Studies on Taxonomy and Evolution of Citrus and Its Related Genera by Isozyme Analysis

The leaf or bark tissue extracts of 108 biotypes of Citrus and its five related genera were analyzed by polyacrylamide gel electrophoresis for isozymes of peroxidase, glutamate oxaloacetate transaminase, phosphoglucose isomerase, phosphoglucose mutase, superoxide dismutase, tetrazolium oxidase, NADP+- dependent malate dehydrogenase and esterase. The inter-or intra-generic differences of isozymograms were compared. Dissimilarities of the isozyme patterns among the samples were calculated by computer based on the UPGMA method to demonstrate the phylogenetic relationship of the biotypes. There are remarkable isozymogramatic differences among the six genera. Generally speaking, each genus possesses its own unique bands. Based on the cluster analysis, the total Citrus biotypes are classified into seven groups, i.e. Honghe papeda, Mauritius papeda, Pummelo, Ichang papeda, Citron, Lemon-lime and Mandarin-orange. Ichang papeda is suggested to be the third subgenus of the genus Citrus. The Fuming trifoliata orange is accepted as a new species of Poncirus-P. polyandra S. Q. Ding. It might be the evolutionary bridge between Poncirus and Citrus, and one of the “missing links” as suggested by W. T. Swingle. Microacrumen (small- fruited mandarin) is the primitive form of the mandarins. It seems better to consid er“ Horse-nose mandarin ”or “Xipigoushigan mandarin” as the typic mandarin originated in China. Euacrumen (large-fruited mandarin) might have arised through two ways. One is hybridization of Microacrumen and C. sinensis, and the other is just the variation of Microacrumen. Ancestors of some biotypes are inferred based on isozyme data. Fortunella obovata Tan. may be a hybrid of Fortunella and Citrus. C. unshiu Marc. arose from the nucellar mutation of“Huangyanbendiguangju mandarin”( C. nobilis Lour.) in Huangyan of Zhejiang Province. C. junos Sieb. came from hybridization of Ichang papeda and mandarin. C. grandis Osbeck and mandarin may have been involved in the origin of C. aurantium L.. C. limonia Osbeck may be a hybrid between C. medica L. and mandarin. The evolutionary trend of citrus fruit trees is proposed, anda tentative phylogenetic diagram is drawn.     

柑桔矮化砧木的生理生化预选指标研究

以７４－１积、紫花宜昌橙、Ｒｕｓｋ枳橙、Ｔｒｏｙｅｒ枳橙、江南柑、９２号红桔和蟹橙为标准系列砧木,研究了它们的生理生化特征,从中筛选出了茎还原性糖含量、叶片还原性糖含量、叶片氨基酸含量、茎蛋白质含量、叶片过氧化物酶活性５个柑桔矮化预选指标和叶片可溶性糖含量、叶片蛋白质含量２个辅助预选指标。     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Production of additional allotetraploid somatic hybrids combining mandarings and sweet orange with pre-selected pummelos as potential candidates to replace sour orange rootstock

Summary Sour orange (Citrus aurantium L.) rootstock has historically been a widely utilized eitrus rootstock throughout the world due to its wide soil adaptability and superior horticultural performance. However, quick-decline isolates of citrus tristeza virus (CTV) have demolished entire industries of sour orange rootstock in some countries, including Brazil and Venezuela. CTV is presently destroying millions of trees of sour orange rootstock in Florida and threatens the citrus industries of Texas and Mexico, where sour orange is the predominant rootstock. Efforts to replace sour orange rootstock are combining traditional breeding and biotechnology approaches, including somatic hybridization and transformation. Molecular techniques have confirmed that sour orange is probably a hybrid of mandarin and pummelo. A major focus of our program continues to be the somatic hybridization of superior mandarins with pre-selected pummelo parents. Here, we report the regeneration of allotetraploid somatic hybrid plants from seven new mandarin+pummelo combinations and one new sweet orange+pummelo combination. All new somatic hybrids were confirmed by leaf morphology, ploidy analysis via flow cytometry, and random amplified polymorphic DNA analysis to show nuclear contributions from both parents in corresponding hybrids. These new somatic hybrids are being propagated by tissue culture and/or rooted cuttings for further evaluation of disease resistance and horticultural performance in field trials.     

