Page 1 2000 9 akiyama@biochem.osakafu-u.ac.jp VA 4 1999 9 2000 2 Plant J., 2, 487-496 (1992) Handberg K. and Stougaard J. Exp. Bot., 48,1357-1365 (1997) Stiller J. (Gresshoff PM) 4 80 5 10 20 ( ) < ecotype > (Lotus japonicus) Gifu B-129 Agrobacterium tumefaciens GV3101 C58C1 Rif r ( ) (pmp90 Gm r, deletion of T-DNA of ptic58) Mol. Gen. Genet., 204, 383-396 (1986) Agrobacterium tumefaciens LBA4404 (Clonetech ) pbgi, pgi ( ) pbgi pbi121 Clonetech gus p35s GUS INT (Mol. Gen. Genet., 220, 245-250 (1990) Max-Planck Willmitzer L. ) gus pgi -gus fusion pbgi 35S CaMV Hind III, Xba I Klenow Xba I, Bam HI, Sma I
Page 2 (1) ENOD12A (2) RD29A 5 0.7% 5 1/10X B5 ( )+0.5 µg/ml BA +0.05 µg/ml NAA + 4 5 1X B5 +0.5 µg/ml BA +0.05 µg/ml NAA +10 mm NH 4 3 +5 µg/ml geneticin base +200 µg/ml ( ) 2 +5 µg/ml geneticin base +100 µg/ml 3 +100 µg/ml 3 1X B5 +0.2 µg/ml BA +100 µg/ml 2 3 1X B5 +100 µg/ml 1 1/2X B5 + 0.5 µg/ml NAA 2 3 1/2X B5 18 20 4.2-4.7
Page 3 (1) 1. 400 2. 70% EtOH-H 2 O 1 2% NaClO 10 4 3. 0.7% 20-30 / *1 4. *2 5. 24 16 /22 8 5 *1 ( 80 ) *2 (2) 1. pbgi pgi Agrobacterium tumefaciens GV3101 C58C1 50% 5 µl 50 µg/ml YEP 2 ml 15 mm 13 cm 2. 28 120 rpm ( 20 ) *3 3. 10 OD660 4. YEP OD660 0.3 8 ml *3 (3) - 1. 9 cm No.2 2 150 1 2. (2)-4. 8 ml 3. (1)-5.
Page 4 4. 5mm 5. No.2 1 4. 80 2 6. 7. 24 16 /22 8 5 8. pbgi 5,6 *4 GUS GUS *4 GUS X/X GUS GUS (2) LBA4404 (4) GUS GUS ( ) (1) 28 120 rpm 22 YEP/Km 50 5 ml in 100 ml OD 660 0.7 ( ) 7 3 5 7 OD660 GV3101 C58C1/p35S GUS 0.3 5/5 4/6 3/6 INT 0.03 0/6 5/6 4/6 (2) 28 2.5 LB/Km 50 9cm 0.3 7 3 4 GV3101 0/6 3/5 C58C1/pBGI LBA4404/pBGI 0/6 0/5 (3) 28 120 rpm 2 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD 660 0.61 (1/10 2.9) OD 660 0.1
Page 5 7 GV3101 C58C1/p35S GUS INT 7 1/5 (4) 28 120 rpm 21 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD 660 0.63 (1/10 ) OD 660 0.3 12 5 7 GV3101 C58C1/pBGI 0/10 0/10 (5) 28 120 rpm 22 YEP/Km 50 2 mlin 15mmI.D.X13 cm OD 660 0.53 (1/10 ) OD 660 0.3 6 4 5 GV3101 C58C1/pBGI 3/6 2/5 *5 (4) 1. 250 mg/l 9 cm 3 9cm 3 2. 3. 1 3 3 4. 5.
