Journal of Medicinal and Aromatic Plant Sciences

Volume: 46 Issue: 3

  • subscription
  • Original Research Article

MetaTopolin-driven breakthrough in lemon beebalm (Monarda citriodora) regeneration: A molecular fidelity study for genome engineering applications

Yadunandan Sen#1,2, Vanila Sharma#2, Suman Singh1,2, Mallesham Bulle3, Prashant Misra1,2, Srinivas Kota1,2*

1Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, 201002, India.
2Plant Sciences and Agrotechnology Division, CSIR-Indian Institute of Integrative Medicine, Canal Road, Jammu, 180001, India.
3Agri Biotech Foundation, Agricultural University Campus, Rajendranagar, Hyderabad-500030, Telangana, India.
#Authors contributed equally
*Corresponding author; Email: [email protected]

Year: 2024, Page: 123-132, Doi: https://doi.org/10.62029/jmaps.v46i3.sen

Received: Sept. 18, 2024 Accepted: Sept. 27, 2024 Published: Feb. 10, 2025

Abstract

Plant cell and tissue regeneration has become the key platform for genome editing and integrated approaches. However, there is a lack of reliable and reproducible in-vitro conservation methods for several plant species. In addition, collecting plant material from natural habitats has decreased species numbers, prompting efforts to create growing methods that support conservation. Biotechnological approaches such as plant tissue culture have increasingly satisfied this growing demand. Thus, optimization of in-vitro regeneration system in Monarda citriodora Cerv. ex-Lag., was done using nodes as explants. The nodal explants were cultured on Murashige and Skoog’s (MS) medium blended with different concentrations of 6-benzylaminopurine (BAP), kinetin (Kn), Thidiazuron (TDZ), metaTopolin (mT) alone or in combination. The highest regeneration efficiency (90.31 ± 0.15%) with multiple shoots (13.22 ± 0.26) induction and shoot length (4.32 ± 0.06 cm) was achieved on shoot regeneration medium (SRM) containing MS salts fortified with 1 mg/L mT. Furthermore, we found that the combined application of 1 mg/L mT+ 0.5 mg/L α-Naphthaleneacetic acid (NAA) resulted in an increased shoot number (25.30 ± 0.45) with a shoot length of 6.13 ± 0.02 cm. A higher rooting percentage (85.51 ± 0.42) was recorded in plants grown in half-strength MS media. The start codon targeted (SCoT) and inter simple sequence repeat (ISSR) markers revealed that the in-vitro regenerated and greenhouse-acclimatized plants showed a similar banding pattern to that of the in-vivo grown mother plant, thus confirming the absence of a polymorphic nature of regenerated M. citriodora plants. These results signify that mT enhances the regeneration and development rate of M. citriodora plants.

Keywords: Molecular markers, Monarda citriodora, MS basal, Nodal explants, Regeneration.

References

Aggarwal, G., Sharma, C., & Srivastava, D. (2012). Thidiazuron: A potent cytokinin for efficient plant regeneration in Himalayan poplar (Populus ciliata Wall.) using leaf explants. Annals of Forest Research, 55(2), 179-188.

Ahmad, A., & Anis, M. (2019). meta-Topolin improves in-vitro morphogenesis, rhizogenesis and biochemical analysis in Pterocarpus marsupium Roxb.: A potential drug-yielding tree. Journal of Plant Growth Regulation, 38(3), 1007-1016. https://doi.org/10.1007/s00344-018-09910-9

Ai, X., Wen, Y., & Wang, B. (2023). Assessment of genetic stability on in-vitro propagation of Ardisia crenata var. bicolor using ISSR markers. International Journal of Plant Biology, 14(1), 218-227. https://doi.org/10.3390/ijpb14010018

Al-Qurainy, F., Khan, S., Nadeem, M., & Tarroum, M. (2015). SCoT marker for the assessment of genetic diversity in Saudi Arabian date palm cultivars. Pakistan Journal of Botany, 47(2), 637-643.

