Adventitious shoot regeneration in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch]

A method for adventitious shoot regeneration from leaves of micropropagated peach shoots has been developed. Apices were excised from in vitro shoot cultures of a seed-derived (juvenile) genotype (P16Cl5) and mature genotypes (Babygold 6, 842 Standard, San Giorgio and Yumyeong). Apices were cultured 21 days in the dark on a medium supplemented with 6-benzyladenine and α-naphthaleneacetic acid and then transferred to an auxin-free medium and incubated in the light for 21 days. The first four apical leaves were excised from these apices and cultured in the same way. During the dark incubation period, leaves developed a callus at the petiolar base. Adventitious shoots appeared on this callus after transfer to auxin-free medium and culture under light conditions. The morphogenic ability of the callus was maintained for several months.     

Expression analysis and genetic mapping of three <Emphasis Type="Italic">SEPALLATA</Emphasis>-like genes from peach (<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch)

SEPALLATA (SEP) MADS box genes play essential and diverse roles in reproductive organ development. To investigate the SEP gene function in peach we isolated three SEP-like genes, PrpMADS2, PrpMADS5, and PrpMADS7, which belong to distinct SEP gene clades. They appeared as single copy genes in the peach genome and were found to preferentially express in flowers and fruits. Arabidopsis transformants expressing 35S: PrpMADS2 were indistinguishable from wild-type plants. Overexpression of PrpMADS5 led to earlier flowering. Through chimeric repressor silencing technology, PrpMADS5 was found to function in floral organ development. Expression of PrpMADS7 in Arabidopsis caused a dramatic attenuation of both juvenile and adult growth phases and, in severely affected plants, it led to flower formation immediately after the embryonic phase. Two microsatellite markers were developed for PrpMADS2 and PrpMADS5 and assigned to the genetic linkage groups 5 and 1, respectively. PrpMADS7, previously identified as PrpAGL2, and PrpMADS5 were identified as potential loci to modify the flowering time and floral organs in Prunus species. Moreover, our results showed the diversification of SEP genes in peach. The gene sequences have been deposited in GenBank and will appear under the accession numbers BQ102369, EF440351, and EF440352.     

Microsatellite variability in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch]: cultivar identification,marker mutation,pedigree inferences and population structure

A collection of 212 peach and nectarine cultivars covering a wide variation of the species were studied with 16 polymorphic single-locus microsatellite, or simple-sequence repeat (SSR), markers. The average number of alleles per locus was 7.3, 35% of the cultivar x locus combinations analyzed were heterozygous and 87% of the cultivars studied could be individually identified. Most of the groups where two or more cultivars had the same SSR fingerprint included known peach mutants or possible synonymies. Pedigree information was tested with the SSR data. Five unexpected genotypes, due to a mutation at five SSR loci were found when comparing the SSR fingerprint of 14 known mutant cultivars and putative synonymous cultivars. The pedigree data were not consistent with the observed data in 11 out of 38 cases that could be analyzed. The group of non-melting fruit flesh cultivars, generally used by the canning industry, was more variable and genetically distant than the rest of the cultivars tested. Based on their level of homozygosity it was possible to separate those cultivars that were obtained by modern breeding technologies from those that were selected from traditional orchards after generations of seed propagation. The former had a distribution of genotypic frequencies close to a random mating model while the latter had a higher level of homozygosity. The implications of these data for the use of SSR fingerprints in breeder's rights protection and peach breeding are discussed.     

Self-compatible peach (<Emphasis Type="Italic">Prunus persica</Emphasis>) has mutant versions of the <Emphasis Type="Italic">S</Emphasis> haplotypes found in self-incompatible <Emphasis Type="Italic">Prunus</Emphasis> species

This study demonstrates that self-compatible (SC) peach has mutant versions of S haplotypes that are present in self-incompatible (SI) Prunus species. All three peach S haplotypes, S ¹ , S ² , and S ^2m , found in this study encode mutated pollen determinants, SFB, while only S ^2m has a mutation that affects the function of the pistil determinant S-RNase. A cysteine residue in the C5 domain of the S ^2m -RNase is substituted by a tyrosine residue, thereby reducing RNase stability. The peach SFB mutations are similar to the SFB mutations found in SC haplotypes of sweet cherry (P. avium) and Japanese apricot (P. mume). SFB ¹ of the S ¹ haplotype, a mutant version of almond (P. dulcis) S ^k haplotype, encodes truncated SFB due to a 155 bp insertion. SFB ² of the S ² and S ^2m haplotypes, both of which are mutant versions of the S ^a haplotype in Japanese plum (P. salicina), encodes a truncated SFB due to a 5 bp insertion. Thus, regardless of the functionality of the pistil determinant, all three peach S haplotypes are SC haplotypes. Our finding that peach has mutant versions of S haplotypes that function in almond and Japanese plum, which are phylogenetically close and remote species, respectively, to peach in the subfamily Prunoideae of the Roasaceae, provides insight into the SC/SI evolution in Prunus. We discuss the significance of SC pollen part mutation in peach with special reference to possible differences in the SI mechanisms between Prunus and Solanaceae.     

