Molecular Analysis of Genetic Fidelity in Micropropagated Plants of Stevia rebaudiana Bert. Using ISSR Marker


Inter-simple sequence repeat (ISSR) markers were used to evaluate the genetic fidelity of in vitro propagated and hardened plants of Stevia rebaudiana Bert. Nodal segments containing axillary buds were used as explant and inoculated on Murashige and Skoog’s (MS) medium containing 3% (w/v) sucrose, 0.8% (w/v) agar supplemented with various concentrations of benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) ranging from 0.20 to 2.00 mg·L-1. Maximum multiple shoots (93%) were obtained in MS medium supplemented with 0.20 mg L-1 TDZ. The best in vitro root induction (87%) was achieved on half strength MS medium without any growth regulator. The rooted plantlets were successfully established in soil and grown to maturity at the survival rate of 96% in the indoor grow room. For ISSR analysis, total genomic DNA was extracted from 20 mg fresh leaves of mother and randomly selected in vitro propagated plants. Out of  fifteen arbitrary primers tested, each produced clear and scorable amplification products ranged in size from about 216 bp in UBC 811 to 1917 bp in (GGGGT)3M with an average of 4.5 products per primer. A total of 45 bands (number of plantlets analyzed multiplied by number of bands with all primers) were generated by the ISSR method. All the ISSR profiles from micropropagated plants were monomorphic and comparable to mother plants, confirming the genetic stability among micropropagated plants and mother plant. Chemical analysis, using high-performance liquid chromatography (HPLC), was done to further confirm the existence of qualitative and quantitative differences in the major secondary metabolites (rebaudioside A, stevioside and steviolbioside) between the mother plant and in vitro propagated plants. Our results clearly show similar chemical profiles and insignificant differences in the major secondary metabolites between the two types of plants. These results suggest that the micropropagation protocol followed in this study is appropriate and applicable for clonal mass propagation of true-to-type elite Stevia rebaudiana plants.

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H. Lata, S. Chandra, N. Techen, Y. Wang and I. Khan, "Molecular Analysis of Genetic Fidelity in Micropropagated Plants of Stevia rebaudiana Bert. Using ISSR Marker," American Journal of Plant Sciences, Vol. 4 No. 5, 2013, pp. 964-971. doi: 10.4236/ajps.2013.45119.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] R. V. Sreedhar, L. Venkatachalam, R. Thimmaraju, N. Bhagyalakshmi, M. S. Narayan and G. A. Ravishankar, “Direct Organogenesis from Leaf Explants of Stevia rebaudiana and Cultivation in Bioreactor,” Biologia Plantarum, Vol. 52, No. 2, 2008, pp. 355-360. doi:10.1007/s10535-008-0073-9
[2] A. S. Richman, M. Gijzen, A. N. Starralt, Z. Yang and J. E. Brandle, “Diterpene Synthesis in Stevia rebaudiana Recruitment and Up Regulation of Key Enzymes from the Gibberellins Biosynthetic Pathway,” The Plant Journal, Vol. 19, No. 4, 1999, pp. 411-421. doi:10.1046/j.1365-313X.1999.00531.x
[3] N. Bondarev, “Peculiarities of Propagation and Development of Stevia rebaudiana Bertoni Plants in Vitro,” Proceedings of 9th International Conference of Horticulture, Lednice, 3-6 September 2001, pp. 431-434.
[4] S. D. Singh and G. P. Rao, “Stevia: The Herbal Sugar of 21st Century,” Sugar Tech, Vol. 7, No. 1, 2005, pp. 17-24. doi:10.1007/BF02942413
[5] N. Lailerd, V. Saengsirisuwan, J. A. Sloniger, C. Toskulkao and E. J. Henriksen, “Effects of Stevioside on Glucose Transport Activity in Insulin-Sensitive and Insulin-Resistant Rat Skeletal Muscle,” Metabolism, Vol. 53, No. 1, 2004, pp. 101-107. doi:10.1016/j.metabol.2003.07.014
[6] P. B. Jeppesen, S. Gregersen, S. E. Rolfsen, M. Jepsen, M. Colombo, A. Agger, J. Xiao, M. Kruhøffer, T. Orntoft and K. Hermansen, “Antihyperglycemic and Blood Pressure- Reducing Effects of Stevioside in the Diabetic Goto-Kakizaki Rat,” Metabolism, Vol. 52, No. 3, 2003, pp. 372- 378. doi:10.1053/meta.2003.50058
[7] J. M. Geuns, “Stevioside,” Phytochemistry, Vol. 64, No. 5, 2003, pp. 913-921. doi:10.1016/S0031-9422(03)00426-6
[8] A. Mitra and A. Pal, “In Vitro Regeneration of Stevia rebaudiana (Bert.) from Nodal Explants,” Journal of Plant Biochemistry and Biotechnology, Vol. 16, No. 1, 2007, pp. 59-62. doi:10.1007/BF03321930
[9] S. Nakamura and Y. Tamura, “Variation in the Main Glycosides of Stevia (Stevia rebaudiana),” Japanese Journal of Tropical Agriculture, Vol. 29, 1985, pp. 109-116.
