Evaluation of in vitro shoot multiplication and ISSR marker based assessment of somaclonal variants at different subcultures of vanilla (Vanilla planifolia Jacks)

  • Miriam Cristina Pastelín Solano
  • Josafhat Salinas Ruíz
  • María Teresa González Arnao
  • Odón Castañeda Castro
  • María Elena Galindo Tovar
  • Jericó Jabín Bello BelloEmail author
Research Article


The effect of subculture cycles on somaclonal variation of V. planifolia using intersimple sequence repeat (ISSR) markers was analyzed. Nodal segments of 2 cm in length were established in vitro and multiplied by 10 subculture cycles in Murashige and Skoog (MS) medium supplemented with 8.86 μM BAP (benzylaminopurine). After 45 days in each culture, the length and number of shoots per explant were evaluated. For ISSR markers, ten shoots per each subculture and the mother plant were used. Ten ISSR primers were used and a total of 118 bands were obtained. The polymorphism (%) was calculated and a dendrogram based on Jaccard’s genetic distance between the subcultures and the donor plant was obtained. These results show that the multiplication rate tends to increase until subculture five, whereas shoot length decreases as the number of subcultures increases. The ISSR markers revealed an increase in the polymorphism percentage after the fifth culture cycle. The dendrogram showed the formation of two groups. The first group, with less genetic variability, is the donor plant and subcultures 1–5; the second group has greater genetic distance and is formed by subcultures 6–10. The results revealed that the number of subcultures with 8.86 μM BAP is a factor that affects the somaclonal variation during in vitro regeneration of V. planifolia. In conclusion, the subculture number affects somaclonal variation and in vitro development of V. planifolia.


