Cryopreservation of Oryza Sativa L. and Evaluation of Genetic stability of the Regenerants.

Abstract

Abstract Cryopreservation, a widely utilized technique for long-term preservation of in vitro cultures, effectively arrests metabolic processes, obviating the need for frequent subcultures and mitigating the risk of somaclonal variation. In this study, we applied cryopreservation methods to intact rice (Oryza sativa L.) calli to determine the optimal age for cryopreservation, investigating the timelines in order to produce chlorophyll, generate chloroplasts, and shoot initiation in R0 plants derived from calli of varying ages. Results revealed that three-month-old calli exhibited the highest regeneration percentage, with chlorophyll development and greening observed within twelve days, and shoot initiation within fifteen days. Subsequent cultivation involved seeds from these matured plants alongside seed-derived plants. Phenotypic and molecular analyses assessed the genetic fidelity of regenerated progenies in R1 and R2. Comparison of twelve qualitative and quantitative characters indicated variations among cryopreserved, control callus, and seed-derived plants. Molecular data, utilizing twenty four rice simple sequence repeat markers, demonstrated a 3.6–7.25% deviation from the control. In the R1 generation, in vitro-derived plants displayed nearly identical characteristics, except for increased plant height and spikelet fertility in seed-derived plants, and extended time to maturity in all in vitro-derived plants. However, these distinctions were absent in the R2 generation.