In vitro anther culture of sweet orange (Citrus sinensis L. Osbeck) genotypes and of a C. clementina × C. sinensis ‘Hamlin’ hybrid

Citrus, and particularly sweet oranges, are very recalcitrant to anther culture. In this paper it was evaluated for the first time the response of 27 genotypes of Citrus sinensis and of one hybrid C. clementina × C. sinensis, to in vitro anther culture. Ten genotypes of sweet oranges showed embryogenic callus induction, mostly blood sweet oranges genotypes, such as Tarocco, Moro and Sanguinelli. In vitro microspore developmental switches from the gamethophytic to the sporophytic pathway were shown by DAPI staining in microspores of these responsive genotypes, after 10 months in culture. However, microsatellite marker analyses showed that these calli were heterozygous. The flow-cytometric analysis of these embryogenic calli showed the presence of two peaks, corresponding to haploid (n) and diploid (2n) genotypes. Differently, anther cultures of the hybrid C. clementina × C. sinensis produced tri-haploid (3n) embryogenic calli and the embryos obtained were homozygous when analyzed by molecular markers (sample sequence repeats), confirming the more responsive characteristic of clementine to microspore embryogenesis through anther culture.     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

Gynogenetic haploids of Citrus after in vitro pollination with triploid pollen grains

This study reports haploid plantlet regeneration through gynogenesis in Citrus clementina Hort. ex Tan., cv. Nules, induced by in vitro pollination with pollen grains of Oroblanco, a triploid cultivar of grapefruit. It indicates that parthenogenesis induced in vitro by triploid pollen can be an alternative method to obtain haploids in monoembryonic cultivars of Citrus. Actually, despite considerable efforts, androgenesis has not been yet successful in many genotypes of Citrus. Pollination and mature stage of pistils was necessary for gynogenic embryo regeneration. Fourteen haploid gynogenic embryos of Nules clementine were obtained. Embryo conversion was high (85.7%) and embryos vigorously germinated producing twelve plantlets. Chromosome counting, performed in the roots of obtained embryos, showed the haploid level (n=x=9). Isozyme analyses confirmed the expected homozygous state of embryos and plantlets. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryogenesis and doubled haploid production from anther culture in gentian (<Emphasis Type="Italic">Gentiana triflora</Emphasis>)

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9–12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH.     

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Induction,germination and shoot development of somatic embryos in cassava

Four Indonesian and two Latin-American cassava genotypes (Manihot esculenta Crantz), were evaluated for their ability to develop somatic embryos from young leaf lobes. All genotypes formed somatic embryos but they differed in the frequency of embryos induced. The best genotypes, M. Col 22 and Tjurug, produced germinating embryos (GE) on 81% (22.1 GE/initial leaf lobe) and 46% (4.3 GE/initial leaf lobe) of the cultured leaf lobes, respectively. Up to 57% of the germinating embryos of M. Col 22 and 12% of Tjurug produced either normal or malformed shoots. Most malformed shoots developed into shoots with normal morphology after prolonged culture. All shoots formed roots after transfer to medium without BAP. Roots of all normal and most malformed regenerants had the original ploidy level (2n=36). Regardless of whether the plants were multipliedin vitro (150 plants) or in the greenhouse (30 plants) there were no morphological differences compared to parent plants.     

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

Endogenous hormone levels in habituated nucellar Citrus callus during the initial stages of regeneration

 Embryogenic nucellar callus cultures of different Citrus species and cultivars growing in hormone-free medium were transferred to medium containing either sucrose or glycerol as the only carbohydrate source. Glycerol has been reported to induce further development of Citrus somatic embryos, while in the presence of sucrose they continue to proliferate in an 'undifferentiated' manner. The endogenous hormone levels of the cultures were evaluated after 2 and 5  days to characterise the initial steps of embryo development. In most cases, differences among treatments were observed only after 5 days of culture. Higher cytokinin levels were found in most of the cultures transferred to the glycerol-containing medium. The effect of ageing sweet orange cultures on their endogenous hormone levels was determined by leaving them in the original culture medium without subculturing for 60 days. While no changes were observed in the free indoleacetic acid and gibberellin contents, lower levels of abscisic acid and cytokinins were found in the aged cultures than in those transferred at the normal interval, every 30 days. The endogenous hormone contents of Citrus callus of different genotypes were compared. Significant differences were observed in the levels of all hormones evaluated, even when the in vitro ontogeny of the different genotypes was very similar. Received: 10 February 2000 / Revision received: 25 August 2000 / Accepted: 29 August 2000     

