Cryopreservation of Bituminaria bituminosa varieties and hybrids.

Bituminaria bituminosa (L.) C.H. Stirton is a drought tolerant, perennial legume pasture species and a source of pharmaceutical compounds. Bituminaria breeding programs aim to develop and conserve hybrids with desirable traits such as high forage quality, tolerance to biotic or abiotic stresses, and high contents of furanocoumarins. In this work we present a cryopreservation study of different B. bituminosa accessions: two varieties and eight intervarietal hybrids resulting from crosses between the three botanical varieties: var. bituminosa, var. crassiuscula, and var. albomarginata. No previous work on cryopreservation of Bituminaria species has been reported. We applied the ultra-fast cooling method, using droplet vitrification on aluminum foil strips. First, we investigated the PVS2 toxicity and cryopreservation damage in two genotypes, comparing three PVS2 treatments and two culture media. An incubation of 30 min in PVS2 resulted in regeneration rates after cryopreservation higher than 80%. The MS medium was selected for optimal meristem outgrowth, in order to avoid the prominent callus formation that was observed in the presence of BAP. These conditions were subsequently used to cryopreserve eight other genotypes. The results were highly variable; 45 days after cryopreservation, survival ranged between 22% and 98% while regeneration ranged between 0% and 96%, depending on the accession. A significant and positive correlation was observed between survival and regeneration. At 90 days post culture plantlets could be recovered from cryopreserved explants of all genotypes. This study shows that the droplet vitrification method is promising for the cryopreservation of eight of the 10 genotypes assayed and the method can thus be applied to develop a cryobank of B. bituminosa.

Accumulation of furanocoumarins by Bituminaria bituminosa in relation to plant development and environmental stress.

Bituminaria bituminosa (L.) C.H. Stirton (Fabaceae) accumulates high concentrations of the furanocoumarins (FCs) angelicin and psoralen, which protect against infection and herbivory. The effects on FC accumulation of the exposure of two populations of B. bituminosa to abiotic stress (cold, heat and drought) under field conditions were determined, as well as the effect of temperature under controlled conditions, in hydroponic culture. In field conditions, psoralen and angelicin levels in the leaf dry matter were 400-6000 and 1500-11,000 µg g(-), respectively. There were significant effects of population on the psoralen concentration and psoralen:angelicin ratio. In hydroponic culture, exposure to a diurnal temperature of 33 °C increased FC levels in population Calnegre but not in Llano del Beal, compared with plants grown at 22 °C; however, high summer temperatures in the field (>30 °C) did not coincide with the highest leaf FC levels, since the plants accumulated FCs preferentially in the fruits. Hence, leaf FC levels were higher in winter. Irrigation, to alleviate water stress in the semi-arid conditions, increased the fruit psoralen concentration but produced only minor decreases in leaf FC levels. There was a significant, positive correlation (P < 0.001) between the FC and nitrogen concentrations in the plant organs analysed (both increased in the order: fruits > growing leaves > mature leaves), reflecting their respective contributions to plant fitness. The genetically- and developmentally-regulated accumulation of FCs by B. bituminosa is altered by short-term variations in environmental conditions, particularly temperature.

Next generation DNA sequencing technology delivers valuable genetic markers for the genomic orphan legume species, Bituminaria bituminosa.

