Title Page

Contents

Abbreviations/terms 17

Abstract 18

I. Introduction 21

II. Materials and Methods 29

1. Nondestructive determination of seed viability using resazurin seed test (RST) 29

1) Establishment of RST methodology 29

(1) Seed sample preparation 29

(2) Artificially ageing treatment of seeds 29

(3) Working conditions with resazurin reagent 30

2) The RST for Brassicaceae seeds 33

(1) Seed sample 33

① Artificially aged seed (AAS) 33

② Intact seeds 33

(2) Resazurin reagent preparation 34

(3) Seed testing method 35

(4) TZ test 36

3) Study on the substances influence on the color change of the RRA 36

(1) Seed sample 36

(2) Sample substances and methods 36

(3) SPME-GC-MS Analysis 37

(4) Ethanol test for the color change of the RRA 38

① Effect of ethanol volatile on the RRA 38

② Effect of ethanol in the RRA 39

2. Nondestructive classification of seed germinability by near infrared spectroscopy (NIR) with single seed base 40

1) Seed samples 40

(1) Corn 40

(2) Radish 40

2) Acquisition of NIR spectra 41

(1) Corn 41

(2) Radish 42

3) Data analysis 43

(1) Corn 43

(2) Radish 43

4) Energy dispersive X-ray analysis 44

III. Results and Discussion 45

1. Nondestructive determination of seed viability using resazurin seed test (RST) 45

1) Establishment of RST methodology 45

2) The RST for Brassicaceae seeds 53

(1) Artificially aged seed (AAS) 53

(2) Intact seeds 62

① Radish 62

② Cabbage 68

③ Broccoli 74

④ Cauliflower 77

(3) Discussion 83

3) Study on the substances influence on the color change of the RRA 92

(1) Effect of glucose and glycine on the color change of the RRA 92

(2) Volatile compounds from seeds 94

2. Nondestructive classification of seed germinability by near infrared spectroscopy (NIR) with single seed base 105

1) Normal and Artificially Aged Corn (Zea mays L.) Seeds 105

2) Viable and nonviable radish (Raphanus sativus L.) seeds 113

References 122

초록 131

Appendix 133

Appendix 1. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 50㎍/㎖, Temperature: 25℃) 133

Appendix 2. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 50㎍/㎖, Temperature: 30℃) 134

Appendix 3. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 50㎍/㎖, Temperature: 35℃) 135

Appendix 4. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 100㎍/㎖, Temperature: 25℃) 136

Appendix 5. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 100㎍/㎖, Temperature: 30℃) 137

Appendix 6. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 100㎍/㎖, Temperature: 35℃) 138

Appendix 7. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 150㎍/㎖, Temperature: 25℃) 139

Appendix 8. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 150㎍/㎖, Temperature: 30℃) 140

Appendix 9. Absorbance changes of RRA at 570nm depending on seed quality, temperature, yeast and resazurin concentration. (Cabbage: cv. Hwangbok, Resazurin: 150㎍/㎖, Temperature: 35℃) 141

Appendix 10. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. Jangbaekminong) 142

Appendix 11. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. R-347) 143

Appendix 12. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. Dynamic) 144

Appendix 13. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. Hongwoljuk) 145

Appendix 14. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. Tara) 146

Appendix 15. Statistic data of absorbance of the RRA from normal, abnormal and dead radish seeds (cv. Greenchallenger) 147

감사의 글 148

Table 1. Experimental design for establishing a methodology. 31

Table 2. Intact seed samples for resazurin seed test. 34

Table 3. The absorbance and color changed in the RRA when the NS and AAS of cabbage are soaked in the 50㎍/㎖ resazurin mixed with different yeast concentrations and incubated at 35℃ for 1,2,3, and 4... 48

Table 4. The absorbance of the RRA, percentage of normal, abnormal and dead seeds, total germination percentage, and mean time germination (MGT) of the radish seeds (cv. Yechanmoo) with different... 57

Table 5. The absorbance difference of the RRA, percentage of the normal, abnormal and dead seeds, and MGT of the three color fractions. 60

Table 6. The absorbance (570nm) of the soaked RRA and percentage of the radish seeds (cv. Jangbaekminong) of the three color fractions. 63

