Criopreservação de calos, ápices caulinares e sementes de barbatimão

Abstract

Barbatimão [Sthyphnodendron adstringens (Mart.) Coville] is a medicinal plant found in the Brazilian Cerrado. The species presents different medicinal functions due to high concentration tannin in different parts of the plant. Cryopreservation is a widely used method in the conservation of vegetative explants of species with medicinal and commercial interest. The aim of the present study was to develop protocols for cryopreservation of barbatimão calluses, shoot tips and seeds. Cotyledon segments were used to induce calluses in MS medium supplemented with different combinations of 2,4-D × TDZ and between concentrations of kinetin × picloram. For cryopreservation, we used calluses with 1 mg L-1 of 2,4-D. For the cryopreservation of the shoot tips, we first tested the influence of BAP and GA3 in the shoot tips’ growth. The "droplet-vitrification" technique was used for shoot tips’ cryopreservation, where the explants were exposed to PVS2 for different periods of time before being plunged into liquid nitrogen. Different BAP and GA3 concentrations were tested in the shoot tips’ cultivation after cryopreservation. For the cryopreservation of the seeds, we first assessed the water content of three different seeds. After plunging the seeds into LN and thawing the percentage of germination, seed vigor and the amount of DNA between different treatments was evaluated. The best results were obtained for the callus formation at the concentrations of 0.5 and 2.0 mg L-1 TDZ, in the absence of 2,4-D or using 2.0 mg L-1 kinetin combined with 0.5 mg L-1 of picloram. The highest amount of phenols were quantified in a medium containing 1.0 mg L-1 of 2,4-D, 2.0 mg L-1 of kinetin and 1.0 mg L-1 of picloram. The growth curve indicated that callus grading must be performed at 49 days of cultivation. The different callus cryopreservation assays did not show regrowth in any of the treatment. The concentration of 0.25 mg L-1 of BAP promoted the largest height of the shoot tips. The periods of 15 and 30 minutes of shoot tips exposure to PVS2 presented higher survival percentages and the combination of GA3 with 2.0 mg L-1 of BAP presented the largest shoot lengths. Cryopreserved seeds showed the same amount of DNA, and seeds with 6 and 9% water content showed greater vigor. We conclude that it is possible to induce calluses from cotyledons using different auxins and cytokinins and that these calluses contain phenols. The cryopreservation methodology used in this study was not successful with calluses. The BAP is essential for shoot tip culture, and GA3 for shoot elongation. The "droplet-vitrification" technique can be used for the cryopreservation of shoot tips. Seeds with low amounts of water can be successfully cryopreserved.