The use of synthetic octoploids in strawberry breeding

Summary A number of methods of producing synthetic octoploids from Fragaria species of lower levels of ploidy are described. Six synthetic octoploids were produced from various combinations involving one hexaploid species, two tetraploid species and four diploid species. Three of the synthetic octoploids are male fertile and were used successfully as male parents in crosses with octoploid strawberry cultivars.     

Breeding barriers between the cultivated strawberry, Fragaria × ananassa, and related wild germplasm

Five-hundred interspecific and intergeneric crosses were performed among accessions of the wild strawberries Fragaria vesca(2x), Duchesnea indica (8x), Potentilla tucumanensis (2x) and 9 genotypes of the cultivated strawberry, Fragaria×ananassa (8x), following an incomplete diallele mating design. Crosses between D. indica and F.×ananassa produced many putative hybrids when D. indica was used as female but a few achenes and plants when used as male; therefore, pollen-pistil compatibility relations were analyzed by fluorescence microscopy in this direction of the cross. Of the genotypic combinations, 78.6% were incompatible at the stigma level and 17.2% at the first third of the style. Only 3.6% were pollen-pistil compatible and produced fruits with achenes (seven did not germinate or originated short-lived plants and nine produced normal plants). F.vesca×F.×ananassa crosses produced 35 hybrid achenes but only 14% germinated, yielding short-lived plants; histological analyses revealed that inviable seeds had less developed (or collapsed) endosperms and smaller embryos than control plump F. vesca seeds. P.tucumanensis was only used as male, with negative results. These species and genera are partially isolated by a complex system of pre- and post-zygotic barriers. Knowledge of their nature would allow the breeder to devise strategies to put the genetic variability available in the group into a useful form. This revised version was published online in July 2006 with corrections to the Cover Date.     

Accelerating and synchronizing germination of strawberry seeds by osmotic pre-treatments

Summary Pre-soaking strawberry seed (Fragaria ananassa Duch.) in osmotic solutions accelerated and partially synchronized their germination. Pre-soaking advanced 50% germination from about 4 weeks to 4 or 5 days. Two to three weeks pre-soaking in a mineral solution of -10⁶Pa nominal osmotic potential at about 20 C was satisfactory. The resulting synchronization of germination could allow seedlings to be selected for rate of growth by directly comparing seedling in bulk sowings.     

Inheritance of resistance to Phytophthora fragariae Hickman in strawberry

Summary It has been found that complete resistance of the strawberry cultivars and selections Earliglow, Guardian, MdUS 2700, MdUS 2929, MdUS 3816 and Redchief to Phytophthora fragariae is inherited in a one-to-one ratio, suggesting that these genotypes have one major resistance gene effective to the races present in the test field.The high level of partial resistance of Cambridge Favourite has clearly been recovered in its progenies. However, from results presented here, no conclusions could be drawn with regard to the number of genes involved in the partial resistance of Cambridge Favourite. It is, however, suggestive that about half the susceptible selections derived from crosses with Cambridge Favourite have shown a relatively high level of partial resistance. The selections concerned were obtained by selection on horticultural characters in seedling populations planted on an uninfested field.     

Improved techniques for the induction and isolation of polyploids in the genus Fragaria

Summary The sensitivity of Fragaria seedlings to colchicine is dependent on the plant organ that is treated. Complete immersion of seedlings in a 1.5% colchicine solution results in total lethality, whereas the survival rates were more than 75% even at concentrations of 3.0% when only shoot apices were treated. High proportions of polyploids were isolated by treating shoot apices of seedlings with a 2.0% colchicine solution for 24–28 h. The dropper method is preferred to the tube method as it involves a minimum of manipulation and requires simple equipment. A differential response to colchicine was observed within and between different diploid species, diploid and tetraploid hybrids.     

A comparison of genetic relationship measures in strawberry (Fragaria × ananassa Duch.) based on AFLPs, RAPDs, and pedigree data

