In Vitro Propagation of Ocimum Gratissimum L. (Lamiaceae) and Its Evaluation of Genetic Fidelity Using RAPD Marker

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DOI: 10.4236/ajps.2012.31006   PDF   HTML     8,044 Downloads   15,672 Views   Citations

Abstract

An efficient plant propagation system through nodal explants was established in Ocimum gratissimum L, a medicinally important herbaceous perennial herb belonging to the family Lamiaceae. Axillary shoot bud proliferation was initiated from nodal explants cultured on Murashige and Skoog (MS) medium supplemented with various concentrations of N6-benzyladenine (BA) (0.5 - 3.0 mg/l), Kinetin (KN) (0.5 - 3.0 mg/l) and 2-isoPentenyladenine (2-iP) (0.5 - 3.0 mg/l). Maximum numbers of shoots (5.17 ± 0.04) with average length (2.50 ± 0.07) were induced on medium containing 1.0 mg/l BA. Shoot multiplication was maintained by repeated subculturing the original nodal explants on shoot multiplication medium after each harvest of newly formed shoots. Histological study shows that the organogenesis occurs directly, without callus formation on epidermal and sub epidermal layer of the explants. Rooting of shoots was achieved on half strength MS medium supplemented with 1.5 mg/1 Indole-3-butyric acid (IBA) and 2% sucrose. Well-developed complete plantlets were transferred to plastic pots containing a mixture of (1:1) soil and vermiculite showed 82.5 % survival rate. Genetic fidelity was assessed by chromosome analysis and DNA fingerprinting using random amplified polymorphic DNA (RAPD) of in vitro and in vivo plants. Nine arbitrary decamers displayed same banding profile showed no genomic alterations, indicating homogeneity among the tissue culture regenerates and genetic uniformity with that of donor plants. The present study provides high fidelity micro-propagated system for efficient and rapid micro-propagation protocol of this important medicinal plant and great use in conserving without risk of genetic instability.

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S. Saha, A. Kader, C. Sengupta and P. Ghosh, "In Vitro Propagation of Ocimum Gratissimum L. (Lamiaceae) and Its Evaluation of Genetic Fidelity Using RAPD Marker," American Journal of Plant Sciences, Vol. 3 No. 1, 2012, pp. 64-74. doi: 10.4236/ajps.2012.31006.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] S. Pei, “Ethnobotanical Approaches of Traditional Medicine Studies: Some Experiences from Asia,” Pharmaceutical Biology, Vol. 39, 2001, pp. 74-79.
[2] S. J. Murch, S. KrishnaRaj, P. K. Saxena, “Phyto-Pharmaceuticals: Mass-Production, Standardization, and Conservation,” Herbal Medicine, Vol. 4, No. 2, 2000, pp. 39-43.
[3] K. C. Wen, “The Turnover Rate of Marker Constituents in Chinese Herbal Medicine,” Journal of Food and Drug Analysis, Vol. 8, No. 4, 2000, pp. 270-277. www.fda.gov.tw/files/publish_periodical/8-4-6.PDF
[4] A. B. Cunningham, “African Medicinal Plants: Setting Priorities at the Interface between Conservation and Primary Healthcare,” People and Plants Working Paper 1, UNESCO, Paris, 1993, p. 92.
[5] M. W. Flower, “Commercial Application and Economic Aspects of Mass Cell Cultures,” In: H. S. Mantel and H. Smith, Eds., Plant Biotechnology, Cambridge University Press, Cambridge, 1983, pp. 3-37.
[6] R. Arora and S. S. Bhojwani, “In Vitro Propagation and Low Temperature Storage of Saussurea lappa C.B. Clarke—An Endangered, Medicinal Plant,” Plant Cell Reports, Vol. 8, No. 1, 1989, pp. 44-47.
[7] G. C. Sudha and S. Seeni, “In Vitro Propagation and Field Establishment of Adhatoda beddomei C.B. Clarke, a Rare Medicinal Plant,” Plant Cell Reports, Vol. 13, No. 3-4, 1993, pp. 203-207. doi:10.1007/BF00239893
[8] D. L. Sulistiarini, “Ocimum gratissimum L,” In: A. P. L. Oyen and D. X. Nguyen, Eds., Plant Resources of South-East Asia No. 19: Essential Oils Plants, Prosea Foundation, Bogor, 1999, pp. 140-142.
