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Raul de la Rosa, Celia M. James and Kenneth R. Tobutt

Eight microsatellite primers were used to distinguish 23 olive cultivars that were parents, or potential parents, in a Spanish breeding program. Four of these microsatellites were particularly informative and were used to check the paternity of 11 olive progenies thought to come from selfings or controlled crosses involving nonemasculated flowers. Seven progenies were found to be highly contaminated, i.e. many seedlings had unexpected alleles, and only four were found to be pure or almost pure. Almost all the contamination detected came from outcrossing, indicating that placing the pollination bags well before anthesis is important and that emasculation to avoid selfing is unnecessary. More than two nonparental alleles per primer were found in each contaminated progeny, showing that more than one cultivar caused contamination. However, the allele data are consistent with `Picual' (the main commercial cultivar growing in the area where the crosses were made) being the contaminant in 48% of the nontrue seedlings (excluding `Picual' self progenies). Some other cultivars planted near the female trees were also found to be sources of contamination. The results obtained show that microsatellite analysis is a convenient technique to assess routinely the crosses made in breeding programs and to check self-incompatibility in olive. The pure progenies identified will be useful for reliable inheritance studies in olive, which have rarely been reported in the literature.

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Lorenzo León, Raúl de la Rosa, Diego Barranco and Luis Rallo

The initial results of a comparative field trial of the first 15 selections of the olive (Olea europaea L.) breeding program of Cordoba, Spain, are presented. These selections came from crosses among ‘Arbequina’, ‘Frantoio’, and ‘Picual’ that were also included in the trial as controls. The trial was planted in July 2001 in a randomized block design with 16 replications and was systematically evaluated for earliness of bearing, vigor, crop, and yield efficiency from 2001 to 2005. Significant differences among selections were found for all characters measured. A greater proportion of early-bearing genotypes than in previous cultivar collections were found, whereas mean accumulated yield was similar to former evaluations. Therefore, the shorter unproductive period obtained in this work seems to indicate that the selection of seedlings for a short juvenile period has provided a shorter unproductive period of the subsequent new cultivars. No correlation between vigor at the seedling stage and vigor in the corresponding adult vegetative propagated selection was found. If the data presented here are confirmed further, some early-bearing cultivars could be suggested as new olive cultivars, the first obtained by cross-breeding in Spain. Additionally, some of them also show a low vigor and could be adapted to high-density hedgerow orchards.

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Aurora Díaz, Antonio Martín, Pilar Rallo and Raúl De la Rosa

Previous analyses of olive (Olea europaea L.) crosses have revealed an important degree of pollen contamination. For this reason, the authors tested the paternity of a set of progenies coming from crosses among different cultivars within the olive breeding program of Córdoba, Spain, using four polymorphic microsatellites. They found that the expected pollen sired 141 (83.4%) of the 169 samples analyzed. The contamination rate was either almost total or almost null within each particular cross considered, not being comparable between different crosses. In a second experiment the authors evaluated the influence of several factors on the success of olive crosses, the type of isolation bag, the timing and number of pollinations, and the cross-compatibility of the parents in a multifactorial assay in 2003. They observed no differences in the type of pollination bag used or the number and timing of pollen additions when they analyzed 145 seeds. The main factor affecting the success of the crosses seems to be the intercompatibility among cultivars, because it had a significant influence on the rate of contamination. In the failed crosses, the authors clearly detected the contribution of more than one cultivar to the paternity. The results obtained here indicate that some knowledge of cross-compatibility relationships in olive is required to design crosses in olive breeding programs effectively. To achieve this objective, the progeny parentage analysis could be of great help because of the high level of pollen contamination found among those studied here.

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Luis Rallo, Diego Barranco, Raúl de la Rosa and Lorenzo León

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Raul De la Rosa, Luis Rallo and Hava F. Rapoport

In the olive (Olea europaea L.), inflorescence and flower differentiation occur in the early spring following a period of winter chilling and dormancy of the potentially reproductive buds. We examined the size, structure, and starch content of these buds during winter rest in the field and during forcing under standard growth-chamber conditions. Basic bud structure and dimensions remained unchanged during the rest period, but starch content increased in the bud's central axis. When cuttings were forced in the growth chamber, the buds followed a morphogenetic pattern similar to that observed in the field, but the sequence of developmental events could be timed more precisely. The first changes observed were the onset of axis growth and the differentiation of axillary primordia within 3 days of transfer to the growth chamber. This was followed by the initiation of new nodes, and, at 15 to 18 days, by the first signs of floral differentiation in the terminal and axillary bud apical meristems. Bud growth and differentiation were accompanied by a decrease in starch content.

