Yeast extract elicitation increases vinblastine and vincristine yield in protoplast derived tissues and plantlets in Catharanthus roseus

Abstract Catharanthus roseus (L.) G. Don, Apocynaceae, is an immensely important medicinal plant, produces a variety of anticancerous compounds. The yield of two most investigated alkaloids vinblastine and vincristine is unfortunately very low. A vast array of technologies including elicitation have recently been used to enrich Catharanthus alkaloid in culture. Yeast extract is a biotic elicitor, the polysaccharide and the peptide moiety have been recognized as a signalling element in enriching secondary metabolites. In this study, the yeast extract elicitation on vinblastine and vincristine was studied in various protoplast derived tissues and plantlets. Four different yeast extract treatments (T1 = 0.5 g/l, T2 = 1.0 g/l, T3 = 1.5 g/l and T4 = 2.0 g/l) were prepared and used. The alkaloid was quantified and a comparative account of yield were presented by the use of High performance thin layer chromatography. The yeast extract amendment in medium improved vinblastine and vincristine yield in cultivating tissues, maximum being in germinating embryos and in in vitro raised leaf. The highest yield was in T3 (1.5 mg/l) in which 22.74% vinblastine and 48.49% vincristine enrichment was noted in germinating embryos; the enhancement was however, treatment-specific. Antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase activities were investigated as addition of yeast extract caused cellular stress and had enriched level of alkaloids.

Somatic Embryogenesis and Plantlet Regeneration from Protoplast Culture of Stevia rebaudiana.

In order to develop a high-efficiency and reproducible regeneration protocol for Stevia protoplasts, various factors such as type and concentration of enzymes, osmoticum, incubation time, plant material type and age were studied. Protoplasts were successfully isolated from leaves of fourweek- old in vitro grown plants using an enzyme mixture comprising of 2% (w/v) Cellulase Onozuka R-10, 1.5% (w/v) Macerozyme Onozuka R-10, 0.2% (w/v) Driselase and 0.1%(w/v) Pectolyase Y- 23 in 0.5 M mannitol, 2.5 mM CaCl2.2H2O and 5 mM 2 (N-morpholino)-ethanesulfonic acid (MES ) at pH of 5.8. Approximately 8.4±0.40x106 protoplasts g-1fresh weight with 98.8±1.39% viability was obtained after incubating in enzyme solution for 4 hours in dark. Viable protoplasts were collected by centrifugation in the presence of 16% sucrose solution. Protoplasts at density of 5x105 mL-1were cultured on modified KM8P medium supplemented with 0.2 mg L-1 2,4-dicholorophenoxyacetic acid (2,4-D), 1 mg L-1 α-naphthalene acetic acid (NAA), 0.5 mg L-1 zeatin, 0.15 M sucrose and 0.3 M mannitol by agarose-bead or thin layer liquid culture technique. The protoplasts regenerated cell walls within 24 hours. First cell division was observed after culturing for 2-3 days and microcolonies were formed within 4 weeks. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Compared to liquid culture, agarose bead culture improved division frequency almost 1.5 times effectively and showing a plating efficiency of 13% and 9.1% respectively with survival rate of 23.5% to 14.8%. Upon transfer to Murashige and Skoog’s medium (MS) with 1 mg L-1BA, alone or in combination with NAA or 2, 4-D at 0.1 mg L-1, protoplast-derived calli produced complete plantlets through somatic embryogenesis in 8-weeks. The regenerated plants survived in soil and all were normal with respect to morphology and growth characters. This protocol might lead to the improvement of the Stevia through somatic hybridization, somaclonal variation and genetic engineering by using protoplast based regeneration System.

Strain improvement of entomopathogenic fungal species beauveria bassiana and metarhizium anisopliae by protoplast fusion.

Protease hyper producing recombinant strains were produced by intergeneric protoplast fusion of entomopathogenic fungi Beauveria bassiana and Metarhizium anisopliae. β-glucuronidase and KCl were used as lysing enzyme and osmotic stabilizer. Along with inter-generic fusion, intra-strain and inter-strain fusions were also carried out using polyethylene glycol (PEG) as fusogen. When the fused protoplasts were regenerated on Czapekdox agar medium, they exhibited fast mycelial growth and abundant sporulation when compared to non-fusants. Pr1 and Pr2 specific activities were found to be increased by two- fold in recombinant strains than the nonfusants and the parental strains.