Quantitative proteomic analysis of short photoperiod and low-temperature responses in bark tissues of peach (<Emphasis Type="Italic">Prunus persica</Emphasis> L. Batsch)

In the temperate climate of the northern hemisphere, winter survival of woody plants is determined by the ability to acclimate to freezing temperatures and to undergo a period of dormancy. Cold acclimation in many woody plants is initially induced by short photoperiod and low, non-freezing temperatures. These two factors (5°C and short photoperiod) were used to study changes in the proteome of bark tissues of 1-year-old peach trees. Difference in-gel electrophoresis technology, a gel-based approach involving the labeling of proteins with different fluorescent dyes, was used to conduct a quantitative assessment of changes in the peach bark proteome during cold acclimation. Using this approach, we were able to identify differentially expressed proteins and to assign them to a class of either ‘temperature-responsive’ or ‘photoperiod-responsive’ proteins. The most significant factor affecting the proteome appeared to be low temperature, while the combination of low temperature and short photoperiod was shown to act either synergistically or additively on the expression of some proteins. Fifty-seven protein spots on gels were identified by mass spectrometry. They included proteins involved in carbohydrate metabolism (e.g., enolase, malate dehydrogenase, etc), defense or protective mechanisms (e.g., dehydrin, HSPs, and PR-proteins), energy production and electron transport (e.g., adenosine triphosphate synthases and lyases), and cytoskeleton organization (e.g., tubulins and actins). The information derived from the analysis of the proteome is discussed as a function of the two treatment factors: low temperature and short photoperiod. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regulation of the expression of lipoxygenase genes in <Emphasis Type="Italic">Prunus persica</Emphasis> fruit ripening

Three genes of the lipoxygenase (LOX) family in peach (Prunus persica var. compressa cv. Ruipan 4) were cloned, and their expression patterns during fruit ripening were analyzed using real-time quantitative PCR. All of the three peach LOX genes had been expressed during fruit ripening; however, their expression patterns were significantly different. During the normal ripening of peach fruits, the expression levels of PpLox1, PpLox2 and PpLox3 increased in varying degrees accompanying upsurge of ethylene evolution. After treated by methyl jasmonic acid (MeJA), the peak of ethylene releasing occurred in advance, and the declining rate of fruit hardness was accelerated, the expression level of the three peach LOX genes in fruits markedly enhanced at the early stage of storage, but significantly decreased at the late storage stage. So, it could be suggested that all three LOXs relate to fruit ripening; however, their functions might be different. PpLox1 expression increase along with the upsurge of ethylene evolution in both control and MeJA-treated peach fruits suggested that PpLox1 probably played a major role in the peach fruit ripening. Expression peak of PpLox2 appeared at the 1 DAH (days after harvest) in both control and MeJA-treated peach fruits, while obvious changes in ethylene evolution and fruit hardness was not observed, which suggested that the rise of PpLox2 expression can be induced by certain stimulation related to ripening, such as harvesting stress and MeJA treatment. The expression of PpLox3 kept a lower level in the natural ripening fruits, whereas raced up at the early stage of storage in the fruits treated with MeJA, which indicated that PpLox3 was expressed inductively and had minor roles during the normal ripening of peach fruits, but when encountered with external stimulation, its expression level would rapidly enhance and accelerate the ripening of peach fruit.     