应用同工酶进行宽皮柑桔分类及其进化研究

利用聚丙烯酰胺凝胶电泳法,分析了50个宽皮柑桔生物型及5个甜橙品种的过氧化物酶、谷氨酸草酰乙酸转氨酶、四唑氧化酶、6-磷酸葡萄糖异构酶、6-磷酸葡萄糖变位酶、超氧化物歧化酶、苹果酸酶以及酯酶等8种酶系统同工酶。运用数量分类学的原理及方法,对同工酶资料进行了聚类分析。结果表明,宽皮柑桔的起源是多元的,小果类桔属于宽皮柑桔的原始类型,在进化过程中起着较为重要的作用。中国原产宽皮柑桔的代表种以道县野桔、马鼻桔或狗屎柑为佳。柑的来源有两条途径,即桔橙杂种和桔的单元演化;蕉柑是栟柑与甜橙的杂种;温州蜜柑来源于黄岩本地广桔的实生变异。     

Study on Peroxidase Isozymes in Citrus and Fortunella

The acidic peroxidase isozymes in petals of 106 Citrus and Fortunella species and cultivars were studied using polyacrylamide gel electrophoresis. The results (Fig. 1; Tables 1 and 2) suggest that these isozymes be of the monomeric nature coded by a single locus. Five alleles were identified and designated as A, B, C, D and F respectively. No genetic difference in peroxidase isozymes was detected among the mandarins orignated from China, Japan and Indian. Man Ju may be a hybrid between Ponkan and Ougan. Ponkan may have not been involved in the origin of Jiao Gan. Zheng Jiang Jin Ju should be an infraspecific hybrid of C. reticulata. C. sinensis has a close relationship with some Chinese mandarins. Changshou Kumquat and Calamondin are shown to be intergeneric hybrids between Citrus andFortunella.     

Chromosomal relationships among cultivars of Citrus reticulata Blanco, its hybrids and related species

Karyotypes of 93 individuals belonging to 18 accessions of mandarins, mandarin hybrids and two related species were analysed with the fluorochromes CMA and DAPI, to identify marker chromosomes. The karyotypes revealed highly differentiated banding patterns and could be classified in four groups (I–IV) according to the presence/absence of chromosomes with three bands (type A) or with two bands (one proximal and one terminal, type B, or both terminal, type C). The accessions of group I exhibited the simplest and homozygous karyotypes (lacking chromosome types A, B and C), represented by `Sunki' and `Cleopatra'. Group II (lacking chromosome types A and B) included three accessions of Mediterranean mandarins and `Cravo' mandarin, all of them with very similar and almost completely homozygous karyotypes. All other karyotypes of groups II and III (lacking chromosome type A) were heterozygous for one or more chromosome pairs and most of them seemed to be hybrid derivatives from non-mandarin accessions. Group IV (with chromosome types A and B) was represented only by two heterozygous hybrids (`Murcott' and `King'). The karyotype of most hybrids agrees with one of the possible combinations resulting from chromosome types segregation from their putative ancestor karyotypes, but at least `Orlando' seemed to be a more complex hybrid. Comparing with banding patterns of other Citrus species, those of group I and the Mediterranean mandarins (group II) are the best candidates to represent C. reticulata (sensu Swingle) as a true species.     

