類 不 兩 都 理 來 類 利 良 例 說 例
良 1) 利 來 不 理 料 省, 率 2) 益 連 3) ; 4) 行 便 F 1 F 2 異 5) (epistasis) (pleiotropy)
Lettuce Linkage Map From: Landry et al. 1987 Genetics 116:335
利 來 (1) 異 (protein and isozyme electrophoresis) (2) DNA 切 (Restriction fragment length polymerphisms, RFLPs) (3) 連 (Arbitrarily primed polymerase chain reaction) DNA (Random amplified polymorphic DNA; RAPD)
異 流 3-9 1-3. 異 流
- 精 異
切 切 說 A bb probe A B A B
流 DNA DNA DNA extraction cdna library Genomic library 切 Enzyme digestion DNA Gel electrophoresis Probe cloning and preparation Southern blotting DNA Pre-hybridization DNA 識 Probe labeling Hybridization Detection. 切 RFLP 流
切 Soybean DNA Restriction Fragment Length Polymorphisms (RFLPs)
連 PCR 流 50mM KCl, 10mM Tris-HCl ph8.3, DNA template DNA 1.5mM MgCl 2, 0.01% gelatine dntp each 200uM(pH 7.0) primer 5-100 pmole DNA template DNA 1 ug-30 ng DNA polymerase 2.5U/100 ul 100 ul PCR cycles DNA denaturation DNA Primer extention Primer annealing 煉. 連 PCR 流
Random Amplified Polymorphic DNA(RAPD)
(FDA)
流 (Cell Flow Cytometry) PMT 5 PMT 4 Sample Flow cell Dichroic Filters PMT 2 PMT 3 Scatter Sensor Bandpass Filters PMT 1 Laser
Utilization of Genetic Transformation in Broccoli (Brassica olercea var. italica) for Postharvest Yellowing Retardation 利 Long-Fang O. Chen INSTITUTE OF BOTANY, ACADEMIA SINICA, NANKANG, TAIPEI 115, TAIWAN, R.O.C.
, (20-25 C ) 易 葉 綠,
不 : 1) 冷 冷 理 ; 2) ; 3) ; 4) ; 5) 裂 類 ; 來 老
例 : (1) 利 冷 冷 不 便 (2) 利 例 易 異
利 不 易 異 (4) 利 理 不 良 (5) 利 裂 Cytokinins 類 易 量 留 不
利 良 狀 利 (Zhong 2001, Euphytica 118:137-144)
. 來 類 類 糧 類,,, 蘿,,,,,,,,,, 類, 梨,, 羅,,, 李,, 理 : 2000
. 類 糧 鈴 狀 類 殺 殺 殺 殺 不 殺 理 : 2000
B Fig. 1. Regeneration and transformation of broccoli (Brassica oleracea var. italica). A: Shoots from ipt transformed peduncle explants; B: Close view of shoot regenerated from peduncle explants under Kanamycin selection.
Studies on the Transformation of Cytokinins synthatase ( isopentenyl transferase, ipt gene )
Plasmid maps (A) psg 529(+) BamHI SpeI NcoI BamHI SacI SpeI RB SAG12 promoter IPT Nos-ter NPTII 1 Kb probe psg529(+) plasmid map LB
Fig. 2. Dot assay for neomycin phosphotransferase (NPTII) activity in crude cell extracts of transformants. positive control: pbi121 (1A,1B); psg529(+) (1C); psg766a (1D); negative control: non-transformed leaf (1E); extraction buffer(1f).
(A) Primers for ipt were 5 -GATTTGATTAAGCTTTTAACTTGC-3 and 5 -GCCCGCCGTTGGCCTCATGAT-3 for psg529(+) coding for a 1 Kb fragment including segment of SAG12 promoter and ipt gene. (B) Primers for nptii were two 21 mers, 5 -GAGGCTATTCGGCTATGACTG-3 and 5 -ATCGGGAGCGGCGATACCGTA-3 coding for a 0.7 Kb fragment[8]. For psg766a, a primer set 5 -ACCCATGGACCTGCATCTA-3 and 5 -GGAGCTCAGGGCTGGCGTAACC-3 [10] was used. Fig 4. PCR identification of the transgenes(ipt and nptii ) for the (A) psg529(+) and (B) psg766a transformants. Thermal cycles for ipt gene were denatured at 94 C 2 min then 25 cycles of 94 C 30 sec, 60 C 30 sec and 72 C 30 sec, 1 cycle of 72 C 7 min. Thermal cycles for nptii gene were denatured at 94 C 5 min then 35 cycles of 94 C 1 min, 55 C 1 min and 72 C 2 min, C 7 min.
