Skip to main content
Log in

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

  • Micropropagation
  • Published:
In Vitro Cellular & Developmental Biology - Plant Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Figure 1.
Figure 2.
Figure 3.
Figure 4.

Similar content being viewed by others

References

  • 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 156:169–175

    Article  CAS  PubMed  Google Scholar 

  • 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 31:79–88

    Article  CAS  Google Scholar 

  • Bajaj YPS (1979) Freeze preservation of meristems of Arachis hypogaea and Cicer arietinum. Indian J Exp Biol 17:1405–1407

    Google Scholar 

  • Barraco G, Sylvestre I, Iapichino G, Engelmann F (2011) Cryopreservation of Limonium serotinum apical meristems from in vitro plantlets using droplet-vitrification. Sci Hortic 130:309–313

    Article  CAS  Google Scholar 

  • 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 52:512–519

    Article  CAS  Google Scholar 

  • Cassells AC (2001) Contamination and its impact in tissue culture. Acta Hortic 560:353–359

    Article  Google Scholar 

  • 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 27:219–231

    Article  CAS  Google Scholar 

  • Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15

    Google Scholar 

  • Dunbar KB, Pittman RN (1992) Adventitious shoot formation from mature leaf explants of Arachis species. Crop Sci 32:1353–1356

    Article  Google Scholar 

  • Dunbar KB, Pittman RN, Morris JB (1993) In vitro culture of embryonic axes from Arachis species for germplasm recovery. J Seed Technol 17:1–7

    Google Scholar 

  • 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 45–113

    Google Scholar 

  • Fontana ML, Mroginski LA, Rey HY (2009) Organogenesis and plant regeneration of Arachis villosa Benth. (Leguminosae) through leaf culture. Biocell 33:179–186

    PubMed  Google Scholar 

  • 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 87:2899–2905

    Article  CAS  PubMed  Google Scholar 

  • 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 94:2042–2050

    Article  CAS  PubMed  Google Scholar 

  • 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 167–184

    Google Scholar 

  • 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 24:103–110

    CAS  Google Scholar 

  • 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 23:61–68

    CAS  PubMed  Google Scholar 

  • 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 35:445–450

    Article  Google Scholar 

  • Gonzalez AM, Cristóbal CL (1997) Anatomía y ontogenia de semillas de Helicteres lhotzkyana (Sterculiaceae). Bonplandia 9:287–294

    Google Scholar 

  • Halmagyi A, Vălimăreanu S, Coste A, Deliu C, Isac V (2010) Cryopreservation of Malus shoot tips and subsequent plant regeneration. Rom Biotechnol Lett 15:79–85

    CAS  Google Scholar 

  • Kanyand M, Peterson CM, Prakash CS (1997) The differentiation of emergences into adventitious shoots in peanut Arachis hypogaea (L). Plant Sci 126:87–95

    Article  CAS  Google Scholar 

  • Krapovickas A, Gregory WC (1994) Taxonomía del género Arachis (Leguminosae). Bonplandia 8:1–186

  • Leunufna S, Keller ERJ (2003) Investigating a new cryopreservation protocol for yams (Dioscorea spp.) Plant Cell Rep 21:1159–1166

    Article  CAS  PubMed  Google Scholar 

  • 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 10:199–212

    Article  Google Scholar 

  • Mallikarjuna N (2002) Gene introgression from Arachis glabrata into A. hypogaea, A. duranensis and A. diogoi. Euphytica 124:99–105

    Article  CAS  Google Scholar 

  • McKently AH, Moore GA, Gardner FP (1991) Regeneration of peanut and perennial peanut from cultivated leaf tissue. Crop Sci 31:833–837

    Article  Google Scholar 

  • Mithila J, Murch SJ, Krishnaraj S, Saxena PK (2001) Recent advances in Pelargonium in vitro regeneration systems. Plant Cell Tissue Organ Cult 67:1–9

    Article  CAS  Google Scholar 

  • 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 23:129–134

    Article  CAS  Google Scholar 

  • Mroginski LA, Kartha KK (1984) Tissue culture of legumes for crop improvement. Plant Breed Rev 2:215–264

    Google Scholar 

  • Mroginski LA, Katha KK, Shyluk JP (1981) Regeneration of peanut (Arachis hypogaea) by in vitro culture of immature leaves. Can J Bot 59:826–830

    Article  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Murthy BNS, Murch SJ, Saxena PK (1998) Thidiazuron: a potent regulator of in vitro plant morphogenesis. In Vitro Cell Dev Biol Plant 34:267–275