Page 6 6. 24 16 /22 8 4 5 *5 YEP YMB ( ) 300 µg/ml 200 µg/ml (5) 1. 2. 3. 24 16 /22 8 3 1 (6) 1. 2. *6 3. 24 16 /22 8 1 4. 5. 6. 24 16 /22 8 1 *6 geneticin base 5 1
Page 7 (7) 1. 2. *7 3. 24 16 /22 8 1 4. 5. *8 6. 24 16 /22 8 *7 ( ) *8 1 (8) 1. 5 mm *9 2. 3. 24 16 /22 8 4. *10 5. 24 16 /22 8 *9 NAA *10 80%
Page 8 (9) *11 1. 2. 9 cm 3. 100 ml 4. 24 16 /22 8 1 *12 5. *11 *12 1 *13 (10) 1. (1 3 ) DNA 2. gus5 (5 CCATCGCAGCGTAATGCTCT3 ), gus6 (5 GCCGACAGCAGCAGTTTCAT3 ) PCR gus *14 95 1min, 95 30sec 55 30sec 72 1 min 30-35 *13 geneticin 50% *14 gus Taq polymerase DNA -gus fusion (5 - CTCGTATGTTGTGTGGAATTGT, 3 - TCACGGGTTGGGGTTTCTAC) 95 1min, 95 30sec 55 30sec 72 X min 30-35
Page 9 1 L 1. (geneticin base, ) ( Phytagel) Milli-Q 2. ph 0.5N KOH, HCl 3. 4. 5. (120 10-15 ) 6. 7. Milli-Q IWAKI SH90-20 90X20 mm 1 20-30 ml IWAKI CUL-JAR300 ( ) 100 ml 2 4 (BA, NAA) DMSO 10mg/ml -20 (NH 4 ) 2 SO 4 4 Phytagel( ) Wako 1 ( ) (J. Exp. Bot., 48,1357-1365 (1997)) Sigma A-7921 0.6 500 g 32,200 Phytagel Sigma P-8169
Page 10 500 g 18,400 0.2% 1/5 Wako Agrobacterium Sigma C-7039 1g 20,400 5 g(0.5 g X10 ) 7,830 (1/10 ) 1. code No. ( ) for 1 L 1/10X B5 Sigma G-5768 (31 g) 0.31 g 1/10X B5 100X 1 ml 5 mm MES DOJINDO 341-01622 (25 g) 1.1 g 0.5 µg/ml BA Wako 020-07621 (1 g) 500 µg 0.05 µg/ml NAA Wako 148-00092 (25 g) 50 µg 0.6% Wako 016-11875(500 g) 6 g ph 5.2 B5 (100X -20 ) code No.( ) X100 for 100 ml 100 µg/ml Wako 092-00282 (25 g) 1 g 1 µg/ml Wako 142-01232 (25 g) 10 mg 1 µg/ml Wako 163-05402 (25 g) 10 mg 10 µg/mlwako 201-00852 (25 g) 100 mg
Page 11 2. 1X B5 3.1 g 1X B5 10 ml 2% ICN 194747(Wako ) 20 g 10 mm (NH 4 ) 2 SO 4 Wako 019-03435 (500 g) 10 ml 400 mm(5.36 g/100 ml) 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel Sigma P-8169 500 g 2 g 200 µg/ml Hoechst 200 mg 0.5 gx10 3. geneticin base (Sigma G-1279, 250 mg) 5 µg/ml 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 200 µg/ml 200 mg 5 µg/ml geneticin base Sigma G-1279 (250 mg) 5 mg
Page 12 4. ( 100 µg/ml ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg 5 µg/ml geneticin base 5 mg 5. ( geneticin base ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 10 mm (NH 4 ) 2 SO 4 10 ml 0.5 µg/ml BA 500 µg 0.05 µg/ml NAA 50 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg
Page 13 6. 1X B5 3.1 g 1X B5 10 ml 2% 20 g 0.2 µg/ml BA 200 µg 0.2% Phytagel 2 g 100 µg/ml 100 mg 7. ( BA ) 1X B5 3.1 g 1X B5 10 ml 2% 20 g 0.2% Phytagel 2 g 100 µg/ml 100 mg 8. 1/2X B5 1.55 g 1/2X B5 5 ml 1% 10 g 0.5 µg/ml NAA 500 µg 0.4% Phytagel 4 g
Page 14 9. ( NAA ) 1/2X B5 1.55 g 1/2X B5 5 ml 1% 10 g 0.4% Phytagel 4 g 10. YEP for Agrobacterium Bacto-peptone 10 g 10 g NaCl 5 g 1M MgCl 2 2 ml for 1 L ph 7.2 MLR-350 24 16 /22 8 40W 15 20,000 lux, 150 µe m -2 s -1