Indução de embriões somáticos em diferentes clones de Coffea canephora

Abstract

With the advent of CRISPR technology, the search for new efficient techniques for tissue culture in coffee intensified, mainly aiming at the improvement of parameters related to the techniques currently used, such as the low efficiency of editing events and the long period of time to obtaining genetically modified plants. Considering that Brazil is the leader in coffee production, with C. Canephora being the second most cultivated species, this work aimed to determine efficient protocols for obtaining somatic embryos of C. canephora, using direct somatic embryogenesis (ESD) and embryogenic cell suspension protoplast (ECS), aiming applications in future work with gene editing via CRISPR/Cas9 technology. For this, different ESD induction experiments were carried out in C. canephora, and tests were also carried out to select the influence of different ESD protocols (for clones 2 and 14 they evaluated the influence of different regions of the leaves) inoculated in medium for ESD clone 2), and several different clones (for clone 2, 14) in the process (for clones 22, 11). Concomitantly, was sought in this work, the induction of somatic embryos from embryogenic cell suspension protoplasts of C. canephora (Clone 14), for this, Yasuda culture medium was used, enriched with of charcoal, varying antioxidant agents, carbohydrates and abscisic acid (ABA) concentrations. The experiments were carried out in a completely randomized design and for the analysis of the ESD experiments, the Tukey test was used at 95% confidence, and for the experiments of induction of embryos in embryogenic cell suspension of protoplasts, regression analysis was used linear. In the ESD experiment, means of regeneration frequency and mean number of embryos per explant were evaluated. For the embryo induction experiment in protoplast callus, the average number of embryos obtained in each treatment was evaluated. The results achieved in this study showed that clone 2 was more responsive in somatic embryogenesis when compared to clones 14 and 22, both in higher explant regeneration frequencies and in number of embryos per regenerated explant, thus indicating that ESD is a highly genotype-dependent technique. For the experiment on the influence of different regions of the leaf on the embryogenic response, no differences were found between the means obtained, as well as for different protocols used. Concerning the induction of formation of ECS somatic embryos, the production of embryos was observed only in the culture medium enriched with 30 gL-1 sucrose and added of ABA, with no significant difference between different concentrations of the growth regulator in the formation of embryos. In this way, it is concluded with this work that it is possible to use ESD as a way of regenerating genetically edited embryos, as well as the application of protoplasts in the coffee edition, although further studies are still needed in relation to the increase in the frequency of regeneration and its further maturation.