BACKGROUND: Bituminaria bituminosa is a perennial legume species from the Canary Islands and Mediterranean region that has potential as a drought-tolerant pasture species and as a source of pharmaceutical compounds. Three botanical varieties have previously been identified in this species: albomarginata, bituminosa and crassiuscula. B. bituminosa can be considered a genomic 'orphan' species with very few genomic resources available. New DNA sequencing technologies provide an opportunity to develop high quality molecular markers for such orphan species. RESULTS: 432,306 mRNA molecules were sampled from a leaf transcriptome of a single B. bituminosa plant using Roche 454 pyrosequencing, resulting in an average read length of 345 bp (149.1 Mbp in total). Sequences were assembled into 3,838 isotigs/contigs representing putatively unique gene transcripts. Gene ontology descriptors were identified for 3,419 sequences. Raw sequence reads containing simple sequence repeat (SSR) motifs were identified, and 240 primer pairs flanking these motifs were designed. Of 87 primer pairs developed this way, 75 (86.2%) successfully amplified primarily single fragments by PCR. Fragment analysis using 20 primer pairs in 79 accessions of B. bituminosa detected 130 alleles at 21 SSR loci. Genetic diversity analyses confirmed that variation at these SSR loci accurately reflected known taxonomic relationships in original collections of B. bituminosa and provided additional evidence that a division of the botanical variety bituminosa into two according to geographical origin (Mediterranean region and Canary Islands) may be appropriate. Evidence of cross-pollination was also found between botanical varieties within a B. bituminosa breeding programme. CONCLUSIONS: B. bituminosa can no longer be considered a genomic orphan species, having now a large (albeit incomplete) repertoire of expressed gene sequences that can serve as a resource for future genetic studies. This experimental approach was effective in developing codominant and polymorphic SSR markers for application in diverse genetic studies. These markers have already given new insight into genetic variation in B. bituminosa, providing evidence that a division of the botanical variety bituminosa may be appropriate. This approach is commended to those seeking to develop useful markers for genomic orphan species.

Cadmium has contrasting effects on polyethylene glycol-sensitive and resistant cell lines in the Mediterranean halophyte species Atriplex halimus L.

Beside a direct toxicity, cadmium impact on plants involves both a secondary-induced water stress and an oxidative stress. Proliferating cell lines of Atriplex halimus were selected for their sensitivity or resistance to polyethylene glycol (PEG 10,000, 20%) and then exposed to 100 microM CdCl2 in the simultaneous presence or absence of PEG 20% or 150 mM NaCl. The PEG resistant cell line exhibited a higher growth in the presence of Cd than the sensitive line, although Cd acccumulation was higher in the former than in the latter. Exogenous PEG induced an increase in Cd concentration in the sensitive but not in the resistant cell line while NaCl induced a decrease in Cd accumulation in both cell lines. In the presence of Cd alone, the water content (WC) was higher and the osmotic potential was lower in PEG-sensitive than in PEG resistant line. The presence of PEG in the Cd-containing medium increased the WC and decreased the osmotic potential in PEG-resistant line comparatively to Cd stress alone, while an inverse trend was observed for the sensitive line. The PEG-resistant cell line displayed a higher ability to cope with oxidative stress in relation to an increase of endogenous antioxidants (glutathione and ascorbic acid), a high constitutive superoxide dismutase (EC 1.15.1.1) activity and an efficient Cd-induced increase in glutathione reductase (GR) (EC 1.6.4.1) and ascorbate peroxidase (APX) (EC 1.11.1.11). Cadmium tolerance in PEG-resistant line is thus not related to any strategy of Cd exclusion or osmotic adjustment but to tolerance mechanisms allowing the tissue to restrict the deleterious impact of accumulated Cd.

Heavy metal accumulation by the halophyte species Mediterranean saltbush.

To identify Cd- and Zn-accumulating plants exhibiting a high growth rate, seeds from the halophyte species Mediterranean saltbush (Atriplex halimus L.) were collected on a heavy-metal-contaminated site in southeastern Spain (Llano del Beal, Cartagena). Seedlings from this ecotype were exposed for 3 wk to 0.1 mM Cd or Zn in a nutrient solution in a fully controlled environment. All plants remained alive and no significant growth inhibition was recorded until the end of the experiment. Mean Cd and Zn accumulation in aerial parts was 830 and 440 mg kg(-1), respectively, and the rate of metal translocation even increased with the duration of stress exposure. Resistance to heavy metals in this species may be partly linked to precipitation of Cd in oxalate crystals in the stems. A Cd-induced decrease in glutathione concentration also suggests that phytochelatins overproduction may occur in these conditions. We conclude that Mediterranean saltbush, which is able to produce up to 5 Mg dry matter ha(-1) yr(-1), may be an effective species for phytoextraction and should be tested for this purpose in field conditions.