Cultivo in vitro de espécies de helicônia (Heliconia spp.) mediante embriogênese somática e embriões zigóticos

Abstract

The aim of this work was to establish protocols for the induction of embryogenic calluses in heliconia species. Two experiments were carried out. The first one had three assays. The first assay consisted of combinations of AIA (indole-3-acetic) (0, 1, 1.5, 2, and 2.5 mg.L-1) with 2,4-D (2,4-dichlorofenoxyacetic acid) (0, 3, 4, and 5 mg.L-1) and the second essay combined BAP (6-benzylamino purine) (0, 1, 1.5, 2, and 2.5 mg.L-1) with 2,4-D (0, 3, 4, and 5 mg.L-1). Sections of ovaries of H. chartacea Lane ex Barreiros cv. Sexy Pink were used in the assays above. The third assay was established from combinations among three levels of AIA (0, 1 and 2 mg.L-1) and five levels of 2,4-D (0, 5, 10, 15, and 20 mg.L-1) within cultivars Sexy Pink and Sexy Scarlet of H.chartacea. The second experiment used explant zygotic embryos of H. bihai cv. (L.) L.Lobster Claw Two in combinations of AIA (0, 1 and 2 mg.L-1) and 2,4-D (0, 5, 10, 15, and 20 mg.L-1). Evaluations in both experiments were carried out on days 30, 60 and 90 after innoculation. Aspects of the callus and the presence of nodules were assessed by visual observations with the help of a stereomicroscope and cytochemical, histologic and scanning electron microscopy analyses were performed. At the end of experiment 1, the index of formation of callus in explants treated with AIA+2,4-D (Assay 1) was greater than that observed with the combinations of BAP+2,4-D (Assay 2). The AIA+2.4-D assay also produced proembryonary cells. In the third assay the Sexy Pink cultivar showed a greater index of formation of callus than the Sexy Scarlet cultivar, but these calluses didn t show somatic embryos at the end of 90 days of cultivation. In the second experiment, formation of somatic proembryos occurred only in zygotic embryos from mature fruit of H. bihai (L.) L. cv. Lobster Claw Two cultivated in the absence of growth regulators. The objective of the second work was to evaluate micropropagation processes for cultivation of zygotic embryos. Initially, an analysis of the internal structure of the embyo was carried out using immature and mature fruits. This was accomplished by embedding the fruits in paraffin and sectioning them with a microtome. Embryos from immature and mature fruits were innoculated in an MS (Murashige and Skoog) environment with different concentrations of salts (full MS and ½ MS), AG3 (0, 2.5 and 5 mg.L-1) and saccharose or glucose (30 g.L-1), and their development was evaluated. Evaluations were carried out 15, 30 and 60 days after innoculation based on visual observations, quantitative parameters, and number of roots. Then, plants were replicated and transferred to an MS environment (full MS or ½ MS), added 0 or 2.5 mg.L-1 BAP, with the objective to evaluate their development after 30 and 45 days after innoculation. The genetic variability of plants from zygotic embryos was estimated using isoenzymatic analyses (PO, EST, ACP, GOT, ADH, and MDH). The internal structure of mature embryos showed tissues in more advanced stages of differentiation than immature embryos, as expected. In the ½ MS environment with saccharose, 85% of the innoculated embryos were converted into plants, just 41% converted into plants in the glucose environment. During the multiplication phase, plants cultivated in an MS environment with a 50% concentration of salts and 2.5 mg.L-1 of BAP showed a greater number of buds. The isoenzymatic analyses showed some changes of isoenzymatic patterns, for example in intensity of coloration and migration of some bands. These results can be associated respectively with the difference of the ages among mother plant and plants obtained in vitro and/or variations in eletrophoretic phenotypes of the plants from in vitro cultivation of zygotic embryos.