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Regulator Stock Solutions 2.3 Antibiotics Stock Solutions

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1. MS vitamin stock (1,000×): 0.5 g/L thiamine HCl, 0.5 g/L pyridoxine HCl, 0.05 g/L nicotinic acid, 2.0 g/L glycine. Store 50 mL aliquots in Falcon tubes at 4 °C for 6 months. 2. Acetosyringone (AS): 300 mM of stock solution is prepared by

dissolving 0.588 g of AS in 10 mL of DMSO, fi lter-sterilize through 0.2 μm membrane, aliquot 1 mL into 1.5 mL sterile Eppendorf tubes, and store at −20 °C for 6 months.

The liquid medium for plant culture is prepared as either 3 mL aliquots in test tubes or 30 mL aliquots in Magenta boxes, pH adjusted with NaOH (1 N) to 5.8, and autoclaved for 30 min at 121 °C.

All solid medium for plant culture is prepared as 500 mL ali- quots in 1,000 mL Erlenmeyer fl asks and pH adjusted with NaOH (1 N) to 5.8 before adding gellan gum (Caisson). After adding 3 g/L gellan gum, the medium is autoclaved for 30 min at 121 °C and poured into 25 petri dishes (9 cm) when medium is cooled down to about 55 °C.

The liquid medium for Agrobacterium culture is prepared as 30 mL aliquots in 125 mL Erlenmeyer fl asks, pH adjusted with 1 N NaOH to 7.0–7.2, and autoclaved for 30 min at 121 °C. For tube culture, aliquots of 3 mL autoclaved medium are added to sterile 14 mL round bottom tubes (Thermo Scientifi c Nalgene).

The solid medium for Agrobacterium culture is prepared as 100 mL aliquots in 300 mL Erlenmeyer fl asks and pH adjusted with 1 N NaOH to 7.0–7.2 before adding agar. After adding 15 g/L agar, the medium is autoclaved for 30 min at 121 °C and poured into fi ve 9 cm diameter petri dishes when medium is cooled down to about 55 °C.

1. Cocultivation medium: Murashige and Skoog (MS) medium [ 14 ]—4.3 g/L 1× MS basal salts, 30 g/L sucrose, 0.1 g/L myoinositol, and 1 ml/L MS stock vitamin.

2. Callus induction medium (M5) [ 7 ]: MS medium plus 2 mg/L BA and 1 mg/L NAA.

3. Shoot induction and multiplication medium (M15) [ 7 ]: MS medium plus 4 mg/L BA.

4. Callus stage selection medium (CSM) [ 7 ]: M5 medium plus 250 mg/L of cefotaxime and 50 mg/L of G418.

5. Stage one shoot selection medium (SSM1) [ 7 ]: M15 medium plus 250 mg/L of cefotaxime and 50 mg/L of G418.

6. Stage two shoot selection medium (SSM2) [ 7 ]: M15 medium plus 125 mg/L of cefotaxime and 25 mg/L of G418.

7. Stage three shoot selection medium (SSM3) [ 7 ]: M15 medium plus 25 mg/L of G418. 2.4 Other Stock Solutions 2.5 Culture Media 2.5.1 Plant Culture Medium Xiaoling He et al.

101 1. YEB media: 5 g/L of tryptone; 1 g/L of yeast extract; 5 g/L

beef extract, 0.24 g/L MgSO 4 , and 5 g/L of sucrose.

1. Disarmed Agrobacterium tumefaciens strain EHA105 [ 15 ] con- taining the binary vector pBI121/ ricchi11 or pBI121/ gf2.8 . All EHA105 culture stocks are maintained in 30 % sterile glycerol (v/v), 1 mL aliquots in 2 mL Eppendorf tubes stored at -80 °C.

2. Both vectors pBI121/ricchi11 [ 7 ] and pBI121/gf2.8 [ 9 ] contain the selectable marker gene neomycin phosphotransfer- ase II ( npt II) for kanamycin and G418 resistance under the NOS promoter and the reporter gene β-glucuronidase ( gus ) driven by the caulifl ower mosaic virus (CaMV) 35S promoter. In addition to these two genes, pBI121/ ricchi11 contains the rice chitinase gene chi11 driven by the CaMV 35S promoter. In a separate construct, pBI121/gf2.8 contains the wheat oxa- late oxidase gene gf2.8 driven by the gf2.8 promoter.

1. Sterilizing solution: 20 % commercial bleach (Clorox) which contains 1.25 % sodium hypochlorite as the active ingredient and one drop of detergent Tween-80.

2. Planting medium: Sunshine Mix 4 (Aggregate Plus): perlite mixture (2:1 v/v).

3. 2, 4, 8, and 14 in. diameter pots and plastic bags.

4. Fertilizer: Osmocote Classic (14:14:14) (Scotts), Professional Horticulture.

3

Methods

In vitro shoot or plantlet cultures are maintained under environ- mental conditions of 25 ± 2 °C, 16 h photoperiod at light intensity of 15 μmol/m 2 _{/s. In vitro shoot or plantlet cultures in liquid}

medium are placed on a shaker with shaking at 95 rpm under the same environmental conditions. In vitro callus cultures are main- tained in the dark at 25 ± 2 °C:

1. Wash the cormels in running tap water for 5 min.

2. Excise approximately 1 cm 3 _{cormel sections containing one}

primary shoot apex or axillary bud each section.

