Indução à ploidia e caracterização em echynochloa polystachya (kunth) hitchc na Amazônia Ocidental

Abstract

Echinochloa polystachya is one of the main forage species present in the lower Amazon region and can be an alternative for animal feed during the flood period of the Amazon River. In vitro propagation studies and obtaining polyploids from diploids of this species can result in individuals with higher productivity, better vegetative development and vigor than the diploids from which they originated for cultivation. The objective of the work was to perform the introduction of Echynoclhoa Polystachya in vitro, induce polyploidy with the use of colchicine and to characterize the genotypes obtained through flow cytometry and stomatal density in order to increase the genetic variability to improve the productivity of this forage. Stems were collected in the city of Manaus, pre-cleaning, pre-aseptic and removal of the leaf sheath and senescent material were performed. They were submitted to the disinfestation process as proposed by Passos and Köpp (2010), then individual test tubes containing the initial culture medium were inoculated: 15 mL of Murashige and Skoog medium (1962) with half the concentrations of salts, free of regulators growth, plus 30 g, sucrose L-1, BAP 4.5 ml.L-1, of the biocide PPM in the concentration of 2 ml.L-1 and 2 gL-1 of Fitagel and adjusted to pH 5, 8 before autoclaving. Thirty days later, the explants were picked and transferred to new individual tubes with new culture media equal to the initial medium and the rates of sprouting and contamination were checked. Experiments were also carried out to evaluate the effects of sucrose, BAP concentrations on the tillering rate of explants. From the increase in the sucrose content from 45 g / L-1, there were responses in the multiplication rate. The doses of BAP and sucrose interfered in the development and multiplication of the in vitro culture, however, for the establishment of an efficient protocol for the in vitro propagation for Echinochloa Polystachya must make adjustments in the sucrose and BAP content during successive subcultures due the decrease in internodes and the consequent loss of vigor. For the study of polyploidy, nodal segments from a single true canarana plant were collected in the municipality of Manaus and subjected to different concentrations of colchicine and exposure times. in a completely randomized design in a 5x4 factorial system. The concentrations of colchicine were: 0; 0.01; 0.025; 0.05 and 0.1. The exposure times were: 0; 12; 24 and 36 hours. Thus, 20 treatments were performed with 3 repetitions, where each repetition was composed of 10 plants, in a total of 600 nodal segments evaluated. The emergence time from planting was evaluated. The evaluation of morphogenesis and growth rate was performed using the technique of "marked tillers" (adapted), whose methodology is detailed by Carrère et al. (1997) and Davies (1993). The best germination receiving colchicine was observed in 50 % at concentrations of 0.01 ml.L-1 and at time 0 (zero) of exposure The percentages of mortality in pre-emergence were affected by the concentrations and time to which they were exposed, in which, at concentration 0.1 ml.L-1 and 36 hours were 85%, with the highest survival at 0.01 ml.L-1 and 0 (zero) exposure times, with a survival rate of 33.1 %. It was also found that anomalous plants did not occur in treatments where the concentration of colchicine was 0 (zero), it can be inferred that the plants were affected in their metabolism by molecular changes, not causing immediate mortality, but affecting cell divisions. In general terms of the 600 nodal segments submitted to the treatments, 172 survived, reflecting a 28% survival rate, in which 159 plants expressed their phenotypic characteristics after exposure to the concentrations and times thus giving rise to supposed polyploids. 20 supposedly polyploid plants were selected for measurements in relation to stomatal density and morphometric characteristics of leaf cell cells. He observed changes in stomatal densities, stomatal functionality, distance between vascular bundles, length and width of epidermal cells and cells of vascular bundles. In the different plants analyzed by flow cytometry, there were changes in both the increase and the elimination in the content of DNA (pg). These changes did not show correlation between the lengths and widths of the cells of the epidermis and the cells of the bundle, however they originated plants with different levels of ploidy and morphometric characteristics. It can be inferred that there was a “rearrangement” of the cells of the leaf blade due to the consequent ploidy. No correlation was observed between the greatest distances between the vascular bundles and the largest amounts of DNA content. For plants whose DNA content was higher than the original plant (P5, P6, P7, P12, P14, P15 and 21), a correlation index (r = 0.86) was found between DNA content (pg ) and the distances between the vascular bundles, a fact that confirms the various studies that mention the largest ploidy, the largest tissue sizes and plant organs. A verified fact was that the plants with the elimination of the DNA content (P4, P8, P9, P10, P11, P13 P16 P17 P18 P19 P20) presented a low correlation with the widths between the vascular bundles (r = 0.1), not corroborating these statements. The plants that stood out in terms of morphometric characteristics, when compared to Plant P1, were plants P5, P7, P8, P9 and P21. Of these, the plants, P8 and P9, stand out, which despite the DNA content, significantly lower, presented morphometric characteristics of interest, superior to the original plant P1. It can be concluded that the concentrations and doses of colchicine in submitted to the species Echinochloa polystachya directly influenced the molecular events, resulting in plants with different levels of ploidy and different morphometric characteristics, indicating potential mutagenic plants that may enter into further breeding work.