GENERACIÓN DE UN SISTEMA DE TRANSFORMACIÓN PARA LOS MUSGOS Bryum billarderi SCHWÄGR Y Pseudocrossidium replicatum (Taylor) R. H. ZANDER

Abstract

The mosses B. billarderi and P. replicatum are species of plants that can tolerate desiccation, salt and other abiotic stresses. In this work, we established a strategy to transform these mosses, with the purpose of developing a technology that allows to understand the mechanisms that confer that tolerance and to explore the opportunity to produce recombinant proteins. First, we developed the conditions to obtain and regenerate the protoplasts from protonema tissues of both species. At this point in B. billarderi, we evaluated the effect of different driselase concentrations (1, 2 and 3 %), exposure times (1, 1.5 and 2 hours) and protonema age (5, 10 and 15 days) on the amount of protoplast isolated and regenerated, this was done only in B. billarderi because we had a large variation in the yield of protoplasts obtained in this specie (from 2.45 x 105 to 2.6 x 106 protoplastos ml-1) whereas in P. replicatum we obtained always a great yield of protoplasts (from 2.05 x 106 to 2.54 x 107 protoplastos ml-1), so it wasn´t necessary to make that assay in this moss. The best treatment to obtain protoplasts (5.35x106 protoplasts g-1) was: age 10 days, concentration 2 % and exposition 2 hours; however, the best regeneration rate (72.08 %) was found at: age 5 days, concentration 2 % and exposition 1 hour. Second, we used the plasmid pPGX8 to transform both species of mosses and the model moss P. patens to compare our results. Transient antibiotic resistant colonies were recovered at high transformation efficiency (82 colonies μg-1 linearized DNA) in the moss B. billarderi, yielding approximately 1640 colonies per genetic transformation event; however, we have only obtained two transient antibiotic resistant colonies in the moss P. replicatum. Comparatively, we obtained less transient antibiotic resistant colonies in P. patens (4.66 colonies μg-1 linearized DNA) than in B. billarderi; nevertheless, we only obtained stable lines in P. patens. The plasmid pPGX8 was made to transform P. patens, because it has homologues regions at the genome of this moss, maybe for this reason we didn´t obtain stables lines in B. billarderi and P. replicatum. Third, we generated the construction pPGX8::CD43 to explore the production of the human protein CD43 in the three species of mosses. We obtained clones only in B. billarderi and P. patens using the construction pPGX8::CD43, but all clones of B. billarderi died after a period of 12 time; however, we obtained 10 stable clones of P. patens, of which 4 were analyzed by PCR obtaining that in all cases it was observed a band correspondent to CD43.