Effect of Nitrogen, Phosphorus and Medium pH to Enhance Alkaloid Production from Catharanthus roseus Cell Suspension Culture

Yıl 2019, Cilt: 6 Sayı: 2, 137 - 153, 15.07.2019

Malay Ranjan Mishra Mishra Rajesh Kumar Srivastava Nasim Akhtar

https://doi.org/10.21448/ijsm.559679

Cited By: 4

Öz

Several elevated levels of nitrogen and phosphate at varying pH of the medium which impart a major influence on callus and biomass development and subsequent production of alkaloids was investigated using suspension culture system of Catharanthus roseus in the present study. The B5 medium was buffered at pH 4.51, 5.82 and 7.32 by addition of different levels of (A) diammonium hydrogen phosphate (NH4)2HPO4 and (B) ammonium dihydrogen orthophosphate (NH4H2PO4) representing the enhanced and varied supply of total nitrogen (NH4++NO3ˉ) and phosphate compared to MS medium (as control) for cell biomass production and alkaloid yield. The pH of the medium have shown significant effects with maximum biomass fresh wt., dry wt. and total alkaloid yield at 5.82 medium pH with elevated phosphate levels and total nitrogen concentration of 3710.10 mg/L compared to control MS medium with 2850 mg/L total nitrogen. At 3667.33 and 3752.48 mg/L of total nitrogen with enhanced phosphate supply showed reduced biomass fresh wt., dry wt. and total alkaloid yield at lower (4.51) and higher (7.32) medium pH respectively. Inclusion of 200 mg/L of tryptophan or phenylalanine as reduced nitrogen source in B5 medium buffered at 5.82 ± 0.2 pH showed enhanced biomass and alkaloid production. Hence, addition of nitrogen, phosphate, tryptophan, phenylalanine as nutrient in suspension culture stimulate their uptake to enhance cell biomass and total alkaloids production but as a function of pH of the medium.

Anahtar Kelimeler

Alkaloid, Catharanthus roseus, medium pH, Nitrogen, Phenylalanine, Phosphorus, Tryptophan

Effect of Nitrogen, Phosphorus and Medium pH to Enhance Alkaloid Production from Catharanthus roseus Cell Suspension Culture

Öz

Several
elevated levels of nitrogen and phosphate at varying pH of the medium which
impart a major influence on callus and biomass development and subsequent
production of alkaloids was investigated using suspension culture system of Catharanthus roseus in the present
study. The B5 medium was buffered at pH 4.51, 5.82 and 7.32 by addition of
different levels of (A) diammonium hydrogen phosphate (NH₄)₂HPO₄
and (B) ammonium dihydrogen orthophosphate (NH₄H₂PO₄)
representing the enhanced and varied supply of total nitrogen (NH₄⁺+NO₃^ˉ)
and phosphate compared to MS medium (as control) for cell biomass production
and alkaloid yield. The pH of the medium have shown significant effects with
maximum biomass fresh wt., dry wt. and total alkaloid yield at 5.82 medium pH
with elevated phosphate levels and total nitrogen concentration of 3710.10 mg/L
compared to control MS medium with 2850 mg/L total nitrogen. At 3667.33 and
3752.48 mg/L of total nitrogen with enhanced phosphate supply showed reduced
biomass fresh wt., dry wt. and total alkaloid yield at lower (4.51) and higher
(7.32) medium pH respectively. Inclusion of 200 mg/L of tryptophan or
phenylalanine as reduced nitrogen source in B5 medium buffered at 5.82 ± 0.2 pH
showed enhanced biomass and alkaloid production. Hence, addition of nitrogen,
phosphate, tryptophan, phenylalanine as nutrient in suspension culture
stimulate their uptake to enhance cell biomass and total alkaloids production
but as a function of pH of the medium.

