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Parthenocarpy and stenospermocarpy are the two mechanisms underlying the seedless fruit set program. Seedless fruit occurs naturally and can be produced using hormone application, crossbreeding, or ploidy breeding. However, the two types of breeding are time-consuming and sometimes ineffective due to interspecies hybridization barriers or the absence of appropriate parental genotypes to use in the breeding process. The genetic engineering approach provides a better prospect, which can be explored based on an understanding of the genetic causes underlying the seedlessness trait. For instance, CRISPR/Cas is a comprehensive and precise technology. The prerequisite for using the strategy to induce seedlessness is identifying the crucial master gene or transcription factor liable for seed formation/development. In this review, we primarily explored the seedlessness mechanisms and identified the potential candidate genes underlying seed development. We also discussed the CRISPR/Cas-mediated genome editing approaches and their improvements.

Seedlessness is one of the highest valued agronomic traits in grapes. Embryo rescue in combination with marker-assisted selection have been widely applied in seedless grape breeding due to the advantages of increasing the ratio of seedless progenies and shortening the breeding cycle. However, the large number of deformed seedlings produced during embryo rescue and the lack of fast, efficient, and low-cost markers severely inhibit the process of seedless grape breeding. In this study, a total of eighty-three grape cultivars (51 seedless and 32 seeded) with diverse genetic backgrounds and two populations derived from embryo rescue, including 113 F1 hybrid individuals (60 seedless and 53 seeded), were utilized. We screened suitable media for converting malformed seedlings into normal seedlings, analyzed the association between the SNP in VviAGL11 and seeded/seedless phenotype, and developed a KASP marker related to stenospermocarpic seedlessness. Our results indicated that the transformation rate of 37.8% was obtained with MS medium supplemented with 2.0 mg·L−1 of 6-BA and 0.5 mg·L−1 of IBA. The presence of an A nucleotide allele at position chr18:26889437 was further confirmed to be fully associated with the stenospermocarpic seedlessness phenotype. The developed KASP marker, based on the verified SNP locus in VviAGL11, successfully distinguished the seedless and seeded genotypes with high precision and throughput. The results will contribute to enhancing the efficiency of embryo rescue and facilitate parent selection and early selection of seedless offspring with molecular markers, thereby accelerating the breeding process in seedless table grapes. [ABSTRACT FROM AUTHOR]

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Datp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Datp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Datp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Datp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment. [ABSTRACT FROM AUTHOR]

Allotopic expression is the term given for the deliberate relocation of gene function from an organellar genome to the nuclear genome. We hypothesized that the allotopic expression of an essential mitochondrial gene using a promoter that expressed efficiently in all cell types except those responsible for male reproduction would yield a cytoplasmic male sterility (CMS) phenotype once the endogenous mitochondrial gene was inactivated via genome editing. To test this, we repurposed the mitochondrially encoded atp1 gene of tobacco to function in the nucleus under the transcriptional control of a CaMV 35S promoter (construct 35S:nATP1), a promoter that has been shown to be minimally expressed in early stages of anther development. The endogenous atp1 gene was eliminated (Δatp1) from 35S:nATP1 tobacco plants using custom-designed meganucleases directed to the mitochondria. Vegetative growth of most 35S:nATP1/Δatp1 plants appeared normal, but upon flowering produced malformed anthers that failed to shed pollen. When 35S:nATP1/Δatp1 plants were cross-pollinated, ovary/capsule development appeared normal, but the vast majority of the resultant seeds were small, largely hollow and failed to germinate, a phenotype akin to the seedless trait known as stenospermocarpy. Characterization of the mitochondrial genomes from three independent Δatp1 events suggested that spontaneous recombination over regions of microhomology and substoichiometric shifting were the mechanisms responsible for atp1 elimination and genome rearrangement in response to exposure to the atp1-targeting meganucleases. Should the results reported here in tobacco prove to be translatable to other crop species, then multiple applications of allotopic expression of an essential mitochondrial gene followed by its elimination through genome editing can be envisaged. Depending on the promoter(s) used to drive the allotopic gene, this technology may have potential application in the areas of: (1) CMS trait development for use in hybrid seed production; (2) seedless fruit production; and (3) transgene containment.

The development of new seedless cultivar represents one of the most important goals in table grape breeding programmes worldwide. The most common technique to obtain new seedless cultivars is embryo rescue, an approach that allows the isolation of immature embryos and their cultivation in vitro. In this study, a total of 23 crosses (developed employing one seeded and one seedless parent) were performed during two seasons (2017 and 2018) for a total of 1140 seedlings. For each cross, the principal parameters related to the efficiency of the pollination were measured (harvested bunches, collected berries, recovered embryos/seeds and plants obtained). Based on these traits, statistical analyses were performed to calculate the female and male parental efficiency and to compare the two techniques of propagation employed: embryo rescue (7.8% of plants obtained) and gamic propagation (8.4%). Finally, the segregation of the SSR marker p3_VvAGL11 was evaluated on the progeny of four crosses in which the same cultivar was used alternately as female or male parent (SugxIta/ItaxSug and CrixIta/ItaxCri). The parameters measured showed a positive correlation between berries, seeds and plants obtained, exclusively in combination with a seeded female parent. The crossing combinations investigated indicate that some genotypes outperformed others when used as female parent in terms of embryos/seeds produced. Therefore, the efficiency in terms of seedlings obtained for the seedless female parents is actually balanced with those obtained for the seeded ones. The proposed research aims to provide useful information to guide the choice of genotypes used in the genetic improvement programs of seedless grapes, to increase their efficiency. [ABSTRACT FROM AUTHOR]

