Sterilization of seeds TIMING 30 min
1 Place around 100 seeds into a sterile 250 ml Erlenmeyer flask.
2 Add 50 ml of hypochlorite/H2O supplemented with Tween-20 while gently mixing and leave for 5 min.
3 Discard hypochlorite/H2O and wash the seeds 4 times with excess (100 ml) of sterile water.
4 Dry seeds on a sterilized paper towel for 5 min.
Germination of sugar beet seeds TIMING 7 days
5 Place the sterilized sugar beets seeds, 2 cm apart (around 10 seeds per plate), with forceps into plates containing solid germination medium. Seal the plates with parafilm and incubate for 7 days in growth chamber adjusted at 21-23 ºC. Seeds of good quality should give 90-100 % germination frequency.
Inoculum preparation (A. rhizogenes strain R1000) TIMING 4 days
6 Start preparing the bacterial strains, carrying the desired constructs, on the 3rd day upon seed sterilization. Streak bacteria from glycerol stock onto the surface of LB plates containing the appropriate antibiotics and incubate at 28 ºC for 2 days.
7 Pick a single colony and suspend in 1 ml liquid LB medium supplemented with antibiotics and incubate with gentle agitation at 28 ºC overnight.
8 Spread 200 μl of the suspension onto two fresh plates containing the appropriate antibiotics and incubate at 28 ºC overnight. After incubation, a bacterial layer is formed which can be used as inoculum for plant transformation.
Induction of hairy roots TIMING 2-3 weeks
9 After 7 days the seeds incubated in Step 5 should have germinated and seedlings should be developed. Select healthy, well developed seedlings for root transformation.
10 Cut the seedlings at the hypocotyl with a sterile blade (Fig. 1a), remove the existing root system and inoculate by holding the seedlings with the forceps and dipping the wounded surface of hypocotyl into the bacterial layer (Fig. 1b).
11 Place inoculated seedlings (5-7 seedlings in line/petri dish) on square petri dishes containing co-cultivation medium, half-covered with sterile filter paper (Fig. 1c). Place a sterile filter paper to cover the wounded surface of plants (Fig. 1d). Seal the dishes with parafilm and place them vertically in growth chamber (16/8 h photoperiod, 21-23 ºC) for 3 days.
12 Transfer the seedlings onto square petri dishes containing emergence medium, supplemented with cefotaxime (250 μg m-1) for the elimination of bacteria and the appropriate antibiotics for the selection of primary transformed roots, half-covered with filter paper. Place a sterile filter paper to cover the wounded surface of plants. Seal the dishes with parafilm and place them vertically in the growth chamber. Monitor regularly for the emergence of hairy roots.
13 After 10 days, transfer the seedlings onto fresh emergence medium, half-covered with filter paper and cover the lower part of seedlings with sterile filter paper. At this time point, hairy roots should be 4-6 cm in length (Fig. 1e).
14 Seedlings with well developed hairy root system can be directly used for functional genomics studies or maintained in liquid MS medium under antibiotic selection or transferred to pots (acclimatize plants by decreasing humidity gradually) or clonally propagated for the establishment of an in vitro hairy root culture (Fig. 1i).