PLANT TISSUE CULTURE Gamma radiation, in vitro selection for salt (NaCl) tolerance, and characterization of mutants in sugarcane (Saccharum officinarum L.) Ashok A. Nikam & Rachayya M. Devarumath & Mahadeo G. Shitole & Vikram S. Ghole & Prahlad N. Tawar & Penna Suprasanna Received: 12 December 2013 /Accepted: 1 July 2014 / Editor: John Forster # The Society for In Vitro Biology 2014 Abstract Gamma-ray-induced mutagenesis and in vitro selec- tion of sugarcane (Saccharum officinarum L. Lam.) plants tolerant to NaCl was achieved using embryogenic calli of sugarcane var. Co740. Sensitivity to gamma radiation and NaCl was studied in vitro in embryogenic calli followed by characterization and evaluation of mutant clones. The results indicated that callus growth and plant regeneration were signif- icantly affected by radiation dose (0, 10, 20, 30, 40, 50, 60, 70, or 80 Gy 60 Co gamma rays) as well as NaCl exposure (0, 50, 100, 150, 200, or 250 mM). Salt tolerance was achieved by culturing irradiated callus on selection medium with different NaCl concentrations through stepwise in vitro selection. Salt- selected embryogenic callus lines were then analyzed for pro- line, glycine betaine, Na + , and K + contents. Salt-selected plants were grown to maturity, and their agronomic performance was evaluated under normal and saline conditions. Twenty-four mutant clones were characterized for proline, glycine betaine, Na + , and K + contents. The mutant clones exhibited improved sugar yield with increments in Brix%, number of millable canes, girth, and yield. The results suggest that in vitro culture and induced mutagenesis offer an effective way to enhance the genetic variation in sugarcane. Keywords Sugarcane . Gamma radiation . In vitro mutagenesis . Salt tolerance . Agronomic evaluation Introduction Plants frequently encounter unfavorable climatic factors that limit plant growth, development, and productivity. It is esti- mated that worldwide 831×10 6 ha of land is affected by salinity (Beltran and Manzur 2005). In this regard, major strategies are being developed including development of new crop varieties, screening and selection of the existing crop germplasm, and introduction of genetic variability through mutation induction (Arzani 2008). Rengasamy (2006) suggested that evaluation of salt-tolerant genetic ma- terial and induction of salt tolerance should be given priority for sustaining crop production in a saline environment. Development of salt-tolerant mutant cultivars can supplement the utilization of salinity-affected regions, but the availability of large populations for mutagenesis and screening are a pre- requisite to obtain sufficient genetic variability. In vitro culture techniques offer a method to generate large populations for mutation induction, and selection. The use of in vitro culture and radiation-induced mutations has become an important method to introduce genetic variability and to rapidly multiply the selected mutants (Jain 2007; Jain et al. 2010; Suprasanna et al. 2012). Methods of chemical and/or radiation-induced in vitro mutagenesis have been successfully used in different A. A. Nikam : P. N. Tawar Tissue culture section, Vasantdada Sugar Institute, Manjari (Bk) Pune 412307, India R. M. Devarumath (*) Molecular Biology and Genetic Engineering Division, Vasantdada Sugar Institute, Manjari (Bk) Pune 412307, India e-mail: rm.devarumath@vsisugar.org.in A. A. Nikam : R. M. Devarumath Department of Biotechnology, University of Pune, Ganeshkhind Pune 411007, India M. G. Shitole Department of Botany, University of Pune, Ganeshkhind Pune 411007, India V. S. Ghole National Institute of Virology, Pashan Pune 411021, India P. Suprasanna Functional Plant Biology, Nuclear Agriculture & Biotechnology Division, Bhabha Atomic Research Centre, Trombay Mumbai 400085, India In Vitro Cell.Dev.Biol.—Plant DOI 10.1007/s11627-014-9630-4