There remains a less space for the medicinal plants to be cultivated at large on the existing agricultural land, as it is mainly used for the production of crops to meet the demand for feeding ever-increasing population. Among the options remaining for the cultivation of medicinal plants are the marginal lands, which are infested with various kinds of stresses such as salt and drought. Molecular biology has proved a strong tool and many gene products have been identified from various organisms which confer to them the resistance and /or tolerance against abiotic stresses. These genes provide an opportunity for the biotechnologists to manipulate and explore the possibilities to improve upon the medicinal plants so that they can be profitably grown on the marginal lands. The work carried out by us is an effort in this direction, where we have mobilized tobacco osmotin gene via Agrobacterium tumefaciens mediated genetic transformation in chicory (Cichorium intybus L.). Our results confirm the integration of osmotin gene in the chicory genome and its role as osmoprotectant has been proved.

To achieve micropropagation of chicory various hormonal combinations (Auxin and Cytokinin) with or without growth adjuvent, Casein Hydrolysate (CH) were tried. Based on the percentage callusing, regeneration in cultures , growth and morphology of plantlets the hormonal combination; IAA(2mM) + KN(5mM) + CH (1000mgl-1) was found optimum.The micropropagated plantlets were transferred to pots for acclimatization so that they can sustain and survive in the natural conditions. The in vitro cultures of transformed and non-transformed chicory were found to accumulate the secondary metabolite, esculin at different morphogenetic stages. The concentration of esculin in in vivo grown plants was compared with different developmental stages of in vitro grown cultures. No significant difference in the esculin content was observed, when comparison was made among cultures of transformed and non-transformed chicory. 1.5 fold increase in esculin content was however, observed in in vitro grown plantlets when compared with in vivo grown plant at flowering stage. The Agrobacterium mediated transformation protocol was optimized considering all the factors for successful transformation. The maximum inhibitory concentration of selectable marker (Kanamycin:150mgl-1) was established. The leaf segments of chicory were co-cultivated with Agrobacterium strain (GV2260) harbouring osmotin gene in the plasmid pBinAR tagged with 35S CaMV promoter. The transformed explants were regenerated on the selection medium optimized for regeneration of chicory. After selection of, the transformants, the putative transgenics were characterized employing molecular biology techniques viz. PCR-utilizing the gene specific primers of osmotin and nptII. These putative transformants were further tested for the presence of osmotin using non-radiolabelled southern blotting of PCR amplified DNA with nptII specific primers, as it was part of the cloning casette of osmotin gene. The southern blotting confirmed the successful integration of the osmotin gene.

To validate the performance of these putative transgenics, physiological and biochemical assessments were made employing leaf disc assay on salt solution. The leaf discs from transgenic chicory plants showed less senescence over the marked period and retained more chlorophyll than those from non-transformed plants. Also proline, the biochemical marker of osmotolerance in plants accumulated upto 90% higher in the transgenic plants as compared to wild type, at 250mM NaCl. These results confirm the over-expression of the osmotin gene in transgenic plants and their improved tolerance when exposed to salt stress.