Transformação genética, propagação em biorreatores e criopreservação de eucaliptos

Abstract

This work carried out the genetic transformation of Eucalyptus grandis x Eucalyptus urophylla with the gene OsCesA4, responsible for cellulose biosynthesis in the secondar y cell wall. In addition, a bibliographical and systematic review on the micropropagation of arboreal species in Temporary Immersion Systems (TISs) was conducted. Afterwards, the efficiency of three TISs in the propagation of E. grandis x E.urophylla was compared regarding the semi solid culture system. Finally, a study was carried out regarding the optimization of the preculture medium, type of cryoprotectant and time of exposure, besides the efficiency of two cryogenic methods for the cryopreservat ion of E. grandis x E. urophylla and the comparison of two sources of explants with different ages in cryopreservation of E. grandis. The insertion of the gene of interest OsCesA4 in the in vitro plants of eucalyptus was confirmed by conventional PCR and visualized in agarose gel. However, there was no reproducibility of this PCR result with the same plants after greenhouse acclimatization. In addition, the confirmation gel of possible contamination with Agrobacterium tumefasciens was inconclusive, since all events and even the control plant showed amplification. These results, together with the non-statistical difference in the cellulose content of the control plants in relation to those that underwent the transformation, raised the hypothesis of the regene ration of possible chimeras plants. In the review on TISs, it was observed that the frequency and time of immersion was one of the main parameters studied in the analyzed papers and 35% of them worked with eucalyptus species. It was also observed that hype rhydricity was the main problem reported, and most reported that plants propagated in TISs were more successful in acclimatization than those propagated in conventional media. In addition, few studies have compared the efficiency of different TISs in the trees´ propagation, which is a possible point to be addressed in future studies. In the experiment using TISs, ElecTIS was the most efficient system for the propagation of E. grandis x E. urophylla plants, presenting average of 14.6 shoots per explant and nonhyperhydric plants. In the cryopreservation experiment of E. grandis x E. urophylla, the preculture medium that was most efficient in plant survival was 0.125; 0.25 and 0.5 M sucrose / 24 h at each concentration. The cryoprotectant that presented the highest percentage of plant survival was PVS2 with exposure to it for 15 and 30 minutes. And the droplet-vitrification method presented higher percentage of plant survival after cryopreservation when compared to cryo plate. When using the optimized parameters for the cryopreservation of E. grandis x E. urophylla, survival of 30% was found, a value not considered ideal for cryobank storage. For E. grandis, explants from 20-day-old plants showed the highest percentage of survival, with a value of 48%, while the explants from 3 -year-old plants did not survive, which shows the great influence of age on the final recovery of the plants´ explants after cryopreservation.