Development of a new SSR-based linkage map in apricot and analysis of synteny with existing Prunus maps

Linkage maps of the apricot accessions ‘Lito’ and ‘BO 81604311’ were constructed using a total of 185 simple sequence repeat (SSR) markers sampled from those isolated in peach, almond, apricot and cherry; 74 were derived from a new apricot genomic library enriched for AG/CT microsatellite repeats (UDAp series), and in total, 98 had never been mapped in Prunus before. Eight linkage groups putatively corresponding to the eight haploid apricot chromosomes were identified for each parent. The two maps were 504 and 620 cM long, respectively, with 96 anchor markers showing a complete co-linearity between the two genomes. As few as three gaps larger than 15 cM were present in ‘Lito’ and six in the male parent; the maps align well with all the available SSR-based Prunus maps through the many common anchor loci. Only occasionally inverted positions between adjacent markers were found, and this can be explained by the small size of cross populations analysed in these Prunus maps and in those reported in literature. The newly developed apricot SSRs will help saturating the existing Prunus maps and will extend the choice of markers in the development of genetic maps for new breeding populations.     

Improved efficiency in apricot breeding: Effects of embryo development and nutrient media on in vitro germination and seedling establishment

Apricot (Prunus armeniaca L.) embryos at three stages of development were cultured on C2d, SBH and WPM media. In vitro culture produced high percentages of germination and seedlings throughout all three developmental stages. Significant media effects were noted for changes in both embryo length and weight during the culture period, as well as number of plants produced. Embryos between 5 and 9 mm (developmental stage I) germinated and developed into plants in a significantly higher percentage than in the other two more mature stages. Therefore, embryo culture can be successfully used as a tool in an apricot breeding program to obtain higher percentages of seedlings from planned hybridization or to overcome a lack of seed germination.     

Different media requirements for micropropagation of apricot cultivars

Factors affecting in vitro propagation of several apricot cultivars were studied. The effect of nutrient media and BA concentration were strongly genotype dependent. Generally, best results were achieved with Quoirin and Lepoivre (1977) and a modification of WPM macronutrients (Lloyd and McCown, 1981). Optimum BA concentration was different for each cultivar but best results were obtained between 1.78 and 3.11 μM. Apricot shoots rooted well with different concentrations of IBA but most shoots showed symptoms of apical necrosis that could be overcome by dipping the shoot tips in solutions of 22.2 or 44.4 μM of BA prior to transfer to rooting medium. This revised version was published online in August 2006 with corrections to the Cover Date.     

Antioxidant enzymes as biochemical markers for sharka resistance in apricot

The activity of antioxidant enzymes in different apricot (Prunus armeniaca L.) cultivars, resistant or susceptible to Plum pox virus (PPV), was analyzed during the years 2002 and 2003. Resistant cultivars showed higher activities of catalase (CAT), ascorbate peroxidase (APX) and dehydroascorbate reductase (DHAR) than susceptible cultivars. Only CuZn-SOD isozymes were detected in the apricot cultivars. However, no correlation was observed between this isozyme pattern and the resistance to PPV. On the other hand, PPV-resistant apricot cultivars could have a greater capability for elimination of H₂O₂ and recycling of ascorbate-glutathione cycle, and they have at least two of these enzymatic activities (CAT, APX and DHAR) over the average. In contrast, this response was not observed in the susceptible cultivars. All these data suggest that the activities of CAT, APX and DHAR could be used as biochemical markers of sharka resistance in apricot.     

Control of hyperhydricity in micropropagated apricot cultivars

Summary The effects of different factors on the control and reversion of hyperhydricity during the in vitro propagation of Prunus armeniaca were studied. Treatments that decreased the hyperhydricity but did not affect micropropagation rates were the use of the bottom cooling system for 1 or 2 wk and agargel as gelling agent in ‘Helena’, whereas the best results were obtained with the bottom cooling system for 2 wk and the use of 0.8% agar as gelling agent in ‘Lorna’. Hyperhydric shoots reverted to normal after keeping them for 3 wk in the bottom cooling system.     

Introduction and establishment of apricot in vitro through regeneration of shoots from meristem tips

Summary In this study different aspects of the in vitro introduction and establishment of apricot cultivars were investigated through meristem tip culture. The best time to introduce the meristems of ‘Canino’ was when buds were starting to swell. Various plant growth regulators were used at different concentrations on four distinct apricot cultivars to promote the development of the meristems to shoots which could then be micropropagated. Very diverse results were obtained depending on the genotype. In general, meristems did not survive without N⁶-benzyladenine. Concentrations of gibberellic acid from 2 to 4 mg 1⁻¹ (5.8–11.4 μM) promoted explant elongation. This step was critically important to obtain apricot shoots large enough to be transferred to proliferation medium.     

