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A diploid hybrid between Citrus clementina and C. sinensis (CDO) was used as male parent in 4x x 2x sexual hybridizations with tetraploid Fina clementine and Moncada mandarin as female parents to produce triploid hybrids. Unexpectedly, in addition to some triploids, a total of seventy-two tetraploid plants were obtained, 62 plants from Fina x CSO and ten from Moncada x CSO. SSR and SNP markers analysis releaved that recovered tetraploid plants were hybrids arising from unreduced pollen gametes. A maximum-likelihood methodology based on parental heterozygosity restitution (PHR) of markers close to the centromeres was used to identify the mechanism underlying 2n pollen gamete formation. Thirty five tetraploid hybrids were identified to arise from FDR (56%) and 18 from SDR (29%) mechanisms. Inconclusive data were obtained for nine tetraploid hybrids. The pattern of variation of PHR values of ten SSR and twi SNP markers located along linkage groups 1 and 2 confirmed our results at population level. To our knowledge this is the first report in citrus of progenies of tetraploid hybrids recovered from 2n pollen gametes and the first description of coexistence of two meiotic mechanisms (SDR and FDR) producing unreduced pollen gametes. (Texte intégral)

Citrus breeding programs focused to produce seedless varieties need to increase the knowledge about the parthenocarpic ability (PA) of the different citrus genotypes used as parents in sexual hybridizations. Current information is insufficient and in some cases contradictory. In this context, ten citrus genotypes were assessed for their PA including Campeona, Imperial, Saltenita, Fortune and Moncada mandarins, Clemenules and Monreal clementines, Tardia Picana satsuma, Ellendale tangor and Pink pummelo. We used terminal flowers at balloon stage and performed the following treatments: 1) hand-cross-pollination with FOrtune and bagged (Clemenules was used for Fortune due the self-incompatibility of Fortune, 2) hand-self-pollination and bagged, 3) emasculated and bagged, 4) bagged and 5) open pollination. About 100 flowers were used for each treatment and were repeated in two consecutive seasons. Fruits were collected when ripe and data of fruit set, number of seeds per fruit and fruit weight were redorded. Different levels of PA were observed; Tardia Picana satsuma showed the highest level of PA followed by Moncada, CLemenules, Imperial and Pink pummelo. Campeona, Ellendale and Saltenita displayed a low level of PA, whereas Fortune and Monreal (a self-compatible Clementine) do not show PA and needs the stimulus of pollination for fruit development. In addition, we observed different relations between fruit size and seed number depending on the genotype. These results have been used for parental combination strategies in our triploid breeding program. (Texte intégral)

Temperature is one of the main factors affecting the sexual reproductive success of plants. Both Pollen Grain Germination (PGG) and Pollen Tube Growth (PTG) play a crucial role on the progamic phase that elapses from pollination to fertilization. We have studied the effects of four temperature regimes (constant 10°C, 20°C, 30°C and field conditions) in PGG and PTG in Citrus. 'Fortunr' mandarin (CItrus clementina x C. tangerina) was used as female recipient. Two adult trees of 'FOrtune' cultivated in containers were placed under each temperature regime and pollinated with three donor genotypes grown under field conditions: Clemenules clementine (C. clementina), Pineapple sweet orange (C. sinensis) and one Papeda spedies (C. ichangensis). Ten sampes per expetimental condition were collected every day during 10 consecutive days starting the day after pollination for histological studies. PGG and PTG were monitored on 13 cross sections along the pistil. This method allowed finding the distance travelled by pollen tubes every day from the surface of the stigma. Unexpectedly we observed both PGG and PTG at 10°C. This result seems to disagree with previous research in citrus which concluded that PGG in vitro does not occur at 10°C. The results also show clear differences in the kinetics of the PTG and their dependence on temperature and genotype. At 10°C Papeda and CLemenules pollen tubes reached the ovary seven days after pollination, while Pineapple pollen tubes only reached half of the style. (Texte intégral)

La obtención de variedades de mandarino que produzcan frutos sin semillas es indispensable para el mercado de consumo en fresco ya que los consumidores no aceptan la presencia de semillas en los frutos. La obtención de híbridos triploides es una estrategia que permite obtener variedades sin semillas ya que presentan muy baja fertilidad y normalmente no producen semillas ni inducen la formación de semillas en otras variedades por polinización cruzada. En este artículo se presenta el programa de mejora genética de mandarino que nuestro grupo está realizando en el IVIA junto con los diferentes avances biotecnológicos desarrollados que nos han permitido establecer y desarrollar un amplio programa de obtención de nuevas variedades de mandarino mediante hibridación sexual.

Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the 4x parents used in our program are doubled-diploid (DD), also known as autotetraploid. The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends on the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNP and SSR markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one displays a tendency for disomy. The PHR in the 2n gametes is 2/3 of that obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x X 2x hybridizations are more efficient than 2x X 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x X 2x hybridisations provides a greater opportunity to select innovative products. (Résumé)

The main application of shoot-tip grafting in vitro (STG) is to control graft-transmissible pathogens that requires the use of healthy trees in the new plantings. The routine application of STG using 0.1-0.2 mm shoot tips is very efficient for elimination of all citrus graft-transmissible pathogens from local or imported varieties. It has allowed the recovery of hundreds of healthy cultivars and the planting of hundreds of millions of healthy certified trees worldwide. Only in Spain about 140 million certified nursery plants propagated from micrografted plants have been planted. STG is also a very useful technique for regeneration of elite genotypes in several research areas. In vitro grafting for these purposes may be done using larger shoots (up to 1 cm). STG is being routinely used for the following purposes: (i) Regeneration of somatic hybrids from embryos difficult to germinate; (ii) Regeneration of plants from irradiated shoots to produce seedless varieties; (iii) Regeneration of plants from haploid embryos that are very difficult to germinate. STG was used to regenerate the 'Clemenules' haploid plant that has been used by the International Citrus Genome Consortium to sequence the whole citrus genome; (iv) Production of stable tetraploid plants of non-apomictic genotypes, which are very useful for triploid breeding; (v) Regeneration of transgenic plants from shoots that are very difficult to root in vitro. STG has become a routine application in citrus genetic transformation.

Polyploidy has long been recognized as an important force in the evolution of flowering plants and often results in novel phenotypes having advantages in adaptation and major interest for agriculture. Many crops, including citrus, are bred to a higher level of ploidy in search of desirable traits. In previous works of our group synthetic clementines with different levels of ploidy, specifically haploid, dihaploid and autotetraploid, were generated. This plant material represents a valuable tool for studying the effects of polyploidization at the molecular level since important traits are expressed in different manners. By using a microarray approach, we have accomplished a comprehensive analysis of transcriptome divergence among the newly created clementine lineages with different genome dosage. Differences in expression in the bark tissue of the four lineages were found significant for 89 (haploid-dihaploid comparison) or 93 genes (diploid-tetrapolid comparison). These genes were classified on the basis of their structure in different families.

The availability of a saturated genetic map of Clementine was identified by the International Citrus Genome Consortium as an essential prerequisite to assist the assembly of the reference whole genome sequence based on a Clementine derived haploid. The primary goals of the present study were to establish a Clementine reference map, and to perform comparative mapping with pummelo and sweet orange. Five parental genetic maps were established with SNPs, SSRs and InDels. A medium density reference map (961 markers for 1084.1 cM) of Clementine was established and used by the ICGC to facilitate the chromosome assembly of the haploid genome sequence. Comparative mapping with pummelo and sweet orange revealed that the linear order of markers was highly conserved. Reasonable inferences of most citrus genomes should be obtained by mapping next-generation sequencing data against the haploid reference genome sequence. Skewed segregations were frequent and higher in the male than female Clementine potentially leading to false interpretation of the genetic determinism of phenotypic traits. The mapping data confirmed that Clementine arose from hybridization between 'Mediterranean' mandarin and sweet orange and identified nine recombination break points for the sweet orange gamete that contributed to the Clementine genome. Introgression of pummelo genome fragments were identified in heterozygosity in each chromosome. Moreover, it appeared that the genome of the haploid Clementine used to establish the citrus reference genome sequence was inherited primarily from the 'Mediterranean' mandarin. The usefulness of this genetic map, anchored in the reference whole genome sequence, is discussed.

Somatic hybridization by protoplast fusion is an effective technique for citrus rootstock breeding. It theoretically allows the accumulation of all genes of the parents irrespective of their heterozygosity level and therefore the addition of complementary dominant traits. In the present work, protoplasts were isolated from callus of C. macrophylla and from leaves of 'Carrizo' citrange (C. sinensis x P. trifoliata). Chemical, electro-chemical and electric fusions were performed and ploidy level of the regenerated plants was evaluated by flow-cytometry. Five tetraploid plants, one pentaploid plant, one mixoploid (3x-6x) plant and one heptaploid plant were recovered. All these plants were analyzed with SSR markers, distributed in the nine chromosomes of citrus. Cytoplasmic genomes were also characterized using chloroplastic and mitochondrial markers. Results showed that mitochondrial genome was inherited from C. macrophylla in all plants while segregation was observed for chloroplastic genome. Nuclear genome analysis revealed the loss of parental alleles in most of the regenerated plants. In tetraploids, it affected mainly C. macrophylla alleles, while the alleles lost in pentaploid and heptaploid plants were mostly from P. trifoliata. Results suggest chromosome instability in this complex intergeneric combination with apparent non-random loss of chromosome fragments. Two allotetraploid somatic hybrids were selected for evaluation as rootstocks.

