Tissue culture experimentation, a first pass trial

[Northern_Loki]

Introduction

This will be my first attempt at tissue culture propagation and preservation. Fail or succeed, here we go.

The current plan is to divide this experiment description into several sections:

General description
Description of materials and equipment
Description of media formulation
Preparing the media for use and sterilization
Preparing for an aseptic (sterile) work area
Preparing plant tissue for culture initiation (stage I)
Preparing the tissue culture for multiplication (stage II)
Preparing the multiplied culture for rooting (stage III)

The documentation of this effort will take a little while, so please be patient.

General Description.

The term “tissue culture” has been used as a catch-all phrase for the technique of using a sterile media along with a small amounts of plant material to propagate and multiply plant tissue in a vial or some sort of enclosed container.
The concept is based on the premise that a single plant cell containing a full copy of the plant DNA has sufficient information to regenerate an entire plant. This is called totipotency - the capacity of a cell (or a group of cells) to give rise to an entire organism.

Tissue culture, being an imprecise term, encompasses several techniques for differing reasons, including:

• Stem propagation, e.g. for cloning
• Micro propagation, e.g. for removal of microbiological contamination / infections using shoot-tips
• Meristem Culture, e.g. for removal of viruses using shoot-tip dissection (meristem)
• Protoplast Culture, e.g. undifferentiated cell culture for many engineering / research techniques

The process follows:

differentiated plant tissue (explant) dedifferentiation callus (heterogenous) redifferentiation (whole plant) cellular totipotency

By emulating different growth conditions and by manipulating the growth media, the tissue cells can be encouraged to regenerate an entire plant following the above process.

For instance, the addition of an auxin to the growth media encourages root development. Likewise, the addition of a cytokinen to the media encourages shoot development. Equal amounts of auxins and cytokinens encourages callus induction.

As such:

Auxin Cytokinen = root development
Auxin Cytokinen = shoot development
Auxin == Cytokinen = callus development

The most common use for tissue culture is micro-propagation but there are several other, namely scientific, uses including the ability to remove any viral or infectious load in the plant material.

The growth media itself has several characteristics:

• Inorganic salts consisting of macro and micro nutrients
• Vitamins
• An organic source of carbon (sucrose, for instance)

Additional adjuncts may be added that have been shown to improve growth, such as:

• Amino acids

To provide support for plant growth, a gelling agent is added. Typically,

• Agar

These comprise the base growing media. To this base growth media, plant auxins and cytokinens are added to encourage cell differentiation as described above.

The function of the culturing media, as a whole, is to:

• Provide water
• Provide mineral nutrition
• Provide vitamins
• Provide growth regulators
• Gas exchange
• Removal of plant metabolite waste

Materials and equipment

There are a variety documented methods, materials, formulations, and equipment required to successfully perform tissue culturing. To simplify sourcing the materials, I’ll be basing this trial off of the PhytoTechnologies “Hemp Multiplication Kit” with a couple of small modifications along with whatever additional equipment I have handy.

What are the materials?

Materials include the chemistry and disposable/use-once items.

• Vinegar
• Sodium Bicarbonate
• Sucrose
• Agar
• IBA
• NAA
• TDZ
• MS Medium with calcium chloride and vitamins
• PPM
• Distilled Water
• Beer and a bong

Vinegar. This is acetic acid. White distilled vinegar from the grocery store is fine. This will be used as a PH down.

Sodium Bicarbonate. This is also known as baking soda available from the grocery store. This will be used as a PH up.

Sucrose. This is also known as plain cane sugar also available from the grocery store.

Agar. Agar is a gelling medium. Alternative gelling mediums can be utilized.

IBA. This is an abbreviation that refers to Indole-3-butyric acid potassium salt (C12H12KNO2) or 4-[3-Indoly]Butyric Acid (C12H13NO2). It is a plant auxin precursor.

NAA. This is an abbreviation that refers to 1-Naphthaleneacetic acid. It is a naphthalene-based phytohormone plant auxin. This material is a skin irritant / sensitizer / and is corrosive.

TDZ. This is an abbreviation for Thidiazuron (C9H8N4OS). TDZ is a cytokinin plant growth regulator. It promotes promotes plant organogenesis (shoot regeneration) and plant regeneration. This material is a skin irritant / sensitizer / and is harmful to an aquatic environment.

