Theoretical and Applied Genetics

, Volume 110, Issue 1, pp 157–166 | Cite as

Detection and quantification of in vitro-culture induced chimerism using simple sequence repeat (SSR) analysis in Theobroma cacao (L.)

  • Carlos M. Rodríguez López
  • Andrew C. Wetten
  • Michael J. WilkinsonEmail author
Original Paper


Mutation rates are often elevated in plants regenerated from in vitro culture, giving rise to so-called ‘somaclonal variation’. Detailed characterisation of mutation profiles that arise during culture should improve our understanding of processes influencing mutation and allow the selection of protocols yielding the fewest/least severe changes. Somatic mutations will usually produce genetic chimeras where unchanged alleles are retained by some cells. Such chimeras are difficult to detect but likely to form a significant proportion of any regenerant population. We present a simple protocol that enables the provisional diagnosis of both homogenous and chimeric mutants among large regenerant populations, together with a semi-quantitative means of estimating the proportion of mutant cells. The assay exploits consistent differential amplification of alternate simple sequence repeat alleles at heterozygous loci. Calibration of the relative amplification of alleles from two genotypes—and the synthetic chimeras created from them—revealed a strong linear relationship between ‘peak heights’ representing alternate alleles following capillary electrophoresis. The assay predicts chimeric composition to a reasonable level of confidence (±5%) so long as the infrequent allele exceeds 15% of the template. The system was applied to 233 regenerants of cocoa somatic embryogenesis and identified 72 (31%) putative chimeric mutants for slippage mutation or allele loss across two loci.


Somatic Embryogenesis Simple Sequence Repeat Marker Somaclonal Variation Allele Loss Peak Ratio 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank Cocoa Research UK for funding this study.


