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Ploidy stability of somatic embryogenesis-derived Passiflora cincinnata Mast. plants as assessed by flow cytometry

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Abstract

In this study, flow cytometric analysis was used to evaluate the genetic stability of Passiflora cincinnata Mast. plants regenerated via primary and secondary somatic embryogenesis. Embryogenic calli obtained from culturing zygotic embryos on Murashige and Skoog (MS) medium containing 18.1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 4.4 μM benzyladenine (BA) were transferred to differentiation medium. Torpedo and cotyledonary embryos were obtained. These primary embryos were maintained on differentiation medium to generate secondary embryos. Conversion of primary and secondary embryos yielded 305 and 138 normal plants, respectively. Almost 90% of plantlets survived following acclimatization. Flow cytometric analysis revealed that seed-derived plants had on average 3.01 pg nuclear DNA (2C), and all plants, except for a single plant regenerated via primary embryogenesis, maintained their ploidy. This single plant contained more than twice the average DNA content: 6.21 pg (4C). Epidermal stomata of leaves of the tetraploid plant were larger but lower in density than those of diploid plants, indicating that stomatal characteristics are useful in distinguishing between diploid and tetraploid plants of passion fruit. In summary, the procedure we employed to regenerated P. cincinnata plants via somatic embryogenesis generated mostly genetically true-to-type plants.

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References

  • Alan AR, Zeng H, Assani A, Shi WI, Mcrae HE, Murch SJ, Saxena PK (2007) Assessment of genetic stability of the germplasm lines of medicinal plant Scutellaria baicalensis Georgi (Huang-qin) in long-term, in vitro maintained cultures. Plant Cell Rep 26:1345–1355. doi:10.1007/s00299-007-0332-9

    Article  CAS  PubMed  Google Scholar 

  • Barbosa LV, Mondin M, Oliveira CA, Souza AP, Vieira MLC (2007) Cytological behaviour of the somatic hybrids Passiflora edulis f. flavicarpa + P. cincinnata. Plant Breed 126:323–328. doi:10.1111/j.1439-0523.2007.01362.x

    Article  CAS  Google Scholar 

  • Beck SL, Dunlop RW, Fossey A (2003) Stomatal length and frequency as a measure of ploidy level in black wattle, Acacia mearnsii (de Wild). Bot J Linn Soc 141:177–181. doi:10.1046/j.1095-8339.2003.00132.x

    Article  Google Scholar 

  • Bennet MD, Bhandol P, Leitch IJ (2000) Nuclear DNA amounts in angiosperms and their modern uses—807 new estimates. Ann Bot 86:859–909. doi:10.1006/anbo.2000.1253

    Article  Google Scholar 

  • Campos JMS, Davide LC, Salgado CC, Santos FC, Costa PN, Silva PS, Alves CCS, Viccini LF, Pereira AV (2009) In vitro induction of hexaploid plants from triploid hybrids of Pennisetum purpureum and Pennisetum glaucum. Plant Breed 128:101–104. doi:10.1111/j.1439-0523.2008.01546.x

    Article  CAS  Google Scholar 

  • Carvalho JFP, Carvalho CR, Otoni WC (2005) In vitro induction of polyploidy in annatto (Bixa orellana L.). Plant Cell Tiss Organ Cult 80:69–75. doi:10.1007/s11240-004-8833-5

    Article  Google Scholar 

  • Clarindo WR, Carvalho CR, Araújo FS, Abreu IS, Otoni WC (2008) Recovering polyploid papaya in vitro regenerants as screened by flow cytometry. Plant Cell Tiss Organ Cult 92:207–214. doi:10.1007/s11240-007-9325-1

    Article  Google Scholar 

  • Cuco SM, Vieira MLC, Mondin M, Aguiar-Perecin MLR (2005) Comparative karyotype analysis of three Passiflora L. species and cytogenetic characterization of somatic hybrids. Caryologia 58:220–228

    Google Scholar 

  • De Verno LL (1995) An evaluation of somaclonal variation during somatic embryogenesis. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants. Kluwer Academic Publishers, Dordrecht, pp 361–377

