Biotecnología y sus efectos en los procesos de producción

Biología y tecnología se unen para ofrecer alternativas eficientes y de calidad e ámbitos como la salud, la industria o la acuicultura.Su gran impulso ha dado lugar a cientos de proyectos de investigación y empresariale

Regeneration of plants from somatic embryos of Verticillium dahliae-resistant wild olive genotypes

Regeneration capacity, via somatic embryogenesis, of several wild olive genotypes differing in their response to Verticillium wilt (resistant genotypes Stop Vert, Out Vert, Ac4 and Ac 18 and the susceptible Ac 15) has been evaluated. To induce somatic embryogenesis, methodologies previously used in cultivated (high ratio cytokinin/auxin) or wild olive (low ratio cytokinin/auxin) were used. Obtained results revealed the importance of genotype, explant type, mineral formulation and hormonal balance in the induction process, ca. use of apical buds obtained from micropropagated shoots following the methodology of Mazri et al. (2013) in cultivated olive (4 days in liquid medium MS ½, 30 µM TDZ – 0.54 µM ANA, 8 weeks in basal medium MS ½, followed by subculturing in ECO basal medium supplemented with 0.5 µM 2iP, 0.44 µM BA and 0.25 µM IBA) was adequate to obtain somatic embryos in 2 genotypes, Stop Vert and Ac18, but no embryogenic response was observed in the other three. An analysis of genetic stability on Stop Vert, using SSR and RAPDs markers, was carried out in embryogenic callus, plants regenerated form this callus and micropropagated shoots in comparison with the mother plant. Polymorphism was only observed in the banding pattern generated by RAPDs in one of the 10 callus samples evaluated, resulting in a variation rate of 0.07%. This is the first time in which plants have been regenerated via somatic embryogenesis in wild olive.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Research project: Junta de Andalucía P11-AGR799

Somatic embryogenesis in explants of adult wild olive trees

In this investigation, somatic embryogenesis in explants of adult wild olive trees, using the protocol developed by Mazri et al. (2013, Sci. Hort. 159: 88-95), was induced. Four genotypes differring in the level of resistance to the fungal pathogen Verticillium dahliae were used: Ac18, StopVert and OutVert (symptomless resistant genotypes, Jiménez-Fernández et al. 2015, Plant Pathology, in press) and Ac15 (susceptible genotype, Jiménez-Díaz, IAS-CSIC, Córdoba, personal communication). Three types of explants from actively growing cultures were used: first pair of leaves, petioles and shoot apex. Firstly, all explants were cultured on a liquid induction medium with MS mineral elements at 0.5X and 30 μM TDZ-0.5 μM NAA, for 4 days at 80rpm. Afterwards, explants were transferred to basal MS with 0.5X macroelements, for 8 weeks. Finally, calli were cultured on expression ECO medium supplemented with 0.25 μM IBA, 0.5 μM 2ip and 0.44 μM BA, for several subcultures. Cultures were incubated under darkness at 25 degrees. Embryogenic calli were observed on shoot apex (StopVert) or leaf primordia (Ac18) explants.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Heterologous expression of AtNPR1 gene in olive for increasing fungal tolerance

The NPR1 gene encodes a key component of SAR signaling mediated by salicylic acid (SA). After a pathogen infection, the accumulation of SA releases NPR1 monomers in the cytosol that are translocated to the nucleus, activating the expression of pathogenesis-related (PR) genes. Overexpression of NPR1 has conferred resistance to fungal, viral and bacterial pathogens in several plant species. The aim of this research was to generate transgenic olive plants expressing the gene AtNPR1 from Arabidopsis thaliana to obtain material resistant to fungal pathogens. Three transgenic lines expressing AtNPR1 gene under the control of the constitutive promoter CaMV35S were obtained following the protocol of Torreblanca et al. (2010), using an embryogenic line derived from a seed of cv. Picual. Level of AtNPR1 expression in transgenic calli varied greatly among the different lines, being higher in the line NPR1-780. The elicitation of embryogenic calli in liquid medium with AS did not increase endochitinase activity, a PR protein. However, jasmonic acid induced a transient increase in chitinase activity after 24 h of treatment in all the lines, being the increment higher in transgenic NPR1 than in control. After maturation and germination of transgenic somatic embryos, plants were micropropagated and acclimated to ex vitro conditions. The expression of AtNPR1 did not alter the growth of transgenic plants neither in vitro nor in the greenhouse. Experiments are in progress to determine the resistance of transgenic AtNPR1 plants to V. dalihae and R. necatrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Research projects: Plan Nacional AGL2014-52518-C2-1-R; AGL2017-83368-C2-1-R and Junta de Andalucía P11-AGR799