Table 1. The effects of seedling ages and explant types on frequencies of shoot regeneration from cotyledon, hypocotyl and peduncle under kanamycin selection after A. tumefaciens infection with ipt genes. Seedling ages or storage time 3 days after in vitro germination 9 days after in vitro germination 2-3 weeks after harvest a Explants Cotyledon Hypocotyl Cotyledon Hypocotyl Peduncle Transformation 4/184 frequency b ( 2.2) 10/175 ( 5.7) 1/ 53 ( 1.9) 1/156 ( 0.6) 18/222 ( 8.1) No. of plantlets 31 65 5 3 372 recovered c % positive NPTII activity 20/31 (64.5) 34/65 (52.3) 1/ 5 (20.0) 1/ 3 (33.3) 264/372 (71.0)
Fig. Southern Hybridization of psg529(a) and (b) psg766 transformants; P: plasmid DNA, a 7 and b 12 are non-transformed control.
Fig. Postponing effect on the yellowing of detached leaves of broccoli transformed with ipt gene. A,B,C: transformants (I-126, I-119, P-88), D: nontransformed control, upper row: pictured at the day of detachment, lower row: pictured after detachment and stored at 25 C for 4 days.
Figure. Plantlets of transformant at the left (L) and nontransformed control at the right (R) indicate leafy yellowing at the bottom part of the control plant at the stage when the plant starts bolting.
Fig. Chromo-meter CR-310, Minolta. 50mm.
140 130 IT53-12 IT53-14 IT53-22 IT53-32 Hue angle (H o ) 120 110 100 90 80 0 1 2 3 4 5 6 7 8 Days after harvest IT53 不 ( and : 0.75 kb band; and :1.0 kb band) 類 LDPE 25 Fig 3. Changes of hue angle of the progeny of transformed line IT53 with two types of insert fragments ( and : 0.75 kb band; and :1.0 kb band). Florets was packaged with LDPE-bag and stored at 25 in the dark. Bars are standard errors.
Fig. Ipt gene expression detected by RT-PCR on the Day detached (Lanes 1-7) and 4days after stored at 25 C(Lanes 8-14), 6,13 are PCR(-) kanamycin survival plant, 7 and 14 are non-transformed control.
(A) (B) ipt 北 (A) psg529(+) ; (B) psg766a Fig. 4. Northern analysis in the progeny of psg529(+) (A) and psg766a (B) transformed. The floret was packaged with a LDPE plastic bag and store at 25 in the dark for 0 day and 5 days. Northern hybridization analysis and the probe was a DIG-labeling 0.75-kb fragment of ipt coding region for the identification of psg766a transformed.
Fig. Postponing effect on the yellowing of detached F2 flowers of broccoli transformed with ipt gene. A,B,C,D: transformants (P16-10, P16-11, P16-15, P1 E: non-transformed control, upper row: pictured at the day of detachment, lower row: pictured after detachment and stored at 25 C for 6 days.
89-106 segregation line Day 5 after detached and stored at 25C in dark
Cytokinin synthesizing ipt transformation lines (A) Self-incompatibility line (B) Self-compatibility line
Studies on the mers(ethylene response sensor mutant gene) Transformation
(A) psmy1h-mersi62f (B) pbi-mersi62f mersi62f (ethylene response sensor, ERS), ERS 列 62 Ile(I) Phe(F). CaMV 35S promoter hygromycin knamycin.
Fig. Leaf yellowing comparison in mers transformantsand nontransformed control after continuously applying of 1 ppm ethylene at room temperature.
Fig. Northern analysis of mers on transformation lines at the day of Detached (D=0) and 3 days after storing at 25ºC(D=3) in dark.
Fig. Leaf yellowing comparison in a mers transformant and non-transformed control after detached and stored at 25 ºC in dark.
Fig. Floret yellowing in mers transformation lines in broccoli after detached and stored at 25 ºC in dark for 4 days.
Studies on the Anti-chlorophyllase Transformation
離 不 行 離 不 狀 不 (Zhong 2001, Euphytica 118:137-144)
論 利 老 行 不 不 不 狀 理 切 了 來