    Google Scholar 

  • de Vries SC, Booij H, Meyerink P, Huisman G, Wilde DH, Thomas TL, van Kammen A (1988) Acquisition of embryogenic potential in carrot cell-suspension culture. Planta 176:196–204. doi:10.1007/BF00392445

    Article  Google Scholar 

  • Doležel J, Bartos J (2005) Plant DNA flow cytometry and estimation of nuclear genome size. Ann Bot 95:99–110. doi:10.1093/aob/mci005

    Article  PubMed  Google Scholar 

  • Doležel J, Lucretti S, Schubert I (1994) Plant chromosome analysis and sorting by flow cytometry. Crit Rev Plant Sci 13:275–309. doi:10.1080/713608061

    Google Scholar 

  • Dornelas MC (1995) Culture and protoplasts fusion in Passiflora spp. Dissertation, Escola superior de agricultura luiz de queiroz (in portuguese)

  • Dudits D, Bogre L, Gyorgyey J (1991) Molecular and cellular approaches to the analysis of plant embryo development from somatic cells in vitro. J Cell Sci 99:475–484

    Google Scholar 

  • Dudits D, Györgyey J, Bögre L, Bakó L (1995) Molecular biology of somatic embryogenesis. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer, Dordrecht, pp 267–308

    Google Scholar 

  • Dunstan DI, Tautorus TE, Thorpe TA (1995) Somatic embryogenesis in woody plants. In: Thorpe TA (ed) In vitro embryogenesis in plants. Kluwer, Dordrecht, pp 471–538

    Google Scholar 

  • Endemann M, Hristoforoglu K, Stauber T, Wilhelm E (2001) Assessment of age-related polyploidy in Quercus robur L. somatic embryos and regenerated plants using DNA flow cytometry. Biol Plant 44:339–345. doi:10.1023/A:1012426306493

    Article  Google Scholar 

  • Evans DA, Sharp WR, Medina-Filho HP (1984) Somaclonal and gametoclonal variation. Am J Bot 77:759–774

    Article  Google Scholar 

  • Fehér A (2005) Why somatic plant cells start to form embryos? In: Mujib A, Šamaj J (eds) Somatic embryogenesis. Springer Verlag, Berlin, pp 85–101. doi:10.1007/7089_019

    Google Scholar 

  • Fehér A, Pasternak T, Ötvös K, Miskolczi P, Dudits D (2002) Induction of embryogenic competence in somatic plant cells: a review. Biologia 57:5–12

    Google Scholar 

  • Fehér A, Pasternak T, Dudits D (2003) Transition of somatic plant cells to an embryogenic state. Plant Cell Tiss Organ Cult 74:201–228. doi:10.1023/A:1024072913021

    Article  Google Scholar 

  • Ferreira FR (2005) Passiflora genetics resources. In: Faleiro FG, Junqueira NTV, Braga MF (eds) Passionfruit: germplasm and breeding. Embrapa Cerrados, Planaltina, DF, Brazil, pp 40–52 (in Portuguese)

    Google Scholar 

  • Filonova L, Bozhkov P, von Arnold S (2000) Developmental pathway of somatic embryogenesis in Picea abies as revealed by time-lapse tracking. J Exp Bot 51:249–264. doi:10.1093/jexbot/51.343.249

    Article  CAS  PubMed  Google Scholar 

  • Fiuk A, Rybczyński JJ (2008) Genotype and plant growth regulator-dependent response of somatic embryogenesis from Gentiana spp. leaf explants In Vitro Cell. Dev Biol Plant 44:90–99. doi:10.1007/s11627-008-9124-3

    CAS  Google Scholar 

  • Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158. doi:10.1016/0014-4827(68)90403-5

    Article  CAS  PubMed  Google Scholar 

  • Gesteira AS, Otoni WC, Barros EG, Moreira MA (2002) RAPD-based detection of genomic instability in soybean plants derived from somatic embryogenesis. Plant Breeding 121:269–271. doi:10.1046/j.1439-0523.2002.00708.x

