Title Page
Contents
LIST OF ABBREVIATIONS 17
GENERAL ABSTRACT 19
GENERAL INTRODUCTION 22
LITERATURE REVIEW 26
A. Araliaceae 26
i. Panax ginseng 26
ii. Aralia elata 27
B. Cytogenomics 27
i. Cell cycle synchronization and metaphase accumulation 29
ii. Fluorescence in situ hybridization 30
iii. Flow cytometry 31
REFERENCES 37
CHAPTER 1. Cell cycle synchronization in Panax ginseng root meristem for cytogenomic research 44
ABSTRACT 45
I. INTRODUCTION 46
II. MATERIALS AND METHODS 49
A. Plant materials 49
B. Optimizing the cell cycle synchronization and treatment conditions 49
C. Selection of an efficient microtubule inhibitor 51
D. Accumulation and condensation of metaphase chromosomes 51
E. Cytological analysis to determine mitotic and metaphase indices 51
III. RESULTS 55
A. Cell cycle synchronization using 1.25 mM hydroxyurea at 25℃ 55
B. Nitrous oxide increased S-type metaphase chromosomes 55
C. Chromosome condensation varied by microtubule inhibitor 57
IV. DISCUSSION 65
A. Hydroxyurea treatment suggests an inherent long cell cycle duration 65
B. Metaphase arrest 66
C. Treatment duration determines chromosome condensation 67
D. Applications of accumulated chromosomes 68
V. CONCLUSION 69
REFERENCES 70
CHAPTER 2. Cytogenomic analyses of Panax ginseng cultivars and an aneuploid in transgenic hairy root line 74
ABSTRACT 75
I. INTRODUCTION 76
II. MATERIALS AND METHODS 78
A. Plant materials 78
B. Genomic DNA extraction 78
C. Assessment of genomic DNA purity and quantity 79
D. PCR amplification 79
E. Chromosome spread preparation 80
F. Fluorescence in situ hybridization 80
G. Karyotyping 82
H. Estimation of DNA content 82
III. RESULTS 87
A. Synchronized cells accumulate more metaphases 87
B. Assessment of nucleic acid purity and successful PCR amplification 87
C. Chromosome loss in hairy root line of ginseng 'Yunpoong' cultivar 87
D. Reduction of DNA content in the hairy root line of ginseng 'Yunpoong' cultivar 88
E. Karyotype analysis of the hairy root line of ginseng 'Yunpoong' cultivar 89
F. FISH karyotypes showing genetic variations in Pg167TR repeats 90
IV. DISCUSSION 120
A. Chromosome loss in the hairy root line of 'Yunpoong' cultivar 120
B. FISH karyotype of the aneuploid hairy root line of 'Yunpoong' cultivar 121
C. The hairy root line of 'Yunpoong' cultivar is a tetra-monosomic line 122
D. Pg167TR markers can be used to discriminate ginseng cultivars 123
E. Reduced DNA content corroborated the chromosome loss in the hairy root line of the 'Yunpoong' cultivar 123
V. CONCLUSION 125
REFERENCES 126
CHAPTER 3. Evaluation of the cytogenomic stability of the regenerated plants of Aralia elata using fluorescence in situ hybridization and flow cytometry 131
ABSTRACT 132
I. INTRODUCTION 133
II. MATERIALS AND METHODS 135
A. Plant materials 135
B. Tissue culture 135
C. Minisatellite mining 136
D. Identification of novel repeats in A. elata 136
E. Genomic DNA extraction 137
F. Assessment of genomic DNA purity and quantity 137
G. PCR amplification 138
H. Chromosome spread preparation 138
I. Fluorescence in situ hybridization technique 138
J. Flow cytometry 140
III. RESULTS 144
A. Tissue culture and regeneration of the plants 144
B. Assessment of nucleic acid purity and PCR amplicons 144
C. Chromosome constitution 144
D. Fluorescence in situ hybridization analysis 145
E. Estimation of nuclear DNA content 146
IV. DISCUSSION 164
A. Distribution of the repeat probes among the regenerants 164
B. Genome size and total chromosome length were not correlated 165
C. Tissue culture-derived regenerants for cytogenomic research 166
V. CONCLUSION 167
REFERENCES 168
국문 초록 171
Table 1.1. Summary of metaphase indices between amiprophos methyl and oryzalin 64
Table 2.1. Summary of Araliaceae species, common names, and tissues used for flow cytometric genome size estimation. 116
Table 2.2. List of oligoprobes and primers used in this study to elucidate the repeat distribution in the chromosome complement of ginseng... 117
Table 2.3. Unpaired T-test between the nine ginseng cultivars. 118
Table 2.4. Table showing the distribution and variation of Pg167TR repeats in the chromosomes of ginseng cultivars and in vitro root cultures. 119
Table 3.1. List of probe sequences used in this analysis. 160
Table 3.2. Chromosome constitution and FISH karyotype analysis of A. elata mother plant. 161
Table 3.3. Chromosome constitution and FISH karyotype data of A. elata regenerants. 162