Seedless fruits are favorable in the market because of their ease of manipulation. Sucrose transporters (SUTs or SUCs) are essential for carbohydrate metabolism in plants. Whether SUTs participate directly in causing stenospermocarpy, thereby increasing fruit quality, remains unclear. Three SUTs , namely, VvSUC11 , VvSUC12 , and VvSUC27 from Vitis vinifera , were characterized and ectopic expression in tomatoes. VvSUC11 - and VvSUC12 -overexpressing lines had similar flower and fruit phenotypes compared with those of the wild type. VvSUC27 -overexpressing lines produced longer petals and pistils, an abnormal stigma, much less and shrunken pollen, and firmer seedless fruits. Moreover, produced fruits from all VvSUC -overexpressing lines had a higher soluble solid content and sugar concentration. Transcriptomic analysis revealed more genes associated with carbohydrate metabolism and sugar transport and showed downregulation of auxin- and ethylene-related signaling pathways during early fruit development in VvSUC27 -overexpressing lines relative to that of the wild type. Our findings demonstrated that stenospermocarpy can be induced by overexpression of VvSUC27 through a consequential reduction in nutrient delivery to pollen at anthesis, with a subsequent downregulation of the genes involved in carbohydrate metabolism and hormone signaling. These commercially desirable results provide a new strategy for bioengineering stenospermocarpy in tomatoes and in other fruit plants. [ABSTRACT FROM AUTHOR]

Main conclusion: The seed coat gene VviAGL11 coordinates with endosperm development genes FIS2, PHERESE1 and IKU2 and functions as the key regulator in seed development and abortion processes in grapevine. Seed development is essential for the reproduction of flowering plants. Seed abortion is a specific characteristic that produces seedless berries and is often observed in cultivated grapevines. Although seedlessness is an important trait for table and dried grapevine production, the mechanism of seed abortion remains poorly understood. This research aimed to analyze the co-expression of the seed coat development gene VviAGL11 and the endosperm development genes FERTILIZATIONINDEPENDENTSEED2 (FIS2), PHERESE1 and HAIKU2 (IKU2) that regulate seedless fruit development in grapevine. The transcript levels of VviAGL11, FIS2, PHERESE1 and IKU2 all decreased during seed abortion in the seedless grape 'Thompson Seedless' plants, compared to those of the seeded grape 'Pinot Noir'. The transcript levels of the salicylic acid (SA)-dependent defense response genes EDS1, NPR1, NDR1 and SID2 were higher in 'Thompson Seedless' than 'Pinot Noir' during seed development. Also, WRKY3, WRKY6 and WRKY52, which participate in the SA pathway, were higher expressed in 'Thompson Seedless' than in 'Pinot Noir', indicating that SA-dependent defense responses may regulate seed abortion. The genes related to synthesis and metabolism of gibberellic acid (GA) and abscisic acid (ABA) also showed differential expression between 'Thompson Seedless' and 'Pinot Noir'. Exogenous applications of plant growth regulators (PGRs) to inflorescences of three stenospermocarpy grapevines before flowering showed that GA3 was critical prominently in seed development. Therefore, the co-expression of seed coat and endosperm development-related genes, SA pathway genes, and genes for the synthesis and metabolism of GA3 together enhance seed abortion in seedless grapes. [ABSTRACT FROM AUTHOR]

Seedless fruits are favorable in the market because of their ease of manipulation. Sucrose transporters (SUTs or SUCs) are essential for carbohydrate metabolism in plants. Whether SUTs participate directly in causing stenospermocarpy, thereby increasing fruit quality, remains unclear. Three SUTs, namely, VvSUC11, VvSUC12, and VvSUC27 from Vitis vinifera, were characterized and ectopic expression in tomatoes. VvSUC11- and VvSUC12-overexpressing lines had similar flower and fruit phenotypes compared with those of the wild type. VvSUC27-overexpressing lines produced longer petals and pistils, an abnormal stigma, much less and shrunken pollen, and firmer seedless fruits. Moreover, produced fruits from all VvSUC-overexpressing lines had a higher soluble solid content and sugar concentration. Transcriptomic analysis revealed more genes associated with carbohydrate metabolism and sugar transport and showed downregulation of auxin- and ethylene-related signaling pathways during early fruit development in VvSUC27-overexpressing lines relative to that of the wild type. Our findings demonstrated that stenospermocarpy can be induced by overexpression of VvSUC27 through a consequential reduction in nutrient delivery to pollen at anthesis, with a subsequent downregulation of the genes involved in carbohydrate metabolism and hormone signaling. These commercially desirable results provide a new strategy for bioengineering stenospermocarpy in tomatoes and in other fruit plants.

