Abstract
Thymus persicus, which grows in the northwest area of Iran, is a valuable natural source of medicinal pentacyclic triterpenoids (PTs), i.e. betulinic acid, oleanolic acid and ursolic acid. An in vitro approach to polyploidy induction in the plant was examined for the first time. Polyploidization was induced using in vitro-grown T. persicus shoot-tips immersed in colchicine (0, 0.05, 0.1, 0.3 and 0.5 %) for 12–48 h. Ploidy levels of regenerates were determined by either flow cytometry or chromosome counting at the metaphase. The results confirmed that the mother diploid plant contained a chromosome number of 2n = 2x = 28, 2C DNA = 1.20 pg, whereas 2n = 4x = 56, 2C DNA = 2.39 pg was detected in the induced autotetraploids (CV% < 4 %). Out of 960 shoot tip segments exposed to colchicine, 7.80 and 1.04 % were tetraploid and mixoploid, respectively. The most efficient conditions for inducing polyploidy were the treatment with 0.3 % colchicine for 12 h, followed by 0.3 % for 24 h. The effect of polyploidization on growth and anatomical characteristics as well as PTs production was also measured. Tetraploids differed markedly from diploids, showing lower plantlet height, shorter roots, thicker stems and darker leaves, as well as longer and wider stomata and reduced stomatal density on the abaxial and adaxial leaf surfaces. Furthermore, a positive trend in triterpenoid production was obtained with the tetraploid and mixoploid T. persicus plants. These findings indicate that tetraploids can potentially be used in further breeding efforts to obtain a wide range of triterpenoids with improved medicinal properties.
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Abbreviations
- PTs:
-
Pentacyclic triterpenoids
- BA:
-
Betulinic acid
- OA:
-
Oleanolic acid
- UA:
-
Ursolic acid
- FCM:
-
Flow cytometry
- MS:
-
Murashige and Skoog (1962)
- PGRs:
-
Plant growth regulators
- HPLC:
-
High performance liquid chromatography
- DMSO:
-
Dimethyl sulfoxide
- WPB:
-
Woody plant buffer
- PI:
-
Propidium iodide
- PVP:
-
Polyvinyl pyrrolidone
References
Abdoli M, Moieni A, Badi HN (2013) Morphological, physiological, cytological and phytochemical studies in diploid and colchicine-induced tetraploid plants of Echinacea purpurea L. Acta Physiol Plant 35:2075–2083. doi:10.1007/s11738-013-1242-9
Abedi R, Babaei A, Karimzadeh G (2015) Karyological and flow cytometric studies of Tulipa (Liliaceae) species from Iran. Plant Syst Evol 301:1473–1484. doi:10.1007/s00606-014-1164-z
Aina O, Quesenberry K, Gallo M (2012) In vitro induction of tetraploids in Arachis paraguariensis. Plant Cell Tiss Organ Cult 111:231–238. doi:10.1007/s11240-012-0191-0
Babalola IT, Shode FO (2013) Ubiquitous ursolic acid: a potential pentacyclic triterpene natural product. J Pharmacogn Phytochem 2:214–222
Bakhtiar Z, Mirjalili MH, Sonboli A, Farimani MM, Ayyari M (2014) In vitro propagation, genetic and phytochemical assessment of Thymus persicus-a medicinally important source of pentacyclic triterpenoids. Biologia 69:594–603. doi:10.2478/s11756-014-0346-z
Caruso I, Lepore L, De Tommasi N, Dal Piaz F, Frusciante L, Aversano R, Garramone R, Carputo D (2011) Secondary metabolite profile in induced tetraploids of wild Solanum commersonii Dun. Chem Biodivers 8:2226–2237
Chen H, Gao Y, Wang A, Zhou X, Zheng Y, Zhou J (2015) Evolution in medicinal chemistry of ursolic acid derivatives as anticancer agents. Eur J Med Chem 92:648–655. doi:10.1016/j.ejmech.2015.01.031
Chudzik M, Korzonek-Szlacheta I, Krol W (2015) Triterpenes as potentially cytotoxic compounds. Molecules 20:1610–1625. doi:10.3390/molecules20011610
Chulalaksananukul W, Chimnoi W (1999) Polyploid induction in Centella asiatica (L.) Urban by colchicine treatment. J Sci Res Chula Univ 24:56–65
Comai L (2005) The advantages and disadvantages of being polyploidy. Nat Rev Genet 6:836–846. doi:10.1038/nrg1711
