Development of Genetic Resources and Breeding of High Value Lines in Codonopsis lanceolata

Transcription

1 Development of Genetic Resources and Breeding of High Value Lines in Codonopsis lanceolata

2 : : : : : : : : : - i -

3 . :..,., 40.,,,, (, ),,,,,. Saponin, Inulin, Triterpene,,,. (C. lanceolata), ( for. emaculata), (C. pilosula), (C. minima), (C. ussuriensis) 4.,,.. - ii -

4 (2n=16), 3.,,,,.,, marker.,.., ,,. 2. Chlorogenic acid (CGA) - iii -

5 . LD50 M1, M2. 3. MS Callus. Embryogenic callus 2,4-D 0.2ppm NAA 0.2ppm MS Subculture,. 2 Callus. MS, Hypocothyl Bar Binary Vector Agrobacterium (20 clone X 10 = 200 ) agar ( ) clone. 24 aniline blue UV-. SEM. S RT-PCR. PCR-RFLP Homo - iv -

6 S. 5. S, , RAPD,. 7.,,,,,,,,. 8.,,,,,,. - v -

7 ),,,,,,.,,,,, ( ) 2 ( ) 2. 2) 386,.,,,,, 2, 3, 4,. / vi -

8 3) DNA marker RAPD, RFLP DNA fingerprint. Sample Group, 9 Group, Genetic variation. DNA marker, Group Group Marker. 4) 5kR, 10kR, 15kR, 20kR, 25kR, 30kR, 35kR, 40kR, 50kR, 60kR kR 70%, 40kR LD 50 54%. Chlorogenic acid(cga), CGA 25kR Control CGA. CGA, CGA. LD50 40kR M1,. - vii -

9 5) 40, 50, 60kR 145 M , g-70g 6.,. 7),, clone 1, 2, 3,, 3, callus 1, 2, 3,,. clone,, viii -

10 1) priming, KNO3, Ca(NO3)2, GA %, KNO3 10. Ca(NO3)2 KNO3, 10 ( 72%). GA3 5 15%, 10 90%. GA3,. 2) 2 50% 50% , %. 120cm X 15cm 120cm X 7cm 3,. - ix -

11 . (NAA, BA ) ,. Aniline blue, 1/3,.,, ( ) Aniline blue. RT-PCR Fragment S4-RNase 42%,,, Sepel Signal. - x -

12 Homo Primer Homo Hetero PCR-RFLP SaSa, SbSb Hetero SaSb. Homo. S- S PCR-RFLP S S Cloning 7 S-genotype.. Callus Callus 2,4-D 1ppm NAA 0.2ppm Callus, MS. 2 Callus Callus.. (97036), 4 (Hypocothyl) bar Agrobacterium tumefaciens LBA mg/L Kanamycine x i -

13 Callus, somatic embryo., 3, 8 Somatic embryo %, % Somatic embryo. Bialaphos 2. bar bar. bar bar Forward primer Reverse primer PCR Wild type Control Band, 6 500bp Band bar. Total RNA Northern blot. Bialaphos Bar. 2 4 Bialaphos T2 Bialaphos 4 3 3( ):1 - x ii -

14 ( ), 15( ): 1 ( ). 1:1 3:1, Single(3:1) Two copy Bar.. Group,,, , 2 21, Group. 52, S Homo. group 8 44, Heterosis,.. Heterosis F1 9777, 9772, Vitamin C Ascobic acid Tocoperol(Vitamin E) Ascobic acid Tocoperol Asparaginic acid % Glutamic acid 9704, Asparagine 9704, Glutamine %, % xiii -

15 . Aruginine %, Heterosis A group(9756, 9772, 9777) B group( 9702, 9713, 9716, 9760, 9773, 9776) C group(9705, 9718) Marker Polymorphism.,,,. 2..., Group, Genetic variation., Screening. M2 5.,,. - x iv -

16 ., KNO3, Ca(NO3)2, GA3 GA % 50% , %. 120cm X 15cm 120cm X 7cm 3,.. (NAA, BA ) xv -

17 .. S4-RNase 42%,,, Sepel Signal. Homo PCR-RFLP SaSa, SbSb Hetero SaSb. Homo. S- S 7 S-genotype. S F1.., 4 (Hypocothyl) bar Agrobacterium tumefaciens LBA4404 bar. 1:1 3:1, Single Two copy Bar., - x vi -

18 .. Group,,, , 2 21, Group. 52, S Homo. group 8 44, Heterosis,.. Heterosis F1 9777, 9772, Vitamin C Ascobic acid Tocoperol(Vitamin E) Ascobic acid Tocoperol Asparaginic acid % Glutamic acid 9704, Asparagine 9704, Glutamine %, % 4.2. Aruginine %, Heterosis A group(9756, 9772, 9777) B - x vii -

19 group( 9702, 9713, 9716, 9760, 9773, 9776) C group(9705, 9718) Marker Polymorphism.,,,. F1. - x viii -

20 SUMMARY After opening the market of agricultural products, the foreign imported agri-products are drastically increased in domestic market. Farmers face a lot of problems to keep the domestic market in spite of the considerable achievement during last decades. They have to cultivate new competitive crops for the open market. Improvement of crop plants have the most important roll in the domestic agricultural industries. Even through the most cultivars have been breeded by the conventional breeding method, however, breeder have faced to limit for supplement of the useful genetic resources in natural environment. Development of genetic resources are essential to get the useful new genetic genes and selection breeding is also effective method to improve crop plants. The techniques of genetic engineering for crop improvement through gene transformation and the method of the conventional plant breeding reached limitation. As the principal purpose of the genetic engineering is the collection, evaluation and breeding of genetic resources and Expression of gene, plant tissue culture have been very important role of to develop the genetic engineering in the world, thus as desease, pest and chemical resistance and species with new useful agronomic characteristics. Application of molecular method for breeding of high value line have been useful to provide an opportunity to acceleration of the plant improvement, this research laid emphasis on the development of transgenic plants, induction and selection of radiation mutation, mechanism of self-incompatibility and evaluation of codonopsis germplasm for the breeding - xix -

21 of high value line. 1. Breeding of high quality variety (1) Evaluation of genetic resources with agronomic characteristics Total 384 lines collected from domestic were evaluated for selection of promising lines. These lines were investigated as the character the flower color, stem color, stigma type, leaf type, flower period, root length and root weight for evaluation. Flower color and stigma type were very different. Also value of root weight was showed from 5g to 25g. (2) Evaluation of Genetic diversity by using DNA markers A total of 340 polymorphic bands were generated on agarose and polyacrylamide gel by 19 primers of abitrary sequence, grouped by cluster analysis using sample matching coefficients of similarity. Among the genetic resources, the minimum genetic distance value was obtained sample no 1 and sample no 2, The largest value between sample no. 11 and no. 17. In cluster dendrograms base on polymorphism band, Genetic resources collected could be divided into 8 groups at below about 0.44 level of distance. These results showed that the evoluation of Codonopsis seemed to be done to north form south in korea. This information will help to realize more potential of germplasm diversity and breeding programs in Codonopsis. (3) Evaluation and selection of promising by using induced mutation for breeding of high value lines Mature seeds M1 generation was planted from treated with gamma rays of 5kr - x x -

22 - 60kr in field and M2 seeds were harvested from the M1 plants. In M2 generation, the yield productivities of root weight were higher than original cultivar. various mutant lines were evaluated as the characters of short culm, desistance. Also mutant lines were selected from the advanced generation. Especially several mutants of Codonopsis had increased 9 times on the root weight than original cultivar. These mutants will help to realize more potential of germplasm diversity and breeding programs in Codonopsis. (4) Chemical component analysis As the result of analysis on Codonopsis component of callus, 1 year root, 2 year root and 3 year ago root, total amount of sucrose and fractose were showed range of 0.20(callus)-5.59%(3years ago root) and 0.23(callus)-5.82%(3 years ago root), respectively, Also vitamin analysis, free amino acid analysis and flavor componants were the largest 3years ago root of the investigated samples. 2. The establishment of Codonopsis cultivation method (1) Optimal plant density As the result of cultivation by separating 120cm x 20cm x 15cm and 120cm x 15cm x 7cm and 120cm x 7cm x 7cm for planting density, root length, root weight had the increase yeild about 40% to 120cm x 15cm x 7cm. (2) Optimal shading method As the result of cultivation by 25% and 50% for shading, root weight have - xx i -

23 the increase, yield 3.3g than control(non shading). However phootosynthasis content was constant to investigated samples. (3) Seedling time and the effect of vinyl mulching When seedling was done on May 20, 3.91g, 16.74g per 1 root was produced. However, when seedling was done on March 20, 16.74g per 1 root was produced, which was 400% higher than that of May 20. When mulching was performed by vinyl, emergence time was short in case they were seeded early, whereas seeding March 20 seeded lastest due to high temperature and much water showed the highest yield increase. 3. Genetic analysis of self-incompetibility Domestic Codonopsis cultivars were selfed by self-pollination and action of pollen tube was investigated. This result have a gametophytic self-incompatibility(si). However, Codonopsis cultivars were not elucidated S-genotypes because it's parents were heterozygotes. The analysis of S-genotype was need for application of pollinizer and breeding of a species. Most of codonopsis plants dependent on insect pollination. Cloning of S-RNase gene from codonopsis was conduct with RT-PCR method using stigma tissue. PCR product was doing cloned, sequenced. Determination of S-genotype of superior clone was performed the PCR amplification by using S-RNase primer. The result of PCR amplification was detected band in S2(1300bp), S1 S8-allele by analysis of the 2% Metaphor agarose gel electrophoresis. Domestic breeding lines were determination of S-genotype by PCR amplification, PCR-RFLP and sequencing. S-genotype is symboled with S1S1, S2S2, S3S3, S4S4, S5S5, S6S6, S7S7, - x xii -

