Sistema de seguridad eléctrica

Hormones

Infect four days old cotyledonary leaf segment with A. tumefaciens EHA105 harboring pBI121

Co-cultivate explants on liquid co-cultivation medium containing 100µM acetosyringone

Transfer the explantson callus induction medium containing 25mg/L meropenem

Transfer the callus to shoot regeneration and selection medium containing 15mg/L kanamycin and 25mg/L meropenem

Transfer the regenerated shoots to shoot elongationand selection medium containing 15mg/L kanamycinand 25mg/L meropenem

Transfer the elongated shoots on root induction medium

Transfer rooted plants to pots and acclimatize Wash the explants with sterile distilled water containing

75mg/L meropenem

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Fig. 1 Schematic represent ation of Agrobacterium -mediated transformation of Jatropha curcas

Jatropha (Jatropha curcas L.)

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1. Kanamycin sulfate: Prepare the stock of 100 mg/ml by dis-solving 500 mg of kanamycin powder in 5 ml of sterile dH 2 O, fi lter sterilize, and store at −20 °C ( see Note 4 ).

2. Rifampicin: Prepare the stock of 10 mg/ml by dissolving 10 mg of rifampicin in few drops of dimethyl sulfoxide (DMSO) and then make up the volume by adding sterile dH 2 O, fi lter sterilize, and store at −20 °C.

3. Meropenem: Prepare the stock of 50 mg/ml by dissolving 1 g of meropenem salt in 20-ml sterile distilled water, fi lter steril-ize, and store at 4 °C.

1. Sterilization solution: 70 % (v/v) ethanol and 0.1 % (v/v) sodium hypochlorite, Tween 20 and 0.2 % (w/v) mercuric chloride ( see Note 5 ).

2. Acetosyringone: Prepare the stock solution of 100-mM aceto-syringone (3′,5′-dimethoxy-4′-hydroxyacetophenone) by dis-solving 0.392-g acetosyringone salt in 10-ml DMSO, fi lter sterilize, and store at −20 °C.

3. ß-Glucuronidase: GUS assay buffer (50–100 μg/ml X-glcA in 20 mM NaPO 4 , pH = 7.0).

4. DNA extraction buffer: 20-μM Tris–HCl, pH 7.5, 250-μM NaCl, 25-μM EDTA, pH 8.0, and 0.5 % sodium dodecyl sulfate (SDS).

5. High-salt TE buffer: 10-mM Tris–HCl, pH 8.0, and 1-mM EDTA, pH 8.0, 1 M NaCl.

6. Polymerase chain reaction (PCR) components: 10× Taq buf-fer, 10-mM dNTPs, 50 pmol/μl each of forward and reverse primer, 3 U/μl Taq polymerase, ~100-ng DNA template, and sterile Milli-Q water to make up the volume up to 25 μl.

All stock solutions are freshly prepared on a monthly basis and stored at 4 °C.

1. Murashige and Skoog (MS) major salts (10×): Dissolve 19.0 g/l KNO 3 , 16.5 g/l NH 4 NO 3 , 3.7 g/l, MgSO 4 · 7H 2 O, 4.4 g/l CaCl 2 · 2H 2 O, and 1.7 g/l KH 2 PO 4 [ 17 ].

2. MS minor salts (100×): Dissolve 2.2 g/l MnSO 4 · 4H 2 O, 83 mg/l KI, 620 mg/l H 3 BO 4 , 860 mg/l ZnSO 4 · 7H 2 O, 2.5 mg/l CuSO 4 · 5H 2 O, 25 mg/l NaMoO 4 · 2H 2 O, and 2.5 mg/l CoCl 2 · 6H 2 O [ 17 ].

3. MS vitamin stock (200×): Dissolve 100 mg/l niacin, 100 mg/l pyridoxine HCl, 400 mg/l glycine, and 20 mg/l thiamine HCl [ 17 ].

4. Iron stock (200×): Dissolve 7.45 g/l of Na 2 EDTA (ethylene-diaminetetraacetic acid, disodium salt) in 500-ml dH 2 O and 5.57 g/l of FeSO 4 · 7H 2 O in 500-ml dH 2 O separately. Boil the 2.3.2 Antibiotics

2.3.3 Other Solution

2.3.4 Culture Medium Stock

Devendra Kumar Maravi et al.

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Na 2 EDTA solution and add FeSO 4 solution to it, gently by stirring [ 17 ].

5. All culture medium stock is stored in amber-colored bottle at 4 °C.

6. Agrobacterium minimal medium (AB) buffer (20×): Dissolve 60 g/l K 2 HPO 4 and 20 g/l NaH 2 PO 4 in dH 2 O, adjust pH to 7.0 with 1 N NaOH or 1 N HCl, as required, then autoclave ( see Note 6 ) [ 18 ].

7. AB salts (20×): Dissolve 20 g/l NH 4 Cl, 6 g/l MgSO 4 · 7H 2 O, 3 g/l KCl, 0.2 g/l CaCl 2 , 50 mg/l FeSO 4 · 7H 2 O in dH 2 O then autoclave ( see Note 7 ) [ 18 ].