MS Medium with calcium chloride and vitamins. MS refers to the Murashige & Skoog (1962) nutrient formulation. It is a common nutrient formulation available pre-made from a variety of sources. This formulation is suggested as being a good formulation for hemp / cannabis. If you want to formulate it your self (MS + calcium chloride + vitamins). The formulation is described later.

PPM (optional). This is an abbreviation for Plant Preservative Mixture. PPM is a commercial product that provides some antiseptic properties to the media that can be useful when preparing tissue cultures in the less than perfectly sterile conditions. When used in the proper proportions, PPM will not harm plant cell growth but will slow or eliminate bacterial / fungal growth.

Distilled Water. Don’t use tap water, use distilled.

Beer. Avoidance of parching.

What equipment?

• Culturing containers
• pH Strips or an accurate PH meter
• Forceps
• Scalpel
• Pipette
• Bunsen Burner
• Balance or scale that is accurate to a minimum of 0.1g (100mg)
• Weigh boats
• Beakers
• Wash containers
• Laminar flow hood or sterile glove box
• Autoclave or pressure cooker
• Latex / Nitrile disposable gloves
• Sterilization Pouches (optional)
• Stir plate and bar (optional)
• Instrument rest (optional)
• Micropore tape (optional)
• Autoclave indicator tape (optional)

Culturing containers of some sort w/lid that can be autoclaved/sterilized (high temperature). Glass can be autoclaved. Polycarbonates and some Polypropylenes are tolerant up to 121 Celcius. Be sure to check whether the material can survive high temperatures. The following images are examples of potential containers. Left-to-right, flip-top containers, petri dish, rectangular culture container with removable lid.

Plastics that cannot handle the high heat of sterilization will not survive the process:

pH Strips or an accurate PH meter You’ll need some method to measure and verify the PH of the media. This can be using the typical techniques and materials as illustrated in the following image. From left-to-right, PH indicator drops, PH test strips, laboratory PH probe, PH pen. As usually, be sure things are set-up and calibrated ahead of time.

Forceps
Scalpel
Basically, a really sharp knife, scissors, or a scalpel. Best being a scalpel. And, some sort of instrument to handle the plant material without damaging it or touching it with your hands. From left-to-right, scalpel blades, scalpel, forceps, tweezers.

Pipette
A pipette is a device for accurately measuring generally small quantities of a liquid. From left-to-right and in order of precision, a “digital” micro-pipette, a glass pipette with pump, a plastic pipette, an “eye-dropper” with graduations.

Bunsen Burner or something for sterilizing instruments while handling plant material (such as a glass bead sterilizer). The following image illustrates an inexpensive bunsen burner that utilizes camping stove isobutane gas canisters. Alternatively, a high-proof alcohol may be sufficient in some circumstance although it may not be sufficient to immediately kill some types of spores.

Balance or scale that is accurate to a minimum of 0.1g (100mg). The following image details an analytical balance, you do not need something this precise. A scale like the ones used for reloading ammunition would be sufficient and are relatively inexpensive to purchase. Although, if you can find or afford a more precise scale / balance, they have many uses. If you plan on dealing with hormones and such, the quantities can be so small you’ll need as much additional precision as possible.

Weigh boats These are simply small containers you can use when measuring and weighing ingredients. The next image details some examples. It could be something a simple as a folded piece of paper.

Beakers
Wash containers
Self explainatory. Have several available.

Laminar flow hood or sterile glove box
This will be described in detail later. A flow box or a sterile glove box are working locations that limits the contamination of your sterile media and equipment. Having a sterile work area (or as clean as possible) is essential. A localized working area that’s kept “extra” clean and free from contamination is what we are after.

Autoclave or pressure cooker This is used to sterilize the media, the containers, and any water that will be used for the tissue culture. The following image details an inexpensive 16-quart Presto brand pressure cooker. Notice this one has removable weights on the top of the cooker. The weight can be removed which will determine the cooking temperature when the unit is at pressure. For instance, 15psi weight will result in 121 Celsius / 250 Fahrenheit. A 10 psi weight results in 113 Celsius / 235 Fahrenheit. Etc.

Latex / Nitrile disposable gloves

Sterilization Pouches (optional)

Stir plate and stir bar (optional)

Instrument rest (optional) An instrument rest is simply a fixture that keeps the working parts of the scalpel, forceps, tweezers, etc off of the work surface. This is to avoid picking up any infectious contaminants that may have settled onto the work surface. Alternatively, placing the tools in a container filled with isopropyl alcohol would also be fine.

