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Wiccan_Seeker
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1.000 cuttings from ONE LEAF ?! The Micropropagation project
#5814940 - 07/03/06 06:24 AM (2 years, 2 months ago)

To start out with a fresh leaf, and turn it into 1.000 live plants in one afternoon of kitchen magic and a few weeks of growth.

Micropropagation is a technique used more and more in the floral and biotech industries.

Basically, for our purposes, an agar medium is prepared containing plantfood and a few plant growth regulators. If a tiny piece of plant matter is placed thereon (such as 1/100 square inch of leaf), and then placed into a lighted incubator (a windowsill greenhouse) the pieces of plant matter will form roots and a stem in a few weeks, and you'll have yourself a cutting. It is much like mushroom growing: any piece of the plant can act as a starting point for a new plant.

This technique, though advanced biotechnology, has to be readily adaptable to the hobby cultivator's needs, because it is not all that hightech.

Let's hack this one together, and try to make a Kitchen TEK for this amazing procedure, that is taking the professional floral industry by storm. Let's do this as a team, learn what is to be learn and adapt what can be adapted, to make amateur micropropagation a reality!

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Anonymous
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5814965 - 07/03/06 06:35 AM (2 years, 2 months ago)

Well I got a small bag of agar and some maxicrop, thats a start, maybe get some fulvic acid too. And I could pick up some basic all purpose plant food (10-10-10), although I'd think higher N fertilizer would be better since we're after vegetive growth, right?

I will try this immediately and post a report about the whole process, but we need to come up with a proper recipe for the media in the next few days, or else I'm not gonna be able to.

Edited by paradis (07/03/06 07:24 AM)

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Wiccan_Seeker
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Anonymous #Anonymous]
#5815029 - 07/03/06 07:29 AM (2 years, 2 months ago)

I think the growth medium will be the biggest hurdle to overcome, Paradis! The technique is remarkably simple as I see it, basically a modified mushroom agar tek.

OK, heres teh quick and dirty:

--Prepare agar media dishes/test tubes, then clean the tabletop and instruments
--Sterilize the leaf and cut it up, then put the leafbits onto the agar
--Let it grow (germination box, indirect light)
--Transfer to potting soil and wean to normal humidity

surely this can be done by amateurs, if they can get the medium nutrients..

____________________________________________________________________

STUDENT INSTRUCTIONS
ASEPTIC TRANSFER OF CULTURES TO FRESH MEDIUM

Micropropagation is the use of tissue culture methods to propagate plants. Micropropagation if a form of clonal propagation that differs from all other conventional propagation methods in one important aspect: aseptic conditions are necessary to achieve success with micropropagation.

In commercial and research laboratories, aseptic transfer is usually done in a hood that excludes particles larger than 3 microns from the work area. Contaminants of plant tissue culture are bacteria that reside on dust particles and fungi, including spores. A hood will effectively filter out all of these contaminants.

It is possible to transfer aseptically without a hood when you take all possible precautions to keep dust and spores from falling in the sterile medium. One simple, but fairly effective method for accomplishing this, is to spray a smooth counter top with alcohol and spray all other possible sources of contamination (e.g., hands, instruments, and culture vessels) before transferring. A second method used by plant pathologists is to work around a small Bunsen burner, which creates an updraft to prevent spores and dust from falling into the open dish. Extreme caution is required with this method because of the danger of burns. A third method involves spraying hands, instruments, and vessels with alcohol and working in a large new clear plastic bag. Because newly manufactured plastic is fairly sterile, working inside this sterile bag, which contains still air, will avoid contamination. An old aquarium or cardboard box lined with aluminum foil can be sprayed with alcohol and used to cut down on drafts. With all methods, it is best to keep traffic around the transfer area to a minimum.

Step 1. Put on a pair of sterile gloves. Clean your gloves with 70% ethanol initially, and reclean them if you touch any nonsterile surface or material.

Step 2. Spray your work surface with 70% ethanol.

Step 3. Spray vessels and all tool packets (forceps and razor blades) with 70% ethanol before placing them on the work surface.

Step 4. Arrange the objects on the work surface so that there is a cleared work area for the transferring process. Avoid placing any object "up wind" of your work area.

