We investigate the regulatory mechanisms of stress-induced plant cell reprogramming to embryogenesis, key biotechnological process for improvement, regeneration and selection of high quality/adapted plants for agroforestry and industrial sectors. Our aim is to identify new intervention pathways to modulate determinant factors by small bioactive compounds to improve in vitro embryogenesis yield for crop/forestry breeding.
The extraordinary plasticity of plant cells permits them to generate new organs and embryos from almost all tissues. We analyze the cell and molecular basis of induction of cell reprogramming by stress, totipotency acquisition, embryogenesis initiation and progression, with the aim to identify new targets and strategies to improve yield, even in recalcitrant species. Specifically, stress-induced microspore embryogenesis is the fastest way to produce haploid and doubled-haploid (DHs) plants, which are important tools in breeding programs as a source of new genetic variability fixed in homozygous plants in only one generation, therefore saving time and costs. We study the model crop species of rapeseed and barley, and analyse the applicability of results to other species of agronomic or forestry interest, like cork oak.
The extraordinary plasticity of plant cells permits them to generate new organs and embryos from almost all tissues. We analyze the cell and molecular basis of induction of cell reprogramming by stress, totipotency acquisition, embryogenesis initiation and progression, with the aim to identify new targets and strategies to improve yield, even in recalcitrant species. Specifically, stress-induced microspore embryogenesis is the fastest way to produce haploid and doubled-haploid (DHs) plants, which are important tools in breeding programs as a source of new genetic variability fixed in homozygous plants in only one generation, therefore saving time and costs.
We study the model crop species of rapeseed and barley, and analyse the applicability of results to other species of agronomic or forestry interest, like cork oak. Through a multidisciplinary and integrated approach using techniques of in situ molecular identification, advanced light (confocal) and electron microscopy, physiology, and cell and molecular biology, we have determined that autophagy and several cell-death proteases are crucial players in the plant cell response to stress, while cell reprogramming and totipotency are regulated by hormones (auxins, cytokinins) and epigenetic modifications (DNA methylation, histone acetylation and methylation). Cell wall remodelling, operated by pectin methylesterases and arabinogalactan proteins (AGPs), is also involved and necessary for embryo development. We also investigate the effects of small compounds, modulators of autophagy, proteases, epigenetic marks, and other enzymes, to reduce cell death and improve embryogenesis yield in several crops; recent findings are opening up the development of new biotechnological approaches for improving in vitro embryo production in breeding programs.
Costa ML, Solís MT, Testillano PS, Coimbra S . In Situ/Subcellular Localization of Arabinogalactan Protein Expression by Fluorescent In Situ Hybridization (FISH). Methods in Molecular Biology 2149, 403-427.
Ibáñez S, Carneros E, Testillano PS, Pérez-Pérez JM . Advances in Plant Regeneration: Shake, Rattle & Roll. Plants, 9, 897.
Pérez-Pérez Y, El-Tantawy AA, Solís MT, Risueño MC, Testillano PS . Stress-induced microspore embryogenesis requires endogenous auxin synthesis and polar transport in barley. Frontiers in Plant Science 10:1200. DOI: 10.3389/fpls.2019.01200.
Testillano PS . Microspore embryogenesis: targeting the determinant factors of stress-induced cell reprogramming for crop improvement. Journal of Experimental Botany 70, 2965-2978.
Berenguer E, Solís MT, Pérez-Pérez Y, Testillano PS . Proteases with caspase 3-like activity participate in cell death during stress-induced microspore embryogenesis of Brassica napus. The EuroBiotech Journal, 3, 152-159.
Pérez-Pérez Y, Bárány I, Berenguer E, Carneros E, Risueño MC, Testillano PS. . Modulation of autophagy and protease activities by small bioactive compounds to reduce cell death and improve stress-induced microspore embryogenesis initiation in rapeseed and barley. Plant Signaling and Behavior 14:2, e1559577..
Pérez-Pérez Y*, Carneros E*, Berenguer E, Solís MT, Bárány I, Pintos B, Gómez-Garay A, Risueño MC, Testillano PS . Pectin de-methylesterification and AGP increase promote cell wall remodeling and are required during somatic embryogenesis of Quercus suber. Frontiers in Plant Science 9:1915. *Both authors contributed equally
Testillano PS, Gómez-Garay A, Pintos B, Risueño MC . Somatic embryogenesis of Quercus suber L. from immature zygotic embryos. Methods in Molecular Biology 1815:247-256.
