Cryopreservation of embryogenic callus in Oryza sativa L.: Assessment of impact of callus age on regeneration; morphological and genetic stability regenerants
IF 2.3 3区 生物学Q3 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
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引用次数: 0
Abstract
Cryopreservation, a widely utilized technique for the 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 for greening and shoot initiation in R0 plants. Results revealed that three-month-old calli exhibited the highest regeneration percentage, with greening observed within twelve days and shoot initiation within fifteen days. Using 3% mannitol in the callus culture medium provided osmotic stress, aiding in the formation of compact calli masses suitable for regeneration. Vitrification with cryoprotectants (DMSO, PEG, and glucose) and gradual dehydration improved cell survival. Thawing and post-thaw damage were minimized using rapid thawing, fast cryoprotectant removal, and gradual rehydration. We assessed the phenotypic variations in R1 and R2 generation and genotypic fidelity of regenerants in R1. Phenotypic variations from seed-derived plants were observed in seed characters both in vitrified and cryopreserved calli-derived plants. However, these variations were unstable and disappeared in the R2. SSR markers were used to detect genetic variations in R1, with results showing a 3.6% change in vitrified calli-derived plants and an 8.61% change in cryopreservation-derived plants, likely due to reversible DNA methylation or SNPs in non-coding region. Our study confirms the feasibility of cryopreservation for rice calli, ensuring high regeneration rates and minimal long-term genetic variations.
期刊介绍:
This journal highlights the myriad breakthrough technologies and discoveries in plant biology and biotechnology. Plant Cell, Tissue and Organ Culture (PCTOC: Journal of Plant Biotechnology) details high-throughput analysis of gene function and expression, gene silencing and overexpression analyses, RNAi, siRNA, and miRNA studies, and much more. It examines the transcriptional and/or translational events involved in gene regulation as well as those molecular controls involved in morphogenesis of plant cells and tissues.
The journal also covers practical and applied plant biotechnology, including regeneration, organogenesis and somatic embryogenesis, gene transfer, gene flow, secondary metabolites, metabolic engineering, and impact of transgene(s) dissemination into managed and unmanaged plant systems.