Optimization of Callus Induction and Plant Regeneration in Sugarcane (Saccharum spp.) for a Study of Sucrose Accumulation in Relation to Soluble Acid Invertase Expression

Abstract

The response of three sugarcane genotypes with differential sucrose accumulation to MS medium + 2, 4-dichlorophenoxyacetic acid (2, 4-D) and/or kinetin (kn) was investigated. On MS + 2, 4-D (2 mg/l) + kn (0.5 mg/l), Louisiana Purple (LAP) had the highest callus induction (95.80 %), followed by H65-7052 (82.68 %) and Molokai (MOL) (62.58 %). Rapid callus induction was optimized by using different concentrations of 2, 4-D (1, 2, 3, 4 and 5 mg/l) or 2, 4-D (2 mg/l) + kn (0.5 mg/l). The results revealed that increased callus weight was significantly different among genotypes and treatments. The value averaged over all the treatments was highest in H65-7052 (1.60 g), followed by LAP (0.93 g) and MOL (0.74 g). MS + 2, 4-D (1-2 mg/l) or MS + 2, 4-D (2 mg/l) + kn (0.5 mg/l) could cause rapid callus growth in all the three sugarcane genotypes. For plant regeneration, all cultured calli on different media exhibited good response to MS. The number of organogenic calli averaged over all the treatments was highest in MOL (61.6), followed by LAP (54.6) and H65-7052 (11.3). The most suitable culture medium for MOL and LAP was MS + 2, 4-D (1 mg/l), while MS + 2,4-D (1 mg/l) or MS + 2,4-D (2 mg/l) + kn (0.5 mg/l) was most appropriate for H65-7052. Moreover, sucrose accumulation in relation to the expression of soluble acid invertase (SAI) gene was measured in calli and plantlets. The highest callus sucrose concentration was found in LAP (141.28 mmol/g FW), followed by H65-7052 (133.79 mmol/g FW) and MOL (41.05 mmol/g FW). The plantlet sucrose concentration showed a similar pattern. The SAI expression in both the callus and plantlet of H65-7052 and LAP was lower than that of MOL. In both systems, sucrose accumulation was negatively correlated with the increase in SAI expression. It may be concluded that callus and plantlet can be used as the model system for investigating sucrose accumulation in sugarcane.