Intergeneric Combinations Research Articles

Inter-specific and Inter-generic Hybridization Compatibility of Eriobotrya Species (Loquat) and Related Genera

We studied the cross-compatibility among 91 inter-specific combinations and 21 inter-generic combinations in 7 Eriobotrya plants and 2 related genera (Raphiolepis indica Lindl. and Photinia serrulata Lindl.) using emasculation, bagging, and artificial pollination. Our results showed that 28 of the 91 inter-specific combinations set no fruit, which means nearly 30% of the combinations were incompatible. In the remaining 63 combinations, most showed partial cross-compatibility, and a few showed complete cross compatibility. Eriobotrya plants were incompatible with plants from their related genera (R. indica Lindl. and P. serrulata Lindl.). Backcrossing produced 5 compatible combinations, which could set fruits and produce F1 progeny but only after embryo rescue. Fruit setting ratios varied among various species used as male or female parents. E. prinoides Rehd. & Wils., common loquat (E. japonica) and Eriobotrya × daduheensis, used as female parents resulted in an average fruit-setting ratio of 36.2%–58.2%. E. deflexa Nakai and its two forms, and E. elliptica Lindl. as female parents resulted in 2.9%–16.3% average fruit-setting ratio; however, the fruit set ratio was higher (22.4%–43.1%) if they were used as male parents. Failure of E. deflexa f. koshunensis Nakai × E. prinoides Rehd. & Wils. hybrids to set fruit could be attributed to sporophytic incompatibility.

Localization and overcoming of hybridization barriers in Delosperma and Lampranthus (Aizoaceae)

The large succulent plant family Aizoaceae offers an enormous diversity of growth forms and flower pigmentations as well as prevalent tolerance to drought. Although many family members have potential for a wider use as ornamental plants, directed breeding of midday flowers is still in its infancy and only few cultivars have been launched onto the market so far. Most probably, this fact can be ascribed to the presence of cross-incompatibilities. The present study aimed at localization of intra- and intergeneric hybridization barriers between different genotypes of the genera Delosperma and Lampranthus by pollen germination tests, fluorescence and DIC microscopy, in vitro culture of seeds and ovules, and AFLP analysis. Aside from distinct self-incompatibility, the analyses revealed that pollen tube growth towards the ovules was not inhibited, even after intergeneric pollinations. Cross-combinations within Delosperma seemed to be compatible to a high extent, while hybridizations of the investigated Lampranthus genotypes and intergeneric combinations were largely unsuccessful. Investigations of embryo sacs following wide crosses using ovule clearing and differential interference contrast microscopy revealed fertilization failures as well as abnormal early embryogenesis. Seedlings obtained after wide crossings, mostly exhibited partial or complete loss of chlorophyll causing frequent hybrid breakdown. We therefore conclude that hybridization barriers between the investigated midday flowers occur both pre- and postzygotically.

DNA sequence analysis confirms the identity of the intergeneric hybrid Argyrotegium mackayi × Leucogenes leontopodium (Asteraceae: Gnaphalieae)

DNA sequencing of the nuclear internal transcribed spacer (ITS) and plastid trnK intron confirms that wild intergeneric hybrids between Argyrotegium mackayi and Leucogenes leontopodium occur in the Tararua Range of the North Island of New Zealand. Both parent species belong to the tribe Gnaphalieae (Asteraceae), already known in New Zealand for numerous intergeneric hybrid combinations. However, an Argyrotegium × Leucogenes hybrid is an especially wide cross within this group. In keeping with the presumed breeding biology of the two parent species, plastid DNA sequences indicate A. mackayi as the maternal parent of the sequenced sample.

CHROMOSOME INSTABILITY IN 'CARRIZO' CITRANGE + CITRUS MACROPHYLLA SOMATIC HYBRIDS

Somatic hybridization by protoplast fusion is a very useful technique for citrus rootstock breeding. Indeed, 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 this work, protoplasts were isolated from callus of Citrus macrophylla and from leaves of 'Carrizo' citrange (C. sinensis × P. trifoliata). Chemical, electro-chemical and electric fusions were performed and the ploidy of 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 and SNP markers, distributed in the nine chromosomes of citrus. Cytoplasmic genomes were characterized with chloroplastic and mitochondrial markers. Mitochondrial genome was inherited from C. macrophylla for all plants while segregation was observed for the 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 P. trifoliata alleles were mostly lost in 5x and 7x plants. The results indicate chromosome instability in this complex intergeneric combination with apparent non random loss of some chromosome fragments. Two allotetraploid somatic hybrids have been selected for evaluation as potential rootstocks. (Resume d'auteur)