Linkage and association mapping for the slow softening (SwS) trait in peach (<Emphasis Type="Italic">P. persica</Emphasis> L. Batsch) fruit

Fruit texture is a crucial quality factor influencing consumer preference and shelf life. Peach (P. persica L. Batsch) is a highly perishable fruit subjected to a rapid softening after harvest. Improvement of peach shelf life is an important breeding objective, stimulating the characterization and exploitation of texture-related traits. Variants of melting (M) texture have captured an increasing interest, following the economic success of “Big Top” nectarine, one of the most cultivated varieties worldwide. “Big Top” fruit maintains a crispy texture for an extended period before the onset of the melting phase, prolonging its shelf life. Genetic determinants regulating this complex trait, defined as slow softening (SwS), are still unknown, mainly because of limitations in phenotyping methods. In this work, a mechanical approach for measuring SwS fruit texture was used to phenotype offspring derived from a cross between “Rebus028” (SwS texture) and “Max10” (M texture). Mechanical parameters were used in linkage mapping, allowing the identification of a major QTL on chromosome 8 (qSwS8.1). The presence of this QTL was validated by a genome-wide association study (GWAS) in a panel of accessions phenotyped for mechanical properties. Less significant signals were also detected by GWAS in other genomic regions, suggesting that additional loci may regulate the SwS trait, possibly depending on the genetic background. The inheritance pattern of the SwS trait and the presence of additional loci are crucial aspects to be addressed in future studies, along with a better characterization of other important textural attributes.     

Identifying SNP markers tightly associated with six major genes in peach [<Emphasis Type="Italic">Prunus persica</Emphasis> (L.) Batsch] using a high-density SNP array with an objective of marker-assisted selection (MAS)

One of the applications of genomics is to identify genetic markers linked to loci responsible for variation in phenotypic traits, which could be used in breeding programs to select individuals with favorable alleles, particularly at the seedling stage. With this aim, in the framework of the European project FruitBreedomics, we selected five main peach fruit characters and a resistance trait, controlled by major genes with Mendelian inheritance: fruit flesh color Y, fruit skin pubescence G, fruit shape S, sub-acid fruit D, stone adhesion-flesh texture F-M, and resistance to green peach aphid Rm2. They were all previously mapped in Prunus. We then selected three F₁ and three F₂ progenies segregating for these characters and developed genetic maps of the linkage groups including the major genes, using the single nucleotide polymorphism (SNP) genome-wide scans obtained with the International Peach SNP Consortium (IPSC) 9K SNP array v1. We identified SNPs co-segregating with the characters in all cases. Their positions were in agreement with the known positions of the major genes. The number of SNPs linked to each of these, as well as the size of the physical regions encompassing them, varied depending on the maps. As a result, the number of useful SNPs for marker-assisted selection varied accordingly. As a whole, this study establishes a sound basis for further development of MAS on these characters. Additionally, we also discussed some limitations that were observed regarding the SNP array efficiency.     

Interspecific hybridization of <Emphasis Type="Italic">Prunus persica</Emphasis> with <Emphasis Type="Italic">P. armeniaca</Emphasis> and <Emphasis Type="Italic">P. salicina</Emphasis> using embryo rescue

Embryo rescue technique was used successfully to produce interspecific hybrids by crossing peach (P. persica) as a female parent with apricot (P. armeniaca) and plum (P. salicica). In those crosses that had ‘Yuhualu’ or ‘Zhonghuashoutao’ as female parents, hybrid embryos aborted from the 7th or 8th week after pollination mainly due to post-pollination incompatibility. An embryo rescue protocol was established to rescue such embryos and recover hybrid plants. Modified half-strength MS medium containing 4 mg l⁻¹ 6-BA and 0.5 mg l⁻¹ IBA produced up to 90% germination in the embryos. Modified MS medium with 1.0 mg l⁻¹ 6-BA and 1.0 mg l⁻¹ IBA gave the highest bud induction and multiplication whereas modified MS medium containing 0.5 mg l⁻¹ IAA and 0.2 mg l⁻¹ NAA gave the best rooting percentage. All the hybrids obtained using this embryo rescue technique were verified using simple sequence repeat (SSR) markers. A series of pollen treatments were carried out to partially overcome pre-pollination incompatibility, and it was found accidentally that pollen treatment with electrostatic field not only improved pollen germination but also increased the multiplication coefficient of embryo-induced shoots.     