Citrus triploid hybrids can be recovered by 2x x 2x hybridisations as a consequence of the formation of 2n gametes or by interploid hybridisation. Most of the tetraploid parents used are doubled-diploid (DD). The genetic structure of diploid gametes and particularly the rate of parental heterozygosity restitution (PHR) depend on the meiotic process by which they were originated. Second-division restitution (SDR) is the 2n gamete formation mechanism involved in 'Fortune' and clementines. With SDR, PHR is positively linked with the distance of each locus to the centromere. With DD parents, PHR depends of the rate of preferential pairing and thus the proportion of disomic versus tetrasomic segregations. We have compared the genetic structure of two populations of diploid gametes of clementine (one population of 2n gametes and one of 2x gametes produced by DD) with SNPs and SSRs markers covering the 9 citrus chromosomes. The DD displays mostly tetrasomic segregation; however three linkage groups present intermediate segregation and one display a tendency for disomy. The PHR in 2n gametes is 2/3 of the one obtained in 2x gametes produced by DD. The two methods of triploid production appear complementary in terms of genotypic variability. 4x x 2x hybridizations are more efficient than 2x x 2x for developing new cultivars phenotypically closer to the diploid parent of the DD. Conversely, 2x x 2x hybridisations provides the opportunity to select more innovative products.

Estimation of allele copy numbers for molecular markers has long been considered a challenge for polyploid species, while this process is essential for most genetic research. With the increasing availability and whole genome coverage of SNP markers, it is important to implement a versatile SNP genotyping method to efficiently assign allelic configuration in polyploids. This work demonstrates the usefulness of the KASPar method, based on competitive allele-specific PCR, for the assignment of SNP allelic configuration in an efficient, accurate, simple, and cost effective way and it has been successfully applied in genotyping citrus triploids.

Estimation of DNA allele doses in polyploid genotypes is essential to assign the allelic configuration for the different types of heterozygosity for parents and segregating progenies. It is of special interest in citrus with the implementation of extensive triploid breeding programs to develop new seedless varieties. Single nucleotide polymorphisms (SNPs) are becoming the most important class of molecular markers and we have tested the potential of KASPar technology (based on competitive allele-specific PCR) to infer allele doses of SNP markers in polyploid citrus. DNAs from haploids Citrus clementina and Citrus maxima were mixed at different proportions to test the accuracy of the technique for quantitative analysis. All analyzed 7 SNPs provided clear clustering related with allele doses. DNA mixes at intermediary proportions revealed high correlation coefficients between observed and expected data (mean = 0.9796; sd = 0.0094). For all SNPs, separated cluster analyses and ANOVA from mixed DNA data formed all expected homogeneous groups, with correct assignation for practically all samples. Moreover, two triploid populations were easily genotyped and results were in agreement with expected segregations. KASPar technology is a routine and a cost-effective technique to assess the allele doses at the DNA level, which is especially interesting in citrus triploid breeding programs. Moreover, it could be also used to correlate genomic and transcriptomic doses in allele specific expression analyses.

Although Citrus is the most globally significant tree fruit, its domestication history is poorlyunderstood. Cultivated citrus types are believed to comprise selections from and/or hybrids of several wild progenitor species, but the identities of these progenitors, and their contribution to modern cultivars, remain controversial. Here we report the genomes of a collection of mandarins, pummelos, and oranges, including a high quality reference sequence from a haploid Clementine mandarin. By comparative genome analysis we show that these cultivated types can be derived from two progenitor species. Cultivated pummelos represent selections from a single progenitor species C. maxima. Unexpectedly, however, we find that cultivated mandarins are introgressions of C. maxima into a distinct second population that we identify with the ancestral wild mandarin species C. reticulata. Sweet and sour oranges are found to be interspecific hybrids. Sweet orange, the most widely cultivated citrus, arose as the offspring of previously admixed individuals. In contrast, sour (or Seville) orange is an F1 hybrid of pure C. maxima and C. reticulata parents, implying that wild mandarins were part of the early breeding germplasm.Surprisingly, we also find that a wild Chinese mandarin from Mangshan, China showssubstantial sequence divergence from C. reticulata and appears to represent a distinct taxon.Understanding the relationships and phylogeny of cultivated citrus through genome analysis will clarify taxonomic relationships and enable previously inconceivable opportunities for sequence-directed genetic improvement.Citrus are widely consumed worldwide as juice or fresh fruit, providing important sources ofvitamin C and other health-promoting compounds. Global production in 2012 exceeded 86million metric tons, with an estimated value of US$9 billion (http://www.fas.usda.gov/psdonline/circulars/citrus.pdf). The very narrow genetic diversity of cultivated citrus makes it highly vulnerable to disease outbreaks, including citrus greening disease (also known as Huanglongbing) that is rapidly spreading throughout the world's major citrus producing regions1. Understanding the population genomics and domestication of citrus will enable strategies for improvements to citrus including resistance to greening and otherdiseases. The domestication and distribution of edible citrus types began several thousand years ago in Southeast Asia and spread globally following ancient land and sea routes. The lineages that gave rise to most modern cultivated varieties, however, are lost in undocumented antiquity, and their identities remain controversial2, 3. Several features of Citrus biology and cultivation make deciphering these origins difficult. Cultivated varieties are typically propagated clonally by grafting and through asexual seed production (apomixis via nucellar polyembryony) to maintain desirable combinations of traits (Fig. 1). Thus many important cultivar groups have characteristic basic genotypes that presumably arose through interspecific hybridization and/or successive introgressive hybridizations of wild ancestral species. These domestication events predated the global expansion of citrus cultivation by hundreds or perhaps thousands of years, with no record of the domestication process. Diversity within such groups arises through accumulated somatic mutations, generally without sexual recombination, either as limb sports on trees or variants among apomictic seedling progeny.Two wild species are believed to have contributed to domesticated pummelos, mandarins and oranges. Based on morphology and genetic markers, pummelos have generally been identified with the wild species C. maxima (Burm.) Merrill that is indigenous to Southeast Asia. Although mandarins are similarly widely identified with the species C. reticulata Blanco 4-6, wild populations of C. reticulata have not been definitively described. Various authors have taken dif

En este trabajo se han localizado los centrómeros en los 9 grupos de ligamiento (GL) del mapa genético de mandarino clementino mediante Half Tetrad Analysis (HTA) y se ha determinadoel mecanismo implicado en la formación de los gametos femeninos no reducidos en 16 genotipos de mandarino y un genotipo de naranjo pigmentado. La determinación del mecanismo implicado en la formación de los gametos 2n sin el conocimiento de la posición de los centrómeros requiere del análisis de un elevado número de plantas con un elevado número de marcadores, mientras que conociendo la posición de los centrómeros es posible determinar el mecanismoa nivel individual y poblacional con un reducido número de marcadores próximos a los centrómeros. La restitución de la segunda división meiótica aparece como el mecanismo implicado en la formación de los gametos femeninos 2n en cítricos.

El análisis genético realizado con marcadores Simple Sequence Repeat (SSR) en dos poblaciones de híbridos tetraploides obtenidos en hibridaciones sexuales 4x X 2x nos ha permitido identificar un híbrido de clementino x naranjo (C. clementina x C. sinensis) que produce gametos masculinos no reducidos y que el mecanismo implicado en la formación de los gametos 2n es la restitución de la primera división meiótica (FDR). Este fenómeno se describe por primera vez en los cítricos-en el presente trabajo.

Citrus triploid hybrids can be recovered by 2x X 2x hybridisations as a consequence of 2n gamete formation. Two main meiotic processes are at the origin of such gametes, the first division restitution (FDR) and the second division restitution (SDR). These two mechanisms lead to very different gamete genetic structures. Particularly, the opposite feature of parental heterozygosity restitution (PHR) is observed in relation with the distance to the centromere. Without previous knowledge on centromere location, the determination of the restitution mechanism requires an analysis of the frequency of PHR within a large population with numerous codominant markers. With this method we determined that SDR is the mechanism involved in clementines and 'Fortune' mandarin. With SDR, PHR is a direct function of the distance of the considered locus to the centromere. Therefore the analysis of the evolution of PHR, within a linkage group (LG) allows mapping of the centromere position. Centromere has been located in the 9 LGs of the clementine genetic map by genotyping triploid hybrids with SSR and SNP markers. Analysis of the 2n gamete allelic configuration with loci close to the centromere can shed light on the restitution mechanism at the individual level and open the way for simplified study of this mechanism in a large range of genotypes. We are applying this approach to analyse the 2n gamete origin in several genotypes of mandarin and one genotype of sweet orange. (Texte intégral)