The development of new seedless cultivar represents one of the most important goals in table grape breeding programmes worldwide. The most common technique to obtain new seedless cultivars is embryo rescue, an approach that allows the isolation of immature embryos and their cultivation in vitro. In this study, a total of 23 crosses (developed employing one seeded and one seedless parent) were performed during two seasons (2017 and 2018) for a total of 1140 seedlings. For each cross, the principal parameters related to the efficiency of the pollination were measured (harvested bunches, collected berries, recovered embryos/seeds and plants obtained). Based on these traits, statistical analyses were performed to calculate the female and male parental efficiency and to compare the two techniques of propagation employed: embryo rescue (7.8% of plants obtained) and gamic propagation (8.4%). Finally, the segregation of the SSR marker p3_VvAGL11 was evaluated on the progeny of four crosses in which the same cultivar was used alternately as female or male parent (SugxIta/ItaxSug and CrixIta/ItaxCri). The parameters measured showed a positive correlation between berries, seeds and plants obtained, exclusively in combination with a seeded female parent. The crossing combinations investigated indicate that some genotypes outperformed others when used as female parent in terms of embryos/seeds produced. Therefore, the efficiency in terms of seedlings obtained for the seedless female parents is actually balanced with those obtained for the seeded ones. The proposed research aims to provide useful information to guide the choice of genotypes used in the genetic improvement programs of seedless grapes, to increase their efficiency.

Background: Grapevine reproductive development has direct implications on yield. It also impacts on berry and wine quality by affecting traits like seedlessness, berry and bunch size, cluster compactness and berry skin to pulp ratio. Seasonal fluctuations in yield, fruit composition and wine attributes, which are largely driven by climatic factors, are major challenges for worldwide table grape and wine industry. Accordingly, a better understanding of reproductive processes such as gamete development, fertilization, seed and fruit set is of paramount relevance for managing yield and quality. With the aim of providing new insights into this field, we searched for clones with contrasting seed content in two germplasm collections. Results: We identified eight variant pairs that seemingly differ only in seed-related characteristics while showing identical genotype when tested with the GrapeReSeq_Illumina_20K_SNP_chip and several microsatellites. We performed multi-year observations on seed and fruit set deriving from different pollination treatments, with special emphasis on the pair composed by Sangiovese and its seedless variant locally named Corinto Nero. The pollen of Corinto Nero failed to germinate in vitro and gave poor berry set when used to pollinate other varieties. Most berries from both open- and cross-pollinated Corinto Nero inflorescences did not contain seeds. The genetic analysis of seedlings derived from occasional Corinto Nero normal seeds revealed that the few Corinto Nero functional gametes are mostly unreduced. Moreover, three genotypes, including Sangiovese and Corinto Nero, were unexpectedly found to develop fruits without pollen contribution and occasionally showed normal-like seeds. Five missense single nucleotide polymorphisms were identified between Corinto Nero and Sangiovese from transcriptomic data. Conclusions: Our observations allowed us to attribute a seedlessness type to some variants for which it was not documented in the literature. Interestingly, the VvAGL11 mutation responsible for Sultanina stenospermocarpy was also discovered in a seedless mutant of Gouais Blanc. We suggest that Corinto Nero parthenocarpy is driven by pollen and/or embryo sac defects, and both events likely arise from meiotic anomalies. The single nucleotide polymorphisms identified between Sangiovese and Corinto Nero are suitable for testing as traceability markers for propagated material and as functional candidates for the seedless phenotype. [ABSTRACT FROM AUTHOR]

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six “seedless × seedless” crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness.

White-brown complex (WBC) transporters, also called half-size ATP binding cassette G (ABCG) transporters, are involved in many biological processes, including seed development; however, the WBC transporters in grapevines received less attention to date. To reveal the molecular characteristics of WBCs and the connection between WBCs and agronomic traits of stenospermocarpic (seedless) grapevine, we carried out a genomic census and analysis of ovule-associated expression for VvWBC genes in grapevine. We identified 30 VvWBC genes and cloned full-length complementary DNAs (cDNAs) for 20 of these. The tissue or organ-specific expression analysis showed that several VvWBCs exhibited distinct expression patterns with some showing tissue specificity. Twelve VvWBC genes were found to be expressed in the developing ovules. Moreover, the results of quantitative real-time PCR (qRT-PCR) suggested that four of twelve ovule-expressed VvWBCs have distinct expression profiles during the development of ovules between seeded and stenospermocarpic grapevines. These four genes might be involved in ovule abortion. Meanwhile, chromosome mapping, multiple sequence alignments, exon/intron structure analyses and synteny analyses were preformed on VvWBC genes. Our experiments provide a new perspective on the mechanism of stenospermocarpic seedlessness and put forward a framework for further study of WBC transporters.