Somatic hybridization for citrus rootstock breeding: an effective tool to solve some important issues of the Mediterranean citrus industry

The prevalence of sour orange rootstock in the southern and eastern part of the Mediterranean Basin is presently threatened by the spread of Citrus Tristeza Virus (CTV) and its main vector Toxoptera citricida, combined with abiotic constraints such as drought, salinity and alkalinity. The search for alternative CTV-resistant rootstocks that also withstand the other constraints is now considered an urgent priority for a sustainable citrus industry in the area. Complementary progenitors can be found in citrus germplasm to combine the desired traits, particularly between Poncirus and Citrus genera. The production of somatic hybrids allows cumulating all dominant traits irrespective of their heterozygosity level, and would appear to be an effective way to solve the rootstock challenge facing the Mediterranean citrus industry. This paper presents the results obtained during a regional collaborative effort between five countries, to develop new rootstocks by somatic hybridization. New embryogenic callus lines to be used for somatic hybridization have been created. Protoplast fusions have been performed at CIRAD and IVIA laboratories, focusing on intergeneric combinations. Analysis of ploidy level by flow cytometry and molecular markers confirmed the acquisition of new interesting tetraploid somatic hybrids for six combinations. Diploid cybrids with intergeneric (Citrus×Poncirus) nucleus and C. reticulata or C. aurantifolia mitochondria were also identified for four combinations. The agronomical performance of a pre-existing somatic hybrid between Poncirus trifoliata and Citrus reticulata was validated in calcareous soils in Morocco. Somatic hybridization is now integrated into the breeding programs of the five Mediterranean countries.

Variability of Seed Traits in Interspecific and Intergeneric Combinations between Different Genotypes of Cactaceae

Based on aesthetic consideration of cacti plants and germination capacity of the pollen grains (more than 30%), 19 genotypes of cacti (including Aylostera narvaecensis, A. buiningiana, Rebutia kupperiana var. spiniflorum, R. donaldiana) have been used in cyclic cross-pollination pattern. Fruits and seeds obtained from 24 interspecific and intergeneric combinations were analyzed by studying their main traits. The highest weight was registered for fruit belonging to R. pseudodeminuta var. schumaniana x R. senilis (23.3 mg) and A. buiningiana x A. vallegardensis (20.4 mg). Coefficient of variability for fruit weight had large amplitude, between 12.3% (A. spinosissima x A. albiflora) and 98.4% (A. favistyla x A. archibuinigiana). The fruits belonging to A. muscula x A. vallegardensis were registered with the highest value of number of seeds/fruit (93.2). In the cross between genera, the greatest value of fruits weights mean was registered on R. senilis x A. archibuinigiana (29.2 mg) and R. cajasensis x A. muscula (23.1 mg). Peculiarities of the fruits and seeds resulted from intergeneric combinations presented closer connections than interspecific ones. The phenotypic traits of fruits and seeds did not influence the seeds germination, but germination was clearly influenced by compatibility between genitors.

Intergeneric hybridization between Hordeum and Secale II. Analysis of meiosis in hybrids

Forty-one (41) intergeneric cross combinations between Hordeum and Secale were analysed for meiotic MI chromosome pairing. Hordeum taxa used in these hybrids included diploid, tetraploid, and hexaploid cytotypes. Differences between the sizes of the chromosomes of Hordeum and Secale allowed distinction between auto- and allosyndetic pairing. Allosyndesis was very rare, indicating lack of homology between any of the genomes of Hordeum (H, I, “X”, “Y”) and the R-genome of Secale. Autosyndetic pairing among Hordeum chromosomes occurred in the hybrids involving polyploid species of Hordeurn. The segmental alloploid nature of H. brachyantherum, H. jubatum, H. lechleri, H. arizonicum, H. procerum, H. capense. and H. secalinum was confirmed, but it is suggested that the annual H. depressum is an autotetrdploid. In general the level of autosyndetic pairing among the Hordeum chromosomes was higher in the intergeneric combinations with Secale than in corresponding Hordeum polyhaploids. It is suggested that the genome of Secale exerts influence during synaptonemal complex formation, allowing a higher level of crossing-over between the chromosomes of Hordeum.

