1JBoctor of ~bi[osopb!' in ~ortitultur

Transcription

1 SOMACLONAL VARIATION IN BLACK PEPPER (~ifjt.\ n"j"'um l.) BY M.R.SHYLAJA THESIS Submitted in partial fulfilment of the requirement for the degree of 1JBoctor of ~bi[osopb!' in ~ortitultur Faculty of Agriculture Kerala Agricultural University Department of Plantation Crops and Spices COLLEGE OF HORTICULTURE Vellanikkara, Thrissur 1996

2 CERTIFICATE Certified that the thesis entitled "Somaclonal variation in black pepper (PipeT nigrum L.)" is a record of research work done independently by Smt.M.R. Shylaja under my guidance and supervision and that it has not previously formed the basis for the award of any degree, diploma, fellowship or associateship to her. V e11anikkara 8'-~-'qt A.5~~l-6 Dr.G. SREEKANDAN NAIR Chairman of Advisory Committee Professor and Head Department of Plantation Crops and Spices College of Agriculture Vellayani

3 ABSTRACT Investigationson the exploitation of somaclonalvariation for screening for resistance/toleranceto Phytophthorafoot rot diseasein bla~kpepper were carried out at the Plant Tissue Culture Laboratory of the Department of Plantation Crops and Spices, College of Horticulture, Vellanikkaraduring September 1991to January Calliclones of different black pepper cultivars viz. Kalluvally, Cheriakanyakkadan, Balankotta, Karimunda and Panniyur-l were produced with and without applying in vitro selection pressure using toxic metabolite(s)of Phytophthora capsid. In vitro induction of mutation using gamma irradiation and partial purification of the toxic metabolite(s) present in the culture filtrate of P. capsid were also attemptedin the present study. Leaf puncture bioassay of the concentratedculture filtrate (CCF) of P. cqpsid showed that toxic metabolite(s) were accumulatedin the culture filtrate. The symptomsproduced by CCF were quite typical of natural and artificial infection by P. capsid. Concentrated culture filtrate induced quick electrolyte leakage from leaves and calluses. Concentrated culture filtrate induced necrosis on susceptible calli. The cultivars showedsignificantvariation 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.

4 In the three direct selection/screeningmethods 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 6OCo source did not give any better responseto 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 tlie filtrate. The response of five different,cultivarsat various stages of development of cultures when compared, it was found that the cultivars differed significantlyin callusing, callus growth, regeneration of shoots, recovery of rootable shoots and root growth. The clones regenerated from screenedand unscreened calli were further tested for resistance/toleranceto P. capsid 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. capsid. None of the regeneratedcallicloneswere 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

5 the screened calli derived ones. The calliclones of different cultivarsdiffered significantly in the tolerance/susceptibility reaction to the disease. The calliclones of Cheriakanyakkadan recorded greater degree of tolerance to the disease when compared to others. somaclonal variation. Among the cultivars studied, Kalluvally exhibited high rate of