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Article|02 Feb 2022|OPEN
Variation burst during dedifferentiation and increased CHH-type DNA methylation after 30 years of in vitro culture of sweet orange
Xia Wang1 , Lili Ke1 , Shuting Wang1 , Jialing Fu1 , Jidi Xu1 , Yujin Hao1 , Chunying Kang1 , Wenwu Guo1 , Xiuxin Deng1 and Qiang Xu,1 ,
1Key Laboratory of Horticultural Plant Biology (Ministry of Education), Huazhong Agricultural University, No. 1, Shizishan Street, Wuhan 430070, China
*Corresponding author. E-mail:

Horticulture Research 9,
Article number: uhab036 (2022)
Views: 607

Received: 02 Jul 2021
Revised: 15 Oct 2021
Accepted: 18 Jan 2022
Published online: 02 Feb 2022


Somaclonal variation arising from tissue culture may provide a valuable resource for the selection of new germplasm, but may not preserve true-to-type characteristics, which is a major concern for germplasm conservation or genome editing. The genomic changes associated with dedifferentiation and somaclonal variation during long-term in vitro culture are largely unknown. Sweet orange was one of the earliest plant species to be cultured in vitro and induced via somatic embryogenesis. We compared four sweet orange callus lines after 30 years of constant tissue culture with newly induced calli by comprehensively determining the single-nucleotide polymorphisms, copy number variations, transposable element insertions, methylomic and transcriptomic changes. We identified a burst of variation during early dedifferentiation, including a retrotransposon outbreak, followed by a variation purge during long-term in vitro culture. Notably, CHH methylation showed a dynamic pattern, initially disappearing during dedifferentiation and then more than recovering after 30 years of in vitro culture. We also analyzed the effects of somaclonal variation on transcriptional reprogramming, and indicated subgenome dominance was evident in the tetraploid callus. We identified a retrotransposon insertion and DNA modification alternations in the potential regeneration-related gene CLAVATA3/EMBRYO SURROUNDING REGION-RELATED 16. This study provides the foundation to harness in vitro variation and offers a deeper understanding of the variation introduced by tissue culture during germplasm conservation, somatic embryogenesis, gene editing, and breeding programs.