Step 5. For African violets, use a fresh, healthy leaf. The disinfestation process (removal of surface contaminants) is as follows:

a) Quickly rinse the leaf under cool tap water, then put the whole leaf into a closed container. A jar with a screw-cap lid is best. Depending on the size of the leaves, either a 500- or 1000-ml jar can be used. Wash the leaf in water with 0.1% detergent for 3-4 minutes. Gently agitate the leaf every 20-30 seconds during this washing step.

b) Rinse off the leaf and rinse out the jar with cool tap water.

c) Gently agitate the leaf in 10% bleach solution for 10 minutes. The jar should be filled three-fourths full with the bleach solution. (The bleach solution disinfests the plant tissue material, killing most fungal and bacterial organisms.)

d) Pour off the bleach while keeping the lid loosely in place over the container. (Be careful not to spill bleach solution on clothing because it will leave white spots after laundering.) Note: At this point, the leaf is considered sterile. All subsequent rinses should be done with sterile water, and all manipulations of the leaf performed with sterile instruments and supplies. Open one container at a time and never leave the lid off of any container longer than necessary.

e) Take the container to the sterile counter space. Spray the container with 70% ethanol before placing it in the sterile environment. Remove the lid and pour sterile water over the leaves. Fill the jar approximately half-way with the sterile water.

f) Replace the lid and gently agitate for 2-3 minutes to rinse the bleach off the leaves.

g) Pour off the rinse water in the sink, as in Step 5, d.

h) Rinse with sterile water a total of 4 times.

Step 6. Using the sterile razor blade and forceps, cut off bleach-damaged portions in an empty, sterile petri dish. With the empty dish open, hold the leaf with forceps and cut into strips 1.5-cm x 1.5-cm wide. Place 2 or 3 explants into a petri dish that contains the fresh medium. Gently press on the explants with a forceps to ensure that they make contact with the medium. Do two dishes per student. (The tissue culture medium contains all the compounds needed for plant growth, including mineral nutrients, sucrose, vitamins, and plant growth regulators that result in shoot or root production.)

Step 7. Replace the cover of the petri dish and wrap with parafilm. Use one 1-inch x 4-inch piece per plate. The parafilm will stretch to seal the dish. (Plant tissue cultures require much longer culture times than microbial cultures, so this additional precaution is usually worth the small extra cost and time.)

Step 8. Place the vessel under lights for 16 hours per day if possible. Keep the temperature between 24 and 26ƒ C (about 75ƒ F). Do not put cultures in direct sunlight, but make sure the area where they are placed is well-lighted by fluorescent bulbs. Growth is slower at lower temperatures.

Step 9. Look for small green shoots forming on or near cut surfaces. It may take 2-3 weeks or more before any visible evidence of new growth is noticeable. You may need a microscope to see these structures as they begin to grow, but after 5-6 weeks, they should be visible to the unaided eye.

If a sterile environment was not maintained, contamination will be obvious within 3 to 4 days. Materials contaminated by fungus will have a fuzzy growth on them, whereas material contaminated by bacteria will have slimy growth on them. Discard contaminated petri dishes promptly to avoid spreading plant diseases to other uncontaminated cultures.

The medium used in this exercise contains a cytokinin, and is specifically formulated to favor the production and multiplication of shoots. Once a sufficient number of shoots has been generated, portions of the explant that contain one or more shoots could be transferred to a medium that contains a higher concentration of an auxin, resulting in root production. Once roots have formed, the plantlets are transferred to pots containing a soil-based or soilless medium, and gradually exposed to conditions of lower humidity and greater light.

Source: Iowa State University, biotech dept.

____________________________________________________________________

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Wiccan_Seeker
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5815066 - 07/03/06 08:04 AM (2 years, 2 months ago)

Quote:

Much subsequent research followed, confirming the above findings for many different plants (carnation, chrysanthemum, cymbidium, primula, potato, strawberry, etc.). It has been established that plants are not only able to grow in vitro on sugar-free media at high light intensity and close to normal carbon dioxide concentration, but their quality and acclimatisation capabilities increase.
here!

