Production of fertile somatic hybrids of Gossypium hirsutum?+?G. bickii and G. hirsutum?+?G. stockii via protoplast fusion

Fertile somatic hybrids were obtained via symmetric electrofusion of protoplasts from two combinations of tetraploid cotton (G. hirsutum cv. Coker 201, AD genome) and diploid wild cottons G. bickii (G genome) and G. stockii (E genome), respectively. Observation by morphological, flow cytometric analysis, chromosome counting and RAPD analysis of the tested hybrids of Coker 201 + G. bickii and Coker 201 + G. stockii confirmed the regenerated plants as hybrid status. Cytological investigation of the metaphase root-tip cells revealed there were 78 chromosomes in the hybrids. Flow cytometric analysis showed the tested plants had a relative DNA contents close to the total DNA contents of the two parents. RAPD analysis revealed the hybrids contained specific genomic fragments from both fusion partners, further confirmed their hybridity. The morphology of the hybrids was intermediate between the two fusion partners. The hybrid plants were successfully transferred to the soil, and they bloomed and set bolls. It is sure that the new hexaploids developed by cell fusion would contribute to cotton breeding through backcrossing with the elite genotypes of G. hirsutum.     

Effects of β-lactam antibiotics, auxins, and cytokinins on shoot regeneration from callus cultures of two hybrid aspens, Populustremuloides?×?P. tremula and P. xcanescens?×?P. gradidentata

The effects of β-lactam antibiotics (penicillin, carbenicillin and cefotaxime), cytokinins, and auxins including phenylacetic acid, a β-lactam breakdown product, were evaluated during in vitro shoot morphogenesis in two hybrid aspens; P. tremuloides × P. tremula (XTTa) and P. x canescens × P. grandidentata (XCaG). Although different callus and shoot induction media were used for both hybrids, the β-lactams often engendered similar responses. At concentrations of 1,000 mg l⁻¹, carbenicillin adversely impacted shoot elongation and, to a lesser degree, shoot regeneration. Cefotaxime enhanced caulogenesis for all of the concentrations evaluated (125–500 mg l⁻¹) especially when the cytokinin thidiazuron was used for shoot induction. The shoots formed faster and in greater numbers; and the improvements were significant (α = 0.05) for both hybrids. However, hyperhydricity was more problematic when cefotaxime was included in the media. The incidence of shoot hyperhydricity for the XCaG hybrid was more than twice as great for the highest cefotaxime concentration evaluated (500 mg l⁻¹) than for the control (>90% vs. ~40%). Penicillin had an opposite effect. Hyperhydricity frequencies for the XCaG hybrid were lower when the media were supplemented with penicllin and the reductions were statistically significant at concentrations of 500–1,000 mg l⁻¹. The effects of the antibiotics were generally not reproduced by the auxins (0.1–100 μM), including phenylacetic acid, or the other potential β-lactam degradation products evaluated (e.g. phenylmalonic acid, aminopenicillanic acid). The antibiotics may have affected shoot hyperhydicity indirectly via changes in the time course of shoot regeneration.     

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.     

Androgenic response of genotypes selected from Capsicum annuum L.?×?C. chinense Jacq. hybrids

In research on androhaploids in the progeny of interspecific hybrids within the Capsicum genus, three genetically stable lines of F₇ generation, selected from the C. annuum L. × C. chinense Jacq. hybrid, were used. In the first line, only callus tissue was formed as a reaction to the conditions of culture. Cytometric analysis of this tissue revealed the presence of cells with DNA content in nuclei at the level of 1C to 16C. The tissue was mixoploid and non-embryogenic. Anthers of the other line did not respond. In the third one, nine embryos were obtained, and they developed into plants. By means of cytometric analysis, the 1C DNA level was found in eight of them and these were androgenic plants. The origin of one of the diploid plant was not established due to the homozygous character of the donor plants. The experiment results confirm the already known diversity of genotype reaction to the conditions of culture. It moreover point to the possibility of selection of the forms with an androgenic potency from interspecific hybrids.     