Dehghan E, Hakkinen ST, Oksman-Caldentey KM, Shahriari-Ahmadi F (2012) Production of tropane alkaloids in diploid and tetraploid plants and in vitro hairy root cultures of Egyptian henbane (Hyoscyamus muticus L.). Plant Cell Tiss Organ Cult 110:35–44. doi:10.1007/s11240-012-0127-8
Dhawan OP, Lavania UC (1996) Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica 87:81–89. doi:10.1007/BF00021879
Dhooghe E, Van-Laere K, Eeckhaut T, Leus L, Van-Huylenbroeck J (2011) Mitotic chromosome doubling of plant tissues in vitro. Plant Cell Tiss Organ Cult 104:359–373. doi:10.1007/s11240-010-9786-5
Doležel J, Bartoš J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110. doi:10.1093/aob/mci005
Doležel J, Sgorbati S, Lucretti S (1992) Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. Physiol Plant 85:625–631. doi:10.1111/j.1399-3054.1992.tb04764.x
Doležel J, Bartoš J, Voglmayr H, Greilhuber J (2003) Nuclear DNA content and genome size of trout and human. Cytometry 51:127–129
Ebadi-Almas D, Karimzadeh G, Mirzaghaderi G (2012) Karyotypic variation and karyomorphology in Iranian endemic ecotypes of Plantago ovata Forsk. Cytologia 77(2):215–223. doi:10.1508/cytologia.77.215
Escandón AS, Alderet LM, Hagiwara JC (2007) In vitro polyploidization of Mecardonia tenella, a native plant from South America. Sci Hortic (Amsterdam) 115:56–61. doi:10.1016/j.scienta.2007.07.006
Fernandes A, Leitao MT (1984) Contribution to the cytotaxonomic investigation of the spermatophyte of Protugal. Mem Soc Brot 27:27–75
Fukushima EO, Seki H, Ohyama K, Ono E, Umemoto N, Mizutani M, Saito K, Muranaka T (2011) CYP716A subfamily members are multifunctional oxidases in triterpenoid biosynthesis. Plant Cell Physiol 52:2050–2061. doi:10.1093/pcp/pcr146
Gao SL, Zhu DN, Cai ZH, Xu DR (1996) Autotetraploid plants from colchicine treated bud culture of Salvia miltiorrhiza Bge. Plant Cell Tiss Organ Cult 47:73–77. doi:10.1007/BF02318968
Gao SL, Chen BJ, Zhu DN (2002) In vitro production and identification of autotetraploids of Scutellaria baicalensis. Plant Cell Tiss Organ Cult 70:289–293. doi:10.1023/A:1016577002039
Gao Y, Li Z, Xie X, Wang C, You J, Mo F, Jin B, Chen J, Shao J, Chen H, Jia L (2015) Dendrimeric anticancer prodrugs for targeted delivery of ursolic acid to folate receptor-expressing cancer cells: synthesis and biological evaluation. Eur J Pharm Sci 70:55–63. doi:10.1016/j.ejps.2015.01.007
Goldblatt P (1979) Index to plant chromosome numbers (IPCN). Missouri Bot Gard, St. Louis
Gomes SS, Saldanha CW, Neves CS, Trevizani M, Raposo NR, Notini MM, Santos MO, Campos JM, Otoni WC, Viccini LF (2014) Karyotype, genome size, and in vitro chromosome doubling of Pfaffia glomerata (Spreng) Pedersen. Plant Cell Tiss Organ Cult 118:45–56. doi:10.1007/s11240-014-0460-1
Hamill SD, Smith, Dodd WA (1992) In vitro induction of banana autotetraploids by colchicine treatment of micropropagated diploids. Aust J Bot 40: 887–896
Hsia CN, Chen UC, Tsao CY, Lee CY, Wu TY (2013) Polyploidy hairy roots induction of Salvia miltiorrhiza. Taiwan Agric Res 62:280–288. doi:10.6156/JTAR/2013.06203.08
Huang MT, Ho CT, Wang ZY, Ferraro T, Lou YR, Stauber K, Ma W, Georgiadis C, Laskin JD, Conney AH (1994) Inhibition of Skin tumorigenesis by rosemary and its constituents carnosol and ursolic acid. Cancer Res 54:701–708
Jäger S, Trojan H, Kopp T, Laszczyk MN, Scheffler A (2009) Pentacyclic triterpene distribution in various plants-rich sources for a new group of multi-potent plant extracts. Molecules 14:2016–2031. doi:10.3390/molecules14062016
Jalili A, Jamzad Z (1999) Red data book of Iran a preliminary survey of endemic, rare and endangered plant species in Iran. Research Institute of Forests and Rangelands Publications, Tehran, pp 1–2
Jamzad Z (2009) Thymus and Satureja species of Iran. Research Institute of Forest and Rangelands, Tehran, p 171
Javadi H, Hejazi SM, Babayev M (2009) Karyotypic studies of three Thymus (Lamiaceae) species and populations in Iran. Caryologia 4:316–325. doi:10.1080/00087114.2004.10589697
