Standard Test Protocols for Active Shoot Propagation (ASP & SASP)
Description:
This protocol defines the testing framework for the Active Shoot Propagation (ASP) process. It is meant to provide guidelines for repeatability and a framework to improve this procedure over time. The ASP concept involves starting actively growing shoots in a high humidity, high nutrient environment to maintain the vigor of the existing meristematic shoot and encourage root production. Root initiation of "rootable" materials can occur within a few days and entire propagation cycles of good rooting materials can be completed within 10 days. The Seedling Active Shoot Propagation (SASP) applies the ASP process to seedlings to select individuals and families with high rooting and shoot growth potential. It may provide a useful tool to quickly identify good rooting clones/families as early as possible. The SASP process may also select for other traits, since a population can be selected over multiple iterations within a few weeks. The speculation is that rooting performance of actively growing shoots may be genetically controlled and may correlate to the dormant hardwood cutting root rates of the same materials.
Materials:
Shoot Tips: Cut softwood cuttings with a clean, razor sharp tool from healthy, actively growing indoor grown shoot tips of the target plant and plant immediately (transport in water as needed) into the soil media. Shoot tips should include the meristematic tip with several leaves and a total shoot length at least 1" long. The largest leaf should be at least 1" in length. The shoot tip size, quality and age will likely affect test results, therefore select shoot tips with consistent characteristics. Later testing should recommend the minimal and maximum tip sizes and age. Shoot tips harvested from a greenhouse or outdoor conditions will need to be cut back and re-established indoors as "mother plants", since they may need to re-adjust to the indoor conditions. The resulting new shoots can then be used in ASP tests.
Soil Media: Mix medium textured perlite and vermiculite media (PVM) at a 50:50 ratio in a clean container and a well ventilated area. Obtain washed construction grade sand for use as a media topping (not mixed with the PVM)
Containers: Use full or 1/2 flats with clear green house domes available at garden centers to increase humidity during root initiation. Use standard sized trays that provide good drainage (eg. 36 hole/flat, 3 cell trays work well). The trays must be able to contact the flat bottom.
Fertilizer Solution Rates: Light Solution: Mix 1/4 tsp of 20:20:20 water soluble fertilizer (eg. Jack's Classic or Miracle-Gro) with 1 gallon of water.
Strong Solution: Mix 1/2 tsp of 20:20:20 water soluble fertilizer (eg. Jack's Classic or Miracle-Gro) with 1 gallon of water.
Fluorescent Lighting: Hang fluorescent shop lights 6-7" over a level bench with new 40 Watt cool white bulbs. Lights should cover the entire target flat(s). Three 4' shop lights could illuminate 4 flats. Use a 24 hour timer and adjust it to allow 6 hours of darkness.
Misc. items: 1 qt adjustable spray bottle with clean water. A small thermometer.
Methods:
Media preparation: Pour enough of the strong fertilizer solution into a container to a depth that will fully absorb the media without spilling over the top of the tray. Then apply sand to the wet media to a depth of about 5mm. The media is now ready for planting.
Planting Shoot tips: Use a small tool (pocket screw driver) to poke a hole in the media. Hold the largest leaves of the shoot tip using your thumb and index finger and place deep into the hole with part of a leaf or its stem. Spray water onto the sand to fill in the planting hole and cover the lower stem/leaf with sand. This will effectively lock the cutting into place. Re-soak the trays with the strong fertilizer solution.
Root initiation period: Place the planted trays in the flat, cover with the plastic dome and put under the lights. Install a small thermometer into each flat. The ideal temperature is 75 - 80 degrees F. and can be adjusted by draping plastic over the lights to increase heat, using a fan or room air conditioning to reduce temperature and raising the light to the 7" max. height. Activate the lights with a 24hr timer to allow 6 hours of darkness. Set the darkness during the heat of the day to reduce temperatures. Root initiation of "rootable" materials can occur within a few days. Removing the dome will generally kill plants that do not have sufficient roots. The root initiation period (number of days) can be set low (eg. 4 days) to select for potentially good rooting clones (SASP) and to score the rooting performance of ASP test materials. Longer periods (eg. 6 days) may allow some clones to grow large root masses that are difficult to score/handle without damage.
Water frequency: Watering frequency may vary depending upon a variety of environmental factors. In general, plant trays should be soaked to 100% saturation levels (immersion just under/over the top of tray) every 2 to 4 days. Be sure to apply the same treatment to all test materials.
SASP Example: Sow seeds in a 288 cell flat and grow indoors (see Indoor-Aspen-Seedlings protocol). Cut actively growing tips when most shoots are 2-2.5" tall (about 3 to 4 weeks). Propagate these as ASP cuttings by dipping the 3 cell flat into the strong fertilizer solution (1/2 rate), then plant the tips and set each shoot with the spray water. Label each cell with the test ID and number of shoots planted then take a macro photo of each tray. Install the dome cover and put under the lights as outlined above. Spray the cuttings with water several times each day to maintain high humidity. Remove the cover after 5 days, soak each tray into the light solution (1/4 rate) and put back onto the flat without the dome. Remove dead (unrooted) cuttings as they wilt. Consider removing additional cuttings that are not as vigorous, tall or some other selection criteria. Make new cuttings after 5 days from removing the cover. Plant them next to the surviving "mother plants", there should be plenty of room if the previous selection was rigorous. Repeat the above 10 day cycle to the 4th or 5th iteration, which will promote the best performers from each cycle. Note that you should soak the trays into the strong solution ONCE whenever you have new cuttings, and the light solution while the cover is removed. Green algae may form on the sand surface, scrape off as needed. The final selections can be ramped up in numbers (via ASP methods) and/or transferred outside when they reach 3 - 4" of growth. Dampning off diseases have not been observed with this process.
ASP Example: Cut actively growing tips from SASP final selections and propagate these as ASP cuttings by dipping the new 3 cell flat into the strong fertilizer solution (1/2 rate), then plant the tips and set each shoot with the spray water and re-soak the tray. Label each cell with the test ID and number of shoots planted then take a macro photo of each tray. Install the dome cover and put under the lights as outlined above. Remove the trays after 4 days and carefully lift the cuttings and place them in water to clean the roots. Score them using the ASP evaluation criteria listed below. Lay out the test cuttings onto a paper or poster board with the applicable text and take a macro photo. Transplant the cuttings as needed if you intend to repeat ASP tests.
Evaluation:
Photos:
References: