A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro
‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange...
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| Format: | Article |
| Language: | English |
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Elsevier
2022-06-01
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| Series: | Journal of Integrative Agriculture |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S209531192163856X |
| _version_ | 1818200275347832832 |
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| author | Tian-gang LEI Yong-rui HE Xiu-ping ZOU Xue-feng WANG Shi-min FU Ai-hong PENG Lan-zhen XU Li-xiao YAO Shan-chun CHEN Chang-yong ZHOU |
| author_facet | Tian-gang LEI Yong-rui HE Xiu-ping ZOU Xue-feng WANG Shi-min FU Ai-hong PENG Lan-zhen XU Li-xiao YAO Shan-chun CHEN Chang-yong ZHOU |
| author_sort | Tian-gang LEI |
| collection | DOAJ |
| description | ‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG). This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×104 to 1.4×108 cells μg–1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×108 CLas cells μg–1 DNA were 30 and 40% at 30 and 40 DAG, respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro. This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus. |
| first_indexed | 2024-12-12T02:35:04Z |
| format | Article |
| id | doaj.art-7d3c80240ae14602ac994e6cf283f9fb |
| institution | Directory Open Access Journal |
| issn | 2095-3119 |
| language | English |
| last_indexed | 2024-12-12T02:35:04Z |
| publishDate | 2022-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Journal of Integrative Agriculture |
| spelling | doaj.art-7d3c80240ae14602ac994e6cf283f9fb2022-12-22T00:41:18ZengElsevierJournal of Integrative Agriculture2095-31192022-06-0121616831693A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitroTian-gang LEI0Yong-rui HE1Xiu-ping ZOU2Xue-feng WANG3Shi-min FU4Ai-hong PENG5Lan-zhen XU6Li-xiao YAO7Shan-chun CHEN8Chang-yong ZHOU9Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCitrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCorrespondence CHEN Shan-chun, Tel: +86-23-683409709; Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.ChinaCorrespondence ZHOU Chang-yong, Tel: +86-23-68349707; Citrus Research Institute, Southwest University/National Citrus Engineering Research Center, Chongqing 400712, P.R.China‘Candidatus Liberibacter asiaticus (CLas)’, which causes citrus Huanglongbing (HLB) disease, has not been successfully cultured in vitro to date. Here, a rapid multiplication system for CLas was established through in vitro regeneration of axillary buds from CLas-infected ‘Changyecheng’ sweet orange (Citrus sinensis Osbeck). Stem segments with a single axillary bud were cultured in vitro to allow CLas to multiply in the regenerating axillary buds. A high CLas titer was detected in the regenerated shoots on an optimized medium at 30 days after germination (DAG). This titer was 28.2-fold higher than in the midribs from CLas-infected trees growing in the greenhouse. To minimize contamination during in vitro regeneration, CLas-infected axillary buds were micrografted onto seedlings of ‘Changyecheng’ sweet orange and cultured in a liquid medium. In this culture, the titers of CLas in regenerated shoots rapidly increased from 7.5×104 to 1.4×108 cells μg–1 of citrus DNA during the first 40 DAG. The percentages of shoots with >1×108 CLas cells μg–1 DNA were 30 and 40% at 30 and 40 DAG, respectively. Direct tissue blot immunoassay (DTBIA) indicated that the distribution of CLas was much more uniform in regenerated plantlets than in CLas-infected trees growing in the greenhouse. The disease symptoms in the plantlets were die-back, stunted growth, leaf necrosis/yellowing, and defoliation. The death rate of the plantlets was 82.0% at 60 DAG. Our results show that CLas can effectively multiply in citrus plantlests in vitro. This method will be useful for studying plant-HLB interactions and for rapid screening of therapeutic compounds against CLas in citrus.http://www.sciencedirect.com/science/article/pii/S209531192163856XCitrus‘Candidatus Liberibacter asiaticus’multiplicationin vitro citrus plantlets |
| spellingShingle | Tian-gang LEI Yong-rui HE Xiu-ping ZOU Xue-feng WANG Shi-min FU Ai-hong PENG Lan-zhen XU Li-xiao YAO Shan-chun CHEN Chang-yong ZHOU A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro Journal of Integrative Agriculture Citrus ‘Candidatus Liberibacter asiaticus’ multiplication in vitro citrus plantlets |
| title | A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro |
| title_full | A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro |
| title_fullStr | A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro |
| title_full_unstemmed | A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro |
| title_short | A rapid multiplication system for ‘Candidatus Liberibacter asiaticus’ through regeneration of axillary buds in vitro |
| title_sort | rapid multiplication system for candidatus liberibacter asiaticus through regeneration of axillary buds in vitro |
| topic | Citrus ‘Candidatus Liberibacter asiaticus’ multiplication in vitro citrus plantlets |
| url | http://www.sciencedirect.com/science/article/pii/S209531192163856X |
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