* Unless otherwise stated all procedures should be carried out aseptically using sterile solutions and equipment.

Transcription

1 TRANSFORMATION OF ARABIDOPSIS THALIANA From Dirk Valvekens et al.(dirk Valvekens, Marc Van Montagu, & Mieke Van Lijsebettens Proc. Natl. Acad Sci. 85, ), as used by Caroline Dean et al (Caroline Dean, Department of Molecular Genetics, The Cambridge Laboratory, The John Innes Centre for Plant Science Research, Colney Lane, Norwich, NR4 7UJ, England.) Contents: MEDIA GROWTH OF PLANTS REGENERATION AND TRANSFORMATION OF ROOT EXPLANTS GERMINATION OF F1 PROGENY * Unless otherwise stated all procedures should be carried out aseptically using sterile solutions and equipment. MEDIA * All media are used in standard plastic disposable petri dishes (e.g., Sterilin) or, for later stages, Magenta GA 7 pots (Magenta Corp., USA). During tissue-culturing, petri dishes are sealed with 1/2" Micropore (3M) medical gas-permeable tape -- 1" tape is used on Magenta pots. (The use of Nescofilm, lowers the efficiency of regeneration at all stages due to reduced gaseous exchange.) Hormones are dissolved in dimethyl sulfoxide (DMSO) as x1000 stocks (DMSO should be taken from either freshly opened or frozen stocks). Antibiotics are dissolved in water as x1000 stocks (except for vancomycin, which is made at x100) and subsequently filter-sterilised. Hormones and antibiotics are added after autoclaving and cooling of the media to 65oC. 1. GERMINATION MEDIUM (GM) 1a. GM K50 1x Murashige and Skoog salt mixture (Flow Labs) As GM, but supplemented with: 1% sucrose 50 mgl-1 kanamycin (Sigma) 100 mgl-1 inositol 1.0 mgl-1 thiamine 0.5 mgl-1 pyridoxine 0.5 mgl-1 nicotinic acid 0.5 gl-1 2-(N-morpholino)ethanesulfonic acid (MES) (adjust to ph 5.7 with 1M KOH) 0.8% Difco Bacto agar /0.05 CALLUS-INDUCING MEDIUM (CIM) 1x Gamborg's B5 medium (without 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, and sucrose) (Flow Labs.) 2% glucose 0.5 gl-1 MES (ph 5.7, adjusted with 1N KOH) 0.5 mgl-1 2,4-D (Sigma) 0.05 mgl-1 kinetin (Sigma) 0.8 % agar (for solid CIM medium) /5 SHOOT-INDUCING MEDIUM (SIM) 3a. SIM V750

2 Gamborg's B5 medium (Flow Labs) As SIM, but supplemented with: 2% glucose 750 mgl-1 vancomycin (Sigma (Vancomycin can be obtained from a pharmacist, this is much cheaper than buying it from Sigma.)) 0.5 gl-1 MES (ph 5.7) 0.8% agar 5 mgl-1 N6-(2-isopentenyl)adenine (2ip) (Sigma) 0.15 mgl-1 indole-3-acetic acid (IAA) (Sigma) 3b. SIM V750 K50 3c. SIM V500 K50 As SIM, but supplemented with: As SIM, but supplemented with: 750 mgl-1 vancomycin 500 mgl-1 vancomycin 50 mgl-1 kanamycin 50 mgl-1 kanamycin 0.8% agar 0.8% agar GROWTH OF PLANTS 1. SEED STERILISATION (i) Place seeds into a 15 ml polypropylene centrifuge tube (Falcon) or similar container. (ii) Add 70% EtOH for 2 min. Remove EtOH with pipette. (iii) Replace with 5% commercial bleach (~0.25% available chlorine) containing one drop of Tween 20 per 50 ml. Leave for 15 min, shaking regularly. (iii) Wash seeds in sterile, distilled water (dh20) x 5. (iv) After last wash, either: (a) Dry seed on sterile filter paper and sprinkle on GM, or (b) Leave seeds in 1.0 ml dh20. Add 1 ml of liquid GM. Invert and pour as a soft overlay. In both cases, ensure that the seeds are evenly distributed at around 100 per plate. 2. GROWTH OF SEEDLINGS (i) Incubate seeds on GM in culture room at 20-25oC (continuous light or 16 h light/8 h dark cycle). (ii) Leave until just before any visible sign of floral buds (approx. 3 weeks). This ensures maximum root growth prior to flowering and subsequent senescence. REGENERATION AND TRANSFORMATION OF ROOTS * For both regeneration and transformation, white roots of any age can be used. 1. DAY ONE (i) Using a scalpel, cut-off all the green (upper) parts of the plantlets to leave only the root systems. (ii) Disrupt agar by pulling it apart with forceps. (iii) Tease out bunches of roots. Excess agar should be removed by rinsing the roots in a petri dish containing liquid CIM. (iv) Lay out the washed bunches of roots onto plates containing solid CIM. Gently press down on each bunch to ensure that the roots are in contact with the surface of the agar. (v) Incubate for 3 d in growth room.

