In vitro plant regeneration and cryopreservation of Arachis glabrata (Fabaceae) using leaflet explants

  • Natalia R. Dolce
  • Mirta M. Faloci
  • Ana M. Gonzalez


Arachis glabrata Benth (perennial peanut) is a rhizomatous legume with high forage value and great potential for soil conservation as well as it displays valuable plant genetic resources for the cultivated edible peanut improvement. In this study, we developed for the first time successful protocols for micropropagation and cryopreservation of A. glabrata. First fully expanded leaflets from greenhouse-growing plants were efficiently established in vitro (93%) and displayed high frequency of bud induction (58%) on MS medium with 6 mg L−1 1-fenil-3-(1,2,3-tiadiazol-5-il)urea [TDZ]. Whole plant regeneration was achieved via direct organogenesis by transferring the induced buds to MS media. Immature unexpanded leaves from micropropagated plants were effectively cryopreserved by using the droplet-vitrification technique. Maximum survival (~ 70%) and further regeneration (60–67%) were obtained by preconditioning immature leaves on semisolid MS with 0.3 M sucrose (1 d), exposing to loading solution consisting of 0.4 M sucrose plus 2 M glycerol (30 min) followed by glycerol-sucrose plant vitrification solution PVS3 (150 min in ice), and direct plunging into liquid nitrogen in droplets of PVS3 deposited on cryoplates. Tissues were rewarmed by plunging the aluminum foils directly in liquid MS enriched with 1.2 M sucrose (15 min) at room temperature. Growth recovery and plant regeneration were efficiently achieved via shoot organogenesis, and somatic embryogenesis by culturing cryostored explants on MS added with 6 mg L−1 TDZ. Genetic stability of plants derived from cryopreserved leaves was confirmed by random amplified polymorphic DNA markers. The protocols established in this study have great potential for rapid multiplication and conservation of selected A. glabrata genotypes.


Shoot organogenesis Somatic embryogenesis Thidiazuron Droplet-vitrification Genetic stability Perennial peanut 



The authors would like to thank CONICET (PIP 11220150100398 CO) and SGCyT-UNNE (PI 12A007 and 16A010) for the financial support. We are also grateful to Dr. H. Rey for helpful collaboration and Dr. G. Lavia for providing the plant material.


