In vitro germination and shoot proliferation of the threatened species Cinchona officinalis L (Rubiaceae).

Abstract

Cinchona officinalis (Rubiaceae) is an endemic species of the Loja Valley in southern Ecuador with medicinal uses. Because of over-exploitation in the nineteenth century and more recent disturbances to its ecosystem, C. officinalis populations are threatened. Currently, natural regeneration of the populations is low, despite its high plant regeneration and seed formation capacity. In the present study, an efficient protocol for germination, shoot proliferation and plantlets regeneration was developed for this species. Phenolic content and germination rate of C. officinalis seeds were compared with a control species, C. pubescens. Nodal segments from seedlings of C. officinalis were cultured on Gamborg medium supplemented with different combinations of plant growth regulators. Because the phenol content is high in C. officinalis, the phenolic should be removed with hydrogen peroxide or water washes to stimulate germination. Shoots and callus developed from nodal segments within 45 days using most of the tested combinations of plant growth regulators. The best rates of shoot proliferation, callus formation and adventitious buds were obtained in medium supplemented with 5.0 mg L⁻¹ 6-benzyl-aminopurine and 3.0 mg L⁻¹ indole-3-butyric acid.

Keywords

Callus / Cinchona officinalis / Germination / Phenols / Rubiaceae / Shoot

References

[1] Acosta-Solís M. Cinchonas del Ecuador. 1946, Quito: Editorial Ecuador, 271.

[2] Acosta-Solís M. La Cinchona o Quina Planta Nacional del Ecuador. Rev Acad Colomb Cien, 1989, 17(65): 305-311.

[3] Allan EJ, Scragg AH. Comparison of the growth of Cinchona ledgeriana Moens suspension cultures in shake flasks and 7 liter air-lift bioreactors. Biotechnol Lett, 1986, 8(9): 635-638.

[4] Andersson L. A revision of the genus Cinchona (Rubiaceae-Cinchoneae). Mem N Y Bot Gard, 1998, 81: 1-75.

[5] Bailly C, El-Maarouf-Bouteau H, Corbineau F. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comptes Rendus Biol, 2008, 331: 806-814.

[6] Baker K, Steadman K, Plummer J, Dixon K. Seed dormancy and germination responses of nine Australian fire ephemerals. Plant Soil, 2005, 277(1–2): 345-358.

[7] Barton L. Effect of different storage conditions on the germination of seeds of Cinchona ledgeriana Moens. Contrib Boyce Thompson Inst, 1947, 15: 1-10.

[8] Blom T, Kreis W, van Iren F, Libbenga K. A non-invasive method for the routine-estimation of fresh weight of cells grown in batch suspension cultures. Plant Cell Rep, 1992, 11(3): 146-149.

[9] Dai W, Su Y, Castillo C. Plant regeneration from in vitro leaf tissues of Viburnum dentatum L. Plant Cell Tiss Organ Cult, 2011, 104(2): 257-262.

[10] Díaz M, Loján M (2004) Fenología y propagación en vivero de especies forestales nativas del bosque protector ¨El Bosque¨. Disertación (Ingeniero Forestal). Loja, Universidad Nacional de Loja, p 119

[11] Ellis R, Hong T, Roberts E. Handbook of seed technology for genebanks. 1985, Rome: International Board for Plant Genetic Resources (IBPGR), 715.

[12] Espinosa C, Ríos G. Patrones de crecimiento de Cinchona officinalis in vitro y ex vitro; respuestas de plántulas micropropagadas y de semillas. Rev Ecuat Med Cienc Biol, 2014, 35(1 y 2): 73-82.

[13] Gamborg O, Miller R, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res, 1968, 50: 151-158.

[14] Garmendia A (2005) El Árbol de la Quina (Cinchona spp.): Distribución, caracterización de su hábitat y arquitectura. Loja, Editorial Universidad Técnica Particular de Loja, p 187

[15] Geerlings A, Hallard D, Martinez A, Lopes I, van der Heijden R, Verpoorte R. Alkaloid production by a Cinchona officinalis “Ledgeriana” hairy root culture containing constitutive expression constructs of tryptophan decarboxylase and strictosidine synthase cDNAs from Catharanthus roseus. Plant Cell Rep, 1999, 19: 191-196.

[16] Giroud C, van der Leer T, van der Heijden R, Verpoorte R, Heeremans C, Niessen W, van der Greef J. Thermospray liquid chromatography/mass spectrometry (TSP LC/MS) analysis of the alkaloids from Cinchona in vitro cultures. Planta Med, 1991, 57(2): 142-148.

[17] Han Y-S, van der Heijden R, Vepoorte R. Improved anthraquinone accumulation in cell cultures of Cinchona robusta by feeding of biosynthetic precursors and inhibitors. Biotechnol Lett, 2002, 24(9): 705-710.

[18] Hay C, Anderson L, Phillipson J, Roberts M. In vitro culture of Cinchona species. Precursor feeding of C. ledgeriana root suspension cultures with L-tryptophan. Plant Cell Rep, 1986, 5(1): 1-4.

[19] Hay C, Anderson L, Phillipson J, Curless D, Brown T. In vitro culture of Cinchona species. Part II Time-course studies on the uptake of radio-labelled alkaloid precursors and alkaloids by C. ledgeriana root suspension cultures. Plant Cell Rep, 1987, 9(3): 197-206.

[20] Hoekstra S, Harkes P, Verpoorte R, Libbenga K. Effect of auxin on cytodifferentiation and production of quinoline alkaloids in compact globular structures of Cinchona ledgeriana. Plant Cell Rep, 1990, 8(10): 571-574.