Nineteen of the major strawberry (Fragaria × ananassa Duch.) cultivars grown in the UnitedStates and Canada were examined for AFLP markerpolymorphisms. For the AFLP reactions, the EcoRI-ACC primer was used in combination with fourMseI primers (MseI-CAC, MseI-CAG,MseI-CAT, or MseI-CTT). Each set ofprimers produced 46–66 scorable fragments ranging insize between 50 and 500 bp. The polymorphic fragmentsproduced from each set of primers were more thansufficient to distinguish among all the cultivars,demonstrating the usefulness of AFLP markers forcultivar identification. Similarity coefficients werecalculated based on data from 228 AFLP markers anddata from 15 previously characterized RAPD markers. The RAPD markers had been specifically selected forfingerprinting purposes because they succesfullydistinguish 41 strawberry cultivars, including the 19cultivars analyzed in this study. Separatedendrograms were constructed based on analysis of theAFLP and RAPD marker data using a neighbor-joiningalgorithm. The dendrograms were compared and found tobe very different. Correlations between similaritycoefficients calculated from AFLP marker data,similarity coefficients calculated from RAPD markerdata, and coefficients of coancestry calculated frompedigree information were evaluated. Interestingly,a better correlation with the coefficients ofcoancestry was observed with the RAPD marker data thanwith the AFLP marker data.     

Use of Vaccinium octoploids to facilitate 4x-6x gene transfers

The use of an 8x Vaccinium produced through doubling a tetraploid hybrid with colchicine was studied as a means of facilitating gene exchange between V. corymbosum L. and V. ashei Reade. Analysis included meiotic observations and crossability studies of the 8x plant, as well as attempting to develop 6x breeding lines. Meiotic analysis revealed the presence of micronuclei in Telphase II products and polyspory in sporads. In the crossability studies five 4x highbush plants were pollinated with 8x o2 4x pollen. Data gathered included: % fruit set, average weight/fruit, average number of seeds/fruit, average number of seedlings/pollination, and number of 6x seedlings. Octoploid pollinations were significantly lower than 4x pollinations in all parameters. One 6x plant was produced from the 4x-8x crosses but was found to be mitotically unstable, having somatic cells with chromosome numbers ranging from 48 to 168.     

Resistance to a single UK isolate ofColletotrichum acutatum in strawberry germplasm from Northern Europe

Summary Colletotrichum acutatum on strawberry was first introduced to the UK on runners of cultivar Brighton, which were imported from California in 1983. Recently further outbreaks have been reported following the importation of infected runner plants from mainland Europe. Phytosanitary precautions have so far been successful in controlling the disease but the current trend in the UK is for increasing late season production. This increases the likelihood of there being fruiting plants when the environmental conditions are favourable toC. acutatum. Fifteen cultivars and 19 HRI breeding lines were tested for susceptibility toC. acutatum using the most pathogenic isolate found on plants imported into the UK. It is likely this isolate was originally of Californian origin. Potted plants were spray inoculated and subsequently rated in the glasshouse using a disease severity rating (DSR) with the range from 0–4, where a rating of 4 indicated severe disease symptoms. In contrast to results from California and Italy standard deviations were variable and often large and consequently it was considered unsafe to classify the susceptibility of the genotypes on the basis of mean DSR alone. Classification was thus based on a combination of the mean value and the distribution of the scores but in some cases the results were inconclusive. It was possible to classify five lines as having a high level of resistance (Elvira, Honeoye, EM99, EM224, EM255); six lines had a useful level of resistance but did show symptoms on some plants (Cambridge Favourite, Gorella, Pandora, Pantagruella, EM17 and EM290); five lines were highly susceptible (Elsanta, Redgauntlet, Tamella, EM237 and EM319). The remaining 18 lines could not be classified with the same degree of certainty but 10 were probably resistant.     

In vitro screening of strawberry plants for cold resistance

Formation of embryo autonomy of strawberry, plant regeneration fro membryo components, plant freezing conditions in vitro and the possibility to differentiate objectively genotypes by freezing them in vitro and in vivo were studied to create strawberry screening technology in vitro for cold resistance. It was established that autonomy of strawberry embryos manifests itself not earlier than on 14–16^th day after pollination and full autonomy is reached on 20–22^nd day. Plants regenerated from 26 days old embryos grew most intensively. At the highest rate strawberry plants regenerated from an isolated embryo axis on MS medium without phytohormones, and from rescued cotyledons x on the medium with 1.0 BA and 0.5 NAA. The temperature interval, at which genotypes differentiated according to cold resistance in vitro, was -8 to 12 ^°C. Differentiation of strawberry genotypes according to this character conformed to their differentiation in vivo, provided hardening proceeded not less than 21 days. The correlation between cold resistance in vitro and in vivo reached 0.93. Domination of cold resistance manifested itself in strawberry seedlings from various crossing combinations. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro selection of strawberry (Fragaria × ananassa Duch.) clones tolerant to salt stress

