See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/237073468 Catharanthus roseus alkaloids: Application of biotechnology for improving yield Data in Plant Growth Regulation · May 2012 DOI: 10.1007/s10725-012-9704-4 CITATIONS READS 31 1,994 8 authors, including: Abdul Mujib Abdul Ilah Jamia Hamdard (Deemed to be University) Shridhar University 100 PUBLICATIONS 1,252 CITATIONS 22 PUBLICATIONS 155 CITATIONS SEE PROFILE SEE PROFILE Zahid Hameed Siddiqui Mehpara Maqsood University of Tabuk 10 PUBLICATIONS 199 CITATIONS 22 PUBLICATIONS 342 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Genes proteins somatic embryogenesis View project Tissue culture embryogenesis of medicinal and ornamental plants View project All content following this page was uploaded by Zahid Hameed Siddiqui on 17 May 2014. The user has requested enhancement of the downloaded file. Plant Growth Regul DOI 10.1007/s10725-012-9704-4 REVIEW PAPER Catharanthus roseus alkaloids: application of biotechnology for improving yield Abdul Mujib • Abdul Ilah • Junaid Aslam • Samar Fatima • Zahid Hameed Siddiqui • Mehpara Maqsood Received: 6 September 2011 / Accepted: 17 April 2012 Ó Springer Science+Business Media B.V. 2012 Abstract Catharanthus roseus (L.) G. Don. is a well SE Somatic embryos known medicinal plant. It produces several phyto- TDC Tryptophan decarboxylase compounds many of which show anticancerous properties. SSS Strictosidine synthetase The yields of these compounds are however, very low. In this present article, the current development of secondary metabolite synthesis in C. roseus involving biotechnology has been reviewed keeping in mind the various basic fac- Introduction tors that influence yield. The importance of cell culture, the role of culturing conditions and other approaches aiming at Catharanthus roseus (L.) G. Don. belonging to the family higher production of alkaloids have been discussed. The Apocynaceae, is an important dicotyledonous medicinal genes encoding important enzymes, proteomics, expressed plant. It has a pantropic distribution, naturalized in conti- sequence tag and transcription factors in relation to alka- nental Africa, America, Asia, Australia, Southern Europe loid yield have also been summarized in order to under- and in some islands of the Pacific Ocean. The plant is stand the regulatory mechanisms of C. roseus. cultivated as an ornamental, used particularly for beddings, borders and for mass effect. It blooms throughout the year Keywords Catharanthus roseus Á Indole alkaloids Á and is propagated by seeds or by cuttings. Biotechnology Á In vitro culture conditions C. roseus has been studied extensively for its remarkable anticancer constituents. Vincristine and vinblastine are two Abbreviations very powerful anticancerous compounds of terpenoid- 2, 4-D 2, 4-Dichlorophenoxyaceticacid indole alkaloids class found in C. roseus. Among other BA/BAP 6-Benzyladenine components, (Table 1) ajmalicine and serpentine are used DMSO Dimethylsulfoxide in relieving hypertension and related cardiac disorders. The GA3 Gibberellic acid yield of these two compounds (vincristine and vinblastine, HPLC High performance liquid chromatography Fig. 1) in particular, is very low, therefore is expensive. KN Kinetin This poses a serious challenge to all including biotech- MS Murashige and Skoog nologists as to improve the yield and to reduce the unit NAA a-Naphthalene acetic acid cost. In biotechnology based alkaloid enrichment program, PGR Plant growth regulator the following two approaches have currently been adopted: (1) use of superior cell lines from various sources and optimization of cultural conditions/factors that control A. Mujib (&) Á A. Ilah Á J. Aslam Á S. Fatima Á synthesis and (2) metabolic engineering and/or over Z. H. Siddiqui Á M. Maqsood expression of key enzymes (Van der Heijden et al. 2004). Cellular Differentiation and Molecular Genetics Section, In recent years, various aspects of C. roseus and its Department of Botany, Hamdard University, New Delhi 110062, India alkaloids were discussed (Van der Heijden et al. 1989; e-mail: [email protected] Moreno et al. 1995). In this present article, we reviewed 123 Plant Growth Regul Table 1 Some important anticancerous alkaloids/derivatives isolated study and improve the yield where several plant parts such from cell cultures of C. roseus as stem-node, root, leaf, flower, anther and seed have been Alkaloid Sources of isolation cultured as primary explants sources (Moreno et al. 1995). Ajmalicine Callus, suspension, shoot, hairy root Alsotonine Callus Anthirine Suspension Culture of callus Cathindine Suspension Serpentine Callus, suspension, shoot, hairy root Callus is an undifferentiated tissue which is a good