Genetic differentiation among populations of Sesleria albicans Kit. ex Schultes (Poaceae) from ecologically different habitats in central Europe.

As observed for many other plant species, the populations of Sesleria albicans in Central Europe are located in habitats, which differ to a high degree from each other with regard to ecological factors such as nutrients, light and water as well as in type of land use. The species colonizes limestone cliffs, pavements, screes, grazed and mown grasslands, heaths, fens and open woodlands. In this study, we used random amplified polymorphic DNA (RAPD) analysis to investigate the genetic differentiation among 25 populations of S. albicans from six different types of habitat (beech forests, alpine and lowland rocky ridges, lowland screes, fens, calcareous grasslands). With RAPD analysis, 344 fragments could be amplified, of which 95.9% were polymorphic. The level of polymorphism ranged from 29.7 to 56.7% polymorphic bands per population and was correlated with population size. In an analysis of molecular variance (AMOVA), used to detect variation among individuals within populations, among populations from the same habitat and among different habitats, most of the genetic variation was found within populations (62.06%) and among populations from the same habitat (33.36%). In contrast, only a very low level of differentiation could be observed among different habitats (4.58%). The results of our study give only little evidence for an ecotypic differentiation of Sesleria albicans. This differentiation is principally conceivable, but obviously not related to the investigated RAPD loci.

Regeneration of fertile interspecific hybrids from protoplast fusions between Helianthus annuus L. and wild Helianthus species.

The use of interesting characteristics from wild Helianthus species in sunflower breeding is limited by poor crossability or sterility of interspecific hybrids. To overcome this barrier, mesophyll protoplasts of Sclerotinia sclerotiorum-resistant clones of Helianthus maximiliani, H. giganteus and H. nuttallii were fused with hypocotyl protoplasts of H. annuus in the presence of polyethyleneglycol and dimethylsulfoxide. Fusion products were embedded in agarose and subjected to a regeneration protocol developed for sunflower protoplasts. Organogenic calli were transferred onto solid medium and emerging shoots were elongated in the absence of plant growth regulators. Rooting of shoots was induced by a 1-naphthaleneacetic acid treatment and putative hybrid plants from fusions between H. annuus + H. maximiliani and H. annuus + H. giganteus were transferred into the greenhouse. All of them exhibited a hybrid phenotype with a high percentage of rhizome producing plants. Their hybrid origin was confirmed by random amplified polymorphic DNA analysis. Plants flowered after 3-4 months and set seeds, of which 70-80% germinated.

Regeneration of fertile plants from Helianthus nuttallii T&G and Helianthus giganteus L. mesophyll protoplasts.

Interspecific hybridisation in the genus Helianthus via somatic cell fusion is thought to play an important role in future sunflower breeding programs. The establishment of this technique requires, however, the development of single-cell-regeneration protocols. For this purpose, we applied a regeneration protocol recently developed for Helianthus annuus L. to mesophyll protoplasts of two wild sunflowers (H. nuttallii T&G, H. giganteus L). Protoplasts of both species were embedded in agarose droplets and covered by liquid mKM medium. After 4-5 weeks, callus was transferred onto solid differentiation medium yielding plating efficencies of 1.5% (H. nuttallii) and 2.5% (H. giganteus). Emerging shoots were elongated on hormone-free medium, and root formation was induced by an NAA treatment. Regenerated plants were transferred to the greenhouse where they grew up to a height of 2 m and flowered after 3 months. Seeds were harvested from regenerated plants of both species.

A regeneration protocol for sunflower (Helianthus annuus L.) protoplasts.

Hypocotyl protoplasts of four different Helianthus annuus genotypes were cultivated for 22-28 days in agarose droplets covered with liquid medium. In the first week, supplementation of the medium with plant growth regulators was at a 0.8/1 ratio of cytokinin and auxin followed by a high auxin concentration in the second week and a cytokinin to auxin ratio of 8/1 in the third and fourth week. Following transfer onto solid medium containing cytokinin and auxin in a proportion of 40/1 morphogenic callus started to form globular structures that developed into leaf primordia. Subsequent shoot elongation and rooting were obtained on hormone free medium after dipping the cut shoots into high auxin solution. Thirteen weeks after protoplast isolation, plantlets could be transferred to the greenhouse. Shoot regeneration was obtained for all four cultivars (Florom-328, Cerflor, Euroflor, Frankasol) at different rates reflecting their regenerative potential.

cis-regulatory elements involved in ultraviolet light regulation and plant defense.

An elicitor-regulated transient expression system was established in soybean protoplasts that allowed the identification of cis-regulatory elements involved in plant defense. The 5' region of an ultraviolet (UV) light-inducible and elicitor-inducible chs gene (chs1) of soybean was subjected to deletion analysis with the help of chimeric chs-nptII/gus gene constructs. This analysis delimited the sequences necessary for elicitor inducibility to -175 and -134 of the chs1 promoter. The same soybean sequences were able to direct elicitor inducibility in parsley protoplasts, suggesting a conserved function of cis-acting elements involved in plant defense. In addition, this region of the soybean promoter also promotes UV light inducibility in parsley protoplasts. However, in contrast to the elicitor induction, correct regulation was not observed after UV light induction when sequences downstream of -75 were replaced by a heterologous minimal promoter. This result indicates that at least two cis-acting elements are involved in UV light induction.