Anahtar Kelimeler

Alkaloid, Catharanthus roseus, medium pH, Nitrogen, Phenylalanine, Phosphorus, Tryptophan

Kaynakça

• [1]. Murashige, T., Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant, 15, 473-497.
• [2]. Kirkby, E.A. (1981). Plant growth in relation to nitrogen supply. Ecological Bulletins (Sweden), 33, 239-267.
• [3]. Cramer, M.D., Lewis, O.A.M. (1993). The influence of NO3- and NH4+ nutrition on the carbon and nitrogen partitioning characteristics of wheat (Triticum aestivum L.) and maize (Zea mays L.) plants. Plant and Soil, 154(2), 289-300.
• [4]. Fabre, F., Planchon, C. (2000). Nitrogen nutrition, yield and protein content in soybean. Plant Science, 152(1), 51-58.
• [5]. Ali, B., Hayat, S., Hayat, Q., Ahmad, A. (2010). Cobalt stress affects nitrogen metabolism, photosynthesis and antioxidant system in chickpea (Cicer arietinum L.). Journal of Plant Interaction, 5, 223–231.
• [6]. Guo, X.R., Zu, Y.G., Tang, Z.H. (2012). Physiological responses of Catharanthus roseus to different nitrogen forms. Acta Physiologiae Plantarum, 34, 589–598.
• [7]. Bhadra, R., Shanks, J.V. (1997). Transient studies of nutrient uptake, growth, and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus. Biotechnology and bioengineering, 55(3), 527-534.
• [8]. Garnier, F., Carpin, S., Label, P., Crèche, J., Rideau, M., Hamdi, S. (1996). Effect of cytokinin on alkaloid accumulation in periwinkle callus cultures transformed with a light-inducible ipt gene. Plant Science, 120(1), 47-55.
• [9]. Morgan, J.A., Barney, C.S., Penn, A.H., Shanks, J.V. (2000). Effects of buffered media upon growth and alkaloid production of Catharanthus roseus hairy roots. Applied microbiology and biotechnology, 53(3), 262-265.
• [10]. Morrison K.M., Simmons, S.J., Stapleton, A.E. (2010). Loci controlling nitrate reductase activity in maize: ultraviolet B signaling in aerial tissues increases nitrate reductase activity in leaf and root when responsive alleles are present. Physiologia Plantarum, 140, 334–341.
• [11]. Marschner, M. (1995). Mineral Nutrition of Higher Plants. 2nd Edn., Academic Press, London, New York, pp. 200-255; ISBN-10: 0124735436.
• [12]. Kaul, K., Hoffman, S.A. (1993). Ammonium ion inhibition of Pinus strobus L. callus growth. Plant Science, 88(2), 169-173.
• [13]. Guo, X.R, Chang, B.W, Zu, Y.G., Tang, Z.H. (2014). The impacts of increased nitrate supply on Catharanthus roseus growth and alkaloid accumulations under ultraviolet-B stress. Journal of Plant Interactions, 9(1), 640 - 646. DOI: 10.1080/17429145.2014.886728
• [14]. Lau, T.S.L., Eno, E., Goldstein, G., Smith, C., Christopher, D.A. (2006). Ambient levels of UV-B in Hawaii combined with nutrient deficiency decrease photosynthesis in near-isogenic maize lines varying in leaf flavonoids: flavonoids decrease photoinhibition in plants exposed to UV-B. Photosynthetica, 44, 394–403.
• [15]. Tang, Z.H, Liu, Y.J., Guo, X.R., Zu, Y.G. (2011). The combined effects of salinity and nitrogen forms on Catharanthus roseus: the role of internal ammonium and free amino acids during salt stress. Journal of Plant Nutrition Soil Science, 174, 135–144.
• [16]. Smith, A.M., Stitt, M. (2007). Coordination of carbon supply and plant growth. Plant Cell & Environment, 30, 1126–1149.
• [17]. Zhong, J.J., Wang, S.J. (1998). Effects of nitrogen source on the production of ginseng saponin and polysaccharide by cell cultures of Panax quinquefolium. Process biochemistry, 33(6), 671-675.
• [18]. Hahn, E.J., Kim, Y.S., Yu, K.W., Jeong, C.S., Paek, K.Y. (2003). Adventitious root cultures of Panax ginseng CV Meyer and ginsenoside production through large-scale bioreactor system. Journal of plant biotechnology, 5(1), 1-6.