3. Immerse cormel cubes in 70 % ethanol with 1 drop of Tween- 80 for 5 min.

4. Peel and remove cormel section outer layer tissues and pick and excise the 0.5–1.5 mm shoot tips from the center of shoot apices or axillary buds using a sterile needle. Microscope can be used to help fi nd the shoot tips ( see Note 1 ).

2.5.2 Agrobacterium Culture Medium 2.6 Bacterial Strains and Vector 2.7 Other Supplies and Solutions 3.1 Establishment of In Vitro Plantlets

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5. Place shoot tips into a sterile petri dish containing surface- sterilizing solution (1.25 % sodium hypochlorite plus 1 drop of Tween-80) for 16 s ( see Note 1 ).

6. Transfer shoot tips to another sterile petri dish containing ster- ile distilled H 2 O; lightly shake the petri dish by hand for 2 min

to rinse the shoot tips.

7. Transfer the shoot tips to the test tubes containing 3 mL M15 liquid media, with one shoot tip per test tube. Shake culture for 2 months. Multiple shoots could be induced after 2 months ( see Fig. 1a ). ELISA assay for DsMV can be conducted at this stage ( see Note 1 ).

8. Excise multiple DsMV-free shoots (approximately 10 mm in length) and transfer them to M5 petri dish plate for inducing calluses. Place these culture plates in the dark for 3–4 months. Subculture every month to remove brown and dead tissues.

Fig. 1 ( a ) The multiple shoots induced from one shoot tip explant in the M15 liquid media. ( b ) The calluses

induced after transferring the DsMV-free shoots onto the M5 plate for 3–4 months. ( c ) The multiple shoots and plantlets induced after transferring the callus onto the M15 plate for 3–4 months. ( d ) The cocultivated calluses selected on the callus selection medium CSM. ( e ) The PCR-positive multiple shoots of the independent line C6 with the rice chitinase gene chi11 further selected on the stage two shoot selection medium SSM2. ( f ) The plantlets of the independent line g5 with the wheat oxalate oxidase gene gf2.8 further selected on the stage three shoot selection medium SSM3

103 Calluses should be induced after 3–4 months and can be maintained on the M5 plates for 2 years with subculturing every month ( see Fig. 1b and Note 2 ).

9. Transfer calluses onto M15 petri dish plates for inducing shoots. Place these culture plates in the light for 3–4 months. Subculture every month to remove brown and dead tissues. Multiple shoots and plantlets can be induced after 3–4 months and can be maintained on the M15 plates for 7 years with sub- culturing every month ( see Fig. 1c and Note 3 ).

1. Thaw the frozen Agrobacterium culture glycerol stocks

EHA105/ ricchi11 or EHA105/ gf2.8 on ice and mix by gently tapping the Eppendorf tubes ( see Note 4 ).

2. Streak a portion of the Agrobacterium EHA105/ ricchi11 or EHA105/ gf2.8 on the YEB media plate supplemented with 25 μg/mL rifampicin and 50 μg/mL kanamycin.

3. Incubate the inoculated plates at 28 °C for 48–72 h. These Agrobacterium culture plates can be used immediately or maintained at 4 °C within 1 month for future use.

4. Pick a single colony from the Agrobacterium culture plate and inoculate it into the 3 mL YEB medium containing 25 μg/mL rifampicin and 50 μg/mL kanamycin. Culture in an incubator shaker at 250 rpm, 28 °C for 48 h until the Agrobacterium suspension becomes turbid (check the OD 600 = 0.8–1 by using

a spectrophotometer).

5. Add 2 μl of 0.3 M acetosyringone (AS) to the Agrobacterium culture and mix well. Incubate in 28 °C, 95 rpm for 30 min ( see Notes 5 and 7 ). Pour this 3 mL Agrobacterium + AS culture into 30 mL YEB medium in an Erlenmeyer fl ask for a dilution of tenfold. Stand in a bio-safe hood for 10 min ( see Notes 6 and 7 ). 1. Pour the 10 mL Agrobacterium + AS culture into a 9 cm sterile petri dish for infection. For control (no Agrobacterium ) treat- ment, retain 10 mL of YEB media in a separate sterile petri dish. 2. Pick white soft calluses of taro and chop them into small pieces as infection targets (0.2–0.5 cm/callus) ( see Note 7 ). Drop the calluses immediately in the Agrobacterium + AS culture.

Immerse treatment calluses into the Agrobacterium + AS cul- ture using sterile forceps. Immerse control calluses in YEB media similarly. Immerse 30–40 calluses in one petri dish at the same time. Immersion time in the Agrobacterium + AS culture is approximately 20 min for 30–40 calluses ( see Note 7 ). 3. Transfer all infected calluses of the same treatment side by side

onto a solid MS medium petri dish plate. Seal plates with Parafi lm and cocultivate at 25 ± 2 °C in the dark for 4 days ( see Notes 5 and 7 ).

3.2 Preparation of Agrobacterium Culture

3.3 Infection