A Numerical Taxonomic Study of Citrus and Its Close Relatives

Fifty nine taxa of Citrus, Fortunella and Poncirus were studied by hierarchical agglomerative clustering analysis and quantas type 3 analysis using 86 morphological characters. Five affinity groups were obtained in Citrus. The first group includes C. medica, C. limon, C. limonia, C. aurantifolia and C. iambhiri; the second includes C. grandis, C. aurantium, C. sinensis and C. paradisi; the third is mandarin (C. reticulata); the forth is C. ichangensis; The last is C. hongheensis. Mandarin oranges show two subgroups: one comprise satsuma, King, Shagan, Tankan and Bendiguangju, and the other consists of the remaining typical mandrins. The two papeda oranges, honghe papeda and ichang papeda, could not be clustered into the same group, indicating that they might have evolved from two compeletly different ancestors. Pumelos(C. grandis) show some relationships to honghe papeda orange. C. limonia is assumed to be a hybrid between rough lemon and mandarin. The sweet orange(C. sinensis) is considered to be the offspring of pomelo and mandarin. Zhekiang jinju a small mandarin generally considered similar to calamondin in China, should be a true mandarin orange based on our results. Poncirus was well distinguished from the other two genera, Citrus and Fortunella, by the numerical methods used in this study. Our results show that citron (C. medica), mandarin (C. reticulata) and pumelo (C. grandis) are the original species of the subgenus Citrus, which was congruent with the re-sults obtained by Barrett and Soost (1976), Potvin (1983) and Handa (1985).     

Chromosomal markers distinguish hybrids and non-hybrid accessions of mandarin

Mandarin is the common name of a heterogeneous group of Citrus species with a large range of variation in morphological and molecular characters as well as in number of species. Aiming to identify chromosome markers and to clarify the relationship within this group, the karyotype of 13 mandarin accessions were analyzed using CMA/DAPI staining and in situ hybridization with 5S and 45S rDNA probes. The CMA band pattern together with the position of rDNA sites revealed that mandarins can be separated karyologically into three groups: a) C. sunki and C. reshni; b) the Mediterranean mandarin, C. deliciosa, and the closely related C. tangerina cv. Dancy and C. reticulata cv. Cravo; c) the remaining cultivars, which are cytologically heterozygous and most probably interspecific hybrids. The former two groups are assumed to be pure species together with C. medica and C. grandis. A chromosome marker for mandarin species was identified and the relationship among the pure species and some hybrids is discussed.     

Isozyme gene markers in Vicia faba L.

Summary This study was conducted to assess the genetic basis of the variability observed for the glutamate oxaloacetate transaminase (GOT), Superoxide dismutase (SOD), esterase (EST), and malate dehydrogenase (MDH) isozyme systems in different open-pollinated Vicia faba varieties. Individual plants showing contrasting zymogram patterns were simultaneously selfed and cross-combined. Crossing was unsuccessful in producing progeny, and only selfed progenies were suitable for genetical analysis of isozyme variability. Three zones of GOT activity were made visible. The isozyme of GOT-2 and GOT-3 zones were dimeric and under the control of three alleles at the Got-2 locus and two alleles at the Got-3 locus, respectively. The isozymes of the GOT-1 zone did not show any variability. Three zones of SOD isozyme activity were made visible. The isozymes occurring in the SOD-1 (chloroplastic isozyme form) and SOD-2 (cytosol isozyme form) zones were dimeric and under the control of two alleles at the Sod-1 and Sod-2 loci. The isozyme visualized in the SOD-3 zone (mitochondrial isozyme form) were tetrameric and under the control of two alleles at the Sod-3 locus. Apparently the isozymes made visible in the most anodal esterase zones EST-1, EST-2, and EST-3 were monomeric, and the occurrence of two alleles at each of two different loci explained the variability observed in the EST-2 and EST-3 zones. For MDH, only two five-banded zymogram pattern types were found, and every selfed progeny showed only one of the two zymogram type, indicating that each individual possessed fixed alleles at the loci controlling MDH isozyme. Got-2, Got-3, Sod-1, Sod-2, and Sod-3 appear to be five new isozyme gene markers that can be useful in Vicia faba breeding for linkage study, varietal fingerprinting, outcrossing rate estimate, and indirect selection for quantitative characters.     