Table 7. The absorbance (570nm) of the soaked RRA and percentage of the radish seeds (cv. R-347) of the three color fractions. 67

Table 8. The absorbance (570nm) of the soaked RRA and percentage of the cabbage seeds (cv. Dynamic) of the three color fractions. 69

Table 9. The absorbance (570nm) of the soaked RRA and percentage of the cabbage seeds (cv. Tara) of the three color fractions. 71

Table 10. The absorbance (570nm) of the soaked RRA and percentage of the cabbage seeds (cv.Hongwaljuk) of the three color fractions. 73

Table 11. The absorbance (570nm) of the soaked RRA and percentage of the broccoli seeds (cv. Greenchallenger) of the three color fractions. 76

Table 12. The absorbance (570nm) of the soaked RRA and percentage of the seeds (cv. 077) of the three color fractions. 78

Table 13. The absorbance (570nm) of the soaked RRA and percentage of the seeds (cv. 078) of the three color fractions. 81

Table 14. The absorbance of the soaked RRA, the percentage of the seeds of normal, abnormal and dead in each color fractions, and MGT from RST in 6 Brassica seed lots. 86

Table 15. The absorbance of the soaked RRA, the percentage of the seeds of normal, abnormal and dead in each color fractions, and MGT from RST in two cauliflower seed lots. 87

Table 16. Comparison of germination percentage with the predicted germination percentage from the equation (2) in several crops. 87

Table 17. Absorbance ranges of the soaked RRA from the normal, abnormal and dead of radish, cabbage and broccoli seeds. 88

Table 18. Volatile compounds from cabbage aged seeds were analyzed based on HS-SPME-GC-MS measurement (corresponding to the largest peak, arrow point of Fig. 36. A). It was verified as ethanol. 99

Table 19. Hit, miss and uncertain of normal and artificially aged corn seeds in the calibration set classified by PLS 2 models from raw, the 1st and 2nd derivative data sets.(이미지참조) 109

Table 20. Hit, miss and uncertain of normal and artificially aged corn seeds in the prediction sets classified by PLS 2 models from raw, the 1st and 2nd derivative data sets.(이미지참조) 109

Table 21. Percentage of viable and nonviable radish seeds in the calibration set classified by PLS 2 models from raw, 1st and 2nd derivative data sets.(이미지참조) 117

Table 22. Percentage of viable and nonviable radish seeds in the prediction set classified by PLS 2 models from raw, 1st and 2nd derivative data sets.(이미지참조) 117

Table 23. Comparison of chemical elements in the surfaces of seed coat and cotyledon of viable and nonviable seeds. 120

Fig. 1. Conversion of resazurin to resorufin and dihydroresorufin by oxidation and reduction (Robert & George, 1950). 26

Fig. 2. Seed soaking system using a 96-well PCR plate with a hole at the bottom. Single seed was placed into each well of a 96 PCR plate and soaked into another 96-well plate containing resazurin solution. 32

Fig. 3. The 96-well planting plate in which seeds were planted after soaking in the RRA. Two fold of blue blotters are in the bottom. 36

Fig. 4. SPME analysis procedure (A) and volatile compounds capturing system from seeds (B). 38

Fig. 5. Incubation system of volatile ethanol with resazurin reagent. 39

Fig. 6. Modified seed holder depending on seed shape and size (A) and schematic diagram for measuring NIR spectra of single corn seed (B). 42

Fig. 7. The absorbance at 570nm in resazurin reagents changed when normal and artificially aged cabbage seeds were soaked in different concentrations of resazurin (A＝50㎍/㎖, B＝100㎍/㎖, and C＝150㎍... 47

Fig. 8. The absorbance of the RRA appeared when NS, AAS and no seed (NoS) of cabbage are soaked in the 50㎍/㎖ resazurin mixed with different yeast concentrations of 0, 0.1, 0.2, 0.3 and 0.4 ㎎/㎖ and... 49

Fig. 9. Yeast effects on color change of resazurin reagent in preliminary resazurin seed tests for NS and AAS. The color of No 11-12 was clearly changed to pink while others remained blue. The 96-well plate was... 50