The method of in vitro selection for increased salt tolerance at the seed germination and early growth phase of strawberry seedlings is proposed. Clone Pau/27 was selected on medium containing 200 mM of NaCl from population obtained by free pollination of cv. ‘Paula2019;. Subsequently, on the same medium 18 salt tolerant clones were attained from the population of seeds collected from self pollinated Pau/27 plants. In the next step we examined the influence of a mild salt stress (75 mM of NaCl) on vegetative growth parameters of selected clones and two cvs.; ‘Paula2019; and ‘Senga Sengana’. All materials in the study on the basis of calculated indexes were divided into four groups differing in reaction to salt. First group (clustered together cv. ‘Paula’, Pau/27 and three second generation clones: Pau/27/11, Pau/27/24, Pau/27/30) represents sensitive genotypes. Second group, including ‘Senga Sengana’, Pau/27/06, Pau/27/10, Pau/27/12, Pau/27/13, Pau/27/15, Pau/27/18, Pau/27/20, Pau/27/21, Pau/27/26, Pau/27/27, Pau/27/31 and Pau/27/32 was designated tolerant. Third group contains only one highly tolerant clone Pau/27/08. The last group comprises two highly sensitive clones (Pau/27/01 and Pau/27/03). This revised version was published online in August 2006 with corrections to the Cover Date.     

Pollen storage of Fragaria and Potentilla

Summary Low temperature and humidity were used for the storage of pollen of four species of Fragaria (2n=14, 42 and 56) and two species of Potentilla (2n=14). The stainability of Fragaria pollen was assessed over a number of years by use of aceto-carmine stain and its viability by cross-pollinations; stainability of Potentilla pollen was assessed by acetocarmine. Fragaria pollen so stored remained stainable for seven years and showed the ability to set seed for three years. over the latter period, aceto-carmine staining gave a reasonable prediction of seed set produced by the stored pollen of Fragaria and so is of value in estimating pollen viability in that genus. Potentilla pollen remained stainable for three years.     

Attempts to hybridize gloxinia and Streptocarpus

Summary Hybridization work was done between gloxinia and streptocarpus cultivars and species. No hybrid seeds were obtained. It is concluded that Sinningia speciosa cultivars and Streptocarpus sp. do not hybridize using conventional methods, in spite of positive reports in literature. The origin of these hybrids may probably be explained by the irregular occurrence of peloric flowers in streptocarpus, and zygomorphic flowers in gloxinia.     

Utilizing wild Fragaria virginiana in strawberry cultivar development: Inheritance of photoperiod sensitivity, fruit size, gender, female fertility and disease resistance

The genetics of photoperiod sensitivity, flowering date, fruit size, gender, female fertility, and disease resistance were investigated in progeny between sets of elite F. virginiana selections and F. × ananassa cultivars and selections planted at sites in Michigan, Minnesota and Ontario. Progeny means varied considerably for all the production traits. Most notable were the large fruit and high fertility observed in crosses with High Falls 22 at all three sites, and Montreal River 10 in Ontario and Michigan. Fragaria virginiana ssp. virginiana parents yielded progeny with much larger fruit than F. virginianassp. glauca parents. General combining ability was significant for all traits at all locations, while specific combining ability was significant for only fruit diameter, ovule set and fruit set in Michigan. Overall, the highest number of day-neutral genotypes were detected in Ontario (mean =44%) compared to Minnesota (31%) and Michigan (26%). In progeny populations of day-neutral F. × ananassa × short-day F. virginiana almost all fit the 1:1 ratio expected if day-neutrality is regulated by a single dominant gene; however, only a few families of short-day F. × ananassa ×day-neutral F. virginianacrosses fit a 1:1 ratio. Likewise, in progeny of day-neutral F. virginiana ×day-neutral F × ananassa crosses, only a few of them fit the 3:1 ratio expected if day-neutrality is regulated by a single dominant gene. These data suggest that it should be relatively easy to useF. virginiana germplasm in strawberry cultivar improvement, and that several different sources of day-neutrality may exist in natural populations. This revised version was published online in August 2006 with corrections to the Cover Date.     

Introgression of the low browning trait from the wild Mexican species Solanum hjertingii into cultivated potato (S. tuberosum L.)