Vanilla planifolia Regeneration Somaclonal variation Micropropagation ISSR 



  1. Akdemir H, Suzerer V, Tilkat E, Onay A, Çiftçi YO (2016) Detection of variation in long-term micropropagated mature pistachio via DNA-based molecular markers. Appl Biochem Biotechnol 180(7):1301–1312CrossRefGoogle Scholar
  2. Azman AS, Mhiri C, Grandbastien MA, Tam S (2014) Transposable elements and the detection of somaclonal variation in plant tissue culture: a review. Malays Appl Biol 43(1):1–12Google Scholar
  3. Babu GA, Vinoth A, Ravindhran R (2018) Direct shoot regeneration and genetic fidelity analysis in finger millet using ISSR markers. Plant Cell Tissue Organ Cult 132:157–164. CrossRefGoogle Scholar
  4. Bairu MW, Fennell CW, Van Staden J (2006) The effect of plant growth regulators on somaclonal variation in Cavendish banana (Musa AAA cv. ‘Zelig’). Sci Hortic 108(4):347–351CrossRefGoogle Scholar
  5. Bairu MW, Aremu AO, Van Staden J (2011) Somaclonal variation in plants: causes and detection methods. Plant Growth Regul 63(2):147–173CrossRefGoogle Scholar
  6. Bello-Bello JJ, Iglesias-Andreu LG, Avilés-Vinas SA, Gómez-Uc E, Canto-Flick A, Santana-Buzzy N (2014) Somaclonal variation in habanero pepper (Capsicum chinense Jacq.) as assessed ISSR molecular markers. HortScience 49(4):481–485CrossRefGoogle Scholar
  7. Bory S, Grisoni M, Duval MF, Besse P (2008) Biodiversity and preservation of vanilla: present state of knowledge. Genet Resour Crop Evol 55(4):551–571CrossRefGoogle Scholar
  8. Divakaran M, Babu KN, Ravindran PN, Peter KV (2015) Biotechnology for micropropagation and enhancing variations in Vanilla. Asian J Plant Sci Res 5(2):52–62Google Scholar
  9. Farahani F, Yari R, Masoud S (2011) Somaclonal variation in Dezful cultivar of olive (Olea europaea subsp. europaea). Gene Conserve 10:216–221Google Scholar
  10. Gantait S, Kundu S (2017) In vitro biotechnological approaches on Vanilla planifolia Andrews: advancements and opportunities. Acta Physiol Plant 39(9):196CrossRefGoogle Scholar
  11. Gantait S, Mandal N, Bhattacharyya S, Das PK, Nandy S (2009) Mass multiplication of Vanilla planifolia with pure genetic identity confirmed by ISSR. Int J Dev Biol 3:18–23Google Scholar
  12. Greule M, Tumino LD, Kronewald T, Hener U, Schleucher J, Mosandl A, Keppler F (2010) Improved rapid authentication of vanillin using δ13C and δ2H values. Eur Food Res Technol 231(6):933–941CrossRefGoogle Scholar
  13. Hammer Ř, Harper DAT, Ryan PD (2001) PAST: paleontological statistics software package for education and data analysis. Palaeontol Electron 4:1–9Google Scholar
  14. Jain SM (2001) Tissue culture-derived variation in crop improvement. Euphytica 118(2):153–166CrossRefGoogle Scholar
  15. Jain SM, Brar DS, Ahloowalia BS (eds) (2013) Somaclonal variation and induced mutations in crop improvement, vol 32. Springer, BerlinGoogle Scholar
  16. Kanwar K, Devi V, Sharma S, Soni M, Sharma D (2015) Effect of physiological age and growth regulators on micropropagation of Aloe vera followed by genetic stability assessment. Nat Acad Sci Lett 38(1):29–35CrossRefGoogle Scholar
  17. Khan S, Saeed B, Kauser N (2011) Establishment of genetic fidelity of in vitro raised banana plantlets. Pak J Bot 43(1):233–242Google Scholar
  18. Kılınç FM, Süzerer V, Çiftçi YÖ, Onay A, Yıldırım H, Uncuoğlu AA, Metin ÖK (2015) Clonal micropropagation of Pistacia lentiscus L. and assessment of genetic stability using IRAP markers. Plant Growth Regul 75(1):75–88CrossRefGoogle Scholar
  19. Krishna H, Alizadeh M, Singh D, Singh U, Chauhan N, Eftekhari M, Sadh RK (2016) Somaclonal variations and their applications in horticultural crops improvement. 3 Biotech 6(1):54CrossRefGoogle Scholar
  20. Larkin PJ, Scowcroft WR (1981) Somaclonal variation—a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60(4):197–214CrossRefGoogle Scholar
  21. Lázaro-Castellanos JO, Mata-Rosas M, González D, Arias S, Reverchon F (2018) In vitro propagation of endangered Mammillaria genus (Cactaceae) species and genetic stability assessment using SSR markers. In Vitro Cell Dev Biol Plant 54(5):518–529CrossRefGoogle Scholar
  22. Lee TSG (1987) Micropropagation of sugarcane (Saccharum spp.). Plant Cell Tissue Organ Cult 10(1):47–55CrossRefGoogle Scholar