Enhancing shoot recovery from transgenic avocado somatic embryos

Use of biotechnological tools in avocado (Persea americana Mill.) is hampered by difficulties in obtaining mature somatic embryos with an acceptable germination capacity. Use of semi-permeable cellulose acetate membranes on top of maturation medium has improved the quality of obtained embryos and their germination rate; however, in the case of transgenic embryos the conversion rate is still rather low. In this investigation, a protocol for recovery of transgenic plants has been developed. Mature avocado somatic embryos, over cellulose acetate membranes, were obtained and induced to germinate following the protocol described in Palomo-Ríos (2012). Some transgenic embryos developed buds ≤ 2 mm, which failed to elongate. For shoot recovery, cotyledons were partially removed and the embryonic axis cultured over 4 weeks in MS medium supplemented with either 1 mg/l BA, 1 mg/l TDZ or 1 mg/l BA and 1 mg/l TDZ. Highest shoot recovery was obtained in medium supplemented with 1 mg/l BA and 1 mg/l TDZ, 53.6±6.7% versus 23.2±5,7% and 39.6±6.4% in media supplemented with 1 mg/l BA or 1 mg/l TDZ, respectively. Afterwards, sprouted embryos were cultured over 4 additional weeks in MS medium supplemented with 1 mg/l BA. In some cases, resulting shoots could be induced to proliferate in GD medium (Gamborg et al., 1968) supplemented with 0.3 mg/l BA, while in other cases, they had to be recovered through micrografting onto in vitro germinated seedlings as described in Pliego-Alfaro and Murashige (1987). MS medium supplemented with 1mg/l BA and solidified with 6 g/l Sigma A-1296 agar, was used for micrografts. Shoots derived from micrografts were either induced to proliferate in GD medium with 0.3 mg/l BA or further grafted and cultured in liquid MS medium supplemented with 0.1 mg/l BA, using perlite as substrate. After 8-12 weeks, micrografts could be transferred to ex vitro conditions. References: Gamborg O.L., Muller, R.A., Iojima, K., (1968). Nutrient requirements of suspension cultures of soybean roots cells. Exp. Cell. Res. 50:151-158. Palomo-Ríos, E., (2012). Thesis: Regeneración y Transformación Genética de Aguacate (Persea americana Mill.). Ph.D. Dissertation, University of Málaga, Málaga Pliego-Alfaro, F., Murashige, T., (1987). Possible rejuvenation of adult avocado by graftage onto juvenile root stocks in vitro. Hort Sci. 22:1321-1324.AGL2011-30354-C02-0