Amoo, S. O., & Van Staden, J. (2012). Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix. Plant Cell, Tissue and Organ Culture (PCTOC), 112(2), 249-256. https://doi.org/10.1007/s11240-012-0230-x

Badhepuri, M. K., Manokari, M., Cokul Raj, M., Jogam, P., Dey, A., Faisal, M.,…Shekhawat, M. S. (2023). Meta-Topolin enhanced direct shoot organogenesis and regeneration from leaf explants of Coleus forskohlii (Willd.) Briq. Industrial Crops and Products, 197, 116584. https://doi.org/10.1016/j.indcrop.2023.116584

Bairu, M. W., Stirk, W. A., Dolezal, K., & Van Staden, J. (2007). Optimizing the micropropagation protocol for the endangered Aloe polyphylla: can meta-topolin and its derivatives serve as replacement for benzyladenine and zeatin? Plant Cell, Tissue and Organ Culture, 90(1), 15-23. https://doi.org/10.1007/s11240-007-9233-4

Bednarek, P. T., Pachota, K. A., Dynkowska, W. M., Machczyńska, J., & Orłowska, R. (2021). Understanding in-vitro tissue culture-induced variation phenomenon in microspore system. International journal of molecular sciences, 22(14), 7546. https://doi.org/10.3390/ijms22147546

Bhuiyan, M. N. H., & Adachi, T. (2002). Efficient regeneration from hypocotyl cultures of betalain forming plant, Portulaca sp. Cv. ‘Jewel’: Stimulatory effect of thidiazuron. Plant Biotechnology, 19(1), 57-61. https://doi.org/10.5511/plantbiotechnology.19.57

Bisht, V., Rawat, J. M., Gaira, K. S., Purohit, S., Anand, J., Sinha, S.,…Rawat, B. (2024). Assessment of genetic homogeneity of in-vitro propagated apple root stock MM 104 using ISSR and SCoT primers. BMC plant biology, 24(1), 240-240. https://doi.org/10.1186/s12870-024-04939-3

Boo, K. H., Cao, D. V., Pamplona, R. S., Lee, D., Riu, K.-Z., & Lee, D.-S. (2015). In-vitro plant regeneration of Aster scaber via somatic embryogenesis. Bioscience, Biotechnology, and Biochemistry, 79(5), 725-731. https://doi.org/10.1080/09168451.2014.996202

BRI. (2024). Thymol Market Size, Share, Growth, and Global Industry Analysis by Type (Natural Thymol and Synthetic Thymol) By Application (Pharmaceutical/Biotechnology, Animal Health and Feedstuff, Flavor & Fragrances, and Others), Regional Insights, and Forecast To 2031. Business Research Insoghts.

Bulya, T. E., Glukhareva, T. V., & Kovaleva, E. G. (2023). Obtaining cell cultures of medicinal plants. In Recent Research and Advances in Soilless Culture. IntechOpen.

Calugaru-Spataru, T. (2023). Micropropagation of Monarda fistulosa L. Plants by axillary bud proliferation. Agrobiodiversity for Improving Nutrition, Health and Life Quality, 7(1). https://doi.org/10.15414/ainhlq.2023.0001

Chen, S., Xiong, Y., Yu, X., Pang, J., Zhang, T., Wu, K.,…Ma, G. (2020). Adventitious shoot organogenesis from leaf explants of Portulaca pilosa L. Scientific reports, 10(1), 3675-3675. https://doi.org/10.1038/s41598-020-60651-w

Collins, J. E., Bishop, C. D., Deans, S. G., & Svoboda, K. P. (1994). Composition of the essential oil from the leaves and flowers of Monarda citriodora var. citriodora grown in the United Kingdom. Journal of Essential Oil Research, 6(1), 27-29. https://doi.org/10.1080/10412905.1994.9698320

Davidson, C. G. (2007). Monarda, Bee-balm. In Flower Breeding and Genetics (pp. 756-779): Springer Netherlands.

