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The influence of culture medium and explant on callus and shoot formation of lentil (Lens culinaris Medik.) has been studied. Three different explants (shoot-tip, first node and first pair of leaves) from three Spanish lentil cultivars were cultivated on two basal media: Murashige and Skoog medium (MS) and medium with mineral salts of MS medium plus vitamins of Gamborg's B5 medium (MSB), supplemented with growth regulators. Media with 2,4-D induced the formation of calli in all explants, but no organ regeneration was obtained from these caUi. Multiple shoot formation was obtained from 33% to 92% of the explants in media supplemented with 2.25 mg 1-~ of BA and 0.186 mg I J NAA + 2.25 mg 1-l BA; in the other media one to two shoots per explant were formed in 10 to 98% of the explants. Root formation from explants was achieved only in media with NAA or IAA. Of the explants tested, the best morphogenetic responses were obtained from nodes and the poorest from leaves.

We tested the hypothesis that shrub canopies interact with monthly rain pulses to control litter decomposition in a sandy Monte desert, in Argentina. We assessed (i) the potential for litter decomposition of soils beneath the canopies of two dominant shrub species ( Larrea divaricata and Bulnesia retama, Zygophyllaceae R. Br . ) and from bare-ground microsites or 'openings'; (ii) litter decomposition at different spatial patches over the summer rainy season; and (iii) the interaction between vegetation patches and monthly rain pulses on shortterm litter decomposition, or decomposition pulses. In a greenhouse experiment, we found buried litter decomposition to be higher in soils from under the canopies of a dominant shrub species compared with soils from openings and sterilized controls. This, and higher nutrient concentration under shrub soils, suggest undercanopy soils may support a microbial community capable of decomposing litter at higher rates than soils in bare openings. However, field trials showed that shrub patches did not affect leaf litter decomposition over the rainy season, at least for short periods. We found an interaction between shrub patches and incubation time at the end of the field experiment, with higher litter decomposition rates under B. retama canopies. In a monthly field experiment, we found monthly rain pulses significantly explained decomposition pulses, irrespective of patch type. Our findings support the hypothesis that shrub soils have a greater potential for litter decomposition, but this is not directly translated to the field possibly due to interactions with abiotic factors. Rain pulses create conditions for decomposition pulses to occur at shorter time scales, whereas rainfall may interact with a dominant shrub undercanopy to control litter mass loss over longer time scales.

As part of a project on lentil molecular genetics, the sequence of the 18S-25S ribosomal RNA gene intergenic spacer (IGS) of Lens culinaris Medik. was determined. DNA was cloned after polymerase chain reaction (PCR) amplification. The spacer of 2939 bp was composed of nonrepetitive sequences and four tandem arrays of repeated sequences, named A to D. C and D arrays were formed by the repetition of very short consensus sequences. Similarity was found between lentil and other legume species, in particular those of the Vicieae tribe. A transcription initiation site, putative sites of termination and processing, and promoter-enhancer sequences were detected by computer-aided searches. These sites resemble motifs conserved in the IGS sequences of other plant species. The conservation of motifs in the otherwise highly variable plant IGS sequences points to the relevance of these motifs as functional sequences.

Amplified fragment length polymorphism (AFLP) analysis of 24 in vitro regenerated rye plants was performed in order to evaluate the somaclonal variation rate in this species and to identify rye genomic regions where mutations are preferentially promoted by in vitro culture processes. Regenerated plants were obtained from cell lines derived from immature embryos and plants were regenerated by somatic embryogenesis. Twenty-three regenerants showed variation when compared against sibling plants obtained from the same cell line. A total number of 887 AFLP markers were scored, and 8.8% identified the same polymorphism in plants obtained independently from different cell lines, revealing putative mutational hot spots. Using controlled crossings and analysis of the corresponding progenies, we were able to verify the genetic stability in the next generation for only five of these polymorphisms. The nucleotide sequence of the AFLP amplicon of four of the polymorphic markers was obtained, but only the sequence of two markers was clearly identified in the databases. The sequence of marker A1-303 was identified as part of a tandemly repeated sequence, the 120-bp family, which is located at telomeric regions and is widely distributed among rye chromosomes. The marker A5-375 showed high similarity with regions of Angela retrotransposons.

We provide evidence that nucleotide sequence and methylation status changes occur in the Arabidopsis genome during in vitro tissue culture at a frequency high enough to represent an important source of variation. Somaclonal variation is a general consequence of the tissue culture process that has to be analyzed specifically when regenerated plants are obtained in any plant species. Currently, there are few studies about the variability comprising sequence changes and methylation status at the DNA level, generated by the culture of A. thaliana cells and tissues. In this work, two types of highly reproducible molecular markers, modified methylation sensitive AFLP (metAFLP) and transposon methylation display (TMD) have been used for the first time in this species to analyze the nucleotide and cytosine methylation changes induced by transformation and tissue culture protocols. We found significantly higher average methylation values (7.5%) in regenerated and transgenic plants when compared to values obtained from seed derived plants (3.2%) and that the main component of the somaclonal variation present in Arabidopsis clonal plants is genetic rather than epigenetic. However, we have found that the Arabidopsis regenerated and transgenic plants had a higher number of non-fully methylated sites flanking transposable elements than the control plants, and therefore, their mobilization can be facilitated. These data provide further evidence that changes in nucleotide sequence and methylation status occur in the Arabidopsis genome during in vitro tissue culture frequently enough to be an important source of variation in this species.

In vitro culture of immature embryo and young leaf tissues was carried out with five cultivars of barley, Hordeum vulgare. Two cultivars (Albacete and Porthos) responded poorly from both types of explants, while the three others (Dissa, Golden Promise and Ingrid) produced a high frequency of embryogenic callus from these explants (25-60%). For 'Dissa' and 'Ingrid', young leaf explants were slightly better than immature embryo explants for embryogenic callus induction, while immature embryo cultures of 'Golden Promise' responded better than young leaf explants. Thus, there appears to be a significant genotype × explant interaction in the initiation of embryogenic callus in barley.Some phenotypic variants were detected among the regenerated plants of 'Golden Promise' and 'Ingrid', most originating by epigenetic changes. Only in one case was the variant phenotype heritable, probably due to a mutation in the chloroplast DNA. Mitotic alterations were not detected. Consequently, somaclonal variation did not appear to be a very frequent event in plants regenerated from 1-to 6-month-old cultures of barley.