ARABIDOPSIS TRANSFORMATION

1. Seed Sterilization. Sterilize WS (Wassilewskija) seed for 10 min in a solution of 50% Chlorox with 0.1% SDS in ependorf tubes. Rinse 3 to 5 times with dH20. Dry thoroughly on sterile filter paper.
2. Root cultures. Place 35 seed into each of 35 250 ml Belco flasks with 50 ml B5 medium. Place on shaker (7080 rpm) at 23!C, 24 hr light for about 3 weeks. Roots will fill flask and provide plenty of tissue for transformation. Use roots while still white and healthy.
3. Preculture on CIM. Removing entire root mass from flask and cut off shoots and any green roots. Using forceps, pull off small bundles of roots and lay them on MSKig medium. Place several root bundles on each 25x100 plate. Seal dishes with filter tape (Carolina Biologicals) and incubate at 23!C, 24 hr light for 2 to 4 days.
4. Inoculation. Pour 4050 ml MSKig liquid medium into a tripour 100 5m filter sitting in a 25x100 petri dish. (We make these filter baskets by cutting off the bottom of tripour beakers and melting a piece of 100 5m mesh to the bottom.) Place Arabidopsis root bundles from MSKig into a petri dish and cut them into 0.5 cm segments. Transfer root pieces into filter units with medium. Pipet 1.5 to 2 ml of an Agrobacterium overnight culture into each filter with roots. Mix gently and let sit a few minutes. Blot the roots on sterile filter paper. Place bundles of roots on MSKig medium containing 100 5M Acetosyringone Our root bundles cover about 1 cm2. Seal with filter tape and incubate at 23!C, 24 hr light for 23 days.
5. Rinse and Transfer to SIM with Selection. Place roots bundles into a tripour filter which is sitting in a 25x100 petri dish. Pour 3050 ml liquid MSKig medium over the roots and shake the filter unit vigorously in the solution. Repeat rinse with a clean petri dish until liquid is clear. Blot the roots on filter paper. Transfer root bundles to MSg v500 containing 50 mg/l kanamycin, 58 bundles per 25 x 100 petri dish. Make sure the roots are in contact with the medium. Seal with filter tape and place at 23!C, 24 hr light for 1020 days.
6. Transfer to No Hormone medium. Green nodules and the first indications of shoots should appear within a few weeks. When shoots are first visible (they can look like dark spots to the naked eye) transfer entire explant, or break up explant into smaller pieces, to GM v500 k50. Secure lid to dish with two pieces of tape. Incubate 7 to 14 days at 23!C, 24 hr light. As the shoots develop, it is helpful to carefully break up the explants to allow the shoots to expand.
7. Root Induction: When shoots are large enough, excise them from the callus and place them on MSRg v500 k50 medium in a 25x100 petri dish. Secure lid to dish with two pieces of tape and incubate for 24 days.
8. Plantlet Growth: Transfer shoots to GM v500 k50 medium in 25x100 petris. Secure lid to dish with two pieces of tape. This ensures good air exchange and helps to harden off the seedlings. Roots should form in 1020 days.
9. Transfer to Soil: When shoots have produced a good root system, transfer them to potting mix as follows: Place MetroMix in 2" pots and moisten with Hoaglands solution. Carefully pull robust plants from the agar. Rinse well with water from squirt bottle and use fingers and/or forceps to gently remove agar and vitreous tissue from base of shoot. Place root in a hole made in the MetroMix and gently press the soil around the root. Water the plantlet into the soil using the squirt bottle. Immediately place the potted plant into a Magenta box with lid. Return the plants to the culture room (23!C, 24 hr light). Within a day or two, the stems should straighten up and start to grow. As quickly as possible, without allowing the plants to wilt, remove the lid from the Magenta box in stages, starting one to two days after transplanting and removing the lid within a week. Water the plants as necessary to keep them moist. We have had up to 80% success rate with this procedure, however, the plants must not be vitreous. When well established, the plants can be placed in a growth chamber.
10. Grow to Seed in Magenta boxes: Transfer rooted plants to GM v500 k50 medium in Magenta boxes (about 100 ml medium per box). To facilitate air exchange and to allow for room to grow, the lid is replaced with a modified Magenta box using the Coupler for Magenta Vessel (Sigma C 0667). The Magenta box is modified by drilling a 6 mm hole in the bottom of the box (which will become the top); this hole is covered by a Suncap Closure (Sigma C6920), held on by a rubber band during autoclaving. If condensation occurs, replace the Magenta box lid with a dry one. About half of the plants will set seed but plants in soil will set seed much faster.

    ARABIDOPSIS MEDIA

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    Basic Medium

    • 1 pkg. Murashige and Skoog Minimal Organics Medium without Sucrose (Gibco #5103118 or Sigma # M6899)
      
    • 10 ml DM Vitamin Supplement
      
    • 0.05% MES (0.5 g/l)
      
    • 0.8% agar (8 g/l)

    pH 5.8

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    DM Vitamin Supplement 100 X Stock

    • 10 mg/l thiamine
      
    • 50 mg/l pyridoxine
      
    • 50 mg/l nicotinic acid

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    GM = Germination Medium

    • Basic Medium 1% sucrose (10 g/l)

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    MSKig Callus Inducing Medium

    • Basic Medium
      
    • 2% glucose (20 g/l)
      
    • 0.5 mg/l 2,4D 2.3 5M
      
    • 0.3 mg/l Kinetin
      
    • 1.4 5M 5 mg/l IAA 28.5 5M

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    MSg Shoot Inducing Medium

    • Basic Medium
      
    • 2% glucose (20 g/l)
      
    • 0.15 mg/l IAA 0.86 5M
      
    • 5.0 mg/l 2iP 24.6 5M

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    MSRg Root Inducing Medium

    • Basic medium
      
    • 2% glucose (20 g/l) 12 mg/l
      
    • IBA 58.8 5M
      
    • 0.1 mg/l Kinetin 0.46 5M

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    Notes v500 = 500 mg/l
    vancomycin B5 medium = Gamborg's B5 medium (Gibco # 5001153)
    Use 50 mg/l kanamycin for selection.

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    Background

    Agrobacterium genetically colonizes wounded plant cells by transferring a defined segment(TDNA) of its Ti plasmid into the plant nuclear genome.Although this natural gene transfer mechanism has been widely adopted in plant transformation,the possibility of generating transformed progeny by Agrobacterium infection in planta has not been successfuly exploited.Here we show stably transformed progenies of a crucifer plant,Arabidopsis ,are efficiently obtained by simple incision of primary shoots at their bases and by subsequent Agrobacterium inoculation at the wound site.Adventitous shoots arising from the infected wound site were transformed at a high frequency and produced transformed progenies later.