La obtención de híbridos triploides es la estrategia principal para la producción de mandarinas sin semillas ya que los consumidores no aceptan la presencia de éstas en los frutos. En los cítricos se pueden obtener híbridos triploides a partir de hibridaciones 2x X 2x como consecuencia de la formación de gametos femeninos no reducidos y mediante hibridaciones interploides (2x X 4x y 4x X 2x). Muchos de los parentales 4x utilizados son dobles diploides (DD). La estructura genética de los gametos diploides y particularmente la restitución de la heterocigosidad materna (RHM) para cada locus, depende de los procesos meióticos que los originan. La restitución de la segunda división meiótica (SDR) es el mecanismo implicado en la formación de gametos femeninos no reducidos en las clementinas y en mandarino `Fortune´ (Cuenca et al., 2011). Con SDR, la RHM varía dependiendo de la posición del locus considerado y del número de entrecruzamientos originados entre el locus considerado y el centrómero. La RHM varía entre 0%, para los loci muy próximos al centrómero y un valor máximo que depende del nivel de interferencia cromosómica. Los datos experimentales obtenidos en cítricos indican que el modelo de interferencia parcial (Cx(Co)4) es el que presenta un mejor ajuste. Para los parentales DD, la RHM depende del grado de asociación preferencial entre los cromosomas y por lo tanto de la relación entre la segregación a nivel disómico (100%) y/o tetrasómico (55,5-66,6%). El objetivo de este trabajo es analizar la variabilidad genotípica de los híbridos triploides obtenidos mediante hibridaciones sexuales 2x X 2x y 4x X 2x y su implicación en el programa de mejora genética de híbridos triploides de cítricos que estamos desarrollando en el IVIA.

En el presente artículo se describen los aspectos más importantes la mancha marrón de las mandarinas causada por Alternaria alternata, indicando las variedades afectadas y los mecanismos genéticos que determinan la susceptibilidad a la enfermedad. Se describen las ventajas y limitaciones de las técnicas disponibles para evaluar la susceptibilidad de las variedades, y su aplicación práctica en el programa de obtención de híbridos triploides del IVIA. Por último, se indican brevemente las medidas agronómicas y los tratamientos fungicidas recomendados para su control. En este apartado se incide especialmente en la importancia del grado de susceptibilidad de las variedades y las características microclimáticas de las parcelas en la eficacia de las estrategias de control.

En la Sesión S17 'Varieties' del XII Congreso Internacional de Citricultura celebrado en Valencia entre los días 18 y 23 de noviembre de 2012, se presentaron un total de ocho comunicaciones orales y 30 comunicaciones en formato póster. La sesión estuvo moderada por los Drs. Pablo Aleza (IVIA, España) y Guiseppe Reforgiato Recupero (CRAACM, ltalia). Las comunicaciones presentadas se pueden dividir en tres grandes grupos: (1) Obtención de nuevas variedades, (II) Evaluación y comportamiento agronómico de variedades comerciales y (III) un tercer grupo genérico que engloba comunicaciones de temáticas más generales; programas de mejora genética mediante irradiación, estado actual de la citricultura de Chile, composición de sustancias volátiles en la corteza de los frutos, análisis con marcadores moleculares, biología reproductiva, etc.

A haploid C. clementina was chosen by the International Citrus Genomic Consortium (ICGC) to establish the reference whole genome sequence for Citrus. Development of a dense clementine linkage map was part of the objectives of this global collaborative project. Two inter-specific populations between C. clementina and C. maxima were used for this purpose. 156 hybrids of Nules clementine x Pink pummelo and 200 hybrids of Chandler pummelo x Nules clementine were genotyped with 1003 markers. 306 were SSRs markers (66 from genomic libraries, 207 from ESTs and 33 from clementine BAC end sequences -BES-), 34 were Indels markers mined from BES and 663 SNPs mined from Clementine BES or identified by candidate gene sequencing. 901 markers were successfully mapped in the 9 clementine linkage groups. Segregation distortion was observed for many loci of clementine when it was used as male parent while it followed Mendelian segregation for most markers when used as female parent. However marker order was mostly conserved between the male and female maps; thus, data of the two populations were joined to establish the reference clementine genetic map. The total clementine linkage map cover 1250 cM with linkage groups from 105 cM until 210 cM. This map is strongly anchored on a large diploid clementine BAC library resource. It is a powerful tool for Citrus genetics and supports the alignment of the haploid clementine whole genome sequence in the framework of the ICGC collaborative project.