24 S8S8 in this studies. 4. Development of Bialaphos-Resistant Codonopsis by Introducing of the bar gene This experiments was conducted to develop bialaphos-resistant transgenic Codonopsis. Optimal shoot regeneration was obtained in MS medium supplemented with 0.1ppm NAA and 1ppm BA. In this condition, Codonopsis leaf disc were cocultivated with Agrobacterium tumefaciens LBA4404 which carries the nptii and the bar gene in the T-DNA for resistance to kanamycin. Codonopsis shoots showing in vitro resistance to 100ppm kanamycin and 10ppm bialaphos were obtained by using a two step selection and regeneration procedure. The presence of 100ppm kanamycin in a rooting medium strongly inhibited root formation but bialaphos did not affect the ability of plantlets to form roots PCR analysis indicate that the nptii and bar genes were stably integrated in the Codonopsis genome. 5. Breeding of high value lines As the result of investigation on agronomic characteristics and yeild of clones selected and breeded for 5years from 1995 to 2000, 9756, 9772 and 9777 lines increased yield higher than 30% of 43.7g per 1 root that is low yield of selected lines. Among them 9772, and 9777 had more yield, and agronomic characteristics was superior. Therefor, they were named Ansung IL Ho and Ansung E Ho, respectively - x x iii -

25 CONTENTS Chaper 1. General Introduction Section 1. Purpose and Categ ory of Study Section 2. Necessity of Study Chaper 2. Development of Genetic Resource S ection 1. Introduction S ection 2. Collection and Charecteristics of Genetic Res ource S ection 3. Evaluation of Diversity by M olecular M arkers S ection 4. Development of Genetic Resource by Gama ray Section 5. Analysis of Biochemical Componant Section 6. Conclusion Chapter 3. Es tablishm ent of Cultivation M ethod S ection 1. Introduction Section 2 Germination rate by Priming treatment. Section 3. Development Standard Cultivation Method Section 4. Conclus ion Chapter 4. Genetic Analys is of S elf- incom patibility S ection 1. Introduction Section 2. Self- incompatibility T ype S ection 3. Gene Cloning and Genetic Analys is of S elf- incom patibility Section 4. Conclus ion Chapter 5. Dev elopm ent of in vitro Breeding m ethod by Cell Culture S ection 1. Introduction Section 2. Embryogenic Callus F ormation and Regeneration Section 3. Development of T rans genic Plant by bar Gene Section 4. Conclus ion Chapter 6. Breeding of High Value Lines S ection 1 Introduction. S ection 2. S election of S uperior Lines on Ag ronom ic Characteristics Section 3. Chemical Componant of Superior Lines Section 4. Molecular Marker of Superior Lines Section 5. Conclusion Chapter 7. Conclus ions Reference - xx iv -

26 1 1 / / 6 2 / 7 3 Marker / 12 4 / 17 5 / 23 6 / / 32 2 Priming / 33 3 / 36 4 / / 48 2 / 49 3 / 59 4 / / 75 2 Embryogenic Callus / 77 3 bar / 82 4 / / 93 2 / 93 3 / Marker / / / 124 / x x v -

27 1 1 1.,..,,,, (, ),,,,,, 40., Saponin, Inulin, Triterpene,,,. (C. lanceolata), ( for. emaculata), (C. pilosula), (C. minima), (C. ussuriensis) 4 1. Komarov( ),, 3, Taquet(1908) - 1 -

28 .,,.,. (2n=16), 3.,,,.,, marker.,,,,., Marker., F

29 2. saponin betalain betacyanin betaxanthins,...,., (nitrates).. priming. 3.,,,., F1, - 3 -

30 in vivo in vitro..... mutagen,,,. 4. (Codonopsis lanceolata)., - 4 -

31 .... S-locus, S (Gametophytic Self-Incompatibillity) (Sporophytic Self-Incompatibility). S-, ribonuclease, (S-RNase) rrna. S- S-RNase

32 2 1.,.,,,, (, ),,,,,, 40., Saponin, Inulin, Triterpene,,,. (C. lanceolata), ( for. emaculata), (C. pilosula), (C. minima), (C. ussuriensis) 4 1. Komarov( ),, 3, Taquet(1908)

33 ,,.,. (2n=16), 3.,,,.,, marker.,,., Marker (1)..,, - 7 -

34 ,, 41, (2). 2 ( ), 2 ( ) 2.. 7cm x 15cm.,,,, ,,,,,,,. 1 Crossing. 3, - 8 -

35 1.,,,,, 41, 5-10 ( 2-2.). 2 ( ) 2 ( ) 2 ( 2-2.) Girisan(Hwaamsa) Girisan(Kure) Girisan(Bemsakol) Sulaksan(Hankeyoung) Sulaksan(Ansan) Sulaksan(Jumbo) Juwangsan Dukyusan(Jangki) Dukyusan(Muju) Dukyusan(Koje) Chiaksan(Sangwonsa) Chiaksan(Namtebong) Keyoungsan Japan(Kyusu) Japan(Tokoku) China(Sandung) 2 2 Total

36 . 386 ( 2-1). 1.,,,,., 2, 3, 4,. / (. 2-2)

37 2-2. / ( )

38 3 Marker RAPD marker Primer 10 base oligonucleotide primer 72 (Operon kit A, B, C, D, E, F) random primer, 0.5 primer 3 genomic DNA, 0.04 Taq DNA polymerase, 1 10 reaction buffer, mM dntp, 1 MgCl PCR Reaction mixture. PCR machine GeneAmp PCR System 9600(Perkin-Elmer Cetus), DNA denaturation; annealing; extension 45cycles, cycle denaturation 2 cycle extension 5. PCR TAE buffer(40 mm Tris-acetate, 1 mm EDTA, ph8.2) 1.5% agarose gel 0.5 / ethidium bromide 40 UV transilluminator type 55 Polaroid film. Molecular standard 1kb DNA ladder.. RFLP marker RFLP probe DNA CoxII, ATP A, 26S-rRNA

39 DNA PsbA, clone P1, P10 B1clone. HindIII, EcoRI BamHI DNA, Southern blot BMS DIG-labelling and detection kit.. DNA fingerprinting 21mer fingerprinting primer sequencing gel Promega Silver staining Band. 2. RAPD marker RAPD DNA (1 Cm2), 100ul Buffer( 10mM Tris-Cl, 450mM EDTA ph8.0, 1mg/ml Proteinase K, 1% Sarocosyl) 50oC 1, Tube 11000rpm 10, 100ug/ml RNase, 100oC 5, Template DNA. RAPD Primer Operon 10-mer Random Primer, PCR 94oC 30, Annealing 37oC-45oC, 72oC 41 Cycle. 2% Agarose gel 6% Polyacrylamide gel, Et-Br Silver Band Pattern Nei and Li(1979), UPGMA. 72 Random Primer

40 Primer, 2% Agarose gel Band OPC-05 14, 5-9 Main band (.2-2). Sample Group, 9 Group, Genetic variation. DNA marker, Group Group Marker RAPD banding pattern OPC06(A), OPG08(B) and OPF06(C) 3. RFLP marker RFLP probe DNA CoxII, ATP A, 26S-rRNA DNA PsbA, clone P1, P10 B1clone. HindIII, EcoRI BamHI DNA, Southern blot BMS

41 DIG-labelling and detection kit. DNA Cox II, ATPA, 26S-rRNA Probe Southern hybridization (. 2-3). DNA Probe Clone Band Southern blot DNA EcoRI(A) HindIII(B), 26s-rRNA. 4. DNA fingerprinting 21mer fingerprinting primer sequencing gel Promega Silver staining Band. Siver staining Band 70 Polymorphism Band

42 PCR Fingerprinting primer Marker (. 2-4). DNA Marker mer primer PCR PCR Polyacrylamide gel 5. band pattern Dendrogram 9 Group (. 2-5)

43 kR, 10kR, 15kR, 20kR, 25kR, 30kR, 35kR, 40kR, 50kR, 60kR

44 , Chlorogenic acid(cga). LD50 M1,. 2. 5kR, 10kR, 15kR, 20kR, 25kR, 30kR, 35kR, 40kR, 50kR, 60kR , 20kR 70%, 40kR LD 50 54%. 70kR ( 2-6)

45 Chlorogenic acid(cga), CGA 25kR Control CGA (. 2-3) Dose (kr) CGA content Fresh wt. ( /plant) Amount Concentration (nmoles/plant) (nmoles/g fresh wt.) 0 5.8(100)b (103) ( 97) ( 88) ( 74) ( 72) ( 62) ( 53) ( 36) -c ( 31) ( 27) - - a; Analysis was carried out for the seedlings 14days after imbibition and the values are the average of 20 samples. b; Value in parenthesis are percent of the nonirradiated control. c; Sample could not develop normally CGA, CGA

46 (. 2-4). LD50 40kR M1, CGA Chlorogenic acid concentration(um) Original After Inbibition exposure (A) (B) to 10 kr(c) (B-C) 50uM chlorogenic acid in water Extract from dry seeds Extacts from germination seeds days after imbibition 6 days after imbibition 8 days after imbibition 10 days after imbibition 12 days imbibition 3. 40, 50, 60kR (10,000 )

47 cm, 15cm 10 15g 14 (. 2-7 ). 2-7., M , g-70g 6, 70g-80g 5, 100g 4. M2. 15, (80g/ )

48 M g 10,.(. 2-5) (M3) 10g-20g 20g-25g 25g-30g 30g-35g 35g-40g 40g-45g 45g-50g 50g M M M M M Total Percentage 39/150 * M2 M3 30 data.,. M

