The potential for early generation selection to identify potato clones with resistance to Verticillium wilt

Verticillium wilt (VW) of potato, caused primarily by the fungus Verticillium dahliae, results in yield loss and is therefore an important soil-borne disease. Resistance to VW exists in potato germplasm and is used by breeders during cultivar development. Breeders could make more rapid progress toward the development of VW resistant clones if they had an effective early generation selection strategy. The purpose of this study was to determine whether selection for VW resistance could be carried out in the first tuber generation on single hills. One hundred and fifty-two clones from 19 families were planted as single hills on a V. dahliae-infested field. Each plant was scored for vine maturity, VW symptom expression, yield, stem colonization (colony forming units (cfu), in dried basal stem segments) and incidence (percent infected stems). In the second clonal generation, which consisted of replicated four-hill plots, stem colonization scores and incidence values were used to identify clones which were more resistant than a moderately resistant cultivar and others which were more susceptible than a susceptible cultivar. The efficiency and reliability of the single-hill selection strategy, based on symptoms and yield, was then determined by comparison to the four-hill results. We determined that the best single-hill selection strategy was negative selection (discard clones with the lowest performance) with low stringency, based on yield.     

Evaluation of resistance to verticillium wilt in diploid,wild potato interspecific hybrids

Summary Verticillium wilt (V. albo-atrum Reinke & Berthold or V. dahliae Kleb) threatens potato (Solanum tuberosum L.) production in most growing areas of the world. Genetic resistance offers the most cost-effective and environmentally-sound control measure. However, there is a dearth of genetic and breeding information on resistance to verticillium wilt in potato, because of obscure parentage of some standard cultivars and the complex segregation at the tetraploid level. The wide range of genetic variability in wild relatives of potatoes can be potentially useful as a source of disease resistance, such as verticillium wilt resistance. Six diploid, wild, interspecific Solanum hybrids involving resistant x resistant and susceptible x resistant crosses, were assayed for verticillium wilt resistance under greenhouse conditions to evaluate their potential as sources of verticillium wilt resistance. The cross between S. gourlayi Oka. and S. chacoense Bitt. and its reciprocal had the most resistant progenies based on mean colony counts. No simple mode of inheritance can be proposed based on the observed segregation ratios. However, the continuous distributions observed on verticillium wilt disease response among hybrid families indicate that inheritance of resistance may be polygenic and complex. In addition, skewness of colony count distributions toward the resistance parents were characteristic of all resistant x susceptible crosses suggesting that resistance may be dominant. By contrast, the susceptible x susceptible cross showed a more normal distribution. Overall, the cross between S. gourlayi and S. chacoense showed the most potential as a source of verticillium wilt resistance.     

Sources of resistance to Verticillium wilt in Capsicum

Summary A total of 125 novel accessions of Capsicum annuum L. and Capsicum baccatum L. were screened for sources of resistance to Verticillium wilt (Verticillium dahliae Kleb). A soil infestation method with 2000 microsclerotia of V. dahliae per gram of planting medium, and a soil temperature of 25±3°C, identified 27 Capsicum accessions with resistance to Verticillium wilt. P.I. 215699 (a mixture of Capsicum baccatum var. microcarpum and Capsicum annuum), P.I. 535616 (Capsicum annuum), and P.I. 555614 (Capsicum annuum), had the lowest disease severity and the highest percentage of resistant plants.     

Transmission of bacterial wilt resistance by First Division Restitution (FDR) 2n pollen via 4x×2x crosses in potatoes

Summary Resistance to bacterial wilt (Pseudomonas solanacearum) found in tuber-bearing Solanum species was transferred into a diploid potato breeding population. Simultaneous selections were made for agronomic characters, production of first division restitution (FDR) 2n pollen, and resistance. Diploid resistant genotypes were identified via inoculation with a virulent isolate (CIP-204) of race 3 of Pseudomonas solanacearum. These resistant diploid genotypes were crossed to susceptible tetraploid potatoes. An investigation was made to assess whether resistant diploid genotypes transmit resistance to bacterial wilt, which is a quantitatively inherited trait, to tetraploid potatoes via FDR 2n pollen. Tetraploid seedlings from 4x×2x crosses were inoculated with the same isolate CIP-204, and the percentage of surviving seedlings was scored. Some 4x×2x families from resistant diploid genotypes demonstrated a high level of survival rate. The transmission of bacterial wilt resistance was achieved by the use of FDR 2n pollen. It was speculated that a female x male interaction effect on the survival rate of the evaluated progeny may exist. Selecting proper 4x and 2x parents would be important for obtaining a higher frequency of transmission of resistance to bacterial wilt in the progeny.     