Or, in other words, the medium can contain only:

inorganic salts (full-range houseplant fert)
vitamins (likely from a vitamin tab)
hormones (working on it. perhaps, maybe, possibly, common rooting powder might do)
medium thickener (agar, but a cottonball/rockwool impregnated with the above liquid will work too)

This mixture, without a carbon source, is hostile to just about anything other than plants and algi and may just be "the cardboard medium of micropropagation" as for contam resistance. "close to normal CO2 levels" is a house that is lived in.

I have to look into the plant hormones bit further, but the substrate might be as simple as houseplant fert + rooting powder + vitamin pill in water, dropped on a cotton ball in a testtube or cotton pad in a petri, this grown in a cuttings box in the windowsill.
If you settle for larger pieces of leaf this might just work with some tweaking..

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Anonymous
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5815071 - 07/03/06 08:10 AM (2 years, 2 months ago)

"Maxicrop" has cytokonin and like 70 other micronutrients. It's extracted from norwegian sea kelp. Might be too much though, I dont know.

Our resident weed guru actually knows how to do this shit successfully, so i'm sure he knows the proper recipe for the agar media, and apparently it takes twice as long as regular cloning does.

I'm just gonna wait for him to chime in.

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Wiccan_Seeker
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Anonymous #Anonymous]
#5815087 - 07/03/06 08:26 AM (2 years, 2 months ago)

The plot thickens!

I found out that you need a dual hormone system, one for roots and one for shoots. The cytokinins I'm working on (maxicrop ei?) but the other hormones, the Auxins, occur abundantly in piss and are in fact the rooting hormones in most garden center rooting powders.

I saw confirmed on another site that eliminating the carbohydrate source from the mix greatly reduces the contamination rate while being no less effective.

The technique I proposed (swapping agar for a cottonball) is in fact known and works, it is used in the technique called "microponics".

Its high time the experts chimed in :grin:

---

Coconut milk and the root tips of sprouting (pop)corn contain high amounts of cytokinins, as it does to a lesser but potentially useful degree in carrot juice.

---

If you elevate Auxins (relative to Cytokinins) you favor root growth, and if you elevate Cytokinins you favor sprouting. The balances occur in the corresponding plant parts almost regardless of species. This means the process can probably be done biologically. If you sprout seeds, lets say beans, you can collect the stems and roots separately, and liquify them by repeated freezing. If you on a sustaining medium "water" your bit of leaf with root juice you'd promote root growth, and if you water with shoot juice you favor shoot formation. A whole sprout (root+shoot) might give interesting results. For the tragically lazy fresh bean sprouts can be bought in the health food store or even the supermarket and juiced in the freezer.

This is bizarre, yet makes sense in a scientifically delirious way :nut:

Biotech results with "organic" techniques!
This is very ghetto but it would be damn elegant if it worked.

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Openminded
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5815445 - 07/03/06 10:44 AM (2 years, 2 months ago)

I'm gonna sprout some beans now :laugh:

. I had been thinking about using plant mush as a basic hormone source, never got round to it before though.

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trauma47645
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Openminded]
#5815471 - 07/03/06 10:53 AM (2 years, 2 months ago)

what about gibberellic acid.. it is a very potent plant growth hormone.. that should really help with speeding the shoot growth up.. just remember a little bit goes a very long way.. and i have a source if anyone needs it.. just PM me... its not hard to find on your own though

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Wiccan_Seeker
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: trauma47645]
#5815497 - 07/03/06 11:03 AM (2 years, 2 months ago)

As far as I know now it has to be cytokinins.

Florists use cytokinin sprays to treat their cutflowers, but there must be a more OTC source!

Anyone got a comment on the leafy bean shoots?

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SubGen1us

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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5818275 - 07/03/06 11:44 PM (2 years, 2 months ago)

i was reading about this in one of my friends horticulture books and i asked some questions here on the shroomery about it but got no good responses.

The one thing i did get to work is taking this gel i got from menards, it was called GeltoRoot.
I placed a leaf on top the gel, i made little incisions in the leaf where the veins were.
kept in humid enviroment with flouro 12 inchs away and shoots came up at incisions.
Picked the shoots outa gel with tweasers when they where about 1/2 an inch long and planted into soil.

