Physiological and genomic variations in rice cells recovered from direct immersion and storage in liquid nitrogen

The use of cryoprotectants and slow cooling rates are routine procedures fo r the cryopreservation of plant cell lines. However, our results with rice (Oryza sativa L,, ev. Taipei 309) show that calli can be cryopreserved by d irect immersion and stored in liquid nitrogen without any cryoprotection, T he efficiency of recovery using this method, as well as a conventional meth od was generally increased with a previous abscisic acid (ABA) treatment. F ollowing cryopreservation, calli demonstrated some differences with respect to unfrozen calli of the same lines, Thus, resistance to freezing stress ( - 20 degrees C for 2 h) increased significantly in all lines tested, irresp ective of their pre-incubation with ABA, Calli that had been directly store d in liquid nitrogen also demonstrated a higher competence for genetic tran sformation than their unfrozen counterparts, as indicated by the transient gene expression levels obtained after particle bombardment, These differenc es might lead to further biotechnological applications, A genetic analysis of amplified DNA polymorphisms was performed with three independent lines t hat had been subjected to four combinations of ABA treatment and direct imm ersion in liquid nitrogen, At the loci screened with the randomly amplified polymorphic DNA (RAPD) markers tested, the genetic variations among lines and among calli of the same line appear to bd more related to tissue-cultur e-induced somaclonal variation than to cryoselection.