The interaction between compatible and self-incompatible pollen of apple and pear as influcenced by their ratio in the pollen cloud

Summary The production of self-seed was investigated in apple and pear with the aid of viable and compatible mentor/ pioneer pollen in relation to the proportion of self-imcompatible pollen present in the pollen cloud.Treatments consisted of mixtures of compatible and self-incompatible pollen at ratios of 1:1, 1:2 and 1:9 applied once or twice or followed by self-pollination. Selfing only, whether once or twice, produced virtually no fruits or seeds, while mixed pollinations did. Generally, fruit set tended to decrease and the self-seed to increase with increasing amounts of self pollen in the pollinations.The pioncer-pollen method (compatible in advance of self pollen) appeared more effective than the mentor pollen method (compatible and self pollen mixed). The use of viable instead of dead mentor/pioneer pollen causes competition for the ovules with the self pollen, but has the advantage that, in addition to some self-seed, other seeds are formed which, e.g. in apple, are necessary to keep the fruits on the tree until harvest.Fruit set was moderately reduced at ratios of compatible pollen to self pollen not exceeding 1:9 with pear and 1:5 with apple which constitute maximal ratios in practice as regards pollinator trees: trees of the main cultivar. In fruit orchards and probably in nature with other species, the interaction between compatible and self pollen may result in more self-seed, and so suggests that inbreeding played a greater part in the evolution of self-incompatible species, than the meager results of articial self-pollination imply.     

Pollen and pollination experiments. I. the contribution of stray pollen to the seed set of depetalled,hand-pollinated flowers of apple

Summary Using the finger nails to remove most of the petals is a fast method of preparing apple flowers for hand pollination in the balloon stage. Such flowers still appeared quite attractive to insects; left to open pollination, the seed set per flower averaged two thirds of that of similarly treated, but hand-pollinated flowers. With the aid of marker pollen of scab or mildew resistant donors and the subsequent screening of the seedling progenies for resistance, it was shown that the stray pollen was responsible on average for one of every three seeds formed by unbagged depetalled and hand-pollinated flowers.     

Pollination with gamma-irradiated pollen and development of fruits,seeds and parthenogenetic plants in apple

Summary Four apple (Malus X domestica) genotypes, Erovan, Golden Delicious, R1-49 and X6677, were pollinated with a marked pollen irradiated by -rays at doses ranging from 125 to 1000 Gy. Pollination with such irradiated pollen affected fruit set, seed number and seed contents, and induced the formation of parthenocarpic fruits and the development of parthenogenetic embryos. The immature embryos extracted from seeds. 2 and 3 months after pollination, were cultured in vitro and germinated after 2 months of cold treatment (3°C). Haploid plants were obtained in all 4 genotypes, after pollination with pollen irradiated at doses from 200 to 500 Gy. The optimum conditions for induction of apple haploids, by irradiated pollen approach, have been established.     

DNA fingerprinting of apple (Malus spp.) rootstocks using Simple Sequence Repeats

Identification of apple clonal rootstocks based on visual morphological characteristics is often difficult because of the genetic similarity of many rootstocks and the restriction of observable characteristics at the nursery stage. In this study, a total of 66 rootstock clones representing a sub‐sample of a collection of apple rootstocks from different countries maintained at NIFTS, Morioka, Japan, was screened with seven simple sequence repeat (SSR) markers to establish genetic identities, estimate genetic diversity, verify parentage and examine genetic relationships. All seven SSR loci produced one or two amplified fragments and made it possible to differentiate among the rootstocks, except between virus‐free subclones and their original parents as well as between ‘Aotea 40’ and ‘Aotea 106’. The parentage of all but one rootstock clone was confirmed. The placement of individual rootstock clones into subgroups in a phenogram generated from un‐weighted pair group average cluster analysis was generally based on parentage or genetic origin. Based upon the ability of the SSR data to differentiate these apple rootstocks and the high level of gene diversity, it is proposed that all commercial apple rootstocks be fingerprinted to provide a mechanism to verify the identity of individual clones.     

Screening for scab resistance by RAPD markers in cultivars of apple (Malus spp.)

Genetic markers are a much faster and more practical alternative to classical methods for the identification of genes for scab resistance present in different apple cultivars. In our study, 28 scab-resistant cultivars, four wild sources of the resistance genes and 10 susceptible cultivars were screened for the presence of the RAPD fragments OPM18/900, OPD20/600 and OPA15/900, which are reported to be linked to the Vf gene. All three marker fragments were successfully amplified with different protocols in Vf-resistant cultivars including ‘M. floribunda 821’. No marker fragments were amplified in susceptible cultivars, three out of four Va-resistant cultivars, three out of four Vm-resistant cultivars, two Vr-resistant cultivars, ‘Antonovka PI 172612’ and ‘M. pumila R 12740-7A’. All three markers were found in the cv. ‘Nova Easygro’, reported to possess the Vr gene, and the cv. ‘Reglindis’, reported to be Va-resistant. M. atrosanguinea of unknown origin showed the presence of OPD20/600 and OPA 15/900 marker bands. The cvs. ‘Nova Easygro’, ‘Reglindis’ and M. atrosanguinea are probably carriers of the VF gene.     