Internal discoloration of tubers resulting from impact damage (blackspot bruise) is a serious quality problem in potato production and utilization, reducing profits to growers and increasing costs for processors. Resistance to blackspot bruise has been identified in the wild species Solanum hjertingii and is therefore a potential germplasm resource for genetic resistance to this problem. A bridging cross between S. hjertingii and a cultivated diploid clone was used to produce a triploid hybrid population that exhibited very low tuber browning potential, indicating a dominant pattern of inheritance for this trait. The triploid progeny were subjected to in vitrochromosome doubling and the resulting hexaploid clones were screened for browning potential. A hexaploid clone selected for low browning was reciprocally crossed with cultivated S. tuberosum cultivars exhibiting high susceptibility to blackspot bruise. Tubers obtained from the seed progeny of these 4x-6x crosses (hereafter referred to as the BC₁ populations) were evaluated for browning potential and polyphenol oxidase (PPO) activity. Tubers from the BC₁ populations displayed a very low potential for melanin production, while PPO activity was quite variable. The low Pearson correlation coefficient (r² = 0.45), between browning potential and PPO activity suggests that the mechanism of blackspot bruise resistance derived from S. hjertingii cannot be explained simply as a reduction in the initial PPO activity. The expression of substantial resistance to browning and dominant expression pattern in these BC₁ progeny indicate that utilizing genetic elements derived from S. hjertingii provides a robust approach for developing blackspot bruise resistant potato varieties. This revised version was published online in August 2006 with corrections to the Cover Date.     

A quantitative classification method for assessing resistance to Phytophthora fragariae var. fragariae in strawberry

Summary Race specific resistance to red core (red stele) root rot, caused by Phytophthora fragariae var. fragariae, is known to occur in the cultivated strawberry (Fragaria × ananassa Duch.), but frequently this resistance does not completely prevent infection. We therefore developed a classification method which distinguishes partial and complete resistance. It accounts for differences in the aggressiveness of isolates and in the appropriateness of experimental conditions for infection. This method is based on the microscopically assessed disease severity of a tested strawberry genotype relative to that of an universally susceptible reference cultivar. If the tested genotype is significantly (P0.01) less diseased, it is considered to possess resistance. Data from 18 genotype-isolate combinations, including five genotypes (Blakemore, Md683, Redgauntlet, Del Norte, Yaquina A) and four North American isolates (A2, A4, A6 and A10) demonstrated the validity of our classification scheme.Abbreviations PIRT Percentage of Invaded Root Tissue     

Introgression of unique characteristics of floral initiation under 24 hour day-length of Fragaria chiloensis ‘CHI-24-1’ into F. × ananassa

A unique flower initiation of the wild Junebearing strawberry strain ‘CHI-24-1’ in Fragaria chiloensis occurs under 24 h DL and high temperature conditions. To introduce the floral initiation characteristics of ‘CHI-24-1’ into cultivated strawberries of F. × ananassa, a cross pollination was conducted between ‘CHI-24-1’ and the Japanese short-day type strawberry cultivar ‘Nyoho’. The floral initiation of ‘CHI-24-1’ was induced in both parent and daughter plants linked with runners under a 24 h DL and 23/20 ^∘C, but not 8 and 16 h DLs at the same temperatures. Of the 21 F1 hybrids grown under the 24 h DL, 12 showed flower truss production in the parent and/or daughter plants linked with runners. Among 64 F1 hybrids, 26 exposed to 8, 16 and 24 h DLs for 30 days produced no flower trusses. However, 32, one and five F1 hybrids produced flower trusses under the 24 h DL alone, 8 h DL alone and both the 8 and 24 h DLs, respectively. The results of the experiments indicated that none of the F1 hybrids were day-neutral plants, but approximately 60% had the characteristics of floral initiation under 24 h DL, which was inherited from the pollen parent of ‘CHI-24-1’. The importance of the unique floral initiation characteristics under 24 h DL with high temperature for strawberry breeding was discussed.     

Synthesis and preliminary characterization of a new species (amphidiploid) in Cucumis

A successful interspecific hybridization between cucumber (Cucumis sativus L., 2n = 14) and a wild it Cucumis species, C. hystrix Chakr. (2n = 24) was made via embryo rescue. Hybrid plants (2n = 19; 7 from cucumber and 12 from C. hystrix) were sterile, but morphologically uniform. Self-pollination and backcrossing of F₁ hybrid plants to either parent confirmed presence of both male- and female-sterility that were likely caused by lack of homology and improper pairing during meiosis. While the multiple-branching habit, densely brown hairs (on corolla and pistil), orange-yellow corolla, and ovate fruit of F₁ hybrid plants were similar to that of the C. hystrix parent, the appearance of the first pistillate flower was more similar to that of C. sativus parent. The diameter and internode length of the stem, and the shape and size of leaves and flowers were intermediate when compared to the parents. The chromosome number in the hybrid was doubled through somaclonal variation during embryo culture and regeneration process to restore the fertility. Pollen grains were released and fruits with viable seeds matured on fertile, synthetic amphidiploid plants. The results from flow cytometry indicated that, on average, 7.3% of the morphologically unique regenerants had the 4C DNA content of 2.35 pg relative to the 2C DNA content of the original F₁ hybrid at 1.17 pg and, therefore, were likely chromosome-doubled F₁ hybrids (2n = 38). Nutrition alanalysis indicated that the synthetic species had higher protein (0.78%)and mineral (0.35%) content compared to the normal pickling cucumber(0.62% and 0.27%, respectively), and could be considered a new Cucumis crop having a special place in the future agriculture. Preliminarily evaluation indicates that C. hystrix possesses a high level of root-knot nematode resistance, and that this resistance is partially expressed in the interspecific F₁ and chromosome-doubled F₁. This and the fact that the fruit morphology of the fertile amphidiploid differs during the growing season (e.g., short and long fruit) suggest that it could be useful in broadening the germplasm base of cucumber. This revised version was published online in July 2006 with corrections to the Cover Date.     

Relationships between African species of the genus Cucumis L. Estimated by the production,vigour and fertility of F1 hybrids

Summary The production, vigour, and fertility of F₁ hybrids between nine African species of the genus Cucumis L. were studied as a measure of the relationships between the species. Hybrid plants were obtained from 29 out of the 72 possible cross combinations. Two F₁ hybrids died as seedlings, and 27 hybrids were raised to maturity. Pollen production and stainability varied greatly amongst these hybrids, as did fruit and seed set following self pollination and backcrossing with either parental species. The fruit shape of the hybrids was always intermediate between that of both parental species.Two species appeared to be closely related: C. prophetarum L. and C. anguria var. longipes A. Meeuse. Most other species produced highly to moderately fertile F₁ hybrids with at least one other species. C. metuliferus Naud. produced only sterile hybrids with C. zeyheri Sond. 2x. The results sustain the recent taxonomic classification of the genus (Jeffrey, 1980).     

Intersubgeneric crosses within the genus Pelargonium

Summary The two cultigens, Pelargonium X domesticum (n=11), subgenus Pelargium, and P. X hortorum (n=9), subgenus Ciconium, were crossed reciprocally and with P. peltatum (n=9), subgenus Dibrachya. Diploid and tetraploid cultivars of P. X hortorum and P. peltatum and tetraploid cultivars of P. X domesticum were used. Viable seeds were obtained from crossing P. X hortorum and P. peltatum and four hybrid plants were produced. Only fruit set occurred in the cross P. X domesticum x P. X hortorum. In all other combinations no fruit set and no seeds were produced.In P. X domesticum x P. X hortorum and P. X domesticum x P. peltatum pollen tubes penetrated the stigma but not the style or ovary. Although the styles elongated in P. X domesticum x P. X hortorum, the ovules did not grow. Fruit set also occurred when the emasculated florets of P. X domesticum were bagged and not pollinated. Fruit set in this cross was apparently a physiological response of the plant, perhaps to the cold temperature, 5°C, used to induce flowering and not a response to either pollination or fetilization.     

Interspecific hybridization of Fragaria moschata with two diploid species, F. nubicola and F. viridis

The crosses Fragaria moschata × F. nubicola, F. moschata × F. viridis, and their reciprocal combinations were done to create tetraploids for eventual introgression into octoploid cultivars of Fragaria × ananassa via the synthetic octoploid system. The combination F. nubicola × F. moschata averaged 3.3 healthy plants/pollination, which was 2.3 times higher than the next best combination, F. moschata × F. viridis. Fragaria viridis × F. moschata crosses averaged only 0.1 healthy plants/pollination and no plants were obtained from F. moschata × F. nubicola hybridizations. The success of the F. nubicola × F. moschata crosses in this study indicated that embryo/endosperm ratios were not as important a factor in producing hybrids with F. moschata × diploids species, as previously suggested.Although 90% of pollinated flowers set fruit with an average of 19 seeds/pollination only 15% of the seeds contained healthy, white embryos and only 38% of these developed into healthy seedlings. The remaining 62% of embryos lacked pigment, had short hypocotyls or arrested development after slight expansion out of the seed coat. Only 39% of the crosses resulted in viable progenies. After one year, 27% of these hybrids bloomed and 71% of the blooming hybrids were tetraploids. All six diploid and six of the seven hexaploid accessions were successfully incorporated into 89 hybrids.