Criopreservação de cultivares de amoreira-preta

Abstract

The blackberry belongs to the Rubus genus and the Rosaceae family and its fruits are appreciated all over the world. Propagation by root cuttings, shoots or new stalks has disadvantages of spreading pests and diseases, in addition to the high cost of maintaining the fields and of labor. Embrapa Clima Temperado, Pelotas-RS, holds the largest ex situ collection of this culture in Brazil. The conservation of genetic materials, however, to be effective, requires the development of biotechnological strategies that can overcome problems such as diseases and environmental damage capable of affecting materials preserved by other methods. In this scenario, cryopreservation emerges because, in addition to providing advantages such as the elimination of genetic damage, reducing the need for periodic assessments and control of viability, provides the preservation of collections for long periods with safety, low maintenance costs and using small spaces. Cryobanks help the genetic breeding programs and the productive sector in the maintenance and availability of genotypes. Cryopreservation protocols for the Rubus genus are already described in other countries; however, trasnfer of technology or repeatability in different laboratories is a challenge to be faced. Therefore, the objective of this work was to study cryopreservation protocols and improve freezing techniques for different blackberry genotypes. Tests were carried out, testing and adapting preparation methodologies and procedures for encapsulation-dehydration and vitrification of plant tissues. Initially, Embrapa's newest releases (‘BRS Xingu’ and ‘BRS Cainguá’) were used, and in a second moment, other materials (‘Tupy’ and ‘Comanche’) were added. These genotypes were subjected to preliminary tests of composition of alginate capsules and regeneration medium, dehydration of the alginate matrix, exposure to cryoprotectants and preconditioning; tests were also carried out with encapsulation-dehydration and vitrification techniques with the application of slow and ultra-fast freezing methodologies, totaling eleven tests presented here. The experiments were conducted using a completely randomized design. Data were subjected to analysis of variance using the F test (p≤0.05). Finding statistical significance, the data were compared by probability tests and polynomial regressions as needed. Preliminary tests showed good results, obtaining up to 95% survival in experiments in which the composition of the capsule and the regeneration medium were tested; however, immersion in liquid nitrogen caused necrosis in most of the material, resulting in 12% survival of the encapsulated explants. In the tests with the vitrification technique and with the application of preconditioning with day/night thermal variation and PVS2 cryoprotectant, when the treatments were carried out at low temperature, good results were observed, with survival of 45, 60, 56 and 63% cultivars ‘BRS Xingu’, ‘BRS Cainguá’, ‘Tupy’ and ‘Comanche’, respectively. Improvements in pre-cryopreservation processes can be made, such as the dehydration time of the explant capsule set, the composition of the capsule and the regeneration medium in the encapsulation-dehydration work. The vitrification techniques, in turn, showed good survival results, but with calluses formation.