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market and mandarin triploid hybrids have this trait. Citrus triploid plants can be recovered by 2x 9 2x sexual hybridisations as a consequence of the formation of unreduced gametes at low frequency. Triploid embryos are found in small seeds that do not germinate under greenhouse conditions. Extensive breeding programs based on this type of hybridisation require very effective methodologies for embryo rescue and ploidy evaluation. In this work, we describe an effective methodology to recover triploid hybrids from 2x 9 2x hybridisations based on in vitro embryo rescue and ploidy level determination by means of flow cytometry. The influence of parents and environmental conditions on obtaining triploid hybrids has been analysed. The strongest effect was associated with the genotype of the female parent while a strong interaction was found between the male parent genotype and environmental conditions. The effect of the female parent genotype on the length of the juvenile phase was also demonstrated by observing a large number of progenies over the last 10 years. The methodology described here has enabled us to obtain over 4,000 triploid hybrids so far, of which 13 have been protected in the European Union and two are being extensively planted by citrus growers to establish new commercial plots. These triploid hybrids have been analysed with simple sequence repeats markers to differentiate all the new triploid varieties and their parents, and thus molecular identification will help defend plant breeders' rights.

En este artículo se presentan las dos nuevas variedades triploides de mandarino Garbí y Safor, obtenidos por hibridación entre el parental femenino diploide Fortune y los parentales masculinos diploides tangor Murcott (C. reticulata x C. sinensis) y mandarino Kara (C. unshiu x C. nobilis), respectivamente. Las dos variedades se han protegido en la Unión Europea, Egipto, Marruecos, Turquía y se han patentado en Estados Unidos.

Adventitious embryony from nucellar cells is the mechanism leading to apomixis in citrus. However, singular cases of polyembryony have been reported in non-apomictic genotypes as a consequence of 2x×4x hybridisations and in vitro culture of isolated nucellus. The origin of the plants obtained as a consequence of these two processes is still unclear. In this work, we systematically analyzed the genetic structure (ploidy and allelic constitution at SSR locus) of plants obtained from polyembryonic seeds arising from 2x×4x sexual hybridisations or regenerated from nucellus culture in vitro of different non-apomictic citrus genotypes. Histological studies were also conduced to try to identify the initiation process of polyembryony in nonapomictic genotypes. We demonstrate that all plants obtained from the same undeveloped seed in 2x×4x hybridisations resulted from fission of the original zygotic embryo. Also, the plants obtained from in vitro culture of nucellus were recovered by somatic embryogenesis from cells having the same genotype as the zygotic embryos of the same seed. It appears that in non-apomictic citrus, proembryos or embryogenic cells are formed by fission of the original zygotic embryo and that the development of these adventitious embryos, normally hampered, can take place in vivo or in vitro as result of two different mechanisms that prevent the dominance of the initial zygotic embryo.

Se describe por primera vez un mecanismo de la biologia reporductiva de los genotipos no apomicticos de citricos. En estos se forman proembiones o células embiogénicas por la fision del embrioon zigotico original. Normalment solo de desarrolla el embrion original, pero si se inhibe la dominancia del mismo mediante procedimientos mecanicos o genéticos, los proembriones se desarrollan produciendo varias plantas a partir de una misma semilla que son idénticas généticamente entre sf. Esta caracteristica tiene importantes implicacions para los programas de mejora genética basdos en la obtension de hibridos triploides.*

Las clementinas son las variedades de mandarino más representativas y más ampliamente cultivadas de la citricultura española. Se recolectan desde mediados de septiembre hasta finales de enero. Los mercados demandan mandarinas hasta el mes de abril, por lo que también se cultivan algunos híbridos tipo mandarino de maduración tardía. Tanto los clementinos como estos híbridos son partenocárpicos y autoincompatibles, por lo que producen frutos sin semillas en cultivos aislados. Sin embargo, tanto el polen como los óvulos de estas variedades son viables y la polinización cruzada causa la formación de frutos con semillas en ambos grupos de variedades, lo que provoca un gran problema comercial porque los consumidores no aceptan las mandarinas con semillas. Como consecuencia de esta situación, la obtención de nuevas variedades de mandarino de maduración tardía que no produzcan semillas y que no induzcan la formación de semillas en las clementinas por polinización cruzada es el objetivo prioritario de la citricultura española.