  1. Akasaka Y, Daimon H, Mii M (2000) Improved plant regeneration from cultured leaf segments in peanut (Arachis hypogaea L.) by limited exposure to thidiazuron. Plant Sci 156:169–175CrossRefPubMedGoogle Scholar
  2. Angelici CMLCD, Gimenes MA, Hoshino AA, Lopes CR, Palmieri DA, Valls JFM, Nobile PM (2008) Genetic diversity in section Rhizomatosae of the genus Arachis (Fabaceae) based on microsatellite markers. Genet Mol Biol 31:79–88CrossRefGoogle Scholar
  3. Bajaj YPS (1979) Freeze preservation of meristems of Arachis hypogaea and Cicer arietinum. Indian J Exp Biol 17:1405–1407Google Scholar
  4. Barraco G, Sylvestre I, Iapichino G, Engelmann F (2011) Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification. Sci Hortic 130:309–313CrossRefGoogle Scholar
  5. Bhattacharya C, Dam A, Karmakar J, Bandyopadhyay TK (2016) Direct somatic embryogenesis and genetic homogeneity assessment of regenerated plants of Anthurium andraeanum Linden cv. Fantasia. In Vitro Cell Dev Biol Plant 52:512–519CrossRefGoogle Scholar
  6. Cassells AC (2001) Contamination and its impact in tissue culture. Acta Hortic 560:353–359CrossRefGoogle Scholar
  7. Crowe JH, Carpenter JF, Crowe LM, Anchordoguy TJ (1990) Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27:219–231CrossRefGoogle Scholar
  8. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  9. Dunbar KB, Pittman RN (1992) Adventitious shoot formation from mature leaf explants of Arachis species. Crop Sci 32:1353–1356CrossRefGoogle Scholar
  10. Dunbar KB, Pittman RN, Morris JB (1993) In vitro culture of embryonic axes from Arachis species for germplasm recovery. J Seed Technol 17:1–7Google Scholar
  11. Evans DA, Sharp WR, Flick CE (1981) Growth and behavior of cell cultures: embryogenesis and organogenesis. In: Thorpe TA (ed) Plant tissue culture. Methods and applications in agriculture. Academic Press, New York, pp 45–113Google Scholar
  12. Fontana ML, Mroginski LA, Rey HY (2009) Organogenesis and plant regeneration of Arachis villosa Benth. (Leguminosae) through leaf culture. Biocell 33:179–186PubMedGoogle Scholar
  13. Foster JL, Adesogan AT, Carter JN, Blount AR, Myer RO, Phatak SC (2009) Intake, digestibility, and nitrogen retention by sheep supplemented with warm-season legume haylages or soybean meal. J Anim Sci 87:2899–2905CrossRefPubMedGoogle Scholar
  14. Foster JL, Carter JN, Sollenberger LE, Blount AR, Myer RO, Maddox MK, Phatak SC, Adesogan AT (2011) Nutritive value, fermentation characteristics, and in situ disappearance kinetics of ensiled warm-season legumes and bahiagrass. J Dairy Sci 94:2042–2050CrossRefPubMedGoogle Scholar
  15. French EC, Prine GM, Ocumpaugh WR, Rice RW (1993) Regional experience with forage Arachis en the United States. In: Kerridge PC, Hardy B (eds) Biology and agronomy of forage Arachis. CIAT, Cali, pp 167–184Google Scholar
  16. Gagliardi RF, Pacheco GP, Carneiro LA, Valls JFM, Vieira MLC, Mansur E (2003) Cryopreservation of Arachis species by vitrification of in vitro-grown shoot apices and genetic stability of recovered plants. Cryo-Lett 24:103–110Google Scholar
  17. Gagliardi RF, Pacheco GP, Valls JFM, Mansur E (2002) Cryopreservation of cultivated and wild Arachis species embryonic axes using desiccation and vitrification methods. Cryo Letters 23:61–68PubMedGoogle Scholar
  18. Gill R, Ozias-Akins P (1999) Thidiazuron-induced highly morphogenic callus and high frequency regeneration of fertile (Arachis hypogaea L.) plants. In Vitro Cell Dev Biol Plant 35:445–450CrossRefGoogle Scholar
  19. Gonzalez AM, Cristóbal CL (1997) Anatomía y ontogenia de semillas de Helicteres lhotzkyana (Sterculiaceae). Bonplandia 9:287–294Google Scholar
  20. Halmagyi A, Vălimăreanu S, Coste A, Deliu C, Isac V (2010) Cryopreservation of Malus shoot tips and subsequent plant regeneration. Rom Biotechnol Lett 15:79–85Google Scholar
