Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.)

  • Elena Palomo-Ríos
  • Araceli Barceló-Muñoz
  • José A. Mercado
  • Fernando Pliego-Alfaro
Original Paper


Key factors influencing the efficiency of transformation of embryogenic cultures, induced from immature zygotic embryos, of avocado cv. ‘Duke 7’ were evaluated. Initially, the sensitivity of somatic embryos to the antibiotics kanamycin, used for selection, carbenicillin, cefotaxime and timentin, all used for elimination of Agrobacterium cells, were evaluated. Isolated globular somatic embryos were more sensitive to kanamycin than embryogenic masses, and 25 mg l−1 kanamycin completely restricted callus proliferation. Cefotaxime at 500 mg l−1 partially inhibited proliferation of embryogenic cultures, while both carbenicillin and timentin did not affect callus growth. For genetic transformation, somatic embryos were infected with A. tumefaciens containing the pBINUbiGUSint plasmid. After 2 days, the embryos were transferred to selection medium supplemented with 50 mg l−1 kanamycin and 250 mg l−1 timentin for 2 months. Then, kanamycin level was increased to 100 mg l−1 for two additional months. The A. tumefaciens strain AGL1 yielded higher transformation rates, 6%, than EHA105 or LBA4404, 1.2%. The percentage of kanamycin resistant calli obtained was significantly influenced by the embryogenic line used as source of explants. Genetic transformation was confirmed by PCR and Southern blot analysis. A significant improvement in the germination rate was obtained when transgenic embryos were cultured in liquid MS medium with 4.44 μM BA and 2.89 μM GA3 for 3 days in a roller drum and later transferred to the same medium gelled with 7 g l−1 agar. Plants from five independent transgenic lines were acclimated and grown in the greenhouse, being phenotipically similar to control plants.