Somaclonal variation for resistance to Verticillium dahliae in potato (Solanum tuberosum L.) plants regenerated from callus

Summary Plant tissue culture is recognized as an important tool to generate useful genetic variability for crop improvement. Regenerated plants from callus induced from stem explants of Solanum tuberosum cv Désirée were assessed by in vitro selection, for resistance to Verticillium dahliae. This fungus is the causal agent of Verticillium wilt, a serious vascular wilt disease both in crops and wild species.The rate of in vitro multiplication by single node cuttings was used as a parameter of screening in two selection cycles with different concentrations of V. dahliae filtrate. One resistant clone was selected and then evaluated by inoculation in the growth chamber. Induced damage, and morphological traits (dry weight, leaf area and tuber production) were estimated. The selected clone was comparable to the resistant control, cv Kondor.The results suggest that genetic variation induced in tissue culture cound be utilized to generate disease resistance.     

The breeding potential of wild potato species resistant to the potato tuber moth,Phthorimaea operculella (Zeller)

Summary The potato tuber moth, Phthorimaea operculella (Zeller) is an important pest of potatoes in the field and in stores in warm environments throughout the world. In this study genetic resistance to potato tuber moth was identified in clones of Solanum sparsipilum (coded MBN) originally developed for resistance to bacterial wilt and root-knot nematode. Resistance to this pest in S. sucrense and S. tarijense as well as S. sparsipilum was exploited in wide crosses with diploid and tetraploid cultivated potatoes, and haploids derived from S. tuberosum; hybrid progenies were produced. Crosses between resistant S. pinnatisectum or S. commersonii and cultivated potatoes failed completely, although S. commersonii did hybridise with two bridging species S. lignicaule and S. capsicibaccatum which are slightly compatible with cultivated potatoes. Resistance to potato tuber moth was transferred to all progenies except those in which S. tarijense was the resistant parent. The development of potatoes resistant to potato tuber moth is discussed in the context of population breeding for the lowland tropics.     

Transmission of a Bacillus thuringiensis cry3Aa transgene from diploid to tetraploid potato using 4x-2x hybridization: effect of ploidy increase on transgene expression and implications for TPS hybrid production

The study examined the effect of ploidy elevation through unreduced gametes on transgene expression in potato. Tetraploid transgenic progenies were obtained from one tetraploid potato cultivar crossed with 2n pollen producing diploid clones harbouring an exogenous transgene (cry3Aa). Both single‐ and multiple‐insert diploid transgenic lines that were regenerated by Agrobacterium tumefaciens leaf disc inoculation were used in crosses. A DAS‐ELISA system and no‐choice feeding bioassay enabled characterization of the parental lines as either ‘high’ or low’ expressers of the Cry3Aa protein. High Cry3Aa expression was observed for both single‐insert transgenic diploids and their 4x‐2x progeny. On the contrary, 68% of 4x‐2x progeny derived from a multiple‐insert, diploid transgenic had significantly reduced Cry3Aa expression compared with the parent, with 32% demonstrating nearly complete silencing of the transgene. Multiple copies of a transgene, like homologous native genes, may be susceptible to transgene silencing following polyploidization. Therefore, incorporation of exogenous transgenes into a true potato seed (TPS) production system is feasible if a single‐insert diploid parent is used. Gene‐centromere mapping of the cry3Aa transgene demonstrated that a non‐transgenic refuge might be naturally created in a TPS hybrid system through genetic recombination.     

