Somatic embryogenesis and in vitro shoot propagation of Gentianautriculosa

Branka Vinterhalter 1 , Nevena Mitić 1 , Dragan Vinterhalter 1 , Branka Uzelac 1  and Dijana Krstić-Milošević 1
  • 1 University of Belgrade, Institute for Biological Research “Siniša Stanković”, Despot Stefan Boulevard 142, 11060 Belgrade, Serbia
Branka Vinterhalter, Nevena Mitić, Dragan Vinterhalter, Branka Uzelac and Dijana Krstić-Milošević

Abstract

Study describes protocols for in vitro propagation of Gentiana utriculosa L. via axillary shoot multiplication and indirect somatic embryogenesis. Shoot cultures were established from seedling epicotyl explants cultured on MS medium supplemented with 0.25 mg L−1 BA and 0.1 mg L−1 IAA. Medium containing 2% sucrose and 0.2 mg L−1 BA improved multiple shoot production, providing 2.3 shoots per explant. The highest rooting (29.6%) was obtained on medium with 1/2 MS mineral salts and 0.5 mg L−1 NAA. Somatic embryogenesis was induced using different explants, including immature seeds as well as leaves and roots from shoot cultures. Following auxin treatment with either 1.0 mg L−1 2,4-D (immature seeds and leaves) or 0.1 mg L−1 NAA (roots), explants produced embryogenic calli which upon transfer to plant growth regulator-free medium allowed embryo conversion into plantlets. The best embryogenic response (82%) was obtained in calli derived from leaves cultured with their abaxial surface in contact with medium, whereas the highest embryo conversion rate (68%) was recorded for calli induced on immature seed explants. Histological analysis in all explant types revealed development of proembryogenic cell complexes at callus periphery, giving rise to somatic embryos. The presence of embryos at various stages of development indicated asynchronous somatic embryogenesis in G. utriculosa. Derooted embryo-derived plantlets placed on medium with 0.2 mg L−1 BA multiplied further as shoot cultures.

  • Bach A. & Pawlowska B. 2003. Somatic embryogenesis in Gentiana pneumonanthe L. Acta Biol. Cracov. 45: 75–86.

  • Butuic-Keul A., S¸uteu A. & Deliu C. 2005. In vitro organogenesis of Gentiana punctata.Not.Bot.Hort. Agrobot. 33: 38–41.

  • Cai Y., Liu Y., Liu Z., Zhang F., Xiang F. & Xia G. 2009. High-frequency embryogenesis and regeneration of plants with high content of gentiopicroside from the Chinese medicinal plant Gentiana straminea Maxim. In Vitro Cell Dev. Biol. Plant 45: 730–739.

  • Casanova E., Moysset L. & Trillas M.I. 2008. Effects of agar concentration and vessel closure on the organogenesis and hyperhydricity of adventitious carnation shoots. Biol. Plant. 52: 1–8

  • Chen J.-Y., Chen Q.-L., Xu D., Hao J.-G., Schläppi M. & Xu Z.-Q. 2009. Changes of gentiopicroside sintesis during somatic embryogenesis of Gentiana macrophylla. Planta Med. 75: 1618–1624.

  • Dević M., Momčilović I., Krstić D., Maksimović V. & Konjević R. 2006. In vitro multiplication of willow gentian (Gentiana asclepiadea L.) and the production of gentiopicrin and mangiferin. Phyton 46: 45–54.

  • Dhawan O.P. & Lavania U.C. 1996. Enhancing the productivity of secondary metabolites via induced polyploidy: a review. Euphytica 87: 81– 89.

  • Drobyk N.M., Lyudmyla R.H., Mel’nyk V.M., Kravets N.B., Konvalyuk I.I., Twardovska M.O. & Kunakh V.A. 2015. In vitro manipulation and propagation of Gentiana L. species from the Ukrainian Flora, pp 45–79. In: Rybczyński J.J., Davey M.R. & Mikuła A. (eds), The Gentianaceae – Volume 2: Biotechnology and Applications, Springer-Verlag, Berlin, Heidelberg.

  • Fiuk A. & Rybczyński J.J. 2007. The effect of several factors on somatic embryogenesis and plant regeneration in protoplast cultures of Gentiana kurroo (Royle). Plant Cell Tissue Org. Cult. 91: 263–271.

  • Fiuk A. & Rybczyński J.J. 2008a. Morphogenic capability of Gentiana kurroo Royle seedling and leaf explants. Acta Physiol. Plant. 30: 157–166.

  • Fiuk A. & Rybczyński J.J. 2008b. Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants. In Vitro Cell Dev. Biol. Plant 44: 90–99.

  • Gamborg O.L., Miller R.A. & Ojima K. 1968. Nutrient requirements of suspension cultures of soybean root cells. Exp. Cell Res. 50: 151–158.

  • Hayta S., Akgun I.H., Ganzera M., Bedir E. & Gurel A. 2011. Shoot proliferation and HPLC-determination of iridoid glycosides in clones of Gentiana cruciata L. Plant Cell Tissue Org. Cult. 107: 175–180.

  • He T., Yang L. & Zhao Z. 2011. Embryogenesis of Gentiana straminea and assessment of genetic stability of regenerated plants using inter simple sequence repeat (ISSR) marker. Afr. J. Biotechnol. 10: 7604–7610.

  • Holobiuc I. & Catana R. 2012. Recurrent somatic embryogenesis in long-term cultures of Gentiana lutea L. as a source for synthetic seed production for medium-term preservation. Arch. Biol. Sci. 64: 809–817.

  • Hosokawa K., Nakano M., Oikawa Y. & Yamamura S. 1996. Adventitious shoot regeneration from leaf, stem and root explants of commercial cultivars of Gentiana. Plant Cell Rep. 15: 578–581.

  • Hosokawa K., Oikawa Z. & Zamamura S. 1998. Mass propagation of ornamental gentian in liquid medium. Plant Cell Rep. 17: 747–751.

  • Hostettmann-Kaldas M., Hostettmann K. & Sticher O. 1981. Xanthones, flavones and secoiridoids of american Gentiana species. Phytochem. 20: 443–446.

  • Hostettmann K. & Jackot–Guillarmod A. 1977. Xanthones et C-glucosides flavoniques du genre Gentiana (section Cyclostigma). Phytochem. 16: 481–482.

  • Ivanova M. & Van Staden J. 2011. Influence of gelling agent and cytokinins on the control of hyperhydricity in Aloe polyphylla. PlantCellTissue Org.Cult. 104: 13–21.

  • Janković T., Krstić-Milošević D., Aljančić I., Šavikin K., Menković N., Radanović D. & Milosavljević S. 2009. Phytochemical re-investigation of Gentiana utriculosa.Nat. Prod. Res. 23: 466–469.

  • Köhlein F. 1991. Gentians. In: Jermyn J. (ed.), Tiber Press, Portland, Oregon, USA.

  • Landrein B., Lathe R., Bringmann M., Vouillot C., Ivakov A., Boudaoud A., Persson S. & Hammant O. 2013. Impaired cellulose synthase guidance leads to stem torsion and twists phyllotactic patterns in Arabidopsis. Curr. Biol. 23: 895–900.

  • Linsmaier E.M. & Skoog F. 1965. Organic growth factor requirement of tobacco tissue cultures. Physiol. Plant. 18: 100–128.

  • Lloyd G. & McCown B. 1980. Commercially-feasible micropropagation of mountain laurel-Kalmia latifolia by use of shoot-tip culture. Proc. Int. Plant. Prop. Soc. 30: 421–427.

  • Mikuła A. & Rybczyński J.J. 2001. Somatic embryogenesis of Gentiana genus I. The effect of preculture treatment and primary explant origin on somatic embryogenesis of Gentiana cruciata (L.), G. panonica (Scop.), and G. tibetica (King). Acta Physiol. Plant. 23: 15–25.

  • Mikuła A., Skierski J. & Rybczyński J.J. 2002. Somatic embryogenesis of Gentiana genus III. Characterization of three-yearold embryogenic suspensions of G. panonica originated from various seedling explants. Acta Physiol. Plant. 24: 311–322.

  • Mikuła A., Tykarska T., Zielińska M., Kura´s M. & Rybczyński J.J. 2004. Ultrastructural changes in zygotic embryos of Gentiana punctata L. during callus formation and somatic embryogenesis. Acta Biol. Cracov. Bot. 46: 109–120.

  • Mikuła A., Olas M., Sliwinska E. & Rybczyński J.J. 2008. Cryopreservation by encapsulation of Gentiana spp. cell suspensions maintains regrowth, embryogenic competence and DNA content. Cryo. Letters 29: 409–418.

  • Momčilović I., Grubišić D. & Nešković M. 1997. Micropropagation of four Gentiana species (G.lutea, G. cruciata, G. purpurea, G. acaulis). Plant Cell Tissue Org.Cult. 49: 141–144.

  • Morgan E.R., Butler R.M. & Bicknell R.A. 1997. In vitro propagation of Gentiana cerina and Gentiana corymbifera. New Zeal. J. Crop. Hort. 25: 1–8.

  • Mujib A. & Samaj J. 2006. Somatic embryogenesis. Springer-Verlag, Berlin, Heidelberg, New York.

  • Murashige T. & Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.

  • Nalawade S.M., Sagare A.P., Lee C.-Y., Kao C.-L. & Tsay H. S. 2003. Studies on tissue culture of Chinese medicinal plant resources in Taiwan and their sustainable utilization. Bot. Bull. Acad. Sin. 44: 79–98.

  • Nassour M., Chassériaux G. & Dorion N. 2003. Optimization of protoplastto-plant system for Pelargonium × hortorum ‘Alain’ and genetic stability of the regenerated plants. Plant Sci. 165: 121–128.

  • Pawlowska B. & Bach A. 2003. In vitro propagation of protected species Gentiana pneumonanthe L. for ornamental horticultural uses. Folia Horticult. 15: 113–122.

  • Petrova M., Stoilova T. & Zagorska N. 2006. Izoenzyme and protein patterns of in vitro micropropagated plantlets of Gentiana lutea L. after application of various growth regulators. Biotechnol. Biotechnol. Equip. 20: 15–19.

  • Rybczyński J.J., Davey M.R., Tomiczak K., Niedziela A. & MikuRybczyński J.J., Davey M.R., Tomiczak K., Niedziela A. & Mikula A. 2015. Systems of plant regeneration in Gentian in vitro cultures, pp. 1–44. In: Rybczyński J.J., Davey M.R. & Mikuła A. 2015. Systems of plant regeneration in Gentian in vitro cultures, pp. 1–44. In: Rybczyński J.J., Davey M.R. & Mikuła A. (eds), The Gentianaceae – Volume 2: Biotechnology and Applications, Springer-Verlag, Berlin, Heidelberg.

  • Sagare A.P., Suhasini K. & Krishnamurthy K.V. 1995. Histology of somatic embryo initiation and development in chickpea (Cicer arietinum L.). Plant Sci. 109: 87–93.

  • Sachs J. 2011. A Text-Book of Botany: Morphological and Physiological. In: Bennett A.W. (ed.), Cambridge University Press, New York

  • Sharma N., Chandel K.P.S. & Paul A. 1993. In vitro propagation of Gentiana kurroo – an indigenous plant of medicinal importance. Plant Cell Tissue Org. Cult. 34: 307–309.

  • Sheng X., Zhao Z., Yu H., Wang J., Xiaohui Z. & Gu H. 2011. Protoplast isolation and plant regeneration of different doubled haploid lines of cauliflower (Brassica oleracea var. botrytis). Plant Cell Tissue Org.Cult. 107: 513–520.

  • Skrzypczak L., Wesolowska M. & Skrzypczak E. 1993. Gentiana species: in vitro culture, regeneration and production of secoiridoid glucosides, pp. 172–186. In: Bajaj Y.P.S. (ed.), Biotechnology in Agriculture and Forestry.

  • Skrzypczak-Pietraszek E., Skrzypczak L. & Wesolowska M. 1993. Gentiana tibetica King from tissue culture. Sci. Pharm. 61: 287–296.

  • Tomiczak K., Mikuła A., Sliwinska E. & Rybczyński J.J. 2015. Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. In Vitro Cell Dev. Biol. Plant 51: 350– 359.

  • Tutin T.G. 1972. Flora Europea. Cambridge University Press, London.

  • Vinterhalter B. & Vinterhalter D. 1998. In vitro propagation of spotted gentian Gentiana punctata L. Arch. Biol. Sci. 50: 177–182.

  • Vinterhalter B., Krstić-Milošević D., Janković T., MilojevićJ. & Vinterhalter D. 2012. In vitro propagation of Gentiana dinarica Beck. Cent. Eur. J. Biol. 7: 690–697.

  • Viola U. & Franz C. 1989. In vitro propagation of Gentiana lutea. Planta Med. 55: 690.

  • Wesolowska M., Skrzypczak L. & Dudzińska R. 1985. Rodzaj Gentiana L. w kulturze in vitro. Acta. Polon. Pharm. 42: 79–83.

  • Yamada Y., Shoyama Y., Nishioka I., Kohda H., Namera A. & Okamoto T. 1991. Clonal micropropagation of Gentiana scabra Bunge var. buergeri Maxim. and examination of the homogenity concerning the gentiopicroside content. Chem. Pharmaceut. Bull. 39: 204–206.

  • Zhang Z. & Leung D.W.M. 2002. Factors influencing the growth of micropropagated shoots and in vitro flowering of gentian. Plant Growth Regul. 36: 245–251.

Purchase article
Get instant unlimited access to the article.
Log in
Already have access? Please log in.


or
Log in with your institution

Journal + Issues

Search