In the present investigation, optimization of the best concentration combinations of BAP (Benzyl Amino Purine), TDZ (Thidiazuron) and Silver Nitrate (AgNO3) for micropropagation of carnation (Dianthus caryophyllus L) cv. Irene was performed. Genetic fidelity in the microplants of different generation was determined with the help of ISSR markers. Results of the present investigation showed that the MS gelled media supplemented with BAP (0.5 mgl–1) and AgNO3 (1.5 mgl–1) showed maximum shoot proliferation, good shoot length and reduced hyperhydricity significantly. Our study also indicates that hyperhydricity decreased by substantially by increasing the concentration of AgNO3 compared to control. High level of AgNO3 in MS medium did not helped to reduce hyperhydricity. Another chemical combination of TDZ (1.0 mgl–1) and AgNO3 (0.5 mgl–1) in MS gelled media gave the maximum shoot proliferation, shoot length with minimum hyperhydricity which showed normal growth and development compared to control and other treatments. This concentration combination proved effective to reduce hyperhydricity and to enhance shoot proliferation. Because TDZ with AgNO3 had pronounced effect to reduce hyperhydricity, therefore, this concentration combination was used to advance the progeny up to subculture (SC10) using donor mother microplants (SC1) selected from the best concentration response to know the genetic fidelity among the different generations. Microplants which were non hyperhydrified were used to assess the genetic fidelity using ISSR markers. Fourteen ISSR primers were used that gave good amplification and produced 616 bands with an average of 44 amplicon per primer from in vitro raised microplants including mother plant where the banding patterns exhibited uniform and homogeneous banding patterns. This study may help us to know applicability of ISSR markers to establish genetic fidelity among in vitro – raised microplants of carnation.
Keywords: Carnation, Clonal fidelity, Hyperhydricity, ISSR markers, Micropropagation, Plant Growth Regulators, Silver Nitrate, TDZ, True to type microplants
Full text and supplemented materials
References
Arif, M., Rauf, S., Din, A.U., Rauf, M., and Afrasiab, H., High frequency plant regeneration from leaf derived callus of Dianthus caryophyllus L., Am. J. Plant Sci., 2014, vol. 5, pp. 2454–2463.
Bhatia, R., Singh, K.P., Sharma, T.R., and Jhang, T., Evaluation of the genetic fidelity of in vitro propagated gerbera (Gerbera jamesonii Bolus) using DNA-based markers, Plant Cell, Tissue Organ Cult., 2011, vol. 104, pp. 131–135.
Chae, S.C. and Park, S.U., Improved shoot organogenesis of Echinacea angustifolia DC treated with ethylene inhibitors, Life Sci. J., 2012, vol. 9, pp. 1725–1728.
Cruz de Carvalho, M.H.B., Van Le, Y., Zuily-Fodil, Y., et al., Efficient whole plant regeneration of common bean (Phaseolus vulgaris L.) using thin-cell-layer culture and silver nitrate, Plant Sci., 2000, vol. 159, pp. 223–232.
Donelly, V.A. and Vidaver, W., Leaf anatomy of red raspberry transferred from culture to soil, J. Am. Soc. Hort. Sci., 1984, vol. 109, pp. 172–176.
Doyle, J.J. and Doyle, J.L., Isolation of plant DNA from fresh tissue, Focus, 1990, vol. 12, pp. 13–15.
Esmaiel, N.N., Al-Doss, A.A., and Barakat, M.N., An assessment of in vitro culture and plant regeneration from leaf base explants in carnation (Dianthus caryophyllus L.), J. Food Agric. Environ., 2013, vol. 1111, pp. 1113–1117.
Gao, H., Xia, X., An, L., Xin, X., and Liang, Y., Reversion of hyperhydricity in pink (Dianthus chinensis L.) plantlets by AgNO3 and its associated mechanism during in vitro culture, Plant Sci., 2017, vol. 254, pp. 1–11.
Gill, A.P.S. and Arora, J.S., Performance of Sim carnations under subtropical climatic conditions of Punjab, Indian J. Hortic., 1988, vol. 45, pp. 329–335.
Goto, S., Thakur, R.C., and Ishii, K., Determination of genetic stability in long-term micropropagated shoots of Pinus thunbergii Parl. using RAPD markers, Plant Cell Rep., 1998, vol. 18, nos. 3–4, pp. 193–197.
Joshi-Saha, A., Valon, C., and Leung, J., A brand new start: Abscisic acid perception and transduction in the guard cell, Sci. Signal., 2011, vol. 4, pp. 645–652.
Kantia, A. and Kothari, S.L., High efficiency adventitious shoot bud formation and plant regeneration from leaf explants of Dianthus chinensis L., Sci. Hortic., 2002, vol. 96, pp. 205–212.
Kaviani, B., Hesar, A.A., and Kharabian-Masouley, A., In vitro propagation of Matthiola incana (Brassicaceae)-an ornamental plant, Plant Omics J., 2011, vol. 4, no. 7, pp. 435–440.
Kevers, C., Franck, T., Strasser, R.J.D., Ommes, J., and Gasper, T., Hyperhydricity of micropropagated shoots: a typically stress-induced change of physiological state, Plant Cell, Tissue Organ Cult., 2004, vol. 77, pp. 181–191.
Lakshmanan, P., Lee, C., and Goh, C., An efficient in vitro method for mass propagation of a woody ornamental Ixora coccinea L., Plant Cell Rep., 1997, vol. 16, pp. 572–577.
Mayor, M., Nestares, G., Zorzoli, R., and Picardi, L., Reduction of hyperhydricity in sunflower tissue culture, Plant Cell, Tissue Organ Cult., 2003, vol. 72, pp. 99–103.
Mujib, A. and Pal, A.K., Inter varietal variation in response to in vitro cloning of carnation, Crop Res. Hisar, 1995, vol. 10, pp. 90–194.
Murashige, T. and Skoog, F., A revised medium for rapid growth and bioassays with tobacco tissue culture, Physiol. Plant, 1962, vol. 15, pp. 473–497.
Nalousi, A.M., Hatamzadeha, A., Azadi, P., et al., A procedure for indirect shoot organogenesis of Polianthes tuberosa L. and analysis of genetic stability using ISSR markers in regenerated plants, Sci. Hortic., 2019, vol. 244, pp. 315–321.
Onamu, R., Obukosia, S.D., Musembi, N., and Hutchinson, M.J., Efficacy of Thidiazuron in In vitro propagation of carnation shoot tips: Influence of dose and duration of exposure, Afr. Crop Sci. J., 2003, vol. 11, no. 2, pp. 125–132.
Patil, V.M., Dhande, G.A., Thigale, D.M., and Rajput, J.C., Micropropagation of pomegranate (Punica granatum L.) ‘Bhagava’ cultivar from nodal explant, Afr. J. Biotech., 2011, vol. 10, pp. 18130–18136.
Qin, Y. and Zhang, S., Response of in vitro strawberry to silver nitrate (AgNO3), Hortic. Sci., 2005, vol. 40, no. 3, pp. 747–751.
Reddy, M.P., Sarla, N., and Siddiq, E.A., Inter simple sequence repeats (ISSR) polymorphism and its application in plant breeding, Euphytica, 2002, vol. 128, pp. 9–17.
Rizwan, H.M., Irshad, M., He, B.Z., et al., Silver nitrate (AgNO3) boosted high-frequency multiple shoot regeneration from cotyledonary node explants of okra (Abelmoschus esculentus L.), Appl. Ecol. Environ. Res., 2018, vol. 16, no. 3, pp. 3421–3435.
Rohela, G.K., Jogam, P., Prasad, B., and Christopher, R., 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. Res. Int., 2019, p. 3698742. https://doi.org/10.1155/2019/3698742
Shi, C., Qi, C., Ren, H., et al., Ethylene mediates brassinosteroid-induced stomatal closure via Gα protein-activated hydrogen peroxide and nitric oxide production in Arabidopsis, Plant J., 2015, vol. 82, pp. 280–301.
Shimizu-Sato, S., Tanaka, M., and Mori, H., Auxin-cytokinin interactions in the control of shoot branching, Plant Mol. Biol., 2009, vol. 69, no. 4, p. 429.
Staden, V.J., Zažímalová, E., and George, E.F., Plant growth regulators II: Cytokinins, their analogues and antagonists, in Plant Propagation by Tissue Culture, George, E.F., Hall, M., and De Kleck, G.J., Eds., Dordrecht: Springer-Verlag, 2008, vol. 1, pp. 205–226.
Van den Dries, N., Giannì, S., Czerednik, A., et al., Flooding of the apoplast is a key factor in the development of hyperhydricity, J. Exp. Bot., 2013, vol. 64, pp. 5221–5230
Vinoth, A. and Ravindhran, R., Reduced hyperhydricity in watermelon shoot cultures using silver ions, In Vitro Cell. Dev. Biol. Plant, 2015. https://doi.org/10.1007/s11627-015-9698-5
Yadav, M.K., Gaur, A.K., and Garg, G.K., Development of suitable protocol to overcome hyperhydricity in carnation during micropropagation, Plant Cell, Tissue Organ Cult., 2003, vol. 72, pp. 153–156.