  21. Kanyand M, Peterson CM, Prakash CS (1997) The differentiation of emergences into adventitious shoots in peanut Arachis hypogaea (L). Plant Sci 126:87–95CrossRefGoogle Scholar
  22. Krapovickas A, Gregory WC (1994) Taxonomía del género Arachis (Leguminosae). Bonplandia 8:1–186Google Scholar
  23. Leunufna S, Keller ERJ (2003) Investigating a new cryopreservation protocol for yams (Dioscorea spp.) Plant Cell Rep 21:1159–1166CrossRefPubMedGoogle Scholar
  24. Luque R, Sousa HC, Kraus JE (1996) Método de coloracao de Roeser (1972) e Kropp (1972) visando a subtituicao do azul do astra por azul de alciao 8GS ou 8GX. Acta Bot Bras 10:199–212CrossRefGoogle Scholar
  25. Mallikarjuna N (2002) Gene introgression from Arachis glabrata into A. hypogaea, A. duranensis and A. diogoi. Euphytica 124:99–105CrossRefGoogle Scholar
  26. McKently AH, Moore GA, Gardner FP (1991) Regeneration of peanut and perennial peanut from cultivated leaf tissue. Crop Sci 31:833–837CrossRefGoogle Scholar
  27. Mithila J, Murch SJ, Krishnaraj S, Saxena PK (2001) Recent advances in Pelargonium in vitro regeneration systems. Plant Cell Tissue Organ Cult 67:1–9CrossRefGoogle Scholar
  28. Mroginski E, Rey HY, Gonzalez AM et al (2004) Thidiazuron promotes in vitro plant regeneration of Arachis correntina (Leguminosae) via organogenesis. J Plant Growth Regul 23:129–134CrossRefGoogle Scholar
  29. Mroginski LA, Kartha KK (1984) Tissue culture of legumes for crop improvement. Plant Breed Rev 2:215–264Google Scholar
  30. Mroginski LA, Katha KK, Shyluk JP (1981) Regeneration of peanut (Arachis hypogaea) by in vitro culture of immature leaves. Can J Bot 59:826–830CrossRefGoogle Scholar
  31. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497CrossRefGoogle Scholar
  32. Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol Plant 34:267–275CrossRefGoogle Scholar
  33. Nishizawa S, Sakai A, Amano Y, Matsuzawa T (1993) Cryopreservation of asparagus (Asparagus officinalis L.) embryogenic suspension cells and subsequent plant regeneration by the vitrification method. Plant Sci 88:67–73CrossRefGoogle Scholar
  34. Pacheco G, Gagliardi RF, Valls JFM, Mansur E (2009) Micropropagation and in vitro conservation of wild Arachis species. Plant Cell Tissue Organ Cult 99:239–249CrossRefGoogle Scholar
  35. Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55CrossRefGoogle Scholar
  36. Panta A, Panis B, Ynouye C, Swennen R, Roca W (2014) Development of a PVS2 droplet vitrification method for potato cryopreservation. Cryo Letters 35:255–266PubMedGoogle Scholar
  37. Pennycooke JC, Towill LE (2000) Cryopreservation of shoot tips from in vitro plants of sweet potato [Ipomoea batatas (L.) Lam.] by vitrification. Plant Cell Rep 19:733–737CrossRefGoogle Scholar
  38. Pittman RN, Banks DJ, Kirby JS, Mitchell ED, Richardson PE (1983) In vitro culture of immature peanut (Arachis spp.) leaves: morphogenesis and plantlet regeneration. Peanut Sci 10:21–25CrossRefGoogle Scholar
  39. Prine GM, Dunavin LS, Glennon RJ, Roush RD (1986) Arbrook rhizoma peanut—a perennial forage legume. Univ. of Florida, Gainesville. Agricultural Experiment Station Circular S-332Google Scholar
  40. Prine GM, Dunavin LS, Moore JE, Roush RD (1981) Florigraze rhizoma peanut—a perennial forage legume. Univ. of Florida, Gainesville. Agricultural Experiment Station Circular S-275Google Scholar
  41. Rey H, Mroginski L (2009) Cryopreservation of Arachis pintoi (Leguminosae) seeds. Seed Sci Technol 37:202–205CrossRefGoogle Scholar
  42. Rey HY, Mroginski LA (2003) Regeneration of plants from apical meristem tips and nodal segments of Arachis pintoi. Peanut Sci 30:75–79CrossRefGoogle Scholar
  43. Rey HY, Faloci M, Medina R, Dolce N, Mroginski L, Engelmann F (2009) Cryopreservation of in vitro grown shoot tips and apical meristems of the forage legume Arachis pintoi. Cryo Letters 30:347–358PubMedGoogle Scholar
  44. Rey HY, Faloci M, Medina R, Dolce N, Mroginski L, Engelmann F (2013) Cryopreservation of Arachis pintoi (Leguminosae) somatic embryos. Cryo Letters 34:571–582PubMedGoogle Scholar
