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Made in the Shade Tissue Culture

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Made in the Shade Tissue Culture

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11/02/2019

Our last tissue culture liner shipment of the year headed out on its way to Amsterdam. Exciting new hosta introductions from Don Dean, Dan Wols, Ed Schulz, Trudy Van Wyk and Jeff Moore.

05/12/2019

Our TC lab continues to grow. Please join me in welcoming our latest staff addition. Thanks to my friend Michael Mellott for the referral.

03/09/2019

I really like this new hosta introduction from Don Dean. Light of Day as a stage 3 TC, a stage 4 plant, and a mature plant.

10/22/2018

Class of 2018 Initiations
It was a tedious but exciting weekend of initiating about 40 new hosta cultivars into tissue culture. Exciting new introductions from hybridizers Dick Barbee, Meg and Jim Dalton, Don Dean, Rick Goodenough, Richard Merritt, Jeff Moore, Marlene Rosenberg, Dennis Savory, Jeff White, Milt Whitmer, Gene Wogoman and Dan Wols...just to name a few.

09/04/2018

Working on some fun new introductions in the lab today. First up is Hosta 'Pure Intentions' from Minnesota hybridizer Don Dean. Named in honor of his BFF and spouse Gayle. It will be offered by the Boyz at Naylor Creek in 2019.

08/29/2018

You would think that propagating plants in the tissue culture lab would be the hard part. Now it comes time to ship the liners from point A to point B. Never mind that "This Side Up" on a box means nothing to the USPS, UPS or FedEx. We slide the trays in a mesh sleeve and then use plastic peanuts (a necessary evil) to immobilize the liners in the box when they inevitably get turned upside down. Shipping to Europe means you get to learn about phytos and air cargo handling.

08/28/2018

Day 8 – Plant Tissue Culture

Today we reach the end of the TC journey. We have been through Initiation (stage 1), Multiplication (stage 2) and Rooting (stage 3). All of these first 3 stages were preformed “in vitro” (i.e. inside a sterile culture vessel). Today we go ex-vitro with Acclimation (stage 4) and move from the lab to the greenhouse.

The key to understanding the acclimation stage is to recall the composition of the TC media. Remember the most prevalent compound in the media? It is water. Plants in vitro have been growing in a 100% humidity environment their entire life. And their roots have formed in water. We have also been feeding the plants sugar so they didn’t need to photosynthesize to produce their own energy source. The leaf stoma are stuck open at this point, not really needing to regulate gas exchange that becomes an important function during photosynthesis. When going ex vitro, these same plants will very quickly desiccate until the stoma becomes functional. At the same time, the roots do not have root hairs. You have probably noticed this if you have ever rooted a cutting in water. The resulting roots don’t have to be very efficient because they are surrounded by water. So the goal of stage 4 is to wean the plants off of a 100% humidity environment, develop root hairs, and to kick start the photosynthesis process.

This is accomplished in a greenhouse on a mist bench where we periodically apply a water mist to keep the foliage moist. Plants are removed from the culture vessel and the media is washed from the roots. They are then transported to the greenhouse. The plants are first potted in 96 cell packs and placed on the mist bench where they remain for a couple of weeks. The goal at this point is good root development that will fill the cell pack. After about 6 weeks in the greenhouse we have a well developed liner that is suitable for potting up and growing on. Commercial hosta growers start with these cell pack liners and typically grow them on for a full growing season before offering them for retail sales.

08/27/2018

Day 7 – Plant Tissue Culture

Begin with the end in mind… A TC lab will always establish a production target for each cultivar. Once we are nearing that required number in the stage 2 multiplication stage, we are ready to move on to stage 3 rooting. We divide the plants one more time in the hood (yes that does require some idea on a typical multiplication factor for a specific cultivar so you don’t overshoot production), but this time we modify the media slightly. The media remains the same except we modify the hormone ratio in favor of auxins. And miraculously in only about 4 weeks we have roots appearing.

In stage 2 multiplication we always transfer one plant to one culture vessel. That way any contamination losses we might encounter along the way are contained. At the stage 3 point we have produced a large number of plants (that are presumably still clean) and so we can now use a larger culture vessel that will contain multiple plants.
In the early days we incorporated activated carbon in the stage 3 media. (That is what makes the media black in color.) There are different theories on why you would do this, but it does create a dark environment for root development and the roots do tend to grown downwards. We also used deli style containers as culture vessels in the early days. (Just make sure they are made of polypropylene so they don’t melt in the autoclave). We no longer use carbon (it is not really required although you will find roots growing in all directions including upwards) and we now use square Caisson culture vessel for better space management.

