High-efficiency organogenesis and evaluation of the regenerated plants by flow cytometry of a broad range of Saccarum spp. hibrids

Abstract

The aim of this study was to evaluate the organogenic potential of Brazilian sugarcane varieties, in addition to verifying the in vitro multiplication rate and genetic stability by flow cytometry over monthly and consecutive subcultures. For that, stem apexes of twenty-two varieties were collected in field conditions and taken to the laboratory where external layers of leaves were removed. After surface sterilization, the innermost portion of the stem segment was sectioned and placed in a MS medium supplemented with 5.0 mg L−1 of NAA and 0.5 mg L−1 of KIN. For the multiplication rate, ten varieties were selected and inoculated in MS medium of liquid or semi-solid consistency plus 0.10 mg L−1 of KIN and 0.20 mg L−1 of BAP. Subcultures were performed every 30 days for a period up to 8 months. Genetic stability was verified by flow cytometry every two subcultures. At the end of the experiment, the sprouts were rooted and acclimatized in a greenhouse. As a result, it was observed that the regeneration occurred both by direct and indirect organogenic pathway. The varieties of sugarcane differed significantly regarding the regeneration capacity and amount of adventitious shoots formed. In multiplication, a significant interaction was observed between variety, consistency of the culture medium and number of subcultures. In general, in the first subcultures, the liquid consistency medium presented similar or superior results when compared with the semi-solid medium, however, from the fourth subculture, the semi-solid medium was superior. Morphological variations were verified from the fourth subculture. In addition, in some varieties, small changes in the relative amount of DNA were detected by flow cytometry. Sprouts of normal-looking sugarcane were successfully rooted and plantlets acclimatized after the eighth subculture.