Genetic transformation Somatic embryogenesis Transgenic fruit trees 





Gibberellic acid


Murashige and Skoog medium


Embryogenic avocado culture medium


Somatic embryo


  1. Álvarez R, Alonso P, Cortizo M, Celestino C, Hernández I, Toribio M, Ordás RJ (2004) Genetic transformation of selected mature cork oak (Quercus suber L.) trees. Plant Cell Rep 23:218–223CrossRefPubMedGoogle Scholar
  2. Barceló-Muñoz A, Encina CL, Simón-Pérez E, Pliego-Alfaro F (1999) Micropropagation of adult avocado. Plant Cell Tiss Organ Cult 58:11–17CrossRefGoogle Scholar
  3. Bartlett JG, Alves SC, Smedley M, Snape JW, Harwood WA (2008) High-throughput Agrobacterium-mediated barley transformation. Plant Methods 4:22CrossRefPubMedPubMedCentralGoogle Scholar
  4. Ceasar SA, Ignacimuthu S (2010) Effects of cytokinins, carbohydrates and amino acids on induction and maturation of somatic embryos in kodo millet (Paspalum scorbiculatum Linn.). Plant Cell Tiss Organ Cult 102:153–162CrossRefGoogle Scholar
  5. Chanderbali AS, Albert VA, Ashworth VETM, Clegg MT, Litz RE, Soltis DE, Soltis PS (2008) Persea americana (avocado): bringing ancient flowers to fruit in the genomics era. BioEssays 30:386–396CrossRefPubMedGoogle Scholar
  6. Chen AH, Yang JL, Niu YD, Yang CP, Liu GF, Yu CY, Li CH (2010) High-frequency somatic embryogenesis from germinated zygotic embryos of Schisandra chinensis and evaluation of the effects of medium strength, sucrose, GA3, and BA on somatic embryo development. Plant Cell Tiss Organ Cult 102:357–364CrossRefGoogle Scholar
  7. Cruz-Hernández A, Witjaksono, Litz RE, Gomez-Lim M (1998) Agrobacterium tumefaciens—mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos. Plant Cell Rep 17:497–503CrossRefGoogle Scholar
  8. Etienne H, Bertrand B (2003) Somaclonal variation in Coffea arabica: effects of genotype and embryogenic cell suspension age on frequency and phenotype of variants. Tree Physiol 23:419–426CrossRefPubMedGoogle Scholar
  9. FAOSTAT (2010) http://faostat.fao.org/
  10. Gamborg OL, Miller RA, Ojima K (1968) Plant cell cultures. I. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res 50:151–158CrossRefPubMedGoogle Scholar
  11. Gordon SP, Heisler MG, Reddy GV, Ohno C, Das P, Meyerowit EM (2007) Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134:3539–3548CrossRefPubMedGoogle Scholar
  12. Hellens R, Mullineaux P, Klee H (2000) A guide to Agrobacterium binary Ti vectors. Trends Plant Sci 5:446–451CrossRefPubMedGoogle Scholar
  13. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti plasmid. Nature 303:179–180CrossRefGoogle Scholar
  14. Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877CrossRefPubMedPubMedCentralGoogle Scholar
  15. Holford P, Newbury H (1992) The effects of antibiotics and their breakdown products on the in vitro growth of Antirrhinum majus. Plant Cell Rep 11:93–96PubMedGoogle Scholar
  16. Hood EE, Helmer GL, Fraley RT, Chilton MD (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J Bacteriol 168:1291–1301CrossRefPubMedPubMedCentralGoogle Scholar
  17. Humara J, Martín MS, Parra F, Ordás RJ (1999) Improved efficiency of uidA gene transfer in stone pine (Pinus pinea) cotyledons using a modified binary vector. Can J For Res 29:1627–1632CrossRefGoogle Scholar
  18. Husaini AM (2010) Pre- and post-agroinfection strategies for efficient leaf disk transformation and regeneration of transgenic strawberry plants. Plant Cell Rep 29:97–110CrossRefPubMedGoogle Scholar
  19. Jefferson RA (1987) Assaying chimaeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  20. Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967CrossRefPubMedGoogle Scholar
  21. Li X, Ahlman A, Yan X, Lindgren H, Zhu L-H (2010) Genetic transformation of the oilseed crop Crambe abyssinica. Plant Cell Tiss Organ Cult 100:149–156CrossRefGoogle Scholar
  22. Litz RE, Witjaksono, Raharjo S, Efendi D, Pliego-Alfaro F, Barceló-Muñoz A (2005) Persea americana avocado. In: Litz RE (ed) Biotechnology of fruit and nut crops. CABI Publishing, Wallingford, pp 326–347CrossRefGoogle Scholar
  23. Litz RE, Raharjo SHT, Gómez-Lim MA (2007) Avocado. In: Pua EC, Davey MR (eds) Transgenic crops V. Biotechnology in agriculture and forestry. Springer, Berlin, pp 167–187Google Scholar
  24. Márquez-Martín B, Sesmero R, Quesada MA, Pliego-Alfaro F, Sánchez-Romero C (2011) Water relations in culture media influence maturation of avocado somatic embryos. J Plant Physiol 168:2028–2034CrossRefPubMedGoogle Scholar
  25. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  26. Nauerby B, Billing K, Wyndaele R (1997) Influence of the antibiotic timentin on plant regeneration compared to carbenicillin and cefotaxime in concentrations suitable for elimination of Agrobacterium tumefaciens. Plant Sci 123:169–177CrossRefGoogle Scholar
  27. Newett SDE, Crane JH, Balerdi CF (2002) Cultivars and rootstocks. In: Whiley AW, Schaffer B, Wolstenholme BN (eds) The avocado: botany, production and uses. CABI Publishing, Wallingford, pp 161–187CrossRefGoogle Scholar