[9] T. T. Adebolu and S. A. Oladimeji, “Antimicrobial Activity of Leaf Extracts of Ocimum gratissimum on Selected Diarrhea Causing Bacteria in Southwestern Nigeria,” African Journal of Biotechnology, Vol. 4, No. 7, 2005, pp. 682-684.
[10] J. A. Lemos, X. S. Passos, O. F. L. Fernandes, J. R. Paula, P. H. Ferri, L. K. H. Souza, A. A. Lemos and M. R. R. Silva, “Antifungal Activity from Ocimum gratissimum L. towards Cryptococcus Neoformans,” Memorias do Instituto Oswaldo Cruz, Vol. 100, No. 1, 2005, pp. 55-58. doi:10.1590/S0074-02762005000100011
[11] C. V. Nakamura, T. U. Nakamura, E. Bando, A. F. N. Melo, D. A. G. Cortez and B. P. Diaz Filho, “Antibacterial Activity of Ocimum gratissimum L. Essential Oil,” Memorias do Instituto Oswaldo Cruz, Vol. 94, No. 5, pp. 675-678. doi:10.1590/S0074-02761999000500022
[12] C. N. Ezekwesili, K. A. Obiora and O. P. Ugwu, “Evaluation of Anti-Diarrhoeal Property of Crude Aqueous Extract of Ocimum gratissimum L. (Labiatae) in Rats,” Biokemistri, Vol. 16, No. 2, 2004, pp. 122-131.
[13] F. B. Holetz, T. U. Nakamura, B. P. D. Filho, D. A. G. Cortez, J. A. M. Diaz and C. V. Nakamura, “Effect of Essential Oil of Ocimum gratissimum on Herpetomonas Samuelpessoai,” Acta Protozoologica, Vol. 42, 2003, pp. 269-276.
[14] L. M. Pessoa, S. M. Morais, C. M. L. Bevilaqua and J. H. S. Luciano, “Anthelmintic Activity of Essential Oil of Ocimum gratissimum Linn. and Eugenol against Haemonchus contortus,” Veterinary Parasitology, Vol. 109, No. 1-2, 2003, pp. 59-63. doi:10.1016/S0304-4017(02)00253.4
[15] D. J. Chitwood, “Phytochemical Based Strategies for Nematode Control,” Annual Review of Phytopathology, Vol. 40, 2002, pp. 221-249. doi:10.1146/annurev.phyto.40.032602.130045
[16] R. E. Veira, R. Fahoer and S. K. Raina, “Genetic Diversity of Ocimum gratissimum L. Based on Volatile Oil Constituents, Flavonoids and RAPD Markers,” Biochemical Systematics and Ecology, Vol. 29. No.3, 2001, pp. 287-304.
[17] Wealth of India, “A Dictionary of India Raw Materials and Industrial Products Vol. VII (N-Pe),” CSRI Publication, New Delhi, 1966, pp. 79-89.
[18] V. H. Heywood, “Flowering Plants of the World,” Oxford University Press, Oxford, 1978, pp. 239-240.
[19] D. Ajitkumar and S. Seeni, “Rapid Clonal Multiplication through in Vitro Axillary Shoot Proliferation of Aegle marmelos (L.) Corr., a Medicinal Tree,” Plant Cell Reports, Vol. 17, No. 5, 1998, pp. 422-426. doi:10.1007/s002990050418
[20] E. Prakash, P. S. Sha Valli Khan, P. S. Reddy and K. R. Rao, “Regeneration of Plants from Seed-Derived Callus of Hybanthus Enneaspermus L. Muell., a Rare Ethnobotanical Herb,” Plant Cell Reports, Vol. 18, No. 10, 1999, pp. 873-878. doi:10.1007/s002990050677.
[21] C. Hu and P. J. Wang, “Meristem Shoot Tip and Bud Culture,” In: A. D. Evans, R. W. Sharp, V. P. Ammirato and Y. Yamada, Eds., Handbook of Plant Cell Culture, Macmillan, New York, 1983, pp. 177-227.
