<Emphasis Type="Italic">Aegilops-Secale</Emphasis> amphiploids: chromosome categorisation,pollen viability and identification of fungal disease resistance genes

The aim of this study was to assess the potential breeding value of goatgrass-rye amphiploids, which we are using as a “bridge” in a transfer of Aegilops chromatin (containing, e.g. leaf rust resistance genes) into triticale. We analysed the chromosomal constitution (by genomic in situ hybridisation, GISH), fertility (by pollen viability tests) and the presence of leaf rust and eyespot resistance genes (by molecular and endopeptidase assays) in a collection of 6× and 4× amphiploids originating from crosses between five Aegilops species and Secale cereale. In the five hexaploid amphiploids Aegilops kotschyi × Secale cereale (genome UUSSRR), Ae. variabilis × S. cereale (UUSSRR), Ae. biuncialis × S. cereale (UUMMRR; two lines) and Ae. ovata × S. cereale (UUMMRR), 28 Aegilops chromosomes were recognised, while in the Ae. tauschii × S. cereale amphiploid (4×; DDRR), only 14 such chromosomes were identified. In the materials, the number of rye chromosomes varied from 14 to 16. In one line of Ae. ovata × S. cereale, the U-R translocation was found. Pollen viability varied from 24.4 to 75.4%. The leaf rust resistance genes Lr22, Lr39 and Lr41 were identified in Ae. tauschii and the 4× amphiploid Ae. tauschii × S. cereale. For the first time, the leaf rust resistance gene Lr37 was found in Ae. kotschyi, Ae. ovata, Ae. biuncialis and amphiploids derived from those parental species. No eyespot resistance gene Pch1 was found in the amphiploids.     

Two-dimensional leaf isozyme patterns of <Emphasis Type="Italic">Aegilops kotschyi ’ Secale cereale</Emphasis> amphiploids

Esterase, peroxidase, shikimic dehydrogenase, malic dehydrogenase and diaphorase isozymes in leaves of the amphiploids Aegilops kotschyi × Secale cereale and their parental forms (Ae. kotschyi and S. cereale) were analyzed using two-dimensional gel electrophoresis in non-denaturing conditions. In the amphiploid isozymess were detected, which were not detected in leaves of parental plants. In contrast, some parental isozymes were not detected in refer to the gels of the amphiploids. Detection of new isoforms in the amphiploids and no detection of some parental isoforms is discussed considering the recombination, gene suppression, acting of inhibitors, chromosome translocation and also refer to the previous results of the electrophoretic analysis of proteins and enzymes of Aegilops sp. × S. cereale amphiploids.     

Pollen proteins after two-dimensional gel electrophoresis and pollen morphology of the amphiploids Aegilops kotschyi and Ae. variabilis with Secale cereale

Proteins from pollen of parent forms and amphiploids Aegilops variabilis ×Secale cereale and Ae. kotschyi×S. cereale, obtained by in vitro propagation or colchicine treatment of F₁ hybrids, were subjected to a study by two-dimensional (2-D) electrophoresis. Qualitative and quantitative diversities of protein patterns were revealed for the amphiploid pollen. The majority of peptides found in the parent forms were also present in the patterns of the amphiploid pollen; however, some of the parent-form-peptides were not expressed and proteins characteristic only of the amphiploids appeared. In the 2-D combined protein pattern obtained for the parent forms, amphiploids Ae. variabilis × S. cereale produced pollen with a poorer spectrum of proteins. In amphiploid 408B, obtained from treated the F₁ generation with colchicine, the 2-D pattern revealed the presence of less than 50% of the proteins recorded for the parent forms. Pollen grain morphology was studied under a scanning microscope. The structure and shape of exines differed from those of the parents. In the parent forms the pollen grains had only one pore, while in amphiploid pollen, one, two or three pores were observed. Possible explanations for the differences in the 2-D patterns of amphiploids and their parent forms (impoverishment of the protein spectrum and appearance of new peptides) are (1) somaclonal variation and mutagenic activity of colchicine, (2) suppression of structural genes, (3) activity of regulators and (4) translocations. Pollen grains with two or even three pores could appear as a result of the independent activity of the genes from three amphiploidal genomes. Received: 6 March 2001 / Accepted: 26 June 2001     