[10] L. Sivaram and U. Mukundan, “In Vitro Culture Studies on Stevia rebaudiana,” In Vitro Cellular and Developmental Biology of Plant, Vol. 39, No. 5, 2003, pp. 520- 523. doi:10.1079/IVP2003438
[11] M. A. Shatnawi, R. A. Shibli, S. M. Abu-Romman, M. S. Al-Mazra’awi1, Z. I. Al Ajlouni, W. A. Shatanawi and W. H. Odeh, “Clonal Propagation and Cryogenic Storage of the Medicinal Plant Stevia rebaudiana,” Spanish Journal of Agricultural Research, Vol. 9, No. 1, pp. 213- 220.
[12] M. Sakaguchi and T. Kan, “Japanese Researches on Stevia rebaudiana,” Ci Cult, Vol. 34, 1982, pp. 235- 248.
[13] M. Debnath, “Clonal Propagation and Antimicrobial Activity of an Endemic Medicinal Plant Stevia rebaudiana,” Journal of Medicinal Plants Research, Vol. 2, No. 2, 2008, pp. 45-51.
[14] P. K. Mishra, R. Singh, U. Kumar and V. Prakash, “Stevia rebaudiana: A Magical Sweetener,” Global Journal of Biotechnology and Biochemstry, Vol. 5, No. 1, 2010, pp. 62-74.
[15] P. J. Larkin and W. R. Scowcroft, “Somaclonal Variation- A Novel Source of Variability from Cell Cultures for Plant Improvement,” Theoretical and Applied Genetics, Vol. 60, No. 4, 1981, pp. 197-214. doi:10.1007/BF02342540
[16] N. D. Salvi, L. George and S. Eapen, “Plant Regeneration from Leaf Base Callus of Turmeric and Random Amplified Polymorphic DNA Analysis of Regenerated Plants,” Plant Cell Tissue and Organ Culture, Vol. 66, No. 2, 2001, pp. 113-119. doi:10.1023/A:1010638209377
[17] M. Moktaduzzaman and S. M. M. Rahman, “Regeneration of Stevia rebaudiana and Analysis of Somaclonal Variation by RAPD,” Biotechnology, Vol. 8, No. 4, 2009, pp. 449-455.
[18] I. A. Ibrahim, M. I. Nasr, B. R. Mohammed and M. M. El-Zefzafi, “Nutrient Factors Affecting in Vitro Cultivation of Stevia rebaudiana,” Sugar Tech, Vol. 10, No. 3, 2008, pp. 248-253. doi:10.1007/s12355-008-0044-7
[19] B. Janarthanam, M. Gopalakrishnan, G. Lakshmisai and T. Sekar, Plant Regeneration from Leaf Derived Callus of Stevia rebaudiana Bertoni,” Plant Tissue Culture and Biotechnology, Vol. 19, No. 2, 2009, pp. 133-141. doi:10.3329/ptcb.v19i2.5430
[20] M. Kalpana, M. Anbazhagan and V. Natarajan, “Utilization of Liquid Medium for Rapid Micropropagation of Stevia rebaudiana Bertoni,” Journal of Ecobiotechnology, Vol. 1, No. 1, 2009, pp. 16-20.
[21] A. Das, S. Gantait and N. Mandal, “Micropropogation of an Elite Medicinal Plant (Stevia rebaudiana Bertoni),” International Journal of Agriculture Research, Vol. 6, No. 1, 2011, pp. 40-48. doi:10.3923/ijar.2011.40.48
[22] H. Lata, S. Chandra, Y. H. Wang, V. Raman and I. A. Khan, “TDZ-Induced High Frequency Plant Regeneration through Direct Shoot Organogenesis in Stevia rebaudiana Bertoni: An Important Medicinal Plant and a Natural Sweetener,” American Journal of Plant Sciences, Vol. 4, 2013, pp. 117-128. doi:10.4236/ajps.2013.41016
[23] E. Zietkiewicz, A. Rafalski and D Labuda, “Genome Fingerprinting by Simple Sequence Repeat (SSR)-Anchored Polymerase Chain Reaction Amplification,” Genomics, Vol. 20, No. 2, 1994, pp. 176-183. doi:10.1006/geno.1994.1151
[24] H. Yang, Y. Tabei, H. Kamada, T. Kayano and F. Takaiwa, “Detection of Somaclonal Variation in Cultured Rice Cells Using Digoxigenin-Based Random Amplified Polymorphic DNA,” Plant Cell Reports, Vol. 18, 1999, pp. 520-526.