Dorman, H. J. D., & Deans, S. G. (2004). Chemical composition, antimicrobial and in-vitro antioxidant properties of Monarda citriodora var. citriodora, Myristica fragrans, Origanum vulgare ssp. hirtum, Pelargonium sp. and Thymus zygis oils. Journal of Essential Oil Research, 16(2), 145-150. https://doi.org/10.1080/10412905.2004.9698679

Doyle, J. J., & Doyle, J. L. (1987). A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin.

Driver, J. A., & Suttle, G. R. L. (1987). Nursery handling of propagules. In Forestry Sciences (pp. 320-335): Springer Netherlands.

El-Mahrouk, M., Dewir, Y. H., & Omar, A. M. K. (2010). In-vitro propagation of adult strawberry tree (Arbutus unedo L.) through adventitious shoots and somatic embryogenesis. 93-98.

Fajinmi, O. O., Amoo, S. O., Finnie, J. F., & Van Staden, J. (2014). Optimization of in-vitro propagation of Coleonema album, a highly utilized medicinal and ornamental plant. South African Journal of Botany, 94, 9-13. https://doi.org/10.1016/j.sajb.2014.05.006

Fatima, N., Ahmad, N., & Anis, M. (2021). meta-Topolin promotes improved micropropagation, photosynthetic performances, biomass and proline levels of an India ipecac (Tylophora indica Burm f.). In Meta-topolin: A Growth Regulator for Plant Biotechnology and Agriculture (pp. 169-186): Springer Singapore.

Gontar, Ł., Geszprych, A., Przybył, J., Buła, M., & Osińska, E. (2022). Chemical variability of lemon beebalm (Monarda citriodora Cerv. ex Lag.) during plant phenology. Journal of Applied Research on Medicinal and Aromatic Plants, 31, 100433. https://doi.org/10.1016/j.jarmap.2022.100433

Grzeszczuk, M., Wesołowska, A., & Stefaniak, A. (2020). Biological value and essential oil composition of two Monarda species flowers. Acta Scientiarum Polonorum Hortorum Cultus, 19(4), 105-119. https://doi.org/10.24326/asphc.2020.4.10

Gupta, S., Kachhwaha, S., Kothari, S. L., & Jain, R. (2020). Synergistic effect of cytokinins and auxins enables mass clonal multiplication of drumstick tree (Moringa oleifera Lam.): a wonder. In-vitro Cellular & Developmental Biology - Plant, 56(4), 458-469. https://doi.org/10.1007/s11627-020-10065-0

Igwe, D. O., Ihearahu, O. C., Osano, A. A., Acquaah, G., & Ude, G. N. (2021). Assessment of genetic diversity of Musa species accessions with variable genomes using ISSR and SCoT markers. Genetic Resources and Crop Evolution, 69(1), 49-70. https://doi.org/10.1007/s10722-021-01202-8

Işlek, C. (2021). Effect of meta-Topolins on senescence. In mMeta-Topolin: A growth regulator for plant biotechnology and agriculture (pp. 59-69): Springer Singapore.

Ivanova, M., & Van Staden, J. (2010). Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. Plant Cell, Tissue and Organ Culture (PCTOC), 104(1), 13-21. https://doi.org/10.1007/s11240-010-9794-5

Katoch, M., Phull, S., Vaid, S., & Singh, S. (2017). Diversity, phylogeny, anticancer and antimicrobial potential of fungal endophytes associated with Monarda citriodora L. BMC microbiology, 17(1), 44-44. https://doi.org/10.1186/s12866-017-0961-2

Katoch, M., & Pull, S. (2017). Endophytic fungi associated with Monarda citriodora, an aromatic and medicinal plant and their biocontrol potential. Pharmaceutical biology, 55(1), 1528-1535. https://doi.org/10.1080/13880209.2017.1309054

Khan, N., Ahmed, M., Hafiz, I., Abbasi, N., Ejaz, S., & Anjum, M. (2015). Optimizing the concentrations of plant growth regulators for in-vitro shoot cultures, callus induction and shoot regeneration from calluses of grapes. OENO One, 49(1), 37. https://doi.org/10.20870/oeno-one.2015.49.1.95

Kher, M., Joshi, D., Nekkala, S., Nataraj, M., & Raykundaliya, P. D. (2014). Micropropagation of Pluchea lanceolata (Oliver & Hiern.) using nodal explant. Journal of Horticultural Research, 22(1), 35-39. https://doi.org/10.2478/johr-2014-0004

Koç, E. (2021). Effects of meta-Topolin on the growth, physiological and biochemical parameters in plant tissue culture. In meta-Topolin: A growth regulator for plant biotechnology and agriculture (pp. 265-278): Springer Singapore.