Comparison of FDR- and SDR-derived tetraploid progeny from 2x×4x crosses using haploids of Solanum tuberosum L. that produce mixed modes of 2n eggs

 The relationship between heterozygosity and heterosis in tetraploid potato (Solanum tuberosum subsp. tuberosum L., 2n=4x=48) was examined in a series of first-division restitution (FDR)- and second-division restitution (SDR)-derived tetraploid subpopulations. The subpopulations were constructed using two 2n egg-producing, mixed-mode haploids (2n=2x=24) crossed to three tetraploid (2x= 4x=48) potato clones. Half-tetrad analysis using a co-dominant electrophoretic marker (Pgm-2), which is closely linked to the centromere, discriminated between FDR- and SDR-derived 4x progeny. The FDR:SDR ratio of the 4x progeny observed was dependent upon the haploid parent used in the 2x×4x cross. Field studies were conducted between 1992 and 1996 to compare the yield and specific gravity of the two subpopulations and their parents from three crosses. There was no difference in the total tuber yield or specific gravity between the FDR- and SDR-subpopulations based upon family means, despite the expectation that FDR-derived progeny would transmit a greater portion of the genome’s heterozygosity intact than SDR-derived progeny. The 4x parent in each family had a higher yield than either 4x progeny subpopulation. Inbreeding, as a consequence of the haploidization process and a lack of genetic diversity, may have negated any advantage of the FDR-derived progenies over the SDR-derived progenies. Received: 14 April 1998 / Accepted: 12 May 1998     

Somatic embryogenesis inAesculus

Summary Somatic embryogenesis was observed with explants taken from four types ofAesculus tissue: (a) shoots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (b) roots of 4-wk-oldin vitro germinated excised embryos (seed fromA.×arnoldiana), (c) shoots from newly forced 3-yr-old seedlings (A. glabra), and (d) newly forced shoots from a 30-yr-old tree (A.×arnoldiana “Autumn Splendor”). Shoots provided three types of explants, single node, shoot apex, and internodal section, and all exhibited embryogenesis. Proembryogenic masses developed in a few cases after 6 wk in culture but were more commonly seen after 3 mo. The yellow, friable proembryogenic masses emerged from proximal cut ends of explants. Almost all cultures that formed embryos possessed leaves, either from developing apical or axillary buds or from adventitious buds, prior to the emergence of proembryogenic masses. Only tissues that had begun to senesce and had been exposed to cytokinin (benzyladenine at 5 or 25 μM) formed somatic embryos. Embryos with distinct cotyledonlike structures and root/shoot axes developed during the 10 to 16 wk following the inital emergence of proembryogenic masses. Enhanced frequency of embryogenesis was obtained by dark culture of root and shoot explants from 4-wk-old germinated embryos (A.×arnoldiana) and by dark and cold (5°C) treatment of shoot tissue cultures derived from 3-yr-old seedlings (A. glabra). Embryogenic potential was greatest in the most juvenile tissue and least in the mature tissue. Five percent of shoot explants taken from the 30-yr-old select treeA.×arnoldiana “Autumn Splendor” produced somatic embryos.     

Somatic embryogenesis and plant regeneration in Triticum aestivum x Leymus angustus F1 hybrids and the parental lines

Somatic embryos and plants were produced from cultured inflorescence and leaf segments of Triticum aestivum X Leymus anaustus F₁ hybrids and the parental lines. Inflorescences showed a better capacity for somatic embryogenesis and plant regeneration than leaves. Leymus anaustus produced the highest number of embryogenic calli, while the hybrids were intermediate between this species and Triticum aestivum. Presence of 2,4-D was shown to be essential for induction and maintenance of somatic embryogenesis. Addition of five amino acids (glutamine, proline, asparagine, aspartic acid and glutamic acid) did not have any marked effect when they were used in the callus induction medium. The regenerated plants had the same morphology as the original plants. No cytological modification was observed in the examined plants.     

Production and molecular characterization of two new Citrus somatic hybrids for scion improvement

Somatic hybridization by protoplast fusion from cell suspension cultures and leaf parent has been a well-established technique holding great potential for citrus variety improvement. In this study, somatic hybrid plants were regenerated from the following two fusion combinations: ‘Murcott’ tangor (Citrus reticulata Blanco × C. sinensis (L.) Osbeck) + Hirado Buntan Pink pummelo (HBP) (C. grandis (L.) Osbeck) and ‘Bingtang’ orange (C. sinensis (L.) Osbeck) + Calamondin (C. microcarpa Bunge). Somatic hybrids were selected at an early stage based on their higher capacity for embryogenesis comparing to non-hybrid cells. Flow cytometry analysis showed that all plants from pre-selected lines of the two combinations were tetraploid. SSR analysis confirmed their hybrid nature, with nuclear DNA from both fusion parents, and absence of parental specific bands was also detected. Cytoplasmic compositions of the recovered plants were further revealed by CAPS and cpSSR analysis. The allotetraploid somatic hybrids from the ‘Murcott’ tangor + HBP combination will be applied to develop triploid seedless cultivars by interploid crossing with diploid seedy citrus cultivars, and those from ‘Bingtang’ orange + Calamondin could be valuable for Asiatic citrus canker-tolerant and ornamental citrus breeding.     

Aegilops-rye amphiploids and substitution rye used for introgression of genetic material into rye (Secale cereale L.)

The valuable genes ofAegilops biuncialis, Ae. ovata, Ae. kotschyi, andAe. variabilis were transferred to rye, by crossingAegilops-rye amphiploids with tetraploid and diploid substitution rye. The C-banded karyotype of the BC₁ and BC₂ generations of amphiploids with 4x substitution rye and BC₁ with 2x substitution rye showed great variation in chromosome number and composition. In the BC₁ generation of amphiploids with 4x and 2x substitution rye, seed set success rate and germination rate varied depending on origin. However, plant sterility in all cross combinations of amphiploids with 4x and 2x substitution rye resulted in their elimination from further experiments in the BC₃ and BC₂ generations, respectively. In backcrosses of 4x substitution rye with amphiploidsAe. variabilis × rye 4x, fertile 4x rye plants containingAegilops chromatin were produced in the BC2 generation.     

Elimination of a tandem repeat of telomeric heterochromatin during the evolution of wheat

 An analysis of accessions of Triticum and Aegilops species (86 diploid, 91 tetraploid and 109 hexaploid) was performed using squash-dot hybridization with the tandem repeat Spelt1 sequence as a probe. The Spelt1 sequence is a highly species-specific repeat associated with the telomeric heterochromatin of Aegilops speltoides Boiss. in which its copy numbers vary from 1.5×10⁵ to 5.3×10⁵. The amounts of Spelt1 are sharply decreased in tetraploid and hexaploid species and vary widely from less than 10² to 1.2×10⁴. Two tetraploid wheats, Triticum timopheevii Zhuk. and T. carthlicum Nevski, are exceptional endemic species and within their restricted geographical distributions maintain the amounts of Spelt1 unaltered. The Spelt1 repetitive sequence was localized on the 6BL chromosome of tetraploid wheat Triticum durum Desf. cv ‘Langdon’ by dot-hybridization using D-genome disomic substitution lines. The possible causes of the loss of the telomere-associated tandem repeat Spelt1 in the process of wheat evolution and polyploidization are discussed. Received: 5 March 1998 / Accepted: 28 May 1998     

Three new hybrids of Ophioglossum (Ophioglossaceae) from Monte Pisano,Tuscany (Central Italy)

Based on general morphology, spore measurements and ornamentation (scanning electron microscope), genome size estimation, and molecular systematics (trnL-trnF IGS), we show the extreme systematic complexity within the European representatives of the genus Ophioglossum. In particular, three hybrids from Tuscany are described: the tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane, seen as the hybrid between diploid O. lusitanicum L. and hexaploid O. azoricum C.Presl; the tetraploid O. × giovanninii Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the homoploid hybrid between tetraploid O. vulgatum L. and tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane; the pentaploid O. × pseudoazoricum Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the hybrid between hexaploid O. azoricum C.Presl and tetraploid O. vulgatum L. All the three new taxa grow in different localities in the Monte Pisano mountain range.     

Enhancement of embryogenic culture initiation from tissues of mature sweetgum trees

 Male inflorescences, female inflorescences, and leaves collected from dormant buds of three sweetgum (Liquidambar styraciflua) trees were tested for induction of somatic embryogenesis following treatment with thidiazuron, naphthaleneacetic acid (NAA) or different combinations of the two. Explants were placed into culture either within a few days after collection or following 2 months of storage at –15  °C. Although embryogenic cultures were obtained from all three trees, embryogenesis induction was strongly affected by genotype (source tree), with 100% of the staminate inflorescence explants from one tree producing embryogenic cultures in one experiment. Embryogenesis induction was also influenced by explant type, with staminate inflorescences up to five times more likely to produce an embryogenic culture than female inflorescences. No embryogenic cultures were obtained from leaf explants. While treatment with plant growth regulators was not required for embryogenesis induction from inflorescence explants, culture on medium with NAA alone resulted in the highest production of repetitively embryogenic cultures and cultures producing proembryogenic masses. Dormant buds stored for 2 months at –15  °C were still able to produce embryogenic cultures, although frozen storage decreased this ability by over one-half for staminate inflorescences. Received: 20 January 1999 / Revision received: 18 April 1999 / Accepted: 29 April 1999     

Somatic hybrid plants produced by electrofusion between Solanum melongena L. and Solanum torvum Sw

Summary Somatic hybrid plants between eggplant (Solanum melongena) and Solanum torvum have been produced by the electrofusion of mesophyll protoplasts in a movable multi-electrode fusion chamber. Using hair structure as a selection criteria, we identified a total of 19 somatic hybrids, which represented an overall average of 15.3% of the 124 regenerated plants obtained in the two fusion experiments. Several morphological traits were intermediate to those of the parents, including trichome density and structure, height, leaf form and inflorescence. Cytological analyses revealed that the chromosome numbers of the somatic hybrids approximated the expected tetraploid level (2n=4x=48). Fifteen hybrid plants were homogeneous and had relatively stable chromosome numbers (46–48), while four other hybrids had variable chromosome numbers (35–48) and exhibited greater morphological variation. The hybridity of these 19 somatic hybrid plants was confirmed by analyses of phosphoglucomutase (Pgm) and esterase zymograms.     

Embryo rescue response and genetic analyses in interspecific crosses of oilseed Brassica species

Embryo rescue technique is widely utilized to save developing embryo from abortion in interspecific hybridization of plants. In Brassica species, success of an embryo rescue depends on the age of embryo, developmental stage of embryo, and media composition. In this study, ten crosses were conducted between diploid oilseed species B. rapa × tetraploid B. napus and B. rapa × tetraploid B. juncea along with their reciprocal crosses. The immature embryos from parents and crosses were cultured at 14 d after pollination in MS and ½ MS medium without any growth regulators for direct embryogenesis. Embryos were found at different stages of development during isolation, from globular to cotyledonary, indicating that rate of embryo development depends on genotype. Embryos isolated at the cotyledonary stage showed the highest regeneration percentage (av. 83.29%) and required the lowest time to regenerate (5.8 d) compared with another type of embryo, torpedo stage, cultured. Embryo rescued in ½ MS media accounted for 10–51%, 4.0–61%, 6.0–21%, and 5.0–43% greater shoot length, root length, number of leaves, and days to flowering, respectively, in different cross combinations compared with full-strength MS media. The results indicated that the rate of regeneration from embryo is higher in ½ MS media compared with full-strength MS media. Percent regeneration from the cultured embryos differed significantly in different cross combinations. Days required for regeneration from embryos, length of siliqua, and the number of ovules per siliqua accounted for high heterosis and broad sense heritability. The findings of this experiment would be useful in developing cost-effective embryo rescue protocol and desired interspecific hybrids through embryo rescue in Brassica species.

