frequency of 2n pollen. Our goal was to examine precisely the relationship between frequency of stainable 2n pollen and seeds per fruit (s/f) in 4x × 2x crosses. A 4x cultivar with high female fertility and 21 2n pollen producing 2x hybrids were used as seed and pollen parents, respectively. 2n pollen frequencies varied from 1 to 99% in the 2x hybrids and seed set ranged from 0 to 305 s/f in 4x×2x crosses. Mean seeds per fruit increased in response to increases from 1 to 50% 2n pollen, but did not increase in response to using a pollen sample with a 2n pollen frequency over 60%. Seed set averaged 11 s/f corresponding to pollen samples with 1–5% 2n pollen, 50 s/f at 10–20%, 125 s/f at 20–30%, and over 170 s/f at 2n pollen frequencies over 60%. Seed set in 210 control 4x × 4x crosses indicated that equivalent seed set occurs in 4x × 2x crosses when 2n pollen frequencies are over 20–30%. The 2x hybrids differed in mean and variation of 2n pollen frequencies and seed set in 4x × 2x crosses. One 2x hybrid with a meiotic aberration (sy-3) resulting in all univalents at Metaphase I (ordinarily leading to male sterility) and parallel spindles (ps) at Anaphase II had close to 100% 2n pollen (of the stainable pollen) and gave equivalent s/f in 4x × 2x crosses to s/f in 4x × 4x crosses. It is apparent that ample seed set occurs for using the 4x × 2x breeding scheme with 2n pollen frequencies over 10% and that even very low 2n pollen frequencies (1–10%) provide seed for the 4x×2x breeding approach.     

Foliar resistance toPhytophthora infestans (Mont.) de Bary (US-8) in 13 Mexican and South AmericanSolarium species having EBNs of 1, 2, and 4 and implications for breeding

The recent US-8 clonal lineage ofPhytophthora infestans, the pathogen causing late blight (LB) disease, is one of the most widespread and difficult to control. Sources of foliar resistance to US-8 were evaluated in 1927 seedling genotypes by sampling 49 plant introductions (PIs) representing 13 Mexican and South AmericanSolanum species having an Endosperm Balance Number (EBN) of 1, 2, or 4, and one species with an unknown EBN. Species evaluated were 2x(1EBN)S. bulbocastanum, S. cardiophyllum, S. commersonii, S. pinnatisectum, andS. trifidum; 2x(2EBN)S. berthaultii, S. megistacrolobum, S. microdontum, andS. verrucosum; 4x(2EBN)S. fendleri andS. stoloniferum; 6x(4EBN)S. guerreroense;2x unknown EBNS. polyadenium; as well as4x S. tuberosum families as controls. Foliar resistance in the field was monitored in 1998 and 1999. Resistance differed relative to species geographic origin, EBN, species themselves, PIs within a species, and genotypes within a PI. In 1998 Mexican germplasm had a lower area under the disease progress curve (AUDPC, 1140) and greater resistance than South American germplasm (1601), while the 1EBN (1119) and unknown EBN (1075) species had greater resistance than 2EBN (1470) and 4EBN (1888) species. AUDPC ranged among species from 555 (S. cardiophyllum) to 1932 (S. megistacrolobum) and among PIs within a species most widely forS. bulbocastanum (267–1007) and least widely forS.polyadenium (1019–1179). In 1999 LB symptoms were not observed on the species before an early freeze; however, all 1999S. tuberosum families showed LB symptoms and segregated for resistance, pointing to greater resistance in the species than in cultivated germplasm. Resistance found in diploid 2EBN germplasm is more accessible due to greater crossability with cultivated germplasm; however, though more difficult to introgress, resistance found in some of the more reproductively isolated 1EBN germplasm is worth pursuing.     

Introgression of cold (4 C) chipping from 2x (2 endosperm balance number) potato species into 4x(4EBN) cultivated potato using sexual polyploidization

Cold-chipping cultivars could reduce microbial spoilage and chemical use due to cold storage of chipping potatoes. Sexual polyploidization breeding schemes that introgress cold chipping from diploid potato species may develop improved cultivars at an accelerated rate. The research objectives were (1) to determine the breeding behavior of cold chipping introgressed from 2× 2 Endosperm Balance Number (EBN) potato species into 4×(4EBN) progeny using sexual polyploidization, and (2) to determine if differences exist between 2n gametes and n tetrasomic gametes for transmission of cold chipping. Experimental families of 4×(4EBN) progeny were synthesized using sexual polyploidization (2× × 4× and 4× × 2×) and 4× × 4× matings using cold-chippingS. phureja and cultivated potato -wild species (C -W) hybrid parents. Control families from 4× × 4× matings using as parents current industry leading germplasm were also made. Progeny were field grown and evaluated for chip color (1 light-10 dark, ≤4.0 is industry acceptable) after 3 and 6 months storage at 4 C. A larger percentage of progeny from experimental families had acceptable chip color compared to control families, likely due to introgression of cold-chipping alleles from diploid potato species. Out of experimental families, the best chip color means, highest phenotypic variance, and greatest percentage of acceptable chipping progeny resulted in 2× × 4× and 4× × 2× matings. The ability of 2n gametes to transmit dominant alleles to high frequencies of 4× progeny may contribute to the superiority of 4× -2× crosses. Breeding schemes that use sexual polyploidization in conjunction with early generation selection should rapidly develop 4× cold-chipping germplasm.     

Variation for 2n pollen production and male fertility in wildSolanum germplasm resistant toPhytophthora infestans (Mont.) de Bary (US-8)

Wild potato species possess genetic variability for valuable traits including resistance toPhytophthora infestans, the causal agent for potato late blight disease. Breeding schemes using 2n gametes are a powerful tool for introgressing these traits. Forty-five plant introductions (PIs) across 12Solanum species representing various Endosperm Balance Numbers (EBN) and having resistance toP. infestans (US-8, isolate ND 95-2) were screened for 2n pollen production and male fertility. Species evaluated were 2x(lEBN)S. bulbocastanum, S. cardiophyllum, S. pinnatisectum, andS. trifidum; 2x(2EBN)S. berthaultii, S. megistacrolobum, S. microdontum, andS. verrucosum; 4x(2EBN)S. fendleri andS. stoloniferum; 6x(4EBN)S. guerreroense; and 2x(unknown EBN)S. polyadenium. Acetocarmine staining and cytological analyses were used to determine 2n pollen production and pollen stainability from genotypes grown across three locations. Based on frequency, genotypes were assigned to 2n pollen production categories (0≤1%, Low=1%–10%, Medium=11%–60%, and High≥60%) and pollen stainability categories (0≤1%, 1=196–5%, 2=6%–10%, 3=11%–20%, 4=21%–50%, and 5≥50%). Eleven of the 12 species and 37 of the 45 PIs examined contained genotypes producing ≥1% 2n pollen with ranges of 0%–63.6% and 0%–81.8%, respectively. Dependence was found between location and pollen stainability, EBN and pollen stainability, but not for location and frequency of genotypes with 2n pollen. Stability for 2n pollen production was observed across environments. Among the 1EBN germplasm, dependence was found between 2n pollen and pollen stainability. Resistance toP. infestans was found to be independent of 2n pollen production; therefore, identifying genotypes combining 2n pollen production, fertility, and resistance toP. infestans was possible.     

The use of bees for the purpose of inter-mating in potato

The pollination behavior of bumblebees and honey bees was studied on potato flowers in screened enclosures and in the field. In enclosures, the domestic honey bee (Apis mellifera L.) and the bumblebee species,Bombus fervidus Fabricius, seemed to lack any “cue” to initiate visitation of the flowers. When honey was placed on a few flowers, visitation was stimulated. The honey bee tore and chewed at the anther cone to collect pollen, whileB. fervidus probed for nectar which was not present. Shortly afterward, both species ceased visitation and could not be induced to visit further, regardless of the honey stimulus. Neither species was effective as a pollinator. It is concluded that neither species will be useful for large-scale crossing of potato populations. However, another bumblebee species,Bombus impatiens Cresson, is very effective in pollinating potatoes in the field. Manipulating the behavior of such indigenous populations of bumblebees is likely to be the most effective method of exploiting insect pollination in the potato.     

Solanum irosinum,new Peruvian tuber bearing species resistant toPhytophthora infestans

The greatest variety of tuberiferousSolanums of the Western Hemisphere is found in the Peruvian Andes, where, in spite of extensive explorations, new material is still found, such as the species which is here presented under the name ofSolanum irosinum. This is a diploid species; that is, it has 2n = 2x = 24 chromosomes and belongs to the Conicibaccata series. It could be used as a new source of breeding because it offers great resistance to attacks byPhytophthora infestans, the most serious disease of potato crops in the world.     

Potato haploid performance in 2x × 4x crosses

Nineteen haploids (2n = 2x = 24) extracted from four tetraploids ofSolanum tuberosum (2n = 4x = 48) with high 2n egg frequencies and known mode of 2n egg formation were evaluated via 4x progeny from 2x × 4x crosses (haploid × cultivar) for total tuber yield, tuber appearance and tuber set. Ninety-eight, 4x families (15-20 genotypes each) were planted in the randomized complete block design with two replications at two locations. Analyses of variance were conducted for all haploids over three common testers, and each of the four groups of haploids individually over a variable number of testers. The performance of haploids, based on general combining ability effects, was consistent for all traits, when tested with either three or more testers. Diploid clones can be evaluated in 2x × 4x crosses using three unrelated adapted 4x testers. Nonorthogonal comparisons indicated no significant differences for all traits among four groups of haploids, and between modes of 2n egg formation. Significant variation within each group of haploids indicates that selection at the haploid level should be carried out among haploids from either the same or different cultivars. Superior haploids can be utilized in 2x × 4x and 2x × 2x breeding schemes to generate 4x clones.     

Analytical methods for quality control of dried potato flakes

A set of analytical methods was used for the characterization of the oxidative degradation of dehydrated potato flakes. Headspace gas analysis for straight chain hydrocarbon (Ethane-hexane) and for oxygen was used to directly follow the oxidation. Bleaching was followed by quantitatively determining the carotenoid concentration. Analytical results obtained showed a strong association with taste testing results. The data reported show the effectiveness of natural and synthetic antioxidants for the stabilization of dried potato flakes.     

Flower strip networks offer promising long term effects on pollinator species richness in intensively cultivated agricultural areas

Background

Intensively cultivated agricultural landscapes often suffer from substantial pollinator losses, which may be leading to decreasing pollination services for crops and wild flowering plants. Conservation measures that are easy to implement and accepted by farmers are needed to halt a further loss of pollinators in large areas under intensive agricultural management. Here we report the results of a replicated long-term study involving networks of mostly perennial flower strips covering 10% of a conventionally managed agricultural landscape in southwestern Germany.

Results

We demonstrate the considerable success of these measures for wild bee and butterfly species richness over an observation period of 5 years. Overall species richness of bees and butterflies but also the numbers of specialist bee species clearly increased in the ecological enhancement areas as compared to the control areas without ecological enhancement measures. A three to five-fold increase in species richness was found after more than 2 years of enhancement of the areas with flower strips. Oligolectic bee species increased significantly only after the third year.

Conclusions

In our long-term field experiment we used a large variety of seed mixtures and temporal variation in seeding time, ensured continuity of the flower-strips by using perennial seed mixtures and distributed the measures over c. 10% of the landscape. This led to an increase in pollinator abundance, suggesting that these measures may be instrumental for the successful support of pollinators. These measures may ensure the availability of a network of diverse habitats and foraging resources for pollinators throughout the year, as well as nesting sites for many species. The measures are applied in-field and are suitable for application in areas under intensive agriculture. We propose that flower strip networks should be implemented much more in the upcoming CAP (common agricultural policy) reform in the European Union and promoted more by advisory services for farmers.

     

Inheritance of glandular trichomes in crosses withSolanum berthaultii

The inheritance of trichome density and droplet size was investigated in diploid and tetraploid crosses. In haploidTuberosum ×Solanum berthaultii and (S.phureja ×haploid Tuberosum) × S.berthaultii crosses, droplet size on type B trichomes was small or droplets were lacking on F₁ hybrids. Parental phenotypes were recovered in the F₂ and backcross generations. Trichome density was more variable within a clone that was droplet size. Density of both type A and type B trichomes appears to be under the control of small numbers of genes, as parental phenotypes were recovered in the F₂ and backcross generations. The variation observed for each of the three traits studied could not be explained by hypotheses based on the segregation of one or two loci. The insect resistance ofSolanum berthaultii appears to be due to the interaction of several chemical and physical factors, thus potato breeders should handle it as a quantitatively inherited trait.     

Detection of potato viruses X and S in tissue culture plantlets

The latex agglutination test (LAT) and enzyme-linked immunosorbent assay (ELISA) were evaluated in separate studies for their ability to detect potato virus X (PVX) and potato virus S (PVS) in tissue culture plantlets. Healthy and infected clonal lines of several potato cultivars were used. LAT was unsatisfactory because only low levels of agglutination were obtained with infected samples, and because variable and inconsistent results were obtained with both healthy and infected clones. ELISA, however, consistently gave high spectrophotometric readings and intense visual reactions for infected but not for healthy clones. The results indicate that ELISA can be used to detect PVX and PVS in tissue culture plantlets, and in programs where tissue culture is employed, early detection and elimination of infected plantlets is possible.     

Inheritance of tuber initiation in tuber bearingSolanum as influenced by photoperiod

Eight potato clones (three day-neutral (DN) Tuberosum, one dayneutral and four short-day (SD) Andigena) were used as parents. Eight genetic sets bearing P₁, P₂, P_1?, P_2?, F₁, F₂, BC₁, and BC₂ generations were developed. Two sets were DN×DN crosses, four SD×DN, and two SD×SD. Tuber initiation (TI) was evaluated in growth chambers with daylengths of 11, 13, and 15 hours. The stolons of each genotype were examined every eight days between the 38th and 86th day after planting. F₁ generation means from SD×DN crosses showed complete dominance of SD reaction (late TI) over DN reaction (early TI). F₁ generation means from DN × DN and SD× SD crosses showed either no dominance or a slight tendency towards the late TI parent. Within plot variances of the selfed generations suggested a variable degree of parental heterozygosity for the loci controlling TI. Andigena clones showed the greatest heterozygosity. Bell shaped frequency distributions in the segregating generations indicated that TI was under the control of several genes. It appears also that major and minor genes were involved. Broad sense heritability estimates of about 90% at 11 hours and 55% at 15 hours of daylength were computed.     

Inheritance of potato chip color at the 24-chromosome level

Potato chip color is an important trait in determining quality and acceptability of cultivars for processing. Therefore, the genetics of potato chip color is important in varietal breeding. The use of 2xS. tuberosum Group Tuberosum haploid × 2x wild species hybrids provides disomic inheritance for genetic analysis of chip color. Two hundred forty haploid-species hybrids representing crosses between 15 haploids and 10 species were evaluated for potato chip color after 21 days reconditioning (18 to 20C) following 6 months at 4C. Phenotypic variation for chip color was found among the haploid-species hybrids. Crosses between good, medium, and poor performing clones were made in all combinations and 136 families were obtained. Progeny were evaluated for reversion resistance (chipping direct from 10C storage) and reconditioning (chipping from 4C after tuber warming) chip color. Variation for chip color was found among the progeny for each trait. Significant correlations between reversion resistance and reconditioning were found. Yet, the use of at least two chip tests (one for reversion resistance and one for reconditioning) is more informative than the use of one test and subsequent predictions made, regarding the other trait. A three locus hypothesis for both reversion resistance and reconditioning is proposed for the inheritance of each trait. One dominant allele at each of the three loci is necessary for good chipping. One or two loci may be common to both characteristics. Good chipping 2x hybrids that produce 2n pollen can be used to transfer this trait to the 4x level through use of 4x × 2x crosses.     

A rapid and simple method for determination of potato chloroplast DNA type

A procedure for restriction enzyme analysis of the potato chloroplast DNA (ctDNA) is described. The advantages of this method are: 1) rapid determination of ctDNA type, 2) no ultracentrifugation, and 3) low cost of analyses. This method makes it easy to distinguish the ctDNA types of wild and cultivated potato accessions.     

Eradication of potato virus X from potato by ribavirin treatment of cultured potato shoot tips

Ribavirin treatment of cultured potato shoot tips was tested as a means of eradicating potato virus X (PVX) from two potato cultivars. Shoot tips were cultured on liquid medium containing 0, 1, 10 or 100μg/ml ribavirin. Cultures were evaluated periodically for viability, and scored for vigor on a relative growth scale. Developed plantlets were assayed for PVX by transmission tests toGomphrena globosa. Ribavirin treatment was phytotoxic at all tested concentrations, and lethal to all cultivars treated at 100 μg/ml. Treatment also delayed plantlet development by up to 2 months at 1 and 10 μg/ml as compared with nontreated controls. PVX assays indicated that 80 and 83% of the plantlets were free of PVX following treatment with 10 μg/ml for cultivars Russet Burbank and Red McClure, respectively. Five and 6% of the plantlets developed from the 1 μg/ml treatment were PVX-free, whereas 0 and 2% of the controls were PVX-free for the same cultivars. Six to 8 months were required to develop plants from shoot-tip cultures treated with 10 μg/ml ribavirin.