Inheritance of early blight resistance in diploid potatoes

Early blight (Alternatia solani) is a fungal disease in hot and humid environments, which causes leaf, stem and tuber lesions. Early blight resistance should be incorporated into potato cultivars because the fungicide spraying is an expensive solution for developing countries. The diploid cultivated species Solanum tuberosum group Phureja and group Stenotomum are sources of resistance alleles. The elucidation of the inheritance for early blight resistance must help to decide what could be the best breeding procedure to improve this diploid germplasm and transfer the resistance to the tetraploid level. Three experiments were carried out under controlled and field conditions to determine the heritability of this trait using nested and diallel mating designs with haploid, species and haploid-species hybrids. The narrow-sense heritability estimates were relatively high (0.64–0.78). This means that additivity was the most important type of gene action for determining resistance to early blight at the diploid level. The results suggested that diploid parents showing highest levels of resistance, throughout the cycle of disease development, can be used in 4x×2x crosses to obtain resistant tetraploid progenies to this fungal disease.     

Introduction of an amylose-free (amf) mutant into breeding of cultivated potato,Solanum tuberosum L.

Summary Using an amylose-free (amf)mutant of diploid potato (Solanum tuberosum), diploid and tetraploid clones with different genotypes at the amf-locus were produced. In order to make use of the diploid material in analytic breeding of amf-potatoes, clones were selected that produced a considerable frequencies of 2n-pollen and 2n-eggs. Successful attempts were made to select normal synaptic as well as desynaptic clones producing 2n-gametes. Based on the phenotype of starch in the microspores, tetraploid clones with nulliplex, simplex and duplex genotypes at the amf-locus were selected. Prospects of using this material in conventional as well as in analytic breeding of potato are discussed.     

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.     

Bilateral sexual polyploidization in potatoes

Summary Bilateral sexual tetraploidization was achieved by intercrossing diploid Phureja × haploid Tuberosum hybrids. Both parents contribute gametes with the somatic number of chromosomes (diplogynoids and diplandroids). More than 100 tetraploid progeny appeared in nine 2x–2x families. The tetraploids are generally more vigorous and significantly outyield their diploid full-sibs. The superiority of the tetraploids is interpreted on the basis of the mode of diplandroid (2n pollen) formation by first meiotic division restitution (FDR). Heterozygosis, epistasis and genetic diversity, if present in the parental diploid genotype, are largely maintained in the 2n gametes formed by FDR. and syngamy brings about more opportunities for heterotic responses in conjunction with polyploidization. Differences in tuber yield among tetraploid clones within families were found to be statistically significant (P <0.05). Thus, unlike somatic chromosome doubling, which can merely increase chromosome numbers, or somatic hybridization, which could in addition bring about heterosis, sexual polyploidization is also associated with genetic variability. The degree of inbreeding and the genotypic structure in the population of newly arisen tetraploids is discussed. The relationship among asexual reproduction, preservation of favorable nonadditive genetic effects by FDR. restoration of sexuality, and polyploidization is explored. The possible evolutionary significance of these factors is emphasized.     

The resistance of Hordeum bulbosum and its hybrids with H. vulgare to common fungal pathogens

Summary Two tetraploid and two diploid clones of Hordeum bulbosum were screened for resistance to five isolates of powdery mildew which are virulent on cultivated barley. All were resistant and this resistance was also expressed in hybrids with H. vulgare. The tetraploid genotypes were also resistant to isolates of yellow rust and brown rust. These results show that H. bulbosum contains useful genes for resistance to these diseases and that there is a potential to transfer these into cultivated barley.     

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.     

Discovery of 2n gametes in tetraploid oat Avena vaviloviana

Summary Sexual polyploidization via the action of 2n gametes (gametes with the sporophytic chromosome number) has been identified as the most important evolutionary mode of polyploidization among plant genera. This study was conducted to determine whether 2n gametes are present in the tetraploid level of the genus Avena (2n=4×=28) Twenty tetraploid Avena lines, representing four species and one interspecific hybrid, were screened for pollen grain size in order to differentiate between n and 2n pollen. Avena vaviloviana (Malz.) Mordv. line PI 412767 was observed to contain large pollen grains at a 1.0% frequency. Cytogenetic analyses of pollen mother cells of PI 412767 revealed cells with double the normal chromosome number (i.e., 56 chromosomes at metaphase I and anaphase I). The mode of chromosome doubling was found to be failure of cell wall formation during the last mitotic division that preceded meiosis. The resulting binucleate cells underwent normal meiotic divisions and formed pollen grains with 28 chromosomes. Based on the formation and function of 2n gametes, three models involving diploid and tetraploid oat lines are proposed to describe possible evolutionary pathways for hexaploid oats. If stable synthetic hexaploid oat lines could be developed by utilizing 2n gametes from diploid and tetraploid oat species through bilateral sexual polyploidization, the resulting hexaploids could be used in breeding programs for transferring genes from diploids and tetraploids to cultivated hexaploids.     

