In Africa and Asia, members of the genus Brachystelma are well-known for their diverse uses, especially their medicinal and nutritional values. However, the use of many Brachystelma species as a valuable resource is generally accompanied by the concern of over-exploitation attributed to their slow growth and general small size. The aim of the current study was to establish efficient micropropagation protocols for three Brachystelma species, namely Brachystelma ngomense (endangered), Brachystelma pulchellum (vulnerable) and Brachystelma pygmaeum (least concern), as a means of ensuring their conservation and survival. This was achieved using nodal segments (~10 mm in length) as the source of explants in the presence of different concentrations of three cytokinins (CK) namely N6-benzyladenine (BA), isopentenyladenine (iP) and meta-topolin riboside (mTR), over a period of 6 weeks. The highest (25 µM) concentration of cytokinin treatments typically resulted in significantly higher shoot proliferation. However, each species differed in its response to specific CK: the optimal concentrations were 25 µM mTR, 25 µM iP and 25 µM BA for Brachystelma ngomense, Brachystelma pulchellum and Brachystelma pygmaeum, respectively. During the in vitro propagation, both Brachystelma ngomense and Brachystelma pygmaeum rooted poorly while regenerated Brachystelma pulchellum generally lacked roots regardless of the CK treatments. Following pulsing (dipping) treatment of in vitro-regenerated shoots with indole-3-butyric acid (IBA), acclimatization of all three Brachystelma species remained extremely limited due to poor rooting ex vitro. To the best of our knowledge, the current protocols provide the first successful report for these Brachystelma species. However, further research remains essential to enhance the efficiency of the devised protocol.

Department of Chemical Biology and Genetics Centre of the Region Haná for Biotechnological and Agricultural Research Faculty of Science Palacký University Šlechtitelů 27 CZ 783 71 Olomouc Czech Republic

Indigenous Knowledge Systems Centre Faculty of Natural and Agricultural Sciences North West University Private Bag X2046 Mmabatho 2790 South Africa

Laboratory of Growth Regulators Faculty of Science Palacký University and Institute of Experimental Botany AS CR Šlechtitelů 11 CZ 783 71 Olomouc Czech Republic

Research Centre for Plant Growth and Development School of Life Sciences University of KwaZulu Natal Pietermaritzburg Private Bag X01 Scottsville 3209 South Africa

See more in PubMed

Bruyns P.V. A new species of Brachystelma (Apocynaceae) from South Tropical Africa. Novon. 2006;16:452–453. doi: 10.3417/1055-3177(2006)16[452:ANSOBA]2.0.CO;2. DOI

Masinde P.S. A revision of Brachystelma Sims (Apocynaceae: Asclepiadoideaeae-Ceropegia) in East Africa. Kew Bull. 2007;62:37–84.

South African National Biodiversity Institute (SANBI) [(accessed on 8 February 2015)]; Available online: http://redlist.sanbi.org/genus.php?genus=2640.

Bruyns P.V. Ceropegia, Brachystelma and Tenaris in South West Africa. Dinteria. 1984;17:3–80.

Pooley E. A Field Guide to Wild Flowers of KwaZulu-Natal and the Eastern Region. 2nd ed. Natal Flora Publications Trust; Durban, South Africa: 2005.

Rajaram M.S., Rathod J., Dilip V. Pharmacognostical studies on the tuber of Brachystelma edulis Coll. and Helmsl.- an endemic to Peninsular, India. World J. Pharm. Pharm. Sci. 2014;3:1958–1965.

Smith N.M. Ethnobotanical field notes from the Northern territory, Australia. J. Adel. Bot. Gard. 1991;14:1–65.

Moteetee A., Van Wyk B.E. The medical ethnobotany of Lesotho: A review. Bothalia. 2011;41:209–228. doi: 10.4102/abc.v41i1.52. DOI

Deshmukh S., Rathod V. Nutritional evaluation of some wild edible tuberous plants. Asian J. Pharm. Clin. Res. 2013;6:58–60.

Pare D., Hilou O., Ouedraogo N., Guenne S. Ethnobotanical study of medicinal plants used as anti-obesity remedies in the Nomad and Hunter communities of Burkina Faso. Medicines. 2016;3:9. doi: 10.3390/medicines3020009. PubMed DOI PMC

Singh A.K. Probable agricultural biodiversity heritage sites in India: XI. The upper gangetic plains region. Asian Agri-Hist. 2012;16:21–44.

Venu P., Prasad K. The existential crisis in Indian Brachystelma (Apocynaceae) Curr. Sci. 2015;109:680–682.

Muthukrishnan S., Franklin Benjamin J.H., Sathishkannan G., Senthil Kumar T., Rao M.V. In vitro propagation of genus Ceropegia and retrosynthesis of Cerpegin—A review. Int. J. Pharm. Sci. Rev. Res. 2013;22:315–330.

Suttle G.R.L. Commercial Laboratory Production. In: Trigiano R.N., Gray D.J., editors. Plant Development and Biotechnology. CRC Press; Boca Raton, FL, USA: 2005. pp. 311–319.

Krishna H., Singh S.K. Biotechnological advances in mango (Mangifera indica L.) and their future implication in crop improvement—A review. Biotechnol. Adv. 2007;25:223–243. doi: 10.1016/j.biotechadv.2007.01.001. PubMed DOI

Murashige T., Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant. 1962;15:473–497. doi: 10.1111/j.1399-3054.1962.tb08052.x. DOI

Doležal K., Popa I., Krystof V., Spíchal L., Fojtíková M., Holub J., Lenobel R., Schmülling T., Strnad M. Preparation and biological activity of 6-benzylaminopurine derivatives in plants and human cancer cells. Bioorganic Med. Chem. 2006;14:875–884. doi: 10.1016/j.bmc.2005.09.004. PubMed DOI

Cassells A.C. In: Problems in tissue culture: Culture contamination In Micropropagation, Technology and Applications. Debergh P.C., Zimmerman R.H., editors. Kluwer Academic Publishers; Dordrecht, The Netherlands: 1991. pp. 31–44.

Titov S., Bhowmik S.K., Mandal A., Alam M.S., Uddin S.N. Control of phenolic compound secretion and effect of growth regulators for organ formation from Musa spp. cv. Kanthali floral bud explants. Am. J. Biochem. Biotechnol. 2006;2:97–104. doi: 10.3844/ajbbsp.2006.97.104. DOI

Kane M.E. Shoot culture procedures. In: Trigiano R.N., Gray D.J., editors. Plant Development and Biotechnology. CRC Press; Boca Raton, FL, USA: 2005. pp. 149–157.

Caponetti J.D., Gray D.J., Trigiano R.N. History of plant tissue and cell culture. In: Trigiano R.N., editor. Plant Development and Biotechnology. CRC Press; Boca Raton, FL, USA: 2005. pp. 9–15.

Ascough G.D., Novák O., Pencík A., Rolcík J., Strnad M., Erwin J.E., Van Staden J. Hormonal and cell division analyses in Watsonia lepida seedlings. J. Plant Physiol. 2009;166:1497–1507. doi: 10.1016/j.jplph.2009.03.009. PubMed DOI

Zaid A., Al Kaabi H. Plant-off types in tissue culture-derived date palm (Phoenix dactylifera L.) Emir. J. Agric. Sci. 2003;15:17–35.

Amoo S.O., Finnie J.F., Van Staden J. The role of meta-topolins in alleviating micropropagation problems. Plant Growth Regul. 2011;63:197–206. doi: 10.1007/s10725-010-9504-7. DOI

Zhao Y., Grout B.W.W., Crisp P. Variations in morphology and disease susceptibility of micropropagated rhubarb (Rheum rhaponticum) PC49, compared to conventional plants. Plant Cell Tissue Organ Cult. 2005;82:357–361. doi: 10.1007/s11240-005-1836-z. DOI

D’Arth S.M., Simpson S.I., Seelye J.F., Jameson P.E. Bushiness and cytokinin sensitivity in micropropagated Zantedeschia. Plant Cell Tissue Organ Cult. 2002;70:113–118. doi: 10.1023/A:1016034030557. DOI

Ivanova M., Van Staden J. Effect of ammonium ions and cytokinins on hyperhydricity and multiplication rate of in vitro regenerated shoots of Aloe polyphylla. Plant Cell Tissue Organ Cult. 2008;92:227–231. doi: 10.1007/s11240-007-9311-7. DOI

Arinaitwe G., Rubaihayo P.R., Magambo M.J.S. Proliferation rate effects of cytokinins on banana (Musa spp.) cultivars. Sci. Hortic. 2000;86:13–21. doi: 10.1016/S0304-4238(00)00124-2. DOI

Dobránszki J., Hudak I., Magyar-Tábori K., Jámbor-Benczúr E., Galli Z., Kiss E. How can different cytokinins influence the process of shoot regeneration from apple leaves in ‘Royal Gala’ and ‘M.26’. Acta Hortic. 2006;725:191–196.

Bogaert I., Van Cauter S., Werbrouck S.P.O., Doležal K. New aromatic cytokinins can make the difference. Acta Hortic. 2006;725:265–270. doi: 10.17660/ActaHortic.2006.725.33. DOI

Aremu A.O., Bairu M.W., Novák O., Plačková L., Zatloukal M., Doležal K., Finnie J.F., Strnad M., Van Staden J. Physiological responses and endogenous cytokinin profiles of tissue-cultured ‘Williams’ bananas in relation to roscovitine and an inhibitor of cytokinin oxidase/dehydrogenase (INCYDE) treatments. Planta. 2012;236:1775–1790. doi: 10.1007/s00425-012-1721-z. PubMed DOI

Baroja-Fernández E., Aguirreolea J., Martínková H., Hanus J., Strnad M. Aromatic cytokinins in micropropagated potato plants. Plant Physiol. Biochem. 2002;40:217–224. doi: 10.1016/S0981-9428(02)01362-1. DOI

Ivanova M., Novák O., Strnad M., Van Staden J. Endogenous cytokinins in shoots of Aloe polyphylla cultured in vitro in relation to hyperhydricity, exogenous cytokinins and gelling agents. Plant Growth Regul. 2006;50:219–230. doi: 10.1007/s10725-006-9139-x. DOI

Holub J., Hanuš J., Hanke D.E., Strnad M. Biological activity of cytokinins derived from ortho- and meta-hydroxybenzyladenine. Plant Growth Regul. 1998;26:109–115. doi: 10.1023/A:1006192619432. DOI

Valero-Aracama C., Kane M., Wilson S., Philman N. Substitution of benzyladenine with meta-topolin during shoot multiplication increases acclimatization of difficult- and easy-to-acclimatize sea oats (Uniola paniculata L.) genotypes. Plant Growth Regul. 2010;60:43–49. doi: 10.1007/s10725-009-9417-5. DOI

Strosse H., Andre E., Sági L., Swennen R., Panis B. Adventitious shoot formation is not inherent to micropropagation of banana as it is in maize. Plant Cell Tissue Organ Cult. 2008;95:321–332. doi: 10.1007/s11240-008-9446-1. DOI

Gentile A., Jàquez Gutiérrez M., Martinez J., Frattarelli A., Nota P., Caboni E. Effect of meta-topolin on micropropagation and adventitious shoot regeneration in Prunus rootstocks. Plant Cell Tissue Organ Cult. 2014;118:373–381. doi: 10.1007/s11240-014-0489-1. DOI

Werbrouck S.P.O., Strnad M., Van Onckelen H.A., Debergh P.C. Meta-topolin, an alternative to benzyladenine in tissue culture? Plant Physiol. 1996;98:291–297. doi: 10.1034/j.1399-3054.1996.980210.x. DOI

Aremu A.O., Bairu M.W., Szüčová L., Doležal K., Finnie J.F., Van Staden J. Shoot and root proliferation in ‘Williams’ banana: Are the topolins better cytokinins? Plant Cell Tissue Organ Cult. 2012;111:209–218. doi: 10.1007/s11240-012-0187-9. DOI

Wojtania A., Gabryszewska E. Effect of cytokinins and amino acids on multiplication of Pelargonium cultivars. Acta Soc. Bot. Pol. 2001;70:203–207. doi: 10.5586/asbp.2001.026. DOI

Vinayak V., Dhawan A.K., Gupta V.K. Efficacy of non-purine and purine cytokinins on shoot regeneration in vitro in sugarcane. Indian J. Biotechnol. 2009;8:227–231.

Vasudevan R., Van Staden J. Cytokinin and explant types influence in vitro plant regeneration of Leopard Orchid (Ansellia africana Lindl.) Plant Cell Tissue Organ Cult. 2011;107:123–129. doi: 10.1007/s11240-011-9964-0. DOI

Bairu M.W., Stirk W.A., Doležal K., Van Staden J. 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 Organ Cult. 2007;90:15–23. doi: 10.1007/s11240-007-9233-4. DOI

Bairu M.W., Stirk W.A., Doležal K., Van Staden J. The role of topolins in micropropagation and somaclonal variation of banana cultivars ‘Williams’ and ‘Grand Naine’ (Musa spp. AAA) Plant Cell Tissue Organ Cult. 2008;95:373–379. doi: 10.1007/s11240-008-9451-4. DOI

Rosales M.S.D., Solís Á.G.A., Méndezy N.L.V., Balch E.P.M. Effect of cytokinins on the in vitro propagation of Mexican Agaves. Rev. Fitotec. Mex. 2008;31:317–322.

Niedz R.P., Evens T.J. The effefects of benzyladenine and meta-topolin on in vitro shoot regeneration of Citrus citrandarin rootstock. Res. J. Agric. Biol. Sci. 2010;6:45–53.

Salvi N.D., George L., Eapen S. Micropropagation and field evaluation of micropropagated plants of turmeric. Plant Cell Tissue Organ Cult. 2002;68:143–151. doi: 10.1023/A:1013889119887. DOI

De Diego N., Montalbán I.A., Moncaleán P. In vitro regeneration of Pinus spp. adult trees: New method for obtaining clonal plants. Acta Hortic. 2010;865:361–366. doi: 10.17660/ActaHortic.2010.865.52. DOI

Moyo M., Finnie J.F., Van Staden J. Topolins in Pelargonium sidoides micropropagation: Do the new brooms really sweep cleaner? Plant Cell Tissue Organ Cult. 2012;110:319–327. doi: 10.1007/s11240-012-0153-6. DOI

Bairu M.W., Novák O., Doležal K., Van Staden J. Changes in endogenous cytokinin profiles in micropropagated Harpagophytum procumbens in relation to shoot-tip necrosis and cytokinin treatments. Plant Growth Regul. 2011;63:105–114. doi: 10.1007/s10725-010-9558-6. DOI

Werbrouck S.P.O., Van der Jeugt B., Dewitte W., Prinsen E., Van Onckelen H.A., Debergh P.C. The metabolism of benzyladenine in Spathiphyllum floribundum schott ‘petite’in relation to acclimatization problems. Plant Cell Rep. 1995;14:662–665. doi: 10.1007/BF00232734. PubMed DOI

Xiao Y., Niu G., Kozai T. Development and application of photoautotrophic micropropagation plant system. Plant Cell Tissue Organ Cult. 2011;105:149–158. doi: 10.1007/s11240-010-9863-9. DOI

Debergh P.C., Read P.E. Micropropagation. In: Debergh P.C., Zimmerman R.H., editors. Micropropagation, Technology and Applications. Kluwer Academic Publishers; Dordrecht, The Netherlands: 1991. pp. 1–13.

Madhulatha P., Anbalagan M., Jayachandran S., Sakthivel N. Influence of liquid pulse treatment with growth regulators on in vitro propagation of banana (Musa spp. AAA) Plant Cell Tissue Organ Cult. 2004;76:189–192. doi: 10.1023/B:TICU.0000007291.31439.6c. DOI

Phulwaria M., Shekhawat N.S., Rathore J.S., Singh R.P. An efficient in vitro regeneration and ex vitro rooting of Ceropegia bulbosa Roxb.—A threatened and pharmaceutical important plant of Indian Thar Desert. Ind. Crop. Prod. 2013;42:25–29. doi: 10.1016/j.indcrop.2012.05.013. DOI

Chandra S., Bandopadhyay R., Kumar V., Chandra R. Acclimatization of tissue cultured plantlets: From laboratory to land. Biotechnol. Lett. 2010;32:1199–1205. doi: 10.1007/s10529-010-0290-0. PubMed DOI

Yang S.H., Yeh D.M. In vitro leaf anatomy, ex vitro photosynthetic behaviors and growth of Calathea orbifolia (Linden) Kennedy plants obtained from semi-solid medium and temporary immersion systems. Plant Cell Tissue Organ Cult. 2008;93:201–207. doi: 10.1007/s11240-008-9363-3. DOI

Micropropagation as a Tool for the Conservation of Autochthonous Sorbus Species of Czechia