Kaensaksiri T, Soontornchainaksaeng P, Soonthornchareonnon N, Prathanturarug S (2011) In vitro induction of polyploidy in Centella asiatica (L.) Urban. Plant Cell Tiss Organ 107:187–194. doi:10.1007/s11240-011-9969-8
Karamian R, Behjou AM, Ranjbar M (2012) Anatomical findings of Onobrychis sect. Heliobrychis (Fabaceae) in Iran and their taxonomic implications. Turk J Bot 36:27–37. doi:10.3906/bot-1010-2
Karimzadeh G, Danesh-Gilevaei M, Aghaalikhani M (2011) Karyotypic and nuclear DNA variations in Lathyrus sativus (Fabaceae). Caryologia 64(1):42–54. doi:10.1080/00087114.2011.10589763
Kobayashi N, Yamashita S, Ohta K, Hosoki T (2008) Morphological characteristics and their inheritance in colchicine-induced Salvia polyploids. J Jpn Soc Hort Sci 77:186–191. doi:10.2503/jjshs1.77.186
Lavania UC (2005) Genomic and ploidy manipulation for enhanced production of phyto-pharmaceuticals. Plant Genet Resour 3:170–177. doi:10.1079/PGR200576
Lin X, Zhou Y, Zhang J, Lu X, Zhang F, Shen Q, Wu S, Chen Y, Wang T, Tang K (2011) Enhancement of artemisinin content in tetraploid Artemisia annua plants by modulating the expression of genes in artemisinin biosynthetic pathway. Biotechnol Appl Biochem 58:50–57. doi:10.1002/bab.13
Liu J (1995) Pharmacology of oleanolic acid and ursolic acid. J Ethnopharmacol 49:57–68. doi:10.1016/0378-8741(95)90032-2
Liu J (2005) Oleanolic acid and ursolic acid: research perspectives. J Ethnopharmacol 100:92–94. doi:10.1016/j.jep.2005.05.024
Liu G, Li Z, Bao M (2007) Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology. Euphytica 157:145–154. doi:10.1007/s10681-007-9406-6
Lopez-Pujol J, Bosch M, Simon J, Blanche C (2004) Allozyme diversity in the tetraploid endemic Thymus loscosii (Lamiaceae). Ann Bot 93:1–10. doi:10.1093/aob/mch039
Loureiro J, Rodriguez E, Doležel J, Santos C (2007) Two new nuclear isolation buffers for plant DNA flow cytometry: a test with 37 species. Ann Bot 100:875–888. doi:10.1093/annbot/mcm152
Mahdavi S, Karimzadeh G (2010) Karyological and nuclear DNA content variation in some Iranian endemic Thymus species (Lamiaceae). J Agr Sci Tech 12:447–458
Majdi M, Karimzadeh G, Malboobi MA, Omidbaigi R, Mirzaghaderi G (2010) Induction of tetraploidy to feverfew (Tanacetum parthenium Schulz-Bip.): morphological, physiological, cytological and phytochemical changes. HortScience 45(1):16–21
Majdi M, Liu O, Karimzadeh G, Malboobi MA, Beekwilder J, Cankar K, de Vos R, Todorović S, Simonović A, Bouwmeester H (2011) Biosynthesis and localization of parthenolide in glandular trichomes of feverfew (Tanacetum parthenium L. Schulz Bip.). Phytochemistry 72:1739–1750. doi:10.1016/j.phytochem.2011.04.021
Majdi M, Karimzadeh G, Malboobi MA (2014) Spatial and developmental expression of key genes of terpene biosynthesis in feverfew (Tanacetum parthenium). Biol Plant 58(2):379–384
Manikrao AM, Khatale PN, Jawarkar RD, Vyas JV, Mahajan DT, Masand VH (2011) Presuming the probable anti-inflammatory mechanism of ursolic acid: a plant derived pentacyclic triterpenoid, using molecular docking. J Comput Method Mol Design 1:9–13
Marin M, Budimir S, Janošević D, Marin PD, Laušević SD, Grbić ML (2008) Morphology, distribution, and histochemistry of trichomes of Thymus lykae Degen & Jav. (Lamiaceae). Arch Biol Sci 60:667–672. doi:10.2298/ABS0804667M
Mishra BK, Pathak S, Sharma A, Trivedi PK, Shukla S (2010) Modulated gene expression in newly synthesized auto-tetraploid of Papaver somniferum L. S Afr J Bot 76:447–452. doi:10.1016/j.sajb.2010.02.090
Morales R (2002) The history, botany and taxonomy of genus Thymus. In: Stahl-Biskup E, Saez F (eds) The Genus Thymus. Taylor and Francis, London, pp 1–43
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol Plant 15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.x
Osborn TC, Pires JC, Birchler JA, Auger DL, Chen ZJ, Lee HS, Comai L, Madlung A, Doerge RW, Colot V, Martienssen RA (2003) Understanding mechanisms of novel gene expression in polyploids. Trends Genet 19:141–147. doi:10.1016/S0168-9525(03)00015-5