49 5 1.., clone clone, callus. clone 1, 2, 3, clone 3., micro,,. 100mg 100ml 70ml, 80oC 30, 1ml 100ml. No.2,.45um membrane filter HPLC. Develosil ODS-HG-%( 4.6 x 150mm) column, ( E x 340nm, Em 450mn).. 20g 500ml 2L diethyleter rotar evaporar. 100rpm 1, 1L load, NaCl diethyeter. diethyeter 50ml NaSO

50 glass filter NaSO4,, 100ml/L 0.5ml ethyl decanoate rotary evaporator 2cm. water bath,., ml, GC-MS.. column PEG20M capillary column DB-WAX(0.25mm x 60m), carrier gas helium, column 30cm/sec, column 40oC-220oC, 3oC/min, 220oC, FID, make up gas.. 500mg 25ml 20ml, 100oC 15, 25ml. No.2, 0.22um membrane filter HPLC. YMC-Pack-PA3 (4.6 x 250mm) column, RI 16x10-6 RIU/FS.. 2.5g 25ml BHT 100mg/L hexan 5 No.5. 10ml Sep Pak Plus minicolumn 5% isoprophylalchol/ hexan 5ml 1.5ml 3.5ml, 5% isoprophylalchol/hexan

51 5ml HPLC. Asahipak ODP-50(4.6x150mm) column, (UV 259nm) Asparaginic acid % clone callus. Glutamic acid Yeonsan #3, Muju clone Free Samples amino acids (%) Callus* Muju #1 Muju #2 Muju #3 Yeonsan #3 Asp Glu Asn Ser Gla Arg T hr T hea Ala T yr nd Met Val nd Phe Ile Leu nd Lys *Callus induced from roots of Muju #3. Muju #1: one years old roots from Muju clone, Muju #2: two years old roots from Muju clone, Muju #3: three years old roots from Muju clone, Yeonsan #3: three years old roots from Yeonsan clone. nd: not determined. Asparagine callus, Muju #3-25 -

52 . Serine Muju #3, callus 1/4. Glutamine callus 1.339%, Muju # % 4.2. Aruginine %, Yeonsan #3. Threonine, Theanin callus. Valine Lysine, (. 2-7) Sucrose Yeonsan#3(5.598%) > Muju #2(5.511%) > Muju #1(3.656%) > Muju #3(2.928%) > Callus(0.202%), callus. Fructose Sucrose, Yeonsan #3 Callus Free sugars(%) Samples Callus* Muju #1 Muju #2 Muju #3 Yeonsan #3 Sucrose Fructose *Callus induced from roots of Muju #3. Muju #1: one years old roots from Muju clone, Muju #2: two years old roots from Muju clone, Muju #3: three years old roots from Muju clone, Yeonsan #3: three years old roots from Yeonsan clone

53 4. Vitamin C Ascobic acid Tocoperol(Vitamin E) 5. Ascobic acid Tocoperol Callus, Muju #3. Ascobic acid Tocoperol Muju #1 > Muju #2 > Muju # Vitamins Samples (mg/g) Callus* Muju #1 Muju #2 Muju #3 Yeonsan #3 Ascobic acid T ocoperol *Callus induced from roots of Muju #3. Muju #1: one years old roots from Muju clone Muju #2: two years old roots from Muju clone, Muju #3: three years old roots from Muju clone, Yeonsan #3: three years old roots from Yeonsan clone. 5. ( 6), Hexanal, 3-Pentenal, 4-methyl-, 2-Hexenal 2-Penten-1-ol, (Z)-; 2-Hexene, 1-(1-ethoxyethoxy)-, (E)-; 1-Hexanol; 3-Hexen-1-ol; 2, 4-Hexadiental, (E, E)- ; 2-Hexen-1-ol, (Z)- ; Acetic acid; Pentane, 2-methoxy-2, 4, 4-trimethyl-; Cyclobutane, 1, 2-diethyl-, cis- ; 1-Butyne, 3-ethoxy-3-methyl- ; 2, 5-Cyclohexadiene-1, 4-dione,; 2, 5-bis(1,1-dimethylpropyl) ; Propane, 2-chloro-2-nitro- ; Silane, butoxytrimethyl- ; 3-(3-Carboxy-4-hydroxyphenyl)-D-alanine ; Cyclotrisiloxane, hexamethyl- ; Hexanoic acid ; Phenol, 3-methyl- ; Phenol, 2, 6-bis(1, 1-dimethyllethyl)-4-methyl-; Benzeneethanol ; Hexanoic acid, 2-ethyl- ; 3-Hexanoic acid, (E)- ; 3-Hexanoic acid ; Cycloheptane, 1, 3,

54 Table 6. Flavor components of three years old Codonopsis lanceolata from Muju clone Peak No.a Compound Retention time b MS spectral data of samples c Hexanal 3- Pentenal, 4- methyl- 2- Hexenal 2- Penten- 1- ol, (Z)- 2- Hexene, 1- (1- ethoxyethoxy)-, (E)- 1- Hexanol 3- Hexen- 1- ol 2, 4- Hexadiental, (E, E)- 2- Hexen- 1- ol, (Z)- Acetic acid Pentane, 2- methoxy- 2, 4, 4- trimethyl- Cyclobutane, 1, 2- diethyl-, cis- 1- Butyne, 3- ethoxy- 3- methyl- 2, 5- Cyclohexadiene- 1, 4- dione, 2, 5- bis(1,1- dimethylpropyl) Propane, 2- chloro- 2- nitro- Silane, butoxytrimethyl- 3- (3- Carboxy- 4- hydroxyphenyl)- D- alanine Cyclotrisiloxane, hexamethyl- Hexanoic acid Phenol, 3- methyl- Phenol, 2, 6- bis(1, 1- dimethyllethyl)- 4- methyl- Benzeneethanol Hexanoic acid, 2- ethyl- 3- Hexanoic acid, (E)- 3- Hexanoic acid Cycloheptane, 1, 3, 5- tris(methylene)- Octanoic acid Phenol, (1, 1- dimethylethyl)- 2- methoxy- Nonanoic acid 1- Naphthalenol, 1, 2, 3, 4, 4a, 7, 8, 8aoctahydro- 1, 6- dimethyl- 4- (1- methyl... Decanoic acid Dodecanoic acid 1, 2- Benzenedicarboxylic acid, butyl 2- methylpropyl ester , 56, 41, 43, 57 41, 69, 39, 55 83, 41,39, 55 57, 41, 86 73, 43, 41,45, 83 56, 55, 41, 43, 69 41, 67, 39, 82, 69 81, 39 57, 41, 39, 67, 82 43, 42, 45, 60 73, 45, 97 41, 56, 55, 70, 43, 84 97, 41, , 192, 57, 248, , 45, 105, , 75, 145, 41, 39, , 151, 77, 68, , 57, 45, 193, 75, 167, , 41, 87, , 79, 77, 51, , 57, 145, 105, 41, 177, 91 91, 122, 65, 43, 92 88, 73, 41, 39 55, 41, 114, 68 73, 42, 41, 135, , 79, 91, 41, 39 60, 73, 41, 101, , 57, 221, 137, 41, 91 60, 57, 41, 129, 115, 73 95, 121, 164, 204, 43, 41, 22 73, 60, 41, 129, 193, 172, 87 73, 60, 41, 200, 129, 157, , 223, 41, 76, 104, 224 athe peak number correspond to the orders of a retention time.bthe components were identified by comparing their mass spectra(ms) and retention times for GC with those of authentic compounds. cthe most itensive peaks(max. seven) are represented

55 5-tris(methylene)- ; Octanoic acid ; Phenol, (1, 1-dimethylethyl)-2-methoxy- ; Nonanoic acid ; 1-Naphthalenol, 1, 2, 3, 4, 4a, 7, 8, 8a- octahydro-1, 6-dimethyl-4-(1-methyl...; Decanoic acid; Dodecanoic acid; 1, 2-Benzenedicarboxylic acid, butyl 2- methylpropyl ester ,.,,,, (, ),,,,,. (C. lanceolata), ( for. emaculata), (C. pilosula), (C. minima), (C. ussuriensis) 4 1.,,,

56 ,,,,,, 37, 30,,,,,,, 388 ( :40, :348). 7, 6 ( 2, 4), 30 ( 25, 5). Marker 19 RAPD Primer., DNA RFLP 26S rrna probe polymerphism. APC Silver staining Marker.,, clone 1, 2, 3,, 3. callus 1, 2, 3,,. clone,,

57 (gama-ray) 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70KR 10,000.. GCA contentent 40kR LD50.,

58 3 1,,,, (, ),,,,,, 40., Saponin, Inulin, Triterpene,,,. saponin betalain betacyanin betaxanthins,...,., (nitrates)

59 .. priming. 2 Priming cm petri dish, AOSA rule ,. Priming 4 1, 2 KNO3(100uM), 2 Ca(NO3)2(150mM), 2 GA3(0.3mM), ( / )8/16, 12/12, 16/8 30. (radicle) 1mm 10 SAS

60 2. priming 10-20%.,, KNO3, Ca(NO3)2, GA %, % (. 3-1) (S) (H) S 1 S 2 S 3 H 4 H 5 H 6 H 7 H 8 H 9 H10 Germination(%) (day) : 4 1. T50 7-8,. KNO3 10, T (.3-2). Ca(NO3)2 KNO3, 10 ( 72%) (. 3-3). GA3 5 15%, 10 90%

61 (. 3-4) (S) (H) KNO3 S 1 S 2 S 3 H 4 H 5 H 6 H 7 H 8 H 9 H 10 Germination(%) (day) : 2 KNO3(100 M) (S) (H) Ca(NO3)2 S 1 S 2 S 3 H 4 H 5 H 6 H 7 H 8 H 9 H 10 Germination(% ) : 2 Ca(NO3)2(150mM)

62 ( %). GA3, (S) (H) GA3 S 1 S 2 S 3 H 4 H 5 H 6 H 7 H 8 H 9 H 10 Germination(%) : 2 GA3(0.3mM) ( ) 3 28, 4 29, cm X 20cm 2, 10, 1. 3.,,,,,