Differential regulation of soybean chalcone synthase genes in plant defence, symbiosis and upon environmental stimuli.

Four independent recombinant lambda clones hybridizing to parsley chalcone synthase (CHS) cDNA were isolated from a soybean (Glycine max) genomic library. Restriction fragment length polymorphism (RFLP) analysis indicated that the CHS gene family comprises six members. The CHS genes were found to be clustered with three genes on a 10 kb segment and pairs on others. DNA sequences of the 5'-, the coding-, and the 3' untranslated regions were determined for three different genes. A consensus alignment of the 5' regions revealed extensive homology between them for up to 150 bp upstream of the TATA box. Developmental regulation of CHS was observed in uninfected and in rhizobium-infected roots. Regulation at the level of transcription by different stimuli was investigated in the root, stem and cotyledons of soybean seedlings. Our results suggest a co-operative induction of CHS genes by wounding and elicitor treatment of cotyledons. The most rapid transcript accumulation, however, was observed in roots and stems. The induction of CHS genes by light was found to be UV dependent. A possible involvement of different members of the CHS gene family in response to elicitor versus UV treatment was analysed by the use of gene specific probes, and unexpectedly revealed that only CHS 1 transcription was induced by either elicitor or UV treatment of seedlings.

Rhizobium meliloti nodA and nodB genes are involved in generating compounds that stimulate mitosis of plant cells.

The nodB gene of Rhizobium meliloti encodes a 23.8-kDa protein that is conserved in several Rhizobium species. Monospecific polyclonal antibodies against NodB were used to localize this protein in the cytosol of R. meliloti and Escherichia coli cells containing nodABC genes. In comparison to the NodA and NodC proteins, NodB is synthesized in a disproportionately low amount. The NodA and NodB proteins are involved in generating small, heat-stable compounds that stimulate the mitosis of various plant protoplasts. Our experiments suggest that NodC is not involved in the synthesis of the factors. On the basis of their properties, we speculate that the factors are cytokinin-like substances.

Toxicity and residue studies in dairy animals with firemaster FF-1 (polybrominated biphenyls).

Two studies (one in Holstein calves and one in Holstein cows) were conducted to determine potential toxicity and residue levels following oral ingestion of polybrominated biphenyls (PBB). The material was FireMaster FF-1. Administration was by gelatin capsules. Doses in calves were 0.1, 1.0, 10, or 100 mg/kg body weight, while doses in cows were equivalent to 0.01, 0.1, 1.0, or 10 ppm in the diet. The calves were sacrificed after 2, 4, 6, or 12 weeks. The cows were fed 158 or 228 days, and were then in a recovery period for 182 or 112 days. In the calf study, signs of toxicity were observed only in animals fed 100 mg/kg-day. Administration of 10 mg/kg-day or less for up to 12 weeks caused no overt signs of toxicity. Histologic studies were conducted upon selected organs and tissues taken at time of sacrifice. The only treatment-induced lesions among animals fed 0.1 mg/kg-day were minimal lesions in the kidney and skin in the one calf fed at this level for 12 weeks. Treatment-induced lesions were present in the kidneys, skin, and/or liver from some animals fed levels of 1.0 mg/kg-day and above. The relative severity of these lesions was related to the level and length of exposure. Treatment-associated changes were observed in the testes of all males in this study. The hypospermatogenesis observed was consistent with the age of the animals due to prepuberal development of the testes. No clinical signs of toxicity or histologic changes attributed to PBB were observed in the cows. Two cows were pregnant at the initiation of the study and give birth to normal, healthy calves during the study. These calves grew normally and appeared healthy when sacrificed at about 6 months of age. Residue levels were quantitated as the hexabromobiphenyl isomer (BP-6) which is the major isomer present in the PBB mixture. Tissue residue levels in calves increased with dose and duration of administration of PBB with highest levels being found in the fat. At 100 mg/kg the levels in fat were about 6000 to 6300 ppm after 6 to 12 weeks. Residue levels of BP-6 in milk and fat of the cows also increased with dose and duration of administration. Maximum levels in milk were about 1/3 the levels in the diet. In general, the levels plateaued after about 4 to 6 weeks and did not go appreciably higher even though administration of the FireMaster FF-1 was continued. Maximum levels in fat were on the order of approximately 4.5 times the levels in the diet, except at the lowest dose level. Residues decreased in milk after administration of PBB was discontinued, but detectable levels were still present 6 months later. Residues were found in the calves born of treated cows indicating passage of the PBBs through the placental barrier and/or ingestion through the milk.