{"title":"利用实时荧光定量PCR和扫描电镜技术监测粗柠檬植株转化后嵌合解离的情况。","authors":"Gautam Chhabra, Manveer Sharma, Anu Kalia, Ajinder Kaur, Jagdeep Singh Sandhu","doi":"10.1007/s11816-023-00877-y","DOIUrl":null,"url":null,"abstract":"<p><i>Citrus</i> spp. are recalcitrant to in vitro shoot regeneration and we report an improved in planta protocol for genetic transformation of rough lemon that bypasses shoot regeneration in tissue culture. The features of the protocol were the use of an <i>Agrobacterium</i> suspension with an OD<sub>600 nm</sub> = 0.6–1.0 supplemented with 100 μg acetosyringone, gentle shaking of embryo axes pricked at shoot apical meristems (from 2-day-old germinating seeds) at 70 rpm during agro-infection, followed by growth and development of plantlets at 30 °C. PCR screening of 2-month-old <i>T</i><sub>0</sub> plants revealed the presence of an amplicon corresponding to the <i>β</i>-1,3-<i>glucanase</i> gene in the primary branches of 25 plants with a transformation efficiency of 7.74%. PCR analysis of the secondary branches of these plants after 18 months showed chimerism, i.e., the coexistence of transformed and untransformed branches in all 25 plants. Quantification of <i>β</i>-1,3-<i>glucanase</i> expression in the transformed secondary branches by qRT-PCR showed that plant number 32 had maximum (3.71-fold) relative transgene expression. The qRT-PCR analysis of all four tertiary branches arising from the transformed secondary branch of plant number 32 showed no significant differences in expression among themselves and from the transformed secondary branch, suggesting restoration of the transformed branches with uniform expression and dissociation of chimerism. Scanning electron microscopy examination of leaves from secondary and tertiary branches that uniformly expressed the transgene showed a smooth, waxy surface with non-significant variation in stomata, which had a narrow opening and a mean pore length of 4.22 ± 0.25–5.09 ± 0.36 µm. In contrast, the leaves of untransformed branch had a rough surface and a significantly large stomatal opening with a mean pore length of 7.82 ± 0.67 µm. The micro-morphological characteristics of the leaves confirmed the dissociation of chimerism in the transformed tertiary branches of plant number 32. The study demonstrates identification of chimerism after in planta transformation using PCR technique, and the novelty relates to monitoring dissociation of chimerism in transformed tertiary branches of <i>T</i><sub>0</sub> generation using qRT-PCR analysis and its corroboration by electron microscopy. The protocol for genetic transformation in plants described in the present study can be used for trait improvement by transgenesis.</p>","PeriodicalId":20216,"journal":{"name":"Plant Biotechnology Reports","volume":null,"pages":null},"PeriodicalIF":1.7000,"publicationDate":"2023-11-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":"{\"title\":\"Monitoring dissociation of chimerism through real-time PCR and scanning electron microscopy following in planta transformation of rough lemon (Citrus jambhiri Lush.)\",\"authors\":\"Gautam Chhabra, Manveer Sharma, Anu Kalia, Ajinder Kaur, Jagdeep Singh Sandhu\",\"doi\":\"10.1007/s11816-023-00877-y\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><i>Citrus</i> spp. are recalcitrant to in vitro shoot regeneration and we report an improved in planta protocol for genetic transformation of rough lemon that bypasses shoot regeneration in tissue culture. The features of the protocol were the use of an <i>Agrobacterium</i> suspension with an OD<sub>600 nm</sub> = 0.6–1.0 supplemented with 100 μg acetosyringone, gentle shaking of embryo axes pricked at shoot apical meristems (from 2-day-old germinating seeds) at 70 rpm during agro-infection, followed by growth and development of plantlets at 30 °C. PCR screening of 2-month-old <i>T</i><sub>0</sub> plants revealed the presence of an amplicon corresponding to the <i>β</i>-1,3-<i>glucanase</i> gene in the primary branches of 25 plants with a transformation efficiency of 7.74%. PCR analysis of the secondary branches of these plants after 18 months showed chimerism, i.e., the coexistence of transformed and untransformed branches in all 25 plants. Quantification of <i>β</i>-1,3-<i>glucanase</i> expression in the transformed secondary branches by qRT-PCR showed that plant number 32 had maximum (3.71-fold) relative transgene expression. The qRT-PCR analysis of all four tertiary branches arising from the transformed secondary branch of plant number 32 showed no significant differences in expression among themselves and from the transformed secondary branch, suggesting restoration of the transformed branches with uniform expression and dissociation of chimerism. Scanning electron microscopy examination of leaves from secondary and tertiary branches that uniformly expressed the transgene showed a smooth, waxy surface with non-significant variation in stomata, which had a narrow opening and a mean pore length of 4.22 ± 0.25–5.09 ± 0.36 µm. In contrast, the leaves of untransformed branch had a rough surface and a significantly large stomatal opening with a mean pore length of 7.82 ± 0.67 µm. The micro-morphological characteristics of the leaves confirmed the dissociation of chimerism in the transformed tertiary branches of plant number 32. The study demonstrates identification of chimerism after in planta transformation using PCR technique, and the novelty relates to monitoring dissociation of chimerism in transformed tertiary branches of <i>T</i><sub>0</sub> generation using qRT-PCR analysis and its corroboration by electron microscopy. The protocol for genetic transformation in plants described in the present study can be used for trait improvement by transgenesis.</p>\",\"PeriodicalId\":20216,\"journal\":{\"name\":\"Plant Biotechnology Reports\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":1.7000,\"publicationDate\":\"2023-11-24\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Plant Biotechnology Reports\",\"FirstCategoryId\":\"5\",\"ListUrlMain\":\"https://doi.org/10.1007/s11816-023-00877-y\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q4\",\"JCRName\":\"BIOTECHNOLOGY & APPLIED MICROBIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Plant Biotechnology Reports","FirstCategoryId":"5","ListUrlMain":"https://doi.org/10.1007/s11816-023-00877-y","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q4","JCRName":"BIOTECHNOLOGY & APPLIED MICROBIOLOGY","Score":null,"Total":0}
Monitoring dissociation of chimerism through real-time PCR and scanning electron microscopy following in planta transformation of rough lemon (Citrus jambhiri Lush.)
Citrus spp. are recalcitrant to in vitro shoot regeneration and we report an improved in planta protocol for genetic transformation of rough lemon that bypasses shoot regeneration in tissue culture. The features of the protocol were the use of an Agrobacterium suspension with an OD600 nm = 0.6–1.0 supplemented with 100 μg acetosyringone, gentle shaking of embryo axes pricked at shoot apical meristems (from 2-day-old germinating seeds) at 70 rpm during agro-infection, followed by growth and development of plantlets at 30 °C. PCR screening of 2-month-old T0 plants revealed the presence of an amplicon corresponding to the β-1,3-glucanase gene in the primary branches of 25 plants with a transformation efficiency of 7.74%. PCR analysis of the secondary branches of these plants after 18 months showed chimerism, i.e., the coexistence of transformed and untransformed branches in all 25 plants. Quantification of β-1,3-glucanase expression in the transformed secondary branches by qRT-PCR showed that plant number 32 had maximum (3.71-fold) relative transgene expression. The qRT-PCR analysis of all four tertiary branches arising from the transformed secondary branch of plant number 32 showed no significant differences in expression among themselves and from the transformed secondary branch, suggesting restoration of the transformed branches with uniform expression and dissociation of chimerism. Scanning electron microscopy examination of leaves from secondary and tertiary branches that uniformly expressed the transgene showed a smooth, waxy surface with non-significant variation in stomata, which had a narrow opening and a mean pore length of 4.22 ± 0.25–5.09 ± 0.36 µm. In contrast, the leaves of untransformed branch had a rough surface and a significantly large stomatal opening with a mean pore length of 7.82 ± 0.67 µm. The micro-morphological characteristics of the leaves confirmed the dissociation of chimerism in the transformed tertiary branches of plant number 32. The study demonstrates identification of chimerism after in planta transformation using PCR technique, and the novelty relates to monitoring dissociation of chimerism in transformed tertiary branches of T0 generation using qRT-PCR analysis and its corroboration by electron microscopy. The protocol for genetic transformation in plants described in the present study can be used for trait improvement by transgenesis.
期刊介绍:
Plant Biotechnology Reports publishes original, peer-reviewed articles dealing with all aspects of fundamental and applied research in the field of plant biotechnology, which includes molecular biology, genetics, biochemistry, cell and tissue culture, production of secondary metabolites, metabolic engineering, genomics, proteomics, and metabolomics. Plant Biotechnology Reports emphasizes studies on plants indigenous to the Asia-Pacific region and studies related to commercialization of plant biotechnology. Plant Biotechnology Reports does not exclude studies on lower plants including algae and cyanobacteria if studies are carried out within the aspects described above.