  45. Rey HY, Scocchi AM, Gonzalez AM, Mroginski LA (2000) Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture. Plant Cell Rep 19:856–862CrossRefGoogle Scholar
  46. Rohlf FJ (1994) NTSYS-pc numerical taxonomy of multivariate analysis system. Version 2.11W. Exeter software, New YorkGoogle Scholar
  47. Rouse RE, Mullahey JJ (1997) Perennial peanut ground cover in citrus orchard row middles and discussion of potential environmental benefits. Proc Fla State Hort Soc 110:79–82Google Scholar
  48. Runthala R, Jana MK, Mohanan K (1993) Cryopreservation of groundnut (Arachis hypogaea L.) embryonic axes for germplasm conservation. Cryo Letters 14:323–334Google Scholar
  49. Sakai A (2000) Development of cryopreservation techniques. In: Engelmann F, Takagi H (eds) Cryopreservation of tropical plant germplasm. Current research progress and application. IPGRI, Rome, pp 1–7Google Scholar
  50. Schäfer-Menuhr A, Schumacher HM, Mix-Wagner G (1997) Cryopreservation of potato cultivars: design of a method for routine application in genebanks. Acta Hortic 447:477–482CrossRefGoogle Scholar
  51. Smartt J, Stalker HT (1982) Speciation and cytogenetics in Arachis. In: Patee HE, Young CT (eds) Peanut science and technology. Yoakum, Texas, pp 21–49Google Scholar
  52. Sullivan ML, Foster JL (2013) Perennial peanut (Arachis glabrata Benth.) contains polyphenol oxidase (PPO) and PPO substrates that can reduce post-harvest proteolysis. J Sci Food Agric 93:2421–2428CrossRefPubMedGoogle Scholar
  53. Tanaka D, Niino T, Isuzugawa K, Hikage T, Uemura M (2004) Cryopreservation of shoot apices of in vitro grown gentian plants: comparison of vitrification and encapsulation-vitrification protocols. Cryo Letters 25:167–176PubMedGoogle Scholar
  54. Towill LE, Bonnart R (2003) Cracking in a vitrification solution during cooling or warming does not affect growth of cryopreserved mint shoot tips. Cryo Letters 24:341–346PubMedGoogle Scholar
  55. Vásquez-Yanes C, Aréchiga MR (1996) Ex situ conservation of tropical rain forest seed: problems and perspectives. Interciencia 21:293–298Google Scholar
  56. Vidoz ML, Klusacek P, Rey HY, Mroginski LA (2006) In vitro plant regeneration of Arachis correntina (Leguminosae) through somatic embryogenesis and organogenesis. Plant Cell Tissue Organ Cult 86:111–115CrossRefGoogle Scholar
  57. Vidoz ML, Rey HY, Gonzalez AM, Mroginski LA (2004) Somatic embryogenesis and plant regeneration through leaf culture in Arachis glabrata (Leguminosae). Acta Physiol Plant 26:59–66CrossRefGoogle Scholar
  58. Vila S, Gonzalez A, Rey H, Mroginski L (2003) Somatic embryogenesis and plant regeneration from immature zygotic embryos of Melia azedarach (Meliaceae). In Vitro Cell Dev Biol Plant 39:283–287CrossRefGoogle Scholar
  59. Wetzstein HY, Baker CM (1993) The relationship between somatic embryo morphology and conversion in peanut (Arachis hypogaea L.). Plant Science 92 (1):81–89Google Scholar
  60. Williams JGK, Kubelic AR, Livak KJ, Rafalski JA, Tingey SW (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535CrossRefPubMedPubMedCentralGoogle Scholar
  61. Zhang N, Wen B, Ji M, Yan Q (2014) Low-temperature storage and cryopreservation of grapefruit (Citrus paradisi Macfad.) seeds. CryoLetters 35:418–426PubMedGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2017

Authors and Affiliations

  • Natalia R. Dolce
    • 1
    • 2
  • Mirta M. Faloci
    • 1
    • 3
  • Ana M. Gonzalez
    • 1
    • 2
  1. 1.Instituto de Botánica del NordesteUniversidad Nacional del Nordeste - Consejo Nacional de Investigaciones Científicas y TécnicasCorrientesArgentina
  2. 2.Facultad de Ciencias AgrariasUniversidad Nacional del NordesteCorrientesArgentina
  3. 3.Facultad de Ciencias Exactas y Naturales y AgrimensuraUniversidad Nacional del NordesteCorrientesArgentina

Personalised recommendations