Somaclonal variation in black pepper (piper nigrum L)

Abstract

Investigations on the exploitation of somaclonal variation for screening for resistance to Phytophthora foot rot disease in black pepper were carried out at the Plant Tissue Culture Laboratory of the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkara during September 1991 to January 1995. Calliclones of different black pepper cultivars viz. Kalluvally, Cheriakanyakkadan, Balankotta, Karimunda and Panniyur – 1 were produced with and without applying in vitro selection pressure using toxic metabolite(s) of Phytophthora capsici. In vitro induction of mutation using gamma irradiation and partial purification of the toxic metabolite(s) present in the culture filtrate of P. capsici were also attempted in the present study. Leaf puncture bioassay of the concentrated culture filtrate (CCF) of P. capsici showed that toxic metabolite(s) were accumulated in the culture filtrate. The symptoms produced by CCF were quite typical of natural and artificial infection by P. capsici. Concentrated culture filtrate induced quick electrolyte leakage from leaves and calluses. Concentrated culture filtrate induced necrosis on susceptible calli. The cultivars showed significant variation in callus necrosis. Prolonged duration of selection/screening with CCF totally inhibited the regeneration potential of the calli. Concentrated culture filtrate was not found to inhibit shoot proliferation and shoot growth in already regenerated cultures but inhibited the root growth. In the three direct selection/screening methods tried for calli viz. growing in CCF incorporated MS medium (Method 1) shaking in CCF incorporated liquid MS medium (Method 2) and double layer culture technique (Method 3), cultivars showed significant differences in callus necrosis and callus growth. Direct screening of calli was not found to inhibit the regeneration of shoots, shoot proliferation and recovery of rootable shoots but affected the root growth adversely. Gamma irradiation of calli using 60Co source did not give any better response to in vitro screening. The toxic metabolite(s) present in the culture filtrate could not be separated by organic solvent fractionation. However ion exchangers like Dowex 1 and Dowex 50 could be used for separation of the toxic fraction from the filtrate. The response of five different cultivars at various stages of development of cultures when compared, it was found that the cultivars differed significantly in callusing, callus growth, regeneration of shoots, recovery of rootable shoots and root growth. The clones regenerated from screened and unscreened calli were further tested for resistance/ tolerance to P. capsici using different methods of screening viz.natural screening (keeping in infected field), screening by electrolyte leakage method and screening by artificial inoculation of culture disc of P. capsici. None of the regenerated calliclones were found to be resistant to the disease in natural screening. When the tolerance level of the regenerated calliclones was looked into, the performance of the unscreened calli derived clones was found better as compared to the screened calli derived ones. The calliclones of different cultivars differed significantly inthetolerance/susceptibility reaction to the disease. The calliclones of Cheriakanyakkadan recorded greater degree of tolerance to the disease when compared to others. Among the cultivars studied, Kalluvally exhibited high rate of somaclonal variation.