Abstract
In this study, an efficient, reproducible, and genetically stable regeneration protocol has been developed in Artemisia maritima L. The experiments were conducted for callus induction, plant regeneration, and somatic embryogenesis using stem and leaf of A. maritima as explants. The optimal callus induction (81.3%) was observed on 2.5 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.5 mg L−1 6-benzylaminopurine (BAP). The shoot regeneration was observed on different concentrations of BAP, α-naphthaleneacetic acid (NAA), and thidiazuron (TDZ), using nodal segments and microshoot tips as explants. The microshoot tips were more responsive compared to nodal segments with the highest induction frequency (90.33%) obtained on 1.5 mg L−1 BAP. Maximum root induction frequency (74.36%) was obtained on 1.5 mg L−1 NAA. The somatic embryogenesis was induced on Murashige and Skoog (MS) medium amended with TDZ and indole-3-butyric acid (IBA) with maximum embryogenic induction frequency on 1.0 mg L−1 TDZ and 2.5 mg L−1 IBA. The somatic embryos developed into globular, heart-shaped, and bipolar plantlet stages on BAP and NAA as revealed through scanning electronic microscopy (SEM) and histological studies. The fully developed plants were acclimatized (75% survival rate) and transferred to natural photoperiod conditions. The DNA content and genetic stability of direct regenerated and somatic embryo–derived plants were analyzed by flow cytometry. The 2C DNA content of in vivo plants, direct regenerated, and somatic embryo–derived plants was 14.89, 14.61, and 14.37 pg, respectively. The genetic stability was maintained in in vitro cultures in comparison to field-grown plants of A. maritima. This study for the first time tried to formulate regeneration protocol via direct and indirect organogenesis and somatic embryogenesis for A. maritima. This paper was also the first report for comparing the 2C DNA content of A. maritima grown in vivo to in vitro cultured plants.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data Availability
All data generated or analyzed during this study are included in this published article.
References
Abdulhafiz F, Mohammed A, Kayat F, Zakaria S, Hamzah Z, Reddy Pamuru R, Gundala PB, Reduan MF (2020) Micropropagation of Alocasia longiloba Miq and comparative antioxidant properties of ethanolic extracts of the field-grown plant, in vitro propagated and in vitro-derived callus. Plants 9:816. https://doi.org/10.3390/plants9070816
Adhikary D, Kulkarni M, El-Mezawy A, Mobini S, Elhiti M, Gjuric R, Ray A, Polowick P, Slaski JJ, Jones MP, Bhowmik P (2021) Medical cannabis and industrial hemp tissue culture: present status and future potential. Front Plant Sci 12:275. https://doi.org/10.3389/fpls.2021.627240
Ali M, Abbasi BH, Ahmad N, Khan H, Ali GS (2017) Strategies to enhance biologically active-secondary metabolites in cell cultures of Artemisia–current trends. Crit Rev Biotechnol 37:833–851. https://doi.org/10.1080/07388551.2016.1261082
Al-Khayri JM (2018) Somatic embryogenesis of date palm (Phoenix dactylifera L.) from shoot tip explants. In: Jain S, Gupta P, (eds) Step wise protocols for somatic embryogenesis of important woody plants. Forestry Sciences vol 85. Springer, Cham, pp 231–244. https://doi.org/10.1007/978-3-319-79087-9_19
Aslam J, Mujib A, Sharma MP (2014) Somatic embryos in Catharanthusroseus: a scanning electron microscopic study. Notulae Sci Biol 6:167–172. https://doi.org/10.15835/nsb629337
Aslam N, Zi M, Chaudhary MF (2006) Callogenesis and direct organogenesis of Artemisia scoparia. Pakistan J Biol Sci 9:1783–1786. https://doi.org/10.3923/pjbs.2006.1783.1786
Bennett MD, Leitch IJ (2005) Nuclear DNA amounts in angiosperms: progress, problems and prospects. Ann Bot 95:45–90. https://doi.org/10.1093/aob/mci003
Bennett MD, Leitch IJ (2011) Nuclear DNA amounts in angiosperms: targets, trends and tomorrow. Ann Bot 107:467–590. https://doi.org/10.1093/aob/mcq258
Bhagat RC, Singh N (1989) Natural resource development of Himalayas. Jay Kay Book House, Jammu
Bharti U, Sharma E, Parihar J, Sharma N (2019) Genetic system of Artemisia maritima L.: an overexploited medicinal species under stress. Proc Natl Acad Sci India Sect B Biol Sci 89:1373–1378. https://doi.org/10.1007/s40011-018-1057-y
Bisht D, Kumar D, Kumar D, Dua K, Chellappan DK (2021) Phytochemistry and pharmacological activity of the genus Artemisia. Arch Pharmacal Res 44:439–474. https://doi.org/10.1007/s12272-021-01328-4
Boo KH, Cao DV, Pamplona RS, Lee D, Riu KZ, Lee DS (2015) In vitro plant regeneration of Aster scaber via somatic embryogenesis. Biosci Biotechnol Biochem 79:725–731. https://doi.org/10.1080/09168451.2014.996202
Bukhori MFM (2013) Improved protocol for high frequency plant regeneration through somatic embryogenesis in Carica papaya. Res Biotechnol 4:9–19
Butiuc-Keul A, Farkas A, Cristea V (2016) Genetic stability assessment of in vitro plants by molecular markers. Stud Univ Babeş-Bolyai Biol 61(1):107–114
Cardoso JC, da Silva JAT (2013) Micropropagation of Zeyheria montana Mart. (Bignoniaceae), an endangered endemic medicinal species from the Brazilian cerrado biome. In Vitro Cell Dev Biol - Plant 49:710–716. https://doi.org/10.1007/s11627-013-9558-0
Chandra S, Bandopadhyay R, Kumar V, Chandra R (2010) Acclimatization of tissue cultured plantlets: from laboratory to land. Biotechnol Lett 32:1199–1205. https://doi.org/10.1007/s10529-010-0290-0
Dangash A, Ram M, Niranjan R, Bharillya A, Misra H, Pandya N, Jain DC (2015) In vitro selection and hormonal regulation in cell culture of Artemisia annua L. plant. JSM Cell Dev Biol 3:1013
Das A, Kesari V, Rangan L (2013) Micropropagation and cytogenetic assessment of Zingiber species of Northeast India. 3 Biotech 3:471–479. https://doi.org/10.1007/s13205-012-0108-y
Deepa AV, Thomas TD (2020) In vitro strategies for the conservation of Indian medicinal climbers. In Vitro Cell Dev Biol - Plant 56:784–802. https://doi.org/10.1007/s11627-020-10084-x
Dolezel J, Greilhuber J, Suda J (2007) Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc 2:2233–2244. https://doi.org/10.1038/nprot.2007.310
Ene AC, Atawodi SE, Ameh DA, Kwanashie HO, Agomo PU (2009) In vivo antiplasmodial effect of chloroform extracts of Artemisia maciverae Linn and Artemisia maritima Linn. African J Biotechnol 8:6612–6616
ENVIS (2003) FRLHT's ENVIS Centre on Medicinal Plants, Bengaluru. http://envis.frlht.org/frlhtenvis.nic.in or http://envis.frlht.org/. Assessed 19 March, 2021
Ghorbani S, Kosari-Nasab M, Mahjouri S, Talebpour AH, Movafeghi A, Maggi F (2021) Enhancement of in vitro production of volatile organic compounds by shoot differentiation in Artemisia spicigera. Plants 10:208. https://doi.org/10.3390/plants10020208
Ghosh A, Igamberdiev AU, Debnath SC (2021) Tissue culture-induced DNA methylation in crop plants: a review. Mol Biol Rep 48:823–841. https://doi.org/10.1007/s11033-020-06062-6
