Parasexual hybridization of piper nigrum and piper colubrinum through protoplast fusion

Abstract

Black pepper is the most important export oriented commodity and foreign exchange earner among the Indian spices. Ravages due to diseases, particularly the most devastating Phytophthora foot rot caused by Phytophthora capsici is one of the major constraints in the production of black pepper all over the world. Piper colubrinum, a wild relative of black pepper is found to be immune to foot rot disease. Non – existence of cultivar level tolerance or resistance against foot rot disease in black pepper necessitated the incorporation of incompatible wild relatives through parasexual hybridization. This study was undertaken in the Department of Plantation Crops and Spices and the Plant Tissue Culture laboratory of the Centre for Plant Biotechnology and Molecular Biology, College of Horticulture, Vellanikkara between September 1998 to April 2000. The cultures of P. nigrum and P. colubrinum were initiated in test tubes and were maintained at 26 + 20C temperature and 60 to 80 per cent humidity. The green leaves excised from axenic cultures of both the species were treated with cell wall degrading enzymes, cellulose and pectinase maintained at proper osmotic concentration. In P. nigrum maximum yield was observed at 1.4 per cent cellulose and 0.34 per cent pectinase. Cellulase and pexctinase at a concentration of 1.0 per cent and 0.217 per cent respectively recorded highest yield in P. colubrinum. In both the species 0.6 M osmoticum was found to be optimum to maintain the osmotic potential of the isolation solution. Highest yield of protoplasts was recorded in both the species during 21 h of digestion. Filtration-centrifugation technique was found to be superior in purifying the Piper protoplasts compared to the sucrose floatation method. Centrifugation at 1000 rpm for three minutes was found to be best for purifying P. nigrum protoplasts. For purifying P. colubrimum protoplasts, 600 rpm for three minutes was found to be optimum. Highest viability was noticed at 0.55 M and 0.65 M osmoticum in 1.0 per cent cellulose and 0.28 per cent pectinase during 21 h of digestion in P. nigrum. In P. colubrinum, maximum viability was observed at 0.4 M osmoticum in the enzyme mixture 1.0 per cent Cellulase and 0.186 per cent Pectinase during 18 h of incubation. Protoplasts of both the species when cultured on modified MS medium formed no cell wall and have not undergone any division. A age of the cultures advanced, proptoplast viability decreased in P. nigrum and P. colubrinum. All the protoplasts died by the second week in both the species. The protoplasts of both the species were heterogenous in terms of size. Fusion of the protoplasts was not observed after PEG treatment in the present study.