Characterisation of field established tissue culture derived black pepper (piper nigrum L.) plants using morphological, cytological and molecular markets

Abstract

Universally acclaimed as the ' King of Spices', black pepper (Piper nigrum L.) enjoys a unique position as a commercial crop of historical importance and assumes great economic importance to several nations of the world. Efforts to evolve high yielding types of black pepper through selection as well as hybridisation started as early as in 1953 (Nambiar et al., 1978) and by now, about ten improved varieties were evolved and released. In order to meet the insatiable demand from farmers for the planting materials of high yielding pepper varieties, mass multiplication protocol through micro-propagation has been standardised (Joseph et al., 1996). Babu (2000) tested the fidelity of TC plantlets of black pepper during the hardening stage using RAPD assay. A thorough evaluation and characterisation of these 'I'C plants in relation to their performance in the field is a prerequisite to establish their heritage and usefulness. In this background, the present study was taken up with the main objectives of establishing the genetic identity of TC derived black pepper plants in field; testing the intra-group, inter-clonal and inter- varietal polymorphism and obtaining a thorough and fool proof finger print of the TC plants using a combination of molecular marker techniques and conventional markers. Each of the experimental vines was characterised using morphological traits (biometric as well as qualitative) based on the descriptor formulated by NBPGR. The cytological studies ruled out any variation caused by a change in the chromosome number, which is possible in in vitro cultured plants. The somatic chromosome number was found uniform in all the vines with 2n = 52, which is characteristic of the species. The molecular markers provided a more specific identity for each of the experimental vines. The , zymogram based on peroxidase as well as RAPD banding pattern has given a very clear fingerprint characteristic of each vine. The biometric observations and visual assessment of the vines based on qualitative traits brought out the intra group (within conventional clones or within TC clones), inter- clonal (CC vs. TC clones) and inter varietal variation of the different characters. Among the 22 biometric observations, 17 were found homogenous within clonal groups and also within the TC groups irrespective of varieties. The remaining five traits were discussed with respect to each variety. Most of the qualitative observations also showed uniformity in. the clones as well as TC plants. The estimation of inter-clonal variablity proved the uniformity and better vigour of TC plants compared to the conventional clones. Another interesting observation was the great variablity in qualitative and quantitative traits between TC Subhakara and clonal Subhakara indicating a possible error in labelling. The molecular markers brought out the variablity in the different vines more specifically. In both isozyme and RAPD analysis, the clones under each variety were found to be monomorphic whereas certain variants could be detected within TC plants with respect to a few bands. The vines TC P2-7 and TC P1-1 were separated out as variants by both isozyme and RAPD markers. Other TC vines, which showed polymorphism with respect to either of these markers were Pl-I, PI-2, P2-10, P4-9, Su-9, Su-lO, P4-4 and P4-8. The assessment of inter clonal variability using molecular markers clearly pointed out the distinctly different traits of TC clones and conventional clones Subhakara. Morphological observations too supported this finding. Using RAPD, it was conclusively proved that the vines labelled as TC Su were in fact TC P 4. This confirmed the possibility of an error in labelling while the TC plantlets were transported from Vellanikkara to Panniyur. The inter-varietal polymorphism brought out by morphology and molecular markers was useful in discriminating the four black pepper varieties and to asses the genetic distance between them, which is an important criterion in the selection of parents for hybridisation. The results exposed the genetic proximity between PI and P2 and between P 4 and Suo The standardisations of various protocols for cytological and molecular marker analysis in black pepper as well as the relative efficiency of the different marker systems were also discussed. The RAPD technique was found most effective in assessing the genetic constitution of the individual vine.