Vicente Moreno

Transfer of the yeast salt tolerance gene HAL1 to Cucumis melo L. cultivars and in vitro evaluation of salt tolerance

An Agrobacterium-mediated gene transfer method for production of transgenic melon plants has been optimized. The HAL1 gene, an halotolerance gene isolated from yeast, was inserted in a chimaeric construct and joined to two marker genes: a selectable-neomycin phosphotransferase-II (nptII)-, and a reporter-β-glucuronidase (gus)-. The entire construct was introduced into commercial cultivars of melon. Transformants were selected for their ability to grow on media containing kanamycin. Transformation was confirmed by GUS assays, PCR analysis and Southern hybridization. Transformation efficiency depended on the cultivar, selection scheme used and the induction of vir-genes by the addition of acetosyringone during the cocultivation period. The highest transformation frequency, 3% of the total number of explants cocultivated, was obtained with cotyledonary explants of cv. ‘Pharo’. Although at a lower frequency (1.3%), we have also succeeded in the transformation of leaf explants. A loss of genetic material was detected in some plants, and results are in accordance with the directional model of T-DNA transfer. In vitro cultured shoots from transgenic populations carrying the HAL1 gene were evaluated for salt tolerance on shoot growth medium containing 10 g l−1 NaCl. Although root and vegetative growth were reduced, transgenic HAL1-positive plants consistently showed a higher level of tolerance than control HAL1-negative plants

Plant regeneration from calli of melon (Cucumis melo L., cv. «Amarillo Oro»

Callus cultures from cotyledon and hypocotyl explants of a Spanish cultivar of melon (‘Amarillo Oro’) have been tested for their growth and morphogenic capacity on a series of media with different concentrations of indole-3-acetic acid (IAA) and 6-furfurylaminopurine (kinetin). Melon tissues were able to undergo morphogenesis both via organogenesis and embryogenesis, depending on culture conditions and explant source. Shoot buds were obtained at high rates in cotyledon explants. In response to 1.5 mg/l IAA and 6.0 mg/l kinetin, more than 90% of the calli produced well-developed shoots. Hypocotyls failed to form shoots but formed somatic embryos on auxin containing media while cotyledon explants usually gave abundant shoots but only rarely formed embryos. It was possible to maintain organogenic callus lines for at least 12 months under defined conditions. Plants were recovered from adventitious shoots produced both in cotyledon-derived calli and from organogenic cell lines.

Overexpression of dehydrin tas14 gene improves the osmotic stress imposed by drought and salinity in tomato

One strategy to increase the level of drought and salinity tolerance is the transfer of genes codifying different types of proteins functionally related to macromolecules protection, such as group 2 of late embryogenesis abundant (LEA) proteins or dehydrins. The TAS14 dehydrin was isolated and characterized in tomato and its expression was induced by osmotic stress (NaCl and mannitol) and abscisic acid (ABA) [Godoy et al., Plant Mol Biol 1994;26:1921-1934], yet its function in drought and salinity tolerance of tomato remains elusive. In this study, transgenic tomato plants overexpressing tas14 gene under the control of the 35SCaMV promoter were generated to assess the function of tas14 gene in drought and salinity tolerance. The plants overexpressing tas14 gene achieved improved long-term drought and salinity tolerance without affecting plant growth under non-stress conditions. A mechanism of osmotic stress tolerance via osmotic potential reduction and solutes accumulation, such as sugars and K(+) is operating in tas14 overexpressing plants in drought conditions. A similar mechanism of osmotic stress tolerance was observed under salinity. Moreover, the overexpression of tas14 gene increased Na(+) accumulation only in adult leaves, whereas in young leaves, the accumulated solutes were K(+) and sugars, suggesting that plants overexpressing tas14 gene are able to distribute the Na(+) accumulation between young and adult leaves over a prolonged period in stressful conditions. Measurement of ABA showed that the action mechanism of tas14 gene is associated with an earlier and greater accumulation of ABA in leaves during short-term periods. A good feature for the application of this gene in improving drought and salt stress tolerance is the fact that its constitutive expression does not affect plant growth under non-stress conditions, and tolerance induced by overexpression of tas14 gene was observed at the different stress degrees applied to the long term.