• [19]. Uozumi, N., Makino, S., Kobayashi, T. (1995). 20-Hydroxyecdysone production in Ajuga hairy root controlling intracellular phosphate content based on kinetic model. Journal of fermentation and bioengineering, 80(4), 362-368.
• [20]. Van Gulik, W.M., Ten Hoopen, H.J.G., Heijnen, J.J. (1993). A structured model describing carbon and phosphate limited growth of Catharanthus roseus plant cell suspensions in batch and chemostat culture. Biotechnology and Bioengineering, 41(8), 771-780.
• [21]. Correia, J.J., Lobert, S. (2001). Physiochemical aspects of tubulin-interacting antimitotic drugs. Current pharmaceutical design, 7(13), 1213-1228.
• [22]. Isah, T., Umar, S., Mujib, A., Sharma, M.P., Rajasekharan, P.E., Zafar, N.,· Frukh, A. (2018). Secondary metabolism of pharmaceuticals in the plant in vitro cultures: strategies, approaches, and limitations to achieving higher yield. Plant Cell, Tissue & Organ Culture (PCTOC), 132, 239–265.
• [23]. El-Sayed M, Verpoorte R. (2007). Catharanthus terpenoid indole alkaloids: biosynthesis and regulation. Phytochemistry Reviews, 62, 277-305. DOI:10.1007/s11101-0069047-8
• [24]. Buchanan, B.B., Gruissem, W., Jones, R.L. (2000). Biochemistry & molecular biology of plants (Vol. 40). Rockville, MD: American Society of Plant Physiologists.
• [25]. Van Der Heijden, R., Jacobs, D.I., Snoeijer, W., Hallared, D., Verpoorte, R. (2004). The Catharanthus alkaloids: Pharmacognosy and Biotechnology. Current Medicinal Chemistry, 11, 607-628.
• [26]. Moreno, P.R., van der Heijden, R., Verpoorte, R. (1994). Elicitor-mediated induction of isochorismate synthase and accumulation of 2, 3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures. Plant cell reports, 14(2-3), 188-191.
• [27]. Shanks, J.V., Rijhwani, S.K., Morgan, J., Vani, S., Bhadra, R., Ho, C.H. (1999). Quantification of metabolic fluxes for metabolic engineering of plant products. In Plant cell and tissue culture for the production of food Ingredients Springer, Boston, MA. 1999; pp. 45-60.
• [28]. Baldi, A., Dixit, V.K. (2008). Yield enhancement strategies for artemisinin production by suspension cultures of Artemisia annua. Bioresource technology, 99(11), 4609-4614.
• [29]. Zeng, Y., Yan, F., Tang, L., Chen, F. (2003). Increased crocin production and induction frequency of stigma-like-structure from floral organs of Crocus sativus L. by precursor feeding. Plant Cell, Tissue and Organ Culture, 72(2), 185-191.
• [30]. Seitz, H.U., Eilert, U., De Luca, V., Kurz, W.G.W. (1989). Elicitor-mediated induction of phenylalanine ammonia lyase and tryptophan decarboxylase: accumulation of phenols and indole alkaloids in cell suspension cultures of Catharanthus roseus. Plant cell, tissue and organ culture, 18(1), 71-78.
• [31]. Mishra, M.R., Srivastava, R.K., Akhtar, N. (2018a). Enhancing alkaloid production from cell culture system of Catharanthus roseus with different carbon sources. European Journal of Biotechnology and Bioscience, 6(5), 12-20.
• [32]. Mishra, M.R., Srivastava, R.K., Akhtar, N. (2018b). Enhanced Alkaloid Production from Cell Culture System of Catharanthus roseus in Combined Effect of Nutrient Salts, Sucrose and Plant Growth Regulators. Journal of Biotechnology and Biomedical Science 1(4). 14-34. DOI: 10.14302/issn.2576-6694.jbbs-18-2475
• [33]. Mishra, M.R., Srivastava, R.K., Akhtar, N. (2019). Abiotic stresses of salinity and water to enhance alkaloids production in cell suspension culture of Catharanthus roseus. Global Journal of Bio-Science and Biotechnology, 9(1), 7-14.
• [34]. Gamborg, O.L., Miller, R., Ojima, K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 50(1), 151-158.
• [35]. Kalidass, C., Ramasamy, M.V., Daniel, A. (2010). Effect of auxin and cytokinin on vincristine production by callus cultures of Catharanthus roseus L.(apocynaceae). Tropical and Subtropical Agroecosystems, 12, 283-288.
• [36]. Sreevidya, N., Mehrotra, S. (2003). Spectrophotometric method for estimation of alkaloids precipitable with Dragendorff's reagent in plant materials. Journal of AOAC International, 86(6), 1124-1127.
• [37]. SPSS Inc. Released (2006). SPSS for Windows, Version 15.0. Chicago, SPSS Inc.
• [38]. Naeem, M., Aftab, T., Khan, M.M.A. (eds) (2017). Catharanthus roseus: Current Research and Future Prospects. Springer International Publishing AG, Cham, 412pp. https://doi.org/10.1007/978-3-319-51620-2
• [39]. Li, Q., Tang, M., Tan, Y., Ma, D., Wang. Y., Zhang, H. (2016). Improved production of chlorogenic acid from cell suspension cultures of Lonicera macranthoids. Tropical Journal of Pharmaceutical Research, 15(5), 919-927. http://dx.doi.org/10.4314/tjpr. v15i5.4
• [40]. Wongchai, C., Chaidee, A., Pfeiffer, W. (2012). Multivariate analyses of salt stress and metabolite sensing in auto- and heterotroph Chenopodium cell suspensions. Plant Biology, 14, 129–141.
• [41]. Nagella, P., Murthy H. N. (2010). Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresource Technology, 101(17), 6735- 6739.
• [42]. Herzbeck, H., Husemann, W. (1985). Photosynthetic carbon metabolism in photosynthetic cell suspension culture of Chenopodium rubrum L. In: Karl-Hermann Neumann, Wolfgang Barz, Ernst Reinhard (eds). Primary and secondary metabolism of plant cell culture: Part 1, Springer-Verlag Berlin Heidelberg. 15-23. 10.1007/978-3-642-70717-9_2.
• [43]. Ramawat, K. G. (1999). Production in Culture: Optimization In: Ramawat K.G. Merillon, J.M. (Eds). Biotechnology Secondary Metabolites, Science Publisher, USA, pp 123-143.
• [44]. Asada, M., Shuler, M. L. (1989). Stimulation of ajmalicine production and excretion from Catharanthus roseus: effects of adsorption in situ, elicitors and alginate immobilization. Applied microbiology and biotechnology, 30(5), 475-481.
• [45]. Mukundan, U., Bhide, V., Singh, G., Curtis, W.R. (1998). pH-mediated release of betalains from ransformed root cultures of Beta vulgaris L. Applied Microbiology and Biotechnology, 50(4), 241-245.
• [46]. Rahimi, Sh., Hasanloo, T. (2016). The effect of temperature and pH on biomass and bioactive compounds production in Silybum marianum hairy root cultures. Research Journal of Pharmacognosy, 3(2), 53-59.
• [47]. Sivakumar, G., Yu, K.W., Hahn, E.J., Paek, K.Y. (2005). Optimization of organic nutrients for ginseng hairy roots production in large-scale bioreactors. Current Science, 89(4), 641-649.
• [48] Wang Y, Ye Q, Zhu Y. (2008). Preliminary study on the cell suspension culture of Eucommia ulmoides and secondary metabolite-chlorogenic acid. Guihaia, 5, 024.
• [49]. Nowacki, E., Jurzysta, M., Gorski,. P., Nowacka, D., Waller, G.R. (1976). Effect of Nitrogen Nutrition on A1kaloid Metabolism in Plants. Biochemie und Physiologie der Pflanzen, 169, 231-240.
• [50]. Liu, Q., Cui, L., Guo, Y., Ni, X., Zhang, Y., Kai, G. (2013). Optimization of nutritive factors in culture media for growth and tropane alkaloid production from Anisodus acutangulus hairy roots. Journal of Applied Pharmaceutical Science, 301, 001-004. DOI: 10.7324/ JAPS.2013.30101
• [51]. Georgiev, V., Berkov, S., Georgiev, M., Burrus, M., Codina, C., Bastida, J., Ilieva, M., Pavlov, A. (2009). Optimized nutrient medium for galanthamine production in Leucojum aestivum L. in vitro shoot system. Zeitschrift für Naturforschung C, 64(3-4), 219-224.
• [52]. McDonald, K. A., Jackman, A. P. (1989). Bioreactor studies of growth and nutrient utilization in alfalfa suspension cultures. Plant cell reports, 8(8), 455-458.
• [53]. Endress, R. (Eds) (1994). Plant Cell as producers of Secondary compounds. In: Plant cell biotechnology Berlin: Springer-Verlag. Pp. 121-251.
• [54]. Nef, C., Ambid, C., Fallot, J. (1987). Influence of External pH on Alkaloid Production and Excretion by Catharanthus Roseus Resting Cell Suspensions. In: Marin B. (eds) Plant Vacuoles. NATO ASI Series (Series A: Life Sciences), Springer, Boston, MA, vol 134.
• [55]. Pitta-Alvarez, S.I., Giulietti, A.M. (1999). Influence of chitosan, acetic acid and citric acid on growth and tropane alkaloid production in transformed roots of Brugmansia candida Effect of medium pH and growth phase. Plant cell, tissue and organ culture, 59(1), 31-38.
• [56]. Whitmer, S., van der Heijden, R., Verpoorte, R. (2002). Effect of precursor feeding on alkaloid accumulation by a strictosidine synthase over-expressing transgenic cell line S1 of Catharanthus roseus. Plant cell, tissue and organ culture, 69(1), 85-93.
• [57]. Aslam, J., Mujib, A., Fatima, S., Sharma, M.P. (2008). Cultural conditions affect somatic embryogenesis in Catharanthus roseus L.(G.) Don. Plant Biotechnology Reports, 2(3), 179.
• [58]. Wielanek, M., Urbanek, H. (2006). Enhanced glucotropaeolin production in hairy root cultures of Tropaeolum majus L. by combining elicitation and precursor feeding. Plant cell, tissue and organ culture, 86(2), 177-186.
• [59]. Ouyang, J., Wang, X.D., Zhao, B., Wang, Y.C. (2005). Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola. Process Biochemistry, 40(11), 3480-3484.
• [60]. Namdeo, A.G., Jadhav, T.S., Rai, P.K., Gavali, S., Mahadik, K.R. (2007). Precursor feeding for enhanced production of secondary metabolites: a review. Pharmacognosy Reviews, 1(2), 227.
• [61]. Yuehua, W. (2011). Effect of different amino acid precursorfeeding on the active ingredient of Fritillaria cirrhosa D. Don culture. Journal of Anhui Agricultural Science, 24, 026.
• [62]. Liang, X., Zhu, X., Li, H. (2009). Effects of precursor and elicitor on isoflavone accumulation in cell-suspension cultures of soybean. J Xiamen Univ (Natural Science), 1, 028.
• [63]. Chang, B.W., Cong, W.W., Chen, Q., Zu, Y.G., Tang, Z.H. (2014). The influence of different forms and concentrations of potassium nutrition on growth and alkaloid metabolism in Catharanthus roseus seedlings. Journal of plant interactions, 9(1), 370-377.
• [64]. Lidon, F.C., Ramalho, J.C. (2011). Impact of UV-B irradiation on photosynthetic performance and chloroplast membrane components in Oryza sativa L. Journal of Photochemistry and Photobiology B: Biology, 104, 457–466.
• [65]. Singh, V.P., Srivastava, P.K., Prasad, S.M. (2012). Differential effect of UV-B radiation on growth, oxidative stress and ascorbate–glutathione cycle in two cyanobacteria under copper toxicity. Plant Physiology and biochemistry, 61, 61–70.
• [66]. Singh, S., Agrawal, M., Agrawal, S.B. (2013). Differential sensitivity of spinach and Amaranthus to enhanced UV-B at varying soil nutrient levels: association with gas exchange, UV-B-absorbing compounds and membrane damage. Photosynthesis Research, 115, 123–138.
• [67]. Ramani. S., Chelliah, J. (2007). UV-B-induced signaling events leading to enhanced-production of Catharanthine in Catharanthus roseus cell suspension cultures. BMC Plant Biology, 7, 61.
• [68]. Fabon, G., Monforte, L., Tomas-Las-Heras, R., Nunez-Olivera, E., Martinez-Abaigar, J. (2012). Dynamic response of UV absorbing compounds, quantum yield and the xanthophylls cycle to diel changes in UV-B and photosynthetic radiations in an aquatic liverwort. Journal of Plant Physiology, 169(1), 20–26.
• [69]. Malik, S., Bhushan, S., Sharma, M., Ahuja, P.S. (2016). Biotechnological approaches to the production of shikonins: a critical review with recent updates. Critical reviews in biotechnology, 36(2), 327-340.
• [70]. Lata, B. (2007). Cultivation, mineral nutrition and seed production of Catharanthus roseus (L.) G. Don in the temperate climate zone. Phytochemistry Review, 6, 403-411.
• [71]. Hassan, R.A., Habib, A.A., El-Din, A.A.E. (2009). Effect of nitrogen and potassium fertilization on growth, yield and alkaloidal content of periwinkle (Catharanthus roseus G. Don). Medicinal and Aromatic Plant Science and Biotechnology, 3(special issue), 24-26.
• [72]. Abdolzadeh, A., Hosseinian, F., Aghdasi, M., Sadgipoor, H. (2006). Effects of nitrogen sources and levels on growth and alkaloid content of periwinkle. Asian Journal of Plant Sciences, 5(2), 271-276.
• [73]. Shangguan, Z.P., Shao, M.A., Dyckmans, J. (2000). Nitrogen nutrition and water stress effects on leaf photosynthetic gas exchange and water use efficiency in winter wheat. Environmental and Experimental Botany, 44(2), 141-149.
• [74]. Singh, A., Agrawal, M. (2015). Effects of ambient and elevated CO2 on growth, chlorophyll fluorescence, photosynthetic pigments, antioxidants, and secondary metabolites of Catharanthus roseus (L.) G. Don. grown under three different soil N levels. Environmental Science and Pollution Research, 22, 3936-3946.
• [75]. Łata, B., Sadowska, A. (1996). Effect of nitrogen level in the substrate on yield and alkaloid content in Catharanthus roseus L. (G) Don. Folia Horticulturae, 8(2), 59-69.
• [76]. An, L., Liu, Y., Zhang, M., Chen, T., Wang, X. (2005). Effects of nitric oxide on growth of maize seedling leaves in the presence or absence of ultraviolet-B radiation. Journal of Plant Physiology, 162, 317–326.
• [77]. Zhang, M., Dong, J.F., Jin, H.H., Sun, L.N., Xu, M.J. (2011). Ultraviolet- B-induced flavonoid accumulation in Betula pendula leaves is dependent upon nitrate reductase-mediated nitric oxide signaling. Tree Physiology, 31(8), 798–807.
• [78]. Yu, F., De Luca, V. (2013). ATP-binding cassette transporter controls leaf surface secretion of anticancer drug components in Catharanthus roseus. Proceedings of the National Academy of Science USA, 110(39), 15830–15835.
• [79]. Monnerat, C.S., Freitas, M.S. ., Vieira, I.J.C., Martins, M.A., Carvalho, A.J.C., de Santos, P.C dos, Lima, T.C. (2018). Ajmalicine bioproduction in Catharanthus roseus (L) G. Don inoculated with arbuscular mycorrhiza and fertilized with nitrogen. Revista Brasileira de Ciência do Solo., 42, e0170057
• [80]. Hashemabadi, D., Sabzevari, F., Kaviani, B., Ansari, M.H. (2018). Organic N-fertilizer, rhizobacterial inoculation and fungal compost improve nutrient uptake, plant growth and the levels of vindoline, ajmalicine, vinblastine, catharanthine and total alkaloids in Catharanthus roseus L. Folia Horticulturae, 30(2), 21-31. DOI: 10.2478/fhort-2018-0018.
• [81]. Karthikeyan, B., Abdul Jaleel, C., Azooz, M.M. (2009). Individual and combined effects of Azospirillum brasilense and Psudomonas fluorescens on biomass yield and ajmalicine production in Catharanthus roseus. Academic Journal of Plant Sciences, 2(2), 69-73.
• [82]. Karthikeyan, B.N., Joe, M.M., Abdul Jaleel, C., Deiveekasundaram, M. (2010). Effect of root inoculation with plant growth promoting rhizobacteria (PGPR) on plant growth, alkaloid content and nutrient control of Catharanthus roseus (L.) G. Don. Natura Croatica, 19(1), 205-212.
• [83]. Attia, F.A., Saad, O.A.O. (2001). Bio-fertilizers as partial alternative of chemical fertilizer for Catharanthus roseus G. Don. J. Agric. Sci., Mansoura Univ., 26(11), 7193-7208.
• [84]. Jaleel, C.A., Manivavannan, P., Sankar, B., Kishorekumar, A., Gopi, R., Somasundaram, R., Panneerselvam, R. (2007). Psudomonas fluorescens enhances biomass yield and ajmalicine production in Catharanthus roseus under water deficit stress. Colloids and Surfaces B: Biointerfaces, 60(1), 7-11.
• [85]. Khalid, A., Arshad, M., Zahir, Z.A. (2004). Screening plant growth-promoting rhizobacteria for improving growth and yield of wheat. Journal of Applied Microbiology, 96(3), 473-480.