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。

IF 1.7 4区 生物学 Q4 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Gautam Chhabra, Manveer Sharma, Anu Kalia, Ajinder Kaur, Jagdeep Singh Sandhu
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引用次数: 0

摘要

柑橘类植物对离体茎再生有抗性,我们报道了一种改进的植物内粗柠檬遗传转化方案,绕过组织培养的茎再生。该方案的特点是,在农业感染期间,使用OD600 nm = 0.6-1.0的农杆菌悬浮液,添加100 μg乙酰丁香酮,以70 rpm的速度轻轻摇动芽尖分生组织的胚轴(来自萌发2 d的种子),然后在30°C下进行植株的生长发育。对2月龄T0植株进行PCR筛选,25株植株一次枝上存在β-1,3-葡聚糖酶基因对应的扩增子,转化效率为7.74%。经过18个月的PCR分析,25株次生枝均嵌合,即转化枝与未转化枝并存。利用qRT-PCR对转化后的次生枝中β-1,3-葡聚糖酶的表达量进行定量分析,结果表明,植株号32的相对转基因表达量最高,为3.71倍。对32号植株转化后的二级分支产生的4个三级分支进行qRT-PCR分析,结果显示它们之间的表达和转化后的二级分支之间的表达没有显著差异,表明转化后的分支恢复了均匀表达和嵌合解离。对均匀表达转基因的二、三枝叶片进行扫描电镜观察,叶片表面光滑、蜡质,气孔变化不显著,气孔开口窄,平均孔长为4.22±0.25 ~ 5.09±0.36µm。未转化枝叶片表面粗糙,气孔开口明显较大,平均气孔长度为7.82±0.67µm。叶片的微观形态特征证实了32号植株三级枝嵌合的解离。该研究利用PCR技术鉴定了植物转化后的嵌合现象,其新颖之处在于利用qRT-PCR分析和电子显微镜证实了T0代转化三级分支中嵌合现象的解离。本研究描述的植物遗传转化方案可用于通过转基因进行性状改良。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Monitoring dissociation of chimerism through real-time PCR and scanning electron microscopy following in planta transformation of rough lemon (Citrus jambhiri Lush.)

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.

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来源期刊
Plant Biotechnology Reports
Plant Biotechnology Reports 生物-生物工程与应用微生物
CiteScore
4.10
自引率
4.20%
发文量
72
审稿时长
>12 weeks
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.
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