The stage 3 culture vessels go back on the growing racks for about 4 weeks under the same lighting regime.

08/26/2018

Day 6 – Plant Tissue Culture

Once a hosta is initiated into culture as a tight little bud, in only a day or two the outer leaves of the bud will start unfurling. Hopefully you start to see some multiplication in the initial 6 to 10 weeks. But it may take a longer period of time for the cytokinins to really kick in and the plant to start multiplying. Hosta do not yield as large of a multiplication rate as many plants. Once they get going, hosta will give a multiplication factor of 3 to 6 (typically, sometimes more, sometimes less) over a typical 4 to 8 week transfer cycle. So every 4 to 8 weeks we move the culture vessel back to the hood, divide the plant, and then each division gets some fresh media in a new tube. The part that still amazes me is that these plants grow and multiply just fine, but have no roots at this point. The plant doesn’t need them at this point. Plus any root formation would be a pain to deal with as we divide and replate each cycle.

Here is a pic of a new Don Dean introduction that we recently propagated for Naylor Creek Nursery – ‘Sapphire Pillows’. These are scheduled for their next division today. If you look closely you will see about 6 potential divisions in the culture tube. Also note the lack of any roots because we are controlling the cytokinin to auxin ratio in favor of bud multiplication.

To illustrate the geometric multiplication in a simple manner, let’s take a 4 week (1 month) transfer cycle with a multiplication factor of 3. So 1 becomes 3 (after 4 weeks); 3 becomes 9 (after another 4 weeks); 9 becomes 27; 27 becomes 81; 81 becomes 243….. and so on. After 12 months you would have theoretically produced in excess of 50,000 plants!!! But that’s if everything goes perfectly. Every time you (or the scalpel) touches the plant you run the risk of introducing contamination. Plus there will be off-types along the way (just like in your own garden). So the actual numbers rarely ramp up as quickly as illustrated, but you can still produce a good number of plants in a relatively short period of time.

And speaking of contamination, getting the initial explant clean in the initiation stage is what makes you or breaks you in this business. Staying clean is relatively easy. Starting clean is the challenge. In the first couple of weeks you will have a good idea if you are clean. If not, the fungi and bacteria grow at a much faster rate and overwhelm the explant. And then at the other extreme, the explant just sits there. There is no bacteria or fungi, but on closer inspection you will find that you bleached the meristem tissue to death. That is the fine line we walk in the initiation stage. It is as much an art form as it is science.

08/25/2018

Day 5 – Plant Tissue Culture

Yesterday we breezed through getting a plant into a culture vessel. The end of the process occurred in a laminar flow hood under sterile conditions. But despite the sterile air flowing through the hood, we must insure all the equipment and material placed in the hood is first surface sterilized with either heat or chemicals. Before we get started we wipe down the interior surface of the hood with a disinfecting wipe. And then everything gets sprayed with alcohol (70% isopropyl alcohol) as it is placed in the hood. That also includes our hands. The culture vessels were heat sterilized in the autoclave so ideally the interior of the vessels has remained sterile, but we still need to chemically sterilize the outer surface of the vessel. Once inside the hood, a scalpel and forceps are used to handle the explants (never touching them directly with our hands). Before and after handling every explant, the scalpel and forceps are heat disinfected inside the hood. Some labs use an open flame along with alcohol to “flame” the tools. We used an infrared Bacti-Cinerator in the early days; now we use a glass bead sterilizer for this purpose. Working with two pairs of utensils is efficient as you let one set cool while using the other.

After we place a plant in a culture vessel it is removed from the hood. Since the culture vessel caps are not air tight, we wrap the seam with plastic. Relatively minor fluctuations in air temperature from day to night will produce a slight vacuum in the tube and we want to make sure that outside “dirty” air does not enter the tube. In the early days we used a relatively expensive material called Parafilm. These days we use inexpensive plastic cling wrap.

The plants are placed on growing racks that are lighted on a 16 hour per day cycle. We started with fluorescent tubes, but they really put off some heat when you have dozens of them going at the same time. Last year we switched out to LED tubes and they have made a remarkable difference in the lab which now runs 5 to 7 degrees F cooler as a result.

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