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*Phytagel rather than agar is critical for success.

Autoclave and then add:

2 ml of 500x Vitamix (see below)

3 mg of indol-3-acetic acid (IAA)

0.15 mg of 2,4-dichlorophenoxyacetic acid (2,4-d)

0,6 mg of 6-benzylair nopurine

0.3 mg of 6-(y,y-dimethylallylamino)-purine (2iP)

Scotch 3M microporous filter tape (Carolina Biologicals)

Use of this air-permeable tape is essential for successful transformation.

LB liquid medium

Agrobacterium stock cultures containing T-DNAs of interest MS liquid medium 500x Vitamix (50 ml) 5 g of myo-inositol 250 mg of thiamin-HCl 2.5 mg of biotin 25 mg of pyridoxine-HCl 25 mg of nicotinic acid 50 mg of glycine Root-regeneration medium (1 liter) lx MS salts (GIBCO) 10 g of sucrose 0.5 g of MES

Adjust pH to 5.7 with 1 n KOH, and then add 1.1 g of Phytagel. Autoclave and then add: 2 ml of Vitamix (see above) 2 mg of indole butyric acid 500 mg of Timentin appropriate antibiotic for selection of T-DNA

(Use elevated levels of antibiotic for good selection of transgenic roots.)

CAUTION: 6-benzylaminopurine, biotin, 2,4-d, HC1, IAA, KH4P04, KOH, MES (see Appendix 3)

Procedure

Growth of Arabidopsis for Root Transformation

1. Place surface-sterilized seeds in a straight dense line on germination medium (see Chapter 5, Figure 5.1). Seal the plates with Parafilm and incubate, n a vertical orientation, for 3 weeks at 22°C, 16 hours light/8 hours dark.

2. After 3 weeks, wrap the plates in aluminum foil to etiolate and transfer to 4°C for a further 2 weeks.

This treatment has been reported to increase transformation efficiency. Plants may be stored at 4°C for up to 3 months.

Agrobacterium Transformation

3. Twenty-four hours prior to harvesting the roots, transfer the plants back to 22°C, 16 hours light/8 hours dark.

Use sterile working techniques for the steps involving root tissue.

4. After 24 hours, sever the roots below the hypocotyl with a sharp sterile razor.

Assume that one plate of roots is sufficient for one transformation.

5. Transfer the roots to callus-inducing medium, separating them with a sterile forceps to ensure that all tissue is in contact with medium. Seal plates with Scotch 3M microporous tape instead of Parafilm.

Use of this air-permeable tape is essential for successful transformation.

6. Incubate plates horizontally for 3-5 days at 23°C, under long days or constant light.

7. During this time, initiate 5-ml cultures of Agrobacterium containing the T-DNAs of interest in LB medium containing the appropriate antibiotics. Grow the cultures for 48 hours at 28°C under vigorous agitation.

8. After 48 hours, use the 5-ml cultures to inoculate fresh cultures in 50 ml of LB. Grow the 50-ml cultures for 24 hours at 28°C.

9. After 24 hours, pellet the Agrobacterium culture by centrifuging at 2000 rpm in a bench-top centrifuge for 15 minutes. Resuspend the cells in 5 ml of liquid MS medium.

10. Collect roots from the callus-inducing medium (see Step 6), and transfer them to a Petri dish containing a small amount of liquid MS medium. Wound the roots by gently squeezing or pressing them against the bottom of the Petri dish with the side of a blunt forceps.

11. Collect the wounded roots nto a bundle and transfer them to 50ml Falcon tubes contain *ng 5 ml of concentrated Agrobacterium culture (obtained in Step 9). Vortex the mixture for 1 minute. Incubate with shaking for 15 minutes at 28°C.

12. Gently blot the root bundles on sterile filter paper. Transfer them to Petri dishes containing fresh filter paper, presoaked with MS liquid. Separate the roots on the filter paper, close the plates, and seal with Parafilm.

13. Incubate the culture for 2-3 days, making sure that the roots do not become dry.

Root Regeneration

14. After cocultivation, collect the roots and transfer them to a fresh plate containing MS medium. Rinse gently. Blot the roots carefully on sterile filter paper and transfer them to callus-inducing medium containing timentin (500 mg/liter). Seal the plates with Scotch 3M filter tape and incubate in the light for 2 days.

Timentin kills the Agrobacterium.

15. Transfer the roots to root-regeneration medium containing timentin (500 mg/liter) and the appropriate antibiotic for selection of transgenic callus.

Selection of transformed regenerating roots requires a higher concentration of antibiotic than selection of resistant shoots. For hygromycin selection, 20 mg/liter is recommended.

16. When roots appear (~1 week later) transfer the calli with regenerating roots to fresh root-regeneration medium every 10-14 days.

Alternatively, remove regenerating roots from the calli and transfer to 5-ml liquid cultures. The medium for liquid culture is root regeneration medium, minus Phytagel.

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