63 ( ) cm X 15cm 120cm X 7cm. 2.,,,,,. ( ) 3 28, ,,,,,. 2. Sink Source, %. 50% (.3-5)

64 .3-5. ======================================================= (g) (cm) ( molco2/m2/s) ======================================================= Control % shading % shading Control:non shading, Sink. Source Sink. 3. 3, , ,,

65 /20 March /20 April /20 May * , %. Source. 2, ,,,,, cm X 15cm 120cm X 7cm 3,.. 120cm X 7cm. 120cm

66 X 15cm X 7cm. 120cm X 20cm X 7cm 120cm X 15cm X 7cm 2, 120cm X 20cm X 7cm 40% (g) (g) ( ) ( ) (g) ( ) cm X 15cm X 7cm cm X 15cm X 7cm

67 ,...,. Bar (cm) (cm) (g) (NAA, BA ) (.3-9). NAA, BA Control,

68 (g) (Cm) (g) NAA BA Control * :,

69 NAA BA Control (cm) (cm) (g)

70 4, Saponin, Inulin, Triterpene,,,...,., (nitrates)... Priming %., KNO3, Ca(NO3)2, GA %, KNO3 10. Ca(NO3)2 KNO3-44 -

71 , 10 ( 72%). GA3 5 15%, 10 90%. GA3, % 50%. Sink. Source Sink. (1 ) ( ).,, , %. Source

72 , cm X 15cm 120cm X 7cm 3,. 120cm X 15cm X 7cm. 120cm X 15cm X 7cm...,. (NAA, BA )

73

74 4 1 (Codonopsis lanceolata).,.... S-locus, S (Gametophytic Self- Incompatibillity) (Sporophytic Self- Incompatibility).,, (parent plant).,. S-locus

75 glycoprotein(slg) cdna Brassica, S- receptor protein kinase SRK(S receptor kinase).,,,, (haploid) S. 2 (codominant). Anderson S S-, McClure ribonuclease, (S-RNase) rrna. S- S-RNase ( ) Clone. clone, 5 (20 clone X 10 = 200 ) agar

76 ( )., clone. 24 (SEM) aniline blue UV-. clone (bud-flower pollination ),,. clone. ( ) SEM % glutaaldehyde (0.1M potsphate ph7.0), 1.0% osmium tetraoxide (0.1M potsphate ph7.0) 1 0.1M potsphate ph7.0 5 ethanol(25, 50, 70, 80, 99, 100%) 2 1. CO2, tray mounting. Gold coating Hitachi S-700, Japan. ( ) Aniline blue Ethanol/acetic acid, 3:1, 5, 1N NaOH, 60 Incubation, 0.01% anniline blue(dissolved 2% K3PO4) 2, (NiKon, EX exiter filter, BA 420 barrier filter)

77 ( ) protein 50, 50. SDS-PAGE -mercaptoethanol, active staining SH -mercaptoethanol. ( ) SDS-PAGE 12% polyacrylamide gel gel Gallagher. Well loading 40, running Coomassie blue. 2. ( 4-1. A, B,). Anther, Roqal 4 ( 4-2 ). Brassica

78 4-1.. A: ; B: 4-2., ( ). ( 4-3)

79 4-3. Aniline blue, 1/3 ( 4-4 A).. ( 4-4. B), 1/3 Transsection ( 4-4 C)

80 4-4. A; Aniline blue B; C; 1/3 Section D;, E; Aniline blue, ( 4-4. D). ( ) Aniline blue ( 4-4. E). 3.. Selfing (

81 4-5). 4-5., 2-3, 1, 3, 5 ( ) 3 ( 4-5). 4. Anther S ( 3 10%), 22-29kDa, (PI) 7.5 S genotype. ribonuclease. Anther Total protein

82 Anther 5 (. 4-6, 4-7) 4-6. IEF,., ( 8 ). Anther.( 4-7 )

83 4-7. IEF ( ), ( ) 5. Anther Anther IEF, Con A-peroxidase Band 2-3, Anther Band ( 4-8 ) IEF ( ), ( )

84 , Anther. ribonuclease 2 S genotype hetero type, S allele. (,, petunia ).. callose. (,, petunia ). SDS-PAGE kDa 2 major band S- (Tomato(20-30kDa), Petunia(24-30kDa), (30-35kDa)). S kDa S- (SLG), SLG SRK 100kDa S

85 S- ribonuclease RNase activity, RNase active staining SDS-PAGE ( ) RT-PCR Fragment Cloning 0.5g Guanidinium (Chirgwin 1979) RNA, oligo(dt)-column poly(a+) RNA Template PCR. PCR 94, 3, 94, 1, 50, 2, 72, 4 1 Cycle 30 Cycle, 1.5% Agarose Gel. Promega Direct PCR Cloning Kit Cloning, E. coli. HB101 Compentent Cell. ( ) DNA

86 DNA 1g. buffer 15ml 2-mercaptoethanol 750, 20% SDS 1ml M potassium acetate 5ml 20. isoprophanol 10ml (O/N ) 1 TE Buffer 500. phenol chloroform isoprophanol 500 3M Na-acetate 75, 70% EtOH 500. pellet 1 TE Buffer. ( )PCR 10 x reaction buffer(500mm KCl, 100mM Tris-HCl, PH 8.3, 15mM MgCl) 2.5, 2.5mM dntp 2, Genomic DNA 0.05, 100pM Primer 0.8, 0.5unit Taq pol (TaKaRa, Japan), Distilled Water Total Vol. 25. PCR Condition Predenaturation(94, 2min), Denaturation (94,1min, 1 Cycle), Annealing (50,2mins, 30 Cycles), Extention (72,3mins, 1 Cycle), Final hold(72, 5mins), store (4 ). 6 1% Agarose gel 25 UV transilluminator. ( ) PCR-RFLP

87 PCR Perkin-elmer PE free denaturation denaturation 93 1, annealing 55 2, extention free extension PCR buffer( : 500mM KCl, 100mM Tris-HCl ph 8.3, 15mM MgCl2, 0.01% gelatin) genomic DNA 2.5 ( 50ng), primer 100pM, dntp 2.5mM Taq polymerase 1 unit. PCR 4 6. total volume x buffer(750mm KCl, 150mM Tris-HCl ph 7.5, 92.5mM MgCl2, 10mM dithiothreitol) PCR 2.5, restriction endonuclease 2.5units, distilled water incubation 1 x TBE buffer 1.5 2% agarose gel 100V, 1 UV transilluminator,. ( ) PCR cloning E. coli(hb101, JM109) competent cell. Promega ligation kit PCR pgem-t Easy Vector cloning. plasmiddml transformation 10 x KCM 30% PEG. dideoxynucleotide chain termination sequencer(applied Biosystems) Lasergene sequence software

88 ( ) Plasmid DNA Plasmid DNA alkaline lysis, PEG RNA. E. coli Plasmid DNA single colony 50 / LB(Luria Broth) 37. OD600=1 50 oak ridge tube 4 3,000 rpm 5 pellet. pellet 2 solution (50mM Glucose, 25mM Tris-Cl,, ph 8.0, 10mM EDTA) 5. 3 sloution (0.2 N NaOH, 1% SDS) M potassium acetate(60 5M potassium acetate, 11.5 glacial acetic acid, 28.5 SDW) 20, 4, 6,000rpm 20. oak ridge tube TE-saturated phenol 3,000rpm 5 phenol : chloroform : isoamyl alcohol (25 : 24 : 1, v/v/v). chloroform : isoamyl alcohol(24 : 1, v/v). oak ridge tube isopropylalcohol 20 4, 10,000 rpm 10. pellet 70% ethyl alcohol 37 TE buffer(10 mm Tris-HCl, 1mM EDTA, ph 8.0) microcentrifuge tube. Tube 2 ribonuclease A(Sigma, 10 / )

89 600 PEG (20% PEG, 2.5 M NaCl), 20 4, 15,000rpm DNA. PEG 70% ethyl alcohol 300 TE. ( ) Sample, 2ml Guanidinium, 18000rpm Homogenage 2. 12,000rpm 10, 1.4ml 0.7g CsCl, 0.8ml 5M CsCl 45,000rpm 12,, Phenol, RNase Northern Blot. Northern Blot RNA 40 /. Formaldehyde MOPs, Hybridization Detection DIG- System X-ray Band. ( ) , Anilin blue,,. Mendel

90 X2-test. 2. RT-PCR Fragment 0.5g mrna ( mrna Kit) cdna ( cdna kit), S Primer( Forword:QLVLTWP, Reverse:ILKDKFDLL) PCR PCR 480bp ( 10 ) RT-PCR Cloning Band Gene Clean Kit, TA-Cloning Vector Cloning ( 10). Clone S4-RNase 42% Homology ( 4-10)