I dont know exactly wat is in the gel but figured it was just a simple rooting hormone.

auxins? b9?

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-={Nite-Crew}=-

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the man
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: SubGen1us]
#5818674 - 07/04/06 02:11 AM (2 years, 2 months ago)

yea i had looked into this before but only foudntaht coconut water canbe used. other hormones are pretty hard to get ahold of at least it seems that way.

peace

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Treat the cops like mushrooms, feed them shit and keep them in the dark

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cricket
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5819712 - 07/04/06 12:03 PM (2 years, 2 months ago)

Anybody else thinking about rainbow Jello?

You could pour a pre-sterilized layer of cytokinin medium . Let it gel, then pour a (thin) layer of pre-sterilized auxin medium on top of that. It might be worth it to try a hormone free, nutrient agar layer at the bottom.
In theory, the auxin would get the roots to form. As they grow they will reach into the cytokinin layer, stimulating the growth of shoots. Once the new plant no longer needs the hormone treatment it would grow into the nutrient layer and start to support it's own growth.
Something else to think about...
Shoot growth requires more water then root growth. If the rooting layer is too wet it will fail. If you can separate the layers with a harmless oil (veggie maybe) you can adjust the moisture content of each layer of agar to meet the needs of each stage of growth. This would allow you to make a dryer layer for rooting, followed with a wet layer for shoots. The nutrient mix could be made dry so the density could balance the weight of the young plant and also start hardening it off so that it will adjust to normal environments faster

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Herbus
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Wiccan_Seeker]
#5819798 - 07/04/06 12:24 PM (2 years, 2 months ago)

I have done micropropagation, briefly, in Plant Propagation classes.

I could probably get the nutrient recipe. Getting the right agar-mix constitution is prime.

As I'm sure many are aware, sterile conditions are a necessity.

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...

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Magash
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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Herbus]
#5820158 - 07/04/06 02:11 PM (2 years, 2 months ago)

Off the top of my head I forget the exact mix. There are a few places that sell kits with the agar all mixed already and with auxin's to add to start the root growth.

Wiccan pretty much summed it up "The medium used in this exercise contains a cytokinin, and is specifically formulated to favor the production and multiplication of shoots. Once a sufficient number of shoots has been generated, portions of the plant that contain one or more shoots could be transferred to a medium that contains a higher concentration of an auxin, resulting in root production. Once roots have formed, the plantlets are transferred to pots containing a soil-based or soilless medium, and gradually exposed to conditions of lower humidity and greater light."

It's a great process if you have to have a high number of plants or a species that doesn't root well the normal way of cloning but takes over 3 times as long so for the home gardner it isn't to practical.

There have been many experiments with cultures. In fact Spice of Life Seeds is doing a experiment now and they are almost done. What they have done is to start the culture then seal it in a type of gel that hardens. These cultures are sold the same way as seeds but have a lower life span but are copies of the plants taken from.
These (they claim )can be treated like seeds and planted in the normal manner.

Then again they have been working on this for about 2 years now and still can't get the final process down which is why they haven't been released yet.

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fastfred

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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: Magash]
#5820454 - 07/04/06 03:30 PM (2 years, 2 months ago)

Awesome thread!

I've been lectured on micropropagation before. Trying to come up with a good home method is very interesting.

I would suggest that rather than getting too gheto someone might make a bulk order of the proper chems once the method is nailed down and distribute them to interested parties.

I would suggest getting a catalog from http://www.phytotechlab.com I have their catalog and it's pretty good. It has useful info and procedures that make it a good read. It doesn't have any prices listed though unfortunately. They seem like a pretty good company. It sounds like they have a good staff that would be willing to help out with a project like this if we were getting our supplies from them. They will make custom media, and I bet they would be willing to give plenty of advice.

I'd like to get a cutting started if anyone has one they can spare. I've got access to quite a bit of lab equipment and could probably help get the protocol nailed down if I had a cutting to work with.

