Otimização da regeneração in vitro de Urochloa spp. via embriogênese somática

Abstract

The establishment of an efficient methodology for regeneration in Urochloa spp via somatic embryogenesis is an essential step for genetic engineering works and to support the breeding programs of the signal grass. The aim of this study was to evaluate different concentrations of growth regulators on callus induction, somatic embryogenesis and plant regeneration of Urochloa spp. using mature seeds as the initial explant. Firstly, eight treatments were tested containing different concentrations (1, 2, 4 and 8 mg L-1) of 2,4-D or picloram, using Marandu cultivar. After, the same concentrations of picloram were tested for others genotypes: cultivars Xaraés and Piata of U. brizantha, U. decumbens cv. Basilisk, U. humidicola. cv. Llanero and U. ruziziensis cv. Ruziziensis. The experiments were conducted in a completely randomized design with four replications per treatment, and each replicate consisted of a petri dish containing 10 seeds each. The experiments were repeated three times. Seeds were sterilized in 5% NaOCl containing three drops of Tween 20, washed five times in sterile distilled water and inoculated onto MS medium containing the treatments described above. The experiments were maintained in growth chamber at 25 ° C ± 2 ° C in the dark, and sub-cultured every 14 days until the emergence of embryogenic calluses. To induce plant regeneration, these calluses were transferred to hormone-free MS medium for 14 days in indirect light followed by transfer to MS/2 medium containing 2 mg/L-BA in direct light with a photoperiod of 16 hours. The number of seeds that produced primary calluses, the number of calluses with shoots and the number of regenerated plants were analyzed in each treatment. To demonstrate embryogenic competence, the different calluses types were cyto-chemically analyzed by double staining with acetic carmine and Evans blue. There was no significant difference between concentration of 2,4D or picloram for production of primary calluses of U. brizantha Marandu ranging from 58.5% to 69.2%. Calluses induced under picloram showed the highest percentage of shoot formation differing significantly from those induced under 2.4D. The genotypes U. decumbens cv. Basilisk, U. brizantha cv. Marandu and Xaraés showed the significant highest number of regenerated plants at 1 mg/L picloram. Green and morphologically normal plants were regenerated and acclimatized, opening the possibility of using this protocol to obtain transgenic plants in Urochloa spp.