Impact of nitric oxide on shelf life and quality of nectarine (<Emphasis Type="Italic">Prunus persica</Emphasis> var. <Emphasis Type="Italic">nucipersica</Emphasis>)

Nectarine is an emerging fruit crop in India which has immense nutritional quality and fairly good amount of antioxidants. In India, peach orchards are being replaced by nectarine primarily because of fuzzless peel. However, shelf life of nectarine is lean due to its climacteric behaviour. Hence, we attempted to observe the effect of nitric oxide (NO), using its donor compound, namely sodium nitroprusside (SNP) on postharvest life and quality of ‘Silver Queen’ nectarine fruit. In this study, fruit of ‘Silver Queen’ nectarine were treated with various concentrations of SNP (0.25 mM, 0.5 mM, 1.0 mM, 1.5 mM) after harvesting at climacteric stage of maturity. Fruit were stored at ambient condition for further analysis on daily basis, after air drying at room temperature. Our results revealed that among the various concentrations of SNP, 0.5 mM was found to be best in reducing physiological loss in weight (PLW), maintaining firmness and retaining higher phenolics, antioxidant activity, exhibiting slower increase in lipoxygenase (LOX) and pectin methylesterase (PME) activity and better quality fruit up to 8 days than 4 days of untreated fruit. Hence, postharvest dip of ‘Silver Queen’ nectarine fruit in 0.5 mM solution of SNP could be recommended for enhancing the shelf life by 4 days.     

Transgenic peach plants (<Emphasis Type="Italic">Prunus persica</Emphasis> L.) produced by genetic transformation of embryo sections using the green fluorescent protein (GFP) as an <Emphasis Type="Italic">in vivo</Emphasis> marker

The main obstacle to genetic engineering of fruit tree species is the regeneration of transformed plantlets. Transformation events in peach (Prunus persica L.) have been reported using particle bombardment or Agrobacteriummediated transformation of immature embryos. However, the regeneration of plants from transgenic tissues is still difficult and the recovery of non-chimeric plants has not been reported to date. In this paper we describe an efficient, reliable transformation and regeneration system to produce transgenic peach plants using embryo sections of mature seeds as starting material. This represents an important advantage due to the availability of such material throughout the year. A. tumefaciens strain C58 (pMP90) containing the binary plasmid pBin19 was used as vector system for transformation. We used the Nospro-nptII-Noster cassette as a selectable marker and the CaMV35Spro-sgfp-CaMV35Ster cassette as a vital reporter gene coding for an improved version of the green fluorescent protein (sGFP). In vitro cultured embryo sections were Agrobacterium-cocultivated and, after selection, transgenic shoots were regenerated. Shoots that survived exhibited high-level of sGFP expression mainly visible in the young leaves of the apex. In vivo monitoring of GFP expression permitted an early, rapid and easy discrimination of both transgenic and escape buds. After elimination of escapes, transgenic shoots were rooted in vitro and the recovered plantlets were screened using PCR amplification. Southern analysis confirmed stable genomic integration of the sgfp transgene. The high levels of GFP expression were also maintained in the second generation of transgenic peach plants.     

High-throughput sequencing of <Emphasis Type="Italic">Prunus ferganensis</Emphasis> indicates that it is a geographical population of <Emphasis Type="Italic">P. persica</Emphasis>

Peach belongs to the genus Prunus, which includes Prunus persica and its relative species, P. mira, P. davidiana, P. kansuensis, and P. ferganensis. Of these, P. ferganensis have been classified as a species, subspecies, or geographical population of P. persica. To explore the genetic difference between P. ferganensis and P. persica, high-throughput sequencing was used in different peach accessions belonging to different species. First, low-depth sequencing data of peach accessions belonging to four categories revealed that similarity between P. ferganensis and P. persica was similar to that between P. persica accessions from different geographical populations. Then, to further detect the genomic variation in P. ferganensis, the P. ferganensis accession “Xinjiang Pan Tao 1” and the P. persica accession “Xia Miao 1” were sequenced with high depth, and sequence reads were assembled. The results showed that the collinearity of “Xinjiang Pan Tao 1” with the reference genome “Lovell” was higher than that of “Xia Miao 1” and “Lovell” peach. Additionally, the number of genetic variants, including single nucleotide polymorphisms (SNPs), structural variations (SVs), and the specific genes annotated from unmapped sequence in “Xia Miao 1” was higher than that in “Xinjiang Pan Tao 1” peach. The data showed that there was a close distance between “Xinjiang Pan Tao 1” (P. ferganensis) and reference genome which belong to P. persica, comparing “Xia Miao 1” (P. persica) and reference ones. The results accompany with phylogenetic tree and structure analysis confirmed that P. ferganensis should be considered as a geographic population of P. persica rather than a subspecies or a distinct species. Furthermore, gene ontology analysis was performed using the gene comprising large-effect variation to understand the phenotypic difference between two accessions. The result revealed that the pathways of gene function affected by SVs but SNPs and insertion-deletions markedly differed between the two peach accessions.     