Micropore tape (optional)

Autoclave indicator tape (optional)

Media formulation

Formulations of the growth media vary depending on 1) the type of plant and 2) the individual theory 3) the goal. Similar to growing tomatoes versus oranges, the nutrient needs will vary. Past experimentation has shown what types of formulations work best for different plant species. As such, formulations are a guideline to what has worked in the past. This does not mean you, personally, shouldn’t vary a formulation. Experiment, succeed, improve.
The media formulation will also vary depending on the current goal. At different stages in the culture propagation, adjustments to the base formula with additional adjuncts will spur differing plant response.

Growth media.

The base media used here is called the Murashige & Skoog nutrient formulation. Vitamins and amino acids are added to this base formula. As noted earlier, this is a common formulation and is available on the commercial market pre-made. However, if you wish to make it yourself or modify the formula, here is one formulation:

MS Macroelements
1650ppm Ammonium Nitrate
332.2ppm Calcium Chloride, Anhydrous
1900ppm Potassium Nitrate
170ppm Potassium Phosphate, Monobasic
180.7 Magnesium Sulfate, Anhydrous

MS Microelements
6.2ppm Boric Acid
0.025ppm Cobalt Chloride•6H2O
0.025ppm Cupric Sulfate•5H2O
37.26ppm Na2EDTA•2H2O
27.8ppm Ferrous Sulfate•7H2O
16.9ppm Manganese Sulfate•H2O
37.26ppm Na2EDTA•2H2O
0.25ppm Molybdic Acid (Sodium Salt)• 2H2O
0.83ppm Potassium Iodide
8.6ppm Zinc Sulfate•7H2O

Vitamins
100ppm myo-Inositol
0.5ppm Nicotinic Acid (Free Acid)
0.5ppm Pyridoxine•HCI
0.1ppm Thiamine•HCl

Amino Acid
2.0ppm Glycine (Free Base)

Further information on the theory of nutrient formulation is referenced here: The Components of Plant Tissue Culture Media I.pdf (1.0 MB) and here: ComponentsOfTissueCultureMedia
Information of the theory of vitamin supplementation in culturing media is referenced here: The Components of Plant Tissue Culture Media ll.pdf (921.4 KB)

–In progress–

Sources for plant hormones:

Auxins - Plant Growth Regulators - Biochemicals

Cytokinins - Plant Growth Regulators - Biochemicals

https://www.bio-world.com/categories/3_43_288_691/Tissue-Culture-Hormones.html
https://www.bio-world.com/productinfo/3_43_288_691/7008/Indole-Butyric-Acid.html
https://www.bio-world.com/productinfo/3_43_288_691/118473/Thidiazuron.html
https://www.bio-world.com/productinfo/3_43_288_691/143688088/Naphthaleneacetic-Acid.html

Source for MS media with vitamins:

Murashige & Skoog Basal Medium with Vitamins

https://www.bio-world.com/productinfo/3_43_288_690/6798/Murashige-and-Skoog-MS-Medium-w-Vitamins.html

Sources for gelling media:

Gelling Agents - Biochemicals

https://www.bio-world.com/categories/3_43_288_841/Gelling-Agents.html

Other methods to find equipment:
Many of these items can also be found on Ebay for much cheaper cost-wise. Glassware, equipment, materials are often at significant discount. Used industrial items such as scales/balances can be found at pennies on the dollar if you know what to look for and are willing to entertain some risk. Like buying a used automobile. Scales and balances are sensitive to transport and are easily damaged, so be sure the vendor knows how to prep and pack the item for gorilla transport. The majority of the equipment items described above were sourced via Ebay. Industrial auctions are also a good source. Universities used to sell items as they are upgraded. I don’t know if they still do this but doesn’t hurt to ask. In each case, it just takes some time to find the good deals.

For chemistry purchased via Ebay, there will be some prep required and it is likewise somewhat difficult to verify the content and purity of chemistry purchased via Ebay. The amounts of hormones utilized at our scale is small so you don’t need much. Consider such tradeoffs if going through Ebay, as well.

[Northern_Loki]

Preparing the media for use and sterilization of items

Here we’ll discuss preparing the growth medium along with sterilization of the media and other items.

Notes on the use of a pressure cooker.

If you’ve never used a pressure cooker, let’s take a look at utilizing an inexpensive pressure cooker that can be had for around $60 USD new. If you purchase one used or already have one for canning, please be sure it has a safety relief valve (a black rubber insert on the lid). If it doesn’t have the safety valve, it is fairly old. Get a different one.

The safety value is rated to an engineered value of somewhere over 2X the rated pressure of the cooker. If the pressure exceeds the vessel pressure rating, the safety value will pop out and quickly relieve the pressure situation. This avoids a potential explosion.

The pop-up value is initially in the down position. When heat is applied, steam will escape through this valve in the beginning. As the rate of steam generation increases, the valve will eventually “pop-up” and prevent the escape of steam and, thus, start building pressure in the vessel.

Illustration of the pop-up valve on the Presto units:

The pressure regulating weight acts as a valve. The weight sits on top of a tube that enters into the pressure vessel. It seals the vessel from escaping steam until the pressure is high enough to move the weight. Once the pressure moves the weight; some of the steam escapes, the pressure drops, the weight again seals the vessel, and the process repeats over and over. This has the effect of regulating the pressure. The amount of mass (weight) on this tube dictates the pressure in the vessel (hence the reason to not place a towel over this).

Example weights, for the Presto series, used to adjust the temperature / pressure. 5 pounds pressure—228°F, 10 pounds pressure—240°F, 15 pounds pressure—250°F (note chart below in this post).

Also, read the instructions. While the cooker is coming up to pressure, it will leak water a bit around the pop-up value and weight. Let it do it’s thing. It will eventually seal itself such that steam will only be escaping from the weight. The only item placed onto the lid should be the pressure weight. Do not place anything else such as towels on the lid/weight/pop-up valve. Read the instructions and understand how to safely operate a pressure cooker.

Otherwise:

Opps. Anyone can make something safe into something unsafe. With that out of the way, they are normally safe to use.

We’ll be looking to target approximately 250 degree Fahrenheit / ~120 degree Celcius. This corresponds to 15 PSI. At this high temperature, bacteria, fungi, and heat resistant spores will be killed.

Here is a chart that details the temperature obtained while under pressure:

[1]

Sterilizing tools and wash water

1.Clean a couple of containers for water. Here I’m using some canning jars and lids:

2. Fill with distilled water, screw on top, and, optionally, add autoclave indicator tape:

3. Optionally, place jars into autoclave pouch. A pouch is another layer of protection against collecting dust, bacteria, and spores on the jar surface while awaiting use:

4. Fill the pressure cooker with water to about 1/4 inch above the spacing plate on the bottom of the cooker:

5. Add other items such as scalpel blades, scalpel, forceps, etc to a pouch (optional). Add the items to be sterilized to the cooker:

6. Inspect and then place the cover onto the pressure cooker along with the weight set for 15 PSI. Turn on the heat to medium-high:

7. As the water begins to boil, the pressure value will pop-up preventing steam from escaping:

8. Eventually, the pressure will rise high enough that it will lift the weight on the center valve. It will start moving around, whistling, sounding like a steam locomotive. At this point, the pressure cooker is at it’s target pressure. Turn down the heat a bit so that it is at what amount to a slow boil. Set a timer for 20 minutes.

9. After the allotted time, approximately 20 minutes, the sterilization process is complete. Turn off the heat and allow the cooker to naturally cool for around 15 minutes or until the pop-up value has dropped back into the down position. Carefully remove the weight, open the cover, and remove the contents.

You’ll note that the indication tape and/or the indicators on the pouch have changed colors. This indicates the the pressure cooker achieved the target temperature:

Preparing the media

There are several methods that can be employed when creating and sterilizing the media that namely have to do with getting the agar melted. You can use an a hot plate with a stirrer, the microwave, or simply during the pressure cooking / autoclave.

