Intraspecific somatic and sexual hybridisation between dihaploid lines of Solanum tuberosum L.: evaluation of morphological traits and resistance to late blight Phytophthora infestans (Mont.) de Bary in the foliage

Intraspecific tetraploid somatic and sexual hybrid plants have been resynthesised following protoplast fusion and by sexual crosses between two dihaploid potato (Solanum tuberosum) lines each possessing complementary agronomic traits. The dihaploid PDH 40 possesses good tuber shape and yield but has foliage susceptibility to late blight (Phytophthora infestans). On the other hand, the dihaploid PDH 727 possesses resistance to blight in the foliage but has a low yield of small and irregular shaped tubers. Since it was only possible to use a partial selection strategy based on culture media to facilitate recovery of somatic hybrid plants-further morphological and esterase isozyme based characterisations were performed to identify somatic hybrid plants from amongst the non-hybrid plant material. When the blight resistance of both the intraspecific somatic and sexual hybrid plants was assessed there was no significant difference in the mean resistance value and it was intermediate between those of their parents. However, the range of resistance was much wider among the sexual hybrids than among the plants derived from somatic fusion. An assessment of tuber yield between tetraploid sexual and somatic hybrids showed no significant difference and it was higher than that of either parent value. The implication of these results in the context of potato genetics and breeding is discussed.     

Production of somatic hybrids between S. tuberosum L. and late blight resistant Mexican wild potato species

Interspecific somatic hybrids between dihaploid breeding clones of potato, S. tuberosum and two accessions of wild Mexican species S. pinnatisectum and the hybrid line S. pinnatisectum × S. bulbocastanum were regenerated following electrofusion of mesophyll protoplasts to combine important agricultural traits of S. tuberosum and a high level of late blight resistance from selected wild accessions. In two fusion combinations 239 calli were regenerated; 162 from 195 calli analysed were identified as hybrids by means of isozyme analysis of peroxidases and, for some hybrid clones, by RAPD analysis. Depending on the fusion combination, 47–89 percent of the somatic hybrids had the expected ploidy level and 7–16 percent were mixoploids. Somatic hybrids were phenotypically intermediate as compared to their parents and some of them were able to be backcrossed sexually with potato. Fertility and crossability depended on combination and ploidy level of the somatic hybrids. In tests with detached leaves the wild partner clones had a high late blight resistance score of 8,6 and 8,9; the susceptible tuberosum-partners of 2,8 and 3,5, respectively. Nearly 25 percent of somatic hybrids had a resistance level of 6 or higher in the first year of assessment. The average resistance value of most somatic hybrids was lower than the average parental level. The reasons for variation in resistance values are discussed in connection with the practical application of fusion hybrids. This revised version was published online in July 2006 with corrections to the Cover Date.     

Differentiation of the high molecular weight glutenin subunit D t x 2.1 of Aegilops tauschii indicated by partial sequences of its encoding gene and SSR markers

The devastating late blight pathogen Phytophthora infestans infects foliage as well as tubers of potato. To date, resistance breeding has often focused on foliage blight resistance, but tuber blight resistance is becoming more and more important in cultivated potatoes. In this study, a reliable tuber assay for resistance assessment was developed and foliage and tuber blight resistance (R) was compared in four mapping populations. In the RH4X-103 population, tuber blight resistance inherited independently from foliage blight resistance. Three specific R genes against P. infestans were segregating. The Rpi-abpt and R3a genes function as foliage-specific R genes, whereas the R1 gene acts on both foliage and tuber. In the segregating populations SHRH and RH94-076, tuber and foliage blight resistance correlated significantly, which suggests that resistance in foliage and tuber is conferred by the same gene (could be R3b) and quantitative trait loci (QTL), respectively. In the CE population neither tuber nor foliage resistance was observed.     

Transfer of resistance to Verticillium dahliae Kleb. from Solanum torvum S.W. into potato (Solanum tuberosum L.) by protoplast electrofusion