Optimización de la transfección de protoplastos para la edición génica en fresa

Esta investigación ha sido financiada por los fondos FEDER EU, el Ministerio de Economía y Competitividad de España (AGL2017-86531-C2-1-R), y el contrato FPI PRE2018-085509.La fresa es un fruto blando de gran importancia económica, particularmente en Andalucía. La mejora de las cualidades organolépticas del fruto y la disminución del reblandecimiento para alargar la vida postcosecha del fruto, son unos de los principales objetivos de los programas de mejora en este cultivo. El reblandecimiento del fruto es consecuencia del desmantelamiento de la pared celular, la disolución de la lámina media y la pérdida de turgencia. En fresa, el silenciamiento mediante la transformación en antisentido de genes que codifican poligalacturonasas (PG) aumenta la firmeza del fruto y la vida postcosecha (Paniagua et al., 2020). Por tanto, estos genes son excelentes dianas para la edición génica con el fin de mejorar la calidad del fruto de la fresa. La transfección de protoplastos con complejos preensamblados Cas9-sgRNA permite la producción de plantas editadas vía CRISPR/Cas9 libres de ADN foráneo, que podrían ser consideradas como no transgénicas. En esta investigación, se ha optimizado un protocolo para la transfección de protoplastos de fresa, con el objetivo final de producir plantas no transgénicas con el gen de poligalacturonasa FaPG1 mutado. Como fuente de material vegetal se utilizaron hojas de plantas de Fragaria x ananassa, cv. ‘Chandler’, micropropagadas en medio Murashige y Skoog (MS) suplementado con 2 mg/L de BA. Para la extracción de protoplastos se utilizó el protocolo descrito por Barceló et al. (2019). A las 24 h del aislamiento, los protoplastos fueron transfectados con el plásmido pHBT-sGFP(S65T)-NOS que contiene el gen marcador GFP, mediante un tratamiento con polietilenglicol (PEG), como se describe en Yoo et al. (2007). Se evaluaron, entre otras variables, el efecto de la concentración y tiempo de incubación en PEG y la concentración de ADN. Los valores más altos de protoplastos con actividad GFP a las 48 h de la transfección, entre el 15-18%, se obtuvieron tras la incubación en 20% de PEG en presencia de 5 µg de ADN.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Agrobacterium-mediated transformation of olive (Olea europaea L.) with an antifungal protein from Aspergillus giganteus.

Broad-spectrum resistance to pests and diseases is difficult to obtain through classical breeding programs, hence, this is a targeted trait for accelerating the development of major olive cultivars using plant trans- formation technologies. Olive Verticillium wilt, caused by Verticillium dahliae, is considered to be an important constraint for cultivation of olive trees (López-Escudero and Mercado-Blanco 2010). Different transgenic approaches have been proposed to engineer plants for resistance to fungal diseases, including production of antifungal proteins (Gurr and Rushton 2005). Regarding this approach, among different anti- fungal compounds, the antifungal protein (AFP) from Aspergillus giganteus can be considered a promising candidate for practical applications in crop protection (Meyer 2007). AFP is a defensin-like protein that belongs to a group of small-sized secretory proteins rich in cysteine residues. The protein possesses in vitro antifungal activity inhibiting the growth of several fungal pathogens. Previous work has already shown that afp gene can be expressed in transgenic rice plants inducing resistance to the fungus Magnaporthe grisea and indicating the usefulness of such approach for protection against rice blast. (Coca et al. 2004). In this work, transgenic olive plants were generated by Agrobacterium-mediated transformation as des- cribed by Torreblanca et al. (2010). The AGL-1 strain containing the pBIN61-afp binary vector was used. This plasmid contains the nptII gene for paromomycin selection and a chemically synthesized codon-op- timized afp gene under the control of the 35S CaMV promoter. Globular somatic embryos derived from a mature seed of cultivar `Picual ́ were transformed obtaining an average success rate around 2%. Plants were regenerated from six independent lines and transgenic nature was confirmed by PCR studying nptII and afp insertion. With the aim of studying whether the afp gene can be used to induce resistance against fungal diseases in olive, susceptibility to the fungal pathogens Rosellinia necatrix and Verticilium dahliae will be evaluated. In addition, the inhibitory effect of proteins extracts from transgenic leaves on the in vitro growth of these fungal pathogens will also be examined.Universidad de Málaga. Campus de Excelencia Internacional Andalucia Tec

Expression analysis of Flowering related genes in olive plants transformed with the "Medicago truncatula" FT gene MtFTa1

