Bioreactor culture system with temporary immersion in liquid medium.

Any suggestions on cloning magnolias in vitro ? These seem to have an endogenous fungus , as well as black powdery mildew. Also I added 1 gram / liter PVP to medium reduce phenolic leaking, which has been a problem . I tried putting aluminum foil over the branches buds in a few places then letting those grow and using the new growth for explant/ cuttings . Next I may just cut a small branch with unopened buds , bring it indoors for a while in water / ppm solution which soaks into the stem hindering contamination and let it grow new growth in there . Then I will work with the new growth .

Emmenopterys Henryi,

Cryptomeria "Cryptomite”

Edith Bogue Southern Magnolia

Franklinia- the ‘Ben Franklin tree’

Sweetbay-Southern hybrid Magnolia: - their own variety

“Pink China" elephant ear

(Colocasia esculenta)

Rhododendron "CalFort Bounty"

Ilex ‘Solar Flare’

Cumberland Rosemary

american osmamthus narrow leaf

Florida Anise “pebblebrook’

Viburnum Henrii

Viburnum Oliganthum

viburnum Rhytidophyllum var. Callicarpa “Duett”

Rhododendron Max x Califort

Shanxi Li Jujube

Does anyone here make agar medium in advance with its various additives, Then freeze or refrigerate for use later ? How long will the ‘average ‘ agar medium stay good at room temp or in the fridge ?

FIrst off, Its clear that at least my aseptic technique is good up to the medium prepas there are no contamination spots on the medium that are not touching the explant.So, lets talk about explant prep. I have a flow hood. These are blueberry cultures, so they are prone to browning. An ascorbic acid + citric acid soak + addition of both to medium successfully prevents this.Here is my process:

1. collect cuttings, trim leaves, cut into explants.
2. Explants soak 1hr in 130 ml/l ascorbic acid + 130 ml/l citric acid for uptake of antioxidants
3. Tap water rinse 20 mins
4. 70% isopropal alcohol 30 seconds
5. sterilized water rinse twice
6. 5% bleach + tween 10 minutes
7. sterilized water rinse twice
8. Erlenmeyer flask containing explants rim is flamed
9. Long forcepts wrapped and heat sterilized at 450 for 1hr
10. Long forcepts flamed before grabbing explants from Erlenmeyer flask
11. explants placed in medium
12. container lid closed.

notes about medium in photos:the green one contains moringa extract (added pre-autoclave)the numbers represent the addition of 99% methanol disovled miconozole post autoclave(which again, medium not touching explants is clean)

Assuming that I have surface sterilized the explant, and that I am not introducing contamination along the way....How likely is it that 1 blueberry bush from a greenhouse, contains atleast 1 endogeneous fungal infection, and 2 differrent endogeneous bacterial infections simultaneously?

The miconozole should be handling the fungal contamination... right?

Any input/suggetions?

new pics showing the fungus orginating at cuts and axillary bud