8. D-glucose (0.5 %): Dissolve 5-g D -glucose in 1 l of dH 2 O then autoclave [ 18 ].

1. Murashige and Skoog medium (MS): MS medium (major salt 50 ml, minor salt 5 ml, vitamin 5 ml, iron 5 ml, and myoino-sitol 100 mg) supplemented with 3 % (w/v) sucrose, adjust pH 5.84 with 1 N NaOH or 1 N HCl. Add agar-agar (3.2 g/l) prior to autoclaving (Subheading 2.3.4 ).

2. Liquid cocultivation media (LCM): MS liquid medium con-taining 6.66-μM BAP and 0.24-μM IBA, adjust pH to 5.7, and supplement with 100-μM acetosyringone ( see Note 8 ).

3. Callus induction medium (CI): MS medium supplemented with 6.66-μM BAP, 0.24-μM IBA, 3 % (w/v) sucrose, adjust pH to 5.84 with 1 N NaOH or 1 N HCl and solidifi ed with 0.8 % (w/v) agar-agar, autoclave and add 25 mg/l meropenem, mix thoroughly, and dispense 25 ml into 90-mm sterile Petri dishes.

4. Shoot regeneration and selection medium (SR): MS medium supplemented with 6.66 μM BAP, 0.24 μM IBA, 1.5 μM GA3, 3 % (w/v) sucrose, adjust pH to 5.84 with 1 N NaOH or 1 N HCl, and solidifi ed with 0.8 % (w/v) agar-agar.

Autoclave and add 15 mg/l kanamycin and 25 mg/l merope-nem during explant inoculation ( see Note 9 ).

5. Shoot elongation and selection medium (SE): MS medium supplemented with 1-μM GA3, 3 % (w/v) sucrose, adjust pH to 5.84 with 1 N NaOH or 1 N HCl and solidify with 0.8 % (w/v) agar-agar, autoclave, and add 15 mg/l kanamycin and 25 mg/l meropenem during explant inoculation.

6. Root induction medium (RI): MS medium consisting of half strength of MS major and MS minor elements, full strength of vitamin, and 2 % (w/v) sucrose; adjust pH to 5.84 with 1 N NaOH or 1 N HCl, solidify with 0.6 % (w/v) agar, autoclave, and add 5-μM IBA and 25 mg/l meropenem during shoot transfer.

7. Sterilize all tissue culture media by autoclaving for 20 min at 121 °C and 15 psi.

2.3.5 Plant Culture Media

Jatropha (Jatropha curcas L.)

30

1. Luria-Bertani (LB) agar medium: Dissolve 25 g of LB powder in 1 l of dH 2 O. Add 15 g/l agar-agar, autoclave, cool to about 55 °C, and add 50 mg/l kanamycin, 10 mg/l rifampicin for the selection of Agrobacterium tumefaciens strain EHA105 harboring plant binary vector pBI121.

2. AB minimal media: Combine 5-ml sterile 20× AB buffer and 5-ml 20× AB salts in 90-ml sterile D -glucose (fi nal concentration of D -glucose is 0.5 %).

3 Methods

1. Remove the seed coat and soak in distilled water for overnight at room temperature.

2. Surface-sterilize the de-coated seeds with 0.1 % sodium hypo-chlorite solution supplemented with few drops of Tween 20 for 15 min and rinse with distilled water several times.

3. Surface-sterilize the seeds with 70 % ethanol for 5 min and rinse three times with sterilized distilled water.

4. Finally, sterilize with 0.2 % mercuric chloride for 2 min and rinse with sterile distilled water 3–4 times under the laminar air fl ow cabinet.

5. Blot dry the seeds using sterile fi lter paper.

6. Carefully dissect the endosperm to expose out embryos with papery cotyledonary leaves using forceps and scalpel.

7. Separate out the papery cotyledonary leaves and germinate on MS basal medium.

8. Cut the papery cotyledonary leaves into four segments (8 mm) with their edges removed and use as explant for Agrobacterium - mediated transformation.

1. Streak Agrobacterium tumefaciens strain EHA105 on solid LB medium supplemented with 50 mg/l kanamycin and 10 mg/l rifampicin for 2 days at 28 °C ( see Note 10 ).

2. Inoculate a single bacterial colony into 25 ml of liquid AB min-imal medium with appropriate antibiotics to select the binary plasmid and agitate overnight at 28 °C on a rotary shaker at 180 rpm, until optical density at 600 nm reaches to 0.8.

3. Transfer the liquid culture to a centrifuge tube, and pellet the cells by spinning for 10–15 min at 4 °C for 4,025 × g . Decant the supernatant and resuspend the cell pellet in liquid coculti-vation media (pH 5.7) supplemented with 100-μM aceto-syringone and 0.24-μM IBA. Adjust the bacterial cell density, if necessary, by further dilution to achieve OD 600 = 0.6–0.8.

Agrobacterium suspension cultures are then ready to use for transformation experiment.

2.3.6 Culture Media for A. tumefaciens

3.1 Preparation