Cryopreservation of Bituminaria bituminosa varieties and hybrids

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.