Induction of triploid Citrus plants from endosperm calli in vitro

Summary Triploid hybrid Citrus plants were regenerated by somatic embryogenesis in vitro from endosperm derived calli. A sequence of media formulations was used to induce and support proliferation of primary callus from endosperm, to induce embryogenesis from primary callus, and to allow embryo development leading to viable plantlets. Calli were induced from cellular endosperm of Citrus sinensis (sweet orange), C. Xparadisi (grapefruit), and C. grandis (pummelo) excised 12–14 weeks post-anthesis. Induction of embryogenesis from sweet orange and pummelo primary calli required gibberellic acid and double mineral nutrient concentrations. Embryogenesis was not induced from grapefruit calli in these experiments. Only sweet orange embryos developed sufficiently to allow plant regeneration. Triploid axillary buds were minigrafted onto etiolated diploid rootstock seedlings in vitro in order to transfer triploid regenerants to soil and the external environment. Triploidy (2n = 3x = 27) was observed consistently in all phases of regeneration and in recovered plants. These results demonstrate that triploid hybrid plant recovery from Citrus endosperm can overcome barriers to sexual hybridization resulting from apomixis.Florida Agricultural Experiment Station Journal Series No. R-00627     

A key to totipotency: Wuschel-like homeobox 2a unlocks embryogenic culture response in maize (Zea mays L.)

The ability of plant somatic cells to dedifferentiate, form somatic embryos and regenerate whole plants in vitro has been harnessed for both clonal propagation and as a key component of plant genetic engineering systems. Embryogenic culture response is significantly limited, however, by plant genotype in most species. This impedes advancements in both plant transformation-based functional genomics research and crop improvement efforts. We utilized natural variation among maize inbred lines to genetically map somatic embryo generation potential in tissue culture and identify candidate genes underlying totipotency. Using a series of maize lines derived from crosses involving the culturable parent A188 and the non-responsive parent B73, we identified a region on chromosome 3 associated with embryogenic culture response and focused on three candidate genes within the region based on genetic position and expression pattern. Two candidate genes showed no effect when ectopically expressed in B73, but the gene Wox2a was found to induce somatic embryogenesis and embryogenic callus proliferation. Transgenic B73 cells with strong constitutive expression of the B73 and A188 coding sequences of Wox2a were found to produce somatic embryos at similar frequencies, demonstrating that sufficient expression of either allele could rescue the embryogenic culture phenotype. Transgenic B73 plants were regenerated from the somatic embryos without chemical selection and no pleiotropic effects were observed in the Wox2a overexpression lines in the regenerated T0 plants or in the two independent events which produced T1 progeny. In addition to linking natural variation in tissue culture response to Wox2a, our data support the utility of Wox2a in enabling transformation of recalcitrant genotypes.     

Microspore embryogenesis induced through in vitro anther culture of almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis> Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25?±?1?°C, without the hot thermal shock at 35?±?1?°C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.     

In vitro propagation of the Egyptian medicinal plant,Echinops spinosissimus Turra

The in vitro propagation of medicinal species is proving to be one successful approach to addressing the issues raised by increasing global demand, overharvest of wild populations and variability in product quality. In this report we describe a protocol for establishing nodal cultures of an Egyptian medicinal plant, Echinops spinosis-simus Turra and for the induction of regeneration in epidermal peels and leaf explants obtained from the nodal cultures. Kinetin was found to be optimal for induction and proliferation of nodal cultures. Transverse slices of leaf blades produced regenerants resembling somatic embryos in the presence of thidiazuron while regeneration was observed on epidermal peels only when the medium contained elevated auxin concentrations.     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

Reflexions sur nos cultures d'antheres de plantes cultivées

Abstract

Considerations about our anther cultures of cultivated plants. – One of the main activities performed at the Casaccia Nuclear Centre, in the framework of a contract between CNEN and the European Communities, centers on the induction of haploid plants by anther culture and the subsequent chromosome doubling in order to obtain completely homozygous diploid plants. In tobacco, it is now possible to obtain haploid plants from any cultivar; we perform in vitro culture of internodes from which homozygous diploid plants are regenerated, taking advantage of natural phenomenon of endopolyploidy. In order to try to generalize this method of producing haploid plants in other plant species, we are studying the mechanism involved in haploid embryogenesis which occurs in vitro in the microspores. Datura, Nicotiana and Atropa are among the genera in which a direct embryogenesis from the microspore is observed; it is interesting to note that all three genera belong to the family Solanaceae and are very rich in alkaloids. In almost all the other cases of in vitro induction of haploids, microspores produce calli from which plantlets can be differentiated, but this way of plant regeneration is less interesting because only few plantlets are obtained and it is not sure that each haploid comes from a single microspore. We examined the factors which could influence the transformation of microspores into embryoids in tobacco, namely: the developmental stage of microspore, the degeneration of tapaetal cells, the genotype of microspore, the composition of cultural media, the physiological conditions of the plant from which the anthers were taken. From a practical point of view, it would be desirable to have informations on methods giving a maximum number of haploid plants from one embryogenic anther and the greatest number of embryogenic anthers from the cultured anthers. Our recent experiments on anther culture in liquid shaken medium have yielded good results (about 7,000 embryoids from 25 embryogenic anthers). Further, we are conducting several experiments in order to synchronize the development of the microspores in the anthers; to this end, we analyse the effect of cold treatment, ionizing radiation and gravity force. Experiments are being performed with other cultivated species, beside tobacco, in order to solve some problems of plant breeding more easily and quickly through haploidy. With the aim of introducing, in cultivated tomato, some desirable characters from the wild species, Lycopersicum peruvianum, (self-incompatibility, disease resistance, simultaneous flowering), we have obtained the interspecific hybrid through in vitro culture of young embryos. Haploid production from this hybrid could allow to quickly obtain various genetic recombinations from these two species. For this purpose we are carrying out anther cultures as well as single microspore cultures. In rice, strawberry and L. peruvianum, several diploid and tetraploid plantlets were obtained from our anther cultures. Work is in progress to ascertain the mode of their origin.     