The Distribution of Coumarins and Furanocoumarins in Citrus Species Closely Matches Citrus Phylogeny and Reflects the Organization of Biosynthetic Pathways

Citrus plants are able to produce defense compounds such as coumarins and furanocoumarins to cope with herbivorous insects and pathogens. In humans, these chemical compounds are strong photosensitizers and can interact with medications, leading to the “grapefruit juice effect”. Removing coumarins and furanocoumarins from food and cosmetics imply additional costs and might alter product quality. Thus, the selection of Citrus cultivars displaying low coumarin and furanocoumarin contents constitutes a valuable alternative. In this study, we performed ultra-performance liquid chromatography coupled with mass spectrometry analyses to determine the contents of these compounds within the peel and the pulp of 61 Citrus species representative of the genetic diversity all Citrus. Generally, Citrus peel contains larger diversity and higher concentrations of coumarin/furanocoumarin than the pulp of the same fruits. According to the chemotypes found in the peel, Citrus species can be separated into 4 groups that correspond to the 4 ancestral taxa (pummelos, mandarins, citrons and papedas) and extended with their respective secondary species descendants. Three of the 4 ancestral taxa (pummelos, citrons and papedas) synthesize high amounts of these compounds, whereas mandarins appear practically devoid of them. Additionally, all ancestral taxa and their hybrids are logically organized according to the coumarin and furanocoumarin pathways described in the literature. This organization allows hypotheses to be drawn regarding the biosynthetic origin of compounds for which the biogenesis remains unresolved. Determining coumarin and furanocoumarin contents is also helpful for hypothesizing the origin of Citrus species for which the phylogeny is presently not firmly established. Finally, this work also notes favorable hybridization schemes that will lead to low coumarin and furanocoumarin contents, and we propose to select mandarins and Ichang papeda as Citrus varieties for use in creating species devoid of these toxic compounds in future breeding programs.     

Characterization of zygotic and nucellar seedlings from sour orange-like citrus rootstock candidates using RAPD and EST-SSR markers

A series of crosses designed for introgression of mandarin (Citrus reticulata Blanco) and pummelo (C. maxima Merr.) germplasm, to develop an alternative rootstock to sour orange (C. aurantium L.), were carried out. It is necessary to identify those hybrids that yield nucellar seedlings for rootstock propagation. Rootstocks can be developed through traditional plant breeding methods; however, the ability to screen and select for economically important traits (such as production of true nucellar seedlings) in an efficient fashion is limited by the difficulties of screening techniques based on whole plant performance. To address these problems, we have used randomly amplified polymorphic DNA (RAPD) and fluorescently labeled expressed sequence tag simple sequence repeat (EST-SSR) molecular markers. A total of 204 individual seedlings obtained from 34 hybrid parental plants were successfully characterized using five RAPD primers. Ten hybrid parents and their progenies, found to be genetically similar among themselves, were selected for more scrutiny using eight EST-SSR primer pairs. The degrees of genetic similarity (nucellars) among progeny seedlings were determined and compared with that of their parents. The mean genetic similarity varied from 67–99% among the selected rootstock candidates screened. The genetic similarity relationship identified using RAPD and EST-SSR molecular markers was highly concordant (p = 0.001). Two elite rootstock candidates (B6R5T56; B6R11T129) that seem to be ideal for future mandarin and pummelo derived rootstock breeding programs have been identified. Our results indicate that either RAPD or EST-SSR analyses could be equally successful in identifying true nucellars among the progenies obtained from introgression crosses of mandarin and pummelo, thus improving the accuracy of early selection in a citrus rootstock breeding program.     