Transformation of androgenic-derived Festulolium plants (Lolium?perenne L.?×?Festuca?pratensis Huds.) by Agrobacterium?tumefaciens

Genetic transformation of androgenic-derived amphidiploid Festulolium plants (Lolium perenne L. × Festuca pratensis Huds., 2n = 4x = 28) by Agrobacterium tumefaciens has been achieved. Anther culture-induced calli of Festulolium “Bx351” were inoculated with Agrobacterium tumefaciens strain LBA4404 carrying pIG121-Hm encoding the hygromycin resistance (hph) and β-glucuronidase (uidA) genes under the control of a CaMV 35S promoter. Twenty-three putative transformants were obtained from the hygromycin selection, 19 of which (82.6%) showed GUS activity. The integration of transgene was detected by using genomic DNA PCR analysis, RT-PCR analysis and Southern blot hybridization, respectively, which revealed that foreign gene was integrated into the genomes of dihaploid transformants (2n = 2x = 14). The haploid embryogenic system offers a stable means of transformation, as the introduced trait can be readily fixed through chromosome doubling. An erratum to this article can be found at     

A ThCAP gene from Tamarix hispida confers cold tolerance in transgenic Populus (P. davidiana?×?P. bolleana)

The ThCAP gene, which encodes a cold acclimation protein, was isolated from a Tamarix hispida NaCl-stress root cDNA library; its expression patterns were then assayed by qRT-PCR in different T. hispida tissues treated with low temperature (4°C), salt (400 mM NaCl), drought (20% PEG6000) and exogenous abscisic acid (100 μM). Induction of ThCAP gene was not only responsive to different stress conditions but was also organ specific. When transgenic Populus (P. davidiana × P. bolleana) plants were generated, expressing ThCAP under regulation of the cauliflower mosaic virus CaMV 35S promoter, they had a greater resistance to low temperature than non-transgenic seedlings, suggesting that ThCAP might play an important role in cold tolerance.     

Comparative study (AFLP and morphology) of three species of Prosopis of the Section Algarobia: P. juliflora, P. pallida, and P. limensis. Evidence for resolution of the “P. pallida–P. juliflora complex”

The problems of delimitation of species of Prosopis originate from the few morphological discontinuities which exist among some of them; some, however, originated as a result of wide distribution of germplasm without proper knowledge of the species, in particular, much material catalogued as P. juliflora, but being of other species, was distributed for reforestation projects worldwide. This work tests the morphological results obtained for P. pallida and P. limensis of the Peruvian–Ecuadorian coast and for P. juliflora of the Caribbean Basin of Colombia and Venezuela utilizing a study of AFLPs and a study of the morphology of plantlets developed in a conventional garden study. The phenogram obtained for the AFLPs demonstrates each of the three species to be a well differentiated cluster and the molecular variance between them is significantly greater than the variance within each species. Study of the plantlets also indicates statistically significant differences for four morphological characters between P. juliflora and the other two species (P. pallida and P. limensis). These results, in addition to the morphological differentiation evident between adult plants of P. pallida and P. limensis and the clear separation of these two species from P. juliflora, corroborate the genetic identity of the three taxa analyzed.     

A wild Marsh Warbler?×?Sedge Warbler hybrid (Acrocephalus palustris?×?A. schoenobaenus) in Norway documented with molecular markers

We present photographic and molecular evidence of a wild Marsh Warbler × Sedge Warbler (Acrocephalus palustris × A. schoenobaenus) hybrid that occurred over three breeding seasons (2007–2009) near Trondheim, Central Norway. The bird had the appearance of a Marsh Warbler but with some typical Sedge Warbler plumage traits. DNA analyses of a few plucked body feathers, using the COI barcode region (mtDNA) and conserved microsatellite loci, confirmed that the bird was a hybrid, with a Marsh Warbler mother and a Sedge Warbler father.     