Influence of plant age,disease incubation time,and host plant resistance on Verticillium wilt symptom expression in lucerne (Medicago sativa)

Summary The most economical means to control Verticillium wilt in lucerne, caused by Verticillium albo-atrum, is through resistant cultivars. Most Verticillium wilt resistance evaluations use some form of root-cut-soak inoculation procedure. In Trial 1, three lucerne cultivars were inoculated at eight plant ages and plant symptoms were scored at ten successive weekly intervals. Trial 2 consisted of two cultivars inoculated at ten plant ages and symptoms were evaluated at eight weekly intervals. In an experiment to study the genetics of resistance to this pathogen, four plants showing no visual symptoms were selected from each of four germplasms. The selected populations were crossed in diallel fashion. The population crosses plus the parents were evaluated for resistance to V. albo-atrum. Results from Trial 1 and Trial 2 showed disease severity increased with increasing inoculation age and with increasing scoring age. Cultivar symptom expression interacted with inoculation age, but not with scoring age. The diallel analysis supported resistance to Verticillium wilt being conferred by additive gene action, as heterosis was not detected and general combining ability mean square was 3.75 times larger than the specific combining ability mean square.     

Cytological studies on adventitious shoots and minitubers of a monoploid potato clone

Summary A three step procedure for adventitious shoot regeneration on leaf explants of monoploid potato clone H7322 and a minituber induction procedure on stem segments have been described. Chromosome counts on 92 adventitious shoots showed that 85% of them had been polyploidized, i.e., 71% were diploid, 1% tetraploid, and 13% were mixoploid. Cytophotometric studies on nuclei of soil grown tubers of tetraploid cv Astarte, of 1x, 2x and 4x adventitious shoots of H7322, and of diploid H²578 showed in all cases polyploidization with prominent classes up to 8C and 16C. However, nuclei of pith cells of 5 weeks old minitubers which had developed on monoploid H7322 itself or on 1x adventitious shoots of H7322 showed predominantly 1C and 2C values. Pith cells of minitubers of monoploid H7322 were screened, after iodine staining, for the presence of variant cells containing reddish-brown staining (amylose-free) starch. In more than 75% of the investigated minitubers one or a few of such variant cells were found indicating that such a variation occurs in minitubers of monoploid potato and that this variant character is expressed in cells of vegetative storage organs like potato tubers.     

Progenies of allotriploids of Oriental × Asiatic lilies (Lilium) examined by GISH analysis

With the aim of utilizing allotriploid (2n = 3x = 36) lily hybrids (Lilium) in introgression breeding, different types of crosses were made. First, using diploid Asiatic lilies (2n = 2x = 24), reciprocal crosses (3x − 2x and 2x − 3x) were made with allotriploid hybrids (AOA) obtained by backcrosses of F₁ Oriental × Asiatic hybrids (OA) to Asiatic cultivars (A). Secondly, the AOA allotriploids were crossed with allotetraploid (OAOA, 2n = 4x = 48), in 3x − 4x combination. Finally, the AOA allotriploids where crossed to 2n gamete producer F₁ OA hybrids (3x − 2x (2n)). Two types of triploids were used as parents in the different types of crosses, derived from: (a) mitotic polyploidization and (b) sexual polyploidization. Ploidy level of the progeny was determined by estimating the DNA values through flowcytometry as well as chromosome counting. The aneuploid progeny plants from 3x − 2x and reciprocal crosses had approximate diploid levels and in 3x − 4x crosses and 3x − 2x (2n) the progeny had approximate tetraploid levels. Balanced euploid gametes (x, 2x and 3x) were formed in the AOA genotypes. Recombinant chromosomes were found in the progenies of all crosses, except in the case of 2x − 3x crosses through genomic in situ hybridization (GISH) analyses. Recombinant chromosomes occurred in the F₁ OA hybrid when the triploid AOA hybrid was derived through sexual polyploidization, but not through mitotic polyploidization with two exceptions. Those recombinant chromosomes were transmitted to the progenies in variable frequencies.     