2004 SIVB Congress Symposium Proceedings “Thinking Outside the Cell”: Applications of Somatic Hybridization and Cybridization in Crop Improvement, with Citrus as a Model

Although somatic hybridization techniques are being ignored by variety improvement programs for most commodities, their contribution to citrus variety improvement continnes to expland and with increasing complexity. Citrus is, one of the few commodities where somatic hybridization is reaching its predicted potential, as somatic hybrids are now possible from most desirable parental combinations. Somatic hybrid citrus plants have been produced from more than 250 parental combinations, including more than 130 at the CREC. The CREC hybrids include 34 from sexually compatible intergeneric combinations, 16 from sexually incompatible combinations, and 81 interspecific combinations. The objective of this report is to demonstrate the impact of somatic hybridization on citrus improvement programs, and to discuss its potential with other commodities. For citrus scion improvement, several applications are aimed at the development of improved seedless fresh fruit varieties, and these include symmetric somatic hybridization, haploid+diploid fusion, targeted cybridization to transfer cytoplasmic male sterility (mtCMS) from Satsuma mandarin, and triploidy via interploid crosses using somatic hybrid allotetrapoid breeding parents. For rootstock improvement symmetric somatic hybridization provides an opportunity to hybridize complementary rootstocks without breaking up successful gene combinations. Rootstock somatic hybridization is providing opportunities for improving disease and inseet resistance, soil adaptation, and tree size control. Wide somatic hybridization provides an opportunity for gene transfer from related species, including some that are sexually incompatible. Extensive field research on citrus somatic hybrid rootstocks combined with emerging molecular analyses of citrus has allowed for the development of additional strategies for rootstock improvement. These include rootstock breeding and selection, at the tetraploid level using somatic hybrid parents, and the resynthesis of important rootstocks at the tetraploid level via fusion of selected superior parents. Ongoing examples of each strategy will be provided, along with ideas for extending the technology to other commodities.

Genetic and epigenetic reprogramming of the wheat genome upon allopolyploidization

In the past few years we have analysed alterations in genome structure and expression that occur in wheat upon allopolyploidization. Our major findings in natural and synthetic allopolyploid wheat are reviewed here. It was found that allopolyploidization brings about rapid genome evolution through the instantaneous generation of a variety of cardinal genetic and epigenetic alterations comprising: (1) non-random elimination of coding and non-coding DNA sequences, (2) epigenetic changes such as DNA methylation of coding and non-coding DNA leading, among others, to gene silencing, and (3) activation of retroelements, which in turn alters the expression of adjacent genes. These changes were reproducible, occurring in the F1 hybrids or in the first generation(s) of a series of nascent allopolyploids corresponding to various interspecific and intergeneric combinations. Moreover, these changes were similar to those that occurred twice in nature: first, at the transition from diploid to tetraploid wheat (-0.5 Mya) and, second, at the transition from tetraploid to hexaploid wheat (-9500 years ago). Elimination of non-coding sequences augments the differentiation of homoeologous chromosomes at the polyploid level, thus increasing the physical divergence between homoeologues and contributing to the diploid-like meiotic behaviour of polyploid wheat. Transcriptional and post-transcriptional alterations of gene activity, including transcriptional activation of retro-elements, led to novel expression patterns. These phenomena emphasize the plasticity of the genome with regard to both structure and gene expression. This plasticity in turn might improve the adaptability of the newly formed allopolyploids and facilitate their rapid and successful establishment in nature.

Self-fertile cybrids Nicotiana tabacum (+ Hyoscyamus aureus) with a nucleo-plastome incompatibility.