Production and molecular characterization of potential seedless cybrid plants between pollen sterile Satsuma mandarin and two seedy <Emphasis Type="Italic">Citrus</Emphasis> cultivars

Cytoplasmic male sterility (CMS) is known to be controlled by mitochondrial genome in higher plants including Satsuma mandarin (Citrus unshiu Marc.). Citrus symmetric fusion experiments often produce diploid cybrids possessing nuclear DNA from the mesophyll parent and mitochondrial DNA (mtDNA) from the embryogenic callus parent. Therefore, it is possible to transfer CMS from Satsuma mandarin as callus parent to seedy citrus cultivars as leaf one by somatic cybridization. Herein, symmetric fusion technique was adopted to create cybrids for potential seedlessness by transferring CMS from Citrus unshiu Marc. cv. Guoqing No. 1 (G1) to two traditional Chinese seedy citrus cultivars, ‘Shatian’ pummelo (C. grandis (L) Osbeck) and ‘Bingtang’ orange (C. sinensis (L) Osbeck). Flow cytometry analysis showed that 19 plants recovered from G1 + ‘Bingtang’ orange and 17 of 35 plants regenerated from G1 + ‘Shatian’ pummelo were diploid. The remaining plants from G1 + ‘Shatian’ pummelo were tetraploid. The diploid plants from the two combinations were confirmed as true cybrids by simple sequence repeat (SSR) and cleaved amplified polymorphic sequence (CAPS) analysis, with nuclear DNA from their corresponding leaf parent and mtDNA from their common suspension parent, G1 Satsuma mandarin. The remaining plants from G1 + ‘Shatian’ pummelo were identified as somatic hybrids with mtDNA from G1. The chloroplast simple sequence repeat (cp-SSR) analysis revealed somatic hybrid/cybrid plants from the two combinations in most cases possessed either of their parental chloroplast type, and two plants from G1 +‘Shatian’ pummelo and all embryoids analyzed from G1 + ‘Bingtang’ orange possessed chloroplast DNA (cpDNA) from both parents. These results demonstrated that we succeeded in introducing mtDNA from G1 Satsuma mandarin into the two target seedy citrus cultivars for potential seedlessness through symmetric fusion.     

Comparative genome-wide segregation analysis and map construction using a reciprocal cross design to facilitate citrus germplasm utilization

Citrus genetic resources are rich but underutilized in breeding because their complex reproductive biology and the scarceness of inheritance studies on agronomic traits. Here, we investigated the genomic distribution of segregation distortion regions, the inheritance of organelle DNA and colinearity between scion citrus linkage maps by using a reciprocal cross design. The parents were Fortune, a hybrid mandarin from C. clementina, and Chandler, a hybrid pummelo from C. grandis that largely differ in fruit size, taste and colour. The inheritance of organelle DNA was studied in 201 hybrids by using four organelle DNA markers, and the linkage maps were based on 174 of those hybrids. Around ten percent of the seedlings derived from the pummelo as female parent showed the same organelle markers as those of the mandarin, indicating a possible exception to their expected maternal inheritance in citrus. Most segregation distortion affects just the allele frequencies, generally representing differences in pollen fertilization success, as a likely consequence of the presence of gametal factors affecting the functionality of gametes and pollen-pistil interactions. The large extension of colinearity found when comparing the C. grandis and C. clementina linkage maps to those previously reported for rootstock species (C. aurantium and P. trifoliata), will be helpful to infer the position of orthologous genes and QTLs in citrus species and for a more useful genetic characterization of citrus germplasm collections.     

Soluble glutamic oxalacetic transaminase (GOT) polymorphism in cattle

Zymograms performed on extracts of bovine white cells revealed two phenotypes in the soluble (anodal) isozyme of glutamic oxalacetic transaminase (GOT). Genetic data are consistent with the hypothesis that two co-dominant, autosomal alleles, GOT-1-A and GOT-1-B , give rise to the phenotypes GOT-1-AA, GOT-1-AB and GOT-1-BB. The phenotype BB has not been encountered. The allele GOT-1-B has been observed in one of the five breeds surveyed, the Central and Upper Belgium breed where it has a frequency of about 0,008.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Inheritance and linkage relationships of glutamate oxaloacetate transaminase isoenzymes in apple