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Inmaculada Moreno-Alías, Hava F. Rapoport, Rafael López, Lorenzo León and Raúl de la Rosa

In recent years, the juvenile period of olive seedlings has been reduced by diverse forcing and pruning-formation techniques, but it is still a major drawback for breeding. Plant height at the time when seedlings are transplanted to the field has been found effective for obtaining earlier flowering, but the value of this parameter varies widely among olive breeding programs. We evaluated different transplant dates, taking into account mean plant height and age, with the aim of determining the optimum parameters for achieving early flowering and easy plant management and, at the same time, for the early elimination of genotypes with a long juvenile period. A mean seedling height of ≈100 cm at the time of planting in the field provided the best results in terms of shortening the juvenile period and facilitating handling of the seedlings. For all dates tested, seedling height at transplanting was effective as a pre-selection criterion for the early elimination of genotypes with long juvenile periods, with slight improvement in effectiveness with advancing transplant date.

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Aurora Díaz, Antonio Martín, Pilar Rallo, Diego Barranco and Raúl De la Rosa

We studied the self-incompatibility of two main Spanish olive (Olea europaea L.) cultivars, `Picual' and `Arbequina', by testing the selfing of the seeds with microsatellites. For this purpose, we used a rapid single-seed DNA extraction method and four highly polymorphic microsatellites. We analyzed seeds produced in branches bagged for selfing from mono- and multi-cultivar orchards in 2002 and 2003. We did not find any seed coming from selfing in the bagged branches, for either cultivar, in the two types of orchards. Additionally, we tested seeds coming from free pollination in mono-cultivar orchards from different locations. In the case of `Picual' olive, only three seeds out of the 70 collected were the product of selfing, although they came from mono-cultivar orchards located in areas where the cultivar used as the female parent was predominant. From the 20 seeds of `Arbequina' olive harvested in the middle of two high-density plantations, not one was a product of selfing. According to this, olive would behave as an allogamous species in mono-cultivar growing conditions and the pollen coming from long distances would be able to produce a normal bearing. Therefore, there is strong evidence to support the idea that the cultivars studied could be self-incompatible. Future experiments in self-compatibility should include a paternity check of the possible self seeds obtained.

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Milad El Riachy, Luis Rallo, Raúl de la Rosa and Lorenzo León

The duration of the juvenile period (JP) before the seedlings reach the adult phase (AP) and begin flowering is the major impediment for fruit breeding in olive. In this work, the effect of soil solarization on the growth and duration of the JP of olive seedlings was studied. Seedlings obtained from open pollination of ‘Manzanilla de Sevilla’ and from crosses between ‘Arbequina’ × ‘Arbosana’ and ‘Picual’ × ‘Koroneiki’ were tested. Solarization treatment consisted on covering the soil around the trees for 3 years by black plastic film in a split-plot design with the three progenies as the main factor and solarization/not solarization as the secondary factor. Solarization resulted in both higher soil temperature and soil moisture in solarized plots with respect to non-solarized. Significant differences between crosses were found for plant vigor and flowering measurements throughout the experiment. Soil solarization increased trunk diameter and the number of seedlings reaching AP in ‘Manzanilla’ open progeny. A positive effect of solarization on flowering index was also observed in ‘Picual’ × ‘Koroneiki’ progenies. These results suggest that soil solarization may be recommended as a tool for shortening the duration of the JP and thus accelerating the selection process in olive breeding programs.

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Fernando Santos-Antunes, Lorenzo León, Raúl de la Rosa, Juán Alvarado, Ana Mohedo, Isabel Trujillo and Luis Rallo

A first attempt to determine the effect of vigor and parents on the length of the juvenile period of olive seedlings is here reported. Vigor seems to have a significant influence on the percentage of flowering seedlings, especially in the first 2 years of bearing. The different parents used have produced differences in the juvenile period of their descendants. A correspondence between the length of the unproductive period of the parents and the length of the juvenile period that they transmit to their descendants has been observed. The seedling forcing growth protocol described here has been able to produce flowering seedlings 28 months after germination, with >93% of seedlings flowering 65 months after germination.

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Raúl De la Rosa, Angjelina Belaj, Antonio Muñoz-Mérida, Oswaldo Trelles, Inmaculada Ortíz-Martín, Juan José González-Plaza, Victoriano Valpuesta and Carmen R. Beuzón

In the present work, a set of eight new hexa-nucleotide simple sequence repeats (SSRs) is reported in olive (Olea europaea L). These SSRs loci were generated on the basis of expressed sequence tag (EST) sequences in the frame of an olive genomic project. The markers showed a high level of polymorphism when tested on a set of cultivars used as genitors in the olive breeding program of Córdoba, Spain. The long-core repeat motif of these markers allows a wider separation among alleles, thus permitting an accurate genotyping. Besides, these markers showed comparable levels of polymorphism to di-nucleotide SSRs, the only ones so far reported in olive. Selected on the basis of their discrimination capacity, four of the eight SSRs were used to test their ability for paternity testing in a total of 81 seedlings coming from 12 crosses. The paternity testing showed that seven crosses matched the alleged paternity and the remaining five were products of illicit pollinations. These results exactly matched with previous paternity testing performed with di-nucleotide SSR markers. These results demonstrate the usefulness of the developed hexa-nucleotide repeated motifs for checking the paternity of breeding progenies and suggest their use on variability studies.