Marián López

Agrobacterium tumefaciens-mediated transformation of Pinus pinea L. cotyledons: an assessment of factors influencing the efficiency of uidA gene transfer

Abstract This study is the first report of a protocol for transfer and expression of foreign chimeric genes into cotyledons excised from Pinus pinea L. embryos. Agrobacterium tumefaciens EHA105 harbouring the plasmid p35SGUSint was more infective than LBA4404 or C58 GV3850, as determined by the percentage of cotyledons showing uidA expression. Factors which significantly affected the T-DNA transfer included: (1) preinduction and concentration of bacteria, (2) days of coculture and (3) the wounding procedure applied. More efficient transfer of the uidA gene was achieved growing the bacteria in YEP medium at pH 7, infecting the cotyledons according to the sonication-assisted Agrobacterium-mediated transformation procedure with a bacterial density of 1 (OD600 nm) for 5 min, and coculture for 72 h. Using this protocol, 49.7% of the cotyledons showed a diffuse blue staining 7 days after infection. However, all were necrotic 30 days after inoculation. Since a decrease in bacterial density to 0.01 allowed the recovery of about 4% of cotyledons forming buds 1 month after inoculation, we conclude that the high mortality associated with the infection may be related to the hypersensitive response of the plant to bacterial infection.

Regeneration of plants from isolated cotyledons of salgareño pine (Pinus nigra Arn. ssp.Salzmannii (Dunal) Franco)

SummaryPlantlet regeneration of salgareño pine (Pinus nigra Arn. ssp.salzmannii (Dunal) Franco) was achieved from cotyledons. The data showed that the best differentiation response occurred when cotyledons, from 1-d-old embryos germinated in darkness, were cultured on Murashige and Skoog medium (half-strength macroelements) with 22 μM N6-benzyladenine (BA) and 0.05 μM α-naphthaleneacetic acid (NAA) for 2–3 wk. Shoot development was obtained by subculturing treated explants on the same medium without growth regulators. Shoots were successfully micropropagated by sequential subculturing them on medium containing growth regulators (5 μM BA and 0.005 μM NAA) and on hormone-free medium for 2 and 12 wk, respectively. To induce adventitious roots, shoots were treated with 3 μM NAA, and 8 μM indole-3-butyric acid for 2 wk, followed by transfer to Murashige and Skoog medium (1/4-strength macroelements, 20 g·L−1 sucrose) without growth regulators. After 3–4 wk, almost all the rooted shoots (65%) could be successfully transplanted and acclimatizated in the greenhouse, where the plants exhibited normal growth habit.

Transient expression of the uidA gene in Pinus pinea cotyledons: A study of heterologous promoter sequences

Transfer and expression of the β-glucuronidase gene (uidA) in cultured cotyledons of stone pine (Pinus pinea L.) was obtained by microprojectile bombardment. Conditions for optimum transient expression were established by using plasmid pBI121 delivered by 1.0 μm-diameter gold particles, into 1-day-old cultured cotyledons. Helium pressure of 6.2 MPa, microcarrier travel distance of 6 cm, and 0.8 μg of plasmid DNA per bombardment, were the best parameters for high levels of transient uidA expression. By using these parameters, 98% of bombarded cotyledons showed β-glucuronidase activity, with a mean of 63 Gus foci per cotyledon. This system was used to study the expression of uidA gene driven by several heterologous promoters. The expression under the control of the sunflower polyubiquitin gene (UbB1) promoter (Δ1 deletion) was higher (99% of GUS positive cotyledons) than under the control of the CaMV35S promoter, whereas the rice actin and the maize alcohol dehydrogenase gene promoters gave lower uidA expression, as determined histochemically. These results were confirmed by using the GUS fluorometric assay. Use of a deletion of the sunflower polyubiquitin promoter resulted in GUS activity detectable 35 days after bombardment, and significant levels of GUS activity were confirmed at the end of that period. The results will be useful to design protocols for stable transformation and high levels of transgene expression in P. pinea.