La presencia de semillas en los frutos de mandarina es una característica indeseable ya que el mercado de consumo en fresco no acepta frutos con semillas. Los híbridos triploides de cítricos son de gran interés ya que no producen semillas ni inducen la formación de semillas en otros genotipos por polinización cruzada. Los híbridos triploides se pueden obtener mediante hibridación somática entre protoplastos haploides y diploides y mediante hibridación sexual entre parentales diploides y entre parentales diploides y tetraploides. El objetivo general de esta tesis doctoral es la obtención de parentales para la producción de híbridos triploides de cítricos, la caracterización de los diferentes tipos de hibridaciones sexuales que permiten obtener híbridos triploides y un mejor conocimiento de la biología reproductiva de los cítricos en relación con la obtención de híbridos triploides. Estos objetivos generales se concretan en los siguientes objetivos específicos: 1.Obtención de plantas haploides mediante la técnica de ginogénesis in situ inducida por polen irradiado y posterior rescate y cultivo de embriones in vitro. Se han obtenido genotipos haploides, dobles haploides y aneuploides de clementina `Clemenules´ y uno de los genotipos haploides ha sido elegido por el Consorcio Internacional de Genómica de Cítricos para la secuenciación del genoma completo de los cítricos. 2.Obtención de plantas tetraploides de diferentes genotipos apomícticos de mandarinos, naranjos dulces y pomelos que puedan ser utilizados como parentales masculinos en hibridaciones 2x X 4x para la obtención de híbridos triploides. Mediante citometría de flujo se han identificado plantas tetraploides espontáneas en 15 genotipos de los 17 estudiados, con frecuencias que oscilaron entre el 9,7% en mandarino `Kinnow´ y el 0,5% en mandarino `Page´. Se demuestra que todos los genotipos tetraploides obtenidos proceden de la duplicación del número de cromosomas de células de la nucela. 3.Obtención de plantas tetraploides de genotipos no apomícticos de mandarino que puedan ser utilizadas como parentales masculinos y femeninos en hibridaciones 2x X 4x y 4x X 2x. Para ello se realizó el microinjerto in vitro de ápices caulinares tratados con colchicina y orizalina. Se han obtenido plantas tetraploides a partir de la brotación directa del ápice microinjertado y a partir de la segregación de citoquimeras, obteniendo plantas tetraploides estables de las clementinas `Clemenules´, `Fina´, `Marisol´ y mandarino `Moncada´. 4.Obtención de híbridos triploides mediante hibridaciones 2x X 2x, 2x X 4x y 4x X 2x. Se demuestra que los embriones triploides se encuentran en diferentes tipos de semillas en función del tipo de hibridación realizada y se propone una mejora en cada etapa del proceso de obtención de híbridos triploides. Se evaluaron las eficiencias de las diferentes estrategias en función de los genotipos parentales. Las hibridaciones 4x X 2x y 2x X 4x presentaron la mayor eficiencia en la obtención de híbridos triploides; sin embargo en aquellos genotipos capaces de producir frecuencias muy elevadas de gametos 2n, como mandarino `Fortune´, la estrategia 2x X 2x es muy eficiente. Los resultados obtenidos también demuestran la gran influencia que tiene el parental masculino en la obtención de híbridos triploides, lo que implica la selección de una estrategia u otra. Por fin, se discuten las implicaciones de los diferentes tipos de hibridación a nivel de las segregaciones y de la diversidad genotípica teórica de las poblaciones triploides. 5.Determinación del origen genético de las plantas regeneradas a partir de semillas parcialmente desarrolladas originadas en las hibridaciones 2x X 4x utilizando genotipos no apomícticos como parentales femeninos. Se demuestra con marcadores microsatélites que el origen de este tipo de poliembrionía ocurre como consecuencia de una embriogénesis secundaria a partir del embrión zigótico original. Así mismo, esta característica se confirma mediante la regeneración de plantas a partir del cultivo de nucelas in vitro de genotipos no apomícticos de cítricos. Se concluye que la formación de embriones adventicios a partir del embrión zigótico original es un fenómeno frecuente en los genotipos no apomícticos de cítricos y que el desarrollo de estos embriones está generalmente inhibido por el embrión principal. Se discuten las implicaciones que tiene este mecanismo en los programas de mejora genética y además se describe por primera vez la ocurrencia de este mecanismo en la biología reproductiva de los genotipos no apomícticos de cítricos. 6.Establecer un perfil molecular con marcadores microsatélites de 15 nuevos híbridos triploides de mandarino presentados por el Instituto Valenciano de Investigaciones Agrarias en el Registro de Variedades Protegidas de la Oficina Española de Variedades Vegetales. El análisis genético se realizó con un secuenciador automático mediante electroforesis capilar que permite calcular para marcadores heterocigóticos la dosis alélica de cada híbrido triploide lo que genera una diferenciación varietal mucho más potente.