91 1 TGG TAA TAA TGG TAT TTC CAT AAA TTG TAT AAA TAT CCT CTG CTT CCA CGG TGG V I M V F P L I V L I S S A S T V G 55 GAC TCA ATC GAA GCG ATC ATG TAG GTT CTG GGA AAG AGA GTG GCT CAA ACA TGG L N R S D H V G F W E R E W L K H G 109 CCC TAC CCC TTG TAA CAA TCC TAC TGA CAA TTT TTT TAC GGT CCA CGG TTT GTG P T P C N N P T D N F F T V H G L W 163 GCC TTC AAA CAG GAA TGG ACC TAA CCC AAA AAA ATG CAA AAC TAC AAC CAT AAA P S N R N G P N P K K C K T T T I N 217 CTC TCA AAA GGT ACA GAT AGG ATG GCC GAA TAT GAC AGC CCA GTT GGA AAT TAT S Q K V Q I G W P N M T A Q L E I I 271 TAA CGT TTC CAA TTA TTT CCA ATT TAC CCA CCA ATA TCA CCC GGC CGT ATG CAA S N Y F Q F T H Q Y H P A V C N S N 325 CTC TAA TAC CTG TGG GTA TCC CAC AAT AAA GGA CGA CAT GCA TTA TTT AAA AAC T C G Y P T I K D D M H Y L K T V I 379 AGT AAT CAA AAC AAC AAT ACG AAA CCA AAA TTA AGT GCC AAA AGA ATA TAG GAC K N N N T K P K X K C Q K N X R T T 433 GAC GAT TCA ACC GAC ATA CGC AGT GGG AAT AAC AGG TCA CTG GTG GAT ATT GAA I Q P X I R S G N N R S L V D I E N 487 AAT GCC A G 4-9. RT-PCR,,, Sepel Total RNA Northern Blot,, Sepel Signal ( 4-10) PCR., Level. 3. PCR-RFLP Homo Homo

92 Primer ( 4-11), Homo Hetero PCR-RFLP F2 21 S-type SaSa 1, 10, 13, 14, 15, 16 6 SbSb 6, 7, 11, 12, Hetero SaSb 2, 3, 4, 5, 8, 9, 17, 18, 20, X2-Test 1 : 2 : 1. Primer 1 Primer 2 Primer Intron Primer 6 Primer 5 Primer 4 S Primer 1 : atgattctgcaaggtcaaacccacg; A Primer 4 : atgaattcatatggataatggtcaaccg S Primer 2 : gccttcagactcgaatggaca; A Primer 5 : tggcatttacaatatctacc S Primer 3 : cagccggctgtctgccactt ; A Primer 6 : cggttcgatcgagtacgttg Primer primer primer

93 4-1 PCR-RFLP{ Male Female SaSa SaSb SbSb SaSa SaSb SbSb : many pollen tubes penetrating, - : no pollen tubes penetrating, +- : less than 5 pollen tubes penetrating. Saand Sballele showed codominant. 21 F2 DNA Primer Single band(.4-12). 4 2% Agarose gel Band 3 Group Group band, Group Band 3, Group Band 4. Homo. System Hetero

94 . M Primer PCR Primer PCR MboI pattern 4. S- S mrna cdna Primer PCR S-RNase. Primer PCR-RFLP S S Cloning 7 S-genotype ( ).,

95 S 45%. 1 TGG TAA TAA TGG TAT TTC CAT AAA TTG TAT AAA TAT CCT CTG CTT CCA CGG TGG V I M V F P L I V L I S S A S T V G 55 GAC TCA ATC GAA GCG ATC ATG TAG GTT CTG GGA AAG AGA GTG GCT CAA ACA TGG L N R S D H V G F W E R E W L K H G 109 CCC TAC CCC TTG TAA CAA TCC TAC TGA CAA TTT TTT TAC GGT CCA CGG TTT GTG P T P C N N P T D N F F T V H G L W 163 GCC TTC AAA CAG GAA TGG ACC TAA CCC AAA AAA ATG CAA AAC TAC AAC CAT AAA P S N R N G P N P K K C K T T T I N 217 CTC TCA AAA GGT ACA GAT AGG ATG GCC GAA TAT GAC AGC CCA GTT GGA AAT TAT S Q K V Q I G W P N M T A Q L E I I 271 TAA CGT TTC CAA TTA TTT CCA ATT TAC CCA CCA ATA TCA CCC GGC CGT ATG CAA S N Y F Q F T H Q Y H P A V C N S N 325 CTC TAA TAC CTG TGG GTA TCC CAC AAT AAA GGA CGA CAT GCA TTA TTT AAA AAC T C G Y P T I K D D M H Y L K T V I 379 AGT AAT CAA AAC AAC AAT ACG AAA CCA AAA TTA AGT GCC AAA AGA ATA TAG GAC K N N N T K P K X K C Q K N X R T T 433 GAC GAT TCA ACC GAC ATA CGC AGT GGG AAT AAC AGG TCA CTG GTG GAT ATT GAA I Q P X I R S G N N R S L V D I E N 487 AAT GCC GGT ATG TAC ATA ACC CAG AAA CAA AAC GTC TTG CAA TCC TCT CAA AGG A G M Y I T Q K Q N V X A I L S K A 541 CGA CGA CTG AAT TAG TTG AGG TCA CTT T T E L V E V T L S-RNAse SaSatype 1 TGG TAA TAA TGG TAT TTC CAT AAA TTT CCA CAG TGG TTT AAA TAT TGT CTC CTG V I M V F P L I S T V V L I L S P G 55 GAT ACA ATA ATT TCC AAT TTA CCC ACC AAT ATC ACC TGG CGG TCT GCA ACT CTA Y N N F Q F T H Q Y H L A V C N S N 109 ATC TTA CCC CTT GTA AGG ATC ACA TCA AAC CCC CGA ACA ATT TTT TTA CGG TCA L T P C K D H I K P P N N F F T V M 163 TGT ACA TCA GCA AGA AAC AAA ACG TCT CTA GAA TCC TCT CAA AGG CGA AGA TTG Y I S K K Q N V S R I L S K A K I E 217 AAC CGC AGA GAG AAA AAA ACG TAT TAC TCG AAC CCC AGC TGG CAA TTA TTT GGC P Q R E K N V L L E P Q L A I I W P 271 CGT TCG ATC GAA CCA AAA ATA AAC TCT TCT GGG ATA AAG AGT GGA TGA AAC ATG F D R T K N K L F W D K E W M K H G 325 GCA CCT GTG GGT ATC CCA CAA TAG ATA ACG AGA ACC ATT ACT TTG AAA CCG TAG T C G Y P T I D N E N H Y F E T V G 379 GTT TGT GGC CCC CAA CCA TGG CAG GAC CTA ACC CAA TTA ATT GCC CAA TAA GGA L W P P T M A G P N P I N C P I R N I 433 ACA TCC GGA AGG ACG GGA AAA AAA GAG CAC TGT TGG ATA TTG AAA ATG CCA TAC R K D G K K R A L L D I E N A I R N 487 GCA ATG GTG CCG ACA ATA AGA AAC CAA CGA CGA CTG AAT TAA AAC TCA AGT GCC G A D N K K P T T T E L K L K C Q K K 541 AAA AGA AGG GTG TTG AGA T G V E I F S-RNAse SbSbtype

96 1 TGG GTA ATA ATG GTA TTT CCA TAA ATT GTT TAA ATA TTG TTT CCG TCC ACG GTG V I M V F P L I V L I L F P S T V G 55 GGA TAC AAT AAT TTC CAA TTT ACC CAC CAA TAT CAC CTG GCT GTC TGC AAC TCT Y N N F Q F T H Q Y H L A V C N S N 109 AAT CTT ACC CTT TGT AAG GAT CCC CCT AAC AAT TTT TTT ACG GTC CAC GGT TTG L T L C K D P P N N F F T V H G L W 163 TGG CCT CCA AAC ATG GTA GGA CCT AAC CCA ATT AAA TGC CCA ATA AAA AAT ATC P P N M V G P N P I K C P I K N I R 217 CGA AAG TAT ACA TCA GCA AGA AAC AAA ACG TCT CTA GAA TCC TCT CAA AGG CGA K I Y I S K K Q N V S R I L S K A K 271 AGA TTG TCG ATC GAA CAA AAA ATA ACC TCT TCT GGG ATA AAG AGT GGA TGA AAC I F D R T K N N L F W D K E W M K H 325 ATG GCT CCT GTG GGT ATC CCA CAA TAG ATA ACG AGA ACC ATT ACT TTG AAA CCG G S C G Y P T I D N E N H Y F E T V 379 TAA TCA AAA GTA CAG AGA GAA AAA TTA CTC GAA CAC CAG CTG GAA ATT ATT TGG I K I Q R E K L L E H Q L E I I W P 433 CCG AAC GTA AAC CGG ACG GGA AAA AAA GAG CAC TGT TGG ATA TTG AAA ATG CCA N V E P D G K K R A L L D I E N A I 487 TAC GCA ATG GTG CCG ACA ATA AGA AAC CAA AAC TCA AGT GCC AAA AGA AGG GTA R N G A D N K K P K L K C Q K K G T 541 CGA CGA CTG AAT TGG TTG AGA TCG CTC T T T E L V E I S-RNAse ScSctype 1 TGG TTA TTA TGG TAT TTT TCA TTA ATG ATT AAT ATT GGT GTT GTT AGG ATG GGT V I M V F S L I V L I S C S S T M G 55 TAA CAA TTA TTT TTC AAT TTA CGC AGC AAT ATT CAG CCG GGC TTT TGC AAT TCT Y N Y F S I Y A A I F S R A Y C N S 109 AAT CCA TCC TTG TAA GGA TCT TCC TGA CAA GTT GTT TAC GTT CAC GGT TTG CGC N P X P C K D L P D K L F T V H G L 163 CTA GGT ACC AAA GAT AAG AAA CCA AAA CTC AAG TGC CAA GAG AAT AAT CAG AAA R L G T K D K K P K L K C Q E N N Q 217 TCT CAG CAG GTA CAG ATA GGG AAT CTG ACT GCC CAG TTG ATA ATT ATT TGG CCG K S Q Q V Q I G N L T A Q L I I I W 271 AAC GTG CTC GAT CGA ACC GAT CAT GTA GGC TTC TGG AAT AGA CAG TGG AAC AAA N V L D R T D H V G F W N R Q W N K 325 CAT GGC AGC TGT GGG AAA GCG CCC ACA ATA AAG GAC GAA ATG CAT TAC TTT AAA H G S C G K A P T I K D E M H Y F K 379 ACA GTA ATC AAA ATG TAC ATA ACC CAG AAA CAA AAC GTT TCT GAA ATC CTC TCA T V I K M Y I T Q K Q N V S E I L S 433 AGG GCG AAG ATT GAA CCG GAG GGG AAA ATC AGG AGA CGG GAT GAT ATT ATA AAT R A K I E P E G K I R R R D D I I N 487 GCC ATA TGC CTT CAA ACG ACG TAG GAG ATG ACC CAA TAT ATG CAA GAA TAA AAC A I C X S N D V G D D P I Y C K N K 541 CAT TAC GAC TGA ATT AGT TGA GGT CAC TAT T I T T E L V E V T S-RNAse SdSdtype

