title: Cryopreservation of Bituminaria bituminosa varieties and hybrids.
abstract: Bituminaria bituminosa (L.) C.H. Stirton is a drought tolerant, perennial legume pasture species and a source of pharmaceutical compounds. Bituminaria breeding programs aim to develop and conserve hybrids with desirable traits such as high forage quality, tolerance to biotic or abiotic stresses, and high contents of furanocoumarins. In this work we present a cryopreservation study of different B. bituminosa accessions: two varieties and eight intervarietal hybrids resulting from crosses between the three botanical varieties: var. bituminosa, var. crassiuscula, and var. albomarginata. No previous work on cryopreservation of Bituminaria species has been reported. We applied the ultra-fast cooling method, using droplet vitrification on aluminum foil strips. First, we investigated the PVS2 toxicity and cryopreservation damage in two genotypes, comparing three PVS2 treatments and two culture media. An incubation of 30 min in PVS2 resulted in regeneration rates after cryopreservation higher than 80%. The MS medium was selected for optimal meristem outgrowth, in order to avoid the prominent callus formation that was observed in the presence of BAP. These conditions were subsequently used to cryopreserve eight other genotypes. The results were highly variable; 45 days after cryopreservation, survival ranged between 22% and 98% while regeneration ranged between 0% and 96%, depending on the accession. A significant and positive correlation was observed between survival and regeneration. At 90 days post culture plantlets could be recovered from cryopreserved explants of all genotypes. This study shows that the droplet vitrification method is promising for the cryopreservation of eight of the 10 genotypes assayed and the method can thus be applied to develop a cryobank of B. bituminosa.
authors.affiliation: Instituto de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universitat Politècnica de València, Camino de Vera, 14 46022 Valencia, Spain. Electronic address: cgisbert@btc.upv.es.
authors.forename: Carmina
authors.initials: C
authors.lastname: Gisbert
authors.affiliation: Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Department of Natural Resources, Calle Mayor s/n, 30150 La Alberca, Murcia, Spain. Electronic address: mercedes.dabauza@carm.es.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Dabauza
authors.affiliation: Instituto Murciano de Investigación y Desarrollo Agrario y Alimentario (IMIDA), Department of Natural Resources, Calle Mayor s/n, 30150 La Alberca, Murcia, Spain. Electronic address: enrique.correal@carm.es.
authors.forename: Enrique
authors.initials: E
authors.lastname: Correal
authors.affiliation: Laboratory of Tropical Crop Improvement, KU Leuven, Willem de Croylaan 42, 3001 Leuven, Belgium; International Institute of Tropical Agriculture, P.O. Box 10, Duluti, Arusha, Tanzania; Bioversity International, Willem de Croylaan 42, 3001 Leuven, Belgium. Electronic address: rony.swennen@biw.kuleuven.be.
authors.forename: Rony
authors.initials: R
authors.lastname: Swennen
authors.affiliation: Bioversity International, Willem de Croylaan 42, 3001 Leuven, Belgium. Electronic address: b.panis@cgair.org.
authors.forename: Bart
authors.initials: B
authors.lastname: Panis
mesh_terms: D002678:Chimera; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology / Q000633:toxicity; D003470:Culture Media; D007887:Fabaceae / Q000145:classification / Q000254:growth & development*; D005838:Genotype; D018519:Meristem / Q000254:growth & development*; D058989:Vitrification
chemical_list: D003451:Cryoprotective Agents; D003470:Culture Media
keywords: Droplet vitrification; Psoralea bituminosa; Tedera; var. Crassiuscula; var. albomarginata