La estructura de las variedades de mandarino cultivadas en España presenta importantes problemas. En la primera parte de la campaña desde mediados de septiembre a finales de Enero, existen diversas variedades de clementina de elevada calidad que permiten un abastecimiento continuo de los mercados internacionales en este periodo, incluso llegando a la saturación de los mismos por un exceso de producción. Las clementinas tienen polen y óvulos viables, pero son sexualmente autoincompatibles, por lo que no se polinizan entre ellas y producen frutos sin semillas. Los mercados internacionales demandan cantidades importantes de mandarinas tardías de calidad más allá del periodo de producción de las clementinas. Para cubrir esta demanda se introdujeron variedades de mandarinos híbridos como Fortune, Nova, Ellendale, Ortanique y más recientemente Moncada y Nadorcott. Estas variedades tienen polen y óvulos viables, pero como en el caso de las clementinas son autoincompatibles y producen frutos sin semillas.

En el IVIA se está realizando un amplio programa de mejora genética de mandarinos que tiene como objetivo la obtención de nuevas variedades de mandarino tardías y que no produzcan semillas, con la finalidad de resolver el problema de la inadecuada estructura de variedades de esta especie. El programa se basa en la obtención de híbridos triploides, para lo que se usan diversas técnicas biotecnológicas de cultivo de tejidos y citometría de flujo. Se han obtenido más de 9.000 híbridos triploides que están en distintas fases de evaluación.

La estructura de las variedades de mandarino cultivadas en España presenta importantes problemas (Navarro, 2005). En la primera parte de la campaña desde mediados de Septiembre a finales de Enero, existen diversas variedades de clementina de elevada calidad que permiten un abastecimiento continuo de los mercados internacionales en este periodo. Las clementinas tienen polen y óvulos viables, pero son sexualmente autoincompatibles, por lo que no se polinizan a sí mismas ni entre ellas y por tanto producen frutos sin semillas. Los mercados internacionales demandaban cantidades importantes de mandarinas tardías de calidad más allá del periodo de producción de las clementinas. Para cubrir esta demanda se introdujeron variedades de mandarinos híbridos como Fortuna, Nova, Ellendale u Ortanique. Estas variedades tienen polen y óvulos viables, pero como en el caso de las clementinas son autoincompatibles y producen frutos sin semillas. Estas variedades tardías, además de problemas de calidad, tienen el gran inconveniente de que su polen puede fertilizar a las clementinas y a su vez ser fertilizadas por el polen de estas, lo que provoca la aparición de semillas en ambos grupos de variedades. Esto produce un importante problema comercial, ya que los consumidores no aceptan las mandarinas con semillas. Las medidas para disminuir la formación de semillas implican la separación de las colmenas de abejas de las parcelas de producción de híbridos, lo que provoca conflictos con el sector de la apicultura y no resuelve el problema. Además, recientemente han surgido problemas importantes debidos a los daños provocados por la Alternaria alternata en Fortune, que está provocando el arranque o sobreinjerto de esta variedad. Debido a esta situación el problema de la inadecuada estructura de variedades de los mandarinos sigue sin resolverse.

Citrus are subject to many biotic and abiotic stresses and markets are continuously requiring fruits of higher quality. These pose important problems in most citrus growing areas that can only be solved with the establishment of citrus improvement programs to recover new and healthy genotypes to be used as rootstocks and varieties. Success of traditional citrus breeding strategies is limited by the peculiar genetic and reproductive characteristics of citrus. Biotechnology offers different approaches that can overcome many of these limitations. In this paper the following technologies used in Spain are reviewed: a) Shoot tip grafting in vitro. It is used worldwide to recover plants free of all known citrus pathogens, and already hundreds of millions of trees originated in this technique have been planted in the field; b) Embryo rescue. It is used in large programs to recover triploid seedless hybrid varieties from aborted seeds from 2n x 4n and 2n x 2n crosses; c) Protoplast fusion. It allows the production of allotetraploid hybrids among sexually compatible and incompatible parents to be used as rootstocks or as parentals for interploid breeding. Fusions between diploid and haploid cell lines are also used to produce triploid hybrids. d) Genetic transformation. Efficient protocols for transformation of several genotypes have been developed and introduction in citrus of genes of potential agronomical interest has been accomplished.