97 1 TGG TAA CAA TGG TAT TTC CAT TAT TTG TAT TAA TAT TTT CTC CTT CCG CGG CAA V T M V F P L F V L I F S P S A A K 55 AAT ACA ATT ATT TCC AAT TTA CCC ACC AAT ATC ACC TGG CTG CCT GCA ATC CTA Y N Y F Q F T H Q Y H L A A C N P N 109 ACC CTA TCC CTT GTA AGG ATC CCC CTG ACA ATT TTT TTA CAA TCC AAA CAG CAA P I P C K D P P D N F F T I Q T A I 163 TCA AAC CAA ACT CAA TTG GAC ATA ACC CAT TAA ATT GCA TTC AAC CAA CCG TGA K P N S I G H N P L N C I Q P T V N 217 ATG CTC AAA AGG TAC AGT TAG GGA ATC TGA CAA CTC AGT TGG AAA TAA TTT GGC A Q K V Q L G N L T T Q L E I I W P 271 CGA ACG TAA TGT ACA TAA CCC AGA AAC AAA ACG TCT CTA AAA TCC TCT CAA AGG N V M Y I T Q K Q N V S K I L S K A 325 CGA AGA TTA CCT GTG GAC ATC CCA CAA TAA TGA ACG ACA TTC ATT ACT TTC ACG K I T C G H P T I M N D I H Y F H G 379 GTT TGT GGC CTT ACA ATC GAG CCG ATC ATA TAA GCT TCT GGA ATA AAC AGT GGA L W P Y N R A D H I S F W N K Q W N 433 ACA AAC ATG GCG AAC CGG AGG GGA AAC CCA GGA AAC AGA TAG ATA TTG TAA ATG K H G E P E G K P R K Q I D I V N A 487 CCA TAC GCA AAG GTA CCG GCG ATA AGG AAC CAA AAC TCA AGT GCC AAA AGA ATA I R K G T G D K E P K L K C Q K N N 541 TTA GTT GAG GTT GGT CT Q V T E L V S-RNAse SeSetype 1 TGG TAA TAA TGG TAT TTT CTG CTT CCA CGG TGC CAT AAA TTG TAT AAA TAT CCG V I M V F S A S T V P L I V L I S G 55 GAT CCA ATT ATT TCC AAT TTA CCC ACC AAT ATC ACC CGG CCG TAT GCA ACT CTA S N Y F Q F T H Q Y H P A V C N S N 109 ATC CTA CCG TCC ACG GTT TGT GGC CTT GTA ACA ATC CTA CTG ACA ATT TTT TTA P T V H G L W P C N N P T D N F F T 163 CGC CTT CAA ACA GGA ATG GAC CTA ACC CAA AAA AAT GCA AAA CTA CAA CCA TAA P S N R N G P N P K K C K T T T I N 217 ACT CTC AAA AGG TAC AGA TAG GAA ATA TGA CAG CCC AGT TGG AAA TTA TTT GGC S Q K V Q I G N M T A Q L E I I W P 271 CGA ACG TTC TCA ATC GAG TAG GTT CTG GAG CGA TAG AGA GTG GCT CCA TGA AAA N V L N R V G F W S D R E W L H E K 325 ACA TGG CAC CTG TGG GCA TTA TTT ATA TCC CAC AAT AAA GGA CGA CAT GAA AAC H G T C G H Y L Y P T I K D D M K T 379 AGT AAT CAA AAT GTA CAT AAC CAA GGC GCA GAA ACA AAA CGT CTT GCA ATC CTC V I K M Y I T K A Q K Q N V X A I L 433 TCA ACG ATT CAA CCG ACG AAA ATG CCA TAC GCA GTG GGA ATA ACA GGT CAC TGG S T I Q P X E N A I R S G N N R S L 487 TGG ATA TTG GTA ACA AGT GCC AAA AGA ATA TAG GAA CAA TAC GAA ACC AAA ATT V D I G N K C Q K N X R N N T K P K 541 ACG ACG ACT GAA TTA GTT GAG GTC ACT T X T T T E L V E V S-RNAse SfSftype

98 1 TGG GTA ATA ATG GTA TTT ATT GTT CCG TCC ACG GTG TAA CCA TAA ATA TTG TTT V I M V F I V P S T V L P L I L F G 55 GGA TAC AAT AAT CAA TAT CAC CTG TTC CAA TTT ACC CAC GCT GTC TGC AAC TCT Y N N Q Y H L F Q F T H A V C N S N 109 AAT CTT ACC CTT TGT AAG GAT CCC CCT AAC AAT TTT TTT ACG GTC CAC GGT TTG L T L C K D P P N N F F T V H G L W 163 TGG CCT CCA AAC ATG CCA ATT AAA TGC CCA ATA AAA AAT GTA GGA CCT AAC ATC P P N M P I K C P I K N V G P N I R 217 CGA AAG GTA CAG AGA GAA CTG GAA ATT ATT TGG CCG AAC GTA AAA TTA CTC GAA K I Q R E L E I I W P N V K L L E H 271 CAC CAG TCG ATC GAA ATA AAG AGT GGA TGA AAC CAA AAA ATA ACC TCT TCT GGG Q F D R D K E W M K T K N N L F W H 325 ATG GCT CCT GTG GGT ATC CCA CAA ATT ACT TTG AAA CCG TAA TAG ATA ACG AGA G S C G Y P T H Y F E T V I I D N E 379 ACC TCA AAA TAT ACG TCT CTA GAA ACA TCA GCA AGA TCT CAA AGG CGA AAC AAA N K I N V S R Y I S K L S K A K Q I 433 TCC AGA TTG AAC CGG ACG GGA AAA AAA GAG CAC TGT TGG ATA TTG AAA ATG CCA K I E P D G K K R A L L D I E N A I 487 TAC GCA ATG GTG CCG ACA GAA TCA AGT GCC AAA AGA ATA AGA AAC CAA AAC ACG R N G A D E L K C Q K N K K P K T T 541 ACG ACT AGG GTT TGG TTG AGA TCG CTC T T K G L V E I S-RNAse SgSgtype 4 S-locus, S... ( ),. Aniline blue, 1/

99 , 1/3 Transsection.,, ( ) Aniline blue. 2-3, 1, 3, 5 ( ) 3. callose. (,, petunia ). RT-PCR Fragment S4-RNase 42%,,, Sepel Signal. PCR.,

100 Level. Homo Primer Homo Hetero PCR-RFLP SaSa, SbSb Hetero SaSb. Homo. System Hetero. S- S PCR-RFLP S S Cloning 7 S-genotype

101 5 1,,,., F1,.....,,,

102 2,4-D 1mg/L BA 1mg/L Murashige & Skoog (MS) Naphythyloxyacetic acid (NAA) 1mg/L Benzylaminopurine(BA) 1mg/L. (Ahn et al. 1986). 2,4-D 1.0mg/L, 6%sucrose MS MS.(Min et al. 1992). saponine riboflavin,,,, 2 3,,.. 5-enolpyruvyl shikimate-3-phosphate synthetase (EPSPS), Basta bialaphos phosphinothricin(ppt). PPT bar Steptomyces hygroscopicus SF1293 bar phosphinothricin acetyltransferase (PAT) PPT. (Takeshi et al. 1986; Thompson et al. 1987; White et al. 1990)Bar

103 (Eliseu etal, 1994; Choi et al. 1996), (Nehra et al. 1994; Vasil et al. 1992), (Viegas et al. 1993), (Spencer et al. 1990; Zhao et al. 1993), (Keertis et al. 1993; Toki et al. 1992), (Aeom, et al. 1996), (Block et al. 1989).,, agrobacterrium biolaphos. 2 Embryogenic Callus 1.. Callus (Codonopsis lanceolata (S. et Z.) Trautv.) 2 3,,, Murashige Skoog(MS) (1962) 6 Callus. Embryogenic callus 2,4-D 0.2ppm NAA 0.2ppm MS,

104 . Callus 2 MS, Callus. Embryogenic callus 2,4-D 0.3ppm Kinetin 0.05ppm MS Subculture,.. MS, 100 /L myo-inositol, 0.4 /L thiamine HCl, 30 /L sucrose, 0.4 Gelrite(MS ), ph , 16 ( 2,000 lx cool-white ) 6 shoot. (RH60 70%, 27 /22, 16 30,000 lx). 2. Callus 2 3,, 2-3,

105 . 2,4-D. 2,. 6, Callus (A) calli, (B) Cotyledomary embryos(root and shoot), (C) 2,4-D MS, 2,4-D 2. MS, Callus ( 5-1).Embryogenic callus Subculture,. Callus 2,4-D 1ppm NAA 0.2ppm Callus. Embryogenic callus

106 2,4-D 1/10 NAA 0.2ppm Embryogenic callus. MS Callus 2 Callus. 2,4-D+Kinetin, 2,4-D+BA, 2,4-D+NAA, 2,4-D+Kinetin 1mg/L 2,4-D 0.1mg/L Kinetin Callus ( 5-1). 2,4-D+BA 2,4-D+Kinetin Callus 2,4-D 0.3mg/L Callus ( 5-2). 2,4-D+NAA 2,4-D+Kinetin 2,4-D+BA 2,4-D mg/L, NAA mg/L Callus ( 5-3). Callus. 2,4-D Callus. 0.3 mg/l 2,4-D 0.05 mg/l NAA

