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

• Published in: In vitro cellular & developmental biology. Plant 2018-04, Vol.54 (2), p.133-144
• Main Authors: Dolce, Natalia R., Faloci, Mirta M., Gonzalez, Ana M.
• Format: Article
• Language: English
• Subjects: Aluminum; Arachis glabrata; Biomedical and Life Sciences; Cell Biology; Cryopreservation; Cultivars; Culture media; Deoxyribonucleic acid; Developmental Biology; DNA; Embryonic growth stage; Embryos; Explants; Fabaceae; Foils; Genetic resources; Genotypes; Germplasm; Glycerol; Leaves; Legumes; Leguminosae; Life Sciences; Liquid nitrogen; MICROPROPAGATION; Multivariate analysis; Nitrogen; Organogenesis; Peanuts; Plant Breeding/Biotechnology; Plant Genetics and Genomics; Plant resources; Plant Sciences; Plants; Polymerase chain reaction; Preconditioning; Random amplified polymorphic DNA; Regeneration; Seeds; Semisolids; Soil conservation; Somatic embryogenesis; Sucrose; Sugar; Urea; Vitrification
• ISSN: 1054-5476; 1475-2689
• DOI: 10.1007/s11627-017-9865-y

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.