Protocol for Poplar Transformation
(Caiping Ma, 1/16/05)
1-2 month-old poplar plants grown in vitro and young fresh shoots from plants grown in
greenhouse are good source materials for poplar transformation. However, the material
from the greenhouse need to be surface sterilized before inoculation with Agrobaterium.

Explants Preparation
1. Explants from in vitro grown plants:
Cut the shoots tip (with 3-4 leaves) off from the plants growing in Magenta
boxes and transfer them into ½ MS propagation medium with auxin
(promote plant to root) or without auxin depend on poplar clone.
2. Explants from ex vitro grown plants:
Cut shoot tip (8-10 cm) off from a poplar plants grown in greenhouse, and
gently wash them in warm, soapy water. Separate leaf, stem, and petiole and
sterilize them in 70% ethanol for 1-3 minutes and 20% chlorine bleach for
10-12 min, and rinse three to five times in sterile water.
3. Collect leaf, stem, and petiole from in vitro plants and place them or sterilized
greenhouse materials in sterile water in Petri dish. Use an autoclaved paper
punch to obtain leaf sections and a scalpel to get wounded stem or petiole
segments (5-8 mm). Pre-culture both leaf disks and stem explants on
Callus Induction Medium (CIM) for 1-2 days in dark. Pre-culture also can be
omitted depend on different poplar genotype.

Inoculation With Agrobacterum and Co-cultivation
1. Plate Agrobacterium from glycerol stock or a fresh colony in 50 ml LB
medium with appropriate antibiotics, grow 1-2 days at 28˚C in dark.
2. Spin down Agrobacterium in a centrifuge for 30 min at 3000-4000 rpm.
3. Dilute it to an OD600nm = 0.4 to 0.6 with MS liquid medium containing
25μM acetosyringon
4. Place 30-40 explants (leaf discs and wounded 3-5 mm stems) in a 50 ml
Falcon tube containing 30-40 ml diluted Agrobacterium.
5. Put the tubes in a shaker at 150-200 rpm for 1 hour.
6. Blot excess Agrobacterium from the explants over sterile paper and cocultivation them on the fresh CIM in dark for 2 days.

Callus Induction And shoot Regeneration
1. Transfer inoculated explants into 50 ml Falcon tube with 30-40 ml sterile
water and rinse 4-5 times and 1 time in washing solution containing 400
mg/L timentin.
2. Blot explants dry on sterile paper and place onto CIM with 50 mg/L
kanamycin and 200 mg/L timentin to control Agrobacterium
3. Incubate explants in dark for 2-3 weeks.
4. Transfer explants onto Shoot Induction Medium (SIM) with 100 mg/L
kanamycin and 200 mg/L timentin and culture in light
5. Subculture on the same fresh SIM every 2-3 weeks until shoots form
6. Transfer the explants with multiple small shoots to Shoot Elongation
Medium (SEM) with 100 mg/L kanamycin and 200 mg/L timentin

Rooting of Resistant Regenerants in Tissue Culture
1. Excise shoots at 0.5-1.0 cm in height , and place individual shoots onto
½ MS rooting medium containing 25-50 mg/L kanamycin and 100 mg/L
timentin for further selection. Make sure to mark shoots obtained from
different explants (representing different transformed events) and shoot
from the same explant (which might not be different regenerants); record
date and original plate for each shoot. 10-12 shoots can be placed in each
magenta box.
2. Place the boxes in the growth room for 1-2 weeks until root emergence
3. Shoots that root on kanamycin are likely to be transformed
4. Perform PCR to verify the transgene in the plants and eliminate escapees
5. Propagate the PCR-positive, resistant plantlets in rooting medium

Transplanting into Greenhouse
1. Choose new, about 1-2 month-old propagated plants (about 5-6 cm in
height)
2. Remove plants from Magenta boxes and wash off excess agar gently with
clean tap water. (Medium removal should be thorough to avoid fungal or
bacterial contamination during plant acclimatization)
3. Place the plants in small pots with potting soil (Perlite: Peat moss 2:1)
4. Transfer pots into zip plastic bags with water for about 3 weeks in
a growth room at 25 ± 1˚C under 16 h photoperiod
5 Gradually open the bags and take the plants out and maintain them in the
greenhouse