Pollen and pollination experiments. III. The viability of apple and pear pollen as affected by irradiation and storage

Summary Apple and pear pollen was irradiated with doses of 0, 50, 100, 250 and 500 krad (gamma rays) and stored at 4°C and 0–10% r.h. From the in-vitro germination percentages an average LD 50 dose of about 220 krad was estimated. For both irradiated and untreated pollen a close and corresponding lineair relationship existed between germination percentage and pollen tube growth.Irradiated pollen was much more sensitive to dry storage conditions than untreated pollen, resulting in less germination and more bursting. Apparently, irradiation caused the pollen cell membrane to lose its flexibility faster than normal. Rehydration of dry-stored, irradiated pollen in water-saturated air restored germination percentages up to their initial levels. The importance of this procedure in germination trials is stressed.     

Early determination of genotypes for apple scab resistance by forced flowering of test cross progenies

Summary Apple selections with different major genes for resistance to apple scab (Venturia inaequalis) derived from Malus floribunda and M. pumila were crossed with each other. The progenies were screened as young seedlings for their reaction to V. inaequalis race 1. A gene for resistance from M. pumila, causing stellate necrotic (SN) lesions, was epistatic to a second gene for resistance from M. floribunda, causing irregular chlorotic (Chl) lesions. Although in most cases SN, Chl and susceptible phenotypes were clearly distinct, occasionally reactions were difficult to characterize or varied from one inoculation to another. Selected seedlings showing resistant or susceptible reactions were forced to flower in 16–20 months in the greenhouse and test crossed with susceptible cultivars. Test cross seedlings were screened for scab reaction. The presence of both genes for resistance in a resistant plant was indicated by presence of both Chl and SN resistant phenotypes in the test cross progeny. Chi-square analysis of four large progenies produced a good fit to the expected ratio. The use of the forced flowering technique to determine scab resistance genotypes in 28 months demonstrated its value in breeding apples with multiple disease resistance.     

Genetic Relationships in Malus Evaluated by RAPD 'Fingerprinting' of Cultivars and Wild Species

The potential use of RAPD markers for taxonomic studies in Malus was investigated using 18 accessions of wild species and 27 apple cultivars. 29 preselected random decamer primers were applied to three sets of Malus genotypes. Random amplified polymorphic DNA (RAPD) ‘fingerprints’ were analysed for polymorphic amplification fragments, and coefficients estimating genetic similarity were calculated on the basis of about 50 polymorphic RAPD loci in each set of genotypes. Cluster analysis by an unweighted pair-group method with arithmetic averages (UPGMA) revealed that, in the cultivars, the molecular classification was in good agreement with the known lineage. A dendrogram generated for the wild species gave relationships that were, in principle, in accordance with the known phylogenetic information. Closely related species from section I were clearly distinguishable from those of sections III and IV. On the molecular level, a high degree of genetic diversity was found among both different apple cultivars and wild species of the genus Malus. The results gave additional evidence for the hypothesis that M. pumila and M. sylvestris were involved in the origin of the cultivated apples.     

Molecular characterisation of cultivars of apple (Malus × domestica Borkh.) using microsatellite (SSR and ISSR) markers

In this study, two microsatellite-based methodologies (SSR and ISSR) were evaluated for potential use in fingerprinting and determination of the similarity degree between 41 commercial cultivars of apple previously characterised using RAPD and AFLP markers. A total of 13 SSR primer sets was used and 84 polymorphic alleles were amplified. Seven ISSR primers yielded a total of 252 bands, of which 176 (89.1%) were polymorphic. Except for cultivars obtained from somatic mutations, all cultivars were easily distinguishable employing both methods. The similarity coefficient between cultivars ranged from 0.20 to 0.87 for SSR analysis and from 0.71 to 0.92 using the ISSR methodology. Dendrograms constructed using UPGMA cluster analysis revealed a phenetic classification that emphasises the existence of a narrow genetic base among the cultivars used, with the Portuguese cultivars revealing higher diversity. This study indicates that the results obtained based on the RAPD, AFLP, SSR and ISSR techniques are significantly correlated. The marker index, based on the effective multiplex ratio and expected heterozygosity, was calculated for both analyses (MI = 1.7 for SSR and MI = 8.4 for ISSR assays) and the results obtained were directly compared with previous RAPD and AFLP data from the same material. The SSR and ISSR markers were found to be useful for cultivar identification and assessment of phenetic relationships, revealing advantages, due to higher reproducibility, over other commonly employed PCR-based methods, namely RAPD and AFLP. This revised version was published online in August 2006 with corrections to the Cover Date.     

