TITLE: Acclimation of in vitro grown individual lines of big sagebrush (Artemisia tridentata) to ex vitro conditions in support of genotype-by-environment experiments and restoration
AUTHORS: Peggy  Martinez, Marcelo      Serpe, Rachael   Barron, Sven Buerki

[DESCRIPTION]
The following protocol describes the ex vitro, hardening and acclimation of in vitro grown genetically identical plantlets (i.e. clones) of Artemisia tridentata ssp. tridentata (Asteraceae). The goal of this protocol is to create functional clonal lines of Artemisia tridentata to be used in genotype-by-environment (GxE) experiments. Overall, the protocol consists of the following four major steps and takes 16 weeks to complete on 11-16-week old in vitro plantlets: i) transfer in vitro plantlets from modified Murashige & Skoog (MMS) media to a sand and vermiculite soil mixture (4:1 ratio; hereafter referred to as sandy soil) in an enclosed, high humidity vessel to initiate plantlet establishment and root growth (four weeks); ii) gradually open vessels to initiate dropping of in vitro leaves and growth of functional leaves (four weeks) associated with decreased of humidity and increased gas exchange; iii) establishing a watering regime to promote and maintain growth of functional plantlets (six weeks) and iv) transfer plantlets into a more complex soil mixture (similar to natural conditions composed of sand, silt and vermiculite at 2:1.5:0.5 ratio; referred to as silt soil) in an open vessel to complete acclimation; especially hardening of the root system (two weeks or more depending on specific needs). Upon completion, the sagebrush plantlets will be exhibiting a similar phenotype as sagebrush seedlings. Finally, although optional, we are encouraging users to conduct stem xylem pressure measurements on acclimatized and well-watered plantlets prior to starting GxE experiments to evaluate their hydraulic conductivity and overall level of stress. 

[GUIDELINES]
In vitro plantlets should be between 11 and 16 weeks old at time of transplant. Individuals younger than 11 weeks have insufficient biomass and root growth to withstand the acclimation.  Individuals older than 16 weeks will have a more extensive root system and are at greater risk for root tearing during transplant procedure.

[STEPS]
SECTION: Prepare sandy soil with nutrient solution
1. Prepare sandy soil

Mix play sand and vermiculite to a 4:1 v/v (sand/vermiculite) in large plastic tub (hereafter referred to as sandy soil). If play sand is very dry, add enough DI water to moisten the sandy soil mixture.
 

2. Tare PhytoCon vessel (946mL) on the balance and add 406 g of the sandy soil mixture.  
 

SECTION: Prepare sandy soil with nutrient solution
2. Media preparation

Using a graduated cylinder, add 500 mL DI water to an Erlenmeyer flask with stir bar and place onto the stir plate. Turn on stir plate to ~300 rpm, no heat is needed. 
On the balance, weigh out 2.2 g Murashige and Skoog + Gamborg vitamins into weigh boat and add to flask. Mix until dissolved. 
Pipette 1 mL Plant Preservative Mixture (PPM) to flask. Add 500 mL DI water to media mixture to bring to 1L volume. 
SECTION: Prepare sandy soil with nutrient solution
3. pH correction of the media

 Place calibrated pH meter into the 1/2MS + PPM solution created in Step 2.
Add 0.1M KOH dropwise to the solution until pH 5.8 is reached. 
 

SECTION: Prepare sandy soil with nutrient solution
4. Add media to sand mixture and autoclave

Pour 40 mL of the 1/2MS + PPM solution into a graduated cylinder and pour into prepared PhytoCon vessels. 
 Place lids lightly on top of vessels. 

 

SECTION: Prepare sandy soil with nutrient solution
4.1. Steam sterilize the prepared PhytoCon vessels at 15 psi, 121ºC, for one hour to sterilize the soil and vessels. 
After sterilization process, press lids down tightly.
Allow soil + media to cool thoroughly before beginning transplant procedure (step 5 onwards).
SECTION: Transfer plantlets from in vitro to ex vitro conditions
5. Sterilize and prepare Laminar flow clean bench for transplant of in vitro plantlets (Fig. 1)

Sterilize the inside of the laminar flow clean bench with 70% v/v ethanol. Wipe the outside of the culture vessels containing in vitro grown plantlets and PhytoCon vessels with 70% v/v ethanol prior to placing inside the laminar flow clean bench. Pour a small amount of 90% v/v ethanol into large petri dishes and allow to dry in the laminar flow clean bench. Place forceps and scoop into 90% v/v ethanol. Heat bead sterilizer to 280°C.
 
 


SECTION: Transfer plantlets from in vitro to ex vitro conditions
6. Transplanting in vitro grown plantlets into ex vitro conditions (Fig. 2)

Working inside the laminar flow clean bench, sterilize scoop by dipping it into 90% v/v ethanol and placing in bead sterilizer for ~30 seconds. Open PhytoCon vessel and dig a small ~3 cm deep hole into the soil medium.
Rinse scoop with DI water to remove excess soil and place back into 90% v/v ethanol. Then, sterilize forceps using the same procedure used to sterilize the scoop.
Allow forceps to cool slightly. 
Open the culture vessel containing an in vitro grown plantlet and grasp the base of the stem (right above the sterile media). Gently pull the plantlet and roots out of the media (Fig. 2). 
If media clings to the roots, place into sterile petri dish and using forceps, scrape off media.
 