[21] Ivanova M, Van Staden J. Nitrogen source, concentration, and NH₄+:NO₃− ratio influences shoot regeneration and hyperhydricity in tissue cultured Aloe polyphylla. Plant Cell Tissue Organ Cult, 2009, 99(2): 167-174.

[22] Jordán M (1975) Histologische und physiologische Untersuchungen zur Kapazität der Androgenese bei in vitro kultivierten Prunus-, Pyrus-, Ribes- und Nicotiana Antheren. Inaugural-Dissertation. Gieben, Justus Liebig-Universität, p 113

[23] Khouri H, Ibrahim R, Rideau M. Effects of nutritional and hormonal factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra. Plant Cell Rep, 1986, 5(6): 423-426.

[24] Koblitz H, Koblitz D, Schmauder H, Gröger D. Studies on tissue cultures of the genus Cinchona L. Alkaloid production in cell suspension cultures. Plant Cell Rep, 1983, 2(3): 122-125.

[25] Koblitz H, Koblitz D, Schmauder H, Gröger D. Studies on tissue cultures of the genus Cinchona L. In vitro mass propagation through meristem-derived plants. Plant Cell Rep, 1983, 2(2): 95-97.

[26] Liu F, Huang L, Yang L, Reinhound P, Jongsma M, Wang C. Shoot organogenesis in leaf explants of Hydrangea macrophylla ‘Hyd1’ and assessing genetic stability of regenerants using ISSR markers. Plant Cell Tiss Organ Cult, 2011, 104: 111-117.

[27] Lu J, Li X, Yang Y, Jia L, You J, Wang W. Effect of hydrogen peroxide on seedling growth and antioxidants in two wheat cultivars. Biol Plant, 2013, 57(3): 487-494.

[28] Madsen J (2002) Historia cultural de la cascarilla de Loja En: Aguirre Z, Madsen J, Cottas E, Balslev H (eds) Botánica Austroecuatoriana: estudios sobre los recursos naturales en las provincias de El Oro, Loja y Zamora Chinchipe. Ediciones AbyaYala. Quito, pp 385–399

[29] McCalley D. Analysis of the Cinchona alkaloids by high-performance liquid chromatography and other separation techniques. J Chromatogr A, 2002, 967(1): 1-19.

[30] Pérez-Jiménez M, Carrillo-Navarro A, Cos-Terrer J. Regeneration of peach (Prunus persica L. Batsch) cultivars and Prunus persica × Prunus dulcis rootstocks via organogenesis. Plant Cell Tiss Organ Cult, 2012, 108: 55-62.

[31] R Core Team (2013) R: a language and environment for statistical computing. Vienna: the R Foundation for Statistical Computing. ISBN: 3-900051-07-0. http://www.R-project.org/

[32] Ramos-Valdivia A, van der Heijden R, Verpoorte R. Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How. Planta, 1997, 203: 155-161.

[33] Rentería J (2002) Ecología y Manejo de la Cascarilla (Cinchona pubescens Vahl), en Santa Cruz, Galápagos, Disertación (Ingeniería Forestal). Loja, Universidad Nacional de Loja, p 101

[34] Rosales M, González R. Comparación del contenido de compuestos fenólicos en la corteza de ocho especies de pino. Madera y Bosques, 2003, 9(2): 41-49.

[35] Schmauder H, Groger D, Koblitz H, Koblitz D. Shikimate pathway activity in shake and fermenter cultures of Cinchona succirubra. Plant Cell Rep, 1985, 4(5): 233-236.

[36] Sharma M, Verma R, Singh A, Batra A. Assessment of clonal fidelity of Tylophora indica (Burm. f.) Merrill “in vitro” plantlets by ISSR molecular markers. SpringerPlus, 2014, 3 400

[37] Siddique I, Anis M. In vitro shoot multiplication and plantlet regeneration from nodal explants of Cassia angustifolia (Vahl.): a medicinal plant. Acta Physiol Plant, 2007, 29(3): 233-238.

[38] Staba J, Chung A. Quinine and quinidine production by Cinchona leaf, root and unorganized cultures. Phytochemistry, 1981, 20(11): 2495-2498.

[39] Stevens L, Giround C, Pennings J, Verpoorte R. Purification and characterization of strictosidine synthase from a suspension culture of Cinchona robusta. Phytochemistry, 1993, 33(1): 99-106.

[40] Thomas A. Cinchona en Uganda. Empire J Exp Agric, 1946, 14: 75-84.

[41] Ulloa C, Jorgensen P. Árboles y Arbustos de los Andes del Ecuador. 1995, Quito: Ediciones Abya-Yala, 329.

[42] Verkhoturov V, Frantenko V. Effect of hydrogen peroxide on anti- and prooxidant status of barley seeds during germination. Russ Agric Sci, 2008, 34(1): 11-13.

[43] Walton N, Parr A, Robins R, Rhodes M. Toxicity of quinoline alkaloids to cultured Cinchona ledgeriana cells. Plant Cell Rep, 1987, 6(2): 118-121.

[44] Warhurst D, Craing J, Adagu I, Meyer D, Lee S. The relationship of physico-chemical properties and structure to the differential antiplasmodial activity of the Cinchona alkaloids. Malar J, 2003, 2 26

[45] Wijnsma R, Go J, van Weerden I, Harkes P, Verpoorte R, BaerheimSvendsen A. Anthraquinones as phytoalexins in cell and tissue cultures of Cinchona spec. Plant Cell Rep, 1985, 4(5): 241-244.

[46] Xuanyu L, Zhijun D, Hongyan Ch, Xinhua H, Songquan S. Nitrite, sodium nitroprusside, potassium ferricyanide and hydrogen peroxide release dormancy of Amaranthus retroflexus seeds in a nitric oxide-dependent manner. Plant Growth Regul, 2011, 64: 155-161.