107 5-1. callus 2,4-D kinetin 2,4- D(mg/ L) Kinetin(mg/ L) / / / / /+ - / : callus, +: callus, +/-: non-embryogenic callus, -/+: callus callus 2,4-D BA 2,4- D(mg/ L) BA(mg /L) / / / / /+ + +/- +/- -: callus, +: callus, +/-: non-embryogenic callus, -/+: callus callus 2,4-D NAA 2,4- D(mg/ L) NAA(mg/L) / /- + +/ / - +/ - +/ - +/ - -: callus, +: callus, +/-: non-embryogenic callus, -/+: callus

108 3 Bar 1.. (Codonopsis lanceolata Trautv.) bar pbi vector series Agrobacterium tumefaciens LBA leaf disk tranformation. pbi-bar Agrobacterium tumefaciens LBA ml YEP ml YEP , 30 1 Agrobacterium tumefaciens LBA filter paper 5. 2,4-D zeatin 1ppm MS 25 2 Agrobacterium. kanamycin 100 /L carbenicillin 500 /L

109 . kanamycin bar polymerase chain reaction (PCR). genomic DNA 1g. buffer 15ml 2-mercaptoethanol 750, 20% SDS 1ml M potassium acetate 5ml 20. isoprophanol 10ml (O/N ) 1 TE Buffer 500. phenol chloroform isoprophanol 500 3M Na-acetate 75, 70% EtOH 500. pellet 1 TE Buffer. RNA Sample, 2ml Guanidinium, 18000rpm Homogenage 2. 12,000rpm 10, 1.4ml 0.7g CsCl, 0.8ml 5M CsCl 45,000rpm 12,, Phenol, RNase Northern Blot. Northern Blot RNA 40 /. Formaldehyde MOPs, Hybridization Detection DIG-System X-ray Band

110 PCR Taq DNA polymerase reaction buffer (50mM Tris-HCL, ph 8.6, 40mM KCL, 0.01 BSA) Mg 1.5 mm, primers 200nM,, 200 M dntps, DNA 5ng Taq polymerase 2 units mineral oil. NPT- 5 -CTGGAATGAAGTGCAGGACGAGG-3'(NPT -F) 5'-GCCAACGCTATGTCCTGATAGC-3' (NPT -R), bar 5'-GGATCCATGAGCCCAGAA-3' (bar-f,18mer) 5 -TCAGATCTCGGTGACGGGCA-3'(bar-r, 20mer) primer.pcr NPT 97 7 denaturation PCR PCR 72 7 extension. bar PCR denaturation 94 60, annealing , extension PCR absoult ethanol 10mMTE buffer(ph 8.0) 0.8 agarose gel. 2. Agrobacterium bar (97036), 4 (Hypocothyl) bar ( 5-2) Agrobacterium tumefaciens LBA mg/L Kanamycine 1 10 Callus

111 , somatic embryo. RB P H P X B S E LB Pnos NPT nos 35S- Pro Bar nos 1Kb 5-2. bar Vector., 3, 8 Somatic embryo %, % Somatic embryo bar, 1 Somatic embryo 1,000ppm Bialaphos 2 ( 5-3-a)

112 bar bar (. 5-3-b). 4. PCR bar 2. PCR bar Forward primer Reverse primer 21mer. PCR annealing 58 (. 5-4) PCR Wild type Control Band, 6 500bp Band bar

113 5. Northern blot PCR bar 35-S Promoter 35S Promoter. Total RNA Northern blot (.5-5). Bialaphos Bar. A B 5-5. Bar A; wild type; B; transgenic plant 5. bar A. tumefaciens bar Forword Reverse primer PCR

114 Bialaphos ( 4) Bar X 2- test W ild type 0 21 T (3:1) T (3:1) T (15:1) T (3:1) T 1/N (1:1) T 2/N (1:1) T 3/N (3:1) T 4/N (1:1) * MS Bialaphos 10ppm 25. T2 Bialaphos 4 3 3( ):1( ), 15( ): 1( ). 1:1 3:1, Single(3:1) Two copy Bar., ( ), Program

115 TF , 40, 80 ( 5-6).. Control ,. Control., ( ) ; ( ) Control. 4,,,., F1,

116 .,,,. 2,4-D 1mg/L BA 1mg/L Murashige & Skoog (MS) Naphythyloxyacetic acid (NAA) 1mg/L Benzylaminopurine(BA) 1mg/L. (Ahn et al. 1986). 2,4-D 1.0mg/L, 6%sucrose MS MS (Min et al. 1992)., Homo. Callus Callus 2,4-D 1ppm NAA 0.2ppm Callus, MS. 2 Callus Callus

117 saponine riboflavin,,,, 2 3,,.. condon usage G+C. A+T polyadenylation signal mrna. transgene silence system.(matzke and Matzke, 1995). codon usage, (Agaisse and Lereclus, 1995). 80, (Lee et al.1996; tumer et al. 1987),

118 (Barton et al. 1987;Cho,1995 ;Perlak et al. 1990;Salm et al. 1994; Vaeck et al.1987)., 1996 FDA, USDA, EPA Monsanto (Round-UPTM soybean),, ,

119 6 1..,, (1) 178 1,,,

120 1997 3,,. (2) ,, ,, (M3). 30,,,. 1,

121 2 3 (4 ),, (M4). 30,,,. 3nd Seledtion 1, Phenotypic recurrent selection Group.,,. 1 15g, 10 cm, 0.6cm. 1 20g, 15cm, 1cm, 2nd recurrent selection 1,, ( 1), 2 21 ( 2),

122 .( 3)

123 Group (. 1, 1). recurrent selection. Genomic, 3 (S Homo ). 3. (1). 1. group

124 6-1. Male Female SaSa SbSb ScSc SdSd SeSe SfSf SgSg ShSh Sa Sa Sb Sb Sc Sc Sd Sd Se Se Sf Sf Sg Sg Sh Sh g 9703, 9705, 9708, 9712, 9714, 9715, 9716, 9732, 9734, 9743, Group.,

125 F1. F1 Hetro Genotype 9701 SaSb SaSb SaSc SaSc SaSc SaSd SaSd SaSe SaSe SaSf SaSf SaSg SaSg SaSh SaSb SaSd SaSe SaSe SaSf SaSf SaSg SaSg SaSh SbSc SbSc SbSc SbSd

126 F1 Hetero Genotyepe 9738 ScSe ScSf ScSg ScSg ScSg ScSh ScSd ScSd ScSe ScSe ScSf ScSf ScSg ScSg ScSg ScSh SdSe SdSe SdSf SdSf SdSg SdSg SdSh SdSe SdSe SdSf SdSf SdSg SdSg SdSh SeSf SeSf SeSg SeSg SeSh SeSf SeSf SeSg SeSg SeSh SgSg

127 F1 Hetero Genotype 9779 SgSg SgSh ShSh ShSh SbSd SbSe SbSe SbSf SbSf SbSg SbSg SbSf ScSd ScSd (2) (2 ) 25-30% 96004, 96021, 96036, 96054, 40%. 3-4,

128 (%) (%) 2 2 /1 /

129 4.,, ( ). 3 8 group , 9705, 9713, 9716, 9718, 9756, 9760, 9772, 9773, 9776, , 9612, 9619, 9649, 9669, 9777, 9620, 9643, 9624, 9625, 9691, 9630, 9642, 9650 (S3 )., Parent lines No. of node Root w eight Parent lines No. of node Root W eig ht 9604 SaSa SdSd SaSa SeSe SbSb SeSe ScSc SeSe ScSc SfSf ScSc SfSf SdSd SgSg , 2,

130 (.6-4). ( ) , 9705, 9713, 9716, 9718, 9756, 9760, 9772, 9773, 9776, cm x 15cm x 7cm 120, ,,.,. Heterosis. 3, 2. F1 Heterosis.6-6.Heterosis,.. Heterosis F

131 9777, 9772, (1998) 2 F1 Hetro Genotype 9702 SaSb SaSc SaSg SaSd SaSe SdSf SdSh SeSh SeSf SeSg SeSh Heterosis r=0.712*, r=-0.103, r= Heterosis 9777, 9772, 9756 F1 ( 6-6), A B, C

132 Heterosis ratio Group A

133 Cross(F1) Root weight Root length Root width M.P. H M.P. H M.P. H (Group A) 9756(SdSf) (SeSf) (SeSh) (Group B) 9702(SaSb) (SaSg) (SaSd) (SdSh) (SeSf) (ScSg) (Group C) 9705(SaSc) (SaSc) Mean of heterosis ratio r between M.P. and H * M.P.=Mid parent values, obtained from the means(percent of Keoyong codonopsis) of experiment in 1996, 1997 and H=Heterosis ratio:(f1/m.p.) X

134 3 1.. (F1) 97004, 97009, 97018, 97036, clone., micro,,.. 100mg 100ml 70ml, 80oC 30, 1ml 100ml. No.2,.45um membrane filter HPLC. Develosil ODS-HG-%( 4.6 x 150mm) column, ( E x 340nm, Em 450mn).. 20g 500ml 2L diethyleter rotar evaporar. 100rpm 1, 1L load, NaCl diethyeter. diethyeter 50ml NaSO4 24. glass filter NaSO4,, 100ml/L 0.5ml ethyl decanoate rotary

