In vitro regeneration, detection of somaclonal variation and screening for mosaic virus in sugarcane (Saccharum spp.) somaclones

Abstract

Three sugarcane accessions susceptible to sugarcane mosaic virus; HSF-240, S-2000-US-359, and S-2003-US-704 were evaluated for callogenesis and regeneration ability. For callogenesis, five different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) was used. The best callogenesis was obtained when Murashige and Skoog (MS) was portified with 3 mg/L 2,4-D and the highest regeneration was obtained on media containing MS + kinetin 0.5 + 0.5 mg/L naphthalene acetic acid (NAA). After succesful regeneration and rooting on half strength MS medium, with 1.5 mg/L indole-3-butyric acid supplementation, plantlets were shifted to green house. Enzyme linked immunosorbent assay (ELISA) test was performed to detect the presence of sugarcane mosaic virus (SCMV) in the regenerated plantlets and simple sequence repeat (SSR) markers were used to evaluate the genetic variation at DNA level between the parent’s plants and regenerated somaclones of the accession HSF-240. A total of 26 parent plants and 64 somaclones, among the regenerated plants were selected for the screening of virus through double antibody sandwich (DAS-ELISA) test. Four (4) parent plants out of the 26, showed negative reaction to the virus test. Ten (10) somaclones showed positive reaction to the disease, 9 somaclones showed mild reaction to virus and 45 somaclones showed negative reaction. For the detection of somaclonal variation, 38 primers pair were used and 15 simple sequence repeats (SSR) primer pairs were found to be polymorphic with 51.61% polymorphism. The study demonstrates that SSR genetic markers are the best tool for the investigation of genetic variation in sugarcane.

Keywords: Callogenesis, somaclones, simple sequence repeats (SSR), genetic markers