The first method, described next, utilizes a hot plate / stirrer. Here, we’ll prepare 500mL of media solution. This is a generic procedure, the formulas follow afterwards:

1. Fill a heat tolerant container / beaker with distilled water to slightly less than 500 ml.
   
   

2. Weight the nutrient mixture, the sucrose, and the agar depending on the formula used.
   
   
   
   

3. Add the nutrient / vitamin mixture, the sucrose, and the agar to the water. Turn on the stirrer.
   
   

4. Check and adjust the PH to approximately 5.8. Use sodium bicarbonate (PH up) or the vinegar (PH down) to adjust.
   
   

5. Adjust the volume to 500ml. Turn on the heat.
   
   

6. Bring temperature of the solution to approximately 85C (185F) in order to melt the agar. This may vary somewhat depending on the type of agar. The solution will appear cloudy and then will become clear once the agar has melted. The agar will begin gelling once the solution reaches approximately 55-60C. So, you have some time to get the solution transferred into the culture containers, once ready. The quantity of the agar into the solution along with the PH will determine how “solid” the media will become once cooled.
   
   
   
   Refer to the specific agar instructions from your vendor as to the suggested quantities. For instance:
   
   

8. Once the agar has melted and the the solution appears clear, allow it to cool but not less than around 55C / 131F (gelling temperature).
9. Using a pipette or other precise liquid measuring device, add the plant preservative mixture (PPM). The suggested ratios for PPM are between 0.05% to 0.2% volume. This means, for instance for 1 liter of solution, 1ml of PPM would amount to 0.1% concentration. 0.05% concentration would be 0.5ml. Etc.
10. Using a pipette or other precise liquid measuring device, add any hormones listed in the formulas.
    
    
11. Ensure solution is completely mixed.
12. Partially fill culture containers / tubes with media solution. Do not fully close / seal. Loosely cover / close the containers.
    Use a glove to remove the hot liquid from the heat plate:
    
    
13. Fill each container to less the 1/4 of the volume (not closing the lid entirely):
    
    
    
    Here we have approximately 25ml in each culturing container:
    
    

13. Optionally place the containers into a sterilization pouch.
    
    
14. Follow the pressure cooker sterilization process as detailed above in the sterilization of water / tools. Once sterilization process has completed, seal/close the lid completely.
    Completed sterilization of media:
    
    

15. Allow the media to cool. The agar should gel. The thickness of the gel is determined by the amount of agar added and the PH of the media. Once at room temperature it is ready for use or for short term storage.
    This is what gelled media looks like (with a bit extra agar) if allowed to cool in the beaker:
    
    
    
    It’s has a jello like consistency:
    
    

Media Formula / Recipes

The following formulas are example formulas for 1L of solution. If preparing a smaller amount, you must adjust the quantities accordingly. For 500mL, divide each quantity in half.

[Northern_Loki]

Preparing for an aseptic (sterile) work area

The adjective “aseptic” is used to describe an environment that is free from microorganism such as bacteria, spores, and yeasts. It is sterile.

The media being used in culturing is delicious. Perhaps not to humans, but to just about everything else. It is a rather optimal growth medium for not just plant cells but for microorganisms. We need to do our best to only add plant cells to the sterile media while avoiding the introduction of the microorganisms.

This can be achieved through the use of a couple of mechanisms to help ensure we minimize the introduction of undesirable contaminants and controlling any organisms that slip by.

• antibiotics
• aseptic techniques
• aseptic work space

Antibiotics / Biocides

Our first goal is to avoid the need for antibiotics by not introducing microorganisms to the media. However, let’s consider the difficulty of this goal.

As a precaution, in our less than perfectly sterile environment, we can employ the use of antibiotic compounds or biocides.

Plant Preservative Mixture
One such product is Plant Preservative Mixture (PPM). This is a patented compound consisting of 5-chloro-2-methyl-3(2H)-isothiazolone and 2-methyl-3(2H)-isothiazolone.

PPM controls microbes by penetrating the microbial cell wall and inhibiting several enzymes in the citric acid cycle and electron transport chain.

PPM generally survives autoclaving which makes this biocide useful for tissue culture and sterile filtration is avoided.

Continuing. In Progress…

Aseptic Work Space

In a controlled laboratory environment, the workspace is designed to limit the possibility of sample contamination through the use of several individual work areas. One work area is utilized to clean plant matter (ex-plant), another area is maintained to prepare the media and prep the materials needed, and a final area is where the aseptic work is performed. Each area is maintained as isolated clean rooms with the final area being maintained at the highest clean room rating that can be reasonably maintained. They are integrated in a ringed defense similar to a russian doll. That is, each inner ring is more hygienic than the outer rings. For instance, the outer ring might be the outdoors, the next ring is plant cleaning, the next ring may be ex-plant preparation and sterilization, the next ring being the final preparation area, and the final ring being an isolated flow hood within the preparation area. Each ring being isolated work areas. The primary intent is to continually reduce the number of viable contaminants on the matter and in the air where each consecutive “ring” being increasingly more sensitive to contaminants.