135 evaporator 2cm. water bath,., ml, GC-MS.. column PEG20M capillary column DB-WAX (0.25mm x 60m), carrier gas helium, column 30cm/sec, column 40oC-220oC, 3oC/min, 220oC, FID, make up gas.. 2.5g 25ml BHT 100mg/L hexan 5 No ml Sep Pak Plus minicolumn 5% isoprophylalchol/hexan 5ml 1.5ml 3.5ml, 5% isoprophylalchol/hexan 5ml HPLC. Asahipak ODP-50(4.6x150mm) column, (UV 259nm). 2.. Vitamin C 2nd selection 3rd selection 9704, 9709, 9718, 9736, 9744 clone Vitamin C Ascobic acid Tocoperol(Vitamin E).6-7. Ascobic acid Tocoperol

136 97036, Control ,, Ascobic acid Tocoperol Vitamin (mg/g) Sample Ascobic acid Tocoperol , 9709, 9718, 9736, 9744 clone.(. 6-8). Check,

137 6-8. Peak No.a 1. Hexanal Compound name of line Pentenal, 4-methyl- 2- Hexenal 2-Penten-1-ol, (Z)- 2- Hexene, 1- (1- ethoxyethoxy)-, (E)- 1- Hexanol 3- Hexen- 1- ol 2, 4- Hexadiental, (E, E)- 2- Hexen- 1- ol, (Z)- Acetic acid Pentane, 2- methoxy- 2, 4, 4- trimethyl- Cyclobutane, 1, 2- diethyl-, cis- 1- Butyne, 3- ethoxy- 3- methyl- 2, 5- Cyclohexadiene- 1, 4- dione, 2, 5-bis(1,1-dimethylpropyl) Propane, 2-chloro-2- nitro- Silane, butoxytrimethyl- 3- (3- Carboxy- 4- hydroxyphenyl)- D- alanine Cyclotrisiloxane, hexamethyl- Hexanoic acid Phenol, 3-methyl- Phenol, 2, 6- bis(1, 1- dimethyllethyl)- 4- methyl- Benzeneethanol Hexanoic acid, 2-ethyl- 3- Hexanoic acid, (E)- 3- Hexanoic acid Cycloheptane, 1, 3, 5- tris(methylene)- Octanoic acid Phenol, (1, 1- dimethylethyl)- 2- methoxy- Nonanoic acid 1- Naphthalenol, 1, 2, 3, 4, 4a, 7, 8, 8aoctahydro- 1, 6- dimethyl- 4- (1- methyl... Decanoic acid Dodecanoic acid 1, 2-Benzenedicarboxylic acid, butyl 2- methylpropyl ester

138 . Asparaginic acid % Asp Glu Asn Ser Gla Arg Thr Thea Ala Tyr Met Val Phe Ile Leu Lys * 1, 2 F1. Glutamic acid 9704, Asparagine 9704, Glutamine %, % 4.2. Aruginine %, Threonine,

139 Theanin Valine Lysine, (.6-9) Level. 4 Marker 1.. F1 marker 34 Marker.. DNA PCR DNA (1 Cm2), 100ul Buffer( 10mM Tris-Cl, 450mM EDTA ph8.0, 1mg/ml Proteinase K, 1% Sarocosyl) 50oC 1, Tube 11000rpm 10, 100ug/ml RNase, 100oC 5, Template DNA

140 . PCR marker Primer 21 base oligonucleotide primer 10, 0.5 primer 3 genomic DNA, 0.04 Taq DNA polymerase, 1 10 reaction buffer, mM dntp, 1 MgCl PCR Reaction mixture. PCR machine GeneAmp PCR System 9600(Perkin-Elmer Cetus), DNA denaturation; annealing; extension 45cycles, cycle denaturation 2 cycle extension 5. PCR TAE buffer(40 mm Tris-acetate, 1 mm EDTA, ph8.2) 1.8% agarose gel 0.5 / ethidium bromide 40 UV transilluminator type 55 Polaroid film. Molecular standard lamda Hind III.. band primer 21mer annealing 58 PCR. Agarose gel, band 100ul 1 x TE buffer 30, PCR Template DNA. PCR Band TA cloning vector

141 . marker sequencing PCR clone RNA DNA, Taq Dye Primer-Sequence Kit (Applied Biosystem ). PCR Applied Biosystem. DNA GENETYX-CD version DNASIS version PCR-Southern hybridization band band PCR band PCR, banding menbrane transfer band labelling Southern blot. 2. Marker -PCR Marker Marker Primer mer, PCR 94oC 30, Annealing 58oC, 72oC 45 Cycle. 2% Agarose gel, Et-Br Banding. 3 Primer, Band Cloning PCR-Southern

142 ( 6-6 ). Southern blot Hybridization band, Pattern ( 6-6). band Cloning ( 6-7)... M M 6-6. Band pattern PCR-Southern blot

143 Specific fragment 1 ATT GTA TAT AAT TCC CCC CTT TAT AGC TGA CGA GGG CGT ACT TTG CAT TTT TGG GGA TTC TAT TCA ACA GCC ATA TTG CTT TTA TAC TGA ACT TGG ATT CAA ACA CCT TTA TGC ATA TAG TTG GTT CTC TTG AAT CTG GTT TGA AGG GCC TGG ATT CAA GTA TCT CAT CAC AGG TAT GTT TCC TTT ATT TTA TTG GAA TTC TTT TCC CCT ATT GAT TAC TGT CCA AGT GTT CAT TCA TTT GAG CAT TAT ATA TTT TTC GGC TGC ACA GTT TGG TCG AAC CAT TGG AAA CAC AAT CTT CCT TAG CAC ATT GGC CTA TCG TGA ATT TCC GAT CCA CAT CTG TAT GTT GTT TGT GTT ATC TAC TTG ATA GGG TGA TCT GGT AGT AGT AAC GGA TAC TTC GAT TAA AGT GCA CAA GAT GAA AGT AGG CGG AAG ATT AGA GGA GAA GGC TTG CCT TAA ATG CTT CTC AAG CAA TCC ACT ATG CTG CTG ATC TAG GTT AGA AGG GCG AAT TCC AGC ACA CTG GCG GCC GTT ACT AGT TTC CCC CCT TAA TAT ATG TTA Specific fragment 2 ATT GTA TAT AAT TCC CCC CTT TTA ATA TAT AAA ATA AAA CAA GTC TGG TAC ATT GCA CTA TTC TTA TCA AGG TTA CTT TTT AGG GTA GTT TTC TAC CTC TTT ATT ATT TGA GTA TTC AAA TGG GAC TCT TTG AGT GGA GTT GGT TAG TAT TAC AAA ATT AGT TGT CAG CAT AAG TTG ATT CGA CAG AGA GAT GCC CTA ATT ATT GCT CTG GCA CCT TTG GCA CCT TTT GTC TGA CAT TGT GGT GAA GTA AGA TGT TGA TGT TCA TTG GCC AAT CCC CAT GTA TAT GTC AAC TTT CTT TGA AGA GAA TAT TGC ATG GAA TAT GAG CCT GAT TCT ATA TTA ATT CTT TCC CTT TAT AGC TGA CGA GGG CGT ACT TTG CAT TTT TGG GAG GTT CTA TTC AAC AGC CAT ATT GGT TTT ATA CTG AAC TTG GAT TCA ACA CCT TTA TGC ATA TAG TTG GGT CTC TTG AAT CTG GTT TGA AGG GCC CAA CAC TAA GGA GAC SGG AAW TTM AAT AAA ATC ATT TCA CGA ACG AGG AGT AAT CGA AWT TGA ATG GAG AAT CAA TCG GAA GCG TGG CTC TTA TAC CTT GTT AGT TTG GCA AAG TTA TCC ATG AAT CAA CGA GCT CAG TTG TGG AGA AAG AAG GAG GTT ATC TTA TTG ATT ATT CAA GTG AAA ATT ACA GCC TAA CAG AAT CTA GAA GAT ACA TTT ATA TGA AGT ATG GCT GGA TTA CAT GTT AAT GAC CAC TAA CAG AAT CAG TTT AAA CCC CAC TCT CCA CTT GTA ACG GAC TCT ACT TTA CTA TGC AAT CAC CTG CAA GTG CAC CCA CTG CGA TTG ATA ATG GTT GTA CCC TTA ATA TAT AAA GTA AAA CAA GTC TGG TAC AAT GCA GTA TTC TTA TCA AGG TTA CTT TTA AGG GTA GTT TTC TAC CTC TTT AAT AAT TGA GTA TTC AAA TGG GAC TCT TTG AGT GGA GTT GAG TAA GTA TTA CAA AAT TAG TTG TAC GCA TAA TTG ATT CGA CAG AGA GAT GCC CTA ATA TTT GCC TTG GCA CCT TTG GCA CCT TTT GTC TGG ACA TTG TGG GTG AAG TAA CAT GTT GAT CTC TAT TGG CCA ATC CCC ATG TAT ATG TCA ACT TTC TTT GAA GAG AAA TAT TGC ATG GGA ATA TGA GCC TGA TTG TAT ATT ATA TCC TCT CCC CTT ATA GCT CKA CGA GGG CGT ACT TTG ACT TTT TTG GGA GGG TCT ATT TCA ACA GCC ATA TTG CGT TTT ATA CAG TTC CCC CCT TAA TAT ATG TTA 6-6. PCR Fragments. 3. Marker 3 (1998 ) 1 11 Marker Operon 10mer primer 21mer Primer, Annealing 37 (10mer), (21mer) RAPD Band(.6-7) -, + (. 6-10) Band, Pattern

144 2 Heterosis A group(9756, 9772, 9777) B group( 9702, 9713, 9716, 9760, 9773, 9776) C group(9705, 9718) Marker Polymorphism. URP-41F OPB12 OPB-11 OPA RAPD markers

145 RAPD markers Band Line Primer bp Line Primer bp OPA OPB OPA OPB OPA OPA URP OPB URP OPB marker. marker RAPD

146 Marker. Primer Operon mer primer. Primer Marker (.6-8) RAPD ( 6-9). 2,

147 Phenotypic recurrent selection Group,,, , 2 21, Group. 52, S Homo