Optimization of Tissue Culture Conditions of Lallementia royleana (Benth.) Benth. and Assessment of Somaclonal Variations

Abstract

Lallementia rayle ana (Benth.) Benth. or Tukhm-e-Balango is an important medicinal plant that is cultivated largely for its seeds which are frequentl y used in traditional system of medicine. In Pakistan due to over exploitation this plant has been declared endangered from the wild but it is being cultivated in some regions. Tissue culture techniques have been exploited for the micropropagation of many plants, including a wide range of threatened medicinal plants. In the present study, an efficient protocol for direct regeneration of this highly medicinal plant was optimized through different explants (leaves, nodes, internodes and petioles) by using different combinations of phytohormones (NAA, BAP, IAA, Kinetin and 2,4-D). The callogenic ability of all these explants was also checked on MS media supplemented with these phytohormones, but they gave poor callogenic response. Of all the explants used nodal explants gave best organogenic response (80 %) -?producing 17. 7S shoots per explant, on MS media supplemented with BAP 1 mg/l. Whereas, NAA 1 mg/l gave best rooting response, producing 24 roots per explant. In vitro propagation often results in genetic and phenotypic variations in regenerated plants. In the present study these somaclonal variations in regenerated plants were analyzed by PCR based RAPD markers, as compared to the seed derived control plant. Random deeamer RAPD primers, from OPC series (OPCI-OPCS) were used for this purpose. OPC primers revealed overall genetic variation of 36.8 % among all the eleven samples of Lallementia royleana. Among all the regenerated plants highest polymorphism was detected in in vitro regenerated shoot on 1.0 mg/l NAA that produced 18 amplified bands out of which eight bands were polymorphic. To construct similarity matrix along with corresponding dandrogram NTSYS PC version 2. 0 was used based on RAPD data. The present study shows that different hormonal combinations and concentrations used for micropropagation of plants can induce genetic variation even in directly regenerated plants, and RAPD is an effective and reliable method to analyze these variations.