Genetic variation for disease and nematode resistances and forage quality in perennial diploid and tetraploid lucerne populations (Medicago sativa L.)

Summary Twenty-five lucerne populations of the Medicago sativa complex, which were either diploid or tetraploid and wild or cultivated, were analysed for their resistance to four different fungal diseases and to stem nematode. Forage quality, including stem digestibility and saponin content, was also tested.Populations varied in susceptibility to the diseases caused by Colletotrichum trifolii, Verticillium albo-atrum, Sclerotinia trifoliorum and Pseudopezizza medicaginis, and to the nematode Ditylenchus dipsaci. Except for Sclerotinia rot, sativa and falcata subspecies differed in susceptibility, but this grouping of populations did not account for the full range of variation among them. However, the resistance to P. medicaginis was much lower in the sativa than in the falcata populations.Populations also varied significantly in stem fiber content and digestibility. Stem digestibility was negatively correlated to forage yield. Wild sativa and falcata populations had lower fiber content and higher digestibility than cultivated sativa populations. The medicagenic acid was the sapogenin responsible for the anti-nutritional effect of the lucerne measured by the yellow mealworm larvae Tenebrio molitor. The medicagenic acid content was lowest for the pure sativa populations, highest for the pure falcata populations, and intermediate for the French sativa varieties that have some traits originating from falcata germplasm. Some populations could be used in breeding programs to improve disease and nematode resistance, and forage quality.     

Analysis of parental effects on mean vine maturity and chip colour of 4x.2x potato progenies

Summary The parental effects on progenies from potato 4x.2x(FDR) crosses were analyzed to determine anether and to what extent selection criteria used for selecting tetraploid breeding lines have to be adjusted when selecting diploid breeding lines. For vine maturity and chip colour multiple regression analysis of progeny means on means of diploid and tetraploid parents revealed high coefficients of determination (R²) for both characters, indicating good predicting power of the performance of both diploid and tetraploid parents on the performance of their 4x.2x progenies. For vine maturity the multiple regression slope for the tetraploid perents was significantly larger than for the diploid parents, and progeny means were towards the tetraploid parents. This indicates that selection criteria for vine maturity may be less stringent at the diploid level. However, diploids may be earlier maturing than tetraploids and therefore selection criteria can better be similar at both ploidy levels. For chip colour, multiple regression slopes for diploid and tetraploid parents were similar and progeny means were towards the darkest coloured parent irrespective of its ploidy level. This indicates that selection of breeding lines for chip colour can be conducted similarly at both ploidy levels.     

Incompatibility in diploid and tetraploid crosses of Cucumis sativus and Cucumis metuliferus

The African horned cucumber (Cucumis metuliferus Naud.; 2x = 2n = 24) contains genes that can confer resistance to many important cucumber (C. sativus L.; 2x = 2n = 14) pests [e.g., root-knotnematode, Meloidogyne incognita (Kofoid & White) Chitwood]. Cucumber is highly susceptible to this root-knot nematode species, and a recent screening of C. sativus accessions in the U.S. National Plant Germplasm collection did not identify sources of resistance. Thus,autotetraploids of Cucumis sativus and C. metuliferus were created to recover fertile resistant interspecific progeny. Autotetraploids were obtained at the highest rate when seeds were immersed in 0.5% colchicine for a period of 6 to 8 hrs. Treatment durations less than 6 hrs produced few tetraploids, and durations of 10 hrs or more were lethal to seeds or developing seedlings. Crosses between C. sativus and C. metuliferus were made using diploid and tetraploid lines in all possible combinations, including reciprocals. Fruit development occurred in crosses when diploid and tetraploid C. sativus were used as the female parent. However, seeds developed only in fruit of C. sativus (4n) ×C. metuliferus (2n) crossings. Seeds from these crosses, however,were flat and not viable. No fruit development occurred in crosses whereC. metuliferus was used as the female parent. This revised version was published online in August 2006 with corrections to the Cover Date.