In vitro Plant Regeneration through Organogenesis and Somatic Embryogenesis of Punica granatum Linn. (Lythraceae)

Abstract

Reliable protocol was developed to obtain healthy plants of imported (Indian) Punica granatum Linn. using in vitro germinated seedlings and juvenile explants. In vitro seed germination of imported seeds was induced on MS medium supplemented with different concentrations of GA3. The best medium was found to be the MS medium supplemented with 5.0 mg/L GA3 and seed stratification induced the seed germination percentage and seedling height. Cotyledon, hypocotyle, internode and leaf explants were excised from in vitro germinated seedlings for calli induction. MS medium supplemented with 2.5 mg/L NAA and 3.0 mg/L BAP was found to be best medium for callus induction from cotyledons, 2.5 mg/L NAA and 4.0 mg/L BAP from hypocotyle, 1.5 mg/L NAA and 2.0 mg/L BAP from internode and 1.5 mg/L NAA and 3.0 mg/L BAP from leaf explants. Cotyledon explants showed the highest percentage of callus induction followed by internode, leaf and hypocotyle explants. MS medium supplemented with 0.5 mg/L NAA and 3.0 mg/L BAP was found to be the best medium for shoot regeneration from calli obtained from all types of explants. Highest shoot regeneration percentage was showed in cotyledon derived callus followed by hypocotyle, internode and leaf derived calli. For somatic embryogenesis leaf disc explants were taken from 45 days old P. granatum Linn. plantlets. MS medium supplemented with 500.0 mg/L L-Glutamine, 2.0 mg/L 2,4-D and 2.0 mg/L BAP was found to be the best medium for embryonic callus induction, MS medium with 500.0 mg/L L-Glutamine, 1.0 mg/L 2,4-D was found to be the most effective medium for embryonic callus formation. The treatment with 0.25 mg/L BAP, 500.0 mg/L Casein hydrolysate, 500.0 mg/L L-Glutamine and 30.0 g/L sucrose under dark conditions gave the highest percentage of matured embryonic callus.