Summary Independent dimeric genes GOT-2 and GOT-4 determining activity for glutamate oxaloacetate transaminase (E.C.2.6.1.1; GOT) in zones GOT-II and GOT-IV respectively were identified. Three alleles were found for GOT-2 and two for GOT-4, including a null allele for GOT-2 which produced detectable heterodimeric bands but not homodimeric bands. Linkage studies with leucine aminopeptidase (E.C.3.4.11.1; LAP) genes suggested linkage of GOT-2 with LAP-2 (r=0.13±0.23) and GOT-4 with LAP-1 (r=0.10±0.40).The results reported in this paper are part of a London University PhD thesis by the first author     

Genetic control of enzyme polymorphisms in the California vole,Microtus californicus

Inheritance of 15 polymorphic isozymes was investigated in captive Microtus californicus. Eleven of the isozymes show patterns consistent with a Mendelian model of inheritance: glycerol-3-phosphate dehydrogenase (GPD), lactate dehydrogenases A and B (LDH-A and LDH-B), malic enzyme 2 (ME-2), isocitrate dehydrogenase 1 (ICD-1), phosphogluconate dehydrogenase (PGD), glutamate-oxaloacetate transaminase 1 (GOT-1) phosphoglucomutase 2 (PGM-2), leucine aminopeptidase (LAP), glucosephosphate isomerase (GPI), and esterase 2 (ES-2, from kidney). four of the isozymes show patterns that cannot be interpreted by a simple genetic model: esterases 1 and 4 (ES-1, ES-4, from hemolysate), esterase 3 (ES-3, from plasma), and protein 1 (PT-1). The following pairs of loci are assorting independently: LAP and PGD, LAP and PGM-2, GOT-1 and PGD, GOT-1 and GPD, LAP and GPD, GPD and PGD, GPI and PGD. Data from one test cross mating indicate that GPD and PGM-2 are loosely linked with recombination about 30%. Additional data are needed to confirm this relationship.This study was conducted while the first author was the recipient of an NIH Traineeship. The Departments of Genetics and Zoology provided financial support for the maintenance of the animal colony. This work was supported in part by an NIH-Biomed grant (3-S05-RR-07006-08S1) to W. Z. Lidicker.     

柑橘体细胞杂种有性后代的创造及杂种鉴定

110 plantlets were obtained from the cross between citrus allotetraploid somatic hybrid [Hamlin sweet orange (Citrus sinensis Osback) + Rough Lemon (C. jambhiri Luss)] (as the pollen parent) and mono-embryonic diploid type Iben No.4 [Huanongbendizao tangerine (C. reticulata Blanco) x Ichang papeda (C. ichangensis Swingle)] by the means of young embryo culture in vitro, and there were 93 triploids and 17 diploids through chromosome counting and the ploidy analyser identification. RAPD analyses showed that all sexual progenies were hybrid.     

Implementation of extensive citrus triploid breeding programs based on 4x?×?2x sexual hybridisations

Seedlessness is one of the most important characteristics for mandarins for the fresh-fruit market, and mandarin triploid hybrids have this trait. Triploid citrus plants can be recovered by 4x?×?2x hybridisations using non-apomictic genotypes as female parents. In this study, we characterise the type of seeds obtained in 4x?×?2x hybridisations and the ploidy level of plants recovered from each type of seed. The majority of the plants recover were triploid (98.3?%), but a few diploid, tetraploid and pentaploid plants were also produced and their genetic origin was analysed by simple sequence repeat (SSR) markers. We also analysed the influence of parents and environmental conditions on the efficiency of recovery triploid hybrids. In this work, we present an effective methodology to recover triploid hybrids from 4x?×?2x hybridisations based on in vitro embryo rescue and determination of ploidy level by flow cytometry that allow us to recover more than 4,400 triploid hybrids from more than 60 parental combinations.