    Article  CAS  Google Scholar 

  • Guerra MP, Dal Vesco L, Ducroquet PHJ, Nodari RO, Reis MS (2001) Somatic embryogenesis in goiabeira serrana: genotype response, auxinic shock and synthetic seeds. Braz J Plant Physiol 13:117–128

    Google Scholar 

  • ITI Tropicals (2008) Available via www.passionfritjuice.com. Accessed 27 Out 2008

  • Jain SM (2001) Tissue culture-derived variation in crop improvement. Euphytica 118:153–166. doi:10.1023/A:1004124519479

    Article  CAS  Google Scholar 

  • Jain SM, De Klerk GJ (1998) Somaclonal variation in breeding and propagation of ornamental crops. Plant Tissue Cult Biotechnol 4:63–75

    Google Scholar 

  • Jiménez VM, Thomas C (2005) Participation of plant hormones in determination and progression of somatic embryogenesis. In: Mujib A, Šamaj J (eds) somatic embryogenesis. Springer Verlag, Berlin, pp 103–118. doi:10.1007/7089_034

    Google Scholar 

  • Kaparakis G, Alderson PG (2008) Role for cytokinins in somatic embryogenesis of pepper (Capsicum annuum L.). J Plant Growth Regul 27:110–114. doi:10.1007/s00344-007-9037-0

    Article  CAS  Google Scholar 

  • Karp A (1991) On the current understanding of somaclonal variation. Oxf Surv Plant Mol Cell Biol 7:1–58

    Google Scholar 

  • Kim SW, Oh SC, Liu JR (2003) Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose. Plant Cell Tiss Organ Cult 74:61–66. doi:10.1023/A:1023355729046

    Article  CAS  Google Scholar 

  • Larkin PJ, Scowcroft WR (1981) Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214

    Article  Google Scholar 

  • Liu Z, Gao S (2007) Micropropagation and induction of autotetraploid plants of Chrysanthemum cinerariifolium (Trev.) Vis. In Vitro Cell Dev Biol Plant 43:404–408. doi:10.1007/s11627-007-9085-y

    Article  CAS  Google Scholar 

  • Liu G, Li Z, Bao M (2007) Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology. Euphytica 157:145–154. doi:10.1007/s10681-007-9406-6

    Article  Google Scholar 

  • Loureiro J, Pinto G, Lopes T, Doležel J, Santos C (2005) Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry. Planta 221:815–822. doi:10.1007/s00425-005-1492-x

    Article  CAS  PubMed  Google Scholar 

  • May RA, Sink KC (1995) Genotype and auxin influence direct somatic embryogenesis from protoplasts derived from embryogenic cell suspension of Asparagus officinalis. Plant Sci 108:71–84. doi:10.1016/0168-9452(95)04117-D

    Article  CAS  Google Scholar 

  • Meletti LMM, Maia ML (1999) Passion fruit: production and commercialization. Technical Bulletim, Instituto Agronômico Estado de São Paulo, Campinas 181:1–62 (in Portuguese)

    Google Scholar 

  • Meletti LMM, Soares-Scott MD, Bernacci LC, da S Passos IR (2005) Passion fruit breeding: past and future. In: Faleiro FG, Junqueira NTV, Braga MF (eds) Passion fruit: germplasm and breeding. Embrapa Cerrados, Planaltina, DF, Brazil, pp 55–78 (in Portuguese)

    Google Scholar 

  • Motoike SY, Skirvin RM, Norton MA, Otterbacher AG (2001) Somatic embryogenesis and long term maintenance of embryogenic lines from fox grapes. Plant Cell Tiss Organ Cult 66:121–131

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Nomura K, Komamine A (1985) Identification and isolation of single cells that produce somatic embryos at a high frequency in a carrot suspension culture. Plant Physiol 79:988–991

    Article  CAS  PubMed  Google Scholar 

  • Oliveira JC, Ruggiero C (2005) Passion fuit species with agronomic potential. In: Faleiro FG, Junqueira NTV, Braga MF (eds) Passionfruit: germplasm and breeding. Embrapa Cerrados, Planaltina, DF, Brazil, pp 142–158 (in Portuguese)

