8/4/2019 Cat On

1/28

Tissue Culture for Challenging

Woody Plants

Lynne Caton

Briggs Nursery LLC

Porter, WA

8/4/2019 Cat On

2/28

Briggs Nursery, LLC

Founded in 1912 in Olympia, WAOwned by Spring Garden Corporate AdvisorsOperate on 400 acres in Porter, WAEmploy a staff of 220+ during the growing seasonMaintain over 450 greenhouses for plant productionParticipant in the USNCP (Nursery Certification Program)Focus is on production of young plants (liners, plugs)

Micropropagation of Ericaceaous plants at our nursery was pioneeredby Bruce Briggs in the late 1960s

What we micropropagate: Woody OrnamentalsHerbaceous Perennials

Ornamental GrassesSmall Fruits

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

8/4/2019 Cat On

3/28

Briggs Nursery

Porter, WAcirca 2002

Propagation Lab& greenhouses

Built in 2006

8/4/2019 Cat On

4/28Briggs Nursery TC Laboratory, built in 2006

8/4/2019 Cat On

5/28Briggs Nursery Conley propagation greenhouse, 52,000 square ft., built in 2006

8/4/2019 Cat On

6/28

stage I stage II stage III stage IV

8/4/2019 Cat On

7/28

New Tools to employ in the pursuit of successful micropropation:

Stage IAn alternate chemical and protocol for surface sterilization

Stage IIA 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 IVAn optimal greenhouse environment for acclimatization

8/4/2019 Cat On

8/28

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 orbitalshaker, to break surface tension on the cutting, then drain off solution

NaOCl (SodiumHypochlorite)

Immerse plant pieces in a 10%solution of Clorox, with a drop of

Tween 20, for the appropriatelength of exposure time (5min 1hr) on an orbital shaker, then drainoff solution

Immerse plant pieces in a 1%solution of Clorox, with a drop ofTween 20, as a final rinse

NaDCC (Dichloroisocyanuric Acid)Immerse plant pieces in a 5gm/L solution

of NaDCC, with a drop of Tween 20, forthe appropriate length of exposure time

(5min 24 hrs) on an orbital shaker (noneed 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

8/4/2019 Cat On

9/28

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 tomaintain 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 ofthe 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, atroom temperature, it can be stored for 365 days and still be 100% effective

8/4/2019 Cat On

10/28

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 naHakonechloa - 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 naRhododendron - new flush 10 min 5 min na

Syringa - new flush 10 min 5 min na

Yucca - toes, below soil 15 min 24 hours na

8/4/2019 Cat On

11/28

NaDCC Dichloroisocyanuric Acid, Sodium Salt

Source:Phytotechnology LaboratoriesProduct # D253

1 kilogram = $72.98Comes as a powder, dissolves in waterTypical published rate 2-5 gm/L (Briggs rate 5 gm/L)

How we prepare solution:75 gm NaDCC15 L deionized wateradd a few drops of Tween 20Product 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

8/4/2019 Cat On

12/28

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

8/4/2019 Cat On

13/28

Stage II : Multiplication

8/4/2019 Cat On

14/28

Stage II : Multiplication : Nutrient Salts

A novel nutrient salt solution attributed to Dr. John Preece, Southern Illinois UniversityCoined Preece media at Briggs Nursery

Preece media is a hybrid of:

WPM, Woody Plant MediaDeveloped for micropropagation of Kalmia latifolia

Lloyd & McCown, 1980

DKWDeveloped for micropropagation of Juglans

Driver, Kuniyuki, 1984

8/4/2019 Cat On

15/28

Lloyd & Driver &

McCown Kuniyuki Preece

Component WPM DKW Hybrid*

media components (mg/ l)

Ammonium Nitrate 400.0 1,416.0 908.0

Boric Acid 6.2 4.8 5.5

Calcium Chloride 72.5 112.5 92.5Calcium Nitrate 386.0 1,367.0 876.5

Cupric Sulfate 0.25 0.25 0.25

EDTA, Na2 37.3 45.4 41.35

Ferrous Sulfate 27.85 33.8 30.825

Magnesium Sulfate 180.7 361.49 271.095Manganese Sulfate 22.3 33.5 27.9

Molybdic Acid, Na 0.25 0.39 0.32

Nickel Sulfate - 0.005 0.0025

Potassium Phosphate 170.0 265.0 217.5

Potassium Sulfate 990.0 1,559.0 1,274.5Zinc Nitrate - 17.0 8.5

Zinc Sulfate 8.6 - 4.3

Glycine 2.0 - 1.0

Nicotinic Acid 0.5 - 0.25

Pyridoxine HCl 0.5 - 0.25

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

PREECE MEDIA

8/4/2019 Cat On

16/28

Plants micropropagated on Preece Media

ActaeaAnemoneArbutusBetula

FothergillaHeucheraHydrangeaKalmia

LiquidambarNandinaPierisRhododendronRibesSyringaVaccinium

8/4/2019 Cat On

17/28

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 vitroand does not inhibit root formation either invitroor post vitro, weve seen promising results with Nandinaand 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 culturesgrown under a BA regime

mT cost Phytotech price

Product # per gram

BA N6-benzyladenine B-800 $4.73

mT 6-(3-hyrdoxybenzylamino)purine T-841 $135.71

8/4/2019 Cat On

18/28

Nandinaon mT

Cotinuson mT Cotinuspost mT

BAmT

BA and its analogue mT

8/4/2019 Cat On

19/28

Culture Room (multiplication):

Hepa-filtered air16 hr photoperiodTemperature 70-75 FCultures are bagged for

contamination control

8/4/2019 Cat On

20/28

Stage III Stage IV

8/4/2019 Cat On

21/28

Stage III Conditioning and/or Rooting in vitro

- Do NothingSend the microplants out on a stage II media, after a full subcultureperiod so that most of the cytokinins have been metabolized by the plant

- Go BasalEliminate cytokinins for last subculture, subculture the plants to a mediafree of any PGRs. Or add activated charcoal to the media to sponge upthe PGRs to allow the plant to root more readily

- Use PGRs and do a proper Stage III thingPrepare a media with the addition of auxins (NAA, IBA) for the lastsubculture to initiate rooting in vitro

Non-Media related plant conditioningChange the culture room environment (limited by facility)

reduce relative humidity in the culture vesselreduce room temperature

increase light levels

8/4/2019 Cat On

22/28

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

8/4/2019 Cat On

23/28

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

8/4/2019 Cat On

24/28

New transplants ofmicropropagatedRhododendrons

8/4/2019 Cat On

25/28

New transplants of micropropagated Azaleas

8/4/2019 Cat On

26/28

New transplants of micropropagated Lilacs

8/4/2019 Cat On

27/28

House full of micropropagated Hakonechloaliners

8/4/2019 Cat On

28/28