     

Molecular identification and the features of genetic diversity in interspecific hybrids of Amur sturgeon ( Acipenser schrenckii × A. baerii, A. baerii × A. schrenckii, A. schrenckii × A. ruthenus , and A. ruthenus × A. schrenckii ) based on variability of multilocus RAPD markers

The method of polymerase chain reaction with random primers (RAPD PCR) was used to identify the progeny of the crosses between three sturgeon species, Amur sturgeon (Acipenser schrenckii Brandt, 1869), Siberian sturgeon (A. baerii Brandt, 1869), and sterlet (A. ruthenus Linnaeus, 1758). Using ten primers, genetic variation in 70 yearlings, produced in seven individual crosses: Acipenser schrenckii × A. schrenckii, A. baerii × A. baerii, A. ruthenus × A. ruthenus, A. schrenckii × A. baerii, A. baerii × A. schrenckii, A. schrenckii × A. ruthenus, and A. ruthenus × A. schrenckii was described and evaluated. It was demonstrated that the samples composed of hybrids from individual crosses were more variable than the samples of parental species. On the other hand, pooled samples of hybrids from two cross directions were genetically less variable than the pooled samples of their parents. The three main features of the hybrid RAPD profiles identified included: (1) preservation of marker DNA fragments of both parents in one genome; (2) presence of specific DNA fragments, absent from both parents; and (3) dependence of the frequency of some DNA fragments from the cross direction. Multidimensional scaling clearly distinguishes in the space of three coordinates the individuals of original species and the hybrid progeny with differentiation in the groups of direct and backcross hybrids. Analysis of relationships (UPGMA and NJ) pointed to substantial differentiation between the species, as well as between the species and hybrid progeny. Close genetic relationships between direct and backcross hybrids were demonstrated. Multilocus RAPD markers in association with statistical methods are considered to be the useful tool for discrimination of interspecific hybrids of sturgeon. Possible reasons for the differences in the hybrid RAPD profiles are discussed. Original Russian Text ? K.V. Rozhkovan, G.N. Chelomina, E.I. Rachek, 2008, published in Genetika, 2008, Vol. 44, No. 11, pp. 1453–1460.     

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.     

Plantlet regeneration via somatic embryogenesis from subcultured callus of mature embryos ofPicea abies (Norway spruce)

Summary Embryogenic callus was initiated from radicles of mature embryos removed from imbibed seeds (24 h). Embryogenic and other nonembryogenic types of callus proliferated on a modified half-strength Murashige-Skoog medium (MS) basal medium (BM) supplemented withmyo-inositol, casein hydrolysate (CH), L-glutamine (gln) and growth regulators kinetin (KN), N⁶-benzyladenine (BAP) each (20×10⁻⁶ M), 2,4-dichlorophenoxyacetic acid (2,4-D) (50×10⁻⁶ M) Embryogenic callus bearing suspensor-like cells in a mucilaginous gel matrix was isolated and maintained by subculture every 10 to 12 days on BM with KN, BAP each (2×10⁻⁶ M) and 2,4-D (5×10⁻⁶ M). Somatic embryos developed spontaneously from the callus on this medium at 23±1° C. Closer examination revealed that numerous polyembryonic clusters, comprised of elongated cells (suspensors) and small dense cells with large nuclei (somatic embryos), occurred in the viscous gel. When this enriched embryonal-suspensor mass was subcultured to low 2,4-D (1×10⁻⁶ M), globular embryos developed by 40 to 60 days. Upon transfer to a liquid medium without growth regulators, the embryos elongated and developed cotyledons and shoots with needles. Plantlet development was completed by 30 days in a basal medium without CH, gln and growth regulators. The total culture time was 150 days. Approximately 40±10 embryos were formed from 500 mg of initial callus. Somatic embryogenesis became aberrant if embryos remained attached to the callus mass and were not subcultured within 10 to 12 days according to the described protocol. Somatic embryos were encapsulated in an alginate gel and stored at 4° C for nearly two months without visible adverse effects on viability. Editor's Statement This paper presents advances in the in vitro regeneration of a commercially useful plant species from stored seeds. In addition, data is presented on short-term storage of the plantlets, and long-term proliferation of the embryonal mass in vitro.     

