{"title":"胚胎胼胝体的低温保存:评估胼胝体年龄对再生的影响;再生体的形态和遗传稳定性","authors":"Patu Khate Zeliang, A. Pattanayak","doi":"10.1007/s11240-024-02821-6","DOIUrl":null,"url":null,"abstract":"<p>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 (<i>Oryza sativa</i> 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.</p>","PeriodicalId":20219,"journal":{"name":"Plant Cell, Tissue and Organ Culture","volume":"14 1","pages":""},"PeriodicalIF":2.3000,"publicationDate":"2024-07-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Cryopreservation of embryogenic callus in Oryza sativa L.: Assessment of impact of callus age on regeneration; morphological and genetic stability regenerants\",\"authors\":\"Patu Khate Zeliang, A. Pattanayak\",\"doi\":\"10.1007/s11240-024-02821-6\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p>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 (<i>Oryza sativa</i> 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.</p>\",\"PeriodicalId\":20219,\"journal\":{\"name\":\"Plant Cell, Tissue and Organ Culture\",\"volume\":\"14 1\",\"pages\":\"\"},\"PeriodicalIF\":2.3000,\"publicationDate\":\"2024-07-25\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Cell, Tissue and Organ Culture\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1007/s11240-024-02821-6\",\"RegionNum\":3,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Cell, Tissue and Organ Culture","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1007/s11240-024-02821-6","RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
引用次数: 0
摘要
低温保存是一种广泛应用的长期保存体外培养物的技术,它能有效地阻止新陈代谢过程,从而避免了频繁的移栽,降低了体细胞变异的风险。在本研究中,我们对完整的水稻(Oryza sativa L.)胼胝体采用了低温保存方法,以确定低温保存的最佳年龄,并研究了 R0 植株的返青和发芽时间。结果显示,三个月龄的胼胝体显示出最高的再生率,在十二天内观察到绿色,在十五天内观察到嫩芽萌发。在胼胝体培养基中使用 3% 的甘露醇可提供渗透压,有助于形成适合再生的紧凑胼胝体块。使用低温保护剂(DMSO、PEG 和葡萄糖)进行玻璃化和逐渐脱水可提高细胞存活率。通过快速解冻、快速移除低温保护剂和逐步脱水,解冻和解冻后的损伤可降至最低。我们评估了 R1 和 R2 代的表型变化以及 R1 代再生体的基因型保真度。在玻璃化和低温保存的胼胝体衍生植株的种子特征中都观察到了与种子衍生植株的表型变异。然而,这些变异并不稳定,在 R2 中消失了。使用 SSR 标记检测 R1 中的遗传变异,结果显示玻璃化胼胝体衍生植株的变化率为 3.6%,冷冻衍生植株的变化率为 8.61%,这可能是由于可逆的 DNA 甲基化或非编码区的 SNP 所致。我们的研究证实了冷冻保存水稻胼胝体的可行性,确保了高再生率和最小的长期遗传变异。
Cryopreservation of embryogenic callus in Oryza sativa L.: Assessment of impact of callus age on regeneration; morphological and genetic stability regenerants
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.