    Google Scholar 

  • Orbovié V, Calovié M, Viloria Z, Nielsen B, Gmitter FG Jr, castle WS, Grosser JW (2008) Analysis of genetic variability in various tissue culture-derived lemon plant populations using RAPD and flow cytometry. Euphytica 161:329–335

    Article  Google Scholar 

  • Otoni WC, Blackhall NW, D’utra Vaz FB, Casali VWD, Power JB, Davey MR (1995) Somatic hybridization of the Passiflora species, P. edulis f. flavicarpa Degener and P. incarnata L. J Exp Bot 46:777–785

    Article  CAS  Google Scholar 

  • Paiva Neto VB, Botelho MN, Aguiar R, Silva EAM, Otoni WC (2003) Somatic embryogenesis from immature zygotic embryos of Annatto (Bixa orellana L.). In Vitro Cell Dev Biol Plant 39:629–634. doi:10.1079/IVP2003465

    Article  Google Scholar 

  • Passos IRS, Bernacci LC (2005) Tissue culture applied to in vitro germplasm conservation and breeding of passionfruit (Passiflora spp.). In: Faleiro FG, Junqueira NTV, Braga MF (eds) Passionfruit: germplasm and breeding. Embrapa Cerrados, Planaltina, DF, Brazil, pp 360–384 (in Portuguese)

    Google Scholar 

  • Perl A, Saad S, Sahar N, Holland D (1995) Establishment of long-term embryogenic cultures of seedless Vitis vinifera cultivars–a synergistic effect of auxins and the role of abscisic acid. Plant Sci 104:193–200

    Article  CAS  Google Scholar 

  • Pfosser M, Amon A, Lelley T, Heberle-Bors E (1995) Evaluation of sensitivity of flow cytometry in detecting aneuploidy in wheat using disomic and ditelosomic wheat-rye addition lines. Cytometry 21:387–393

    Article  CAS  PubMed  Google Scholar 

  • Pinto G, Park YS, Silva S, Neves L, Araújo C, Santos C (2008) Factors affecting maintenance, proliferation, and germination of secondary somatic embryos of Eucalyptus globulus Labill. Plant Cell Tiss Organ Cult 95:69–78

    Article  CAS  Google Scholar 

  • Pinto-Sintra AL (2007) Establishment of embryogenic cultures and plant regeneration in the Portuguese cultivar ‘Touriga Nacional’ of Vitis vinifera L. Plant Cell Tiss Organ Cult 88:253–265

    Article  Google Scholar 

  • Raghavan V (2004) Role of 2, 4-dichlorophenoxyacetic acid (2, 4-D) in somatic embryogenesis on cultured zygotic embryos of Arabidopsis: cell expansion, cell cycling, and morphogenesis during continuous exposure of embryos to 2, 4-D. Am J Bot 91:1743–1756

    Article  CAS  Google Scholar 

  • Rakoczy-Trojanowska M (2002) The effects of growth regulators on somaclonal variation in rye (Secale cereale L.) and selection of somaclonal variants with increased agronomic traits. Cell Mol Biol Lett 7:1111–1120

    CAS  PubMed  Google Scholar 

  • Rauf S, Khan IA, Khan FA (2006) Colchicine-induced tetraploidy and changes in allele frequencies in colchicine-treated populations of diploids assessed with rapid markers in Gossypium arboreum L. Turk J Biol 30:93–100

    CAS  Google Scholar 

  • Silva ML, Paim Pinto DL, Guerra MP, Floh EIS, Bruckner CH, Otoni WC (2009) A novel regeneration system for a wild passionfruit (Passiflora cincinnata Mast.) species based on somatic embryogenesis from mature zygotic embryos. Plant Cell Tiss Organ Cult 99:47–54. doi:10.1007/s11240-009-9574-2

    Article  Google Scholar 

  • Souza MM, Palomino G, Pereira TNS, Pereira MG, Viana AP (2004) Flow cytometric analysis of genome size variation in some Passiflora species. Hereditas 141:31–38

