Tissue Culture for Challenging Woody Plants

Lynne Caton Briggs Nursery LLC Porter, WA

Briggs Nursery, LLC

Founded in 1912 in Olympia, WA Owned by Spring Garden Corporate Advisors Operate on 400 acres in Porter, WA Employ a staff of 220+ during the growing season Maintain over 450 greenhouses for plant production Participant in the USNCP (Nursery Certification Program) Focus is on production of young plants (liners, plugs)

Micropropagation of Ericaceaous plants at our nursery was pioneered by Bruce Briggs in the late 1960s What we micropropagate: Woody Ornamentals Herbaceous Perennials Ornamental Grasses Small Fruits

Production is approximately 9 million micro-plants & 1 million conventionally propagated plants annually

Briggs Nursery Porter, WA circa 2002

Propagation Lab & greenhouses Built in 2006

Briggs Nursery TC Laboratory, built in 2006

Briggs Nursery Conley propagation greenhouse, 52,000 square ft., built in 2006

stage I

stage II

stage III

stage IV

New Tools to employ in the pursuit of successful micropropation:

Stage I An alternate chemical and protocol for surface sterilization Stage II A novel nutrient salt solution for woody plants A BA (cytokinin) alternative for woody plants

Stage III Altering the environment for hardening off in vitro Stage IV An optimal greenhouse environment for acclimatization

Explant : Initiation : Stage I

-Plant reduced to nodal pieces or basal rosettes/buds -Immerse plant pieces in a soap solution (soap = Tween 20) for a 10 rinse on an orbital shaker, to break surface tension on the cutting, then drain off solution

NaOCl (Sodium Hypochlorite)

Immerse plant pieces in a 10% solution of Clorox, with a drop of Tween 20, for the appropriate length of exposure time (5min 1 hr) on an orbital shaker, then drain off solution Immerse plant pieces in a 1% solution of Clorox, with a drop of Tween 20, as a final rinse

NaDCC (Dichloroisocyanuric Acid)

Immerse plant pieces in a 5gm/L solution of NaDCC, with a drop of Tween 20, for the appropriate length of exposure time (5min 24 hrs) on an orbital shaker (no need to drain off solution)

(no final rinse required)

-Working in a laminar flow hood, make a basal cut to plant piece and place into media

Advantages of NaDCC over NaOCl

Both chemicals, when diluted in water, form Hypochlorous Acid (HOCl) NaDCC has a more potent sterilant action since the compound dissociates to maintain a more constant level of HOCl in solution NaDCC solution has a pH of 6.8 compared to NaOCls pH of 10.0, so high levels of active chlorine are maintained at a plant physiologically friendly pH NaDCC phototoxicity to plant material is minimal (this is THE major advantage) Low toxicity of NaDCC permits culture of shoots without rinsing, so higher levels of the sterilant are in contact with the plant material for a longer period if time NaDCC solution has a long shelf life. If the solution is kept in a closed container, at room temperature, it can be stored for 365 days and still be 100% effective

Sample surface sterilization times

Tween 20 NaDCC soap rinse 5 gm/ L Plant - explant condition w/ shaking w/ shaking rinse Agapanthus - flower buds 10 min 20 min na Anemone - flower buds 10 min 10 min na Azalea - new flush 10 min 5 min na Cordyline - crown, above soil 10 min 6 hours na Hakonechloa - emerging shoots, below soil 15 min 24 hours na Hosta - emerging shoots 10 min 24 hours na Hydrangea - new flush 10 min 15 min na Magnolia - new flush 10 min 10 min na Rhododendron - new flush 10 min 5 min na Syringa - new flush 10 min 5 min na Yucca - toes, below soil 15 min 24 hours na

NaDCC Dichloroisocyanuric Acid, Sodium Salt

Source: Phytotechnology Laboratories Product # D253 1 kilogram = $72.98 Comes as a powder, dissolves in water Typical published rate 2-5 gm/L (Briggs rate 5 gm/L)

How we prepare solution: 75 gm NaDCC 15 L deionized water add a few drops of Tween 20 Product dissolves readily in water, wear protective gear, its an oxidizer We use about 400 mls of solution per Ball jar We store in a closed container, at room temperature

