Title Page
Abstract
Contents
Ⅰ. Introduction 14
Ⅱ. Literature Review 17
1. Carrot (Daucus carota L.) 17
1.1. Breeding and phytonutrients of carrot 17
2. Carotenoid biosynthesis 18
2.1. Carotenoid biosynthesis pathway in plants 18
2.2. Carotenoid biosynthesis in carrot 19
3. Genome editing of plant 20
3.1. Genome editing - ZFN, TALEN, CRISPR/Cas9 21
3.2. CRISPR/Cas9 system 22
3.3. Plant genome editing using the CRISPR/Cas9 system 23
3.4. Genome editing using the CRISPR/Cas9 system in carrot 23
3.5. Genome editing of genes related to carotenoid biosynthesis 24
Ⅲ. Materials and Methods 26
1. Plant materials 26
2. Design of CRISPR/Cas9 vector 26
2.1. Selection of target gene and sgRNA target sequence 26
2.2. Vector construct and Agrobacterium strain 27
3. Agrobacterium-mediated transformation based on CRISPR/Cas9 system 27
3.1. Bacterial cultures 31
3.2. Inoculation & co-cultivation 31
3.3. Regeneration of transgenic plants 32
4. Polymerase chain reaction (PCR) & CRISPR/Cas9 induced mutation detection 35
4.1. Genomic DNA Extraction 35
4.2. PCR 36
4.3. Sanger sequencing 36
Ⅳ. Results 39
1. Agrobacterium-mediated transformation by the CRISPR/Cas9 system 39
1.1. Agrobacterium-mediated transformation and regeneration of transgenic plants 39
1.2. Confirmation of T-DNA insertion through PCR analysis 48
1.3. Mutation patterns in the target genes induced by CRISPR/Cas9 54
Ⅴ. Discussion 62
Ⅵ. Abstract in Korean 68
Ⅶ. Literature 71
Table 1. Target gene sequences used in this study. 29
Table 2. Culture media used for transformation and regeneration of transgenic plants in carrot inbred line 19-1218, 1254. 33
Table 3. Sequences of the primers used for PCR. 37
Table 4. Sequences of the primers used for Sanger sequencing. 38
Table 5. Agrobacterium-mediated transformation using CRISPR/Cas9 system and generation of transgenic plants in carrot inbred line 19-1218. 44
Table 6. Agrobacterium-mediated transformation using CRISPR/Cas9 system and generation of transgenic plants in carrot inbred line 19-1254. 46
Table 7. Confirmation of PCR positive plants through agarose gel electrophoresis analysis of PCR products of Cas9 and nptII genes in transgenic plants of carrot inbred line 19-1218. 51
Table 8. Confirmation of PCR positive plants through agarose gel electrophoresis analysis of PCR products of Cas9 and nptII genes in transgenic plants of carrot inbred line 19-1254. 53
Table 9. Percentages of transgenic plants of carrot inbred line 19-1218 and 19-1218 with different mutation types in the target sequence and sgRNA. 61
Fig. 1. The selection of target genes and designing of CRISPR/Cas9 binary expression cassette. 28
Fig. 2. Schematic representation of the T-DNA region of binary vector pKAtC used in the transformation of carrot. 30
Fig. 3. Schematic diagram for the steps of Agrobacterium-mediated transformation and the generation of transgenic plants in carrot... 34
Fig. 4. Agrobacterium-mediated transformation and regeneration of transgenic plants in carrot inbred line 19-1218. 40
Fig. 5. Agrobacterium-mediated transformation and regeneration of transgenic plants in carrot inbred line 19-1254. 41
Fig. 6. Agarose gel electrophoresis analysis of PCR products of Cas9 and nptII genes in carrot transgenic plants of carrot inbred line 19-1218. 49
Fig. 7. Agarose gel electrophoresis analysis of PCR products of Cas9 and nptII genes in carrot transgenic plants of carrot inbred line 19-1254. 52
Fig. 8. Mutation analysis by Sanger sequencing of DcSGRL sgRNA33 transgenic plants in carrot inbred line 19-1218 and 19-1254. 55
Fig. 9. Mutation analysis by Sanger sequencing of DcSGRL sgRNA34 transgenic plants in carrot inbred line 19-1254. 57
Fig. 10. Mutation analysis by Sanger sequencing of DcLCYB2 sgRNA45 transgenic plants in carrot inbred line 19-1254. 58
Fig. 11. Mutation analysis by Sanger sequencing of DcLCYB2 sgRNA47 transgenic plants in carrot inbred line 19-1218 and 19-1254. 59