Cortés-Eslava J, Gómez-Arroyo S, Risueño MC, Testillano PS . The effects of organophosphorus insecticides and heavy metals on DNA damage and programmed cell death in two plant models. Environmental Pollution 240, 77-86.
Avin-Wittenberg T, Baluška F, Bozhkov PV, Elander PH, Fernie AR, Galili G, Hassan A, Hofius D, Isono E, Le Bars R, Masclaux-Daubresse C, Minina EA, Peled-Zehavi H, Sánchez-Coll N, Sandalio LM, Satiat-Jeunemaitre B, Sirko A, Testillano PS, Batoko H . Review of Autophagy-related Approaches for Improving Nutrient Use Efficiency and Crop Yield Protection. Journal of experimental Botany 69, 1335-1353.
Bárány I*, Berenguer E*, Solís MT, Pérez-Pérez Y, Santamaría ME, Crespo JL, Risueño MC, Díaz I, Testillano PS . Autophagy and cathepsins are activated and involved in cell death during stress-induced microspore embryogenesis in barley. Journal of Experimental Botany 69, 1387-1402. *Both authors contributed equally.
Pérez-Pastrana J, Islas-Flores I, Bárány I, Álvarez-López D, Canto-Flicka A, Canto-Canché B, Peña-Yama L, Muñoz-RamíreZ L, Avilés-Viñas S, Testillano PS*, Santana-Buzzy N* . Development of the ovule and seed of Habanero chili pepper (Capsicum chinense Jacq.): Anatomical characterization and immunocytochemical patterns of pectin methyl-esterification. J. Plant Physiology 230, 1-12. *Co-corresponding authors.
Berenguer E, Bárány I, Solís MT, Pérez-Pérez Y, Risueño MC, Testillano PS . Inhibition of histone H3K9 methylation by BIX-01294 promotes stress-induced microspore totipotency and enhances embryogenesis initiation. Frontiers in Plant Sciences 8:1161.
Corredoira E, Cano V, Bárány I, Solís MT, Rodríguez H, Vieitez AM, Risueño MC, Testillano PS . Initiation of leaf somatic embryogenesis involves high pectin esterification, auxin accumulation and DNA demethylation in Quercus alba. J. Plant Phys. 213, 42-54.
Solís MT, Berenguer E, Risueño MC, Testillano PS . BnPME is progressively induced after microspore reprogramming to embryogenesis, correlating with pectin de-esterification and cell differentiation in Brassica napus. BMC Plant Biology 16, 176.
Projects currently in progress
AEI/FEDER, AGL2017-82447-R (2018-2020), PI: PS Testillano
COST Action CA15138 (TRANSAUTOPHAGY), EU (2015-2020) Chair: C. Vindis
H2020 MC-ITN 675132 (MASSTRPLAN), EU (2015-2019) CSIC Chair: D. Pérez-Sala.
Contract CSIC-Alcaliber I+D+i N.20171981 (2017-2020) PI: PS Testillano
Past projects (last years)
MINECO, AGL2014-52028-R (2015-2018), PI: PS Testillano
MINECO, BFU2015-71869-REDT, Network of Excellence in Autophagy Research, NEAR (2015-2017), PI: P. Boya.
MICINN, BFU2011-23752 (2012-2015), PI: PS Testillano
MICINN, AGL2008-04255 (2009-2011), PI: MC Risueño
MICINN, BFU2008-00203 (2009-2011), PI: PS Testillano
MICINN, Network of In vitro Culture and Genetic Transformation of Fruit Species, BIO2007-30945-E (2008-2011)
MICINN, Network of Genomics and Genetic Diversity in Forestry, GEN2FOR (2007-2011)
MEC, Network of Plant Development (2002-2011)
COST ACTION FA0903 (HAPRECI), EU (2011-2014). Chair: E. Albertini.
MINECO/BioFIG-Univ. Lisboa (Portugal), PRI-AIBPT-2011-0763. (2012-2015). PIs: PS Testillano/S Coimbra.
Univ. Palermo (Italy) CORI 66444 (2012-2014). PIs: PS Testillano/MA Germana.
CSIC/CNR/Univ. Palermo (Italy) 2008IT0046. (2012-2014) PIs: PS Testillano/L Baldoni.
CSIC/UNAM (Méjico) Programa Ignacio Bolívar (2010-1011) PIs: PS Testillano/J Cortés.