Bruce Briggs & Comet cleanser (everything old is new again)

Stage II : Multiplication

Stage II : Multiplication : Nutrient Salts

A novel nutrient salt solution attributed to Dr. John Preece, Southern Illinois University Coined Preece media at Briggs Nursery Preece media is a hybrid of: WPM, Woody Plant Media Developed for micropropagation of Kalmia latifolia Lloyd & McCown, 1980 DKW Developed for micropropagation of Juglans Driver, Kuniyuki, 1984

PREECE MEDIA
Component Ammonium Nitrate Boric Acid Calcium Chloride Calcium Nitrate Cupric Sulfate EDTA, Na2 Ferrous Sulfate Magnesium Sulfate Manganese Sulfate Molybdic Acid, Na Nickel Sulfate Potassium Phosphate Potassium Sulfate Zinc Nitrate Zinc Sulfate Glycine Nicotinic Acid Pyridoxine HCl L loyd & Driver & McCown Kuniyuki Preece WPM DKW Hybrid* media components (mg/ l) 400.0 1,416.0 908.0 6.2 4.8 5.5 72.5 112.5 92.5 386.0 1,367.0 876.5 0.25 0.25 0.25 37.3 45.4 41.35 27.85 33.8 30.825 180.7 361.49 271.095 22.3 33.5 27.9 0.25 0.39 0.32 0.005 0.0025 170.0 265.0 217.5 990.0 1,559.0 1,274.5 17.0 8.5 8.6 4.3 2.0 1.0 0.5 0.25 0.5 0.25

* Preece Hybrid is 1/ 2 WPM, 1/ 2 DKW

Plants micropropagated on Preece Media

Actaea Anemone Arbutus Betula Fothergilla Heuchera Hydrangea Kalmia Liquidambar Nandina Pieris Rhododendron Ribes Syringa Vaccinium

Stage II : Multiplication : Cytokinins

Meta-Topolin (mT) is a BA analogue derived from Populus x robusta BA (N6-Benzyladenine) is a widely used cytokinin in micropropagation systems, but can result in root inhibition during acclimatization mT results in good multiplication in vitro and does not inhibit root formation either in vitro or post vitro, weve seen promising results with Nandina and Cotinus

Other possible advantages of mT vs BA (we have not tested these yet): mT may result in more stable micropropagation of plant chimeras (variegation) mT may have an anti-senescence activity in plants susceptible to tip die-back in culture Disadvantages of mT vs BA that weve encountered in our lab: mT seems to encourage bacterial growth in cultures, as opposed to the same cultures grown under a BA regime mT cost
Phytotech price Product # per gram B-800 $4.73 BA N6-benzyladenine T-841 $135.71 mT 6-(3-hyrdoxybenzylamino)purine

BA and its analogue mT

BA

mT

Nandina on mT

Cotinus on mT

Cotinus post mT

Culture Room (multiplication): Hepa-filtered air 16 hr photoperiod Temperature 70-75 F Cultures are bagged for contamination control

Stage III

Stage IV

Stage III Conditioning and/or Rooting in vitro

- Do Nothing Send the microplants out on a stage II media, after a full subculture period so that most of the cytokinins have been metabolized by the plant - Go Basal Eliminate cytokinins for last subculture, subculture the plants to a media free of any PGRs. Or add activated charcoal to the media to sponge up the PGRs to allow the plant to root more readily - Use PGRs and do a proper Stage III thing Prepare a media with the addition of auxins (NAA, IBA) for the last subculture to initiate rooting in vitro

Non-Media related plant conditioning Change the culture room environment (limited by facility) reduce relative humidity in the culture vessel reduce room temperature increase light levels

Stage IV- after planting to soil, place in tented benches with mist, bottom heat, shade
and 16 hour day-length (supplemental HPS lighting)

Move out of tented benches after rooting begins (3-4 weeks), continue to grow for 2-3 months until well established, then move out to the nursery

New transplants of micropropagated Rhododendrons

New transplants of micropropagated Azaleas

New transplants of micropropagated Lilacs

House full of micropropagated Hakonechloa liners