    Article  CAS  Google Scholar 

  • 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 88:67–73

    Article  Google Scholar 

  • Pacheco G, Gagliardi RF, Valls JFM, Mansur E (2009) Micropropagation and in vitro conservation of wild Arachis species. Plant Cell Tissue Organ Cult 99:239–249

    Article  Google Scholar 

  • Panis B, Piette B, Swennen R (2005) Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae. Plant Sci 168:45–55

    Article  CAS  Google Scholar 

  • Panta A, Panis B, Ynouye C, Swennen R, Roca W (2014) Development of a PVS2 droplet vitrification method for potato cryopreservation. Cryo Letters 35:255–266

    CAS  PubMed  Google Scholar 

  • 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 19:733–737

    Article  CAS  Google Scholar 

  • 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 10:21–25

    Article  Google Scholar 

  • 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-332

  • 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-275

  • Rey H, Mroginski L (2009) Cryopreservation of Arachis pintoi (Leguminosae) seeds. Seed Sci Technol 37:202–205

    Article  Google Scholar 

  • Rey HY, Mroginski LA (2003) Regeneration of plants from apical meristem tips and nodal segments of Arachis pintoi. Peanut Sci 30:75–79

    Article  Google Scholar 

  • 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 30:347–358

    CAS  PubMed  Google Scholar 

  • Rey HY, Faloci M, Medina R, Dolce N, Mroginski L, Engelmann F (2013) Cryopreservation of Arachis pintoi (Leguminosae) somatic embryos. Cryo Letters 34:571–582

    CAS  PubMed  Google Scholar 

  • Rey HY, Scocchi AM, Gonzalez AM, Mroginski LA (2000) Plant regeneration in Arachis pintoi (Leguminosae) through leaf culture. Plant Cell Rep 19:856–862

    Article  CAS  Google Scholar 

  • Rohlf FJ (1994) NTSYS-pc numerical taxonomy of multivariate analysis system. Version 2.11W. Exeter software, New York

  • 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 110:79–82

    Google Scholar 

  • Runthala R, Jana MK, Mohanan K (1993) Cryopreservation of groundnut (Arachis hypogaea L.) embryonic axes for germplasm conservation. Cryo Letters 14:323–334

    Google Scholar 

  • 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 1–7

    Google Scholar 

  • Schäfer-Menuhr A, Schumacher HM, Mix-Wagner G (1997) Cryopreservation of potato cultivars: design of a method for routine application in genebanks. Acta Hortic 447:477–482

    Article  Google Scholar 

  • Smartt J, Stalker HT (1982) Speciation and cytogenetics in Arachis. In: Patee HE, Young CT (eds) Peanut science and technology. Yoakum, Texas, pp 21–49

    Google Scholar 

  • 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 93:2421–2428

    Article  CAS  PubMed  Google Scholar 

  • 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 25:167–176

    PubMed  Google Scholar 

  • 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 24:341–346

    PubMed  Google Scholar 

  • Vásquez-Yanes C, Aréchiga MR (1996) Ex situ conservation of tropical rain forest seed: problems and perspectives. Interciencia 21:293–298

    Google Scholar 

  • 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 86:111–115

    Article  Google Scholar 

  • Vidoz ML, Rey HY, Gonzalez AM, Mroginski LA (2004) Somatic embryogenesis and plant regeneration through leaf culture in Arachis glabrata (Leguminosae). Acta Physiol Plant 26:59–66

    Article  Google Scholar 

  • 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 39:283–287

    Article  CAS  Google Scholar 

  • Wetzstein HY, Baker CM (1993) The relationship between somatic embryo morphology and conversion in peanut (Arachis hypogaea L.). Plant Science 92 (1):81–89

  • 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 18:6531–6535

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhang N, Wen B, Ji M, Yan Q (2014) Low-temperature storage and cryopreservation of grapefruit (Citrus paradisi Macfad.) seeds. CryoLetters 35:418–426

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

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.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Natalia R. Dolce.

Additional information

Editor: Ewen Mullins

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dolce, N.R., Faloci, M.M. & Gonzalez, A.M. In vitro plant regeneration and cryopreservation of Arachis glabrata (Fabaceae) using leaflet explants. In Vitro Cell.Dev.Biol.-Plant 54, 133–144 (2018). https://doi.org/10.1007/s11627-017-9865-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11627-017-9865-y

Keywords

Navigation