Estabelecimento de protocolo para isolamento de protoplastos a partir de raiz de Jatropha curcas

Abstract

Jatropha curcas J. is a species popularly known as physic nut. It serves several economic applications, but it stands out mainly as an alternative to produce biofuel made with the oil extracted from its seeds. The "cake" generated during this extraction contains from 24 to 28% of protein in a dry basis and could be used to feed animals, however this practice is not performed because the seed has a high concentration of toxic substances due to the presence of diterpenes (phorbol esters). Molecular biology offers the possible blockade of the phorbol esters synthesis by silencing the genes responsible for the development of these compounds, especially in casbene synthase (CS), an enzyme that acts on the reaction that generates casbene, the precursor of most diterpenes of Euphorciaceae. There are 3 genes identified with proven activity for diterpene synthase, one perspective for this line of work is to use the CRISPR / Cas9 technique to block such genes, thus regenerating J. curcas plants with no or at least low toxicity. But for the accomplishment of this procedure is necessary to use protoplasts of the plant to be modified, such protocol is not available for the physic nut yet. Therefore, in this research the isolation of root protoplasts of J. curcas was the focus, since both the gene and the protein for the CS were found more significantly in the roots and it is expected that the gene expression of the isolated protoplasts maintain the patterns of the original tissue. Isolation of protoplasts was based on the protocol published by Deryckere et al. (2012) for plants of the Cichorium genus, with certain adaptations to fit the needs of J. curcas. The plants were developed in 3 conditions: in vivo, in vitro and hydroponics. Pieces from 1.5 to 2 cm of the root tip were cut longitudinally into 2 mm pieces under a stethoscope microscope, removing the central stele, and placed in P0 solution (pH 5.5). The roots were then placed in P0 solution with 1% cellulase and 0.5% macerozyme. These roots were left in the dark with gentle shaking for 16 hours, so that the digestion of the cell wall of the tissue could occur. The results did not present great discrepancies among the individual tests: low yield of viable protoplasts and high concentration of debris (cell and tissue fragments). Although the expected results have not been obtained, this work will pose as a basis for possible future researches that wish to isolate protoplasts from roots of J. curcas, since it defines some of its many peculiarities.