[22] K. A. Vincent, K. M. Mathew and M. Hariharan, “Micropropagation of Kaemferia galanga L.—A Medicinal Plant,” Plant Cell Tissue and Organ Culture, Vol. 28, No. 2, 1992, pp. 229-230. doi:10.1007/BF00055522
[23] S. R. Bhat, K. P. S. Chandel and S. K. Malik, “Plant Regeneration from Various Explants of Cultivated Piper Species,” Plant Cell Reports, Vol. 14, No. 6, 1995, pp. 398-402. doi:10.1007/BF00238605
[24] Y. Sahoo, S. K. Pattnaik and P. K. Chand, “In Vitro Clonal Propagation of an Aromatic Medicinal Herb Ocimum basilicum L. (Sweet Basil) by Axillary Shoot Proliferation,” In Vitro Cellular and Developmental Biology—Plant, Vol. 33, No. 4, 1997, pp. 293-296. doi:10.1007/s11627-997-0053-3
[25] C. Gopi, Y. S. Nataraja and P. Ponmurugan, “In Vitro Multiplication of Ocimum gratissimum L. through Direct regeneration,” African Journal of Biotechnology, Vol. 5, No. 9, 2006, pp. 723-726.
[26] T. Murashige and F. Skoog, “A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures,” Physiologia Plantarum, Vol. 15, No.3, 1962, pp. 473-497. doi:10.111/j.1399-3054.1962.tb08051.x
[27] M. G. Murray and W. F. Thompson, “Rapid Isolation of High Molecular Weight Plant DNA,” Nucleic Acids Research, Vo. 8, No. 19, 1980, pp. 4321-4325. doi:10.1093/nar/8.19.4321
[28] J. Smbrook and D. W. Russed, “Molecular Cloning. A Laboratory Manual,” 3rd Edition, Spring Harbor Laboratory Press, Cold Spring Harbor, 2001.
[29] D. B. Duncan, “Multiple Range and Multiple F Test,” Biometrics, Vol. 11, No. 1, 1955, pp. 1-42. doi:10.2307/3001478
[30] R. M. Skirvin, O. McMeans and W. L. Wang, “Storage Water Is a Source of Latent Bacterial Contamination in Vitro,” Trends of Agricultural Science, Vol. 1, 1993, pp. 63-63.
[31] J. Gopal, J. L. Minocha and H. S. Dhaliwal, “Microtuberization in Potato (Solanum tuberosum L.),” Plant Cell Reports, Vol. 17, No. 10, 1998, pp. 794-798. doi:10.1007/s002990050485
[32] S. K. Guru, R. Chandra, S. Khetrapal, A. Raj and R. Palisetty, “Protein Pattern in Differentiating Explants of Chick Pea (Cicer arietinum L.),” Indian Journal of Plant Physiology, Vol. 4, 1999, pp. 147-151.
[33] J. Sen and A. K. Sharma, “Micropropagation of Withania somnifera from Germinating Seeds and Shoot Tips,” Plant Cell Tissue and Organ Culture, Vol. 26, No. 2, 1991, pp.71-73. doi:10.1007/BF00036108
[34] P. S. George, G. A. Ravishankar and L. V. Venkataraman, “Clonal Multiplication of Gardenia jaminoides Elllis through Axillary Bud Culture,” Plant Cell Reports, Vol. 13, No. 1, 1993, pp. 59-62. doi:10.1007/BF0023237
[35] A. Ahuja, M. Verma and S. Grewal, “Clonal Propagation of Ocimum Species by Tissue Culture,” Indian Journal of Experimental Biology, Vol. 20, 1982, pp. 455-458.
[36] F. Begum, N. Amin and M. A. K. Azad, “In Vitro Rapid Clonal Propagation of Ocimum basilicum L.,” Plant Tissue Culture and Biotechnology, Vol. 12, No. 1, 2002, pp. 27-35.
[37] S. Saha and P. D. Ghosh and C. Sengupta, “An Efficient Method for Micropropagation of Ocimum basilicum L.,” Indian Journal of Plant Physiology, Vol. 15, No. 2, 2010. pp. 168-172.
[38] L. V. Hiregoudar, H. N. Murthy, J. G. Bhat, A. Nayeem, B. P. Hema, E. J. Hahn and K. Y. Paek, “Rapid Clonal Propagation of Vitex trifolia,” Biologia Plantarum, Vol. 50, No. 2, 2006, pp. 291-294. doi:10.1007/s10535-006-0023-3
[39] S. Saha and P. D. Ghosh and C. Sengupta, “In Vitro Multiple Shoot Regeneration of Mentha piperita,” Journal of Tropical Medicinal Plants, Vol. 11. No. 1, 2010, pp. 89-92.