• VviABCG20 expressed differently in seed between seeded and seedless grapes. • Sequences of VviABCG20 between seeded and seedless varieties were similar. • Suppression of VviABCG20 homolog gene in tomato led to reduction in seed number. • Expression level of VviABCG20 is related to seed development in grapevines. In grapevine (Vitis vinifera L.), seedlessness is classified into two distinct types: parthenocarpy and stenospermocarpy. Stenospermocarpy (seedlessness due to seed abortion) in grapevine is an agronomically valuable trait which is completely hereditary and not influenced by various environmental factors. Many hypotheses have been proposed for stenospermocarpy. However, the molecular mechanisms underlying development or abortion of the seed are still poorly understood. To identify genes involved in seed development or abortion in grape, we carried out Sequence-Related Amplified Polymorphism (SRAP)-cDNA analysis of grape seed development, comparing the seedless cultivar Youngle with the seeded cultivar Pinot noir. We identified VviABCG20 , which encodes a putative ATP binding cassette G transporter, as a gene more highly expressed in 'Pinot noir'. VviABCG20 is highly expressed in root, floral bud, seed and the expression level of VviABCG20 in all of five seeded grapevine cultivars tested was generally higher than that observed in seedless cultivars. Sequencing of VviABCG20 cDNAs from six stenospermocarpic seedless and seven seeded genotypes revealed that the seed-associated expression pattern of VviABCG20 was unrelated to sequence difference within the transcribed regions. Suppression of the tomato homolog of VviABCG20 in developing tomato fruit led to reduction in seed numbers, suggesting that LeABCG20 plays an essential role in seed development. Our experiments suggest that regulation of VviABCG20 is associated with seed development, and provide a new insight into the mechanism underlying seed abortion in stenospermocarpic grapes. [ABSTRACT FROM AUTHOR]

The breeding of new stenospermocarpic seedless grape cultivars is of particular importance to the growing table grape market and grape processing industry. However, the developmental and genetic architectures of soft‐seed and seedlessness traits are unclear. In this study, we performed anatomical, histochemical and gene expression analyses to characterize the morphological and genetic basis of the seed hardness of 'Hongju', a stenospermocarpic soft‐seed grape cultivar developed by crossbreeding maternal 'Italia' and paternal 'Perlon' cultivars. We found intensively lignified medium integument layers in the seeds of hard‐seed cultivars, but these layers were degraded in the stenospermocarpic seedless cultivars beginning 35 days after flowering. The expression of genes related to the lignin and cellulose biosynthesis pathways was downregulated, whereas that of several cell‐wall loosening‐related genes was upregulated in 'Hongju' compared with 'Italia' beginning at 21 days after flowering. These results suggest that transcriptional suppression of the lignin and cellulose biosynthesis pathways coupled with reduced formation of medium integument plays an important role in the development of soft seeds in stenospermocarpic seedless grape cultivars. [ABSTRACT FROM AUTHOR]

A native Croatian grapevine 'Grk bijeli', sharing a parent-offspring relationships with 'Tribidrag' (aka 'Zinfandel'), is grown exclusively on the Adriatic island of Korčula. It is one of the grape cultivars with female-only functional flowers, causing reduced fertilisation and leads to problems in grape production and wine quality. A typical cluster of 'Grk bijeli' at maturity contains fully developed seeded berries, in addition to a highly variable proportion of undersized seedless berries. The aim of this study was to identify the cause of the reduced reproduction potential of 'Grk bijeli' in order to establish a better growing environment for improved yield and grape quality. 'Grk bijeli' female gametophyte develops normaly and at maturity contains both egg and central cell, together with two synergid cells and three antipodal cells. On the other hand, 'Grk bijeli' pollen grains show developmental deviations. Specifically, most of the pollen grains undergo the first pollen mitosis and contain one vegetative cell and one generative cell, while 20 % of ungerminating grains also accomplish the second pollen mitosis, giving rise to two sperm cells and one vegetative cell. Morover, 'Grk bijeli' pollen has acolporate morphology, which prevents germination and contributes to reduced reproduction. Furthermore, fertilisation after pollination with other varieties results in various degrees of ovule abortion depending on the pollinator, revealing Croatian cultivars 'Plavac mali crni' and 'Pošip bijeli' as favourable varieties. Although this study describes a highly valuable cultivar of local importance, it also contributes to fundamental knowledge of grapevine reproductive biology and offers a strategy for improvement of wine production and oenological performance of semi-fertile varieties in general. [ABSTRACT FROM AUTHOR]