Kumar, S., Mangal, M., Dhawan, A. K., & Singh, N. (2011). Assessment of genetic fidelity of micropropagated plants of Simmondsia chinensis (Link) Schneider using RAPD and ISSR markers. Acta Physiologiae Plantarum, 33(6), 2541-2545. https://doi.org/10.1007/s11738-011-0767-z

Lata, H., Chandra, S., Khan, I., & ElSohly, M. A. (2008). Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L. In-vitro Cellular & Developmental Biology - Plant, 45(1), 12-19. https://doi.org/10.1007/s11627-008-9167-5

Lata, H., Chandra, S., Khan, I., & ElSohly, M. A. (2009). Thidiazuron-induced high-frequency direct shoot organogenesis of Cannabis sativa L. In-vitro Cellular Developmental Biology-Plant, 45, 12-19. https://doi.org/https://doi.org/10.1007/s11627-008-9167-5

Lata, H., Chandra, S., Techen, N., Khan, I. A., & ElSohly, M. A. (2016). In-vitro mass propagation of Cannabis sativa L.: A protocol refinement using novel aromatic cytokinin meta-topolin and the assessment of eco-physiological, biochemical and genetic fidelity of micropropagated plants. Journal of Applied Research on Medicinal and Aromatic Plants, 3(1), 18-26. https://doi.org/10.1016/j.jarmap.2015.12.001

Lu, Z. G., Li, X. H., & Li, W. (2011). Chemical composition of antibacterial activity of essential oil from Monarda citriodora flowers. Advanced Materials Research, 183-185, 920-923. https://doi.org/10.4028/www.scientific.net/amr.183-185.920

Manushkina, T., Kachanova, T., Samoilenko, M., & Petrova, O. (2022). Clonal micropropagation in-vitro of essential oil plants of the family Lamiaceae Lindl. UKRAINIAN BLACK SEA REGION AGRARIAN SCIENCE, 26(4). https://doi.org/10.56407/2313-092x/2022-26(4)-5

Martín, C., & González-Benito, M. E. (2024). Molecular Markers for the Detection and Analysis of Somaclonal Variation. In Somaclonal Variation: Basic and Practical Aspects (pp. 57-82): Springer International Publishing.

Mattarelli, P., Epifano, F., Minardi, P., Di Vito, M., Modesto, M., Barbanti, L., & Bellardi, M. G. (2017). Chemical composition and antimicrobial activity of essential oils from aerial parts of Monarda didyma and Monarda fistulosa cultivated in Italy. Journal of Essential Oil Bearing Plants, 20(1), 76-86. https://doi.org/10.1080/0972060x.2016.1278184

Meena, S. R., Aga, F. A., Chandra, S., Gochar, R., Koli, B., Khan, M. H., & Rasool, S. (2017). Studies on the phytochemical traits and their correlation with quantitative characters of Monarda citriodora Cerv. ex Lag. Annals of Phytomedicin, VI(I), 88-94. https://doi.org/10.21276/ap.2017.6.1.13

Mir, M. A., Mansoor, S., Sugapriya, M., Alyemeni, M. N., Wijaya, L., & Ahmad, P. (2021). Deciphering genetic diversity analysis of saffron (Crocus sativus L.) using RAPD and ISSR markers. Saudi journal of biological sciences, 28(2), 1308-1317. https://doi.org/10.1016/j.sjbs.2020.11.063

Mood, K., Jogam, P., Sirikonda, A., Shekhawat, M. S., Rohela, G. K., Manokari, M., & Allini, V. R. (2022). Micropropagation, morpho-anatomical characterization, and genetic stability studies in Lippia javanica (Burm.f.) Spreng: a multipurpose medicinal plant. Plant Cell, Tissue and Organ Culture (PCTOC), 150(2), 427-437. https://doi.org/10.1007/s11240-022-02294-5

Moyo, M., Finnie, J. F., & Van Staden, J. (2012). Topolins in Pelargonium sidoides micropropagation: do the new brooms really sweep cleaner? Plant Cell, Tissue and Organ Culture (PCTOC), 110(3), 319-327. https://doi.org/10.1007/s11240-012-0153-6

Murashige, T., & Skoog, F. (1962). A Revised Medium for Rapid Growth and Bio Assays with Tobacco Tissue Cultures. Physiologia Plantarum, 15(3), 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Murthy, B. N. S., Murch, S. J., & Saxena, P. K. (1998). Thidiazuron: A potent regulator of in-vitro plant morphogenesis. In-vitro Cellular & Developmental Biology - Plant, 34(4), 267-275. https://doi.org/10.1007/bf02822732

Naaz, A., Hussain, S. A., Anis, M., & Alatar, A. A. (2019). meta-Topolin improved micropropagation in Syzygium cumini and acclimatization to ex vitro conditions. Biologia plantarum, 63(1), 174-182. https://doi.org/10.32615/bp.2019.020

Pathania, A. S., Guru, S. K., Verma, M. K., Sharma, C., Abdullah, S. T., Malik, F.,…Bhushan, S. (2013). Disruption of the PI3K/AKT/mTOR signaling cascade and induction of apoptosis in HL-60 cells by an essential oil from Monarda citriodora. Food and Chemical Toxicology, 62, 246-254. https://doi.org/10.1016/j.fct.2013.08.037

Pivovarov, V., & Dryagin, V. (1991). Introduction of Monarda species in the Moscow area. 35-37.

Rathore, N. S., Rai, M. K., Phulwaria, M., Rathore, N., & Shekhawat, N. S. (2013). Genetic stability in micropropagated Cleome gynandra revealed by SCoT analysis. Acta Physiologiae Plantarum, 36(2), 555-559. https://doi.org/10.1007/s11738-013-1429-0

Rohela, G. K., Jogam, P., Bylla, P., & Reuben, C. (2019). Indirect Regeneration and Assessment of Genetic Fidelity of Acclimated Plantlets by SCoT, ISSR, and RAPD Markers in Rauwolfia tetraphylla L.: An Endangered Medicinal Plant. BioMed research international, 2019, 3698742-3698742. https://doi.org/10.1155/2019/3698742

Saha, S., Kader, A., Sengupta, C., & Ghosh, P. (2012). In-vitro propagation of Ocimum gratissimum L. (Lamiaceae) and its evaluation of genetic fidelity using RAPD marker. American Journal of Plant Sciences, 03(01), 64-74. https://doi.org/10.4236/ajps.2012.31006

Sen, Y., Chouhan, R., Meena, S. R., Dhar, R. S., & Gandhi, S. G. (2023). Development of micropropagation protocol for Colebrookea oppositifolia Sm. using nodal segments. Vegetos, 37(4), 1565-1573. https://doi.org/10.1007/s42535-023-00697-z

Sharma, M. M., Verma, R. N., Singh, A., & Batra, A. (2014). Assessment of clonal fidelity of Tylophora indica (Burm. f.) Merrill “in-vitro” plantlets by ISSR molecular markers. SpringerPlus, 3, 400-400. https://doi.org/10.1186/2193-1801-3-400

Sharma, P., Wajid, M. A., Fayaz, M., Bhat, S., Nautiyal, A. K., Jeet, S.,…Misra, P. (2023). Morphological, phytochemical, and transcriptome analyses provide insights into the biosynthesis of monoterpenes in Monarda citriodora. Planta, 258(3). https://doi.org/10.1007/s00425-023-04207-y

Singh, A. K., Vandana, T., Nidhi, V., Manibhushan, Naresh, C., & Bharati, R. (2009). Monarda-a potential floricultural plant for temperate India. 67681.