[25] W. L. Guo, Y. Li, L. Gong, F. Li, Y. Dong and B. Liu, “Efficient Micropropagation of Robinia ambigua and Detection of Genomic Variation by ISSR Markers,” Plant Cell Tissue and Organ Culture, Vol. 84, No. 3, 2006, pp. 343-351. doi:10.1007/s11240-005-9043-5
[26] J. B. Thomas, D. Vijayan, S. D. Joshi, S. J. Lopez and R. R. Kumar, “Genetic Integrity of Somaclonal Variants in Tea (Camellia sinensis (L.) O Kuntze) as Revealed by Inter Simple Sequence Repeats,” Journal of Biotechnology, Vol. 123, No. 2, 2006, pp. 149-154. doi:10.1016/j.jbiotec.2005.11.005
[27] T. Murashige and F. Skoog, “A Revised Medium for Rapid Growth and Bioassays with Tobacco Tissue Cultures,” Physiologia Plantarum, Vol. 15, No. 43, 1962, pp. 473- 497. doi:10.1111/j.1399-3054.1962.tb08052.x
[28] X. Leroy, K. Leon, G. Charles and M. Branchard, “Cauli- Flower Somatic Embryogenesis and Analysis of Regenerants Stability by ISSRs,” Plant Cell Report, Vol. 19, No. 11, 2000, pp. 1102-1107. doi:10.1007/s002990000252
[29] M. H. Rahman and O. P. Rajora, “Microsatellite DNA Somaclonal Variation in Micropropagated Trembling Aspen (Populus tremuloides),” Plant Cell Report, Vol. 20, No. 6, 2001, pp. 531-536. doi:10.1007/s002990100365
[30] P. Joshi and V. Dhawan, “Assessment of Genetic Fidelity of Micropropagated Swertia chirayita Plantlets by ISSR Marker Assay,” Biologia Plantarum, Vol. 51, No. 1, 2007, pp. 22-26. doi:10.1007/s10535-007-0005-0
[31] M. Chandrika, Thoyajaksha, V. Ravishankar Rai and K. Ramachandra Kini, “Assessment of Genetic Stability of in Vitro Grown Dictyospermum ovalifolium,” Biologia Plantarum, Vol. 52, No. 4, 2008, pp. 735-739. doi:10.1007/s10535-008-0142-0
[32] M. Chandrika, V. Ravishankar Rai and Thoyajaksha, “ISSR Marker Based Analysis of Micropropagated Plantlets of Nothapodytes foetida,” Biologia Plantarum, Vol. 54, No. 3, 2010, pp. 561-565. doi:10.1007/s10535-010-0100-5
[33] H. Lata, H. S. Chandra, N. Techen, I. Khan and M. A. ElSohly, “Assessment of Genetic Stability of Micropropagated Cannabis sativa Plants by ISSR Markers,” Planta Medica, Vol. 76, No. 1, 2010, pp. 97-100. doi:10.1055/s-0029-1185945
[34] H. Lata, S. Chandra, N. Techen, I. Khan and M. A. El- Sohly, “Molecular Analysis of Genetic Fidelity in Cannabis sativa L. Plants Grown from Synthetic Seeds Following in Vitro Storage Conditions,” Biotechnology Letter, Vol. 33, No. 12, 2011, pp. 2503-2508. doi:10.1007/s10529-011-0712-7
[35] K. Hatano, K. Kamura, Y. Shoyama and I. Nishioka, “Clonal Propagation of Aconitum carmichaeli by Tip Tissue Culture and Alkaloid Contents of Clonally Propagated Plants,” Planta Medica, Vol. 54, No. 2, 1988, pp. 152-155. doi:10.1055/s-2006-962375
[36] S. Ahuja, B. B. Mandal, S. Dixit and P. S. Srivastava, “Molecular, Phenotypic and Biosynthetic Stability in Dioscorea floribunda Plants Derived from Cryopreserved Shoot Tips,” Plant Science, Vol. 163, No. 5, 2002, pp. 971-977. doi:10.1016/S0168-9452(02)00246-7
[37] C. Z. Liu, M. Gao and B. Guo, “Plant Regeneration of Erigeron breviscapus (vant.) Hand. Mazz. and Its Chromatographic Fingerprint Analysis for Quality Control,” Plant Cell Report, Vol. 27, No. 1, 2008, pp. 39-45. doi:10.1007/s00299-007-0466-9
[38] H. R. Shilpashree and R. Rai, “In Vitro Plant Regeneration and Accumulation of Flavonoids in Hypericum mysorense,” International Journal of Integrative Biology, Vol. 8, No. 1, 2009, pp. 43-49.

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