Isolamento e regeneração de protoplastos de Coffea canephora a partir de suspensão de células embriogênicas

Abstract

The coffee fruit is a product of high aggregate value from which one of the most consumed drinks all over the world is extracted. Its high production cost, long crop cycle and high market demand attract strong investment in the sector for crop genetic improvement that aims to develop new cultivars with desirable agronomic characteristics. Recently, the CRISPR-Cas9 gene editing tool has been helped breeders in the generation of genotypes of agronomic interest through the possibility of genetic modification without the insertion of transgenes in the host DNA, simplifying the commercialization of these specimens. Besides to the application of CRISPR-Cas 9 technology, protoplasts also enable studies of heterologous expression and somatic hybridization, but the best way to explore the potential of protoplasts in researches is to associate them with an efficient plant regeneration protocol. Thus, the current work aimed to elaborate an efficient and reproducible method specific for coffee protoplast obtention in the diploid (2n=2x=22) genotype clone 14 Coffea canephora targeting the application of precision molecular technology for plant breeding. For this, suspensions of embryogenic calluses of C. canephora clone 14 were selected as biological material, due to its high capacity of regeneration in plants, which is the principal limiting factor of the technique. Enzymatic digestion of the cell wall was performed using two different enzyme solutions: 1% cellulase, 0.5% driselase and 0.5% pectinase; and 0.5% cellulase, 0.5% Macerozyme and 0.2% pectinase, that showed similar values of performance and viability. The pH value of the enzymatic solution was also evaluated in this work, and it was found that pH 5,6 provided greater enzymatic efficiency and cell viability simultaneously. After, tests were carried out to optimize the regeneration of the protoplasts, and the plated cells were subjected to a gradual reduction in osmotic pressure throughout cell culture. After 50 days of plating, the cell pellets were transferred to erlenmeyers where the tests in cell suspension started using shaking and different means of culture. It was also observed that the cultivation conditions adopted did not affect the physical embryogenic appearance and embryogenic potential, besides favouring cell cluster multiplication. Finally, it can be concluded that the purpose of this study was successfully achieved, but it is necessary to continue research aimed at plant regeneration from the cell suspension obtained in order to establish a complete regeneration protocol for C. canephora protoplasts in plants, capable of assisting the generation of new elite genotypes.