Molecular genetic characterization of plant somatic hybrids

An efficient and easy method for genetic characterization of plant somatic hybrids is proposed. In a first qualitative approach, four somatic hybrids and their parental species (Nicotiana tabacum andN. plumbaginifolia) were characterized by DNA fingerprinting and Random Amplification of Polymorphic DNA (RAPD). After this, a quantitative estimation of the degree of parental contribution to the hybrids was carried out by means of a slot-blot analysis. Both qualitative methods, showed one hybrid identical toN. tabacum, two almost identical toN. plumbaginifolia, and a fourth similar to this parental species, but with someN. tabacum admixture. The quantitative method, for the same hybrids, gave 83%, 7%, 7%, and 37%N. tabacum DNA contribution, respectively.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

Le comportement reproducteur de Sminthurus viridis (L.) (Collembola,Symphypleona)

Different phases of the reproductive behaviour of Sminthurus viridis are analysed. Stalked spermatophores are deposited by the ♂, but the sperm is transmitted during a primitive courtship. Oviposition consists of characteristic sequences, present in all species which deposit coated eggs. Here, however, the shortness of the sequences results from the eggs being laid in batches and from the frequently dry conditions in the natural biotope of this species. The anatomy of the genital organs of ♂♂ and ♀♀ is described, with a particular study of the genital plate of the ♂ by SEM.     

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Summary Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10⁶ mature pollen protoplasts were fused with 7·10⁶ mesophyll protoplasts using a PEG/Ca²⁺ method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l^-1). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l^-1). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.Abbreviations AAT aspartate aminotransferase - BCP bromocresol purple - EST esterase - MES 2-(N-morpholino) ethanesulfonic acid - AP acid phosphatase - PEG polyethyleneglycol - PER peroxydase     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Selection of transgenic flax plants is facilitated by spectinomycin

Regeneration of transformed flax shoots after inoculation withAgrobacterium tumefaciens carrying a binary vector with either a neomycin phosphotransferase (nptII) gene and a -glucuronidase (GUS) reporter gene or a spectinomycin resistance gene was examined. Hypocotyls from 4-day-old seedlings were inoculated with either of the twoA. tumefaciens strains. Selection and regeneration were achieved on a medium containing 0.1 M thidiazuron, 0.01 M napthalene acetic acid, 100 mgl^–1 kanamycin sulphate or spectinomycin sulphate and 300 mgl^–1 cefotaxime. Use of different neomycins for the selection of transformed tissues did select transformed calli but not transformed shoots either directly or via a callus phase. Selection based on spectinomycin resistance allowed the growth of transformed shoots. Transgenic shoots were rooted on a medium containing 100 mgl^–1 spectinomycin sulphate. Integration of the spectinomycin resistance gene into the flax genome was confirmed by Southern blot hybridizations and spectinomycin resistance was shown to be inherited as a dominant Mendeliant trait. Therefore, spectinomycin resistance is more suitable for genetic engineering of flax than aminoglycoside resistance.     

Selection for spontaneous tomato haploids using a conditional lethal marker

We describe a method for the isolation of spontaneous haploid tomato plants from greenhousegrown seedlings obtained from crosses involving a transgenic parental line in which a counter-selectionable chimeric gene has been introduced. Transgenic seeds transformed with the aux2 gene, a gene of Agrobacterium rhizogenes that transforms naphthalene acetamide (NAM) into naphthalene acetic acid (NAA), did not develop roots in the presence of NAM, whereas wildtype tomato seeds developed a normal rooting system in its presence. Transgenic plants homozygous for aux2 (cv UC82b) were used to pollinate male-sterile (ms322) tomato plants (cv Apedice). Using NAM as a toxic substrate to kill heterozygous diploid plants carrying aux2, we selected for three maternal haploid plants resulting from the development of the female nucleus without fertilization. Maternal haploid selection using the aux2 marker was less efficient than the visual screening of haploid plants displaying recessive morphological markers of the female parent, but provided evidence for the feasibility of haploid selection in species for which no morphological markers are available.     

Experimental evidence of Wolbachia introgressive acquisition between terrestrial isopod subspecies

Wolbachia are the most widespread endosymbiotic bacteria in animals. In many arthropod host species, they manipulate reproduction via several mechanisms that favor their maternal transmission to offspring. Among them, cytoplasmic incompatibility (CI) promotes the spread of the symbiont by specifically decreasing the fertility of crosses involving infected males and uninfected females, via embryo mortality. These differences in reproductive efficiency may select for the avoidance of incompatible mating, a process called reinforcement, and thus contribute to population divergence. In the terrestrial isopod Porcellio dilatatus, the Wolbachia wPet strain infecting the subspecies P. d. petiti induces unidirectional CI with uninfected individuals of the subspecies P. d. dilatatus. To study the consequences of CI on P. d. dilatatus and P. d. petiti hybridization, mitochondrial haplotypes and Wolbachia infection dynamics, we used population cages seeded with different proportions of the 2 subspecies in which we monitored these genetic parameters 5 and 7 years after the initial setup. Analysis of microsatellite markers allowed evaluating the degree of hybridization between individuals of the 2 subspecies. These markers revealed an increase in P. d. dilatatus nuclear genetic signature in all mixed cages, reflecting an asymmetry in hybridization. Hybridization led to the introgressive acquisition of Wolbachia and mitochondrial haplotype from P. d. petiti into nuclear genomes dominated by alleles of P. d. dilatatus. We discuss these results with regards to Wolbachia effects on their host (CI and putative fitness cost), and to a possible reinforcement that may have led to assortative mating, as possible factors contributing to the observed results.