IN-VITRO PLANT REGENERATION AND COMPARATIVE STUDY OF SECONDARY METABOLITE FROM TRANSFORMED AND NON-TRANSFORMED PLANT OF PIPER SPP.
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2011
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The genus Piper (piperaceae) is largely distributed in tropical and sub-tropical regions of the world. It is famous as the spices king due to its pungent quality. The genus Piper has more than 1000 species but the most well known species are P. nigrum, P. longum and P. betle. In P. nigrum, about 51 cultivars have been reported from the tropical and sub-tropical regions of the India. P. nigrum fruits are also used to produce white pepper and green pepper and valued due to presence of Piperine. It has high amount of secondary compounds such as unsaturated amides, flavonoids, lignans, long and short chain esters, terpenes, steroids and alkaloids (Parmer et al., 1997,1998; Navickiene et al. 2000; Facundo et al., 2005) and also having insecticidal activity (Boll et al. 1994). On the view of above fact, the present investigation is to standardize the in vitro protocol on plant regeneration from different explants of Piper species and comparative analysis of secondary metabolite associated in different explants as well as transformed and non-transformed callus and its impact on microbial activity. The shoot multiplication was achieved in MS medium supplemented with 1.5 mg/l BAP, 0.5 mg/l IAA and 100 mg/l adenine sulphate in Piper longum. However, the same composition did not effect any regeneration efficiency in other species like P.betle and P.nigrum. Among the three cytokinins used, it was observed that BAP was most suitable for shoot proliferation & multiplication. Inclusion of 0.5 mg/l IAA in the culture medium showed higher percentage of shoot proliferation (83.3%) and multiplication within 8 weeks of culture. The number of multiple shoots per explant varied from 1.0 – 3.4 on different culture medium.Callus induction and proliferation was achieved from leaf and stem explants on MS media supplemented with 2.5 mg/l 2, 4-D within 4 weeks of culture. Sub culturing was made every 4 weeks interval to enhance the production of multiple shoots. The elongated microshoots were separated and transferred to different rooting media for induction of root. The maximum percentage of rooting was achieved on ½ strength MS medium supplemented with 0.25 mg/l IBA and 2% (W/V) sucrose. The rooted plantlets were successfully transferred to greenhouse and grown normally. Further, the green friable calli developed from leaf tissues were used for transformation study to compare the Piperine content in transformed and non-transformed calli, fruits, leaf and root through TLC anf HPTLC method. Agrobacterium strain A4 harbouring root inducing plasmid (Ri- plasmid) was used for in vitro transformation. The percentage of Piperine content in transformed callus was 1.35% more than the non-transformed calli. The results also indicate that the root having more Piperine content than leaf and fruits. The phytochemical constituents such as alkaloid, steroid, Triterpenoids, glycosides, Flavonoids, Tannins and carbohydrates were present in the Piper species. But, Saponin and proteins were absent on the basis of chemical test. Further, the crude extracts of P.betle, P. longum and P.nigrum were used for antimicrobial activity. The positive zone of inhibition was observed in bacteria like Micrococcus luteus, Streptomyces epidermidis, E.coli and fungus like Aspergillus flavus and Rhizoctonia solani. Further work is necessary to standardize the protocol on propagation of elite clone of Piper species and enhance the secondary metaboliote by using biotechnological tools.