  1. Aderkas P von, Pattanavibool R, Hristoforoglu K, Ma Y (2003) Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch. Plant Cell Tissue Organ Cult 75:27–34CrossRefGoogle Scholar
  2. Albani MC, Wilkinson MJ (1998) Inter-simple sequence repeat polymerase chain reaction for the detection of somaclonal variation. Plant Breed 117:573–575Google Scholar
  3. Alemanno L, Berthouly M, Michaux-Ferriere N (1996) Histology of somatic embryogenesis from floral tissues in cocoa. Plant Cell Tissue Organ Cult 46:187–194Google Scholar
  4. Cammareri M, Errico A, Filippone E, Esposito S, Conicella C (2002) Induction of variability in chimeric Aster cordifolius ‘White Elegans’ through somaclonal variation. Euphytica 128:19–25CrossRefGoogle Scholar
  5. Clayton TM, Whitaker JP, Sparkes R, Gill P (1998) Analysis and interpretation of mixed forensic stains using DNA STR profiling. Forensic Sci Int 91:55–70CrossRefPubMedGoogle Scholar
  6. Cuatrecasas J (1964) Cacao and its allies: a taxonomic review of the genus Theobroma. Contrib U S Nat Herb 35:379–614Google Scholar
  7. De Schepper S, Leus L, Mertens M, Debergh P, Van Bockstaele E, De Loose M (2001) Somatic polyploidy and its consequences for flower coloration and flower morphology in azalea. Plant Cell Rep 20:583–590CrossRefGoogle Scholar
  8. Diwan N, Cregan PB (1997) Automated sizing of fluorescent-labeled simple sequence repeat (SSR) markers to assay genetic variation in soy bean. Theor Appl Genet 95:723–733CrossRefGoogle Scholar
  9. Evans DA (1998) Somaclonal variation—genetic basis and breeding applications. Trends Genet 5:46–50CrossRefGoogle Scholar
  10. Feuser S, Meler K, Daquinta M, Guerra MP, Nodari RO (2003) Genotypic fidelity of micropropagated pineapple (Ananas comosus) plantlets assessed by isozyme and RAPD markers. Plant Cell Tissue Organ Cult 72:221–227CrossRefGoogle Scholar
  11. Fourré JL, Berger P, Niquet L, André P (1997) Somatic embryogenesis and somaclonal variation in Norway spruce: morphogenetic, cytogenetic and molecular approaches. Theor Appl Genet 94:159–169CrossRefGoogle Scholar
  12. Franks T, Botta R, Thomas MR (2002) Chimerism in grapevines: implications for cultivar identity, ancestry and genetic improvement. Theor Appl Genet 104:192–199CrossRefPubMedGoogle Scholar
  13. Gill PD, Sparkes R, Kimpton CP (1997) Development of the guidelines to designate alleles using an STR multiplex system. Forensic Sci Int 89:185–197CrossRefPubMedGoogle Scholar
  14. Gimenez C, De Garcia E, De Enrech NX, Blanca I (2001) Somaclonal variation in banana: cytogenetic and molecular characterization of the somaclonal variant CIEN BTA-03. In Vitro Cell Dev Biol Plant 37:217–222Google Scholar
  15. Harding K (2004) Genetic integrity of cryopreserved plant cells: a review. Cryoletters 1:3–22Google Scholar
  16. Jaligot E, Rival A, Beule T, Dussert S, Verdeil JL (2000) Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep 19:684–690CrossRefGoogle Scholar
  17. Joyce SM, Cassells AC (2002) Variation in potato microplant morphology in vitro and DNA methylation. Plant Cell Tissue Organ Cult 70:125–137CrossRefGoogle Scholar
  18. Joyce SM, Cassells AC, Jain SM (2003) Stress and aberrant phenotypes in in vitro culture. Plant Cell Tissue Organ Cult 74:103–121CrossRefGoogle Scholar
  19. Kaeppler SM, Kaeppler HF, Rhee Y (2000) Epigenetic aspects of somaclonal variation in plants. Plant Mol Biol 43:179–188CrossRefPubMedGoogle Scholar
  20. Kennedy AJ, Mooleedhar V (1992) Conservation of cocoa in field genebanks—the International Cocoa Genebank, Trinidad. In: ICGT (ed) Proc Int Workshop Conservation Characterization Utilization Cocoa Genet Resources in the 21st Century. The University of The West Indies, St. Augustine, Trinidad, pp 21–26Google Scholar
  21. Lanaud C, Risterucci AM, Pieretti I, Falque M, Bouet A, Lagoda PJL (1999) Isolation and characterization of microsatellites in Theobroma cacao L. Mol Ecol 8:2141–2143CrossRefPubMedGoogle Scholar
  22. Larkin PJ, Scowcroft WR (1981) Somaclonal variation: a novel source of variability from cell cultures for plant improvement. Theor Appl Genet 60:197–214Google Scholar
  23. Leroy XJ, Leon K, Hilyc JM, Chaumeil P, Branchard M (2001) Detection of in vitro culture-induced instability through inter-simple sequence repeat analysis. Theor Appl Genet 102:885–891CrossRefGoogle Scholar
  24. Li Z, Traore A, Maximova S, Gupta PK (1998) Somatic embryogenesis and plant regeneration from floral explants of cacao (Theobroma cacao L.) using thidiazuron. In Vitro Cell Dev Biol Plant 34:293–299Google Scholar
  25. Lim WL, Loh CS (2003) Endopolyploidy in vanda miss Joaquim (Orchidaceae). New Phytol 159:279–287CrossRefGoogle Scholar
  26. Lopez-Baez O, Bollon H, Eskes A (1993). Embryogenèse somatique de cacaoyer Theobroma cacao L. à partir de pièces florales. C R Acad Sci Paris 316:579–584Google Scholar
  27. Lygo JE, Johnson PE, Holdaway DJ, Woodroffe S, Whitaker JP, Clayton TM, Kimpton CP, Gill P (1994) The validation of short tandem repeat (STR) loci for use in forensic casework. Int J Legal Med 107:77–89PubMedGoogle Scholar
  28. Martin C, Gonzalez-Benito E, Iriondo JM, Perez C (2001) Somaclonal variation in tissue cultures: implications in plant breeding and genetic conservation. Agro Food Industry Hi Tech 12:11–13Google Scholar
  29. Matthes M, Singh R, Cheah SC, Karp A (2001) Variation in oil palm (Elaeis guineensis Jacq.) tissue culture-derived regenerants revealed by AFLPs with methylation-sensitive enzymes. Theor Appl Genet 102:971–979CrossRefGoogle Scholar
  30. Maximova SN, Alemanno L, Young A, Ferriere N, Traore A, Guiltinan MJ (2002) Efficiency, genotypic variability, and cellular origin of primary and secondary somatic embryogenesis of Theobroma cacao L. In Vitro Cell Dev Biol Plant 38:252–259Google Scholar
  31. Mohan JS (2001) Tissue culture-derived variation in crop improvement. Euphytica 118:153–166CrossRefGoogle Scholar
  32. Plader W, Malepszy S, Burza W, Rusinowski Z (1998) The relationship between the regeneration system and genetic variability in the cucumber (Cucumis sativus L.). Euphytica 103:9–15CrossRefGoogle Scholar
  33. Rani V, Singh KP, Shiran B, Nandy S, Goel S, Devarumath RM, Sreenath HL, Raina SN (2000) Evidence for new nuclear and mitochondrial genome organizations among high-frequency somatic embryogenesis derived plants of allotetraploid Coffea arabica L. (Rubiaceae). Plant Cell Rep 19:1013–1020CrossRefGoogle Scholar
  34. Risterucci AM, Grivet L, N’Goran JAK, Pieretti I, Flament MH, Lanaud C (2000) A high density linkage map of Theobroma cacao L. Theor Appl Genet 101:948–955CrossRefGoogle Scholar
  35. Rolf B, Wiegand P, Brinkmann B (2002) Somatic mutations at STR loci—a reason for three-allele pattern and mosaicism. Forensic Sci Int 126:200–202CrossRefPubMedGoogle Scholar
  36. Sparkes R, Kimpton CP, Gilbard S, Carne P, Anderson J, Oldroy N, Thomas D, Urquhart A, Gill PD (1996) The validation of 7-locus multiplex STR test for use in forensic casework. (I) Mixtures, degradation and species studies. Int J Legal Med 109:186–194PubMedGoogle Scholar
  37. Traore A, Maximova SN, Guiltinan MJ (2003) Micropropagation of Theobroma cacao L. using somatic embryo-derived plants. In Vitro Cell Dev Biol Plant 39:332–337CrossRefGoogle Scholar
  38. Tremblay L, Levasseur C, Tremblay FM (1999) Frequency of somaclonal variation in plants of black spruce (Picea mariana, Pinaceae) and white spruce (P. glauca, Pinaceae) derived from somatic embryogenesis and identification of some factors involved in genetic instability. Am J Bot 86:1373PubMedGoogle Scholar
  39. Ziegle JS, Su Y, Corcoran KP, Nie L, Mayrand PE, Hoff LB, McBride LJ, Kronick MN, Diehl SR (1992) Application of automated DNA sizing technology for genotyping microsatellite loci. Genomics 14:1026–1031PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2004

Authors and Affiliations

  • Carlos M. Rodríguez López
    • 1
  • Andrew C. Wetten
    • 1
  • Michael J. Wilkinson
    • 1
    Email author
  1. 1.School of Plant SciencesThe University of ReadingReadingUK

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