Mercedes Dabauza

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 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.

Somatic hybridization between an albino Cucumis melo L. mutant and Cucumis myriocarpus Naud.

Abstract Cotyledon-derived protoplasts of an albino Cucumis melo L. mutant were electrofused with protoplasts of Cucumis myriocarpus Naud. Putative somatic hybrids were selected on the basis of albinism complementation and the different behaviour of the protoplast-derived cells of the parental species in a definite sequence of culture media. Somatic hybrids were characterized at a molecular level and we confirmed that the green calli recovered after fusion were interspecific hybrids. RFLP patterns that identify C. melo albino mutant and C. myriocarpus genomes could be obtained by digestion of PCR-derived DNA fragments containing the 18S–25S ribosomal DNA (rDNA) spacer region with the endonuclease Sau3A. This procedure revealed that all of the putative somatic hybrids had the C. melo Sau3A pattern band and only one showed the Sau3A pattern bands corresponding to both parents. In order to confirm that the selected calli were hybrids, a specific oligonucleotide MYR2 was designed according to the sequence diversity within the internal transcribed spacer (ITS) regions of C. melo and C. myriocarpus. ITS1 and MYR2 primers were used to specifically amplify an rDNA fragment of 457 bp from C. myriocarpus. As expected, the selected calli recovered after the fusions, contained the C. myriocarpus genome. Since these calli also showed the RFLP melon pattern it was clear that these hybrids combined both parental genomes.

Regeneration and characterisation of Cucumis melo L. (+) Cucumis anguria L. var. longipes (Hook. fil.) Meeuse somatic hybrids

Cotyledon protoplasts of an albino Cucumis melo L. ‘Cantaloup Charentais’ somaclonal variant were electrofused with protoplasts of the wild species Cucumis anguria L. var. longipes (Hook. fil.) Meeuse. Selection of putative somatic hybrids was based on competence of the albino melon to grow and regenerate shoots together with the ability of the wild species to synthesise chlorophyll. The ITS region of C. anguria ribosomal DNA was sequenced to design species-specific primers, which allowed us to distinguish between parental lines and fusion products by PCR amplification. By using this method, all the organogenic lines characterised proved to be somatic hybrids. Three of sixteen selected lines produced shoots with albino and green sectors. Eleven lines remained green but shoots developed abnormally and did not produce roots in vitro. Two hybrid lines regenerated normal shoots but with a limited ability to produce roots in vitro and in vivo. Applicability of molecular characterisation to optimise the quick recovery of fusion products is discussed.

Micropropagation from apical and nodal segments of Bituminaria bituminosa and the furanocoumarin content of propagated plants

Summary An efficient micropropagation protocol has been developed for Bituminaria bituminosa, a species considered to be of pharmaceutical interest for its furanocoumarin (FC) content. Four accessions of B. bituminosa with different FC contents were used: variety albomarginata (“albo-tedera”), variety bituminosa (“bitu-Calnegre”), var. albomarginata × var. bituminosa (“albo-hybrid”), and var. bituminosa × var. albomarginata (“bitu-hybrid”). Different concentrations of sodium troclosene solution were evaluated for in vitro establishment of apical and nodal explants cultured on modified Murashige and Skoog (MS) medium. The effects of the type of agar and the addition of activated charcoal (AC) were also evaluated. The best treatment for surface sterilising the explants was 3.5 g l–1 sodium troclosene. The lowest frequency of hyperhydrated shoots was achieved using 0.8% (w/v) American bacteriological agar. Apical explants resulted in greater shoot proliferation than nodal explants. The addition of AC increased the quality and development of regenerated shoots. The highest average numbers of shoots ≥ 5 mm per explant were obtained from apical explants of “albo-tedera” (7.8 shoots) and “albo-hybrid” (7.6 shoots) on a modified MS medium supplemented with plant growth regulators (T2 media) and for “bitu-Calnegre” (6.3 shoots) and “bitu-hybrid” (5.3 shoots) on a modified MS medium without plant growth regulators (T1 media). The regenerated shoots rooted efficiently on different media depending on the plant material. Plantlets were acclimatised and maintained in a greenhouse until they were transferred to the experimental field. The total FC contents in micropropagated field grown plants were found to be higher or equal to those in the non-micropropagated mother plants. The accession “bitu-Calnegre” was found to have higher, or equivalent FC levels to those in other species such as Psoralea corylifolia and Ruta graveolens, reported as sources of FC. All micropropagated plants maintained the characteristics of the non-micropropagated mother plants.

Development of highly efficient genetic transformation protocols for table grape Sugraone and Crimson Seedless.

Genetic engineering of grapevine is a powerful tool to study gene function as well as to introduce new traits into existing Vitis cultivars without altering their essential characters and identity. Agrobacterium-mediated transformation is one of the most efficient methods for gene transfer, but the efficiency of the procedure depends on several parameters such as the grapevine genotype, the selection strategy, the Agrobacterium strain, and concentration used to infect as well as the culture method among others. This chapter describes highly efficient genetic transformation protocols for seedless table grapevine cultivars Sugraone and Crimson Seedless by co-culturing embryogenic calli with Agrobacterium tumefaciens. The procedures are specific for each cultivar by adjusting the kanamycin concentration used to select transformed cells (20 mg/L and 50 mg/L kanamycin for Crimson Seedless and Sugraone, respectively) and the low Agrobacterium density used to infect the embryogenic calli (0.06 OD(600) being more effective for the transformation of Crimson Seedless and 0.2 OD(600) for Sugraone). Other factors that affect the transformation efficiency are the initial amount of embryogenic calli used to co-culture with Agrobacterium and the culture method of calli.

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.

Embryogenesis and plant regeneration of the perennial pasture and medicinal legume Bituminaria bituminosa (L.) C.H. Stirton

Abstract. Bituminaria bituminosa (common name tedera) is a drought-tolerant perennial pasture species of agronomic and pharmaceutical interest for Mediterranean climates. Considering the importance of this legume, in vitro experiments were conducted to develop protocols for plant regeneration from embryogenic calli of leaves, petioles and anthers to efficiently exploit and maintain selected important clones from the tedera breeding program. The type of explant was a key factor in the frequency of embryogenesis and the number of embryos per callus. For plant regeneration from cultured anthers, appropriate anther physiological state (uninucleate stage of microsporogenesis), stress treatments (electroporation, 25 Ω, 25 µF, 1500 V) and culture conditions were determined. A robust flow-cytometry method was developed to analyse the ploidy status of callus, in vitro shoots and in vivo acclimatised plants derived from anther and leaf explants.