Abstract
Crinum brachynema (Amaryllidaceae) is an endemic and critically endangered bulbous medicinal plant of the Northern Western Ghats of India. This species contains an important and promising alkaloid called galanthamine (GAL), an anti-Alzheimer drug. This investigation aimed to establish a reproducible protocol for in vitro regeneration of C. brachynema using twin scales as initial explants for the first time. Thus, cultures were established on Murashige and Skoog (MS) medium with cytokinins (6-benzyladenine (BA), and meta-Topolin (mT). The establishment and growth of shoots from the explants was achieved on MS medium supplemented with BA alone and in combination with 1-naphthaleneacetic acid (NAA). In vitro shoots were cultured on MS medium containing 0.5–10 µM of mT, of which 5.0 µM mT gave the highest shoot regeneration frequency (11.10). In general, mT proved superior as cultures produced a maximum number of regenerants compared to the control. Rooted plants with bulblets were successfully acclimatized in the greenhouse where they were phenotypically similar to the mother plants. Antioxidant activity was estimated using oxygen radical scavenging capacity (ORAC) model. Cultures on mT exhibited a significant increase in antioxidant capacity compared to the control. These findings highlight the beneficial effect and validate the rising importance of mT for in vitro regeneration studies. This study will serve as a potential protocol to conserve and restore the medicinally important C. brachynema, while the regenerated C. brachynema may be subsequently manipulated to further increase the accumulation of antioxidant potential.
Key Message
An efficient regeneration strategy was developed for conservation of Crinum brachynema, a critically endangered medicinal plant, using twin-scale explants; meta-Topolin enhanced in vitro shoot proliferation and improved regenerants antioxidant activity.
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Abbreviations
- AAPH:
-
2,2’-Azobis (2-methylpropionamidine) dihydrochloride
- AChE:
-
acetylcholinesterase
- ANOVA:
-
analysis of variance
- BA:
-
6-benzyladenine
- DMRT:
-
Duncan’s multiple range test
- GAL:
-
galanthamine
- IAA:
-
indole-3-acetic acid
- IBA:
-
indole-3-butyric acid
- IUCN:
-
international union for conservation and nature
- LED:
-
light-emitting diode
- mT:
-
meta-Topolin
- MS:
-
Murashige and Skoog
- NAA:
-
1-naphthaleneacetic acid
- ORAC:
-
oxygen radical absorbance capacity
- PGRs:
-
plant growth regulators
- PPFD:
-
photosynthetic photon flux density
- TE:
-
Trolox equivalents
References
Akhtar G, Jaskani MJ, Sajjad Y, Akram A (2016) Effect of antioxidants, amino acids and plant growth regulators on in vitro propagation of Rosa centifolia. Iran J Biotechnol 14:51–55. https://doi.org/10.15171/ijb.1152
Amoo SO, Aremu AO, Van Staden J (2012) In vitro regeneration, secondary metabolite production and antioxidant activity of micropropagate Aloe arborescens Mill. Plant Cell Tiss Organ Cult 111:345–358. https://doi.org/10.1007/s11240-012-0200-3
Amoo SO, Van Staden J (2013) Influence of plant growth regulators on shoot proliferation and secondary metabolite production in micropropagated Huernia hystrix. Plant Cell Tiss Organ Cult 112:249–256. https://doi.org/10.1007/s11240-012-0230-x
Aremu AO, Gruz J, Subrtova M, Szucova L, Dolezal K, Bairu MW, Finnie JF, VanStaden J (2013) Antioxidant and phenolic acid profiles of tissue cultured and acclimatized Merwilla plumbea plantlets in relation to the applied cytokinins. J Plant Physiol 170:1303–1308. https://doi.org/10.1016/j.jplph.2013.04.008
Argade MD, DeCristofano L, Bhattarai N, Schulte MK, Dukat M (2022) Evaluation of galantamine and deconstructed analogs as α7 nAChR and AChE ligands. Results in Chemistry 4:100286. https://doi.org/10.1016/j.rechem.2022.100286
Ayuso M, García-Pérez P, Ramil-Rego P, Gallego PP, Barreal ME (2019) In vitro culture of the endangered plant Eryngium viviparum as dual strategy for its ex-situ conservation and source of bioactive compounds. Plant Cell Tiss Organ Cult 138:427–435. https://doi.org/10.1007/s11240-019-01638-y
Bairu MW, Stirk WA, 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 Tiss Organ Cult 90:15–23. https://doi.org/10.1007/s11240-007-9233-4
Braca A, Politi M, Sanogo R, Sanou H, Morelli I, Pizza C, Tommasi ND (2003) Chemical composition and antioxidant activity of phenolic compounds from wild and cultivated Sclerocarya birrea (Anacardiaceae) leaves. J Agric Food Chem 51:6689–6695. https://doi.org/10.1021/jf030374m
Chahal S, Kaur H, Lekhak MM, Shekhawat MS, Goutam U, Singh SK, Ochatt SJ, Kumar V (2022) Meta-topolin-mediated regeneration and accumulation of phenolic acids in the critically endangered medicinal plant Crinum malabaricum (Amaryllidaceae): A potent source of galanthamine. S Afr J Bot. https://doi.org/10.1016/j.sajb.2022.01.016
Chahal S, Lekhak MM, Kaur H, Shekhawat MS, Dey A, Jha P, Pandey DK, Kumar V (2021) Unraveling the medicinal potential and conservation of Indian Crinum (Amaryllidaceae) species. S Afr J Bot 136:7–15. https://doi.org/10.1016/j.sajb.2020.04.029
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
Cheesman L, Finnie JF, Van Staden J (2010) Eucomis zambesiaca baker: factors affecting in vitro bulblet induction. S Afr J Bot 76:543–549
Christenhusz MJM, Byng JW (2016) The number of known plants species in the world and its annual increase. Phytotaxa 261:201–217. https://doi.org/10.11646/phytotaxa.261.3.1
De Bruyn M, Ferreira D, Slabbert M, Pretorius J (1992) In vitro propagation of Amaryllis belladonna. Plant Cell Tiss Org Cult 31:179–184. https://doi.org/10.1007/bf00036221
Diop MF, Ptak A, Chrétien F, Henry M, Chapleur Y, Laurain-Mattar D (2006) Galanthamine content of bulbs and in vitro cultures of Leucojum aestivum L. Nat Prod Commun 1:475–479. https://doi.org/10.1177/1934578X0600100609
Dragassaki M, Economou A, Vlahos J (2001) Bulblet formation in vitro and plantlet survival extra vitrum in Pancratium maritimum L. In: I International symposium on acclimatization and establishment of micropropagated plants. Acta Hortic 616: 347–352. https://doi.org/10.17660/ActaHortic.2003.616.51
Erişen S, Kurt-Güra G, Servi H (2020) In vitro propagation of Salvia sclarea L. by meta-Topolin, and assessment of genetic stability and secondary metabolite profiling of micropropagated plants. Ind Crops Prod 157:1122892
Fennell CW, Crouch NR, Van Staden J (2001) Micropropagation of the River Lily, Crinum variabile (Amaryllidaceae). S Afr J Bot 67:74–77
Ghane SG, Attar UA, Yadav PB, Lekhak MM (2018) Antioxidant, anti-diabetic, acetylcholinesterase inhibitory potential and estimation of alkaloids (lycorine and galanthamine) from Crinum species: An important source of anticancer and anti-Alzheimer drug. Ind Crops Prod 125:168–177. https://doi.org/10.1016/j.indcrop.2018.08.087
Haque M, Jahan S, Rahmatullah M (2014) Ethnomedicinal uses of Crinum asiaticum: a review. World J Pharm Pharm Sci 3:119–128
Hassan AKMS, Roy SK (2005) Micropropagation of Gloriosa superba L. through high frequency shoot proliferation. Plant Tissue Cult 15:67–74
Heinrich M (2010) Galanthamine from Galanthus and Other Amaryllidaceae Chemistry and Biology Based on Traditional Use. In: Cordell GA (ed) The Alkaloids. Academic Press, Chennai, pp 157–165
Heinrich M, Teoh HL (2004) Galanthamine from snowdrop—The development of a modern drug against Alzheimer’s disease from local Caucasian knowledge. J Ethnopharmacol 92:147–162. https://doi.org/10.1016/j.jep.2004.02.012
Jagtap UB, Lekhak MM, Fulzele DP, Yadav SR, Bapat VA (2014) Analysis of selected Crinum species for galanthamine alkaloid: an anti-Alzheimer drug. Curr Sci 107:2008–2010. https://doi.org/10.18520/CS%2FV107%2FI12%2F2008-2010
Jayaprakash K, Manokari M, Badhepuri MK, Raj MC, Dey A, Shekhawat MS (2021) Influence of meta-topolin on in vitro propagation and foliar micro-morpho-anatomical developments of Oxystelma esculentum (Lf) Sm. Plant Cell Tiss Organ Cult 147:325–337. https://doi.org/10.1007/s11240-021-02126-y
Jiang W, Hua S, Zhou X, Han P, Lu Q, Qiu Y (2018) Assessment of genetic stability and analysis of alkaloids potential in micropropagated plants of Croomia japonica Miquel, an endangered, medicinal plant in China and Japan. Plant Cell Tiss Organ Cult 135:1–12. https://doi.org/10.1007/s11240-018-1422-9
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
Joseph S, Kumar L, Bai NV (2016) Micropropagation of Cyrtanthus mackenii Hook. F.: from tri scales. Eur J Biotechnol Biosci 4:67–72
Kaur H, Chahal S, Jha P, Lekhak MM, Shekhawat MS, Naidoo D, Arencibia AD, Ochatt SJ, Kumar V (2022) Harnessing plant biotechnology-based strategies for in vitro galanthamine (GAL) biosynthesis: a potent drug against Alzheimer’s disease. Plant Cell Tiss Organ Cult 149, 81–103 (2022). https://doi.org/10.1007/s11240-022-02229-0
Khanam MN, Javed SB, Anis M, Alatar AA (2020) meta-Topolin induced in vitro regeneration and metabolic profiling in Allamanda cathartica L., Ind Crops Prod 145:111944. https://doi.org/10.1016/j.indcrop.2019.111944
Kshirsagar PR, Mohite A, Suryawanshi S, Chavan JJ, Gaikwad NB, Bapat VA (2021) Plant regeneration through direct and indirect organogenesis, phyto-molecular profiles, antioxidant properties and swertiamarin production in elicitated cell suspension cultures of Swertia minor (Griseb.) Knobl. Plant Cell Tiss Organ Cult 144:383–396. https://doi.org/10.1007/s11240-020-01962-8
Kumar V, Moyo M, Gruz J, Subrtova M, Van Staden J (2015) Phenolic acid profiles and antioxidant potential of Pelargonium sidoides callus cultures. Ind Crops Prod 77:402–408. https://doi.org/10.1016/J.INDCROP.2015.09.009
Lekhak MM, Yadav SR (2011) Karyotype studies in two critically endangered and endemic Crinum species (Amaryllidaceae) from Northern-Western Ghats of India. Nucleus 54:25–30. https://doi.org/10.1007/s13237-011-0024-2
Lekhak MM, Patel SB, Otari SS, Lekhak UM, Ghane SG (2022) Bioactive potential and RP-HPLC detection of phenolics and alkaloids (lycorine and galanthamine) from ultrasonic-assisted extracts of Crinum roots. S Afr J Bot. https://doi.org/10.1016/j.sajb.2021.07.024
Maelicke A (2000) Allosteric modulation of nicotinic receptors as a treatment strategy for Alzheimer’s disease. Dement Geriatr Cogn Disord 11:11–18. https://doi.org/10.1159/000051227
Malá J, Máchová P, Cvrčková H, Karady M, Novák O, Mikulík J, Hauserová E, Greplová J, Strnad M, Doležal K (2009) Micropropagation of Wild Service Tree (Sorbus torminalis [L.] Crantz): The regulative role of different aromatic cytokinins during organogenesis. J Plant Growth Regul 28:341–348
Manokari M, Mehta SR, Priyadharshini S, Badhepuri MK, Dulam S, Jayaprakash K, Cokul Raj M, Dey A, Rajput BS, Shekhawat MS (2021) Meta-Topolin mediated improved micropropagation, foliar micro-morphological traits, biochemical profiling, and assessment of genetic fidelity in Santalum album L. Ind Crops Prod 171:113931. https://doi.org/10.1016/j.indcrop.2021.113931
Manokari M, Priyadharshini S, Cokulraj M, Dey A, Shekhawat MS (2022a) Meta-topolin induced morphometric and structurally stable bulblets in Malabar River Lily (Amaryllidaceae). Plant Cell Tiss Organ Cult 148:377–385. https://doi.org/10.1007/s11240-021-02195-z
Manokari M, Badhepuri MK, Cokulraj M, Sandhya D, Dey A, Kumar V, Faisal M, Alatar AA, Singh RK, Shekhawat MS (2022b) Validation of meta-Topolin in organogenesis, improved morpho-physio-chemical responses, and clonal fidelity analysis in Dioscorea pentaphylla L. – an underutilized yam species. S Afr J Bot 145:284–292. https://doi.org/10.1016/j.sajb.2021.12.039
Meiners J, Schwab M, Szankowski I (2007) Efficient in vitro regeneration systems for Vaccinium species. Plant Cell Tiss Organ Cult 89:169–176. https://doi.org/10.1007/s11240-007-9230-7
Mood K, Jogam P, Sirikonda A, Shekhawat MS, Rohela GK, Manokari M, Allini VR (2022) Micropropagation, morpho-anatomical characterization, and genetic stability studies in Lippia javanica (Burm. f.) Spreng: a multipurpose medicinal plant. Plant Cell Tiss Organ Cult 14:1–1. https://doi.org/10.1007/s11240-022-02294-5
Moyo M, Amoo SO, Aremu AO, Gruz J, Šubrtová M, Doležal K, Van Staden J (2014) Plant regeneration and biochemical accumulation of hydroxybenzoic and hydroxycinnamic acid derivatives in Hypoxis hemerocallidea organ and callus cultures. Plant Sci 227:157–164. https://doi.org/10.1016/j.plantsci.2014.08.003
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Nair JJ, Van Staden J (2021) The Amaryllidaceae, a chemically and biologically privileged plant family. S Afr J Bot 136:1–6. https://doi.org/10.1016/j.sajb.2020.09.018
Nazir R, Kumar V, Dey A, Gupta S, Yousuf M, Husaain S, Pandey DK (2021) In vitro propagation and assessment of genetic fidelity in Dioscorea deltoidea, a potent diosgenin yielding endangered plant. S Afr J Bot 140:349–355. https://doi.org/10.1016/j.sajb.2020.07.018
Niedz RP, Evens TJ (2010) The effects of benzyladenine and meta-topolin on in vitro shoot regeneration of a Citrus citrandarin rootstock. Res J Agric & Biol Sci 6:45–53
Ozel CA, Khawar KM, Unal F (2015) Factors affecting efficient in vitro micropropagation of Muscari muscarimi Medikus using twin bulb scale. Saudi J Biol Sci 22:132–138. https://doi.org/10.1016/j.sjbs.2014.09.007
Patel M, Patel H (2019) Crinum reddyi sp. nov. (Amaryllidaceae) from Gujarat, India. Nordic J Bot 37:1–5. https://doi.org/10.1111/njb.02172
Patel SB, Otari SS, Kumar V, Rastogi A, Lekhak MM, Ghane SG (2022) Optimization of lycorine using Response Surface Methodology, extraction methods and in vitro antioxidant and anti-diabetic activities from the roots of Giant Spider Lily: A medicinally important bulbous herb. S Afr J Bot. https://doi.org/10.1016/j.sajb.2022.04.022(In Press)
POWO (2019) ‘’Plants of the World Online. Facilitated by the Royal Botanic Gardens, Kew. https://powo.science.kew.org/
Priyadharshini S, Kannan N, Manokari M, Shekhawat MS (2021) In vitro regeneration using twin scales for restoration of critically endangered aquatic plant Crinum malabaricum Lekhak & Yadav: a promising source of galanthamine. Plant Cell Tiss Organ Cult 141:593–604. https://doi.org/10.1007/s11240-020-01818-1
Punekar SA, Mandar DN, Lakshminarasimhan P (2001) Crinum brachynema Herb. (Amaryllidaceae)-An endemic species found again in Mahabaleshwar, Maharashtra State. J Econ Taxon Bot 25:629–630. https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names
Khonakdari MR, Rezadoost H, Heydari R, Mirjalili MH (2020) Effect of photoperiod and plant growth regulators on in vitro mass bulblet proliferation of Narcissus tazzeta L. (Amaryllidaceae), a potential source of galantamine. Plant Cell Tiss Organ Cult 142:187–199. https://doi.org/10.1007/s11240-020-01853-y
Reis A, Magne K, Massot S, Tallini LR, Scopel M, Bastida J, Ratet P, Zuanazzi JA (2019) Amaryllidaceae alkaloids: identification and partial characterization of montanine production in Rhodophiala bifida plant. Sci Rep 9:1–1. https://doi.org/10.1038/s41598-019-44746-7
Revathi J, Manokari M, Priyadharshini S, Mafatlal MK, Mahipal SS (2019) In vitro propagation, in vitro flowering, ex vitro root regeneration and foliar micro-morphological analysis of Hedyotis biflora. (Linn) Lam Vegetos 32:609–619. https://doi.org/10.1007/s42535-019-00066-9
Rice LJ, Finnie JF, Van Staden J (2011) In vitro production of Brunsvigia undulata from twin-scales. S Afr J Bot 77:305–312. https://doi.org/10.1016/j.sajb.2010.08.011
Santos A, Fidalgo F, Santos I, Salema R (2002) In vitro bulb formation of Narcissus asturiensis, a threatened species of the Amaryllidaceae. J Horticult Sci Biotechnol 77:149–152. https://doi.org/10.1080/14620316.2002.11511471
Shekhawat MS, Priyadharshini S, Jogam P, Kumar V, Manokari M (2021) Meta-topolin and liquid medium enhanced in vitro regeneration in Scaevola taccada (Gaertn.) Roxb. Vitro Cell Dev Biol-Plant 57:296–306. https://doi.org/10.1007/s11627-020-10156-y
Shirin F, Rana PK, Mandal AK (2005) In vitro clonal propagation of mature Tectona grandis through axillary bud proliferation. J For Res 10:465–469. https://doi.org/10.1007/s10310-005-0173-8
Sikder L, Alim MI, Saieda B, Islam MT, Martorell M, Sharifi-Rad J, Khan SA (2021) Pharmacological activities of Crinum viviparum: a laboratory-based study. J Herbs Spices Med Plants 27:177–187. https://doi.org/10.1080/10496475.2021.1891177
Skała E, Grąbkowska R, Sitarek P, Kuźma Ł, Błauż A, Wysokińska H (2015) Rhaponticum carthamoides regeneration through direct and indirect organogenesis, molecular profiles and secondary metabolite production. Plant Cell Tiss Organ Cult 123:83–98. https://doi.org/10.1007/s11240-015-0816-1
Slabbert M, De Bruyn M, Ferreira D, Pretorius J (1993) Regeneration of bulblets from twin scales of Crinum macowanii in vitro. Plant Cell Tiss Org Cult 33:133–141. https://doi.org/10.1007/BF01983226
Wojtania A (2010) Effect of meta-topolin on in vitro propagation of Pelargonium x hortorum and Pelargonium x hederaefolium cultivars. Acta Soc Bot Pol 79:101–106. https://doi.org/10.5586/asbp.2010.013
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The authors thank the Science and Engineering Research Board, Department of Science and Technology (SERB-DST), Government of India for providing the financial support under the Startup Research Scheme (File No. SRG/2019/001279).
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VK and SO are the advisors of this project, planned all experiments and edited the manuscript. HK, SC, PJ and MML conducted the experiments, performed the analysis, acclimatization of plants and wrote the manuscript. All contributors read and approved the final draft of the manuscript.
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Kaur, H., Chahal, S., Lekhak, M.M. et al. Meta-topolin induced in vitro regeneration in Crinum brachynema (Amaryllidaceae): a critically endangered and endemic medicinal plant of India. Plant Cell Tiss Organ Cult 151, 663–672 (2022). https://doi.org/10.1007/s11240-022-02380-8
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DOI: https://doi.org/10.1007/s11240-022-02380-8