source Akuammicine Callus, suspension, shoot of secondary metabolites and other natural products. It is Lochneridine Callus, suspension, hairy root also used to obtain suspension of cells, a key source to get embryos in masses and help in isolating protoplasts at ease. Horhammericine Suspension, shoot In C. roseus, various explants have been tested on several Tabersonine Callus, suspension basal media with or without plant growth regulators Vindoline Suspension, shoot, crown gall (PGRs) for obtaining callus (Akcam and Yurekli 1995; Vindolinine Suspension, shoot Mujib et al. 1995; Moreno et al. 1995; Van der Heijden Catharanthine Suspension, shoot, root et al. 2004). The agar-solidified medium was found 3,4 anhydro vinblastine Shoot more responsive compared to liquid, in which 2, 4-D Leurosine Shoot (0.5–2.0 mg/L) induced fast growing callus from nearly all Catharine Shoot the explant sources. It becomes necessary to transfer cal- Vinblastine Callus, crown gall, shoot, somatic embryo luses on fresh nutrient medium for further growth. The Vincristine Shoot, somatic embryo alteration of media, its components and manipulation of Sources Van der Heijden et al. (1989, 2004); Moreno et al. (1995) PGRs are also often essential to restore growth of declining callus. afresh the current biotechnological developments that aim to enrich alkaloids in C. roseus. Cell suspension Explant and other primary cultures Since early 1980s it has been noted that the plant cell’s Various undifferentiated and differentiated tissues such as growth is fast in agitated-suspension compared to solid callus, suspension, shoot, hairy root have been induced to medium because of easier uptake of nutrients by the Fig. 1 Chemical structure of vinblastine and vincristine Vinblastine Vincristine 123 Plant Growth Regul cultured tissues. The biomass growth could easily be An optimized hairy root culture protocol was established improved by using a variety of bioreactors (Schlatmann on MS with sucrose as carbon and lactalbumin as nitrogen et al. 1994). The process has generally been accomplished source, in which, enhanced level of total alkaloids was by transferring friable (1.0 g) callus to the liquid medium noticed compared to other explants and calli (Sun and Zeng on gyratory shaker (120 rpm). At this suspension period of 2005). Treated with antagonists (verapamil and CdCl2) that growth, early sub culturing (10–15 days interval) promotes blocked Ca?2 fluxes across the plasma membrane over faster growth by forming free cells and cell aggregates as 25 % increase of total alkaloid content in hairy root cul- new cultures. The cell suspension has often been used for tures of C. roseus was observed by Moreno-Valenzuela alkaloid synthesis studies in C. roseus as the extraction of et al. (2003). Higher concentration of ajmalicine and alkaloids is far more simple, it also uptake precursors and catharanthine was found in genetically transformed Cath- other nutrients more rapidly that favours enhanced accu- aranthus hairy root cultures (Vazquez-Flota et al. 1994). It mulation of alkaloids (Junaid et al. 2009; Zhao et al. 2009). was also noted that vindoline and catharanthine producing hairy roots were thin and slow in growth (Palazo´n et al. 1998). The study also indicated that early sub culturing Shoot and root culture induced fast growth of hairy tissues that accumulated enhanced level of lochnericine. Genetically transformed The cultivation of plant parts, i.e., shoot and root has also (with Agrobacterium rhizogenes) roots grew well in simple been practised for in vitro biosynthesis of secondary hormone-free medium and high accumulation of secondary metabolites (Pietrosiuk et al. 2007). A high accumulation metabolite was noticed in Catharanthus root culture. of ajmalicine in multiple shoot cultures of C. roseus was Various bioreactors and complex fermenters were tried to reported in Murashige and Skoog medium on high con- improve the biomass of hairy-roots that accumulated better centration of IAA (11.42 lM) and a low (2.22 lM) con- production of secondary metabolites (Nuutila 1994). centration of BA (Satdive et al. 2003). Vindoline synthesis Efforts to engineer C. roseus hairy roots to produce com- in multiple shoot culture of C. roseus was investigated in mercially significant amounts of valuable compounds like various (continuous light, 16 h) photoperiod regimes and vinblastine and vincristine may require the development of the authors observed that the vindoline biosynthesis was tools to study the effects of over expressing key metabolic very related with shoot morphogenesis (Campos-Tamayo and regulatory genes. In this direction, a glucocorticoid- et al. 2008). Similar protocol was earlier described