Fig. 10. A preliminary trial of the RST. Mixtures of 48 NS and 48 AAS were randomly placed in a 96-well plate and were soaked in the RRA. The 48 of pink color represented the AAS and the 48 of blue color... 51

Fig. 11. The time course germination of seven seed lots of radish seeds (cv. Yechanmoo) treated with different time of artificially ageing treatment. 55

Fig. 12. The time course absorbance change in the RRA at 570 nm from the seven seed lots of radish seeds (cv. Yechanmoo) treated with different time of artificially ageing treatment. 55

Fig. 13. The color changes of the soaked RRA from the RST depending on the length of ageing time in the radish seeds (cv. Yechanmoo). 56

Fig. 14. Three color fractions of the RRA. 56

Fig. 15. Linear regression relationship between absorbance and ageing time (A), germination percentage (B), dead seeds (C), and MGT (D) in radish (cv. Yechanmoo) seeds. 58

Fig. 16. Time course absorbance changes of soaked RRA (A) and percentage of normal, abnormal and dead seeds (B) of the three color fractions. 60

Fig. 17. Absorbance changes (A) and percentage of radish seeds (cv. Jangbaekminong) of the three color fractions of the soaked RRA. 64

Fig. 18. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. Jangbaekminong). 64

Fig. 19. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. R-347). 67

Fig. 20. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. Dynamic). 69

Fig. 21. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. Tara). 71

Fig. 22. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. Hongwaljuk). 73

Fig. 23. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv. Greenchallenger). 76

Fig. 24. Time course absorbance changes of the soaked RRA (A) and the percentage of the normal, abnormal and dead seeds of cauliflower(cv.077) belong to the three color fractions (B). 79

Fig. 25. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv.077). 79

Fig. 26. Time course absorbance changes of the soaked RRA (A) and the percentage of the normal, abnormal and dead seeds of cauliflower (cv. 078) (B) belong to the three color fractions. 82

Fig. 27. Individual absorbance (A) and the absorbance ranges (B) of the soaked RRA measured from the normal, abnormal and dead radish seeds (cv.078). 82

Fig. 28. The conductivity in the soaking solution with the normal and artificially aged seeds cauliflower seed lots (cv. 077 and 078) and non-dormant cabbage seeds (cv. Hwangbok). 91

Fig. 29. TZ test of the non-germinated cauliflower seeds (cv.077) of the blue color fraction. 91

Fig. 30. Effects of glucose and glycine on the color change of RRA soaked with normal and aged cabbage seeds (cv. Hwangbok). Glucose and glycine concentrations were 10, 100 and 1000 ppm. (1,2＝control,... 93

Fig. 31. Absorbance of glucose (A) and glycine (B) containing RRA soaked with normal and artificially aged cabbage seeds (C) (cv. Hwangbok) 94

Fig. 32. A capturing diagram of volatile compounds from the seeds in the RRA using 96-well PCR plate. 94

Fig. 33. Effects of volatile compounds from seeds on the color change in RRA. Color was not changed from the volatiles from control (no seed) and normal radish seeds, but greatly changed from that of aged... 95

Fig. 34. An absorbance of the RRA affected by volatile compounds from the control, normal seeds and aged seeds of radish (cv. Yechanmoo). 95

Fig. 35. SPME-GC spectra of the volatile compounds from the normal and artificially aged cabbage seeds. 96

Fig. 36. HS-SPME-GC spectra of the volatile compounds captured from artificially aged cabbage seeds (A) and normal seeds (B). 98

Fig. 37. SPME-GC spectra from pure ethanol (A), aged cabbage seeds (B) and normal seeds (C). 100

Fig. 38. The color changes of the RRA contacted with ethanol volatiles at different concentrations after incubation for 6 hr at 35℃. 102

Fig. 39. Absorbance differences of the RRA affected by the ethanol volatiles with different concentrations. 102

Fig. 40. Absorbance changes of the RRA added by different concentrations of ethanol. 104

Fig. 41. NIR spectra of corn seeds (A: original, B: mean spectra of the original, C: the 1st derivative of the mean spectra, D: the 2nd derivative of the mean spectra). The patterns of the spectra...(이미지참조) 107

