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Chloroplast transformation vectors require an expression cassette flanked by homologous plastid sequences to drive plastome recombination. The 16-23 plastome region was selected and using this region, a new species-specific plastid transformation vector CuIA was developed with pKSII as a backbone by inserting the 16- and -23 sequences from L. An independent expression cassette with gene encoding aminoglycoside 3'-adenylyltransferase with controlling elements is added into the- intergenic region that confers resistance to spectinomycin. An efficient plastid transformation in bitter melon ( L.) was achieved by bombardment of petiole segments. The frequency of transplastomic plants yielded using standardized biolistic parameters with CuIA vector was two per 15 bombarded plates, each containing 20 petiole explants. Integration of gene was verified by PCR analysis in transplastomes. Transplastomic technology developed may be a novel approach for high level expression of pharmaceutical traits.

Here, we report the optimized conditions for biolistic particle delivery-mediated genetic transformation of bitter melon using petiole segments. In this study, DNA-coated gold particles of 0.6 µm were used for optimizing the parameters of transformation and eventually regeneration of bitter melon putative transgenics. Initially, biolistic parameters namely helium pressure and macrocarrier to target tissue distance, were optimized using binary vector pBI121 carrying both β-glucuronidase gene () and neomycin phosphotransferase II gene ( II) as a reporter and as a selectable marker gene, respectively. The effect of optimized physical parameters on the frequency of transient (79.2 ± 1.52%) and stable (41.9%) expressions has been investigated. The optimized biolistic parameters for petiole segments of L. were determined as follows: 650 psi helium pressure and 6 cm target distance. Using the optimized parameters, transformation of bitter melon was carried out for generation of putative transformants from bombarded tissues on SRM-K medium, with a mean number of 50.3 explants surviving at the end of the final selection (50 mg l kanamycin) round. Finally, the transformants produced were subjected to histochemical assay, and integration of the transgenes ( and II) into the nuclear genome was confirmed by PCR analysis. DNA blot analysis confirmed the transgene integration in the transformed plantlet genomes. The present study may be used for developing transplastomic technology in this valuable medicinal plant for enhanced metabolic engineering pathways and production of biopharmaceuticals.

All the reagents and solvents were purchased from Aldrich/Merck and used without further purifications. Melting points were determined in open capillaries using Stuart SMP30 apparatus and are uncorrected. The progress of the reactions as well as purity of compounds was monitored by thin layer chromatography with F 254 silica-gel precoated sheets using hexane/ethyl acetate 8/2 as eluent; UV light and iodine vapours were used for detection. IR spectra were recorded on a Perkin-Elmer 100S spectrometer utilizing KBr pellets. 1 H NMR and 13 C NMR spectra were obtained at 400 MHz and 100 MHz respectively on Bruker using DMSO-d 6 as solvent and TMS as internal standard. Elemental analyses were performed on a Carlo-Erba model EA1108 analytical unit. Mass spectra were recorded on a Jeol JMSD-300 spectrometer. General procedure for the synthesis of ethyl-2-(3-acetyl-2-oxo-2H-chromen-6-yl)-4methylthiazole-5-carboxylate (3)A mixture of ethyl 2-(3-formyl-4-hydroxyphenyl)-4-methylthiazole-5-carboxylate (1) (0.1 mol) and ethylacetoacetate (2) (0.12 mol) were dissolved in ethanol and cooled at 0-5 0 C. Piperidine (1 mL) was added drop wise to this mixture while stirring. The reaction mixture left overnight, resulting in the formation of yellow colored solid. Purification by recrystallization from ethanol to afford analytically pure product. General procedure for the synthesis of 3,6-disubstituted coumarin derivatives (6a-l)A mixture of ethyl-2-(3-acetyl-2-oxo-2H-chromen-6-yl)-4-methylthiazole-5-carboxylate (3, 1 mmol), thiosemicarbazide (1 mmol) and various phenacyl bromides (5a-g) / 3-(2-bromoacetyl)-2H-chromen-2-ones (5h-k) / 2-(2-bromoacetyl)-3H-benzo[f]chromen-3-one (5l) (1 mmol) were dissolved in 10 mL of absolute ethanol in the presence of catalytic amount of acetic acid and heated at refluxing temperature for 2-4 hrs. Monitored the reaction by TLC and poured contents in an ice cold water, the solid obtained was filtered, dried and recrystallised in ethanol.

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