Microspore embryogenesis and fertile plantlet regeneration in a salt susceptible × salt tolerant rice hybrid

Shed microspore embryogenesis and fertile plantlet regeneration were observed in a salt susceptible × salt tolerant indica rice F₁ hybrid involving IR 24 and CRM 30. The in vitro culture response and regeneration of green plantlets in the hybrid were superior to those of the parents. Direct embryogenesis and plantlet regeneration with multiple tillers were observed in shed microspore embryos. In intact anther culture, plantlet development from microspore involved a callus phase. The number of multiple tillers developed through secondary embryogenesis was almost equal in both the cases. However, the results indicate that regeneration of green plantlets was higher in case of shed microspore culture in liquid medium containing the synthetic polymer Ficoll 400 than from intact anthers cultured on a semi-solid system. Shed microspore culture produced a number of double haploids, which may result in far reaching consequences in genetic improvement of rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryos and Plantlets from Cultured Anthers of Hybrid Grapevines

Embryos and plantlets were produced in large numbers from callusformed by cultured anthers of hybrid grapevines (Vitis viniferax Vitis rupestris). Anthers of Vitis vinifera produced smallamounts of callus or failed to grow in vitro. For embryo formationanthers containing uninucleate microspores were chilled (4 °C)for 72 h before culture with Nitsch medium containing 2, 4-D(5µM) and benzyladenine (1 µM). Highest yields ofembryos were with anthers cultured in darkness. For productionof normal plantlets embryos required chilling (4 °C) for2 weeks. Unchilled embryos produced mainly abnormal plantlets.Chilling was effective in promoting plantlet growth when appliedat any stage of embryogeny. In grapes ability to produce plantlets from cultured anthersis a genetically-determined trait and maleness, as distinctfrom hermaphroditism, may be a predisposing factor. Callus derivedfrom anthers contained both haploid and diploid cells but allplantlets produced so far are diploid. The genetic constitutionof plantlets, whether they are diploids of somatic origin ordiploids from spontaneously doubled haploid cells, is not yetknown and is being determined by standard genetic methods.     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.     

Somatic embryogenesis of a seedless sweet orange (<Emphasis Type="Italic">Citrus sinensis</Emphasis> (L.) Osbeck)

Somatic embryogenesis is an important in vitro technique used to obtain Citrus sinensis (L.) Osbeck (sweet orange) plantlets for conservation, sanitation, propagation, and breeding. The induction of somatic embryogenesis from adult tissues of sweet orange could be an alternative to in vitro organogenesis from epicotyl segments, especially in seedless cultivars, where the latter is not feasible. The aim of this study was to obtain plantlets from ovary-derived somatic embryos of sweet orange cv. ‘Washington Navel’, an important seedless cultivar for citrus fresh fruit production. The explants used were pistils from flower buds, pre-anthesis, from 20-y-old plants cultivated in the field. Forty plantlets from 47 somatic embryos were obtained, in vitro-grafted, and acclimatized in greenhouse conditions. Ploidy evaluation through flow cytometric analysis, as well as the results of target region amplification polymorphism (TRAP) molecular markers confirmed the somatic origin of embryos as genetically similar to donor plants. This technique could be used for obtaining embryogenic cell suspension cultures or regenerated plants from mature tissues other than seed-derived tissues, especially for seedless genotypes.     

Proteomic analysis from haploid and diploid embryos of Quercus suber L. identifies qualitative and quantitative differential expression patterns

Quercus suber L. is a Mediterranean forest species with ecological, social and economic value. Clonal propagation of Q. suber elite trees has been successfully obtained from in vitro‐derived somatic and gametic embryos. These clonal lines play a main role in breeding and genetic studies of Q. suber. To aid in unravelling diverse genetic and biological unknowns, a proteomic approach is proposed. The proteomic analysis of Q. suber somatic and gametic in vitro culture‐derived embryos, based on DIGE and MALDI‐MS, has produced for the first time proteomic data on this species. Seventeen differentially expressed proteins have been identified which display significantly altered levels between gametic and somatic embryos. These proteins are involved in a variety of cellular processes, most of which had been neither previously associated with embryo development nor identified in the genus Quercus. Some of these proteins are involved in stress and pollen development and others play a role in the metabolism of tannins and phenylpropanoids, which represent two of the major pathways for the synthesis of cork chemical components. Furthermore, the augmented expression levels found for specific proteins are probably related to the homozygous state of a doubled‐haploid sample. Proteins involved in synthesis of cork components can be detected at such early stages of development, showing the potential of the method to be useful in searching for biomarkers related to cork quality.