Modifying transient β-glucuronidase expression in pine species using introns

The effect of introns on gene expression was evaluated. Several intron-promoter combinations were introduced by microparticle bombardment into two pine species, stone pine (Pinus pinea L.) and salgareño pine (Pinus nigra Arn. ssp. Salzmannii (Dunal) Franco). Gene expression was evaluated by measuring transient GUS expression. Two promoters (CaMV35S and double CaMV35S modified) and two introns (intron 1 from maize genes alcohol dehydrogenase-1 and Shrunken-1) were used in our study. In both pine species tested, the Sh1-int1 increased transient GUS expression from 2 to 6-fold compared to the intron-less construction. On the contrary, the inclusion of the Adh1-int1 associated with the double CaMV35S modified resulted in a dramatic decrease in the expression in both pine species analyzed. Our results suggest that Sh1-int1 may be useful for the acquisition of the required levels of genetic activity of new agronomic traits introduced into pines.

Factors involved in Agrobacterium tumefaciens-mediated gene transfer into Pinus nigra Arn. ssp. salzmannii (Dunal) Franco.

Cotyledons from dissected sterile embryos of salgareño pine (Pinus nigra Arn. ssp. salzmannii (Dunal) Franco) were inoculated with different disarmed Agrobacterium tumefaciens strains harbouring the binary vector p35SGUSint. The transient expression of a β-glucuronidase gene (uidA) was studied, using a histochemical staining procedure. Nineteen days after inoculation, the activity of β-glucuronidase was detected in epidermal and subepidermal layers of cotyledonary explants. The EHA105 strain harbouring a disarmed agropine-type Ti-plasmid (pTiBO542) was the most effective for gene transfer of the uidA gene. The effects of exudates and extracts from 0-day-old embryos on induction of vir gene expression in A. tumefaciens were also examined. The results of this study showed that salgarño pine embryo exudates contain a substance(s) that induce vir gene expression, in similar way to that observed with 100 μM acetosyringone (AS).All these findings suggest that T-DNA processing and transfer might take place when Agrobacterium infects suitable tissues of salgareño pine.

Foreign Gene Expression in Pinus nigra, P. radiata and P. pinea Following Particle Bombardment

Using particle bombardment we have developed efficient protocols for transient gene expression in excised cotyledons of three pine species, Pinus nigra Arn. ssp. salzmanii (Dunal) Franco (black pine), P. radiata D. Don (radiata pine), and P. pinea L. (stone pine). We have used the plasmid pBI121 to set up the bombardment parameters for transient foreign gene expression, by means of histochemical s-glucuronidase (GUS) assays 24 h after bombardment. Although gusA gene expression was obtained for all pine species, bombardment parameters were different for each one. In black pine, the best results of gusA gene expression were obtained using 1 μm gold particles covered with 0.8 μg plasmid DNA per bombardment, 4.5 MPa of helium pressure and 6 cm of bombardment distance. In radiata pine, 1.6 μm gold particles gave the best results, using 0.8 μg plasmid DNA, 7.6 MPa and 6 cm. In addition, 8-day-old cultured cotyledons gave better results than younger expiants. In stone pine, the best levels of gusA gene expression were obsserved when 1 μm gold particles, 0.8 μg plasmid DNA, 6.2 MPa, and 6 cm were used. In addition, we have used this approach to test the effect of different promoters on gusA gene expression in pine cotyledons, those of the sunflower ubiquitin, the maize alcohol dehydrogenase, and the rice actin genes, as well as the doubled CaMV 35S promoter. The sunflower ubiquitin gene promoter gave the best results, confirmed by using fluorometric GUS assay. On the other hand, the use of the sunflower ubiquitin gene promoter allowed to last the expression of the gusA gene until 20 days of culture of radiata pine cotyledons after bombardment.

Induction of a virulence response in Agrobacterium tumefaciens by exudates of Pinus pinea cotyledons

As a preliminary step in efforts to develop a successful protocol for Agrobacterium-mediated transformation of cotyledonary explants of Pinus pinea L. embryos, we tested the ability of embrionary exudates of this species to induce the expression of the virulence genes virA, virB, virC, virD, virE and virG in Agrobacterium tumefaciens containing vir: lacZ fusion constructs. The results obtained in the vir induction assay indicated the absence of bactericidal or bacteriostatic plant compounds affecting A. tumefaciens growth, and showed that cotyledonary and embrionary exudates of P. pinea are able to induce all virulence genes studied, except virG. The data suggest that A. tumefaciens can be used for gene transfer into this important forest and fruit species.