Cytoplasmic hybrids (cybrids) in a novel inter-generic combination, Nicotiana tabacum (+ Hyoscyamus aureus), were generated by fusion of protoplasts from a plastome tobacco albino mutant (line R100a1) and gamma-irradiated green protoplasts of H. aureus. Cybrids possessed a plastome of H. aureus and a rearranged mitochondrial DNA. The cybrids displayed a syndrome of nucleo-plastome incompatibility expressed as a partial chlorophyll-deficiency of cotyledonary and true leaves at the early stage of vegetative development of plants grown from seeds in soil. During later development, the plants restored a normal green coloration. This character is phenotypically indistinguishable from the same syndrome in previously generated cybrids N. tabacum (+ H. nigrum). In contrast to the cybrids N. tabacum (+ H. nigrum), cybrids N. tabacum (+ H. aureus) were self-fertile, and did not manifest other features that were interpreted as nucleo-mitochondrial incompatibilities in N. tabacum (+ H. nigrum) plants. Therefore, the cybrids N. tabacum (+ H. aureus) present a self-propagating system of Nicotiana (+ Hyoscyamus) nucleo-plastome incompatibility in its pure form.

ROOTSTOCK BREEDING BY SOMATIC HYBRIDISATION FOR THE MEDITERRANEAN CITRUS INDUSTRY

The predominance of sour orange rootstock for citrus in the Mediterranean area is currently threatened by the spread of citrus tristeza virus (CTV). As a result, the search for alternative rootstocks resistant to CTV and able to withstand other constraints, such as drought, alkalinity, salinity, Phytophthora and nematode problems, is now considered an urgent priority. Progress in genetic improvement of citrus rootstock by conventional sexual breeding is difficult, mainly because of the reproductive biology and heterozygosity of genitors. These induce important segregations of characters in progeny and a low probability of obtaining recombinant hybrids that combine all the desirable genes and traits of the two parents. In this context, the creation of somatic hybrids is very promising. Indeed, it allows for the accumulation of all the dominant genes of both parents for tolerance to biotic and abiotic factors, irrespective of their level of heterozygosity. This strategy has now been mastered by CIRAD in France with a view to launching a Mediterranean citrus rootstock challenge. Protoplast electrofusions were used for 11 interspecific and intergeneric combinations. Regenerated embryos and plants are studied by flow cytometry and molecular markers in order to select allotetraploid hybrids. 'Me somatic hybrids then undergo an accelerated propagation step, which uses softwood cuttings for further multilocal evaluation of tolerance to biotic and abiotic factors and agronomic rootstock trials. (Resume d'auteur)

A high frequency of intergenomic mitochondrial recombination and an overall biased segregation of B. campestris or recombined B. campestris mitochondria were found in somatic hybrids made within Brassicaceae

Mitochondrial segregation and rearrangements were studied in regenerated somatic hybrids from seven different species combinations produced using reproducible and uniform methods. The interspecific hybridizations were made between closely or more distantly related species within the Brassicaceae and were exemplified by three intrageneric, two intergeneric and two intertribal species combinations. The intrageneric combinations were represented by Brassica campestris (+) B. oleracea, B. napus (+) B. nigra and B. napus (+) B. juncea (tournefortii) hybrids, the intergeneric combinations by B. napus (+) Raphanus sativus and B. napus (+) Eruca sativa hybrids, and the intertribal combinations by B. napus (+) Thlaspi perfoliatum and B. napus (+) Arabidopsis thaliana hybrids. In each species combination, one of the two mitochondrial genotypes was B. campestris since the B. napus cultivar used in the fusions contained this cytoplasm. Mitochondrial DNA (mtDNA) analyses were performed using DNA hybridization with nine different mitochondrial genes as probes. Among the various species combinations, 43-95% of the hybrids demonstrated mtDNA rearrangements. All examined B. campestris mtDNA regions could undergo intergenomic recombination since hybrid-specific fragments were found for all of the mtDNA probes analysed. Furthermore, hybrids with identical hybrid-specific fragments were found for all probes except cox II and rrn18/rrn5, supporting the suggestion that intergenomic recombination can involve specific sequences. A strong bias of hybrids having new atp A-or atp9-associated fragments observed in the intra- and intergeneric combinations could imply that these regions contain sequences that have a high reiteration number, which gives them a higher probability of recombining. A biased segregation of B. campestris-or B. campestris-like mitochondria was found in all combinations. A different degree of phylogenetic relatedness between the fusion partners did not have a significant influence on mitochondrial segregation in the hybrids in this study.