6. In the PhytoCon vessel, feed roots into the small whole previously dug into the soil media. Gently cover roots, and press soil down lightly. Replace lid tightly and label vessel.
7. Continue steps until all plantlets are transplanted.

  
SECTION: Acclimation Phase
7. Plant Tissue Culture Chamber (Fig. 3)

After plantlets are transplanted, place PhytoCon vessels into the plant tissue culture chamber to begin acclimatization. Tissue chamber environment should be at 24°C and PAR ~128 µmol photons × s⁻¹ × m⁻² (= same abiotic conditions as with the in vitro plantlets). The particular chamber pictured uses fluorescent bulbs.

  
SECTION: Acclimation Phase
7.1. Move closed vessels into the lab (Fig. 4)

Move closed PhytoCon vessels into the laboratory to adjust plantlets to variable light intensity. Growth lights are fluorescent bulbs with PAR ~82 µmol photons × s⁻¹ × m⁻² and temperature is between 22 and 24°C. Vessels stay closed during this 2-week period and you will observe condensation on the sides (Fig. 4).

  

SECTION: Acclimation Phase
7.2. Crack open vessels in the laboratory (Fig. 5)

Open PhytoCon vessels (see Fig. 5) to allow for gas exchange to occur. Remove lids and replace slightly off center to minimize water loss and encourage phenotype changes (morphogenesis) to occur.
 
 
    
SECTION: Acclimation Phase
7.3.  Fully open vessels and water as needed (Figs. 6,7, 8)

During this step, lids will be fully removed as determined by plantlet morphology change (see Fig. 6,7 for an example), if there is not a sufficient phenotypic change (= new adapted leaves, which are sage color and covered of trichomes), removing the lid completely will cause plantlet death. 
  
 

Water will be given on an as needed basis when soil water potential falls below -0.2 MPa and no more condensation can be seen on the vessel walls.
Plantlet can be maintained at this stage indefinitely, watering ~50mL 2-3 times per week. We advise using Teros-21 water potential probes to maintain soil water potential under -0.5 MPa (Fig. 8).  
 

After hardening (ca. 15 weeks of growth), water with 1/2 strength Hoagland modified basal salt mixture one time per week. 

 
 
 
   
 
 


SECTION: Completing the hardening process
8. Prepare silt soil

Mix sand/silt/vermiculite (2:1.5:0.5 v/v/v) in large a plastic tub (referred to as silt soil). Add enough DI water to just moisten soil.
Fill smaller PhytoCon vessel (473 mL) approximately 3/4 of the way full with the silt soil mixture (Fig. 8).
 

SECTION: Completing the hardening process
8.1. Transfer plantlet into silt soil

Gently remove plantlet from larger PhytoCon vessel (946 mL). Roots are fine at this stage and tear easily.
Place plantlet in smaller PhytoCon vessel (473 mL) containing the silt soil mixture. Once plantlet is in, fill to the top with additional soil.
Water with 1/2 strength Hoagland Modified basal salt mixture until soil is moist, but no standing water is observed.
SECTION: Completing the hardening process
8.2. Hardening root system (Fig. 8)
The soil water potential should be kept between -0.2 MPa and -0.9 MPa (using Teros-21 METER probe) to harden roots and complete acclimatization (Fig. 8). 
 

  

 

SECTION: Prepare plantlets for controlled laboratory GxE experiment
9. Well-water plantlets
Maintain the soil water potential below -0.1 MPa using Teros-21 METER probes to ensure plantlets are not stressed (Fig. 8). 
SECTION: Prepare plantlets for controlled laboratory GxE experiment
9.1. Acclimate plantlets to growth chamber conditions
Set plant tissue growth chamber conditions to a 16-hr photoperiod, 24°C and PAR ~128 µmol photons × s⁻¹ × m⁻² under fluorescent bulbs. 
Continue with watering regime (using Teros-21 probes) to keep plantlets in well-watered conditions (see step above and Fig. 8).
SECTION: Prepare plantlets for controlled laboratory GxE experiment
9.2. Measure shoot water potentials
Measuring shoot water potentials verifies plantlets are not experiencing water stress.
Follow instuctions on the pressure chamber to properly attach gas hoses to the Scholander-type pressure chamber.
Measure plantlet during mid-day.
Cover leaves with reflective material and allow to equilibriate for 20 minutes.
Leaving leaves covered, excise a stem from the plantlet using a razor blade.
Feed stem through the gasket from the pressure machine. Once stem is fed through, tighten down the gasket and place back onto the pressure chamber.
Turn the lever to chamber and adjust the rate valve to a rate that makes it easy to record values.
When the end of the stem is wet, record the pressure displayed on the screen.
 

SECTION: Prepare plantlets for field GxE experiments
10. Transfer plantlets to new vessles and soil mix
Gently remove plantlets from large PhytoCon vessels and transfer to a (1:1 v/v/v) soil mix comprised of one part soil conditioners (one-part volcanic cinder: two-parts vermiculite: one-part peat moss) and one-part greenhouse potting mix (one-part top soil: one-part compost) into 983cm3 Deepot containers (Fig. 9).
Move plantlets into greenhouse with 16-hr photoperiod maintained at 23° C.
 Water plantlets deeply (i.e. water drips from the bottom of the pots) every two days.