Singh, M., Saharan, V., Rajpurohit, D., Sen, Y., Joshi, A., & Sharma, A. (2017). Thidiazuron induced direct shoot organogenesis in Stevia rebaudiana and assessment of clonal fidelity of regenerated plants by RAPD and ISSR. International Journal of Current Microbiology and Applied Sciences, 6(8), 1690-1702. https://doi.org/10.20546/ijcmas.2017.608.203

Sirikonda, A., Jogam, P., Mood, K., & Allini, V. R. (2023). Micropropagation of 2-methoxy-4-vinyl phenol rich Flemingia strobilifera and assessment of genetic and biochemical fidelity by SCoT and GC-MS analysis. Plant Cell, Tissue and Organ Culture (PCTOC), 154(3), 541-550. https://doi.org/10.1007/s11240-023-02526-2

Sivanesan, I., Song, J. Y., Hwang, S. J., & Jeong, B. R. (2010). Micropropagation of Cotoneaster wilsonii Nakai: A rare endemic ornamental plant. Plant Cell, Tissue and Organ Culture (PCTOC), 105(1), 55-63. https://doi.org/10.1007/s11240-010-9841-2

Strnad, M., Hanuš, J., Vaněk, T., Kamínek, M., Ballantine, J. A., Fussell, B., & Hanke, D. E. (1997a). Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus × Canadensis moench., cv. Robusta). Phytochemistry, 45(2), 213-218. https://doi.org/10.1016/s0031-9422(96)00816-3

Strnad, M., Hanuš, J., Vaněk, T., Kamínek, M., Ballantine, J. A., Fussell, B., & Hanke, D. E. (1997b). Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus× canadensis Moench., cv. Robusta). Phytochemistry, 45(2), 213-218. https://doi.org/https://doi.org/10.1016/S0031-9422(96)00816-3

Su, Y.-H., Liu, Y.-B., & Zhang, X.-S. (2011). Auxin-cytokinin interaction regulates meristem development. Molecular plant, 4(4), 616-625. https://doi.org/10.1093/mp/ssr007

Sujatha, M., & Mukta, N. (1996). Morphogenesis and plant regeneration from tissue cultures of Jatropha curcas. Plant Cell, Tissue and Organ Culture, 44(2), 135-141. https://doi.org/10.1007/bf00048191

Thakur, J., Dwivedi, M. D., Sourabh, P., Uniyal, P. L., & Pandey, A. K. (2016). Genetic homogeneity revealed using SCoT, ISSR and RAPD markers in micropropagated Pittosporum eriocarpum Royle: An endemic and endangered medicinal plant. PloS one, 11(7), e0159050-e0159050. https://doi.org/10.1371/journal.pone.0159050

Waseem, K., Jilani, M., & Khan, M. (2009). Rapid plant regeneration of chrysanthemum (Chrysanthemum morifolium L.) through shoot tip culture. African Journal of Biotechnology, 8(9).

Werbrouck, S. P. O., van der Jeugt, B., Dewitte, W., Prinsen, E., Van Onckelen, H. A., & Debergh, P. C. (1995). The metabolism of benzyladenine in Spathiphyllum floribundum ‘Schott Petite’ in relation to acclimatisation problems. Plant Cell Reports, 14(10), 662-665. https://doi.org/10.1007/bf00232734

Zietkiewicz, E., Rafalski, A., & Labuda, D. (1994). Genome fingerprinting by simple sequence repeat (SSR): Anchored polymerase chain reaction amplification. Genomics, 20(2), 176-183. https://doi.org/10.1006/geno.1994.1151

Cite this article

Sen, Y., Sharma, V., Singh, S., Bulle, M., Misra, P., & Kota, S. (2024). MetaTopolin-driven breakthrough in lemon beebalm (Monarda citriodora) regeneration: A molecular fidelity study for genome engineering applications. Journal of Medicinal and Aromatic Plants, 46(3), 123-132. https://doi.org/10.62029/jmaps.v46i3.sen

Views
142
Downloads
1
Citations