A Protocol for In-vitro direct plant regeneration from leaf tissues for micropropagation of sugarcane

Abstract

An efficient protocol to produce higher number of shoots was developed for Sugarcane (Saccharum Hybrid spp.) variety SL 96 328. In-vitro direct shoot regeneration from sugarcane leaf spindle tissues was achieved in modified Murashige and Skoog medium supplemented with 1.5 mg/L of Benzylaminopurine, 0.5 mg/L of Kinetin, 0.5 mg/L of 2,4-D, 1 mg/L of Indole-3-acetic acid and 400 mg/L of Cysteine Hydrochloride. The survival rate of explants and number, length and vigour of shoots generated from explants, and minimal number of chlorophyll-mutated shoots per explant were recorded. Explants incubated in the dark for two weeks enhanced direct shoot regeneration and produced the highest number of shoots (25) from a single explant. The best diameter and the thickness of the explant were 3 mm and 2 mm, respectively. The amplification of Simple Sequence Repeat primer using Polymerase Chain Reaction (PCR) revealed that the plants obtained through in-vitro directly-regenerated shoots were genetically more identical than those generated from stem cuttings. Nested PCR confirmed that the plantlets obtained from direct shoot regeneration were free from sugarcane white leaf disease (WLD) phytoplasma. Thus, the in-vitro protocol suggested for rapid micropropagation of sugarcane through this study can be adopted in producing genetically-homogenous and WLD-free planting material in establishing sugarcane nurseries.