Micropropagation ofPanax notoginseng by somatic embryogenesis and RAPD analysis of regenerated plantlets

Somatic embryogenesis was induced in callus tissues derived from young flower buds ofPanax notoginseng via callus within 18 weeks of culture. The mature somatic embryos were germinated on half-strength Murashige and Skoog's (MS) medium supplemented with gibberellic acid A₃(GA) and 6-benzyladenine (BA). The most suitable medium for optimal root formation proved to be MS medium supplemented with 1-naphthaleneacetic acid (NAA). Total DNA was extracted from the leaves of the regenerated plantlets ofP. notoginseng. Analysis of random-amplified polymorphic DNA (RAPD) using 21 arbitrary oligonucleotide 10-mers, showed the genetic homogeneity ofP. notoginseng. The amplification products were monomorphic for all of the plantlets ofP. notoginseng regenerated by embryogenesis, suggesting that somatic embryogenesis can be used for clonal micropropagation of this plant.     

Regeneration and large-scale propagation of bamboo (Dendrocalamus strictus Nees) through somatic embryogenesis

A complete protocol for large-scale propagation of Dendrocalamus strictus Nees by somatic embryogenesis has been developed. Seeds cultured on agar-solidified Murashige and Skoog (MS) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D; 3×10^–5 m) produced embryogenic callus from proliferation of the embryo. Somatic embryos formed in vitro multiplied rapidly (two- to five fold every 5 weeks) on semi-solid MS medium containing 2,4-D (1×10^–5 m), kinetin (Kn) (5×10^–6 m), 1-indolebutyric acid (IBA) (2×10^–6 m) and soluble polyvinylpyrrolidone (PVP) (250 mg l^–1), or MS with 2,4-D (1×10^–5 m), 6-benzylaminopurine (BAP) (1×10^–5 m), and soluble PVP (250 mg l^–1). Upon transfer to MS containing 1-naphthaleneacetic acid (NAA) (5×10^–6 m), Kn (5×10^–6 m) and soluble PVP (250 mg l^–1), the dark-green embryos developed into healthy plantlets. Unrooted shoots, if any, obtained on the multiplication media were rooted on MS major salts reduced to half strength supplemented with NAA (3×10^–6 m) and IBA (2.5×10^–6 m). The rooted plants were successfully transferred to soil in polythene bags with over 80% survival. Using this methodology, more than 100,000 plants have been produced. Received: 16 April 1998 / Revision received: 25 September 1998 / Accepted: 10 October 1998     

Genetic Analysis of Putative Triploid Miscanthus Hybrids and Tetraploid M. sacchariflorus Collected from Sympatric Populations of Kushima, Japan

Miscanthus ?×giganteus, a triploid hybrid between tetraploid M. sacchariflorus and diploid M. sinensis, has considerable potential as a bioenergy crop. Currently only one genotype is widely cultivated, increasing its vulnerability to diseases during production. Finding new hybrids is important to broaden genetic resources of M. ×giganteus. Three putative triploid hybrids were discovered in a sympatric population of tetraploid M. sacchariflorus and diploid M. sinensis in Kushima, Japan. The hybrid nature of the triploids was determined by morphological analysis and sequencing the ribosomal DNA internal transcribed spacer (ITS) region. The triploids had awns on their florets, which is a common characteristic of diploid M. sinensis, and sheath hairs, which is typical of tetraploid M. sacchariflorus. All triploids showed heterozygosity in their ribosomal DNA ITS sequences. Based on these results, it is confirmed that the triploids are hybrids and novel genotypes of M. ×giganteus. Natural crossing between tetraploid M. sacchariflorus × diploid M. sinensis may also lead to the production of tetraploid hybrids. ITS analysis of tetraploid plants showed that one maternal parent of the triploid hybrids, K-Ogi-1, had heterozygous ITS, which was different than the other analyzed tetraploid, M. sacchariflorus. Thus, K-Ogi-1 was likely of hybrid origin. These tetraploid hybrids can also be utilized as parents in M. ×giganteus breeding. Since all hybrids identified in this study had tetraploid M. sacchariflorus as maternal parents, collecting and analyzing seeds from tetraploid M. sacchariflorus in sympatric areas could be an effective strategy to identify natural Miscanthus hybrids that can be used as bioenergy crops.     

Ploidy stability of somatic embryogenesis-derived <Emphasis Type="Italic">Passiflora cincinnata</Emphasis> Mast. plants as assessed by flow cytometry

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.