Giorgetti L, Ruffini Castiglione M, Turrini A, Martini G, Nuti Ronchi V, Geri C (2011) Cytogenetic and histological approach for early detection of “mantled’’ somaclonal variants of oil palm regenerated by somatic embryogenesis: first results on the characterization of regeneration system. Caryologia 64:223–234. https://doi.org/10.1080/00087114.2002.10589787
Guan Y, Li SG, Fan XF, Su ZH (2016) Application of somatic embryogenesis in woody plants. Front Plant Sci 7:938. https://doi.org/10.3389/fpls.2016.00938
Hapsoro D, Hamiranti R, Yusnita Y (2020) In vitro somatic embryogenesis of superior clones of robusta coffee from Lampung, Indonesia: effect of genotypes and callus induction media. Biodiversitas 21:3811–3817. https://doi.org/10.13057/biodiv/d210849
Haradzi NA, Khor SP, Subramaniam S, Chew BL (2021) Regeneration and micropropagation of Meyer lemon (Citrus x meyeri) supported by polymorphism analysis via molecular markers. Sci Hortic 286:110225. https://doi.org/10.1016/j.scienta.2021.110225
Hooker JD (1882) The flora of British India, vol 3. L. Reeve and Co., London. https://doi.org/10.5962/bhl.title.678
Irum S, Ahmed H, Mukhtar M, Mushtaq M, Mirza B, Donskow-Łysoniewska K, Qayyum M, Simsek S (2015) Anthelmintic activity of Artemisia vestita Wall ex DC. and Artemisia maritima L. against Haemonchus contortus from sheep. Vet Parasitol 212:451–455. https://doi.org/10.1016/j.vetpar.2015.06.028
Jogam P, Sandhya D, Shekhawat MS, Alok A, Manokari M, Abbagani S, Allini VR (2020) Genetic stability analysis using DNA barcoding and molecular markers and foliar micro-morphological analysis of in vitro regenerated and in vivo grown plants of Artemisia vulgaris L. Ind Crops Prod 151:112476. https://doi.org/10.1016/j.indcrop.2020.112476
Johansen DA (1940) Plant microtechnique. McGraw-Hill, New York
Koul B, Taak P, Kumar A, Khatri T, Sanyal I (2017) The Artemisia genus: a review on traditional uses, phytochemical constituents, pharmacological properties and germplasm conservation. J Glycomics Lipidomics 7:142. https://doi.org/10.4172/2153-0637.1000142
Ku N, Chan L (2013) Somatic embryogenesis: an alternative for propagating selected highland clone of Artemisia annua L. of Vietnam origin. J Biol Agricul Healthcare 3:131–135
Kulus D (2015) Selected aspects of ornamental plants micropropagation in Poland and worldwide. Life Sci 4:10–25. https://doi.org/10.13140/RG.2.1.5086.8082
Lema-Rumińska J, Kulus D, Tymoszuk A, Varejao JM, Bahcevandziev K (2019) Profile of secondary metabolites and genetic stability analysis in new lines of Echinacea purpurea (L.) Moench micropropagated via somatic embryogenesis. Ind Crops Prod 142:111851. https://doi.org/10.1016/j.indcrop.2019.111851
Lualon W, De-Eknamkul W, Tanaka H, Shoyama Y, Putalun W (2008) Artemisinin production by shoot regeneration of Artemisia annua L. using thidiazuron. Z Naturforsch C J Biosci 63:96–100. https://doi.org/10.1515/znc-2008-1-218
Miler N, Kulus D, Sliwinska E (2020) Nuclear DNA content as an indicator of inflorescence colour stability of in vitro propagated solid and chimera mutants of chrysanthemum. Plant Cell Tiss Org Cult 143:421–430. https://doi.org/10.1007/s11240-020-01929-9
Mohajer S, Taha RM, Khorasani A, Yaacob JS (2012) Induction of different types of callus and somatic embryogenesis in various explants of Sainfoin ('Onobrychis sativa’). Aust J Crop Sci 6(8):1305–1313
Nabi N, Singh S, Saffeullah P (2021) Responses of in vitro cell cultures to elicitation: regulatory role of jasmonic acid and methyl jasmonate: a review. In Vitro Cell Dev Biol - Plant 57:341–355. https://doi.org/10.1007/s11627-020-10140-6
Naing AH, Kim SH, Chung MY, Park SK, Kim CK (2019) In vitro propagation method for production of morphologically and genetically stable plants of different strawberry cultivars. Plant Methods 15:36. https://doi.org/10.1186/s13007-019-0421-0
Nazir R, Kumar V, Gupta S, Dwivedi P, Pandey DK, Dey A (2021) Biotechnological strategies for the sustainable production of diosgenin from Dioscorea spp. Appl Microbiol Biotechnol 105:569–585. https://doi.org/10.1007/s00253-020-11055-3
Ochatt SJ (2008) Flow cytometry in plant breeding. Cytometry 73A:581–598. https://doi.org/10.1002/cyto.a.20562
Ochatt SJ, Conreux C, Jacas L (2013) Flow cytometry distinction between species and between landraces within Lathyrus species and assessment of true-to-typeness of in vitro regenerants. Plant Syst Evol 299:75–85. https://doi.org/10.1002/cyto.a.20562
Pandey AK, Singh P (2017) The genus Artemisia: a 2012–2017 literature review on chemical composition, antimicrobial, insecticidal and antioxidant activities of essential oils. Medicines (basel) 4:68. https://doi.org/10.3390/medicines4030068
Parihar J, Hamal U, Hamal IA, Sharma N (2011) Restricted distribution of Artemisia maritima L., an endangered plant in Kishtwar Himalaya, J&K-role of edaphic factors. Natl Acad Sci Lett 34:317–320
Qadir M, Dangroo NA, Shah SWA (2019) Bioactivity-guided phytochemical investigations of Artemisia maritima: isolation and characterization of chemical constituents. Asian J Pharm Clin Res 12:269–274. https://doi.org/10.22159/ajpcr.2019.v12i1.28600
Ribeiro T, Barrela RM, Bergès H, Marques C, Loureiro J, Morais-Cecílio L, Paiva JAP (2016) Advancing Eucalyptus genomics: cytogenomics reveals conservation of Eucalyptus genomes. Front Plant Sci 7:510. https://doi.org/10.3389/fpls.2016.00510
Sainz P, Cruz-Estrada Á, Díaz CE, González-Coloma A (2017) The genus Artemisia: distribution and phytochemistry in the Iberian Peninsula and the Canary and Balearic Islands. Phytochem Rev 16(5):1023–1043. https://doi.org/10.1007/s11101-017-9516-2
Samarina L, Gvasaliya M, Koninskaya N, Rakhmangulov R, Efremov A, Kiselyova N, Ryndin A, Hanke MV (2019) A comparison of genetic stability in tea [Camellia sinensis (L.) Kuntze] plantlets derived from callus with plantlets from long-term in vitro propagation. Plant Cell Tiss Org Cult 138:467–474. https://doi.org/10.1007/s11240-019-01642-2
Sharada D, Krishna PS, Swamy NR (2019) Plant regeneration via somatic embryogenesis in Solanum nigrum L. (Black nightshade) (Solanaceae). Biotechnol J Int 23:1–9. https://doi.org/10.9734/bji/2019/v23i130070
Shashi, Bhat V (2021) Enhanced somatic embryogenesis and plantlet regeneration in Cenchrusciliaris L. In Vitro Cell Dev Biol - Plant 57:499–509. https://doi.org/10.1007/s11627-020-10148-y
Shinde S, Sebastian JK, Jain JR, Hanamanthagouda MS, Murthy HN (2016) Efficient in vitro propagation of Artemisia nilagirica var. nilagirica (Indian wormwood) and assessment of genetic fidelity of micropropagated plants. Physiol Mol Biol Plant 22:595–603. https://doi.org/10.1007/s12298-016-0379-6
Sikdar SU, Zobayer N, Azim F, Ashrafuzzaman M, Prodhan SH (2012) An efficient callus initiation and direct regeneration of Stevia rebaudiana. African J Biotechnol 11:10381–10387. https://doi.org/10.5897/AJB11.2363
Singh P, Bajpai V, Khandelwal N, Varshney S, Gaikwad AN, Srivastava M, Kumar B (2021) Determination of bioactive compounds of Artemisia Spp. plant extracts by LC–MS/MS technique and their in-vitro anti-adipogenic activity screening. J Pharm Biomed Anal 193:113707. https://doi.org/10.1016/j.jpba.2020.113707
Sliwinska E (2018) Flow cytometry - a modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species. Folia Hortic 30:103–128. https://doi.org/10.2478/fhort-2018-0011
Sliwinska E, Thiem B (2007) Genome size stability in six medicinal plant species propagated in vitro. Biol Plant 51:556–558. https://doi.org/10.1007/s10535-007-0121-x
Stappen I, Wanner J, Tabanca N, Wedge DE, Ali A, Khan IA, Kaul VK, Lal B, Jaitak V, Gochev V, Girova T, Stoyanova A, Schmidt JL (2014) Chemical composition and biological effects of Artemisia maritima and Artemisia nilagirica essential oils from wild plants of western Himalaya. Planta Med 80:1079–1087. https://doi.org/10.1055/s-0034-1382957
Varis S, Klimaszewska K, Aronen T (2018) Somatic embryogenesis and plant regeneration from primordial shoot explants of Picea abies (L.) H. Karst. somatic trees. Front Plant Sci 9:1551. https://doi.org/10.3389/fpls.2018.01551
Walia S, Rana A, Singh A, Sharma M, Eswara Reddy SG, Kuma R (2019) Influence of harvesting time on essential oil content, chemical composition and pesticidal activity of Artemisia maritima growing wild in the cold desert region of western Himalayas. J Essent Oil Bear Plants 22:396–407. https://doi.org/10.1080/0972060X.2019.1610077
Wetzstein HY, Porter JA, Janick J, Ferreira JF, Mutui TM (2018) Selection and clonal propagation of high artemisinin genotypes of Artemisia annua. Front Plant Sci 9:358. https://doi.org/10.3389/fpls.2018.00358
Zadoks JC (2013) Crop protection in medieval agriculture: studies in pre-modern organic agriculture. Sidestone Press, Leiden
Zayova E, Nedev T, Petrova D, Zhiponova M, Kapchina V, Chaneva G (2020) Tissue culture applications of Artemisia annua L. callus for indirect organogenesis and production phytochemical. Plant Tiss Cult Biotechnol 30:97–106. https://doi.org/10.3329/ptcb.v30i1.47795
Zhang M, Wang A, Qin M, Qin X, Yang S, Su S, Sun Y, Zhang L (2021) Direct and indirect somatic embryogenesis induction in Camellia oleifera Abel. Front Plant Sci 12:451. https://doi.org/10.3389/fpls.2021.644389
Acknowledgements
The authors are thankful to Dr. Shwetanjali Nimker (Application Scientist) at BD FACS, Jamia Hamdard, New Delhi, for assistance in flow cytometry.
Funding
The first author received research grant no. 191000913 from the University Grants Commission, India.
Author information
Authors and Affiliations
Contributions
All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Neelofer Nabi and Peer Saffeullah. The first draft of the manuscript was written by Neelofer Nabi and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of Interest
The authors declare no competing interests.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Nabi, N., Saffeullah, P. & Singh, S. Micropropagation using direct and indirect organogenesis in Artemisia maritima L.: scanning electron microscopy of somatic embryos and genome size analysis by flow cytometry. In Vitro Cell.Dev.Biol.-Plant 58, 1012–1024 (2022). https://doi.org/10.1007/s11627-022-10291-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11627-022-10291-8