Table 3.4. Chromosome length and genomic DNA content estimation in A. elata mother plant and regenerants. 163
Fig. 1. A model distribution of repetitive elements and genes in plant chromosomes. 33
Fig. 2. Schematic diagram showing the biological classifications of different repetitive... 34
Fig. 3. Simplified concept of fluorescence in situ hybridization. 35
Fig. 4. Schematic representation of a flow cytometry system. 36
Fig. 1.1. Mitotic activity of the hairy root culture of P. ginseng 'Yunpoong' cultivar. 53
Fig. 1.2. A graphical diagram to illustrate the process of cell synchronization in ginseng. 54
Fig. 1.3. Mitotic activity of P. ginseng seedling and hairy root meristems after release from... 58
Fig. 1.4. Aceto-orcein stained mitotic cells in the hairy roots culture of P. ginseng... 59
Fig. 1.5. Responses of the hairy root cells to a 3-hour treatment with amiprophos methyl... 60
Fig. 1.6. Comparison of the metaphase indices of seedling roots treated with either... 61
Fig. 1.7. Metaphase accumulation in synchronized roots after incubation in 0.6 MPa... 62
Fig. 1.8. Degree of chromosome condensation by different microtubule inhibitors. 63
Fig. 2.1. Diagram illustrating the process of cell synchronization and metaphase accumulation in ginseng. 84
Fig. 2.2. A graphical diagram to illustrate the probe preparation and fluorescence in situ hybridization analysis. 85
Fig. 2.3. A graphical diagram to illustrate the estimation of genomic DNA. 86
Fig. 2.4. Gel analysis of P. ginseng gDNA extract and PCR amplicons. 92
Fig. 2.5. FISH metaphase spreads of the different ginseng cultivars and in vitro root cultures. 94
Fig. 2.6. Flow cytogram of cultivar 'Yunpoong' in vitro roots. 95
Fig. 2.7. FISH karyogram of 'Yunpoong' cultivar and its hairy root culture TYP1. 96
Fig. 2.8. FISH ideogram of 'Yunpoong' and its hairy root culture TYP1. 98
Fig. 2.9. FISH metaphase karyogram of the landrace ginseng "Geumsan' with chromosome complement of 2n=48. 99
Fig. 2.10. FISH metaphase karyogram of cultivar 'Chungson' with chromosome complement of 2n=48. 100
Fig. 2.11. FISH metaphase karyogram of cultivar 'Chunpoong' with chromosome complement of 2n=48. 101
Fig. 2.12. FISH metaphase karyogram of cultivar 'Geumpong' with chromosome complement of 2n=48. 102
Fig. 2.13. FISH metaphase karyogram of cultivar 'Gupoong' with chromosome complement of 2n=48. 103
Fig. 2.14. FISH metaphase karyogram of cultivar 'Sunhyang' with chromosome complement of 2n=48. 104
Fig. 2.15. FISH metaphase karyogram of cultivar 'Sunpoong' with chromosome complement of 2n=48. 105
Fig. 2.16. FISH metaphase karyogram of cultivar 'Sunun' with chromosome complement of 2n=48. 106
Fig. 2.17. FISH metaphase karyogram of cultivar 'Sunwon' with chromosome complement of 2n=48. 107
Fig. 2.18. FISH metaphase karyogram of cultivar 'Yunpoong' with chromosome complement of 2n=48. 108
Fig. 2.19. FISH metaphase karyogram of cultivar 'Yunpoong' adventitious root culture YP1 with chromosome complement of 2n=48. 109
Fig. 2.20. FISH metaphase karyogram of cultivar 'Yunpoong' adventitious root culture YP2 with chromosome complement of 2n=48. 110
Fig. 2.21. FISH metaphase karyogram of the aneuploid line of 'Yunpoong' hairy root culture TYP1 with chromosome complement of 2n=44. 111
Fig. 2.22. FISH metaphase karyogram of 'Yunpoong' hairy root culture TYP3 with chromosome complement of 2n=48. 112
Fig. 2.23. Recombination of Pg167TRa and Pg167TRb repeats in ginseng cultivars and in... 113
Fig. 2.24. Repeat distribution ideogram of the nine ginseng cultivars and four in vitro root cultures. 115
Fig. 3.1. A graphical diagram to illustrate the probe preparation and fluorescence in situ hybridization analysis. 142
Fig. 3.2. A graphical diagram to illustrate the estimation of genomic DNA. 143
Fig. 3.3. Micropropagation of A. elata from somatic embryogenesis cultures. 147
Fig. 3.4. Gel analysis of A. elata gDNA extract and PCR amplicons. 148
Fig. 3.5. Chromosome counting in 50 tissue-culture-derived regenerants of A. elata. 150
Fig. 3.6. Variation on the total chromosome length measured from A. elata regenerants. 151
Fig. 3.7. Repeat markers distribution in A. elata mother plant and the regenerants. 152
Fig. 3.8. FISH ideogram illustrating the repeat distribution on the diploid chromosomes of A. elata mother plant and the regenerants. 153
Fig. 3.9. Chromosomal mapping of repeats in 50 somatic embryogenesis-derived regenerants. 158
Fig. 3.10. Flow cytogram of A. elata. 159