Spatial root distribution of apricot trees in different soil tillage practices

Root and soil water distribution was studied in a mature drip-irrigated apricot (Prunus armeniaca L. cv. Búlida) orchard with different soil tillage practices, in a loamy textured soil with a 7% slope, located in Murcia (SE Spain). Three treatments were applied between tree rows:control (no-tillage), whereby, following the common practice in the area, weeds were cut back to ground level by a blade attached to a tractor; perforated treatment, where the soil surface was mechanically perforated with an adapted-plough; and mini-catchment treatment, consisting of mini-catchments with low banks manually raised perpendicular to the line of emitters. Almost all of the apricot root system was located in the first 0.75 m of soil depth, with 91% in the first 0.50 m. More than 75% of the roots corresponded to thin roots, with a diameter less than 0.2 mm. Both tillage treatments decreased runoff compared with the control treatment, while the mini-catchment treatment showed the highest change in soil water content after rainfall events. The mini-catchment treatment was performed in an attempt to reduce the rainwater running down the slope, leaving the accumulated water near plant roots, an effect which was responsible for the higher root length density (RLD) values found in this treatment. In addition, roots were distributed over a wider area, providing higher RLD values up to 1 m from the emitter, meaning that a higher soil volume was explored. For these reasons, the mini-catchment treatment was seen to be the most beneficial soil tillage treatment for optimising water use in semiarid conditions.     

Daily variations in water relations of apricot trees under different irrigation regimes

Mature apricot (Prunus armeniaca L. cv. Búlida) trees, growing under field conditions, were submitted to two drip irrigation treatments: a control (T1), irrigated to 100 % of seasonal crop evapotranspiration (ETc), and a continuous deficit (T2), irrigated to 50 % of the control throughout the year. The behaviour of leaf water potential and its components, leaf conductance and net photosynthesis were studied at three different times during the growing season, when they revealed a diurnal and seasonal pattern in response to water stress, evaporative demand of the atmosphere and leaf age. The deficit-irrigated trees showed, among other effects, a pronounced decrease in leaf water potential (ψ_w), decreased in leaf conductance (g_s) and no osmotic adjustment. For this reason, g_l and ψ_w can be considered good indicators of mature apricot tree water status and can therefore be used for irrigation scheduling.     

The effect of bacterial contamination on the growth and gas evolution ofIn vitro cultured apricot shoots

Summary Shoots of “San Castrese” and “Portici” apricots (Prunus armeniaca L.) free of cultivable bacteria, shoots of the same origin exhibiting bacterial contamination after repeated subcultures, and contaminated shoots treated with cefotaxime were compared for gas exchange, proliferation rate, and fresh and dry weight. Cultures of San Castrese contaminated byBacillus circulans andSphingomonas paucimobilis, and of Portici contaminated withStaphylococcus hominis andMicrococcus kristinae, including those treated with cefotaxime, showed comparable shoot weights and lower proliferation rates than healthy cultures. Bacteria, even if not visible until the end of subculture, markedly influenced the gaseous composition of the jar headspace. Healthy cultures clearly showed photosynthetic activity at 60 μM·m⁻²·s⁻¹ photosynthetically active radiation; in contrast, oxygen quickly decreased and carbon dioxide increased in contaminated cultures, including those treated with cefotaxime, in which bacteria became visible in the culture medium only after repeated subcultures.     

Regeneration of plants from callus and embryos of ‘Royal’ apricot

Immature embryos of apricot (Prunus armeniaca L.) cv. Royal with a PF index of 25–100 were used to regenerate plants in vitro using two methods. In the first case, callus was initiated on MS medium with 4.5 M 2, 4-D plus 0.44 M BA and regeneration of shoots from the callus occurred on MS medium with 4.4 M BA plus 1.0 M 2, 4-D. In the second case, adventitious buds were directly regenerated from the cotyledons on MS medium with 4.4 M BA plus 1.0 M 2, 4-D.Abbreviations BA 6-benzyladenine - IBA dole-3-butyric acid - NAA -naphthylacetic acid - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PF (embryo length/seed length) x 100     

The effect of aminoglycoside antibiotics on the adventitious regeneration from apricot leaves and selection of <Emphasis Type="Italic">npt</Emphasis>II-transformed leaf tissues

The effect of different concentrations of the aminoglycoside antibiotics, geneticin, paromomycin and streptomycin on adventitious regeneration from leaf explants of apricot was tested to design an alternative procedure for selecting transgenic shoots. Streptomycin and paromomycin reduced shoot regeneration percentage with increasing concentration of antibiotics. Almost a complete inhibition of regeneration was reached when 20M paromomycin was used, although up to 40M streptomycin was necessary to completely inhibit regeneration. Geneticin had a very toxic effect on apricot leaves and regeneration was inhibited at almost all concentrations tested. Addition of kanamycin hastened the development of adventitious buds although silver thiosulfate and not kanamycin was responsible for the observed increase in the consistency of the results from independent experiments. Kanamycin and paromomycin at the concentrations tested improved selection of transformed cells and resulted in a larger number of gfp-expressing regions. Paromomycin at 40 and 25.7M kanamycin improved proliferation of transformed tissues as compared with the other antibiotics used and non-selected controls.     

Auxin pulses and a synergistic interaction between polyamines and ethylene inhibitors improve adventitious regeneration from apricot leaves and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of leaf tissues

The effect, on adventitious regeneration from apricot leaf explants and transformation of leaf tissues, of auxins pulses with NAA and 2, 4-D was tested. Addition of the polyamines putrescine and spermidine to the regeneration medium, alone or in combination with the ethylene inhibitors silver thiosulphate and aminoethoxyvinylglycine, were also tested to design a procedure that improved transformation efficiency. Spermidine at 2 mM in combination with 0.5 M aminoethoxyvinylglycine and four-day pulses with two different concentrations of 2, 4-D increased significantly shoot regeneration. Spermidine at the same concentration but in combination with 60 M silver thiosulphate and four-day pulses with 9 M 2, 4-D also increased stable transformation events and GFP-expressing calluses probably by inducing a larger amount of dividing cells where Agrobacterium transferred its T-DNA. Since regeneration from apricot leaves occurs mostly from developing calluses, it is important to obtain many GFP-expressing calluses and, given that transformation efficiencies (number of transformed shoots per total number of explants) in woody plants are generally very low, approaches that allow the optimization of T-DNA transfer and total number of transformed cells obtained, will improve probabilities of obtaining transformed shoots.     

Low sugar and osmotic requirements for shoot regeneration from leaf pieces of Solanum melongena L.

Uniform leaf pieces of egg-plant, Solanum melongena L., were cultured in Murashige and Skoog's medium containing 2 mg l^-1 kinetin and varying sugar levels. Glucose or fructose at 44 mM was optimal in inducing shoot regeneration compared to sucrose. Sucrose at 11 and 22 mM induced more shoot organogenesis than at lower or higher levels. An additional 22 mM mannitol with 22 mM sucrose enhanced shoot regeneration significantly more than 22 mM sucrose alone. The dual role of sugar as carbon and osmotic source in shoot regeneration from leaf explants of egg-plant is discussed.     

Stamen development and winter dormancy in apricot (Prunus armeniaca)

Background and Aims

In temperate woody perennials, flower bud development is halted during the winter, when the buds enter dormancy. This dormant period is a prerequisite for adequate flowering, is genetically regulated, and plays a clear role in possibly adapting species and cultivars to climatic areas. However, information on the biological events underpinning dormancy is lacking. Stamen development, with clear differentiated stages, appears as a good framework to put dormancy in a developmental context. Here, stamen developmental changes are characterized in apricot (Prunus armeniaca) and are related to dormancy.

Methods

Stamen development was characterized cytochemically from the end of August to March, over 4 years. Developmental changes were related to dormancy, using the existing empirical information on chilling requirements.

Key Results

Stamen development continued during the autumn, and the flower buds entered dormancy with a fully developed sporogenous tissue. Although no anatomical changes were observed during dormancy, breaking of dormancy occurred following a clear sequence of events. Starch accumulated in particular places, pre-empting further development in those areas. Vascular bundles developed and pollen mother cells underwent meiosis followed by microspore development.

Conclusions

Dormancy appears to mark a boundary between the development of the sporogenous tissue and the occurrence of meiosis for further microspore development. Breaking of dormancy occurs following a clear sequence of events, providing a developmental context in which to study winter dormancy and to evaluate differences in chilling requirements among genotypes.     

Microsatellite markers in the hexaploid Prunus domestica species and parentage lineage of three European plum cultivars using nuclear and chloroplast simple-sequence repeats

In a previous study, cDNA microsatellite markers were described in apricot (Prunus armeniaca L.). Specific PCR primers were designed to amplify the microsatellite-containing regions from genomic DNA in different Prunus species. In the present work, cDNA microsatellite markers were developed in the hexaploid Prunus domestica L. species and polymorphism was ascertained in a segregating plum population. Co-dominant mendelian segregation of alleles was demonstrated and microsatellite polymorphism displayed up to 6 alleles per SSR locus per individual. Parentage lineage of three full-sib European plum cultivars (cv. Cacanska najbolja, Cacanska rana and Cacanska lepotica) was reconstructed by the analysis of the above nuclear SSR markers, completed by four chloroplastic microsatellite loci. The six most informative nuclear loci enabled discrimination between the three Cacak cultivars and unrelated individuals as well as the previously proposed parents, Wangenheim and Pozegaca. Data obtained support previous evidence that these cultivars originated from the Stanley cultivar. However, SSR analysis finally excluded Wangenheim as the other possible parent. Based on the results obtained with nuclear and chloroplast SSR loci, we propose the origin of those three Cacak cultivars in a cross between Stanley as the mother plant and Ruth gerstetter as the pollinator. Furthermore, we demonstrate the utility of these apricot SSR markers for genotype fingerprinting of the hexaploid plum cultivars.     

3种杏抗旱性和雌蕊发育比较研究

在陕西省淳化县对大扁杏、梅杏和山杏的抗旱性和雌蕊发育情况进行比较,发现在荒坡条件下大扁杏的叶绿素含量高于山杏和梅杏,山杏的抗旱性最强,大扁杏次之,梅杏最差。同时,3种杏雌蕊发育有明显的差异,大扁杏完全花的数量远大于梅杏和山杏,山杏雌蕊发育最差。     

Use of sugarcane bagasse as an alternative low-cost support material during the rooting stage of apple micropropagation

Summary Studies were carried out to evaluate sugarcane bagasse as an alternative to agar for micropropagation of apple clones to reduce the cost of micropropagation and improve the quality of the propagules. Significant improvement in the in vitro rooting process, coupled with cost reduction, were obtained by the use of sugarcane bagasse as a substitute for the traditionally used agar-gelled medium. The tests were undertaken with micro-cuttings of the apple rootstock Marubakaido (Malus prunifolia Borkh.) using a rooting medium composed of half-strength Murashige and Skoog salts and vitamins, 3% (w/v) sucrose, and 0.49 μM indole-3-butyric acid. The plants grown on sugarcane bagasse yielded a 22% increase in root length, 20% increase in plant length, and 63% increase in the number of roots, compared with agar-grown micro-cuttings. Particle size of the sugarcane bagasse had a significant impact on all those parameters, and the best results were obtained with bagasse comprising particles smaller than 0.18 mm. The results demonstrated that the sugarcane bagasse could be used effectively as a substitute for agar during rooting of apple shoots.     

Improved micropropagation in Polygala myrtifolia

Summary Stem segments from apical shoot tips of Polygala myrtifolia were used as primary explants to establish in vitro cultures. Axillary shoots produced on non-contaminated explants were excised and recultured in the same medium to increase the stock of shoot cultures. Equal molar concentrations of five cytokinins [2-isopentenyladenine, kinetin, zeatin, N ⁶-benzyladenine (BA), and adenine] were tested for ability to induce axillary shoot development from double-node stem segments. The highest rate of axillary shoot proliferation was induced on Murashige and Skoog agar medium supplemented with 1.8 μM BA. Seven indole-3-acetic acid (IAA) concentrations (0, 2.9, 5.7, 8.6, 11.4, 14.3, 17.1 μM) were tested to determine the optimum conditions for in vitro rooting of microshoots. Up to 72% of the microshoots rooted with 14.3 μM IAA. Other auxins tested, α-naphthaleneacetic acid and indole-3-butyric acid, were less effective than IAA in inducing adventitious root formation. All rooted plantlets having more than three roots were successfully established in soil.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .     

Role of carbohydrates in micropropagation of cork oak

The influences of carbon sources, fructose, glucose, sorbitol and sucrose on shoot proliferation and in vitro rooting of cork oak (Quercus suber L.) were compared at a wide range of concentrations (1–6%, w/v). The highest number of shoots occurred on glucose-containing medium. Nevertheless, we have chosen 3% sucrose which induced a similar rate of proliferation but favoured shoot elongation, permitting an effectively higher number of shoots during transfers. Sorbitol and autoclaved fructose did not stimulate shoot proliferation. Adventitious root formation was strongly dependent on carbohydrate supply. Sorbitol and autoclaved fructose were completely ineffectively on rooting induction. Glucose was the most effective carbon source on rooting promotion followed by sucrose and filter-sterilized fructose. The rooting response induced by fructose was dependent on the sterilizing procedure. The number of adventitious roots produced per shoot increased with increasing glucose and sucrose concentration. The content of reducing sugars in leaves of proliferation cultures and in leaves and roots of rooted plantlets was more dependent on carbon concentration than on glucose or sucrose supplement. The results presented here show that carbohydrate requirements during cork oak micropropagation depend upon the phase of culture. Sucrose (3%) and glucose (4%) were the best carbon sources respectively during proliferation and rooting phases.