The increasing social demand for global food security in the face of a changing climate, decreasing natural resources and increasing world population, is pushing the search for new crop varieties, with increasing yield and better adapted to new environmental conditions. Present agriculture faces significant economic pressure worldwide for more efficient and accelerated breeding of crops. In this context, breaking knowledge and efficient technologies to exploit plant cell in vitro reprogramming potential for selected plant regeneration and propagation are highly valuable and needed in plant breeding and biotechnology, as well as in applied and basic plant research.
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. We also analyzed plant cell responses to stress conditions. Our aim is to identify new pathways and molecular targets for chemical intervention by small bioactive compounds to improve in vitro cell response to stress, embryogenesis and plant regeneration yield for crop/forestry breeding.
When reprogramming is induced in the haploid microspore (immature pollen), the process is named stress-induced microspore embryogenesis and constitutes the fastest way to produce haploid and doubled-haploid (DHs) plants. DHs are widely used by seed and horticulture companies as a source of new genetic variability fixed in homozygous plants in only one generation, therefore saving time and costs of breeding programs. When in vitro embryogenesis is induced in other somatic cells, somatic embryogenesis is very useful for large-scale clonal propagation and cryopreservation of elite genotypes in species with long reproductive cycles or low seed set, as many forest species.
We perform our investigation in the model crop species rapeseed and barley, and analyse the transfer of results to other species of agronomic or forestry interest, like cork oak. We use a multidisciplinary and integrated approach with techniques of plant physiology, molecular and cell biology.
MAIN RESEARCH LINES
Stress-induced cell death: We investigate the role of autophagy and cell-death proteases as crucial players in the balance between death and life in the plant cell response to stress conditions required to induce reprogramming towards embryogenesis. The aim is to modulate this response to increase cell viability and embryogenesis yield.
Hormonal regulation of stress-induced cell reprogramming and embryogenesis: Our studies aim to characterize the involvement of phytohormones, mainly auxins and cytokinins, in regulation of cell reprogramming, totipotency acquisition and embryogenesis initiation and progression. Their biosynthesis, intercelular transport, signalling and appropriate auxin/cytokinin balance are required for correct embryogenesis induction and progression.
Epigenetic control of cell reprogramming to embryogenesis: We analyze the role of epigenetic marks, particularly DNA methylation, histone H3-H4 acetylation and H3K9 methylation, among other modfications, in the regulation of the swicht of cell fate. We have identified genome-wide changes of these chromatin modifications that accompany cell reprogramming, and found that define epigenetic modulators can induce such changes and enhance reprogramming.
Cell wall remodelling: Our studies have revealed that remodelling of cell wall, operated by pectin methylesterases and arabinogalactan proteins (AGPs), is induced and neccessary for cell reprogramming and embryo formation.
Small molecules to improve reprogramming of cell fate for new biotech strategies: Recent advances in chemically-controlled reprogramming of specialized cells the great potential of application of small chemical molecules to regulate cellular reprogramming. We investigate the effects of small compounds, by screening of chemical libraires and with modulators of autophagy, proteases, epigenetic marks, and other enzymes, to reduce cell death and improve embryogenesis yield. These small molecules are opening up the development of new biotechnological approaches for improving in vitro embryo production in breeding programs.
Pérez-Pastrana J, Testillano PS, Barany I, Canto-Flick A, Álvarez-López D, Pijeira-Fernández G, Avilés-Viñas SA, Peña-Yam L, Muñoz-Ramírez L, Nahuat-Dzib S, Islas-Flores I, Santana-Buzzy N . Endogenous auxin accumulation/localization during zygotic and somatic embryogenesis of Capsicum chinense Jacq. J. Plant Physiology, Published on line 15-Dec-2020. DOI: 10.1016/j.jplph.2020.153333.
Berenguer E, Minina EA, Carneros E, Bárány I, Bozhkov PV, Testillano PS . Suppression of metacaspase and autophagy-dependent cell death improves stress-induced microspore embryogenesis in Brassica napus. Plant and Cell Physiology, DOI: 10.1093/pcp/pcaa128.
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.
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
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.
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.
Rodríguez-Sanz H, Manzanera JA, Solís MT, Gómez-Garay A, Pintos B, Risueño MC, Testillano PS . Early markers are present in both embryogenesis pathways from microspores and immature zygotic embryos in cork oak, Quercus suber L. BMC Plant Biology 14, 224.
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.
Rodríguez-Sanz H, Solís MT, López MF, Gómez-Cadenas A, Risueño MC, Testillano PS . Auxin biosynthesis, accumulation, action and transport are involved in stress-induced microspore embryogenesis initiation and development in Brassica napus L. Plant and Cell Physiology 56, 1401-1417.
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.
Solís MT, El-Tantawy AA, Cano V, Risueño MC, Testillano PS . 5-azacytidine improves microspore embryogenesis initiation by decreasing global DNA methylation but prevents subsequent embryo development in rapeseed and barley. Frontiers in Plant Science 6, 472. Doi: 10.3389/fpls.2015.00472.
Solís MT, Chakrabarti N, Corredor E, Cortés-Eslava J, Rodríguez-Serrano M, Biggiogera M, Risueño MC, Testillano PS . Epigenetic changes accompany developmental programmed cell death in tapetum cells. Plant Cell and Physiology 55, 16-29.
Projects currently in progress
AEI/FEDER, AGL2017-82447-R: New strategies with small molecule modulators of key processes in stress-induced microspore reprogramming to embryogenesis for crop and forest breeding (2018-2021), PI: PS Testillano
COST Action CA19125 (EPI-CATCH: EPIgenetic mechanisms of Crop AdapTation to Climate cHange) EU (2020- 2024). Chair: F. Martinelli, Managing Committee and Core group member: PS Testillano
COST Action CA15138 (TRANSAUTOPHAGY):European Network of Multidisciplinary Research and Translation of AutophagyKnowledge , EU (2015-2020) Chair: C. Vindis
Contract CSIC-Alcaliber I+D+i N.20171981: Analyses of key factors in the in vitro culture of anthers and embryogenesis induction for doubled-haploid production in Papaver somniferum. (2017-2021) 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, 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)
H2020 MC-ITN 675132 (MASSTRPLAN), EU (2015-2019) CSIC Chair: D. Pérez-Sala.
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.
EP19383042. Mammal kinase inhibitors to promote in vitro embryogenesis induction of plants. Priority: Europe, Date: 27-Nov-2019. Authors: TESTILLANO PS, MARTÍNEZ A, GIL C, BERENGUER E, CARNEROS E, PÉREZ-PÉREZ Y.
Contracts with Companies
Contract CSIC-Alcaliber I+D+i (N.20171981): Analyses of key factors in the in vitro culture of anthers and embryogenesis induction for doubled-haploid production in Papaver somniferum. (2017-2021) PI: PS Testillano