Summary Interspecific somatic hybrid plants were regenerated after electrofusion of mesophyll protoplasts with the objective of transferring resistance to Verticillium dahliae from Solanum torvum into potato. Early selection of the putative hybrids was based on differences in cultural behaviour of the parental and hybrid calli (particularly the ability of the latter to regenerate early) in combination with morphological markers. Four putative hybrids were recovered from hundreds of calli, probably resulting from complementation of the two parental genomes. The regenerates were tetraploids (2n=4×=48 chromosomes) and exhibited intermediate traits including leaf form, plant morphology and the presence of anthocyanin. The hybrid nature of the four selected plants was confirmed by examining isoenzyme patterns for isocitrate dehydrogenase (Idh), malate dehydrogenase (Mdh), phosphoglucoisomerase (Pgi) and 6-phosphogluconate dehydrogenase (6-Pgd). While the hybrid plants rooted readily and grew vigorously under in vitro conditions, in the greenhouse their development and growth were retarded by difficulties in rooting. When grafted on potato or S. torvum rootstocks, the hybrid plants recovered normal development and growth. Again, they exhibited intermediate morphological traits. Tests for resistance realized in vitro with medium containing 50% Verticillium wilt filtrate showed that all the somatic hybrids were resistant to the fungus filtrate.     

Identification of somatic hybrids of dihaploid Solanum tuberosum lines and S. brevidens by species specific RAPD patterns and assessment of disease resistance of the hybrids

Summary Symmetric somatic hybrids were produced by electrofusion of protoplasts of two dihaploid tuber-bearing potato (Solanum tuberosum L.) lines and Solanum brevidens Phil., a diploid non-tuber-bearing wild potato species. A total of 985 plants was obtained. Verification of nuclear hybridity of putative hybrids was based on additive RAPD patterns, general morphological characteristics and chromosome counts. 53 (90%) calli regenerated into plants which were identified as somatic hybrids. Most of the hybrids were aneuploids at the tetraploid (4×) or hexaploid (6×) level. The 20 hybrids tested expressed a high level of resistance to potato virus Y (PVY ^N ) characteristic of the S. brevidens parent. Resistance to late blight (Phytophthora infestans (Mont.) de Bary) varied between hybrids, but was on average better than that of the fusion parents. Resistance of hybrids to bacterial stem rot (Erwinia carotovora subsp. atroseptica (van Hall) Dye) was not superior to that of commercial potato cultivars.     

Somatic Fusion for Breeding of Tetraploid Potatoes

From two tetraploid, one Transformed tetraploid, one triploid and 11 dihaploid clones of Solanum tuberosum somatic hybrids were produced by polyethylene glycol mediated somatic fusion. The inter-dihaploid clones comprised clones of agronomic value, homozygous doubled monohaploids, and in vitro selected clones resistant t0 Fusarium or Phytophthora toxins. Presumptive hybrids were enriched at the callus Stage in vitro by using differentiating media and by growth characteristics; further identification was performed by chromosome counting in vitro shoots and by isozyme analysis of in vitro plants. Final analysis was made from morphological characteristic of plant and tuber phenotypes. From 15 different combinations, 6009 plantlets have been regenerated. From five combinations, 310 reentrants were checked for hybrid nature by morphology and cytology and 88 by peroxidase and esterase isozyme analyses. Amongst these, from two combinations, a total of 17 different hybrids were confirmed by all methods. The procedures described are general enough to allow genome combination of interdihaploids resulting in tetraploids of practical breeding value.     

Mapping polygenes for tuber resistance to late blight in a diploid Solanum phureja × S. stenotomum hybrid population

Potato tuber blight is a disease caused by the oomycete Phytophthora infestans (Mont.) de Bary. Due to the significant economic impact of this disease, introgression of durable resistance into the cultivated potato is one of the top priorities of breeding programmes worldwide. Though numerous resistance loci against this devastating disease have already been mapped, most of the detected loci are contributing towards foliar resistance while specific information on tuber resistance is limited. To identify the genetic components of tuber resistance and its relationship to foliar resistance and plant maturity we have investigated the host‐pathogen interaction in a segregating diploid hybrid Solanum phureja × S. stenotomum family. Mature tubers from this mapping family were inoculated with a sporangial suspension of P. infestans (US‐8 clonal lineage) and evaluated for lesion expansion. No significant correlation was detected between late blight resistance in foliage and tubers, and between plant maturity and tuber resistance. Four chromosomal regions were significantly associated with tuber resistance to the disease. The largest effect was detected near the marker locus PSC (LOD 10.7) located on chromosome 10. This locus explained about 63% of the total phenotypic variation of the trait. The other three resistance‐related loci were mapped on chromosomes 8 (GP1282, LOD 4.4), 6 (CP18, LOD 4.0) and 2 (CP157, LOD 3.8). None of the four tuber resistance loci coincides with the foliage resistance loci detected in this same family. Tuber blight resistance quantitative trait loci (QTL) on chromosomes 2, 8 and 10 are distinct from the maturity QTLs and have an additive effect on tuber resistance. These results indicate that different genes are involved in foliar and tuber resistance to P. infestans in the present family and that some of the resistance genes might be associated with late maturity.     

四川省马铃薯种质资源材料引进、评价及利用

引进国内外不同基因类型种质资源材料117套家系实生种籽、试管苗、早代无性系及品种材料等,经过多年培育从中直接筛出新品种3个和一批不同基因类型的主要亲本材料,创新培育2个新品种及一批新的无性系,以供育种和四川省生产应用,有的已成为四川省主栽品种。经筛选出的资源材料其基因类型丰富,具有属高抗晚疫病、病毒病以及抗其它病害,丰产性强,食口及加工品质好,耐贮、休眠性浅等不同特性,有相当利用价值,可进一步探索和应用。     

分子标记技术在马铃薯晚疫病研究中的应用

马铃薯是世界上重要的粮食作物之一,晚疫病则是当今危害马铃薯生产最为严重的病害。重点介绍了四种常用的分子标记技术RFLP、RAPD、AFLP和SSR,以及国内外利用这些标记技术在马铃薯晚疫病研究中的应用。分析了马铃薯晚疫病菌的遗传多样性、菌株抗药性、有性杂交后代的遗传分离以及抗病种质资源的遗传多样性,介绍了遗传图谱的构建、抗性基因及与晚疫病抗性相关的QTL定位、体细胞杂种及回交后代的晚疫病抗性检测。这些对今后中国学者开展相关领域的研究具有重要的借鉴意义。     

Increased frost tolerance and amino acid content in leaves,tubers and leaf callus of regenerated hydroxyproline resistant potato clones

Summary A number of previously selected hydroxyproline (hyp) resistant cell lines of a diploid potato (Solanum tuberosum L., clone H²578, 2n=2x=24) could be regenerated into plants which were further analysed. Hyp resistance, although lower than in the originally selected calli, was still present in regenerated shoots and in callus initiated from these shoots and it was not lost upon (mini)tuber propagation. Regenerated shoots showed a wide range of phenotypic variation. The chromosome number, analysed in 4 clones, appeared to be hypotetraploid (44 or 45). Tuber-propagated regenerants generally showed increased frost tolerance both at the plant and the cell level. In leaves this seemed to be associated with increased levels of both proline and total amino acid content. However, in callus only the total amino acid content but not proline was still elevated. In the wild type the frost tolerance of the tubers appeared to be lower than that of the leaves. Between tubers of wild type and the hyp resistant regenerants no differences in frost tolerance were found, although proline and total amino acid content tended to be higher in tubers from the regenerants.     

Transfer of disease resistance within the genus Brassica through asymmetric somatic hybridization

Summary Asymmetric somatic hybrid plants between Brassica napus L. (oilseed rape genome AACC) and a transgenic line of Brassica nigra L. Koch (black mustard genome BB) were tested for their resistance against rapeseed pathogens Phoma lingam (black leg disease) and Plasmodiophora brassicae (club root disease). The transgenic B. nigra line used (hygromycin-resistant, donor) is highly resistant to both fungi, whereas B. napus (recipient) is highly susceptible. The asymmetric somatic hybrids were produced using the donor-recipient fusion method (with X-irradiation of donor protoplasts) reported by Zelcer et al. (1978) for the production of cybrids. Using hygromycin-B for selection, a total of 332 hybrid calli were obtained. Regenerants, resistant or susceptible to both diseases, were selected. Many hybrids expressed resistance to only one pathogen. Dot blot experiments showed that the asymmetric hybrid plants contained varying amounts of the donor genomic DNA. Furthermore, a correlation was detected between the radiation dose and the degree of donor DNA elimination.     

Half-sib progeny evaluation and selection of potatoes resistant to the US8 genotype of Phytophthora infestans from crosses between resistant and susceptible parents