Bu araşt ı rmada stenospermokarpik Sultani Çekirdeksiz ve Perlette üzüm çe şitlerinde BAP benzylaminopurine ve CCC cycocel 'in çekirdek izi geli şimi ve çimlenmesini uyarmadaki etkileri ara şt ı r ı lm ışt ı r. Sultani Çekirdeksiz çeşidinde çiçeklenme öncesi 500 ppm dozundaki BAP ve CCC uygulamalar ı ile çiçeklenme sonu 500 ppm CCC uygulamalar ı n ı n çekirdek izi gelişimini artt ı rd ığı belirlenmi ştir. Sultani Çekirdeksiz'e göre daha iri rudimenter çekirdeklere sahip olan Perlette çeş idinde ise, tam çiçeklenmede 500 ppm BAP ile, çiçeklenme öncesi 1000 ppm CCC uygulamalar ı çekirdek izi geli şimini artt ı rm ışt ı r. Uyar ı lm ış ve batan çekirdeklerin in vitro çimlendirilmesinde tam bir başar ı sağ lanamam ışt ı r. Bununla birlikte, Perlette'de çiçeklenme sonu 500 ppm BAP ve CCC ile 1000 ppm CCC uygulamalar ı nda s ı ras ı yla, %68.2, %55.2, %40.0 oranlar ı nda kökçük oluş umu gerçekleş miştir. Ancak, kotiledon ç ı kışı na ilişkin bir gelişme olmamışt ı r.

In this study, we documented whether pollen parents are required for fruiting and their effects on fruit set and seed characteristics in field-grown '73-S-20' litchi (Litchi chinensis Sonn.). The effects of preventing female (F) flower pollination to induce parthenocarpy, selfing, and outcrossing with 'Haak Yip' and open-pollinated (OP) fruits derived from F flowers and the second wave of the male (M2) flowers of 'Haak Yip' blooming in synchrony were assessed. The correlation between the percentage of fruit set and shriveled seed/seed weight in self-crossed and outcrossed fruits was calculated. At harvest, the absence of pollination induced a high proportion of parthenocarpic fruit without seed, although the fruit weighed only 4.2 g. Fruit set with outcrossing was greater than that with selfing and the percentage of shriveled seed obtained in self-crossed fruits was much greater than that in outcrossed fruits but not different from that in OP fruits, suggesting that outcrossing increased but selfing decreased the cluster yield and seed weight. However, 33.3% and 23.0% of seed contained embryos in selfed and OP fruits, respectively. No positive correlation between the percentage of fruit set and shriveled seed/seed weight was found, indicating that seed abortion during development was not the key factor leading to low yield. We concluded that tiny parthenocarpic fruit could be obtained but that pollination is essential for the good yield of '73-S-20'. Outcrossing markedly increased fruit set and seed weight and decreased the percentage of shriveled seed. Factors other than the pollen parents may be involved in the seed development of '73-S-20'. [ABSTRACT FROM AUTHOR]

VviAGL11, the Arabidopsis SEEDSTICK homolog, has been proposed to have a causative role in grapevine stenospermocarpy. An association between a mutation in the coding sequence (CDS) and the seedless phenotype was reported, however, no working mechanisms have been demonstrated yet. Previous studies have demonstrated that VviAGL11 has a causative role in stenospermocarpy in grape. Indeed, a full correlation was shown of a missense mutation Arg197Leu in exon 7 of VviAGL11 with seedlessness in cultivated grapevine. The mutated seedless version has a partial dominance over seeded alleles, allowing breeding for seedlessness based in heterozygous seedlings. Moreover, sequence data on the regulative region of VviAGL11 showed low recombination rates between the promoter and the CDS regions, thus suggesting the existence of a Linkage disequilibrium in that region. The existence of incomplete dominance of some alleles in the promoter region has been also observed. Besides this genetic and transcriptional data confirming a major role of VviAGL11 in grape stenospermocarpy, no functional evidence nor a working mechanism has yet been defined. To this aim, we sequenced and analyzed the complete VviAGL11 gene in grapevine varieties representing different seedlessness classes. We demonstrated the existence of specific promotercoding sequence (CDS) combinations self-activation that directly affect the expression level demonstrating a dominant-negative effect of the mutated CDS (Arg197Leu). Transcriptomic analyses on ovule and developing seeds in seeded and seedless varieties highlighted the role of VviAGL11 in hormone signaling and phenylpropanoid metabolism. In this regard, we identified and further functionally validated through luciferase assay and in situ hybridization a Methyl jasmonate esterase, an Indole-3-acetate beta-glucosyltransferase, and an Isoflavone reductase, as direct targets of VviAGL11. The dominant-negative effect of the mutated CDS was also functionally validated in target induction. All our findings allowed us to define a regulatory mechanism correlating the haplotype assortment, the VviAGL11 expression level, and seedlessness class in grapevine. [ABSTRACT FROM AUTHOR]

Seedlessness is a highly valuable trait in the EU and global grape markets. Genotyping and phenotyping of seedless varieties are an efficient tool for assessing the genetic diversity, determining the type of seedlessness, and optimizing their use in table breeding programs. In this work, a total of 42 Armenian Vitis vinifera L. seedless grapevine accessions were sampled from the Armavir region, mainly from the grape collections at Etchmiadzin (ARM006) and Nalbandyan (ARM011). True-to-type genotyping was performed with seven nuclear microsatellite loci and the molecular profiles obtained were compared to those stored in the Vitis International Variety Catalogue (VIVC, www.vivc.de), and the Armenian Vitis database (www.vitis.am). Phenotypic analysis of berries and seeds was carried out according to OIV descriptors. Molecular analysis of seedlessness was performed by the targeted sequencing of VviAGL11 MADS-box gene, responsible for embryo and endosperm development and seed coat lignification. Genetic profiling identified 11 unique genotypes, namely 'Anush', 'Eskeri', 'Hrushaki', 'Karmir Kishmish', 'Karmir Yerevani', 'Kishmish Chernyi', 'Kishmish Khishrau', 'Kishmish Moldavskii', 'Parvana', 'Sultanina' and 'Ushahas Nazeli'. According to their pedigrees in the databases the majority of investigated varieties are genetically Sultanina derived: 'Karmir Yerevani', 'Eskeri', 'Hrushaki' and 'Kishmish Moldavskii' are offspring of Sultanina, while 'Anush' and 'Ushahas Nazeli' have a second-degree relationship. Phenotypic analysis revealed a wide variation in berry size, as well as in the formation of seeds (from very small, to large and noticeable). Mean berry size ranged between 10.5 mm and 20.1 mm in length, and 9.5 mm to 16.9 mm in width. Berries with ovule residues and small rudimental seeds were detected in 'Karmir Yerevani', 'Karmir Kishmish', 'Sultanina' and 'Hrushaki'. Plants of the rest of cultivars produced large, but empty floating seeds, and rarely up to 0.4 well-developed seeds per berry. By contrast, berries of 'Ushahas Nazeli' showed large size 1.5 well-developed potentially viable sinker seeds per berry. Targeted sequencing of VviAGL11 gene revealed heterozygosity for the seedless dominant point mutation Arg-197Leu (A:C) in ten investigated seedless genotypes, which causes the stenospermocarpy in Sultanina-type seedless cultivars. The only exception was found for 'Ushahas Nazeli' which was homozygous for the seeded allele (C:C). We are currently investigating gametes viability, as well as endosperm and embryo development features, in order to integrate-phenotypic and genotypic data. Acknowledgement: This research is supported by MSCA Individual Fellowship (IF-EF-ST/0685-896290, GRAPEINNOVATION). [ABSTRACT FROM AUTHOR]

Seedlessness is a highly desirable characteristic in fresh fruits. However, post-fertilization seed abortion of cross-pollinated citrus fruit is uncommon. The factors regulating stenospermocarpy in citrus are unknown. In this research, we induced stenospermocarpy interfering in newly fertilized ovule cell division. The research also elucidates the most sensitive stage for ovule/seed abortion in citrus. Experiments were conducted with 'Afourer' mandarin that cross-pollinates with several cultivars and species. Cross-pollinated fruitlets were treated with maleic hydrazide (MH), a systemic growth regulator that specifically interferes in cell division. MH reduced ovule growth rate, the number of cell layers in nucella and inhibited embryo sac expansion; moreover, the treatment increased callose accumulation in nucella and surrounding the embryo sac. Fruits developed an early-aborted seed type with an immature, soft and edible seed coat. Seed number (-80%) and seed weight (-46%) were reduced in mature fruits. MH also hampered cell division in ovary walls, mesocarp and endocarp, thus reducing daily fruitlet growth and increasing fruit abscission. Stenospermocarpy could only be induced for a short period of time in the progamic phase of fertilization, specifically, when ovules are ready to be fertilized (7 days after anthesis) to early stages of embryo sac development (14 days after anthesis). [ABSTRACT FROM AUTHOR]

BACKGROUND Since 1982, the embryo rescue technique has been widely applied to embryo germination of stenospermic grapes in cross-breeding programmes. This project aimed to: (1) use embryos to breed new seedless cultivars of Vitis vinifera as the female parents utilising wild Chinese Vitis spp. as the male parents; and (2) develop an efficient method for in vitro embryo rescue and plant development. RESULTS Among the different genotypes, the productions of hybrid plants were significantly different, ranged from 21.1% ('Ruby Seedless' × 'Beichun') to only 1.1% ('Pink Seedless' × 'Beichun'), except for the combinations from which no surviving seedlings were obtained. We collected hybridisation fruits from 28 June to 3 August, and obtained their best sampling times described within days after flowering. The highest rates of embryo formation (24.3%) and plant development (91.4%) were found when ovules of 'Ruby Seedless' were cultured in MM4 + 500 mg L−1 mashed banana. CONCLUSION Seven new hybrids of V. vinifera with wild Chinese Vitis spp. were obtained. As a result of early nuclear-free character identification, 17 seedless grape lines were obtained. An efficient system of seedless grape breeding through embryo rescue was also established. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

DNA markers technology, derived from research in molecular biology and genomics, offers great promise for plant breeding, allowing the 'molecular breeding' via marker-assisted selection. Grapevine genomic resources allowed, in recent years, the characterization at molecular level of genes involved in interesting phenotypes such as stenospermocarpic seedlessness, a trait really appreciated by consumers. Recent studies in table grapes revealed that the VvAGL11 gene, member of the D-lineage MADS-box family, controls the ovule identity, and thus potentially playing an important role in stenospermocarpy. Intragenic markers of VvAGL11 have been found and tested for breeding purposes. In the present paper, we describe an in deep assay on a total of 475 genotypes derived by our own grape germplasm and seeded × seedless crosses F offspring, to evaluate and verify the 'diagnostic' power of VvAGL11 in marker-assisted selection. We found only 8/475 that were seeded and carried the seedless-associated allele in the STS p3_VvAGL11. However, and most importantly, there were no seedless varieties without such allele. We validated the marker as a 100 % effective tool for early negative selection of stenospermocarpy in Vitis vinifera L. crosses. [ABSTRACT FROM AUTHOR]

Abstract: Prevailing ambient temperature during the reproductive phase is one of important factors for seed and fruit set in different plant species. In mango (Mangifera indica L.), natural low temperatures during flowering induced seedless fruits. Here the sexual reproduction process of ‘Tainong 1’ mango at low temperatures (diurnal maximum temperature<20°C) was studied. For comparison, we also examined this process at “normal” temperatures (diurnal maximum ranging from 25 to 30°C, diurnal average temperature>20°C). Results showed: natural low temperatures significantly affected pistil and male gametophyte development, resulting in pollen grains with low viability. Meiotic chromosomal irregularities, including univalents, multivalents, laggards, bridges and micronuclei were detected at higher incidences and significantly greater proportions of nucleolus fragmentation and dissolution were detected when temperatures were low. Pollen tube growth was retarded under low temperature stress, either in vivo or in vitro. The virtual absence of sexual reproduction of ‘Tainong 1’ mango at low temperatures appears to be due largely to slow growth of pollen tube in vivo and to a low rate of successful fertilization. [ABSTRACT FROM AUTHOR]

Conventional crossing of stenospermocarpic grapes for the obtainment of seedless cultivars presents some technical constraints causing embryo abortion in the early berry developmental stages. Embryo rescue technique partially overcomes these limitations, but the obtainment of viable plantlets relies on the optimization of several genetic and methodological issues. This work aimed to regenerate viable plants from immature ovules of stenospermocarpic table grape hybrids by applying a three-step in vitro culture protocol consisting of embryo development, embryo germination-rooting, and plantlet formation. The influence of parental genotypes (six "seedless × seedless" crosses), ovule sampling time (30, 40, 50 days after pollination (DAP)), and extent of embryo germination induction (4, 6, 8 weeks) was assessed on ovule fertilization, embryo development and germination, rooting, and plantlet formation to establish the best rescue time for each combination hybrid. Our optimized protocol included immature ovule isolation for 40 DAP and embryo germination induction for 8 weeks. As for genotypes, the most efficient embryo germination was recovered from hybrids of Thompson, Superior, and Regal cultivars, whereas the highest percentage of viable plants was derived from 50-DAP ovules of Luisa × Thompson progeny. Such an optimized protocol could be useful to maximize the efficiency of future breeding programs for grape seedlessness. [ABSTRACT FROM AUTHOR]

Abstract: The effect of diurnal maximum/minimum (20/10 or 25/15°C) temperatures on seed and fruit development of ‘Irwin’, ‘Kensington’ and ‘Nam Dok Mai’ mangoes (Mangifera indica L.) was studied in a controlled-environment glasshouse. Exposure to low temperatures (20/10°C day/night) 3 days after hand pollination significantly increased the percentage of stenospermocarpic fruit (nubbins), in which embryos were aborted at some stage during early fruit development. There were significant differences between cultivars in the percentage of nubbins produced out of the total fruit set following overnight exposure to 10°C with 21% for ‘Nam Dok Mai’, 11% for ‘Kensington’ and 3% for ‘Irwin’. At 45 days after pollination, nubbin fruits were much smaller in size and weighed ca. 50% less than normal fruits. The lower percentage of nubbin fruits in ‘Irwin’ implies a greater adaptation to cool temperatures by this cultivar during fruit set and early embryo development. [Copyright &y& Elsevier]

