ABSTRACT: ABSTRACT: BACKGROUND: Podophyllotoxin (PTOX), the precursor for semi-synthesis of cancer therapeutics like etoposide, teniposide and etophos, is primarily obtained from an endangered medicinal herb, Podophyllum hexandrum Royle. PTOX, a lignan is biosynthetically derived from the phenylpropanoid pathway. The aim of this study is to investigate changes in the P. hexandrum cell proteome potentially related to PTOX accumulation in response to MeJA elicitation. High-resolution two-dimensional gel electrophoresis (2-DE) followed by colloidal Coomassie staining and mass spectrometric analysis was used to detect statistically significant changes in cell's proteome. Result: The HPLC analysis showed approximately 7-8 fold change in accumulation of PTOX, in the 12d old cell suspension culture (i.e. after 9d of elicitation) elicited with 100 uM MeJA as compared to the control. Using 2-DE a total of 233 spots was detected, out of which 105 spots were identified by MALDI TOF-TOF MS/MS. Data were subjected to functional annotation from a biological point of view through KEGG. The phenylpropanoid and monolignol pathway enzymes were identified, amongst these, chalcone synthase, polyphenol oxidase, caffeoyl CoA 3-O-methyltransferase, S-adenosyl-L-methionine-dependent methyltransferases, caffeic acid-O-methyl transferase etc. are noted as important. The relation of other differentially accumulated proteins with varied effects caused by elicitors on P. hexandrum cells namely stress and defence related protein, transcription and DNA replication and signaling are also discussed. CONCLUSION: Elicitor-induced PTOX accumulation in P. hexandrum cell cultures provides a responsive model system to profile modulations in proteins related to phenylpropanoid/monolignol biosynthesis and other defense responses. Present findings form a baseline for future investigation on a non-sequenced medicinal herb P. hexandrum at molecular level.
Proteome Science 05/2012; 10(1):34. · 2.33 Impact Factor
ABSTRACT: Plant cell culture provides an alternative means for producing secondary metabolites. In this study, experiments were carried
out to study the impact of several parameters, independently and in combination, on the stimulation of menthol production
in the cell suspension culture of Mentha piperita. Callus was obtained from leaf segments of in vitro grown plantlets on Murashige and Skoog (MS) medium supplemented with 0.2mg l−1of 2,4-dichlorophenoxy acetic acid to initiate cell suspension culture. This culture was maintained in half-strength MS medium
supplemented with 0.2mg l−1of 2,4-dichlorophenoxy acetic acid at 15d interval and used for further studies. Precursor feeding alone, i.e., menthone,
at 35μM concentration showed slightly improved productivity. γ-Cyclodextrin alone at 60μM concentration and in combination
with menthone feeding at 35μM increased menthol yield up to 92 and 110mg l−1 in comparison to 77mg l−1 of control culture. Synergistic potentiation effect of menthone feeding at 35μM and γ-cyclodextrin at 60μM treatment followed
by in situ adsorption with RP-8 also showed potential stimulation of menthol production in M. piperita cell culture. Fungal elicitor treatment showed enhanced production level up to 140.8mg l−1 in comparison to that of control. Further studies were carried out with the establishment of Agrobacterium tumefaciens (Ach5) gall-mediated calli, and consequently, cell suspension culture and results showed the significant enhancement of menthol
yield up to 278mg l−1.
In Vitro Cellular & Developmental Biology - Plant 04/2012; 44(6):518-524. · 1.50 Impact Factor
ABSTRACT: Morphologically identical transgenic mint (Menthaarvensis L.) with bacterial glutathione synthetase gene has been developed. Transformed plants were obtained by co-cultivation of
leaf disks with Agrobacteriumtumefaciens strain LBA 4404 harbouring a binary vector pCAMBIA-CpGS that carried E.coli glutathione synthetase (GS), β-glucuronidase as reporter gene and nptII as selective marker gene for kanamycin resistance. Using a constitutive double CaMV 35S promoter and an rbcS transit peptide, we successfully addressed CpGS to the chloroplasts through pJIT 117 vector. Preculture and the presence of AS in the co-cultivation medium played a significant
role in enhancing transformation frequency. The highest transformation frequency was achieved with MS selection medium supplemented
with 25% coconut water, 1.12mgl−1 BAP, 0.2mgl−1 NAA, 50mgl−1 kanamycin and 125mgl−1 cefotaxime. Robust rooting of regenerated shoots was obtained in half-strength liquid MS medium containing 0.2mgl−1 NAA and 50mgl−1 kanamycin. The presence and expression of transgenes in transgenics (T0) was evidenced by GUS histoenzymatic assay, PCR and RT-PCR analysis of nptII and the gene of interest, i.e., GS of putative transgenic leaves. Chromosomal integration of GS gene was confirmed by Southern blot analysis. Transgenic plants were successfully acclimatized in the greenhouse. An overall
transformation frequency of 15% was achieved in approximately 3months of time period. These results are discussed in relation
to heavy metal trafficking pathways in higher plants and to the interest of using plastid expression of PCS for biotechnological applications.
Plant Cell Tissue and Organ Culture 01/2009; 96(2):117-126. · 3.09 Impact Factor