3 2. DAY FOUR (i) After ensuring that there is no visible sign of contamination, stack several (5-30) callus-induced roots into an empty petri dish. (ii) Cut the roots into 0.5 cm explants. (iii) Transfer the root explants into a sieve in a petri dish containing 20 ml of liquid CIM medium. Swirl with blunt-nosed forceps to ensure that all the pieces are bathed. * Custom-made, autoclavable and re-usable, sieves are used to hold the explants during Agrobacterium infections and washings of root explants. These sieves are made from 100 ml plastic three-cornered beakers and 100 um nylon mesh. The top 3-4 cm is cut off the beaker and the mesh held in place across the bottom of this piece by pushing a ring 2 cm high cut from just above the base of the beaker into the lower portion of the top part. The two pieces are sealed together by pushing a hot metal rod through them in several places (Sieves can also be purchased from: Wilson Sieves, 2 Long Acre, Common Lane, Hucknall, Nottingham, NG15 6QD, UK. Tel: (6-11p.m.); FAX: (f.a.o. "Wilson Sieves")). (iv) Add ml of Agrobacterium culture grown overnight at 28oC in Luria broth at 200rpm. Swirl with blunt-nosed forceps. Leave to co-cultivate for 2 min. * We have found that C58C1RifR(pGV2260) Agrobacterium strains transform Arabidopsis with higher efficiency than LBA4404. Binary as well as co-integration vectors may be used (Valvekens et al., 1988). Selection using kanamycin (50 mgml-1) with 35S-neo/pNos-neo or ptr-neo constructs has given significantly more transformants than selection on hygromycin using a ptr- hyg construct. (v) Lift sieve out of buffer and drain. Press the explants down onto the mesh to remove as much excess buffer and agar as possible. Push the semi-dried explants together into a lump. (vi) Blot this root mass dry on double-thick sterile filter-paper ( Whatman No.1) in a petri dish. (This may have to be repeated once or twice.) * It is important to remove as much of the Agrobacterium inoculum as possible, or the plates will be overgrown with bacteria. (vii) If the root mass is large, divide it into two or three. Place each piece onto solidified CIM medium in a petri dish. Tease each lump into about 30 small explants each approx. 0.5 x 0.5 x 1.0 cm. Evenly distribute the explants over the plate and gently press them onto the surface to ensure contact with the medium. * Ensure that the plates are very dry to start with or the agrobacteria will grow too quickly. (viii) Incubate plates in the growth room for 2 d to allow co-cultivation of Agrobacterium. Plates overgrown with agrobacteria after this period should be rejected as is is unlikely that the plant material will regenerate. 3. DAY SIX (i) Transfer the explants to a sieve (as above) in ml of liquid SIM. Agitate with blunt-nosed forceps to wash off the agrobacteria. The bathing

4 medium should become somewhat turbid. Repeat and then lift sieve out of buffer and drain. Press down the explants down onto the mesh to remove as much buffer and agrobacteria as possible. Push the semi-dried explants together into a mass. (ii) Blot this on double-thick sterile filter-paper in a petri dish until the root mass is as dry as possible. (This may have to be repeated once or twice.) Re-divide the lump into small explants again and transfer to solidified SIM V750 K50 medium, taking care that root explants are in close contact with the medium. (iii) Incubate in growth room. * Do the controls first. Split the root mass into two. One half, re-divide onto SIM V750; the other onto SIM V750 K50. The former is the positive control (masses of green shoots should regenerate) whilst the latter is the negative control (no shoots should regenerate). * For non-control plates, transfer all divided explants onto SIM V750 K DAY n (i) In growth room, the positive control plates will start to green within 10 d. On the non-control plates, tiny green calli appear on the yellowish root-explants after 3 weeks. (ii) Every 10 d, transfer explants to fresh SIM V750 K50. Shoots (often initially vitreous) will intermittently appear from the green calli over the next several weeks. (iii) Transfer these shoots to GM, where they will lose their vitreous structure within a few days. (iv) On GM, the excised shoots will develop into multiple rosette-like strucures. Every d, disrupt these structures and select the most normal-looking plant for transfer to fresh GM plates. * Transfer only one rosette per clump of tissue in order to ensure that all transformants are independent T-DNA insertions. * Regenerants from the ecotype Landsberg erecta, root very poorly compared with other ecotypes. The L. erecta transformants will, nevertheless, flower without rooting. (v) At the first sign of flower-bud formation, transfer individual plants to single Magenta GA7 pots. Leave the lids of these pots very loosely in place, but prevented from falling off by securing with 1" Micropore tape. * As anthers will not dehisce in a damp atmosphere, it is essential to keep the humidity low in these pots. (vi) Harvest seed pods when they turn yellowish-brown and are therefore mature. If this is done aseptically, it obviates the need to sterilise the seeds for the next generation. GERMINATION OF F1 PROGENY * To test for transformation, F1 seedlings are germinated on GM K50. (i) Put seeds on GM K50. Seal with Micropore tape. (ii) Put petri dishes in the dark at 4oC (refrigerator) for 3-5 d, to break seed dormancy.

5 * Step (ii) is not necessary if seeds were stored for more than a month. (iii) Incubate petri dishes in growth room for 2 weeks. Sensitive seedlings form neither roots nor leaves, and have white cotyledons. Transformed seedlings are phenotypically normal.