Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants

A protocol for the Agrobacterium-mediated transformation of tomatillo was developed. Up to 40 transgenic plants could be obtained in experiments using 60 cotyledon expiants. The transformed nature of the regenerated plants was confirmed by NPT II and Southern blot hybridization analysis. Using the b-glucuronidase system the tissue specific and developmental patterns of expression of the Cauliflower Mosaic Virus 35S promoter were determined in transgenic tomatillo plants. It was found that this promoter is developmentally regulated during fruit and seed formation.     

Spatial Biodiversity Patterns of Madagascar's Amphibians and Reptiles

Madagascar has become a model region for testing hypotheses of species diversification and biogeography, and many studies have focused on its diverse and highly endemic herpetofauna. Here we combine species distribution models of a near-complete set of species of reptiles and amphibians known from the island with body size data and a tabulation of herpetofaunal communities from field surveys, compiled up to 2008. Though taxonomic revisions and novel distributional records arose since compilation, we are confident that the data are appropriate for inferring and comparing biogeographic patterns among these groups of organisms. We observed species richness of both amphibians and reptiles was highest in the humid rainforest biome of eastern Madagascar, but reptiles also show areas of high richness in the dry and subarid western biomes. In several amphibian subclades, especially within the Mantellidae, species richness peaks in the central eastern geographic regions while in reptiles different subclades differ distinctly in their richness centers. A high proportion of clades and subclades of both amphibians and reptiles have a peak of local endemism in the topographically and bioclimatically diverse northern geographic regions. This northern area is roughly delimited by a diagonal spanning from 15.5°S on the east coast to ca. 15.0°S on the west coast. Amphibian diversity is highest at altitudes between 800–1200 m above sea-level whereas reptiles have their highest richness at low elevations, probably reflecting the comparatively large number of species specialized to the extended low-elevation areas in the dry and subarid biomes. We found that the range sizes of both amphibians and reptiles strongly correlated with body size, and differences between the two groups are explained by the larger body sizes of reptiles. However, snakes have larger range sizes than lizards which cannot be readily explained by their larger body sizes alone. Range filling, i.e., the amount of suitable habitat occupied by a species, is less expressed in amphibians than in reptiles, possibly reflecting their lower dispersal capacity. Taxonomic composition of communities assessed by field surveys is largely explained by bioclimatic regions, with communities from the dry and especially subarid biomes distinctly differing from humid and subhumid biomes.     

Differential organ infection studies, potyvirus elimination, and field performance of virus-free garlic plants produced by tissue culture

Presence of potyvirus in single garlic (Allium sativum L.) cloves from the same bulb, and in five single leaves excised from commercial field-grown individual plants was studied using ELISA. It was found that the viruses were not present in all organs of the same plant, since some cloves of the same bulb were infected with potyvirus but some others were potyvirus-free. Analyzed leaves from a given plant also exhibited irregular distribution of potyvirus. This study also aimed to obtain potyvirus-free plants from two commercial garlic cultivars (Taiwan and Chileno) using cloves subjected to thermotherapy, chemotherapy or meristematic dissection followed by in vitro culture. Thermotherapy (sequential treatment at 32°C for a week, 36°C for 2 weeks, and 38°C for 3 weeks) was found to affect survival of explants and 36.5% cloves from Taiwan and 26.8% from Chileno cultivars were recovered after the treatment. ELISA tests showed that 63% of the cloves of Taiwan that survived the treatment and 70.9% of Chileno explants were potyvirus-negative. Regarding chemotherapy (205 μM Ribavirin solution), the explants (cloves) survived, but only an average of 27.0–34.8% were negative for the presence of potyvirus. When meristematic dissection was applied, an average of 41.7% explants of Taiwan and 34.2% of Chileno survived the treatment, and approximately 64% of these explants from both cultivars were potyvirus-negative. Potyvirus-free garlic plants grown in field conditions showed longer stems with a major fresh and dry weight per bulb, and also exhibited a higher yield than non-treated plants.     

In vitro propagation of three <Emphasis Type="Italic">Agave</Emphasis> species used for liquor distillation and three for landscape

In vitro bud clusters of Calathea orbifolia (Linden) Kennedy were obtained and subcultured in semi-solid (agar) medium and temporary immersion system (TIS) for 12 weeks. Uniform young plants were selected and transferred to soilless mix in a growth chamber for ex vitro acclimatization during 35 days, followed by growing in a shaded greenhouse for 65 days. Comparison of in vitro leaf anatomy, ex vitro photosynthetic behaviors and growth was made between two cultural systems. Plants in TIS produced thicker leaf chlorenchyma and aquiferous parenchyma, lower stomatal frequency and more epicuticular wax than did those in semi-solid medium. Plants from semi-solid medium had consistently lower leaf Fv/Fm values than plants from TIS. Leaf Fv/Fm value in plants from TIS decreased to 0.65 at day 7 after transfer and increased soon up to 0.76 thereafter. In contrast, leaf Fv/Fm value in plants from semi-solid medium reduced to 0.27 at day 7 after transfer and increased slowly up to 0.68 at day 35. During ex vitro acclimatization, plants in TIS had substantial higher photosynthetic rates than plants in semi-solid medium. Plants from TIS had subsequent higher leaf area, fresh and dry weights than plants from semi-solid medium.     

Biodiversity and Land uses at a regional scale: Is agriculture the biggest threat for reptile assemblages?

The human exploitation of land resources (land use) has been considered the major factor responsible for changes in biodiversity within terrestrial ecosystems given that it affects directly the distribution of the fauna. Reptiles are known to be particularly sensitive to habitat change due to their ecological constraints. Here, the impact of land use on reptile diversity was analysed, choosing Catalonia (NE Iberia) as a case study. This region provides a suitable scenario for such a biogeographical study since it harbours: 1) a rich reptile fauna; 2) a highly diverse environment showing strong variation in those variables usually shaping reptile distributions; and 3) good species distribution data. Potential species richness was calculated, using ecological modelling techniques (Ecological Niche Factor Analysis – ENFA). The subtraction of the observed from the potential species richness was the dependent variable in a backwards multiple linear regression, using land use variables. Agriculture was the land use with the strongest relation with the non-fulfilment of the potential species richness, indicating a trend towards a deficit of biodiversity. Deciduous forest was the only land use negatively related with the subtracted species richness. Results indicate a clear relationship between land use and biodiversity at a mesoscale. This finding represents an important baseline for conservation guidelines within the habitat change framework because it has been achieved at the same spatial scale of chorological studies and management policies.     

Molecular biology of capsaicinoid biosynthesis in chili pepper (Capsicum spp.)

Capsicum species produce fruits that synthesize and accumulate unique hot compounds known as capsaicinoids in placental tissues. The capsaicinoid biosynthetic pathway has been established, but the enzymes and genes participating in this process have not been extensively studied or characterized. Capsaicinoids are synthesized through the convergence of two biosynthetic pathways: the phenylpropanoid and the branched-chain fatty acid pathways, which provide the precursors phenylalanine, and valine or leucine, respectively. Capsaicinoid biosynthesis and accumulation is a genetically determined trait in chili pepper fruits as different cultivars or genotypes exhibit differences in pungency; furthermore, this characteristic is also developmentally and environmentally regulated. The establishment of cDNA libraries and comparative gene expression studies in pungent and non-pungent chili pepper fruits has identified candidate genes possibly involved in capsaicinoid biosynthesis. Genetic and molecular approaches have also contributed to the knowledge of this biosynthetic pathway; however, more studies are necessary for a better understanding of the regulatory process that accounts for different accumulation levels of capsaicinoids in chili pepper fruits.     

In vitro embryo formation and plant regeneration from anther culture of different cultivars of Mexican husk tomato (<Emphasis Type="Italic">Physalis ixocarpa</Emphasis> Brot.)

Eight cultivars and two accessions of Physalis ixocarpa Brot. were tested for their capacity to regenerate embryos and plants from anther cultures. Anthers were pretreated at 4°C for 2 days and then at 35°C for 8 days in the dark while cultured on MS medium supplemented with 0.045 μM 2,4-D + 0.03 mg l⁻¹ vitamin B₁₂ (MS1) or with 2.26 μM 2,4-D + 0.1 mg l⁻¹ vitamin B₁₂ (MS3). Anther incubation proceeded under a 16 h photoperiod at 25 ± 2°C. Embryo formation occurred after 6 weeks of incubation in these conditions. Androgenetic responses were cultivar- and culture medium-dependent, with the greatest embryo yields recorded for cv. Chapingo (36.3%) on MS1 medium, and with wild-type 2 (21.8%) on MS3. Further development of regenerated embryos was promoted on MS medium supplemented with 0.54 μM NAA, 8.88 μM BA and 50 mg l⁻¹ casein hydrolysate. The regenerated plants were cultured on half-strength mineral salts MS medium with 2.85 μM IAA to enhance root formation. Rooted plantlets were transferred to pots and acclimatized to the greenhouse. Ploidy analysis of regenerated plants using flow cytometry revealed 72% diploids, 15% haploids and 7% triploids. AFLP analysis of regenerated plants from anthers of a single parental plant showed different polymorphic patterns indicating their gametophytic origin.     

Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues

Transgenic Mexican lime [Citrus aurantifolia (Christm.) Swing] plants were regenerated from tissues transformed by Agrobacterium rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. Transgenic shoots were generated by two different approaches. The first approach used internodal stem segments cocultured with A. rhizogenes. These were placed onto regeneration medium containing Murashige and Skoog salts and B5 organic compounds supplemented with 8 g ⋅ l^–1 agar, 7.5 mg ⋅ l^–1 6-benzylaminopurine, 1.0 mg ⋅ l^–1 -naphthaleneacetic acid, 300 mg ⋅ l^–1 cefotaxime and 80 mg ⋅ l^–1 kanamycin as a selective agent, and incubated under continuous light at 25 °C. Under these conditions, 76% of the explants produced shoots directly with no hairy root phase, with a mean of 1.3 shoots per explant, and 88% of these shoots were genetically transformed as determined by β-glucuronidase (GUS) assays. In the second approach, segments of transformed roots (15 mm long) obtained from internodal stem segments cocultured with A. rhizogenes were cultured on the above regeneration medium under similar conditions. Forty-one percent of these transformed root segments produced adventitious shoots, with a mean of 2.2 shoots per explant and with 90% of shoots transformed. GUS activity was evident in the transformed roots and in all parts of both transformed shoots and regenerated plants. The presence of the npt II and rolB genes in the regenerated plants was confirmed by PCR analysis. The presence of the npt II gene in the regenerated plants was also confirmed by Southern blot. Using these transformation systems, more than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil. Received: 15 March 1997 / Revision received: 30 December 1997 / Accepted: 19 January 1998     

AnthropoAge,a novel approach to integrate body composition into the estimation of biological age

Aging is believed to occur across multiple domains, one of which is body composition; however, attempts to integrate it into biological age (BA) have been limited. Here, we consider the sex-dependent role of anthropometry for the prediction of 10-year all-cause mortality using data from 18,794 NHANES participants to generate and validate a new BA metric. Our data-driven approach pointed to sex-specific contributors for BA estimation: WHtR, arm and thigh circumferences for men; weight, WHtR, thigh circumference, subscapular and triceps skinfolds for women. We used these measurements to generate AnthropoAge, which predicted all-cause mortality (AUROC 0.876, 95%CI 0.864–0.887) and cause-specific mortality independently of ethnicity, sex, and comorbidities; AnthropoAge was a better predictor than PhenoAge for cerebrovascular, Alzheimer, and COPD mortality. A metric of age acceleration was also derived and used to assess sexual dimorphisms linked to accelerated aging, where women had an increase in overall body mass plus an important subcutaneous to visceral fat redistribution, and men displayed a marked decrease in fat and muscle mass. Finally, we showed that consideration of multiple BA metrics may identify unique aging trajectories with increased mortality (HR for multidomain acceleration 2.43, 95%CI 2.25–2.62) and comorbidity profiles. A simplified version of AnthropoAge (S-AnthropoAge) was generated using only BMI and WHtR, all results were preserved using this metric. In conclusion, AnthropoAge is a useful proxy of BA that captures cause-specific mortality and sex dimorphisms in body composition, and it could be used for future multidomain assessments of aging to better characterize the heterogeneity of this phenomenon.