Rapid in Vitro Micro-Propagation of Clean Sugarcane Seed.

Sugarcane (Saccharum spp.) is a major contributor in the world sugar industry and an emerging bioenergy crop. It would benefit tremendously from the development of high-throughput in vitro propagation methods that would allow clean seed production and facilitate varietal commercialization.  Conventional propagation of sugarcane from seedcane pieces with bud sets is slow and accompanied by the high risk of disease transmission to the subsequent crop. In vitro micro-propagation of sugarcane presents a good alternative, since it produces large quantities of disease-free planting material in a timely manner. A method of direct shoot organogenesis of sugarcane using apical meristems as explants is described herein. Sugarcane tops were collected from the field and surface sterilized (10% sodium hypochlorite solution for 15 min). Apical meristems were recovered and cultured in basal MS (Murashige and Skoog) liquid media containing sucrose (20 g/L), and a combination of the cytokinin benzylaminopurine (0.9 mg/L) and the auxin α-naphthylacetic acid (NAA) (1.86 mg/L) for shoot initiation. After 21 days in culture, the initiated plantlets were transferred to solid shoot proliferation media composed of MS, sucrose (20 g/L), NAA (2.0 mg/L), and the cytokinin kinetin (2.0 mg/L). Multiplying shoots were separated and sub-cultured at 3-week intervals.  After reaching a sufficient number, plantlets were transferred to solid rooting media containing MS, sucrose (20 g/L), and the auxin indole-3-butyric acid (2.0 mg/L). The in vitro grown plantlets were transferred to the greenhouse where a survival rate of 81% was recorded in 6 weeks.  The present in vitro tissue culture method provides a simple, high-throughput propagation system for sugarcane. It enables the rapid generation of disease-free planting stocks, thus eliminating the need for field collection of plant material.