A revision of Chorizema (Leguminosae: Mirbelieae)

A taxonomic revision is presented for the genus Chorizema Labill. The circumscription is enlarged slightly to include three species formerly in Oxylobium Andrews. Of the 26 taxa recognised, three species and one subspecies are described as new: C. circinale, C. retrorsum, C. ulotropis, and C. aciculare subsp. loxum respectively. Three new intergeneric combinations are made: C. carinatum, C. obtusifolium, C. spathulatum; one new infrageneric combination is made: C. racemosum; and three names are reinstated: C. humile, C. nanum, and C. rhynchotropis. A key, descriptions, illustrations and distribution maps and notes are provided for all taxa. Lectotypes are chosen where necessary. Relationships between species are analysed cladistically and, on this basis, a new infrageneric classification including three sections, of which two are new (Parviflores and Hirtistylis), is constructed.

Highly asymmetric intergeneric nuclear hybrids between Nicotiana and Petunia: evidence for recombinogenic and translocation events in somatic hybrid plants after “gamma”-fusion

Extremely asymmetric nuclear hybrids have been obtained via protoplast fusion in an intergeneric combination. Irradiated (cobalt(60),100 krad) kanamycinresistant Petunia hybrida mesophyll protoplasts were chemically fused with wild-type mesophyll protoplasts of Nicotiana plumbaginifolia. Eighty-six hybrid colonies were selected on kanamycin-containing medium, and twenty-four of these could be induced to regenerate numerous shoots. Cytological analysis of the regenerants showed the presence of a few chromosome fragments in some lines, and even a metacentric chromosome in yet another line. Besides additional chromosome fragments some lines only possessed typical Nicotiana chromosomes, and this at the diploid (2n = 2X = 20) as well as the tetraploid (2n = 2X = 40) level. Biochemical analysis showed that all regenerants had neomycin phosphotransferase activity (NPTII), which suggests that intergenomic recombination and or translocation events took place at least in those lines where no additional chromosome fragments could be detected. The presence of the NPTII gene was shown by Southern hybridization. All regenerants tested were fertile, and the segregation ratios for the kanamycin gene (for self and backcross pollinations to the recipient partner) for some of the regenerants correspond with Mendelian rules for a monogenic dominant marker. Most of the regenerants showed abnormal segregation ratios; in this case, no correlation could be made between segregation ratio and chromosome composition.Our results demonstrate the existence of intergenomic recombination and translocations evens in nuclear somatic hybrid plants obtained via "gamma"-fusion.

Intergeneric hybridization between Hordeum and Secale (Poaceae). I. Crosses and development of hybrids

A total of 241 intergeneric combinations between Hordeum and Secale were attempted. It was only possible to obtain hybrid offspring from combinations with Hordeum as the female parent. By the aid of embryo culture technique 81 intergeneric combinations were synthesized, 72 of them not reported earlier. Generally hybridization with the polyploid species of Hordeum was most successful. Morphologically the hybrids were intermediate to the parents in most characters, though an obvious effect of genome dosage could be observed. Only few deviations from the expected chromosome numbers of the hybrids were found. All hybrid plants showed complete male sterility.

Intergeneric hybridization in Sparidae: 1. Sparus aurata ♀ × Diplodus puntazzo ♂ and Sparus aurata ♀ × Diplodus vulgaris ♂

Eggs of gilthead sea bream ( Sparus aurata) were inseminated by the sperm of sharp-snout sea bream ( Diplodus puntazzo) and common two-banded sea bream ( Diplodus vulgaris). Under controlled conditions, at 19.5°C, embryogenesis and yolk sac resorption in both intergeneric combinations took less time than embryogenesis and yolk sac resorption in Sparus aurata. Survival and growth of larvae of Sparus aurata, Sparus aurata ♀ × Diplodus puntazzo ♂ and Sparus aurata ♀ × Diplodus vulgaris ♂ were established during yolk sac resorption. During the active feeding period, between days 6 and 30 after hatching, neither growth nor survival differed significantly among the studied species and crosses. Sparus aurata ♀ × Diplodus puntazzo ♂ larvae grew slightly but not significantly faster.

Intergeneric hybridization between Psathyrostachys and Hordeum

A total of 38 intergeneric combinations with Hordeum and Psathyrostachys was attempted. Generally the seed set was high, and 23 new intergeneric hybrid combinations were synthesized by the aid of embryo culture technique. All combinations showed complete sterility. The meiotic pairing behaviour in 8 combinations is reported. On the basis of these data it is clear that the N genome in Psathyrostachys is distinct and shows no genomic relationship, neither with the “X” and I nor with the H genomes in Hordeum.