Department of Biotechnology, Gulbarga University, Gulbarga- 585 106, Karnataka, India

Background and objectives
Eyespot-susceptible sugarcane cultivar Co 419 is one of the promising varieties in terms of high yield and sucrose content in Karnataka state, India. However, it is susceptible to Helminthosporium sacchari (Van Breda de Haan). The loss in yield due to this disease is being ignored by local farmers and no effort has been made in this region to eradicate the disease. In vitro techniques have been used to develop sugarcane lines resistant to eyespot disease [1]. However, there are no such reports in India. Hence the objective of the present investigation is to optimise in vitro strategies for the selection of sugarcane lines (Co 419) resistant to H. sacchari.

Results and conclusions
Fries' medium supplemented with 25 g/l of the susceptible host extract produced 10-25 mg/l of purified toxin (Heiminthosporoside) in 8-10 days of incubation as compared to 1-9 mg/l when cultured on Fries' medium for 22-24 days. Thus addition of the susceptible host extract enhances toxin production and this can be used even in other plant-pathogen systems. Here the objective is to develop disease resistant plants. Callus and leaf explant grown on MS medium [2] supplemented with different concentrations of culture filtrate (1-15% v/v) and partially purified toxin (0.001-0.5% v/v) showed differential tolerance. Callus cultures showed maximum inhibition at 15% culture filtrate concentration and 0.5% partially purified toxin concentration, respectively. LD50 value was 90.3 ml/250 ml and 2.5 g/250 ml for culture filtrate and partially purified toxin, respectively. Similarly leaf explant inoculated on MS medium did not show callus initiation at 10 and 15% culture filtrate, and 0.1 and 0.5% partially purified toxin concentration, respectively. It is assumed that the surviving callus and the callus initiated at the sublethal doses of culture filtrate and partially purified toxin concentration may show some degree of resistance to H.sacchari. Since the disease in question is caused by a toxin, millions of protoplasts can be screened for resistance in a small flask, they are equivalent to thousands of acres of growing plants. Hence, in a rapid and low caste method sugarcane protoplasts were isolated using crude enzyme preparations of cellulase and pectinase (Novo Nordisk, Bangalore). A satisfactory yield of protoplasts was obtained (1x106 protoplasts) in 4 h, incubated at 28-29C in still culture. The enzyme cost is less (Rs 1200/25 l) and ideally suits the working conditions for such studies. The protoplasts when stained with FDA, fluoresced when observed under the fluorescent microscope, thus indicating their viable nature. Hence the above in vitro strategies can be efficiently used for developing disease resistant sugarcane lines.

1. Larkin PJ, WR Scovvcroft, 1983. Plant Cell Tissue Organ Culture 2, 111-121.
2. Heinz DJ, Mea GWP, 1969. Crop Sci. 9, 346-348.