-FF

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fastfred

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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: fastfred]
#5820465 - 07/04/06 03:33 PM (2 years, 2 months ago)

I OCR'd the glossary from the phytotechnology catalog. Maybe this will help with the project.
-----------------------------------------------

Adventitious---Developing from unusual points of origin, such as shoot or root tissues, from callus or embryos, from sources other than zygotes.

Agar---a polysaccharide powder derived from algae used to gel a medium. Agar is generally used at a concentration of 6-12 g/liter.

Aseptic---Free of microorganisms.

Aseptic Technique---Procedures used to prevent the introduction of fungi, bacteria, viruses, mycoplasma or other microorganisms into cultures.

Autoclave---A machine capable of sterilizing wet or dry items with steam under pressure. Pressure cookers are a type of autoclave.

Auxin---A group of plant growth regulators that promotes callus growth, cell division, cell enlargement, adventitious buds, and lateral rooting. Endogenous auxins are auxins that occur naturally. Indole-3-acetic (IAA) is a naturally occurring auxin. Exogenous auxins are auxins that are man-made or synthetic. Examples of exogenous auxins included 2,4-Dichlorophenoxyacetic acid (2,4-0), Indole-3Butyric acid (IBA), ct-Naphthaleneacetic acid (NAA), and 4-Chlorophenoxyacetic acid (CPA).

Callus---An unorganized, proliferate mass of differentiated plant cells, a wound response.

Chemically Defined Medium---A nutritive solution for culturing cells in which each component is specifiable and ideally of known chemical structure.

Clone---Plants produced asexually from a single source plant.

Clonal Propagation---Asexual reproduction of plants that are considered to be genetically uniform and originated from a single individual or explant.

Contamination---Being infested with unwanted microorganisms such as bacteria or fungi.

Culture---A plant growing in vitro.

Cytokinin---A group of plant growth regulators that regulate growth and morphogenesis and stimulate cell division. Endogenous cytokinins, cytokinins that occur naturally, include zeatin and 6-y,~’-dimethyIaIlylaminopurine (2iP). Exogenous cytokinins, cytokinins that are man-made or synthetic, include 6-furfurylaminopurine (kinetin) and 6benzylaminopurine (BA).

Differentiated---Cells that maintain, in culture, all or much of the specialized structure and function typical of the cell type in vivo. Modifications of new cells to form tissues or organs with a specific function.

Explant---Tissue taken from its original site and transferred to an artificial medium for growth or maintenance.

Gibberellins---A plant growth regulator that influences cell enlargement. Endogenous growth forms of gibberellin include Gibberellic Acid (GA3).

Horizontal laminar flow unit---An enclosed work area that has sterile air moving across it. The air moves with uniform velocity along parallel flow lines. Room air is pulled into the unit and forced through a HEPA (High Energy Particulate Air) filter, which removes particles 0.3 i.tm and larger.

Hormones---Growth regulators, generally naturally occurring, that strongly affect growth (i.e., cytokinins, auxins, and gibberellins).

Internode---The space between two nodes on a stem

In vitro---To be grown in glass (Latin). Propagation of plants in a controlled, artificial environment using plastic or glass culture vessels, aseptic techniques, and a defined growing medium.

In vivo---To be grown naturally (Latin)

Media---Plural of medium

Medium---A nutritive solution, solid or liquid, for culturing cells.

Micropropagation---In vitro Clonal propagation of plants from shoot tips or nodal explants, usually with an accelerated proliferation of shoots during subcultures.

Node---A part of the plant stem from which a leaf, shoot or flower originates.

Passage---The transfer or transplantation of cells or tissues with or without dilution or division, form one culture vessel to another.

Passage Number---The number of times the cells or tissues in culture have been subcultured or passaged.

Pathogen---A disease-causing organism.

Pathogenic---Capable of causing a disease.

Petiole---A leaf stalk; the portion of the plant that attaches the leaf blade to the node of the stem.

Plant Tissue Culture---The growth or maintenance of plant cells, tissues, organs or whole plants in vitro.

Regeneration---ln plant cultures, a morphogenetic response to a stimulus that results in the products of organs, embryos, or whole plants.

Shoot Apical Meristem---Undifferentiated tissue, located within the shoot tip, generally appearing as a shiny dome-like structure, distal to the youngest leaf primordium and measuring less that 0.1 mm in length when excised.