Correlation of stylar ribonuclease isoenzymes with incompatibility alleles in apple

Fifty-six cultivars of apple were analysed for stylar ribonucleases; proteins were extracted from styles, separated by non-equilibrium pH gel electrofocusing and stained for activity. Excellent correlation was found between the ribonuclease bands revealed and the 11 known incompatibility, S, alleles, in 14 diploid cultivars genotyped in the classic work of Kobel by monitoring pollen tube growth after test crossing, and in 20 cultivars genotyped, at least partially, by more recent DNA methods. For 12 triploid cultivars studied by Kobel, the correlation was good but not perfect. Two apparent minor electrophoretic variants for S10 were noted and, to distinguish them from each other and also from the electrophoretically similar S3, isoelectric focusing was used. Ten cultivars were genotyped for the first time. In all, 14 ribonuclease bands that may correspond to the ‘new’ S alleles, S12 to S 25, were detected but these alleles should be regarded as provisional until confirmed by pollination tests, especially when the electrophoretic differences were only slight. Analysis of stylar ribonucleases is a convenient method of predicting S alleles in flowering material and thereby investigating incompatibility relationships. The polymorphism of the S locus makes it useful for checking the identity and parentage of cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

The performance of double or mixed pollinations with compatible and self-incompatible or incongruous pollen of pear and apple

Summary Six pear and five apple trials were carried out to ascertain the outcome of combinations of compatible pollen (C) with self (S) or incongruous pollen (I) as to the pollination index (PI=seeds/pollinated flower). The PI of the mixture C+I (1:5) was consistently depressed as compared to that of the control C. The results of the double pollinations S/C and I/C were affected by the temperature at pollination; their PI's at <15°C were twice as high as those at >15°C, being well above and below the PI of C in the former and latter case respectively. The opposite was true for the C/S combination, the PI of which increased with the pollination temperature; the PI of C/I did not differ much from the PI of C, irrespective of temperature. The conclusion was reached that the interaction previously and presently found between compatible and self-incompatible pollen also exists to a fair extent between compatible and incongruous pollen. However, in pear neither the mentor nor the pioneer pollen technique proved to aid its hybridization with apple, the formation of self seed was not observed either. In apple the production of apple × pear hybrids was likewise doubtful, but the double pollinations S/C and C/S formed 4–10% self seed.     

The use of a modified bulk segregant analysis to identify a molecular marker linked to a scab resistance gene in apple

Almost two-hundred random sequence decamer-primers were used to screen a pair of bulked samples and the donor parent Malus floribunda clone 821 for markers linked to the V_f gene conferring resistance to apple scab (Venturia inaequalis (Cke.) Wint.). A single primer was identified which generated a PCR fragment, OPK16/1300, from the donor parent M. floribunda clone 821 and the scab-resistant selections/cultivars bulk, but not from the scab-susceptible recurrent parent bulk. Co-segregation analysis using a segregating apple progeny and polymorphism analysis of individual scab-resistant Coop selections/cultivars confirmed that this marker was linked to the scab-resistance gene V_f with a recombination frequency of 4.3%. OPK16/1300 was then cloned and sequenced. Sequence-specific primers of 25 oligonucleotides based on the marker were synthesized, and used in turn to screen M. floribunda clone 821, scab-susceptible apple cultivars, scab-resistant apple cultivars, and scab-resistant Coop selections. A pair of sequence-specific primers of clone OPK16/1300 amplified a distinct single band of the same size as the RAPD clone. Thus, a sequence characterized amplified region (SCAR) marker was developed which can be used to identify polymorphisms of OPK16/1300 based on the presence or absence of a single band. This revised version was published online in July 2006 with corrections to the Cover Date.     

Pollen grain germination inside the anthers of two chasmogamous angiosperms: Almond (Prunus amygdalus L. Batsch) and apple (Malus pumila Mill.)

Summary Pollen grain germination inside anthers has so far been known in only eleven chasmogamous angiospermous species. The discovery of this phenomenon in some varieties of Prunus amygdalus (almond) and Malus pumila (apple) is therefore significant. The anomaly appears to be genetically controlled, the gene expression occurring under specific environmental conditions.     

