A protocol is presented for regenerating plants from protoplasts of tropical mulberry. Leaves from seedling node cultures maintained in vitro were used as donor tissue. Optimal cell wall digestion was achieved with a combination of cellulase (2%) and macerozyme (1%). The plant growth regulator (PGR) combination zeatin (2.3 lM) and 2,4-dichlorophenoxyacetic acid (2,4-D D ) (2.3 lM) resulted in the highest number (29%) of cell divisions. First cell divisions were observed at day 4 after plating. Only zeatin (2.3 lM) and 2-methoxy-3,6-dichlorobenzoic acid (dicamba) (13.5 lM) supplemented medium supported subsequent divisions in protoplast cultures. Microcolonies reached a cell number of approximately 50, after 40 to 42 days of culture. The cells of these colonies continued dividing, leading to formation of microcalli. Whole plants were obtained after culture of microcalli on Murashige and Skoog (MS) medium containing thidiazuron (TDZ) (4.5 lM) and indole-3-acetic acid (IAA) (17.1 lM). The regenerated shoots were rooted on MS medium supplemented with 4.9 lM indole butyric acid (IBA). With a low survival rate during acclimation, regenerated plants were established in the greenhouse.

Here, we report for the first time, the optimized conditions for microprojectile bombardment-mediated genetic transformation in Vassourinha (Scoparia dulcis L.), a Plantaginaceae medicinal plant species. Transformation was achieved by bombardment of axenic leaf segments with Binary vector pBI121 harbouring β-glucuronidase gene (GUS) as a reporter and neomycin phosphotransferase II gene (npt II) as a selectable marker. The influence of physical parameters viz., acceleration pressure, flight distance, gap width & macroprojectile travel distance of particle gun on frequency of transient GUS and stable (survival of putative transformants) expressions have been investigated. Biolistic delivery of the pBI121 yielded the best (80.0 %) transient expression of GUS gene bombarded at a flight distance of 6 cm and rupture disc pressure/acceleration pressure of 650 psi. Highest stable expression of 52.0 % was noticed in putative transformants on RMBI-K medium. Integration of GUS and npt II genes in the nuclear genome was confirmed through primer specific PCR. DNA blot analysis showed more than one transgene copy in the transformed plantlet genomes. The present study may be used for metabolic engineering and production of biopharmaceuticals by transplastomic technology in this valuable medicinal plant.

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Curcuma longa L. is a spice crop with enormous medicinal and cosmetic properties cultivated across the world. It was propagated vegetatively by means of rhizomes, as these were underneath soil prone to soil borne fungal diseases. Treatment of such diseases using chemical fungicides would hinder their nutritional and medicinal value. To overcome such challenges, there are a few alternatives, the major among them was the deployment of disease-free in vitro raised plantlets and the other was biopriming of these plantlets with Trichoderma viride. Besides fungicidal action, T. viride has a prominent role in alleviating several biotic/abiotic stresses and it was more often used during acclimatization. In this study, we studied the role of T. viride on successful acclimatization of in vitro C. longa plantlets by modulating plant antioxidant defense systems. We determined the stress levels (MDA and chlorophyll contents) and monitored the antioxidant enzyme activities (CAT, APX and GR) in both T. viride-treated and untreated micropropagated plantlets at four different time points (0, 15, 30, 45, 60 days) of acclimatization period. Our results depicted clear enhancement in the antioxidant enzyme activities in T.viride treated than untreated plantlets, which signify the role of T. viride in activating defense mechanisms to combat against oxidative stress. Thus this study would give clear understanding of the influence of T. viride on in vitro raised plantlets in sustaining adverse acclimatization conditions.