  23. Lee-Espinosa HE, Murguía-González J, García-Rosas B, Córdova-Contreras AL, Laguna-Cerda A, Mijangos-Cortés JO, Santana-Buzzy N (2008) In vitro clonal propagation of vanilla (Vanilla planifolia ‘Andrews’). HortScience 43(2):454–458CrossRefGoogle Scholar
  24. Mamedes-Rodrigues TC, Batista DS, Vieira NM, Matos EM, Fernandes D, Nunes-Nesi A, Cruz CD, Viccini LF, Nogueira FTS, Otoni WC (2018) Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogeic calli as affected by successive subcultures. Protoplasma 255:655–667CrossRefGoogle Scholar
  25. Martínez-Estrada E, Caamal-Velázquez JH, Salinas-Ruíz J, Bello-Bello JJ (2017) Assessment of somaclonal variation during sugarcane micropropagation in temporary immersion bioreactors by intersimple sequence repeat (ISSR) markers. In Vitro Cell Dev Biol Plant 53(6):553–560CrossRefGoogle Scholar
  26. Mendes BMJ, Filippi SB, Demetrio CGB, Rodriguez APM (1999) A statistical approach to study the dynamics of micropropagation rates, using banana (Musa spp.) as an example. Plant Cell Rep 18(12):967–971CrossRefGoogle Scholar
  27. Miller MP (1997) Tools for population genetic analyses (TFPGA) 1.3: a windows program for the analysis of allozyme and molecular population genetic data. Computer software distributed by authorGoogle Scholar
  28. Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15(3):473–497CrossRefGoogle Scholar
  29. Peng X, Zhang TT, Zhang J (2015) Effect of subculture times on genetic fidelity, endogenous hormone level and pharmaceutical potential of Tetrastigma hemsleyanum callus. Plant Cell Tissue Organ Cult 122(1):67–77CrossRefGoogle Scholar
  30. Ramírez-Mosqueda MA, Iglesias-Andreu LG (2015) Indirect organogenesis and assessment of somaclonal variation in plantlets of Vanilla planifolia Jacks. Plant Cell Tissue Organ Cult 123(3):657–664CrossRefGoogle Scholar
  31. Ramos-Castellá A, Iglesias-Andreu LG, Bello-Bello JJ (2014) Improved propagation of vanilla (Vanilla planifolia Jacks. ex Andrews) using a temporary immersion system. Vitro Cell Dev Biol Plant 50(5):576–581CrossRefGoogle Scholar
  32. Rani V, Raina SN (2000) Genetic fidelity of organized meristem-derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36(5):319–330CrossRefGoogle Scholar
  33. Reddy MP, Sarla N, Siddiq EA (2002) Inter simple sequence repeat (ISSR) polymorphism and its application in plant breeding. Euphytica 128(1):9–17CrossRefGoogle Scholar
  34. Salazar-Rojas VM, Herrera-Cabrera BE, Delgado-Alvarado A, Soto-Hernández M, Castillo-González F, Cobos-Peralta M (2012) Chemotypical variation in Vanilla planifolia Jack. (Orchidaceae) from the Puebla–Veracruz Totonacapan region. Genet Resour Crop Evol 59(5):875–887CrossRefGoogle Scholar
  35. Sales EK, Butardo NG (2014) Molecular analysis of somaclonal variation in tissue culture derived bananas using MSAP and SSR markers. Int J Biol Vet Agric Food Eng 8(6):615–622Google Scholar
  36. Soliman HIA, Metwali EMR, Almaghrabi OAH (2014) Micropropagation of Stevia rebaudiana Betroni and assessment of genetic stability of in vitro regenerated plants using inter simple sequence repeat (ISSR) marker. Plant Biotechnol 31(3):249–256CrossRefGoogle Scholar
  37. Soto Arenas MA, Cribb P (2010) A new infrageneric classification and synopsis of the Genus Vanilla plum. ex mill. (Orchidaceae: Vanillinae). Lankesteriana 9:355–398Google Scholar
  38. Sreedhar RV, Venkatachalam L, Bhagyalakshmi N (2007) Genetic fidelity of long-term micropropagated shoot cultures of vanilla (Vanilla planifolia Andrews) as assessed by molecular markers. Biotechnol J 2(8):1007–1013CrossRefGoogle Scholar
  39. Stewart CN, Via LE (1993) A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques 14(5):748–750Google Scholar

Copyright information

© Prof. H.S. Srivastava Foundation for Science and Society 2019

Authors and Affiliations

  • Miriam Cristina Pastelín Solano
    • 1
  • Josafhat Salinas Ruíz
    • 2
  • María Teresa González Arnao
    • 3
  • Odón Castañeda Castro
    • 3
  • María Elena Galindo Tovar
    • 1
  • Jericó Jabín Bello Bello
    • 4
    Email author
  1. 1.Faculty of Biological and Agropecuary SciencesUniversity of VeracruzAmatlán de los ReyesMexico
  2. 2.Postgraduate College-Campus CórdobaAmatlán de los ReyesMexico
  3. 3.Faculty of Chemistry SciencesUniversity of VeracruzOrizabaMexico
  4. 4.CONACYT- Postgraduate College-Campus CórdobaAmatlán de los ReyesMexico

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