The stress-induced cell reprogramming towards embryogenesis of the pollen is a powerful biotechnological tool for plant breeding, as the fastest way for rapid generation of new varieties through double haploid plants. The characterization of its regulating mechanisms will permit to exploit the process more efficiently for sustainable agriculture, selection of forest resources and environment control. Cellular and molecular determinants of the process that we are currently investigating are: AUTOPHAGY, PROGRAMMED CELL DEATH, EPIGENETIC REGULATION, PHYTOHORMONE DYNAMICS and CELL WALL REMODELLING.
As a complementary research line, we are also studying new possibilities for the treatment of phytopathologies with NANOBIOTECHNOLOGY approaches, by using magnetic nanoparticles and Quantum Dots for diagnosis and treatments with minimum doses.
Stress-induced pollen reprogramming to embryogenesis: biotechnology tool for plant breeding
We analyze the mechanisms that control the reprogramming and totipotency of the microspore and embryogenesis initiation through a multidisciplinary and integrative study of the physiology of key events that limit its efficiency and correct progression. We use model herbaceous species and plant of agroforest interest (horticultural, cereals, fruit and forest trees). We characterize the determinant factors of cell totipotency acquisition, reprogramming and embryogenesis initiation, in response to stress, for their use as selective targets in strategies for optimization of the system and application in breeding programs of crops, agronomic and environmental. By means of a multidisciplinary and integrated approach with techniques of in situ molecular identification, correlative microscopy, molecular and cell biology and plant physiology, we studied the following elements and processes, which have a key role in the pollen reprogramming:
a) Autophagy and Programmed cell death (PCD)
Regulating the balance between cell survival and death during the process
b) Epigenetic regulation
Operated by DNA methylation and histone modifications, activators and repressors of transcriptional activity.
c) Dynamics of phytohormones
Auxins and their balance with cytokinins.
d) Cell wall remodelling
Mediated by pectin methyl esterases and arabinogalactan proteins (AGPs).
Nanoparticles (NPs) for diagnosis and treatments in crop protection:
We study the mechanisms of internalization and transport in planta of magnetic NPs and Quantum Dots (QDs), as well as their possible cytotoxicity, and the potential uptake of NPs and QDs by the plant. Moreover, we analyze how the dynamics of NPs entrance and movility changes when NPs are functionalized with biomacromolecules that label pathogens (for diagnosis) or with herbicides, fertilizers and pesticids (as controlled delivery systems for plant treatments). We use Medicago, as model, and different crops like pumpkin, pea, tomate, sunflower and wheat.
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
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.
Testillano PS, Risueño MC . Detection of epigenetic modifications during microspore embryogenesis: analysis of DNA methylation patterns dynamics. Methods in Molecular Biology 1356, 491-502.
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, 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.
Chiancone B, Gniech-Karasawa MM, Gianguzzi V, Abdelgalel A, Bárány I, Testillano PS, Botta R, Marinoni DT, Germanà MA . Early embryo achievement through isolated microspore culture in Citrus clementina Hort. ex Tan., cvs. Monreal Rosso and Nules. Frontiers in Plant Science 6, 413. DOI: 10.3389/fpls.2015.00413.
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.
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
MINECO, AGL2017-82447-R (2018-2020), PI: PS Testillano
MINECO, AGL2014-52028-R (2015-2018), PI: PS Testillano
MINECO, Network of Excellence for Autophagy Research, NEAR, BFU2015-71869-REDT (2016-2017). Coord: P Boya
COST Action CA15138 (TRANSAUTOPHAGY), EU (2015-2019) Chair: C Casas
Past projects (last years)
MICINN, BFU2011-23752 (2012-2015), PI: PS Testillano
MICINN, AGL2008-04255 (2009-2011), PI: MC Risueño
MICINN, BFU2008-00203 (2009-2011), PI: PS Testillano
MEC, AGL2005-05104 (2006-2008)PI: MC Risueño
MEC, BFU2005-01094 (2006-2008) PI: PS Testillano
CSIC, PIF2005-NANOAGRO (2005-2007), Coord: D Rubiales
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 Functional Genomics of Forests Species (2002-2006)
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.