Functional Analysis of the Arlequin Mutant Corroborates the Essential Role of the ARLEQUIN/TAGL1 Gene during Reproductive Development of Tomato

Reproductive development of higher plants comprises successive events of organ differentiation and growth which finally lead to the formation of a mature fruit. However, most of the genetic and molecular mechanisms which coordinate such developmental events are yet to be identified and characterized. Arlequin (Alq), a semi-dominant T-DNA tomato mutant showed developmental changes affecting flower and fruit ripening. Sepals were converted into fleshy organs which ripened as normal fruit organs and fruits displayed altered ripening features. Molecular characterization of the tagged gene demonstrated that it corresponded to the previously reported TOMATO AGAMOUS-LIKE 1 (TAGL1) gene, the tomato ortholog of SHATTERPROOF MADS-box genes of Arabidopsis thaliana, and that the Alq mutation promoted a gain-of-function phenotype caused by the ectopic expression of TAGL1. Ectopic overexpression of TAGL1 resulted in homeotic alterations affecting floral organ identity that were similar to but stronger than those observed in Alq mutant plants. Interestingly, TAGL1 RNAi plants yielded tomato fruits which were unable to ripen. They displayed a yellow-orange color and stiffness appearance which are in accordance with reduced lycopene and ethylene levels, respectively. Moreover, pericarp cells of TAGL1 RNAi fruits showed altered cellular and structural properties which correlated to both decreased expression of genes regulating cell division and lignin biosynthesis. Over-expression of TAGL1 is able to rescue the non-ripening phenotype of rin and nor mutants, which is mediated by the transcriptional activation of several ripening genes. Our results demonstrated that TAGL1 participates in the genetic control of flower and fruit development of tomato plants. Furthermore, gene silencing and over-expression experiments demonstrated that the fruit ripening process requires the regulatory activity of TAGL1. Therefore, TAGL1 could act as a linking factor connecting successive stages of reproductive development, from flower development to fruit maturation, allowing this complex process to be carried out successfully.

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.

Responses to NaCl stress of cultivated and wild tomato species and their hybrids in callus cultures

SummaryIf in vitro culture is to be used for evaluating the salt tolerance of tomato hybrids and segregant populations in a breeding programme, it is previously necessary to get quick and reliable traits. In this work, growth and physiological responses to salinity of two interspecific hybrids between the cultivated tomato (Lycopersicon esculentum Mill) and its wild salt-tolerant species L pennellii are compared to those of their parents. The leaf callus of the first subculture was grown on media amended with 0, 35, 70, 105, 140, 175 and 210 mM NaCl for 40 days. Relative fresh weight growth of callus in response to increased salinity in the culture medium was much greater in L pennellii than in the tomato cultivars, and greater in the hybrids than in the wild species. Moreover, the different salt tolerance degree of hybrids was related to that of female parents. At high salt levels, only Cl− accumulation was higher in L pennellii than in tomato cultivars, whereas in the hybrids both Cl−, and Na+ accumulation were higher than in their parents. Proline increased with salinity in the callus of all genotypes; these increases were much higher in the tomato cultivars than in L pennellii, and the hybrids showed a similar response to that of the wild species. Salt-treated callus of the tomato cultivars showed significant increases in valine, isoleucine and leucine contents compared to control callus tissue. In contrast, these amino acids in callus tissues of the wild species and hybrids showed a tendency to decrease with increasing salinity.

A multisite gateway‐based toolkit for targeted gene expression and hairpin RNA silencing in tomato fruits

A collection of fruit promoters, reporter genes and protein tags has been constructed in a triple-gateway format, a recombination-based cloning system that facilitates the tandem assembly of three DNA fragments into plant expression vectors. The new pENFRUIT collection includes, among others, the classical tomato-ripening promoters E8 and 2A11 and a set of six new tomato promoters. The new promoter activities were characterized in both transient assays and stable transgenic plants. The range of expression of the new promoters comprises strong (PNH, PLI), medium (PLE, PFF, PHD) and weak (PSN) promoters driving gene expression preferentially in the fruit, and covering a wide range of tissues and developmental stages. Together, a total of 78 possible combinations for the expression of a gene of interest in the fruit, plus a set of five reporters for new promoter analysis, was made available in the current collection. Moreover, the pENFRUIT promoter collection is adaptable to hairpin RNA strategies aimed at tissue/organ-specific gene silencing with only an additional cloning step. The pENFRUIT toolkit broadens the spectrum of promoter activities available for fruit biotechnology and fundamental research, and bypasses technical difficulties of current ligase-dependent cloning techniques in the construction of fruit expression cassettes. The pENFRUIT vector collection is available for the research community in a plasmid repository, facilitating its accessibility.

