An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis)
Moso bamboo ( Phyllostachys edulis ) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regenerat...
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| Published in | Frontiers in plant science Vol. 13; p. 822022 |
|---|---|
| Main Authors | , , , , , , |
| Format | Journal Article |
| Language | English |
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11.02.2022
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| Abstract | Moso bamboo (
Phyllostachys edulis
) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L
–1
2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L
–1
1-naphthylacetic acid (NAA), 2.0 mg⋅L
–1
6-benzylaminopurine (BAP), and 3.0 mg⋅L
–1
zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L
–1
abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the
Agrobacterium
concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the
PeU3.1
promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous
pds1pds2
mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo. |
|---|---|
| AbstractList | Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L–1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L–1 1-naphthylacetic acid (NAA), 2.0 mg⋅L–1 6-benzylaminopurine (BAP), and 3.0 mg⋅L–1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L–1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo. Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4-6 mg⋅L-1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L-1 1-naphthylacetic acid (NAA), 2.0 mg⋅L-1 6-benzylaminopurine (BAP), and 3.0 mg⋅L-1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L-1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo.Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4-6 mg⋅L-1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L-1 1-naphthylacetic acid (NAA), 2.0 mg⋅L-1 6-benzylaminopurine (BAP), and 3.0 mg⋅L-1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L-1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo. Moso bamboo ( Phyllostachys edulis ) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4–6 mg⋅L –1 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L –1 1-naphthylacetic acid (NAA), 2.0 mg⋅L –1 6-benzylaminopurine (BAP), and 3.0 mg⋅L –1 zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L –1 abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the Agrobacterium concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the PeU3.1 promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous pds1pds2 mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo. Moso bamboo ( ) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of genes controlling important traits and conduct molecular breeding in moso bamboo. Here, we established a plant regeneration system from immature embryos. Calli were induced on MS medium added 4-6 mg⋅L 2,4-dichlorophenoxyacetic acid (2,4-D) with high efficiency (>60%). A plant growth regulator combination of 0.5 mg⋅L 1-naphthylacetic acid (NAA), 2.0 mg⋅L 6-benzylaminopurine (BAP), and 3.0 mg⋅L zeatin (ZT) was suitable for shoot differentiation, and the shoot induction frequency was increased to 43% after 0.5 mg⋅L abscisic acid (ABA) pretreatment. An effective antibiotic screening concentration was determined by hygromycin sensitivity test. We further optimized the concentration and added vacuum infiltration for infection, which improves the transient expression efficiency. A genetic transformation system was established for the first time in moso bamboo, with the transformation efficiency of approximately 5%. To optimize genome editing, two endogenous U3 small nuclear RNA (snRNA) promoters were isolated and used to drive small guide RNA (sgRNA) expression. The results showed that the promoter exhibited higher efficiency, and it was used for subsequent genome editing. Finally, homozygous mutants were obtained by an efficient CRISPR/Cas9 genome-editing system. These technical systems will be conducive to gene functional validation and accelerate the molecular breeding process of moso bamboo. |
| Author | Jin, Kangming Xu, Jing Fan, Huijin Wang, Yujun Zhuo, Renying Huang, Biyun Qiao, Guirong |
| AuthorAffiliation | 1 State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry , Beijing , China 2 Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of Subtropical of Forestry, Chinese Academy of Forestry , Hangzhou , China |
| AuthorAffiliation_xml | – name: 2 Key Laboratory of Tree Breeding of Zhejiang Province, Research Institute of Subtropical of Forestry, Chinese Academy of Forestry , Hangzhou , China – name: 1 State Key Laboratory of Tree Genetics and Breeding, Chinese Academy of Forestry , Beijing , China |
| Author_xml | – sequence: 1 givenname: Biyun surname: Huang fullname: Huang, Biyun – sequence: 2 givenname: Renying surname: Zhuo fullname: Zhuo, Renying – sequence: 3 givenname: Huijin surname: Fan fullname: Fan, Huijin – sequence: 4 givenname: Yujun surname: Wang fullname: Wang, Yujun – sequence: 5 givenname: Jing surname: Xu fullname: Xu, Jing – sequence: 6 givenname: Kangming surname: Jin fullname: Jin, Kangming – sequence: 7 givenname: Guirong surname: Qiao fullname: Qiao, Guirong |
| BackLink | https://www.ncbi.nlm.nih.gov/pubmed/35222480$$D View this record in MEDLINE/PubMed |
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| CitedBy_id | crossref_primary_10_3389_fgene_2022_989199 crossref_primary_10_1111_nph_19398 crossref_primary_10_1007_s10725_024_01236_9 crossref_primary_10_1111_tpj_16420 crossref_primary_10_1186_s12864_024_10176_7 crossref_primary_10_1007_s13237_024_00498_y crossref_primary_10_1111_nph_20386 crossref_primary_10_1186_s13007_023_00993_4 crossref_primary_10_3390_ijms24065103 crossref_primary_10_1093_treephys_tpad086 crossref_primary_10_1360_TB_2023_1125 crossref_primary_10_3390_horticulturae9060685 crossref_primary_10_1111_jipb_13468 crossref_primary_10_1186_s12864_024_09966_w crossref_primary_10_1007_s00709_022_01765_y crossref_primary_10_1186_s12870_024_05717_x crossref_primary_10_3390_plants11101297 crossref_primary_10_3390_f15071180 |
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| ContentType | Journal Article |
| Copyright | Copyright © 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao. Copyright © 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao. 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao |
| Copyright_xml | – notice: Copyright © 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao. – notice: Copyright © 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao. 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao |
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| Keywords | plant regeneration genome editing Phyllostachys edulis genetic transformation immature embryo culture |
| Language | English |
| License | Copyright © 2022 Huang, Zhuo, Fan, Wang, Xu, Jin and Qiao. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
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| Notes | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Reviewed by: Kan Wang, Iowa State University, United States; Jitesh Kumar, University of Minnesota Twin Cities, United States; Weiguo Zhang, Northwest University, China This article was submitted to Plant Biotechnology, a section of the journal Frontiers in Plant Science These authors have contributed equally to this work Edited by: Peng Zhang, Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences (CAS), China |
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| Snippet | Moso bamboo (
Phyllostachys edulis
) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to... Moso bamboo ( ) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to verify the functions of... Moso bamboo (Phyllostachys edulis) is the most important monopodial bamboo species worldwide. Without a genetic transformation system, it is difficult to... |
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| SubjectTerms | genetic transformation genome editing immature embryo culture Phyllostachys edulis plant regeneration Plant Science |
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| Title | An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis) |
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