The objectives of this study were to evaluate the use of potato (Solanum tuberosum L.) late blight (Phytophthora infestans (Mont.) de Bary) resistant parents in cultivar development and identify superior clones possessing moderate to high late blight resistance combined with acceptable maturity and tuber quality. Ninety-five crosses were made between eight unadapted parents with reported late blight resistance (B0718-3, Bertita, Bzura, Greta, Libertas, Stobrawa, Tollocan and Zarevo) and susceptible parents (cultivars or advanced breeding clones) adapted to North American growing conditions. A total of 408 field selected clones were assessed for late blight resistance in the greenhouse and in the field using a mixture of US8 P. infestans isolates (A2 mating type, metalaxyl resistant) that overcame all known R-genes except R8 and R9. Clones with ≤ 10% infected foliar area in the greenhouse test or ≤ 0.30 RAUDPC (relative area under the disease progress curve) value in the field in 1998 were re-tested in 1999. A total of 118 (29% of 408) putative late blight resistant clones were selected. The eight late blight resistant parents differed in both the ability to transmit late blight resistance and in the level of resistance transmitted to the progeny. The Tollocan and B0718-3 families (half-sib progeny) had the greatest degree of resistance and frequency of resistant clones. Scott-Knott cluster analysis ranked 79 clones (67% of 118) in the high and moderate late blight resistant groups. Among these 79 clones, 19 clones had vine maturity equal to or earlier than mid-season combined with acceptable tuber quality. Further selection in 2000 resulted in eight advanced selected clones (six from Tollocan and two from B0718-3 families) with the same level of resistance as the parent combined with vine maturity and tuber quality equivalent to Atlantic, a standard cultivar for chip processing in North America. The results indicate that this breeding approach can be used to select parents for late blight resistance breeding and to identify superior clones with high levels of late blight resistance and marketable vine maturity and tuber quality. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of morphological variation and cold resistance in interspecific somatic hybrids between potato (Solanum tuberosum L.) and S. brevidens Phil.

Summary Somatic hybrids between Solanum tuberosum L. cv. Gracia (2n=4x=48) and Solanum brevidens Phil. (2n=2x=24) were produced via fusion of mesophyll protoplasts. Selection of the protoplast derived putative hybrid calli was based on their vigorous growth. Additive isozyme patterns and chromosome numbers as well as the expression of parental morphological characters have proved the hybrid origin of the selected regenerants. Extensive chromosome loss during the regeneration process resulted in aneuploid hybrids with high frequency. Genomic instability could not be detected in these plants during the period of vegetative propagation. Regenerants from hybrid tissues exhibited wide morphological variation especially in tuber formation. The detailed morphological analysis based on the use of multivariate method (principal component analysis, PCA) enabled to identify morphological groups among the hybrid clones. The positioning of hybrid clones in the PCA space could not be correlated with chromosome numbers. The genomic ratio represented by the tetraploid and diploid parents influenced the morphology of somatic hybrid population according to the applied analytical system. Two selected hybrid clones have exhibited an intermediate degree of frost tolerance compared to the parents, based on the recovery of plants from lower buds after freezing of potted plants.     

Anther culture of tropical japonica × indica hybrids of rice (Oryza sativa L.)

Summary Nine japonica × indica F1 hybrids of rice involving 6 indica and 3 japonica tropical varieties, were large scale anther cultured. The frequency of callusing anthers averaged 18.7%. The microspore-derived calli produced green plants with a mean frequency of 8.7%. Albino plants represented 61% of the shoot forming calli. Monitoring of the green and albino plant regenerating capabilities of calli arising between week 4 and week 8 of incubation of the anthers showed no increase of the albino/green ratio and a slow decrease of the shoot forming ability of the transferred calli after the sixth week of culture. Spontaneous doubled haploids (SDH) represented 46% of the regenerated green plants in 4 hybrids. However, a high frequency of partially sterile regenerants was noticed among 132 SDH plants generated from a hybrid.     

Utilization of Asymmetric Somatic Hybridization for the Transfer of Disease Resistance from Brassica nigra to Brassica napus

Asymmetric somatic hybrid calli were produced between Brassica napus and a transgenic (Hyg ^R) line of B. nigra using a donor recipient fusion method for the production of cybrids. The transgenic line of B. nigra used as a donor also possessed genetic resistance to the pathogenic fungi Phoma lingam and Plasmodiophora brassicae. Using hygromycin for selection, 332 hybrid calli were obtained from which 30 produced shoots (1—-20 per callus) which were rooted on a hormone-free culture medium. The rooted shoots were transferred into soil and cultivated in a growth chamber where the plants were tested for resistance against the two pathogens. Out of 129 hybrid plants tested for resistance against P. brassicae, 30 (23.3 %) plants proved to be resistant and from 78 plants tested for resistance against P. lingam, 41 (52.6 %) plants remained disease-free after infection.     

