Characterization of Podophyllotoxin Yielding Cell Lines ofPodophyllum hexandrum

Callus cultures of Podophyllum hexandrum were initiated and established from juvenile explants isolated from axenic seedlings and mature explants isolated from 1 year old plants on B5 basal medium (at half strength) and modified MS medium supplemented with growth regulators. For initiating cell lines, cell suspension cultures were established with selected callus lines in B5 basal medium supplemented with growth regulators and cell lines were established by cell plating technique. Eight cell lines (RC1, RC2, RC3, RC4, RC5, RC6, RC7 and ZE) maintained as callus stocks in the laboratory have been characterized and compared on the basis of growth rate, morphology, podophyllotoxin content, ploidy, karyotype analysis and RAPD analysis. Morphologically the cell lines were of six different types like globular and dark brown, friable with rough surface and light brown, compact and yellow, compact and creamish, compact and dark brown in colour and creamish and highly friable in nature. The cell lines had variable growth rates with line RC1 being the fastest growing line. The lines also differed in podophyllotoxin accumulating capacity; line RC4 accumulated the highest amount of podophyllotoxin in 4 years of analysis followed by line RC2. Chromosome analysis from 2 and 4 year old cultures revealed the lines displayed the presence of diploid, hypodiploid and hyperdiploid cells in varying frequencies. An assessment of karyomorphology of the cell lines (except cell line ZE) indicated that the chromosomes in general were long (16.0 to 5.33μm) with median to subterminal or terminal region primary constrictions. Cell lines RC3, RC6 and RC7 showed the presence of chromosomes with secondary constrictions. The observed polymorphic banding pattern of the cell lines obtained with eleven RAPD markers reflected the genomic variability present in the cell lines. Primers OPA 01, OPA 02, OPA 04, OPA 12 and OPX 01 generated unique bands in some of the cell lines viz. RC1, RC2 and RC6, indicating that cell lines of P. hexandrum can be characterized and identified on basis of RAPD markers.