Carmina Gisbert

A plant genetically modified that accumulates Pb is especially promising for phytoremediation.

From a number of wild plant species growing on soils highly contaminated by heavy metals in Eastern Spain, Nicotiana glauca R. Graham (shrub tobacco) was selected for biotechnological modification, because it showed the most appropriate properties for phytoremediation. This plant has a wide geographic distribution, is fast-growing with a high biomass, and is repulsive to herbivores. Following Agrobacterium mediated transformation, the induction and overexpression of a wheat gene encoding phytochelatin synthase (TaPCS1) in this particular plant greatly increased its tolerance to metals such as Pb and Cd, developing seedling roots 160% longer than wild type plants. In addition, seedlings of transformed plants grown in mining soils containing high levels of Pb (1572 ppm) accumulated double concentration of this heavy metal than wild type. These results indicate that the transformed N. glauca represents a highly promising new tool for use in phytoremediation efforts.

Expression of the yeast HAL2 gene in tomato increases the in vitro salt tolerance of transgenic progenies

Abstract Agrobacterium -mediated transformation has been used to introduce the yeast halotolerant HAL2 as well as the npt II and uid A marker genes into tomato ( Lycopersicon esculentum ) cv. UC82B. Five to six percent of the explants produced transgenic plants. HAL2 expressing transformants were allowed to self-pollinate and salt tolerance assays were performed in vitro on progenies from two independent transgenic plants with different levels of expression of the transgene. In vitro salt tolerance was evaluated according to the level of growth of hypocotyl-derived calli as well as the rooting capability of isolated shootlets on MS-modified medium supplemented with NaCl. Under salt stress, callus formation from hypocotyl explants was higher on both transgenic-derived progenies than in the control. In addition, progenies from the plant with the highest expression of the transgene (2H20b), also showed a higher level of root production on NaCl-supplemented medium. These results suggested a positive effect of the yeast HAL2 gene on the level of salt tolerance in progenies derived from transgenic plants.

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.

Localization of QTLs for in vitro plant regeneration in tomato

BackgroundLow regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato.ResultsWe developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration.ConclusionsIn this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation.

Efficient regeneration in two potential new crops for subtropical climates, the scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants

Abstract The scarlet (Solanum aethiopicum) and gboma (S. macrocarpon) eggplants are two African crops with potential for vegetable crop diversification in subtropical climates. Here we carried out the in vitro regeneration in two accessions of each species. In a comparison of five media, we found that those containing the cytokinin thidiazuron (TDZ) induced the greatest regeneration response from both cotyledonary and true leaf explants. After organogenesis induction, explants were transferred to Murashige & Skoog (MS) basal medium without growth regulators for 20 days. Isolated shoots rooted after culture on MS medium for 4–6 days. Optimisation of TDZ concentration was studied in a range of concentrations from 0.05 to 0.8 μM. In general, increases in the concentration of TDZ in the medium resulted in a higher density of buds, but they had a slower development. Although differences in regeneration ability were observed among varieties, TDZ concentrations of 0.1 or 0.2 μM gave the best results, with 70–100% explants with shoots and a mean of 2–7 shoots per explant. The efficient regeneration obtained for these species allows the propagation of important genotypes and their improvement through in vitro techniques.

Identification of as Accumulation Plant Species Growing on Highly Contaminated Soils

Soils from the alluvial flats of the Turia River, Valencia, Spain, which were highly contaminated by decades of industrial activity, were surveyed for native plant species that could be candidates useful in phytoremediation. Concentrations of heavy metals and arsenic (As) in soils reached 25,000 mg Kg−1 Pb, 12,000 mg Kg−1 Zn, 70 mg Kg−1 Cd, and 13500 mg Kg−1 As. The predominant vegetation was collected and species identified. Soil samples and the corresponding plant shoots were analyzed to determine the amount of As accumulated by the various plant species. Several were able to tolerate more than 1000 mg Kg−1 As in the soil. Bassia scoparia (Chenopodiaceae) survive in soil with 8375 mg Kg−1 As. Arsenic accumulation in shoots of the various plant species investigated ranged from 0.1 to 107 mg Kg−1 dw. Bassia scoparia (Chenopodiaceae), Inula viscosa (Asteraceae), Solanum nigrum (Solanaceae), and Hirschfeldia incana (Brassicaceae) had the highest values for As accumulation.

In vitro plant regeneration and gene transfer in the wild tomato Lycopersicon cheesmanii

Summary This paper reports on protocols for in vitro plant regeneration, gene transfer and on shoot salt tolerance from two accessions of L. cheesmanii (Riley). Shoot organogenesis was achieved on media with zeatin or BA/IAA combinations. The two accessions tested, LA1401 and LA530, showed differences in both their aptitude to regenerate and their salt tolerance in vitro. The accession LA530 had a higher organogenic potential than LA1401 (97% vs. 80% of organogenic explants). In contrast, none of the LA530 isolated shoots rooted when cultured in full strength MS-modified medium supplemented with 200.mM NaCl while 93% of shoots from LA1401 formed roots. Finally, Agrobacterium-mediated transformation was used to introduce the nptII and uidA marker genes into the salt tolerant accession LA1401. Eight to nine per cent of the cotyledon explants produced transgenic plants. After self-pollination, the inheritance analysis showed a Mendelian segregation of both transgenes. The collection of transgenic plants will be an useful tool in experiments of asymmetric somatic hybridization with L. esculentum.

Fruit quality assessment of watermelons grafted onto citron melon rootstock

BACKGROUND The grafting of watermelons (Citrullus lanatus) is a common technique that increases yield under stressful soil conditions. The most common rootstocks for watermelons are Cucurbita hybrids. However, they often have a negative impact on fruit quality. Exploiting novel Citrullus germplasm such as citron melon (Citrullus lanatus var. citroides) is an alternative to avoid these quality problems. RESULTS Citron melon has been validated as watermelon rootstock, comparing its effects on watermelon quality to those of Cucurbita hybrids. Larger fruits with thicker rinds were observed in fruits from plants grafted onto both citron and Cucurbita rootstocks. The citron melon had no significant effect on flesh sugars or acid profiles compared to non-grafted watermelons, except for an increase in glucose and malic acid content, which also occurred in the Cucurbita rootstocks. The aroma profile of fruits produced on citron melon was similar to that of the non-grafted and self-grafted controls. The citron rootstock did not display the increased levels of (Z)-6-nonen-1-ol (a compound associated with pumpkin-like odors) found in fruits produced with Cucurbita hybrids. CONCLUSION The low impact of citron melon rootstock on fruit quality, along with the enhanced resistance against nematodes, make the citron a promising alternative to Cucurbita rootstocks.

Vigor for In Vitro Culture Traits in S. melongena × S. aethiopicum Hybrids with Potential as Rootstocks for Eggplant

Hybrids of Solanum melongena and S. aethiopicum are of interest as rootstocks of eggplant, as they are highly vigorous and can incorporate resistance to several diseases. However, hybridization between both species is difficult. Therefore, protocols for in vitro culture are of great interest for their micropropagation and biotechnological breeding. We assessed the organogenesis response from leaf explants in four interspecific hybrids and in their parents testing two organogenic media: SIM-A, containing 6-benzylaminopurine and kinetin, and SIM-B, which contains thidiazuron. A higher regeneration capacity in the hybrids compared to their parents was observed. Whereas in interspecific hybrids and in one accession of S. melongena similar regeneration rates were observed for SIM-A and SIM-B, higher regeneration was found in the rest of genotypes when thidiazuron was used. Rooting ability in the interspecific hybrids was lower in in vitro micropropagated plants (35–60%) than in plants regenerated from explants (100%). The addition of indolbutiric acid (1 mg L−1) induced roots in nonrooted genotypes. In summary, we have adjusted in vitro culture conditions for regenerating and rooting S. melongena × S. aethiopicum hybrids. We have also demonstrated that these hybrids are heterotic for regeneration, which may be of interest for basic science studies.

Evaluation of conditions for in vitro storage of commercial and minor grapevine (Vitis vinifera L.) cultivars

ABSTRACT In vitro culture represents a tool for the ex situ conservation of a high number of sanitised plants in a reduced space. However, the culture media and/or other growing conditions need to be optimised to minimising plant growth and storage cost. Growth on MW medium was evaluated in the commercial cultivars ‘Airén’, ‘Bobal’, ‘Chardonnay’, ‘Garnacha Blanca’, ‘Moscatel de Alejandría’, ‘Moscatel de Grano Menudo’, ‘Pedro Ximénez’, ‘Pinot Blanc’, ‘Pinot Gris’, ‘Sauvignon Blanc’, and ‘Tempranillo’; the minor cultivars ‘Chelva’, ‘Valencí Negre’, ‘Valencí Blanc’, and ‘Verdil’; and the endangered cv. ‘Esclafacherre’. Different growth rates were observed among cultivars: those with faster growth need to be subcultured every 1.5–2.0 months; those with the slowest growth every 3.5–4.0 months. The effect of halving the sucrose in MW reduced the growth of the cultivars that grew faster without compromising survival. When IBA was removed from MW, growth was also reduced in some cultivars. Therefore, small modifications of the MW composition are adequate for grapevine in vitro storage under standard incubation conditions. This is an advantage with respect to the change of temperature used in other work to achieve growth reduction, and allows the use of the same chamber for different in vitro culture procedures.