TC process.jpg

photo courtesy Made in the Shade Tissue Culture

(1-17-19) Made in the Shade Tissue Culture is allowing us to share how hostas are multiplied by  tissue culture and grown from start-to finish.

The following information appeared as individual postings (by day-to-day of the process) on Made in the Shade Tissue Culture Facebook page-

A number of you have expressed an interest in how the tissue culture (TC) process works.  It really is pretty simple and more art than science. Before we initiate a plant we must first prepare the TC media. You don’t need to be a chemist to understand the general nature of the chemicals involved in TC media. It is simply fertilizer (both major and minor components) along with vitamins and hormones. The hormones used replicate what Mother Nature does to trigger shoot and root development.

Day 2 – Plant Tissue Culture
Today we will prepare some TC media. We use two different media recipes in the lab. One is for multiplication (often referred to as Stage 2) and one for rooting (often referred to as Stage 3). Both recipes involve the various macro and micro fertilizer components, vitamins, sugar and gelling agent (agar). The only difference between the two involves the choice of hormones. Two types of plant hormones are used in tissue culture – cytokinens (for shoot growth and multiplication) and auxins (for rooting). If you have ever experimented with BAP (also referred to as BA), this is a cytokinen and it has the ability to overcome apical dominance and force otherwise dormant buds into active growth. Conversely if you have ever used Rootone, it is an auxin and triggers root development and growth. What is amazing to me is that it only takes a few parts per million of these hormones in the TC media to do the job.

The formulation for the various fertilizer compounds in TC media is known as MS Basal Salt Mixture (MS refers to two researchers Murashige and Skoog that developed this formulation back in the early 1960’s). It is the starting point for most plant TC media recipes. Different plants prefer slightly different media recipes. These are all determined empirically by trial and error. There is some published data on specific plant media recipes, but the big labs maintain their media recipes as confidential and proprietary. You can purchase a premixed media for hostas (including vitamins and hormones). It is generally based on an article published in 1980 by Mark Zilis when he first attempted to TC hostas. The recipe will work although most experts will point out it is far from optimum. So while a premixed media is a good starting point when you are new to TC, most labs will ultimately custom mix their media from scratch which allows them to tweak everything to their liking. Some of the compounds are used in very small quantities so a highly accurate scale is required. Some compounds are prepared in concentrated solutions and then dispensed with a pipettor.

The various media compounds are dissolved in distilled water. The mixture tend to be naturally acidic. The pH is adjusted upward to between 5.5 and 6. The agar is then added and the solution heated to near boiling which dissolves the agar. The mixture is then dispensed into culture vessels. We use test tubes for stage 2 media and larger Caisson MK-5 vessels for stage 3. An adjustable pipettor is very useful in dispensing a constant volume of media into a culture vessel.

Day 3 – Plant Tissue Culture

Today we will sterilize the media that we prepared yesterday. An autoclave is nothing but a big, automated pressure cooker that allows us to heat the TC media and culture vessels to 250 deg F (121 deg C). The elevated temperature is required to not only kill any bacteria and fungi present, but also any of their heat resistant spores. The goal when complete is to have a sealed culture vessel that is completely sterile inside.

If you wondering why sterile is so important, recall what happens when you leave a bowl of Jello setting out on the counter for a few days. That is what we want to avoid. Bacteria and fungi are everywhere – in us, on us and all around us. They cling to dust particles in the air. For the most part we peacefully co-exist together. However bacteria and fungi love the nutrient rich TC media. Once plants are placed in the media, any bacteria and fungi will multiply much faster than the plants. They can also produce side products that alter media pH and are even fatal to the plant. At the very minimum they compete for the same nutrients that we are providing for the plant.
Once the media has been autoclaved, we store the trays in large bins.

Next we will get to the “fun” part. We will initiate a hosta into culture.
Day 4 – Plant Tissue Culture

Now to the fun stuff. Today we will initiate a hosta into culture. This is commonly referred to as Stage 1 (the first of the four stages of TC). At the inner base of every hosta petiole is a bud (often referred to as a dormant bud). The largest buds are along the outer perimeter of a hosta eye. We will remove a well developed bud with small portion of crown (basal plate) to place into culture. The goal is to sterilize this bud by killing all the bacteria and fungi, but not killing the bud in the process. In the world of plant tissue culture, starting with a part of a plant (known as the explant) that resides below ground is the most challenging. I once heard a statistic that really puts this into perspective – one tablespoon of soil contains more organisms than there are people on the face of the earth. Even well seasoned hosta TC lab technicians often kill the bud while attempting to disinfect it. On the other extreme (really just as bad) they don’t kill the plant, but also don’t kill the bacteria or fungi.

We start by scrubbing the explant under a steady flow of water. From there we peel back multiple layers of primordial leaves. Underneath those outer layers there are bacteria, fungi and even nematodes hiding. This is really the art of TC – knowing how far to peel the layers back, but not going too far. At the center of the bud is the meristem which we want to protect. The remaining bud will be successively cleaned in a detergent wash and then under a steady flow of water for a period of time. It is then moved to a sterile transfer area. A laminar flow hood is nothing but a fancy HEPA filter with a blower and an enclosed area into which the sterilized air flows.

The bud is soaked in a dilute bleach solution in the hood for a period of time. Hopefully the bleach will kill the bacteria and fungi, but not the bud. Since even a 10% bleach solution has a pH of about 10, it is important to then wash off the bleach from the bud in sterile water before placing the bud in a culture vessel containing multiplication media. From there the culture vessel is moved into a lighted growing area.

Tomorrow we talk a little more about sterile technique and what goes on inside a laminar flow hood.

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.

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.

We recently propagated a hosta’. These are scheduled for their next division today. There are about 6 potential divisions in the culture tube. 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.
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.
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.

SHIPPING

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.

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