  28. Padilla IMG, Burgos L (2010) Aminoglycoside antibiotics: structure, functions and effects on in vitro plant culture and genetic transformation protocols. Plant Cell Rep 29:1203–1213CrossRefPubMedGoogle Scholar
  29. Parimalan R, Venugopalan A, Giridhar P, Ravishankar GA (2011) Somatic embryogenesis and Agrobacterium-mediated transformation in Bixa orellana L. Plant Cell Tiss Organ Cult 105:317–328CrossRefGoogle Scholar
  30. Pérez-Barranco G, Torreblanca R, Padilla IMG, Sánchez-Romero C, Pliego-Alfaro F, Mercado JA (2009) Studies on genetic transformation of olive (Olea europaea L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for nptII selection. II. Transient transformation via particle bombardment. Plant Cell Tiss Organ Cult 97:243–251CrossRefGoogle Scholar
  31. Pliego C, Kanematsu S, Ruano-Rosa D, de Vicente A, López-Herrera C, Cazorla FM, Ramos C (2009) GFP sheds light on the infection process of avocado roots by Rosellinia necatrix. Fungal Gen Biol 46:137–145CrossRefGoogle Scholar
  32. Pliego-Alfaro F, Murashige T (1987) Possible rejuvenation of adult avocado by graftage on to juvenile rootstocks in vitro. HortSci 22:1321–1324Google Scholar
  33. Pliego-Alfaro F, Murashige T (1988) Somatic embryogenesis in avocado (Persea americana Mill.) in vitro. Plant Cell Tiss Organ Cult 12:61–66CrossRefGoogle Scholar
  34. Raharjo SHT, Witjaksono, Gomez-Lim MA, Padilla G, Litz RE (2008) Recovery of avocado (Persea americana Mill.) plants transformed with the antifungal plant defensin gene PDF1.2. In Vitro Cell Dev Biol Plant 44:254–262CrossRefGoogle Scholar
  35. Sánchez-Romero C, Márquez-Martín B, Pliego-Alfaro F (2005) Somatic and zygotic embryogenesis in avocado. In: Mujib A, Samaj J (eds) Somatic embryogenesis. Springer, Berlin, pp 271–283Google Scholar
  36. Sarma KS, Evans NE, Selby C (1995) Effect of carbenicillin and cefotaxime on somatic embryogenesis of Sitka spruce (Picea sitchensis (Bong.) Carr.). J Exp Bot 46:1779–1781CrossRefGoogle Scholar
  37. Shehata AM, Wannarat W, Skirvin RM, Norton MA (2010) The dual role of carbenicillin in shoot regeneration and somatic embryogenesis of horseradish (Armoracia rusticana) in vitro. Plant Cell Tiss Organ Cult 102:397–402CrossRefGoogle Scholar
  38. Sokal RR, Rohlf FJ (1995) Biometry. W. H. Freeman and Company, New YorkGoogle Scholar
  39. Srinivasan C, Scorza R (1999) Transformation of somatic embryos of fruit trees and grapevine. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, vol 5. Kluwer, Berlin, pp 313–330CrossRefGoogle Scholar
  40. Tang H, Zen R, Krczal G (2000) An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants. Plant Cell Rep 19:881–887CrossRefGoogle Scholar
  41. Torreblanca R, Cerezo S, Palomo-Ríos E, Mercado JA, Pliego-Alfaro F (2010) Development of a high throughput system for genetic transformation of olive (Olea europaea L.) plants. Plant Cell Tiss Organ Cult 103:61–69CrossRefGoogle Scholar
  42. ur Rahman L, Ikenaga T, Kitamura Y (2004) Penicillin derivatives induce chemical structure-dependent root development, and application for plant transformation. Plant Cell Rep 22:668–677CrossRefPubMedGoogle Scholar
  43. Von Arnold S (2008) Somatic embryogenesis. In: George EF, Hall MA, De Klerk G-J (eds) Plant propagation by tissue culture, 3rd edn, vol. 1, the background. Springer, Dordrecht, pp 335–354Google Scholar
  44. Weir B, Gu X, Wang M, Upadhyaya N, Elliott AR, Brettell RIS (2001) Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aust J Plant Physiol 28:807–818Google Scholar
  45. Witjaksono, Litz RE (1999a) Maturation of avocado somatic embryos and plant recovery. Plant Cell Tiss Organ Cult 58:141–149CrossRefGoogle Scholar
  46. Witjaksono, Litz RE (1999b) Induction and growth characteristics of embryogenic avocado cultures. Plant Cell Tiss Organ Cult 58:19–29CrossRefGoogle Scholar
  47. Yang Y, Bao M, Liu G (2010) Factors affecting Agrobacterium-mediated genetic transformation of embryogenic callus of Parthenocissus tricuspidata Planch. Plant Cell Tiss Organ Cult 102:373–380CrossRefGoogle Scholar
  48. Youssef SM, Jiménez-Bermúdez S, Luz Bellido M, Martín-Pizarro C, Barceló M, Abdal-Aziz SA, Caballero JL, López-Aranda JM, Pliego-Alfaro F, Muñoz J, Quesada MA, Mercado JA (2009) Fruit yield and quality of strawberry plants transformed with a fruit specific strawberry pectate lyase gene. Sci Hort 119:120–125CrossRefGoogle Scholar
  49. Zhang B-H, Liu F, Liu Z-H, Wang H-M, Yao C-B (2001) Effects of kanamycin on tissue culture and somatic embryogenesis in cotton. Plant Growth Regul 33:137–149CrossRefGoogle Scholar
  50. Zhao W, Zheng S, Ling H-Q (2011) An efficient regeneration system and Agrobacterium-mediated transformation of Chinese upland rice cultivar Handao297. Plant Cell Tiss Organ Cult 106:475–483CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Elena Palomo-Ríos
    • 1
  • Araceli Barceló-Muñoz
    • 2
  • José A. Mercado
    • 1
  • Fernando Pliego-Alfaro
    • 1
  1. 1.Departamento de Biología Vegetal, Instituto de Hortofruticultura Subtropical y Mediterránea “La Mayora” (IHSM-UMA-CSIC)Universidad de MálagaMálagaSpain
  2. 2.IFAPA Centro de ChurrianaChurriana, MálagaSpain

Personalised recommendations