In vitro co-culture system using a fiber-supported liquid approach

Establishment of plant cultures from dormant shoots

Collect dormant shoots, cut them (3 cm in length),and cleanse them by submerging in 70% ethanol for 1 minute, followed by rinsing with sterile deionized water. Then, immerse the shoots in a 10% bleach solution for 10 minutes, and rinse them twice with deionized water (Figure 1).

Peel the vegetative shoot buds and transfer them into culture tubes containing 20 mL of Murashige and Skoog agar media. Place the shoot vertically ensuring that the bud is 1 cm above the agar media.

Establishment of plant cultures from seeds

Clean fruit exocarps with 20% bleach for 10 minutes, followed by a 10-minute immersion in 70% ethanol.

Extract seeds within a laminar flow hood and transfer them aseptically into culture tubes containing Woody Plant Medium. Allow them to undergo stratification for ten weeks in darkness at 4 ºC.

Upon germination (Figure 2), micropropagate shoot tips in culture vessels(Magenta GA-7) using an agar-based medium

Maintenance of stock plants

Sustain stock plants in Magenta GA-7 vessels by transferring shoot tips every five weeks onto a fresh medium.

Maintain vessels under a photosynthetic photon flux density of 20 μmol/s/m2, with a 16-hour photoperiod at 24°C.

Optional: When the When the presence of hyper multiplication, an occasional resting cycle with 16 μM indole-3-acetic acid (resting media) is recommended.

Propagate fungal cultures in Petri plates by placing two plugs (0.5 × 0.5 cm) from the youngest part of the colony (Figure 3).

Seal plates with parafilm and maintain in the dark at 20 ºC.

Refresh every 14 days by transferring mycelial plugs to fresh MEA plates to ensure active fungal growth.

Extract three ten-millimeter-diameter plugs from the edge of two-week-old colonies

Remove most of the agar plug and homogenize mycelium with 5 mL of sterile water using a sterilized 15 mL tissue grinder (Figure 4).

Place a sterile ultra-clear porous cellophane sheet on top of a Petri dish containing malt extract agar media and pour 600 µL of homogenate

Spread homogenate uniformly over the entire plate using a cell spreader and incubate the plate in the dark for 14 days.

Prepare the mycelium suspension for inoculum by taking a 2 × 2 cm plug from the previously prepared

plate and homogenate with 7 mL of sterile water using a tissue grinder, and aliquoted in 1.5 mL Eppendorf tubes (Figure 5).

Autoclave (121 ºC for 20 min) rectangular plastic vessels (110 × 297 mm; Southern Sun

BioSystems) and after cooling down to room temperature, set the fiber-supported

paper on the floor of each vessel (Figure 6).

Add 175 mL of plant growth regulator-free liquid ‘New Prunus Medium’ to each vessel.

Transfer fifteen in vitro plants from an agar-based medium to each vessel removing the agar and sealing vessels with polyvinyl chloride film (Figure 7).

Place vessels on a rocker's arm with an articulated shelf that providesone swing every 15 min.

Use another set of rectangular Southern Sun BioSystems vessels containing 175 mL of ‘New Prunus Medium’, fiber matte, and germination paper and add 1 mL of the mycelium suspension for inoculum.

Seal the vessels with PVC film and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC.

After ten and seventeen days of inoculation with A. mellea and D. caespitosa , respectively, transfer the in vitro rooted plants from the liquid media to the inoculated vessels.

Add 60 mL of ‘New Prunus Medium’ without any plant growth regulator just before plant transferring.

Seal the vessels with PVC film and place them on an automatic rocker arm at 5 rpm under μM/m2/s LED light 2 red 1 blue and 16 h/day photoperiod at 24 ºC (Figure 8).

Collect tissues when needed.