This “seems” extreme. As humans we regularly go about our lives with minimal indication or infection from the environment sight unseen. But consider this, according to, Aaron J. Prussin II, et. al., “Bacteria and fungi concentrations of approximately 10^2 to 10^6 CFU m−3 and 10^2 to 10^3 spores m−3, respectively, are typical.”

So, on average, we could assume upwards of one million viable bacterium and upwards of 10,000 spores per cubic meter of air. A single bacteria or a single spore could potentially infect the media since it is an optimal food source for not only plants but also these bacteria and spores.

We have to do our best to limit the number of bacteria and spores within the space where sample is being prepared by creating a “clean room”.

When we say clean room, we are referring to an area where cleanliness protocols are followed that may include:

1. ULPA / HEPA air filtration
2. Sample and preparation isolation
3. Regular disinfection of surfaces
   Continuing. In Progress…

Aseptic Techniques

Prepare 100mL of an ex-plant disinfection solution

This solution should be prepared near the time of use. This should not be prepared as a stock solution.

1. Outside of the aseptic work enclosure, in a small clean container with a cap add 10 mL of 6-8% sodium hypochloride solution (household bleach).
2. Add 90 mL of sterilized water as prepared in the earlier step.
3. Add a four drops of Tween 20 or, if you don’t have that, a couple drops of unscented/clean dish soap.

Once combined, seal the container and give it a swirl / shake to fully mix the solution. Spray the exterior of the container thoroughly with 70% isopropyl alcohol. Place the container into the aseptic work enclosure.

Continuing. In Progress…

[Northern_Loki]

Placeholder: Preparing plant tissue for culture initiation (stage I)

[Northern_Loki]

Placeholder: Preparing the tissue culture for multiplication (stage II)

[Northern_Loki]

Placeholder: Preparing the multiplied culture for rooting (stage III)

[beacher]

This is really interesting, I’ll definitely be following.

I remember breeder Steve talking about tissue culture cloning in the original OG days and how it would revolutionize genetics, but apparently nothing came of it. I figured it was too complex to be done outside of a professional lab. Although to be fair it looks like your setup is practically a professional lab!

Out of curiosity, could this be used on dried plant material? That is, could someone potentially buy smokeable bud and recreate the original plant using these techniques? If so, the implications of this could be really huge.

Good luck with the project and thanks for sharing this with us!

[allotment]

Very interesting. Thanks for letting us take part, I’ll definitely be watching this.

[Northern_Loki]

@beacher

I figured it was too complex to be done outside of a professional lab.

We are going to find out if it’s possible

@beacher

how it would revolutionize genetics

There are some interesting possibility beyond micro-propagation but mostly pipe-dream stuff for us. Currently, that is.

@beacher

your setup is practically a professional lab!

Cameras can be wonderful liars This is situated in an unfinished basement. So, we shall see…

@beacher

Out of curiosity, could this be used on dried plant material?

Not likely using this technique. At least one plant cell has to be viable (able to split and multiply). Dried / desiccated cells are not viable. You’d need a university scale lab to extract DNA from a dried cell and then try to reinsert it into a viable cell, I’d think.

[Badfishy1]

Good luck my man! Watching and pulling for you! This will be next adventure in growing but don’t have the time atm to dedicate to experimenting

[Jellypowered]

Will be following along as well. Thank you for such a great presentation!

[Jdem075]

beer was my favorite!

[MBVapester]

Above my pay scale but I’m interested and following.

[Heritagefarms]

Try a dry nug in the name of science lol but for real I couldn’t resist

[Jay]

This. Is going to be interesting. Pulling up a chair brother. Grow on!

[BubbaRay]

I’ll be enthusiastically watching this!

[Fuel]

Impressive and very interresting, thanks a lot for this exhaustive show of the whole process.

[ReikoX]

[knownotno]

Yea I’m gonna hang-out with this as well. Thanks for sharing the ride.

[50State]

I have been interested in this for some time and am glad you spent the time and effort sharing this with all of us.