    Article  PubMed  Google Scholar 

  • Steiner N, Vieira FN, Maldonado S, Guerra MP (2005) Carbon source affects morphogenesis and histodifferentiation of A. angustifolia embryogenic cultures. Braz Arch Biol Technol 48:895–903

    Article  CAS  Google Scholar 

  • Vieira MLC, Oliveira EJ, Matta FP, Pádua JG, Monteiro M (2005) Biotechnological methods applied to passionfruit breeding. In: Faleiro FG, Junqueira NTV, Braga MF (eds) Passionfruit: germplasm and breeding. Embrapa Cerrados, Planaltina, DF, Brazil, pp 411–453 (in Portuguese)

    Google Scholar 

  • Winkelmann T, Sangwan RS, Schwenkel HG (1998) Flow cytometric analyses in embryogenic and non-embryogenic callus lines of Cyclamen persicum Mill.: relation between ploidy level and competence for somatic embryogenesis. Plant Cell Rep 17:400–404

    Article  CAS  Google Scholar 

  • Yang XM, Cao ZY, An LZ, Wang YM, Fang XW (2006) In vitro tetraploid induction via colchicine treatment from diploid somatic embryos in grapevine (Vitis vinifera L.). Euphytica 152:217–224

    Article  Google Scholar 

  • Yang XM, An LZ, Xiong YC, Zhang JP LIY, Xu SJ (2008) Somatic embryogenesis from immature zygotic embryos and monitoring the genetic fidelity of regenerated plants in grapevine. Biol Plant 52:209–214

    Article  CAS  Google Scholar 

  • Zerbini FM, Otoni WC, Vieira MLC (2008) Passionfruit. In: Kole C, Hall TC (eds) A compendium of transgenic crop plants, v.5, tropical and subtropical fruit and nuts, 1st edn. Wiley, Berlin, pp 213–234

    Google Scholar 

  • Zimermann JL (1993) Somatic embryogenesis: a model for early development in higher plants. Plant Cell 5:1141–1423

    Google Scholar 

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Acknowledgments

The authors would like to thank Fundação de Amparo à Pesquisa do Estado de Minas Gerais - FAPEMIG (Research grant number 1,527/2005), CAPES, and CNPq for financial support, and Prof. Zinmay Renee Sung for valuable suggestions regarding the manuscript.

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Authors and Affiliations

  1. Laboratório de Cultura de Tecidos Vegetais, Departamento de Biologia Vegetal/BIOAGRO, Av. Peter Henry Rolfs s/n, Campus Universitário, 36570-000, Viçosa, MG, Brazil

    Daniela Lopes Paim Pinto & Wagner Campos Otoni

  2. Laboratório de Biologia Molecular e Filogeografia/BIOAGRO, Av. Peter Henry Rolfs s/n, Campus Universitário, 36570-000, Viçosa, MG, Brazil

    Beatriz de Almeida Barros

  3. Laboratório de Genética, Instituto de Ciências Biológicas, Departamento de Biologia, Universidade Federal de Juiz de Fora, 36036-900, Juiz de Fora, MG, Brazil

    Lyderson Facio Viccini & José Marcello Salabert de Campos

  4. Laboratório de Genética, Departamento de Ciências Biológicas, Universidade do Estado do Mato Grosso, 78300-000, Tangará da Serra, MT, Brazil

    Maurecilne Lemes da Silva

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  1. Daniela Lopes Paim Pinto
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  2. Beatriz de Almeida Barros
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  3. Lyderson Facio Viccini
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  4. José Marcello Salabert de Campos
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  5. Maurecilne Lemes da Silva
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  6. Wagner Campos Otoni
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Correspondence to Wagner Campos Otoni.

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Pinto, D.L.P., de Almeida Barros, B., Viccini, L.F. et al. Ploidy stability of somatic embryogenesis-derived Passiflora cincinnata Mast. plants as assessed by flow cytometry. Plant Cell Tiss Organ Cult 103, 71–79 (2010). https://doi.org/10.1007/s11240-010-9756-y

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