Urochloa brizantha is one of the most important warm season forage grasses in tropical countries. Despite its importance, there are few studies on gene expression in this species under stressful conditions. Real-time (RT-qPCR) is an accurate technique for gene quantification analysis, but reference genes must be validated under the same conditions used to assess the expression of the target genes. Here, we evaluated the stability of nine reference genes: Actin 12, Eukaryotic initiation factor 4 A, Elongation factor-1 alpha, FTSH protease 4, U2 auxiliary fator, Succinol Co-enzyme A, Tubulin alfa-5, Tubulin beta-6, Ubiquitin conjugating enzyme. Total RNA was extract from leaf tissues of U. brizantha subjected to 6, 12 and 24 h of cold and heat stresses (10 and 45 °C, respectively), and drought, including moderate (−0.5 to −0.7 MPa), severe (−1.1 to −1.8 MPa) and recovery after re-watering. The RefFinder web-based tool was used to rank the most stable reference genes for each stress. Elongation factor-1 alpha, Elongation factor-1 alpha or Ubiquitin conjugating enzyme, and Eukaryotic initiation factor 4 A were the most stable genes for heat, cold and drought stress, respectively. The expression of Rubisco large subunit gene was normalized against the most stable gene selected by ReFfinder for each stress.

The establishment of an efficient methodology for regeneration in Urochloa spp. via somatic embryogenesis is an essential component of genetic engineering technology. 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, different concentrations (1, 2, 4, and 8 mg/L) of 2,4-D or picloram were tested using U. brizantha cv. Marandu. In a second step, the above concentrations of picloram were also used to evaluate its effect on the morphogenetic potential in different genotypes: U. brizantha cvs. Xaraés and Piatã, U. decumbens cv. Basilisk, U. humidicola cv. Llanero, and U. ruziziensis cv. Ruziziensis. There was no significant difference between concentrations of 2,4-D or picloram for the production of primary calluses in U. brizantha cv. Marandu, ranging from 58.5 to 69.2%. However, embryogenic calluses induced under picloram showed higher percentage of shoot formation after transferring to MS medium with 2 mg/L BA. A higher number of shoots were produced in mature seed cultures of U. decumbens cv. Basilisk and U. brizantha cvs. Marandu and Xaraés supplemented with 1 mg/L picloram. No albino plants were regenerated using picloram. The successful regeneration of green and morphologically normal plants opens the possibility of using this protocol to obtain transgenic plants in Urochloa spp.

The key agricultural species of Urochloa P.Beauv. (signal grass), important as tropical forage grasses, are characterised by asexual seed formation (apomixis), and this presents a challenge for breeding programs. Biotechnological approaches could be an option to develop improved cultivars. We evaluated the regenerative potential from three commercial genotypes, U. brizantha cv. Marandu, U. decumbens cv. Basilisk and U. ruziziensis cv. Ruziziensis, by using leaf-base segments as explants. We tested two auxins (2,4-D and picloram) and one cytokinin (TDZ) at four concentrations (1, 2, 3 and 4 mg L–1). Seeds were scarified, peeled and disinfected before inoculation on half-strength MS media in the dark for 14 days. Leaf-base explants were sectioned in thin slices and inoculated into the media. We analysed the number of primary calluses, number of calluses with shoots clusters and the average of regenerated plants. The lowest concentration of auxins tested (1 mg L–1) yielded the highest number of regenerated plants for Marandú and Basilisk, whereas the optimum for Ruziziensis was 2 mg L–1. Medium with higher concentrations of TDZ (4 mg L–1) was required to produce high frequency of plants for all genotypes. Explants cultured on media with TDZ produced very few calluses. These results indicate that the auxins and cytokinin tested can induce plant regeneration from Urochloa leaf-base segments, and may be used to produce transgenic plants in genetic transformation studies.