Somatic hybrids between Gossypium hirsutum L. (4×) and G. davidsonii Kellog (2×) produced by protoplast fusion

Somatic hybrids were obtained from electrofused protoplasts derived from embryogenic suspension cultures of tetraploid cotton (G. hirsutum L. cv. Coker 201) and embryogenic callus of diploid wild cotton G. davidsonii. The regenerants were initially identified as hybrids by RAPD (random amplified polymorphic DNA) analysis. Subsequently, observation on chromosome counting, morphology and SSR (simple sequence repeat) confirmed the hybrid status. Cytological investigation of the metaphase root-tip cells of the regenerated plants revealed there were 74 to 84 chromosomes in the plants, close to the expected 78 chromosomes. SSR analysis revealed the regenerated plants contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the plants was intermediate between the two fusion partners. The regenerants were difficult to develop into mature plants because their roots browned and they wilted from the stem apex before forming 3 to 5 true leaves. The hybrid plants were transferred to soil by grafting in vitro onto rootstocks.     

Production and characterization of intergeneric diploid cybrids derived from symmetric fusion between Microcitrus papuana Swingle and sour orange (Citrus aurantium)

Plants were regenerated from intergeneric somatic hybridization between embryogenic protoplasts of Microcitrus papuana Swingle and leaf-derived protoplasts of sour orange (Citrus aurantium L.) via electrofusion. The regenerated plants were morphologically similar to the leaf parent in growth vigor, leaf and branch structure. FCM analysis showed that they were diploids. Simple-sequence-repeat (SSR) and cleaved-amplified-polymorphic-sequence(CAPS) were employed for hybridity characterization. SSR banding patterns of the regenerated plants were identical to the leaf parent, sour orange, indicating that they possessed nuclear component derived from sour orange. DNA amplification with chloroplast and mitochondrial universal primers, followed by restriction endonuclease digestion, revealed polymorphism between the fusion parents. Therefore, this method was used to determine the cytoplasmic compositions of the regenerated plants. Banding patterns for all the polymorphic primer/enzyme combinations of the regenerated plants were similar to those of the embryogenic parent, M. papuana, suggesting that only the cytoplasmic components derived from the embryogenic parent were present in the regenerated plants. FCM, SSR and CAPS demonstrated that intergeneric diploid cybrids have been successfully obtained by symmetric fusion. Related results concerning nuclear and cytoplasmic composition of previous diploid somatic hybrids and potential mechanism for regeneration of such kind of plants are discussed herein. This revised version was published online in July 2006 with corrections to the Cover Date.     

Potato germplasm development for warm climates: genetic enhancement of tolerance to heat stress

Complex potato hybrids were derived through unilateral sexual polyploidization between Solanum tuberosum cv. Atlantic and 11 diploid hybrids that produced 2n pollen through co-orientation of second division spindles. The hybrids represented the following genomic compositions: TAPB, TAPC, and TAPM where T = S. tuberosum, A = S. andigena, P = S. phureja, B = S. berthaultii, C = S. chacoense, and M = S. microdontum. The B, C, and M components of the hybrids had been selected from heat tolerant accessions. The heat tolerance and agronomic performance of the hybrids were assessed under both controlled environments and field tests. The hybrids exhibited good tuberization potential (tuber number, tuber weight and percent plants producing tubers) under heat stress conditions in controlled environments. Under severe heat stress conditions in the field in Israel, many of the hybrids tuberized when Atlantic failed to produce any tubers at all. Under more moderate heat stress in Virginia, the complex hybrids exhibited total tuber yield not significantly different from Atlantic, although the tuber set per plant was greater. However, the complex hybrids were more tolerant to heat necrosis and hollow heart than Atlantic. Total glycoalkaloids in field-grown tubers did not exceed the acceptable limit for 13 of 26 selections examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Spontaneous and induced loss of chromosomes in slow-growing somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia

Summary Rate and extent of spontaneous and induced chromosome loss have been determined at the callus level of somatic hybrids of mutants of Solanum tuberosum and Nicotiana plumbaginifolia. AEC (amino ethyl cystein) resistance in potato and Nitrate-Reductase deficiency in N. plumbaginifolia have been used as genetic markers and chromosome morphology as a cytological marker. In this combination, development of hybrid callus was late and slow. Only a limited number of non-regenerable hybrid calli have become available. Chromosome loss could clearly be established in these hybrids from loss of markers and from chromosome cytology. Loss of markers occurred independantly.The best conditions to induce loss of chromosomal material in donor cells by irradiation were found by cytological investigations. A very drastic reduction in chromosome transfer by fusion could be effected by irradiation of plant tissue and subsequent preparation of protoplasts after a few days. Following fusion, hybrid callus was recovered with the potato genome drastically reduced. The amount of loss was deduced from the presence of a few fragments in metaphase cells or from interphase nuclei after in situ hybridization with a repetitive potato DNA probe.