基因敲除SRK的芜菁纯合株系的创制及叶片特征研究

	基因敲除SRK的芜菁纯合株系的创制及叶片特征研究; Creation of homozygous lines based on genome editing of BrrSRK and studies of leaf character of turnip (Brassica rapa var.rapa）
	张洁
导师	杨永平
摘要	Turnip (Brassica rapa var. rapa) is traditionally cultivated on the Qinghai-Tibet Plateau and adjacent highlands, but employments of landraces in cultivation by local people lead to the lack of cultivars with uniform traits. Furthermore, it is difficult to get homozygous cultivars with stable genetic characters due to the strong self-incompatibility of turnip. Several methods could be used to obtain homozygous cultivars, and the CRISPR/Cas9 system could be an efficient method via knocking out the genes related to self-incompatibility. In this thesis, this study mainly introduces the process of creating self-compatible lines by genome editing and the classification and identification of regenerated plants from free microspore culture. We obtained WUSCHEL transgenic plants through overexpression of WUSCHEL gene, and successfully established the genetic transformation system of turnip. We further optimized the genetic transformation system by studying the induction conditions, including variety, preculture time of explants, bacterial fluid concentration during infection, antibiotic concentration in induction medium and plant growth hormone. The results showed that the regeneration frequency and the sensitivity to bacteria was highest for turnip variety of KTRG-B-103. The best pre-culture time for explant were 48 h, and the best bacterial infection concentration was OD6000.3. Firstly, the explants were placed in the medium with 50 mg/L Kanamycin (Kana) inside for one week, and then the explant were transferred to the medium with 10 mg/L Kana for subculture to improve the regeneration rate of adventitious buds, which could reduce the probability of false positive regenerated buds. The best hormonal ratio of the transformation system was 4 mg/L 6-benzylaminopurine (6-BA) and 1.5 mg/L Naphthaleneacetic acid (NAA). Through the established transformation system, we used the petioles of the WUSCHEL transgenic plants as explants and completed the transformation mediated by Agrobacterium tumefaciens carrying CRISPR-BrrSRK vector. The transformation rate of adventitious buds was 43.6%. In the identification of 9 adventitious buds, hygromycin marker genes could be identified in 7 adventitious. In the early stage of the laboratory work, several regenerated plants had been cultured from microspores of five turnip varieties. In order to determine whether morphological characteristics could be used to identify the relationship between microspore culture plants and their parent plants and the differences between different turnip varieties, we used machine language to classify and identify the leaves of turnip plants. First of all, 2450 photos of 44 turnip landraces were collected by digital camera to establish a data set of turnip leaves, and convolution neural network classifier (CNN) was used to construct classification model by training and reorganization. The recognition accuracy of CNN model was the highest and the training loss is the lowest, and the accuracy
	2021-05
文献类型	学位论文
条目标识符	http://ir.kib.ac.cn/handle/151853/74543
专题	昆明植物所硕博研究生毕业学位论文
推荐引用方式 GB/T 7714	张洁. 基因敲除SRK的芜菁纯合株系的创制及叶片特征研究, Creation of homozygous lines based on genome editing of BrrSRK and studies of leaf character of turnip (Brassica rapa var.rapa）[D],2021.

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