Olive tree (Olea europaea L.) forms inflorescences in lateral buds that flower in spring. Flowering occurs due to the presence of a mobile flower-promoting factor called florigen, the product of “FLOWERING LOCUS T” (FT). In many plants, FT and TERMINAL FLOWER 1 (TFL1) genes encode related proteins with opposite functions, i.e. FT induces flowering, while TFL1 represses it. Olive flower induction seems to be mediated by an increase in FT levels in response to cold winters. Because of climate change, warmer winters are expected, which can alter flowering time. Three olive transgenic lines containing the MtFTa1 gene from Medicago truncatula were obtained (FT5, FT7 and FT15) to study the effect of FT on flowering time (Haberman et al., 2017). The embryogenic line P1 from a seed of cv. Picual was used for transformation, and also as control (CP1). FT7 flowered continuously; FT5 did not flower and showed a dwarf branching phenotype, and FT15 had a dwarf-branching habit and developed abnormal flowers. The expression of the transgene and three endogenous genes (OeFT1, OeFT2 and OeTFL1-1) was analyzed in these juvenile plants, as well as in the control (CP1), throughout the year (autumn, winter and spring).Proyecto de Excelencia P11-AGR-7992, Junta de Andalucí

Evaluación del efecto del filtrado crudo del hongo Rosellinia necatrix en el crecimiento de brotes y cultivos embriogénicos de olivo

Rosellinia necatrix es un patógeno muy extendido en plantaciones de aguacate del sur de la Península Ibérica, afectando también, ocasionalmente, a plantaciones de olivo. Nuestro grupo está abordando la obtención de variantes somaclonales de olivo y aguacate, que muestren mayor tolerancia a este patógeno. En este trabajo se presentan los resultados en olivo, tras la exposición de cultivos embriogénicos a dosis crecientes de filtrado crudo del hongo. Se utilizó callo embriogénico, obtenido a partir de radícula, cultivado en medio líquido ECO (Pérez-Barranco et al. 2009 PCTOC 97: 243-251) durante 3 semanas en agitación, y posteriormente filtrado (malla de 2 mm) para separar la fracción fina. Esta fracción fue cultivada durante otras 3 semanas en medio ECO líquido conteniendo un 20, 40, 60 u 80% de filtrado crudo de Rosellinia necatrix, tras su esterilización en frio. Finalmente, el callo embriogénico fue cultivado en placa en medio ECO sólido para su recuperación. El filtrado del hongo al 20% no afectó al crecimiento del callo embriogénico; sin embargo, a partir del 40% el crecimiento fue inhibido, salvo en algunas placas, donde se observó proliferación de callo tras 2 meses en medio de cultivo sin filtrado. Se han obtenido varias líneas que han sobrevivido tras la exposición al 40, 60 y 80% de filtrado del hongo. Estas líneas están siendo cultivadas de nuevo en presencia de la misma concentración de filtrado del hongo para evaluar su comportamiento. Posteriormente, se abordará la recuperación de plantas. Paralelamente, brotes de olivo regenerados a partir de la línea embriogénica P1 han sido cultivados en medio de multiplicación (Vidoy-Mercado et al. 2012, Acta Hort. 949: 27-30) suplementado con filtrado crudo del hongo (60%), durante varios subcultivos, para evaluar el efecto del filtrado en el crecimiento de los brotes.Universidad de Málaga, Campus de Exelencia Internac ional Andalucía Tech. Proyecto AGR-7992 (P11-Junta de Andalucía

Caracterización de variantes somaclonales de olivo obtenidos tras la exposición al filtrado crudo del hongo Rosellinia necatrix.

Líneas embriogénicas de olivo tolerantes al filtrado crudo (FC) del hongo Rosellinia necatrix, un patógeno muy extendido por el sur de España y que afecta al aguacate y al olivo, fueron previamente obtenidas (Palomo-Ríos et al. 2017). Dos de estas líneas fueron seleccionadas para la regeneración de plantas, R2-L3 y R2-L4, ambas tolerantes al 60% (v/v) del FC del hongo. En este trabajo se ha llevado a cabo una caracterización del crecimiento de estas plantas y de su comportamiento tras la inoculación con R. necatrix.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech