Production of compact petunias through polyploidization

  • J. J. Regalado
  • E. Carmona-Martín
  • V. Querol
  • C. G. Veléz
  • C. L. Encina
  • S. I. Pitta-AlvarezEmail author
Original Article


Petunia, a commercially important ornamental plant worldwide, has been subjected to breeding programs that have yielded a high number of varieties. One of the key factors in the commercial value of these varieties is plant compactness. Currently, compact petunias are obtained through the application of expensive, harmful and short-lasting chemicals. To avoid the use of these chemicals, transgenic plants that over-express dwarf-inducing genes have been recently proposed as an alternative, but the current legislation regarding transgenic plants restricts their commercialization. In this work, we studied the effect of polyploidization in the plant architecture of Petunia axillaris, an Argentine native petunia. We developed a new polyploidization protocol that consisted in culturing petunia leaves in RL medium (MS medium supplemented with 30 g l−1 sucrose, 1 mg l−1 BA and 0.2 mg l−1 IAA) supplemented with 0.2 g l−1 colchicine for 15 days. This protocol allowed the regeneration of stable autotetraploid petunias (polyploidization rate: 29.0 ± 8,2%), which were 54% more compact than the diploid ones. Furthermore, they exhibited no variations in agronomical traits compared to the initial genotypes, except for a short delay in blooming. These autotetraploid plants can be used in different breeding programs and the polyploidization method developed can be tested in others cultivars of the genus Petunia for the same purpose.


Colchicine Petunia axillaris Autotetraploids Plant architecture Ornamentals Organogenesis Plant compactness, dwarfing 



This work was supported by funding from CONICET (Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina) Grant PIP 2011–2013 0295. Doctor Regalado Gonzalez is a Post-Doctoral Fellow at CONICET.


  1. Andersen HR, Vinggaard AM, Rasmussen TH, Gjermandsen IM, Bonefeld-Jorgensen EC (2002) Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro. Toxicol Appl Pharmacol 179:1–12CrossRefPubMedGoogle Scholar
  2. Beck MJ, Camper ND (1991) Shoot regeneration from petunia leaf discs as a function of explant size, configuration and benzyladenine exposure. Plant Cell Tissue Organ Cult 26:101–106CrossRefGoogle Scholar
  3. Bhattacharya A, Kourmpetli S, Davey MR (2010) Practical applications of manipulating plant architecture by regulating gibberellin metabolism. J Plant Growth Regul 29:249–256CrossRefGoogle Scholar
  4. Broertjes C, Keen A (1980) Adventitious shoots: do they develop from one cell? Euphytica 29:73–87CrossRefGoogle Scholar
  5. Cai X, Cao Z, Xu S, Deng Z (2015) Induction, regeneration and characterization of tetraploids and variants in “Tapestry” caladium. Plant Cell Tissue Organ Cult 120:689–700CrossRefGoogle Scholar
  6. Carmona-Martin E, Regalado JJ, Raghavan R, Encina CL (2015) In vitro induction of autooctoploid asparagus genotypes. Plant Cell Tissue Organ Cult 121:249–254CrossRefGoogle Scholar
  7. Chen Y, Jiang P, Wilde HD (2014) A self-pollinating mutant of Petunia hybrid. Scientia Hortic 177: 10–13CrossRefGoogle Scholar
  8. Detrez C, Sangwan RS, Sangwan-Norreel BS (1989) Phenotypic and karyotypic status of Beta vulgaris plants regenerated from direct organogenesis in petiole culture. Theor Appl Genet 77:462–468CrossRefPubMedGoogle Scholar
  9. Doleẑel J, Lucretti S, Schubert I (1994) Plant chromosome analysis and sorting by flow cytometry. Crit Rev Plant Sci 13:275–309CrossRefGoogle Scholar
  10. Escandón AS, Alderete LM, Hagiwara JC (2007) In vitro polyploidization of Mercadonia tenella, a native plant from South America. Scientia Hortic 115:56–61CrossRefGoogle Scholar
  11. Fernández R, Morisigue D, Facciuto G (2008) El INTA y la floricultura argentina. Actas de Horticultura 52. Innovación y futuro en la jardinería. I Simposio Iberoamericano- IV Jornadas Ibéricas de Horticultura Ornamental. Pontevedra (España).Google Scholar
  12. Francescangeli N, Zagabria A (2008) Paclobutrazol for height control of petunias. Chil J Agric Res 68:309–314CrossRefGoogle Scholar
  13. Gallone A, Hunter A, Douglas GC (2014) Polyploid induction in vitro using colchicine and oryzalin on Hebe “Oratia Beauty”: Production and characterization of the vegetative traits. Scientia Hortic 179:59–66CrossRefGoogle Scholar
  14. Gantait S, Mandal N, Bhattacharyya S, Kanti Das P (2011) Induction and identification of tetraploids using in vitro colchicine of Gerbera jamesonii Bolus cv. Sciella. Plant Cell Tiss Organ Cult 106:485–493CrossRefGoogle Scholar
  15. Gargul JM, Mibus H, Serek M (2013) Constitutive overexpression of Nicotiana GA2ox leads to compact phenotypes and delayed flowering in Kalanchoë blossfeldiana and Petunia hybrida. Plant Cell Tiss Organ Cult 115:407–418CrossRefGoogle Scholar
  16. González-Roca L, Iannicelli J, Coviella A, Bugallo V, Bologna P, Pitta-Álvarez S, Escandón A (2015) A protocol for the in vitro propagation and polyploidization of an interspecific hybrid of Glandularia (G. peruviana x G. scrobiculata). Scientia Hortic 184:46–54CrossRefGoogle Scholar
  17. Gudesblat GE, Iusem ND, Morris PC (2007) Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide. N Phytol 173:713–721CrossRefGoogle Scholar
  18. Hannweg K, Sippel A, Bertling I (2013) A simple and effective method for the micropropagation and in vitro induction of polyploid and the effect on floral characteristics of the South African iris, Crocosmia aurea. S Afr J Bot 88: 367–372CrossRefGoogle Scholar
  19. Hoshino Y, Eiraku N, Ohata Y, Komaj F (2016) Dynamics of nuclear phase changes during pollen tube growth by using in vitro culture in Petunia. Scientia Hortic 210: 143–149CrossRefGoogle Scholar
  20. Huang R, Liu D, Zhao M, Li Z, Li M, Sui S (2015) Artificially induced polyploidization in Lobularia maritima (L.) Desv. and its effect on morphological traits. HortScience 50:636–639Google Scholar
  21. Kelly RO, Deng Z, Harbaugh BK (2007) Evaluation of petunia cultivars as bedding plants for Florida. ENH 1078. Series of the Environmental Horticulture Department, Florida Cooperative Extension Service, Institute of Food and Agricultural Sciences, University of Florida GainesvilleGoogle Scholar
  22. Khan RH, Thirukkumaran G, Nakamura I, Mii M (2010) Rol (root loci) gene as a positive selection marker to produce marker-free Petunia hybrida. Plant Cell Tissue Organ Cult 101:279–285CrossRefGoogle Scholar
  23. Lee HJ, Kim YE, Yoon YJ, Jeong CS, Lian ML, Paek KY, Park SY (2016) Highly endoreduplicated floral organs of somaclonal variants in clonally propagated Phalaenopsis ‘Spring Dancer’. Plant Cell Tissue Organ Cult 126:67–77CrossRefGoogle Scholar
  24. Mata DA, Botto JF (2009) Manipulation of light environment to produce high-quality poinsettia plants. HortScience 44:702–706Google Scholar
  25. Meyer L, Serek M, Winkelmann T (2009) Protoplast isolation and plant regeneration of different genotypes of Petunia and Calibrachoa. Plant Cell Tissue Organ Cult 99:27–34CrossRefGoogle Scholar
  26. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  27. Ning GG, Shi XP, Hu HR, Yan Y, Bao MZ (2009) Development of a range of polyploid lines in Petunia hybrid and the relationship of ploidy with the single-/double-flowe trait. HortScience 44:250–255Google Scholar
  28. Ochatt SJ (2006) Flow cytometry: (ploidy determination, cell cycle analysis, DNA content per nucleus). In: Medicago truncatula handbook. Accessed Nov 2006
  29. Ochatt SJ (2008) Flow cytometry in plant breeding. Cytom A 73:581–598CrossRefGoogle Scholar
  30. Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tissue Organ Cult 104:329–341CrossRefGoogle Scholar
  31. Oh MH, Choi DW, Kwon YM, Kim SG (1995) An assessment of cytological stability in protoplast cultures of tetraploid Petunia hybrida. Plant Cell Tissue Organ Cult 41:243–248CrossRefGoogle Scholar
  32. Rademacher R (2000) Growth retardants: effects on gibberellin biosynthesis and other metabolic pathways. Annu Rev Plant Physiol 51:501–531CrossRefGoogle Scholar
  33. Radi A (2005) Over-expression of pumpkin GA-oxidases in Arabidopsis thaliana. PhD thesis, University of Braunschweig, GermanyGoogle Scholar
  34. Regalado JJ, Carmona-Martín E, Castro P, Moreno R, Gil J, Encina CL (2015) Study of the somaclonal variation produced by different methods of polyploidization in Asparagus officinalis L. Plant Cell Tissue Organ Cult 122:31–44CrossRefGoogle Scholar
  35. Reuveni M, Evenor D (2007) On the effect of light on shoot regeneration in petunia. Plant Cell Tissue Organ Cult 89:49–54CrossRefGoogle Scholar
  36. Schnelle RA, Barret JE (2010) Paclobutrazol concentration and substrate moisture status impact efficacy of liner dips for size control of three bedding plants. HortTechnology 20:735–739Google Scholar
  37. Tomiczak K, Mikuła A, Sliwinska A, Rybczynski JJ (2015) Autotetraploid plant regeneration by indirect somatic embryogenesis from leaf mesophyll protoplasts of diploid Gentiana decumbens L.f. In Vitro Cell Dev Biol Plant 51:350–359CrossRefPubMedPubMedCentralGoogle Scholar
  38. Tomiczak K, Sliwinska E, Rybczyński JJ (2016) Comparison of the morphogenic potential of five Gentiana species in leaf mesophyll protoplast culture and ploidy stability of regenerated calli and plants. Plant Cell Tissue Organ Cult 126: 319–331CrossRefGoogle Scholar
  39. United States Department of Agriculture (USDA)-National Agricultural Statistics Service (NASS) (2016) Floriculture Crops 2015 Summary. Accessed 21 Jul 2016
  40. van Iersel M, Nemali K (2004) Drought stress can produce small but not compact marigolds. HortScience 39:1298–1301Google Scholar
  41. van Duren M, Morpurgo R, Doleẑel J, Afza R (1996) Induction and verification of autotetraploids and diploid banana (Musa acuminata) by in vitro techniques. Euphytica 88:25–34CrossRefGoogle Scholar
  42. Walworth AE, Song G, Warner RM (2014) Ectopic AtCBF3 expression improves freezing tolerance and promotes compact growth habit in petunia. Mol Breeding 33:731–741CrossRefGoogle Scholar
  43. Winefield C, Lewis D, Arathoon S, Deroles S (1999) Alteration of Petunia plant form through the introduction of the rolC gene from Agrobacterium rhizogenes. Mol Breed 5:543–551CrossRefGoogle Scholar
  44. Ye YM, Tong J, Shi XP, Yuan W, Li GR (2010) Morphological and cytological studies of diploid and colchicine-induced tetraploid lines of crape myrtle (Lagerstroemia indica L.) Scientia Hortic 124: 95–101CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • J. J. Regalado
    • 1
  • E. Carmona-Martín
    • 2
  • V. Querol
    • 1
  • C. G. Veléz
    • 1
  • C. L. Encina
    • 2
  • S. I. Pitta-Alvarez
    • 1
    Email author
  1. 1.Departamento de Biodiversidad y Biología Experimental, Facultad de Ciencias Exactas y NaturalesInstituto de Micología y Botánica, UBA-CONICET, CABABuenos AiresArgentina
  2. 2.Laboratorio de Cultivo de Tejidos y BiotecnologíaInstituto de Horticultura Subtropical y Mediterránea La Mayora, CSIC-UMAAlgarrobo-CostaSpain

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