[40] S. Chattopadhyay, S. K. Datta and S. B. Mahato, “Rapid Micropropagation for Mucuna pruriens f. Pruriens L.,” Plant Cell Reports, Vol. 15, No. 3-4, 1995, pp. 271-273. doi:10.1007/BF00193734
[41] M. Faisal, I. Siddique and M. Anis, “An Efficient Plant Regeneration System for Mucuna Pruriens L. (DC.) Using Cotyledonary Node Explants,” In Vitro Cellular and Developmental Biology—Plant, Vol.42, No. 1, 2006, pp. 59-64. doi:10.1079/IVP2005717
[42] P. K. Tewary, M. K. Raghunath, M. Venkateshwarulu and A. Sarkar, “Genotypic Difference in Response to in Vitro Shoot Development of Mulberry (Morus Spp.),” Indian Journal of Sericulture, Vol. 35, No. 2, 1996, pp. 104-106.
[43] C. Suresh and K. S. Ajay, “In Vitro Shoot Regeneration from Cotyledonary Node Explants of a Multipurpose Leguminous Tree, Pterocarpus marsupium Roxb,” In Vitro Cellular and Developmental Biology-Plant, Vol. 40, No. 2, 2004, pp. 167-170. doi:10.1079/IVP2003488
[44] M. Hossain, B. K. Biswas, M. R. Karim, S. Rahman, R. Islam and O. I. Jorder, “In Vitro Organogenesis of Elephant Apple (Feronia limonia),” Plant Cell Tissue and Organ Culture, Vol. 39, No. 3, 1994, pp. 265-268. doi:10.1007/BF00035980
[45] L. V. Hiregoudar, H. N. Murthy, B. P. Hema, E. J. Hahn and K. Y. Peak, “Multiple Shoot Induction and Plant Regeneration of Feronia limonia (L.) Swingle,” Scientia Horticulturae, Vol. 98, No. 4, 2003, pp. 357-364. doi:10.1016/S0304-4238(03)00018-9
[46] V. Tiwari, K. N. Tiwari and B. D. Singh, “Comparative Studies of cytokinins on in Vitro Propagation of Bacopa monniera,” Plant Cell Tissue and Organ Culture, Vol. 66, No. 1, 2001, pp. 9-16. doi:10.1023/A:1010652006417
[47] I. Siddique and M. Anis, “In Vitro Shoot Multiplication and Plantlet Regeneration from Nodal Explants of Cassia angustifolia (Vahl): A Medicinal Plant,” Acta Physiologiae Plantarum, Vol. 29, No. 3, 2007, pp. 233-238. doi:10.1007/s11738-007-0029-2
[48] P. C. Debergh and L. J. Maene, “A Scheme for Commercial Propagation of Ornamental Plants by Tissue Culture,” Scientia Horticulturae, Vol. 14, 1981, pp. 335-345.
[49] R. H. Wetmore and C. W. Wardlaw, “Experimental Morphogenesis in Vascular Plants,” Annual Review of Plant Physiology, Vol. 2, 1951, pp. 269-292. doi:10.1146/annurev.pp.02.060151.001413
[50] C. Detrez, R. S. Sangwan and B. S. Sangwan-Norreel, “Phenotypic and Karyotypic Status of Beta Vulgaris Plants Regenerated from Direct Organogenesis in Petiole Culture,” Theoretical and Applied Genetics, Vol. 77, No. 4, 1989, pp. 462-468. doi:10.1007/BF00274264
[51] L. B. Andrade, S. Echeverrugaray, F. Fracaro, G. F. Pauletti and L. Rota, “The Effect of Growth Regulators on Shoot Propagation and Rooting of Common Lavender (Lavandula vera DC),” Plant Cell Tissue and Organ Culture, Vol. 56, No. 2, 1999, pp. 79-83. doi:10.1023/A:1006299410052
[52] S. Saha, T. Dey and P. D. Ghosh, “Micropropagation of Ocimum Kilimandscharicum Guerke (Labiatae),” Acta Biologica Cracoviensia Series Botanica, Vol. 52, No. 2, 2010, pp. 50-58. doi:10.2478/v10182-010-0023-7
[53] J. A. Driver and G. R. Suttle, “Nursery Handling of Propagules,” In: J. M. Bonga and D. J. Durzan, Eds., Cell and Tissue Culture in Forestry, Dordrecht, Netherlands, 1987, pp. 320-335. doi:10.1007/978-94-009-4484-8_17