Somaclonal Variation---Phenotypic variation, either genetic or epigenetic in origin, displayed among somaclones.

Somaclones---Plants derived from any form of cell culture involving the use of somatic plant cells.

Stage I---A step in- in vitro propagation characterized by the establishment of an aseptic tissue culture of a plant.

Stage II---A step in in vitro propagation characterized by the rapid numerical increase of organs or other structures.

Stage III---A step in in vitro propagation characterized by preparation of propagules for successful transfer to soil, a process involving rooting of shoot cuttings, hardening of plants, and initiating the change from the heterotrophic to the autotrophic state.

Stage IV---A step in in vitro plant propagation characterized by the establishment in soil of a tissue culture derived plant, either after undergoing a Stage III pretransplant treatment, or in certain species, after the direct transfer of plants from Stage II into soil.

Sterile--- (A) Without life. (B) Inability of an organism to produce functional gametes. (C) A culture that is free of viable microorganisms.

Sterile Techniques---The practice of working with cultures in an environment free from microorganisms.

Subculture---See “Passage”. With plant cultures, this is the process by which the tissue or explant is first subdivided~ then transferred into fresh culture medium.

Tissue Culture---The maintenance or growth of tissue, in vitro, in a way that may allow differentiation and preservation of their function.

Totipotency---A cell characteristic in which the potential for forming all the cell types in the adult organism are retained.

Undifferentiated---With plant cells, existing in a state of cell development characterized by isodiametric cell shape, very little or no vacuole, a large nucleus, and exemplified by cells comprising an apical meristem or embryo.

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fastfred

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Re: 1.000 cuttings from ONE LEAF ?! The Micropropagation project [Re: fastfred]
#5820560 - 07/04/06 04:02 PM (2 years, 2 months ago)

Here is some more good stuff from the catalog...

SURFACE STERILIZING PLANT MATERIAL

To avoid bacterial and fungal growth, which is detrimental to culture growth, explants are surface sterilized before they are used to establish in vitro axenic cultures. The most common disinfectants are listed below with the concentration and exposure times that preserve the explants but at the same time destroy any microbial contamination.

PROCEDURES:
1. Wash explants in a mild detergent before treatment with the disinfecting solution. (Herbaceous material may not require this step.)
2. Rinse explants thoroughly under running tap water for 10-30 minutes.
3. Submerge explants into the disinfectant solution. Seal bottle and gently agitate.
4. Under sterile conditions, decant the solution and rinse explants several times with sterile distilled water.

STERILIZATION PROCEDURES MAY BE ENHANCED BY:

1. Placing the material in a 70% ethyl alcohol solution prior to treatment with another disinfectant solution. The use of a two step (two source) sterilization procedure has proven beneficial with certain species.

2. Using a wetting agent such as Tween 20 or 80 (PhytoTechnology Product Nos. P720 or P738, respectively) can be added to the disinfectants to reduce surface tension and allow better surface contact.

3. Conducting the sterilization process under vacuum. This results in the removal of air bubbles and provides a more efficient sterilization process.

PLANT GROWTH REGULATORS

The importance of plant growth regulators in plant tissue culture is well documented. PhytoTechnology offers a broad range of plant growth regulators specifically tested for plant cell culture. Each product is assayed for physical and chemical characteristics then is biologically tested following the criteria established for powdered media. Each auxin is tested for enhancement of callus growth and/or root initiation in vitro. Each cytokinin is tested for stimulation of shoot production.

FOR LABORATORY USE, PLANT TISSUE CULTURE
MEDIA PREPARATION, AND PLANT RESEARCH
PURPOSES ONLY. NOT FOR USE AS A PLANT
GROWTH REGULATOR ON DEVELOPED PLANTS.
NOT FOR DRUG OR HOUSEHOLD USE.

PRODUCT USE

Auxins: Auxins are generally used in plant cell culture at a concentration range of 0.01-10.0 mg/L. When added in appropriate concentrations they may regulate cell elongation, tissue swelling, cell division, formation of adventitious roots, inhibition of adventitious and axillary shoot formation, callus initiation and growth, and induction of embryogenesis.

Cytokinins: Cytokinins are generally used in plant cell culture at a concentration range of 0.1-10.0 mg/L. When added in appropriate concentrations they may regulate cell division, stimulate auxiliary and adventitious shoot proliferation, regulate differentiation, inhibit root formation, activate RNA synthesis, and stimulate protein and enzyme activity.

Gibberellins: Gibberellins are generally used to promote stem elongation, flowering, and breaking dormancy of seeds, buds, corms, and bulbs. There are over 90 forms of gibberellins, but GA3 is the most commonly used form. Compounds like paclobutrazol and ancymidol inhibit the synthesis of gibberellins.

Abscisic Acid: Abscisic Acid (ABA) plays a role in dormancy development in embryos, buds and bulbs, and in leaf abscission. When used in tissue culture, ABA inhibits the growth of shoots and the germination of embryos. Fluridone may inhibit ABA synthesis.
Polyamines: Polyamines are compounds that occur in high levels within plants and are used in tissue culture media at concentrations of 10-1000 mM. Polyamines may enhance regeneration of roots, shoots and embryos, delay or prevent senescence, and regulate flowering.

METHODS OF PREPARATION

To prepare a I mg/mL stock solution: Add 100 mg of the plant growth regulator to a 100 mL volumetric flask or other glass container. Add 3-5 mL of solvent to dissolve the powder. Once completely dissolved, bring to volume with distilled/deionized water. Stirring the solution while adding water is recommended to keep the material in solution. Store the stock solution as recommended in the tables. One mL of the stock solution in I liter of medium will yield a final concentration of 1.0 mg/L of the plant growth. (See conversion tables).

TISSUE CULTURE MEDIA COMPOSITION

MEDIA COMPONENTS

One of the most important factors governing the growth and morphogenesis of plant tissues in culture is the composition of the culture medium. The basic nutrient requirements of cultured plant cells are very similar to those of whole plants.
Plant tissue and cell culture media are generally made up of some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or other nitrogen supplements, sugar(s), other undefined organic supplements, solidifying agents or support systems, and growth regulators. Several media formulations are commonly used for the majority of all cell and tissue culture work. These media formulations include those
described by White (1963), Murashige and Skoog (1982), Gamborg, et al. (1968), Schenk and Hildebrandt (1972), Nitsch and Nitsch (1969), and Lloyd and McCown (1981). Murashige and Skoog (MS) medium, Schenk and Hilderbrandt (SH) medium, and Gamborg’s B-5 medium are all high in macronutrients, while the other media formulations contain considerably less of the macronutrients.

Macronutrients

The macronutrients provide the six major elements-nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), magnesium (Mg), and sulfur (S)-required for plant cell or tissue growth. The optimum concentration of each nutrient for achieving maximum growth rates varies considerably among species.

Culture media should contain at least 25-60 mM inorganic nitrogen for adequate plant cell growth. Plant cells may grow on nitrates alone. However, considerably better results are obtained with most plant species when the medium contains both a nitrate and ammonium nitrogen source. Certain species require ammonium or another source of reduced nitrogen for cell growth to occur. Nitrates are usually supplied in the range of 10-40 mM; typical ammonium concentrations range between 2 and 20 mM. However, ammonium concentrations in excess of 8 mM may be deleterious to cell growth of certain species. Cells can grow on a culture medium containing ammonium as the sole nitrogen source if one or more of the TCA cycle acids (e.g., citrate, succinate, or malate) are also included in the culture medium at concentrations of approximately 10 mM. When nitrate and ammonium sources of nitrogen are utilized together in the culture medium, the ammonium ions typically will be utilized more rapidly and before the nitrate ions.

Potassium (K) is required for cell growth of plant species. Most media contain K in the nitrate, sulfate, or chloride form, at concentrations of 20-30 mM. The optimum concentrations of phosphorus (P), magnesium (Mg), sulfur (S), and calcium (Ca) range from 1-3 mM when all other requirements for cell growth are satisfied. Higher concentrations of these nutrients may be required if deficiencies in other nutrients exist.