In nonparthenocarpic cucumber, analysis of the interaction between season and female receptivity demonstrated two different modes of fruit development and seed set. Spring climatic conditions induced significantly longer ovaries at anthesis with an autonomous fruit set as in natural parthenocarpy. Autumn climatic conditions induced smaller ovaries with fruit set influenced by the pollination stage of the female flower. These differences involve, during fruit development, a regulation of the empty seed number especially at both ends of female receptivity period. Indeed, seasons have no effect on ovule receptivity and fertility measured by full seed number. It is supposed that priority is given to seeds with an embryo in the competition for limited maternal resources in spring as well as in autumn. In spring, the use of irradiated pollen during the whole female receptivity period did not modify the seed set profile. Moreover, a cytological study of 6-day-old ovules pollinated by irradiated pollen has revealed a similar precocious embryogenesis compared to the control. In cucumber, pollination with irradiated pollen involves stenospermocarpy rather than 'induced parthenocarpy.' However, we have not been able to determine whether the early abortive embryogenesis induced by irradiated pollen is gynogenetic or abnormal zygotic. [ABSTRACT FROM AUTHOR]

Mexico is the leading exporter of mangos worldwide, and 'Ataulfo' is one of the most popular cultivars. However, their production has dramatically dropped in recent years due to the high incidence of nubbins. One of the possible causes is the presence of a delayed self-incompatibility found in this cultivar; thus, proximity to compatible cultivars may help to reduce this incidence. Nevertheless, there is a lack of studies that have rigorously tested this hypothesis in this cultivar. For two consecutive years, the present study evaluated the incidence of nubbins, as well as the quality and quantity of commercial fruits of 'Ataulfo' trees located at 10, 30, and 50 m away from 'Haden' cultivar. Additionally, the yield and economic income of different planting designs were estimated. During both sampling periods, our results clearly indicated that at 10 m away from 'Haden' individuals, 'Ataulfo' trees presented a lower incidence of nubbins and higher production of commercial fruits, and higher yield and total income per hectare than at 30 or 50 m away from them. These results indicate that planting designs of 'Ataulfo' trees located 10 m away from 'Haden' will help to satisfy the increasing demand for mangos of this cultivar in the international market. [ABSTRACT FROM AUTHOR]

Reduced expression of MADS-box gene AGAMOUS-LIKE11 (VviAGL11) is responsible for stenospermocarpic seedlessness in bunch grapes. This study is aimed to characterize the VviAGL11 orthologous gene (VroAGL11) in native muscadine grapes (Vitis rotundifolia) at the molecular level and analyze its divergence from other plants. The VroAGL11 transcripts were found in all muscadine cultivars tested and highly expressed in berries while barely detectable in leaves. RT-PCR and sequencing of predicted ORFs from diverse grape species showed that AGL11 transcripts were conservatively spliced. The encoded VroAGL11 protein contains highly conserved MADS-MEF2-like domain, MADS domain, K box, putative phosphorylation site and two sumoylation motifs. The muscadine VroAGL11 proteins are almost identical (99%) to that of seeded bunch cultivar, Chardonnay, except in one amino acid (A79G), but differs from mutant protein of seedless bunch grape, Sultanina, in two amino acids, R197L and T210A. Phylogenetic analysis showed that AGL11 gene of muscadine and other Vitis species formed a separate clade than that of other eudicots and monocots. Muscadine grape cultivar "Jane Bell" containing the highest percentage of seed content in berry (7.2% of berry weight) had the highest VroAGL11 expression, but almost none to nominal expression in seedless cultivars Fry Seedless (muscadine) and Reliance Seedless (bunch). These findings suggest that VroAGL11 gene controls the seed morphogenesis in muscadine grapes like in bunch grape and can be manipulated to induce stenospermocarpic seedlessness using gene editing technology. [ABSTRACT FROM AUTHOR]

Seedless inheritance has been considered a quasi-monogenic trait based on the VvAGL11 gene. An intragenic simple sequence repeat (SSR) marker, p3_VvAGL11, is currently used to opportunely discard seeded progeny, which represents up to 50% of seedlings to be established in the field. However, the rate of false positives remains significant, and this lack of accuracy might be due to a more complex genetic architecture, some intrinsic flaws of p3_VvAGL11, or potential recombination events between p3_VvAGL11 and the causal SNP located in the coding region. The purpose of this study was to update the genetic architecture of this trait in order to better understand its implications in breeding strategies. A total of 573 F1 individuals that segregate for seedlessness were genotyped with a 20K SNP chip and characterized phenotypically during four seasons for a fine QTL mapping analysis. Based on the molecular diversity of p3_VvAGL11 alleles, we redesigned this marker, and based on the causal SNP, we developed a qPCR-HRM marker for high-throughput and a Tetra-ARMS-PCR for simple predictive analyses. Up to 10 new QTLs were identified that describe the complex nature of seedlessness, corresponding to small but stable effects. The positive predictive value, based on VvAGL11 alone (0.647), was improved up to 0.814 when adding three small-effect QTLs in a multi-QTL additive model as a proof of concept. The new SSR, 5U_VviAGL11, is more informative and robust, and easier to analyze. However, we demonstrated that the association can be lost by intragenic recombination and that the e7_VviAGL11 SNP-based marker is thus more reliable and decreases the occurrence of false positives. This study highlights the bases of prediction failure based solely on a major gene and a reduced set of candidate genes, in addition to opportunities for molecular breeding following further and larger validation studies. [ABSTRACT FROM AUTHOR]