Cytoplasmic relatedness of apple landraces and cultivars: a molecular analysis

Summary Mitochondrial DNA (mtDNA) diversity has been determined in apple landraces and cultivars, based upon restriction fragment length polymorphisms (RFLPs) for several mitochondrial genes. Our analysis includes three cultivars, Golden Delicious, McIntosh and Delicious, which represent the various patterns of mtDNA polymorphisms previously described (Ishikawa et al., 1992). A total of five RFLPs were detected, allowing classification of the apple genotypes into four cytoplasmic groups: GroupI, Golden Delicious-type; GroupII, McIntosh-type; GroupIII, Delicious-type; and Group IV, Dolgo Crab-type. European landraces and cultivars were assigned to Groups I, II, and III, while chinese crab apples were placed in either Group III or IV.     

Meiotic chromosome and pollen morphological studies of polyploid Cucumis species

summary Meiotic chromosome studies of polyploid Cucumis species revealed the presence of bivalent chromosome configurations in the tetraploid C. aculeatus and C. zeyheri and hexaploid C. figarei, while a maximum of ten quadrivalents were observed in pollen mother cells of a tetraploid species, C. heptadactylus.Allotetraploidy was not accompanied by an increase in the number of pores per pollen grain, but the autotetraploid and hexaploid species showed a relatively high percentage of 4-porate pollen. The pollen fertility of the tetraploid species was normal.Contribution from the Department of Horticulture and Department of Agronomy. Supported by the Colorado State University Experiment Station and published as Scientific Series Paper No 2403.Former Graduate Research Assistant, Department of Horticulture (present address, 1030 Sanders St., Auburn, Alabama 36830 USA) and Professor, Department of Agronomy, respectively.     

Identification of marker alleles linked to fire blight resistance QTLs in apple genotypes

Molecular marker analysis can be an effective tool when searching for new fire blight resistance donors. It can speed up the breeding process as well, even though many of the available markers linked to fire blight resistance QTLs have not yet been tested by screening a large number of cultivars. The aim of this study was to search for alternate sources of the three major QTLs of fire blight resistance; FBF7, FB_MR5 and FB_E, as well as to test the efficiency of some markers linked to minor QTLs. Altogether, nine primer pairs were used on 77 genotypes including new Hungarian cultivars and old apple cultivars from the Carpathian basin. Several marker alleles of FB resistance QTLs have been detected in the screened genotypes, most importantly the alleles coupling with FB_MR5 in the old cultivars ‘Kéresi muskotály’, ‘Szabadkai szercsika’ and ‘Batul’. We propose these cultivars as the first available resistance donors of FB_MR5 instead of the crabapple Malus × robusta 5. The results also bring new information regarding the resistance alleles of new Hungarian cultivars and selections.     

Carbamate-induced flowering in asparagus (Asparagus officinalis L.) seedlings: optimization of treatment and cultivar variation in flowering response and pollen germination

Optimal carbamate treatment conditions were studied for flower induction in seedlings of an asparagus cultivar ‘Mary Washington 500 W’. Flower induction was most accelerated by soaking seeds in 50 mg l⁻¹ carbamate solution for 12 days at 25 ^°C under the fluorescent light. Longer exposure to carbamate over a 12 day period induced a higher percentage of seedlings to flower. A higher percentage of flowering seedlings were male. Flower induction frequency among seven cultivars through carbamate treatment widely ranged from 13 to 67%. ‘Geynlim’, ‘Mary Washington 500 W’ and ‘Welcome’ exhibited a high percentage of flowering seedlings, while ‘Larac’ and ‘Vulkan’ showed low values. Only male flowers were induced in all-male cultivars. Variation in pollen germination was found within all cultivars. ‘Geynlim’, ‘Cito’ and ‘Mary Washington 500 W’ showed high values of average pollen germination. Application of carbamate compound to induce flower production can rapidly produce homogenic cultivars which include both sexes. This is necessary for genetic studies and breeding purposes. This revised version was published online in August 2006 with corrections to the Cover Date.     

Pollen and pollination experiments. II. The influence of the first pollination on the effectiveness of the second one in apple

Summary Pollinating apple cultivars twice with compatible pollen at an interval of one or two days produced about twice as many seeds per pollinated flower as a single pollination. With the aid of scab- or mildew resistant marker pollen, it could be shown that the second pollen formed on average twice as many seeds as the first. The first pollen appears to pave the way-partly at its own cost-for the second and was therefore called pioneer pollen.     

Some methodological aspects of apple transformation by Agrobacterium

Summary Leaf explants of apple cvs Gala and Golden Delicious were infected with the Agrobacterium tumefaciens strain AGL0(pMOG410). The effects of a 2 d preculture of the explants before infection and the addition of 5-azacytidine to the selection medium were studied. The percentages of GUS-positive explants after 5 w did not significantly alter due to these treatments. One of the Gala shoots, which was removed from a leaf explant cultured for 8 w on selection medium, proved to be GUS-positive and will be analyzed further. In general, however, it should be concluded that regeneration of transgenic shoots directly from leaf tissue was not very effective.Abbreviations MS Murashige and Skoog medium - GD Golden Delicious