An approach on the in vitro maintenance of sugarcane with views for conservation and monitoring of plant nuclear DNA contents via flow cytometry

Abstract

In vitro conservation techniques can be utilized for germplasm maintenance. However, few reports on the in vitro conservation of sugarcane species are present in the literature. The objective of this study was to subject sugarcane plants to in vitro under minimal growth conditions and to evaluate the survival, regeneration, and the monitoring of nuclear DNA content levels of the plants. Shoots from 10 sugarcane varieties (Saccharum spp.) were introduced into two media: MC1, consisting of half-strength Murashige and Skoog (MS) salts and 3% sorbitol, or MC2, similar to the first formulation, but additionally supplemented with 3.8 μM abscisic acid (ABA). The shoots were maintained for up to 12 mo at 18°C in the presence of light. At the end of the period, the explants were inoculated onto multiplication medium containing 0.9 μM 6-benzylaminopurine (BAP) and 0.47 μM kinetin (Kin) for growth recovery. Flow cytometry analysis of shoots was verified at every 6 mo of storage. As a result, we found distinct behaviors of the varieties studied over the storage time, but in general, MC1 provided the greatest explant survival rates, with an average of approximately 80% cultures being able to recover. Once in the recovery media, the explant regrowth was fast, and the ability to multiply shoots was reestablished from the second 30-d subculture. However, by flow cytometry analysis, we observed a decrease in the estimated relative amount of DNA at 12 mo storage for most varieties examined, which was not observed when the monitoring was done at 6 mo. From these results, we conclude that sugarcane plants survived the minimal growth condition; however, maintaining the genotypes for extended periods in vitro may lead to variations in the estimated amount of nuclear DNA and, thus, be at risk of somaclonal variation.