Pansuksan K, Sangthong R, Nakamura I, Mii M, Supaibulwatana K (2014) Tetraploid induction of Mitracarpus hirtus L. by colchicine and its characterization including antibacterial activity. Plant Cell Tiss Organ Cult 117:381–391. doi:10.1007/s11240-014-0447-y
Rechinger KH (1982) Flora Iranica, Labiatae. NO. 150. Akademic Druck-u. Verlagsanstalt, Graz
Sanguthai O, Sanguthai S, Kamemoto H (1973) Chromosome doubling of a Dendrobium hybrid with colchicine in meristem culture. Na Pua Okika O Hawaii Nei 12:12–16
Sheng H, Sun H (2011) Synthesis, biology and clinical significance of pentacyclic triterpenes: a multi-target approach to prevention and treatment of metabolic and vascular diseases. Nat Prod Rep 28:543–593. doi:10.1039/c0np00059k
Song C, Liu S, Xiao J, He W, Zhou Y, Qin Q, Zhang C, Liu Y (2012) Polyploid organisms. Sci China Life Sci 55:301–311. doi:10.1007/s11427-012-4310-2
Stahl-Biskup E (2002) Essential oil chemistry of the genus Thymus-a global view. Taylor and Francis, London. pp. 73–346
Švehlikova V, Repčak M (2000) Variation of apigenin quantity in diploid and tetraploid Chamomilla recutita (L.) Rauschert. Plant Biol 2:403–407
Urwin NA, Horsnell J, Moon T (2007) Generation and characterisation of colchicine-induced autotetraploid Lavandula angustifolia. Euphytica 156:257–266. doi:10.1007/s10681-007-9373-y
Viehmannová I, Trávníčková M, Špatenková E, Černá M, Trávníček P (2012) Induced polyploidization and its influence on yield, morphological, and qualitative characteristics of microtubers in Ullucus tuberosus. Plant Cell Tiss Organ Cult 109:83–90. doi:10.1007/s11240-011-0076-7
Wallaart TE, Pras N, Quax WJ (1999) Seasonal variations of artemisinin and its biosynthetic precursors in tetraploid Artemisia annua plants compared with the diploid wild-type. Planta Med 65:723–728. doi:10.1055/s-1999-14094
Werker E (1993) Function of essential oil secreting glandular hairs in aromatic plants of the Lamiaceae—a review. Flav Frag J 8:249–255. doi:10.1002/ffj.2730080503
Xing SH, Guo XB, Wang Q, Pan QF, Tian YS, Liu P, Zhao JY, Wang GF, Sun XF, Tang KX (2011) Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L) G. don. J Biomed and Biotechnol 2011:1–10. doi:10.1155/2011/793198
Xu C, Tang T, Chen R, Liang C, Liu X, Wu C, Yang Y, Yang D, Wu H (2013) A comparative study of bioactive secondary metabolite production in diploid and tetraploid Echinacea purpurea (L.) Moench. Plant Cell Tiss Organ Cult 116:323–332. doi:10.1007/s11240-013-0406-z
Zahedi AA, Hosseini B, Fattahi M, Dehghan E, Parastar H, Madani H (2014) Overproduction of valuable methoxylated flavones in induced tetraploid plants of Dracocephalum kotschyi Boiss. Bot Stud 55:1–10. doi:10.1186/1999-3110-55-22
Zhang J, Zhang M, Deng X (2007) Obtaining autotetraploids in vitro at a high frequency in Citrus sinensis. Plant Cell Tiss Organ Cult 89:211–216. doi:10.1007/s11240-007-9240-5
Zhang QY, Luo FX, Liu L, Guo FC (2010) In vitro induction of tetraploids in crape myrtle (Lagerstroemia indica L.). Plant Cell Tiss Organ Cult 101:41–47. doi:10.1007/s11240-009-9660-5
Acknowledgments
The authors thank the Shahid Beheshti University Research Council and the Iran National Science Foundation (INSF, Grant No. 92013885) for financial support of this project. We also thank Ziba Bakhtiar and Fatemeh Goudarzi for their kind collaboration in polyploidization experiment and HPLC analysis, respectively. This work was a part of Mansoureh Tavan’s M.Sc. thesis.
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Tavan, M., Mirjalili, M.H. & Karimzadeh, G. In vitro polyploidy induction: changes in morphological, anatomical and phytochemical characteristics of Thymus persicus (Lamiaceae). Plant Cell Tiss Organ Cult 122, 573–583 (2015). https://doi.org/10.1007/s11240-015-0789-0
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DOI: https://doi.org/10.1007/s11240-015-0789-0