Fig. 42. Principle component score plot for corn seeds (+: normal seeds, ㅁ: aged seeds). The classification was very clear between the two groups of the normal and aged seeds, indicating that the... 108

Fig. 43. NIR spectra of radish seeds. (A: original, B: mean spectra of original, C: first derivative of the mean spectra). 115

Fig. 44. Principle component score plot for radish seeds (+: viable seed, ㅁ: nonviable seed). 116

Fig. 45. X-ray energy spectra of seed coat and cotyledon surfaces of viable and nonviable seeds using EDX detector (Seed coat- a: viable, b: nonviable, Cotyledon- c: viable, d: nonviable). 118

레저주린과 근적외선을 이용한 광학적인 방법으로 종자를 비 파괴적으로 단일 종자단위에서 활력을 검정하는 연구를 수행하였다. 종자를 침지하면 침지액의 색이 변하는 레저주린-효모 시약(RRA)을 개발하였고, RRA를 이용하여 레저주린 종자검정법(RST)을 확립하였다. 십자화과 채소종자의 RST 검정 조건은 RRA의 조성이 50μg/ml의 레저주린-0.4mg/ml의 효모 용액에 종자를 침지하여 35℃에서 4시간 정도 두었을 때 가장 우수하였다. 종자침지를 위하여 96 웰풀레이트(96-well plate)를 이용하였고 발색된 RRA는 멀티웰풀레이트리더(multi-plate reader)를 이용하여 흡광도를 단시간에 측정하였다. 발아율과 흡광도의 상관관계는 무 종자의 정상종자와 여러 단계의 인위퇴화종자 시험에서 상관계수 0.92의 정의 관계를 보였고, 죽은 종자와 흡광도는 상관계수 0.92의 부의 관계, 중간발아 시간(MGT)과 흡광도는 상관계수 0.96의 높은 정의 관계를 각각 나타내었다. 또한 RRA의 색에 따라 청색, 분홍색 및 무색으로 구분하고, 6 품종의 십자화과 채소종자를 실험한 결과에서 RRA 색과 품질과의 관계를 나타내는 방정식을 도출하였다. 이 방정식을 이용할 때 94.3-99.9%의 정확성으로 종자발아율을 예측할 수 있었다. 예측정확성이 낮은 콜리플라워 2품종은 전기전도도 및 테트라졸리움 시험에서 휴면종자로 판명되었다.

글루코스와 글라이신은 RRA의 색을 변하게 하는 물질이 아닌 것으로 나타났다. 십자화과 인위퇴화 종자에서 다수의 휘발성물질이 나오는 것을 발견하였고 정상종자에서는 나오지 않았다. 휘발성물질 중 알콜이 가장 많은 것으로 나타났고 알콜이 RRA의 색을 변하게 하는 물질 중 하나로 판명 되었다.

근적외선 분광분석계(NIR)을 이용하여 옥수수의 정상종자와 인위적 퇴화종자를 단일종자 단위로 스켄하여 주성분분석(PCA) 및 부분최소제곱(PLS) 분석 방법으로 판별하였다. 옥수수의 정상종자와 퇴화종자는 100% 정확도의 검량식(calibration)을 얻었고, 미지의 종자를 100% 정확도로 판별하였다.

또한 NIR을 이용하여 정상적인 무 종자를 분석한 결과 발아종자와 불발아종자에 대하여 100% 정확도의 검량식을 얻었고, 미지의 종자를 판별한 결과 발아종자 94%, 불 발아종자 95%의 정확도로 판별하였다. 불발아 무종자의 종피에서 알루미늄(Al), 규소(Si), 황(S)이 발아종자 종피보다 많았고, 또한 불발아 무 종자의 배유 표면에서 마그네슘(Mg), 규소(Si), 인(P), 황(S), 염소(Cl), 칼리(K) 및 칼슘(Ca)이 발아 배유 표면보다 많았다.

결론적으로 RST 방법과 NIR 종자검정 방법은 간단하고 빠르게 비 파괴적으로 종자의 활력을 검정할 수 있으며, 발아율과 죽은 종자 및 정상종자를 예측하는데 유용하게 이용할 수 있는 기술로서 개발 되었다.