[54] S. Cuenca, J. B. Amo-Marco and R. Parra, “Micropropagation from Inflorescence Stems of the Spanish Endemic Plant Centaurea Paui Loscos ex Willk. (Compositae),” Plant Cell Reports, Vol. 18, No. 7-8, 1999, pp. 674-679. doi:10.1007/s002990050641
[55] Y. Sahoo and P. K. Chand, “Micropropagation of Vitex negundo L., a Woody Aromatic Medicinal Shrub, through High-Frequency Axillary Shoot Proliferation,” Plant Cell Reports, Vol. 18, No. 3-4, 1998, pp. 301-307. doi:10.1007/s002990050576
[56] N. Komalavalli and M. V. Rao, “In Vitro Micropropagation of Gymnema Sylvestre—A Multipurpose Medicinal Plant,” Plant Cell Tissue and Organ Culture, Vol. 61, No. 2, 2000, pp. 97-105. doi:10.1023/A:1006421228598
[57] H. R. Juliani Jr., A. R. Koroch, H. R. Juliani and V. S. Trippi, “Micropropagation of Lippia junelliana (Mold.) Trone,” Plant Cell Reports, Vol. 59, No. 3, 1999, pp. 175-179. doi:10.1023/A:1006396531647
[58] M. Faisal and M. Anis, “Rapid Mass Propagation of Tylophora Indica Merrill via Leaf Callus Culture,” Plant Cell Tissue and Organ Culture, Vol. 75, No. 2, 2003, pp. 125-129. doi:10.1023/A:1025084203958
[59] J. E. Simon, J. Quinn and R. G. Murray, “Basil: A Source of Essential Oils,” In: J. Janick and J. E. Simon, Eds., Advances in New Crops, Timber, Portland, 1990, pp 484-989.
[60] G. J. De Klerk, “How to Measure Somaclonal Variation,” Acta Botanica Neerlandica, Vol. 39, 1990, pp. 129-144.
[61] M. K. Smith, “A Review of Factors Influencing the Genetic Stability of Micropropagated Bananas,” Fruits, Vol. 43, No. 4, 1988, pp. 219-223.
[62] Y. R. Dutta, G. Gangopadhyay, S. Das, B. K. Dutta and K. K. Mukherjee, “Esterase as a Marker to Study the Genetic Fidelity of Micropropagated Banana,” Biologia Plantarum, Vol. 47, No. 3, 2003, pp. 421-424. doi:10.1023/B:BJOP.0000023886.9365.b4
[63] M. K. U. Chawdhury and J. K. Vasil, “Molecular Analysis of Plants Regenerated from Embryogenic Cultures of Hybrid Sugarcane Cultivars (Saccharum spp.),” Theoretical and Applied Genetics, Vol. 86, No. 2-3, 1993, pp. 181-188. doi:10.1007/BF00222077
[64] V. Rani, A. Parida and S. N. Raina, “Random Amplified Polymorphic DNA (RAPD) Markers for Genetic Analysis in Micropropagated Plants of Populus Deltoides Marsh,” Plant Cell Reports, Vol. 14, No. 7, 1994, pp. 459-462. doi:10.1007/BF00234055
[65] R. K. Chaudhuri, A. Pal and T. B. Jha, “Production of Genetically Uniform Plants from Nodal Explants of Swertia Chirata Buch. Ham. Ex Wall—A Critically Endangered Medicinal Herb,” In Vitro Cellular and Developmental Biology, Vol. 43, No. 5, 2007, pp. 467-472. doi:10.1007/s11627-007-9095-9
[66] S. Bhattacharya, T. K. Bondopadhyay and P. D. Ghosh, “Somatic Embryogenesis in Cymbopogon Pendulus and Evaluation of Clonal Fidelity of Regenerants Using ISSR Marker,” Scientia Horticulturae, Vol. 123, No. 4, 2010, pp. 505-513. doi:10.1016/j.scienta.2009.10.011
[67] J. G. K. Williams, A. R. Kubelik, K. J. Livak, J. A. Rafalski and S. V. Tingey, “DNA Polymorphisms Amplified by Arbitrary Primers Are Useful as Genetic Markers,” Nucleic Acids Research, Vol. 18, No. 22, 1990, pp. 6531-6535. doi:10.1093/nar/18.22.6531

  
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