Micro nutrients

The essential micronutrients for plant cell and tissue growth include iron (Fe), manganese (Mn), zinc (Zn), boron (B), copper (Cu), and molybdenum (Mo). Chelated forms of iron and zinc are commonly used in preparing culture media. Iron may be the most critical of all the micronutrients. Iron citrate and tartrate may be used in culture media, but these compounds are difficult to dissolve and frequently precipitate after media are prepared. Murashige and Skoog used an ethylene diaminetetraacetic acid (EDTA)-iron chelate to circumvent this problem.

Cobalt (Co) and iodine (I) may also be added to certain media, but strict cell growth requirements for these elements have not been established. Sodium (Na) and chlorine (CI) are also used in some media but are not essential for cell growth. Copper and cobalt are normally added to culture media at concentrations of 0.1 1.tM, Fe and Mo at 1 ~tM, (at 5 riM, Zn at 5-30 tiM, Mn at 20-90 riM, and B at 25-100 ~tM.

Carbon and Energy Source

The preferred carbohydrate in plant cell culture media is sucrose. Glucose and fructose may be substituted in some cases, glucose
being as effective as sucrose and fructose being somewhat less effective. Other carbohydrates that have been tested include lactose, galactose, rafinose, maltose, and starch. Sucrose concentrations of culture media normally range between 2 and 3 percent. Use of autoclaved fructose can be detrimental to cell growth.

Carbohydrates must be supplied to the culture medium because few plant cell lines have been isolated that are fully autotropic, i.e., capable of supplying their own carbohydrate needs by CO2 assimilation during photosynthesis.

Vitamins

Normal plants synthesize the vitamins required for their growth and development. Vitamins are required by plants as catalysts in various metabolic processes. When plant cells and tissues are grown in vitro, some vitamins may become limiting factors for cell growth. The vitamins most frequently used in cell and tissue culture media include thiamine (B1), nicotinic acid, pyridoxine (B6), and myo-inositol. Thiamine is the one vitamin that is basically required by all cells for growth. Thiamine is normally used at concentrations ranging from 0.1 to 10.0 mg/liter. Nicotinic acid and pyridoxine are often added to culture media but are not essential for cell growth in many species. Nicotinic acid is normally used at concentrations of 0.1-5.0 mg/liter; pyridoxine is used at 0.1-10.0 mg/liter.

Myo-inositol is commonly included in many vitamin stock solutions. Although it is a carbohydrate not a vitamin, it has been shown to stimulate growth in certain cell cultures. Its presence in the culture medium is not essential, but in small quantities myo-inositol stimulates cell growth in most species. Myoinositol is generally used in plant cell and tissue culture media at concentrations of 50-5000 mg/liter.

Other vitamins such as biotin, folic acid, ascorbic acid, pantothenic acid, vitamin E (tocopherol), riboflavin, and p-aminobenzoic acid have been included in some cell culture media. The requirement for these vitamins by plant cell cultures is generally negligible, and they are not considered growth-limiting factors. These vitamins are generally added to the culture medium only when the concentration of thiamine is below the desired level or when it is desirable to grow cells at very low population densities.

Amino Acids or Other Nitrogen Supplements

Although cultured cells are normally capable of synthesizing all of the required amino acids, the addition of certain amino acids or amino acid mixtures may be used to further stimulate cell growth. The use of amino acids is particularly important for establishing cell cultures and protoplast cultures. Amino acids provide plant cells with an immediately available source of nitrogen, which generally can be taken up by the cells more rapidly than inorganic nitrogen.

The most common sources of organic nitrogen used in culture media are amino acid mixtures (e.g., casein hydrolysate), Lglutamine, L-asparagine, and adenine. Casein hydrolysate is generally used at concentrations between 0.05% and 0.1%. When amino acids are added alone, care must be taken as they can be inhibitory to cell growth. Examples of amino acids included in culture media to enhance cell growth are glycine at 2 mg/liter, glutamine up to 8 mM, asparagine at 100 mg/liter, L-arginine and cysteine at 10 mg/liter, and L-tyrosine at 100 mg/liter. Tyrosine has been used to stimulate morphogenesis in cell cultures but should only be used in an agar medium. Supplementation of the culture