Molecular evidence of a reed warbler × great reed warbler hybrid ( Acrocephalus scirpaceus × A. arundinaceus) in Belgium

We report on the second case of a reed warbler × great reed warbler hybrid (Acrocephalus scirpaceus and A. arundinaceus). The bird was captured during a standardised ringing session in Belgium in autumn 1999, and fell between the parental species in all measurements. Molecular analyses of two microsatellite loci verified that the bird was a female that had a reed warbler father and a great reed warbler mother.     

Trichoderma harzianum strain T-22 induces changes in phytohormone levels in cherry rootstocks (Prunus cerasus?×?P. canescens)

The aim of this research was to explain the direct plant growth-promoting activity of Trichoderma harzianum strain T-22 (T22), hypothesizing the involvement of different classes of plant growth regulators. Seven days after the transfer to root-inducing medium, in vitro-cultured shoots of GiSeLa6^? (Prunus cerasus  × P. canescens) were inoculated with T22. Root and shoot growth were significantly affected by T22 (+76 and +61%, respectively). Ten days after inoculation, the levels of indole-3-acetic acid (IAA), trans-zeatin riboside (t-ZR), dihydrozeatin riboside (DHZR), gibberellic acid (GA3) and abscisic acid (ABA) were analyzed by high performance liquid chromatography coupled with mass spectrometry. The results showed that after T22-inoculation, IAA and GA3 significantly increased in both leaves (+49 and +71%, respectively) and roots (+40 and +143%, respectively) whereas t-ZR decreased (−51% in leaves and −37% in roots). Changes in DHZR were observed in T22-inoculated roots (−32%) but not in leaves, whereas the levels of ABA did not differ between the two treatments. The extraction method allowed the simultaneous extraction of phytohormones. There is evidence that the change in phytohormone levels is one of the direct mechanism by which T22 promotes rooting and shoot growth, with notable advantages for rootstock production during nursery processes.     

Are hyperhydric shoots of Prunus avium L. energy deficient?

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus aviumL. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.     

Over-expression of the Arabidopsis Na+/H+ antiporter gene in Populus deltoides CL?×?P. euramericana CL “NL895” enhances its salt tolerance

Soil salinity is a serious problem worldwide. It is necessary to improve the salt tolerance of plants to avoid the progressive deterioration of saline soil. We showed that the over-expression of AtNHX1 improves salt tolerance in a transgenic poplar (Populus deltoides CL × P. euramericana CL “NL895”) under mannose selection. Four transgenic poplar plants were obtained. Southern blot analysis showed that the pmi gene had integrated into the genome of the poplar. RT-PCR confirmed that AtNHX1 could be expressed normally in the transgenic plants. When tested for salt tolerance by NaCl stress, we measured a 100% increase in Na⁺ content in the three transgenic lines (T18, T50, T98) significantly higher than the 33% increase seen in wild-type plants. The chlorophyll content of the transgenic plants was not altered significantly, while the chlorophyll content in the control plants showed a small decrease. MDA content was decreased in the transgenic plants. These results show that the AtNHX1 gene may enhance salt tolerance due to increased vacuolar compartmentalization of sodium ions.     

Screening of IR50?×?Rathu Heenati F7 RILs and Identification of SSR Markers Linked to Brown Planthopper (Nilaparvata lugens St?l) Resistance in Rice (Oryza sativa L.)

Brown planthopper (Nilaparvata lugens St?l) is one of the major insect pests of rice. A Sri Lankan indica rice cultivar Rathu Heenati was found to be resistant to all biotypes of the brown planthopper. In the present study, a total of 268 F₇ RILs of IR50 and Rathu Heenati were phenotyped for their level of resistance against BPH by the standard seedbox screening test (SSST) in the greenhouse. A total of 53 SSR primers mapped on the chromosome 3 were used to screen the polymorphism between the parents IR50 and Rathu Heenati, out of which eleven were found to be polymorphic between IR50 and Rathu Heenati. The eleven primers that have shown polymorphism between the IR50 and Rathu Heenati parents were genotyped in a set of five resistant RILs and five susceptible RILs along with the parents for co-segregation analysis. Among the eleven primers, two primers namely RM3180 (18.22 Mb) and RM2453 (20.19 Mb) showed complete co-segregation with resistance. The identification of SSR markers linked with BPH resistant could be used for the maker assisted selection (MAS) program in rice breeding and to map the resistant genes on rice chromosomes for further gene cloning.     

Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides × P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence

Background  

Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR.     

Analysis of storage proteins (prolamines, puroindolines and Waxy) in common wheat lines Triticum aestivum L. × (Triticum timopheevii Zhuk. × Triticum tauschii) with complex resistance to fungal infections

Storage proteins (prolamines, puroindolines, and Waxy) were studied in common wheat introgression lines obtained with the use of the Saratovskaya 29 (S29) cultivar line and synthetic hexaploid wheat (Triticum timopheevii Zhuk. × T. tauschii) (Sintetik, Sin.) displaying complex resistance to fungal infections. Comparative analysis of storage proteins in the introgression lines of common wheat Triticum aestivum L. and in the parental forms revealed the only line (BC5) having a substitution at the Gli-B2 locus from Sintetik. Hybrid lines subjected to nine backcrosses with the recurrent parental form S29 and selections for resistance to pathogens can be considered as nearly isogenic for the selected trait and retaining the allelic composition of (1) prolamines responsible for the bread-making qualitiy, (2) puroindolines associated with grain texture, and (3) Waxy proteins responsible for nutritive qualities. These lines are valuable as donors of immunity in breeding programs without the loss of the quality of flour and grain as compared to the S29 line and are also important in searching for genes determining resistance to leaf and stem rust and to powdery mildew. The amphiploid has a number of characters (silent Glu-A1 locus and Ha genotype) that can negatively affect the quality of flour and grain and thus should be taken into account when choosing this donor.     

A framework map from grapevine V3125 (Vitis vinifera ‘Schiava grossa’?×?‘Riesling’)?×?rootstock cultivar ‘B?rner’ (Vitis riparia?×?Vitis cinerea) to localize genetic determinants of phylloxera root resistance

Grapevine rootstock cultivar ‘B?rner’ is a hybrid of Vitis riparia and Vitis cinerea Arnold that shows high resistance to phylloxera (Daktulosphaira vitifoliae Fitch). To localize the determinants of phylloxera root resistance, the susceptible grapevine V3125 (Vitis vinifera ‘Schiava grossa’ × ‘Riesling’) was crossed to ‘B?rner’. Genetic framework maps were built from the progeny. 235 microsatellite markers were placed on the integrated parental map. They cover 1,155.98 cM on 19 linkage groups with an average marker distance of 4.8 cM. Phylloxera resistance was scored by counting nodosities after inoculation of the root system. Progeny plants were triplicated and experimentally infected in 2 years. A scan of the genetic maps indicated a quantitative trait locus on linkage group 13. This region was targeted by six microsatellite-type markers newly developed from the V. vinifera model genome sequence. Two of these appear closely linked to the trait, and can be useful for marker-assisted breeding.     

Overexpression of Arabidopsis thaliana Na+/H+ antiporter gene enhanced salt resistance in transgenic poplar (Populus?×?euramericana ‘Neva’)

Salinity is a major abiotic stress factor limiting plant growth and productivity. One possible method to enhance plant salt-resistance is to compartmentalize sodium ions away from the cytosol. In the present work, a vacuolar Na⁺/H⁺ antiporter gene AtNHX1 from Arabidopsis thaliana, was transferred into Populus × euramericana ‘Neva’ by Agrobacterium tumefaciens in order to enhance poplar salt-resistance. The results showed that the transgenic poplar were more resistant to NaCl than the wild-type (WT) in greenhouse condition. Compared with the WT, plant growth and photosynthetic capacity of the transgenic plants were enhanced, and the transgenic plants accumulated more Na⁺ and K⁺ in roots and leaves under the same NaCl condition, whereas malondialdehyde and relative electrical conductivity were lower. All of these properties of the transgenic poplar were likely to be a consequence of the overexpression of AtNHX1 caused Na⁺ sequestration in the vacuoles and improved K⁺ absorption, thus reducing their toxic effects. These results indicated overexpression of the AtNHX1 enhanced salt-resistance of poplar, and AtNHX1 played an important role in the compartmentation of Na⁺ into the vacuoles. Therefore, this study provides an effective way for improving salt resistance in trees.     