Effects of low light on phloem ultrastructure and subcellular localization of sucrose synthase in <Emphasis Type="Italic">Prunus persica</Emphasis> L. var. <Emphasis Type="Italic">nectarina</Emphasis> Ait. fruit

Using electron microscopy, the ultrastructure of phloem unloading zone was examined in the Prunus persica L. var. nectarina Ait. fruit. Our study showed that, in the SE/CC (sieve element/companion cell) complexes, CC developing under low light had a thin cytoplasm layer with few mitochondria and numerous small vacuoles, and not clearly seen nuclei. The cytoplasm vacuolation indicated that the cytoskeleton was destroyed at low light. The effects of low light on CC development suggest that unloading evidently linked to the low accumulation of soluble sugars by fruit. At the young fruit stage, flesh parenchyma around the phloem tissue had no starch grains in the plastids in fruit developing under low light. This is a further indication that less photoassimilates was translocated from source leaves to fruit sinks under low light during the young fruit developmental stage. The activity of sucrose synthase (SuSy), the key enzyme of sucrose metabolism in fruit, increased dramatically during fruit maturation. The highest SuSy activity during the rapid fruit growth phase suggests that sink strength could be correlated with the SuSy activity. The high SuSy activity under normal light possibly indicates that fruit had a capacity to utilize sucrose irrespective of their site of phloem unloading. Immunogold electron microscopy showed that SuSy was localized mainly in the vacuole of flesh parenchyma cells. The vacuole-localized SuSy can hydrolyze sucrose imported from the phloem, which may explain the apparent correlation between SuSy activity and phloem unloading. The double sieve element (SE/SE) complexes occurred in a greater number and had thicker cell walls under normal light intensity than under low light intensity. These data demonstrate clearly that low light decreased SuSy activity in the control of phloem unloading. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 509–517. This text was submitted by the authors in English.     

Effect of light quality on leaf photosynthetic characteristics and fruit quality of peach (<Emphasis Type=Italic>Prunus persica</Emphasis> L. Batch)

Different light filters affect leaf photosynthetic features and fruit quality. Consequently, selecting the appropriate covering filter for rain-shelter cultivation of peaches is a key part of successful production. We used a late-maturing peach variety ‘Xiahui 8’ to study differences in leaf photosynthetic features, chlorophyll fluorescence characteristics, and fruit quality under neutral, red, yellow, green, and blue filter, with natural light as control. The results showed that the leaf photosynthetic ability and internal quality under the neutral filter treatment were elevated compared with the control, and the appearance color was the same as the control. Leaves under neutral filter could maintain higher photosynthetic ability than other filter treatments. In addition, the fruits could also keep higher quality when treated with neutral filter. Therefore, the application of neutral filter in rain-shelter cultivation of ‘Xinhui 8’ peaches is recommended for maintaining high photosynthetic capacity and for improving fruit quality.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in <Emphasis Type="Italic">Plantago major,Plantago maritima</Emphasis>, <Emphasis Type="Italic">Prunus persica</Emphasis>, and <Emphasis Type="Italic">Apium graveolens</Emphasis>

Sugar and sugar alcohol concentrations were analyzed in subcellular compartments of mesophyll cells, in the apoplast, and in the phloem sap of leaves of Plantago major (common plantain), Plantago maritima (sea plantain), Prunus persica (peach) and Apium graveolens (celery). In addition to sucrose, common plantain, sea plantain, and peach also translocated substantial amounts of sorbitol, whereas celery translocated mannitol as well. Sucrose was always present in vacuole and cytosol of mesophyll cells, whereas sorbitol and mannitol were found in vacuole, stroma, and cytosol in all cases except for sea plantain. The concentration of sorbitol, mannitol and sucrose in phloem sap was 2- to 40-fold higher than that in the cytosol of mesophyll cells. Apoplastic carbohydrate concentrations in all species tested were in the low millimolar range versus high millimolar concentrations in symplastic compartments. Therefore, the concentration ratios between the apoplast and the phloem were very strong, ranging between 20- to 100-fold for sorbitol and mannitol, and between 200- and 2000-fold for sucrose. The woody species, peach, showed the smallest concentration ratios between the cytosol of mesophyll cells and the phloem as well as between the apoplast and the phloem, suggesting a mixture of apoplastic and symplastic phloem loading, in contrast to the herbal plant species (common plantain, sea plantain, celery) which likely exhibit an active loading mode for sorbitol and mannitol as well as sucrose from the apoplast into the phloem.