Plant regeneration and Agrobacterium-mediated transformation of cotyledon explants of Citrullus colocynthis (L.) Schrad.

Abstract The effect of 6-benzylaminopurine (6-BA) alone or in combination with naphthaleneacetic acid or indoleacetic acid on the morphogenetic response of cotyledon explants of Citrullus colocynthis (L.) Schrad. was tested. The best results were obtained with a medium containing 25 µm 6-BA, which yielded organogenic calli at a frequency of 81.8%. When these organogenic calli were transferred to elongation medium (basal medium supplemented with 0.5 µm 6-BA), 80% produced well-developed shoots. These shoots rooted normally when cultured on rooting medium containing indolebutyric acid at 2.5 or 5.0 µm. Plants grew to maturity under greenhouse conditions and gave normal fruits. Cotyledon explants were transformed by cocultivation with Agrobacterium tumefaciens LBA4404 carrying the binary vector pBI121 which bears the reporter gene β-glucuronidase (gus) and the marker gene neomycin phosphotransferase (nptII). Transformants were selected for growth capacity on medium with 100 mgl–1 of kanamycin. On the basis of β-glucuronidase expression, the transformation frequency was 14.2%. Molecular characterization by polymerase chain reaction confirmed the presence of the two genes transferred (gus, nptII) in the transgenic plants. Sexual transmission of both genes was also confirmed by studying their expression in progenies from several transgenic plants.

A method for obtaining callus cultures from mesophyll protoplasts of melon (cucumis melo L.)

Abstract Calli were obtained from protoplasts of plants of three cultivars of Cucumis melo L. grown under axenic conditions. The composition of the culture medium in which the axenic plants were grown greatly influenced the rate of recovery of protoplasts capable of undergoing sustained divisions. Under the best conditions, cell wall regeneration was accomplished by up to 90% of the protoplasts and the first divisions were observed within 3–4 days of culture. After 20 days, the percentage of dividing cells was 26% in the cultivar ‘Valenciano Pinyonet’, 21% in the cv. ‘Pinyonet Piel de Sapao’ and 52% in the cv. ‘Cantaloup Charentais’. Two subsequent cultures on media with lower molarity led to calli with elevated growth rates. When plants of the first two cultivars were cultivated in a suboptimal medium, preculture of the leaves was essential in order to obtain protoplasts with mitotic activity.

Neutralizing antibodies against rotavirus produced in transgenically labelled purple tomatoes

Edible fruits are inexpensive biofactories for human health-promoting molecules that can be ingested as crude extracts or partially purified formulations. We show here the production of a model human antibody for passive protection against the enteric pathogen rotavirus in transgenically labelled tomato fruits. Transgenic tomato plants expressing a recombinant human immunoglobulin A (hIgA_2A1) selected against the VP8* peptide of rotavirus SA11 strain were obtained. The amount of hIgA_2A1 protein reached 3.6 ± 0.8% of the total soluble protein in the fruit of the transformed plants. Minimally processed fruit-derived products suitable for oral intake showed anti-VP8* binding activity and strongly inhibited virus infection in an in vitro virus neutralization assay. In order to make tomatoes expressing hIgA_2A1 easily distinguishable from wild-type tomatoes, lines expressing hIgA_2A1 transgenes were sexually crossed with a transgenic tomato line expressing the genes encoding Antirrhinum majus Rosea1 and Delila transcription factors, which confer purple colour to the fruit. Consequently, transgenically labelled purple tomato fruits expressing hIgA_2A1 have been developed. The resulting purple-coloured extracts from these fruits contain high levels of recombinant anti-rotavirus neutralizing human IgA in combination with increased amounts of health-promoting anthocyanins.