Versuche mit Klangattrappen zur Kl?rung der Verwandtschaft der Bauml?uferCerthia familiaris L.,C. brachydactyla Brehm undC. americana Bonaparte

Zusammenfassung Wildlebende Gartenbaumläufer- und Waldbaumläufer- werden durch ihren arteigenen, vom Tonband vorgespielten Gesang (Abb. 1) angelockt und zum Singen und Rufen veranlaßt (Tab. 1). Der Tonbandgesang der Zwillingsart löst dagegen keine, eine schwache oder in seltenen Fällen eine starke Reaktion aus. Ebenso schlecht, beim Waldbaumläufer vielleicht geringfügig besser, ist die Reaktion der Wald- und Gartenbaumläufer auf den Gesang des Amerikanischen Baumläufers und des Gartenbaumläufers auf seinen rückwärts vorgespielten Gesang. Ein Waldbaumläufer antwortete nicht nur auf den Gesang seiner Zwillingsart, sondern auch auf seinen arteigenen mit Gartenbaumläufer-Strophen; nur ganz selten sang er wie ein Waldbaumläufer (Abb. 2).Aus den Versuchsergebnissen wird gefolgert: Der Gesang der Baumläufer hat auf die der eigenen Art eine aggressive Wirkung. Die Kämpfe zwischen artverschiedenen Baumläufer- werden vermutlich optisch ausgelöst. Angehörige des Waldbaumläufers und des Amerikanischen Baumläufers würden sich in der Natur, wenn überhaupt, nicht ohne Einschränkung kreuzen; der Amerikanische Baumläufer sollte deshalb wieder als eigene Art geführt werden. Einige Einwände gegen die Art der Methode und die Folgerungen aus den Versuchen werden diskutiert.SummaryWild Short-toed Tree Creepers and Tree Creepers of the male sex, enticed by the song of their own species played on the tape recorder, can be motivated to sing and call in reply. In contrast, the recorded song of the sibling species evokes no, a slight, or in very few cases, a strong response. The reaction of the European creepers to the recorded song of the American Brown Creeper, and that of the Short-toed Tree Creeper to his song played in reverse is slight; the reaction of the Short-toed species to the song of the Brown-Creeper may be weaker than that of the Tree Creeper.A given individual of the Tree Creeper responded to the song of the sibling species, as well as to his own recorded song, with the song of the Short-toed species. Only rarely he sang like a Tree Creeper.As a result of these trials the following conclusions may be drawn: The songs of the creepers have an aggressive effect on the male members of the species. The fights between the males of the two creepers is supposedly optically released. Members of the European Tree Creepers and members of the American Brown Creepers would only cross-breed with limitations, if at all; the American Brown Creeper should therefore be treated as a separate species. Some objections to the methods used in the study and to the results which are concluded from the trials are discussed.Mit Unterstützung der Deutschen Forschungsgemeinschaft. Herrn Prof. Dr.B. Hassenstein danke ich für den Arbeitsplatz in seinem Institut, Herrn Prof. Dr.O. Koehler für die Bereitstellung der Geräte und Herrn Dr.H. Löhrl für seine Kritik.     

Gibberellin mediates daylength-controlled differentiation of vegetative meristems in strawberry (Fragaria × ananassa Duch)

Background  

Differentiation of long and short shoots is an important developmental trait in several species of the Rosaceae family. However, the physiological mechanisms controlling this differentiation are largely unknown. We have studied the role of gibberellin (GA) in regulation of shoot differentiation in strawberry (Fragaria × ananassa Duch.) cv. Korona. In strawberry, differentiation of axillary buds to runners (long shoot) or to crown branches (short shoot) is promoted by long-day and short-day conditions, respectively. Formation of crown branches is a prerequisite for satisfactory flowering because inflorescences are formed from the apical meristems of the crown.