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Thread: Tissue Culture

  1. #1
    oldmac's Avatar
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    Tissue Culture

    Has anyone here done successful tissue culture with marijuana, or knows of someone who has.

    I am told by experts that mj should be no problem to culture, but I have not been able to find any research or studies into mj tissue culture.

    Anybodies help here would be appreciated

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    I haven't done it personally but you should be able to get results using a standard medium- google 'murasaki tissue culture' and you will find many papers by one of the leading researchers investigating this.

    Also google 'kitchen culture kit'

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    The "kitchen culture kit" is what I came across awhile ago, and what peaked my interest.
    I'm going to look up "murasaki" and see what I can learn.

    Thanx stinkyattic for the info.

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    this came from a pdf i found. hope it helps any


    PLANT TISSUE CULTURE
    Background
    Plant research often involves growing new plants in a controlled environment. These may be plants that we have genetically altered in some way or may be plants of which we need many copies all exactly alike. These things can be accomplished through tissue culture of small explants of the plant of interest. These explants may come from a single mother plant or they may be the result of genetic transformation of plant cells which are then encouraged to grow and to ultimately develop into a whole plant. Tissue culture techniques are often used for commercial production of plants as well as for plant research.
    Tissue culture involves the use of small pieces of plant tissue (explants) which are cultured in a nutrient medium under sterile conditions. Using the appropriate growing conditions for each explant type, plants can be induced to rapidly produce new shoots, and, with the addition of suitable hormones new roots. These plantlets can also be divided, usually at the shoot stage, to produce large numbers of new plantlets. The new plants can then be placed in soil and grown in the normal manner.
    Many types of plants are suitable for use in the classroom. Cauliflower, rose cuttings, african violet leaves and carnation stems will all easily produce clones (exact genetic copies) through tissue culture. Cauliflower florets in particular give excellent results since they can be grown into a complete plant in the basic tissue culture media, without the need for additional growth or root hormones. Green shoots are generally observable within three weeks, and roots develop within six weeks.
    The most important part of this activity, however, is to maintain as sterile an environment as possible. Even one fungal spore or bacterial cell that comes into contact with the growth media will rapidly reproduce and soon completely overwhelm the small plant piece that you are trying to clone.
    Materials
    1 Vial of Murishige Skoog (MS) media. (If you wish to make up your own growing medium you could use the recipe for the Murashige medium given at the end of this sheet.)
    1 L sterile distilled water
    10 g of agar/L
    30 g sucrose/L
    1.5 L or 2 L container in which to prepare the growth medium
    small amounts of 1M NaOH and 1M HCl to adjust the pH of the media
    60 flat bottom culture tubes with closures.
    Glass aquarium or box lined with plastic
    Plastic sheet to cover the top of the aquarium
    Adhesive tape
    10% Bleach in a spray bottle
    70% alcohol in a spray bottle
    Forceps or tweezers
    Gloves
    Cutting equipment such as a scalpel blade or razor blade
    2 bottles of sterile distilled water
    Pressure cooker
    Your chosen plant (cauliflower, rose, African violet or carnation)
    Scalpel or razor blade
    paper towel for cutting on or sterile petri dishes if available
    Beaker or jar in which to wash the plant material
    Detergent-water mixture - 1ml detergent per liter of water
    Sterilizing solution such as a 1 in 4 dilution of chlorox bleach (4% available chlorine as sodium hypochlorite) in sterile distilled water in a large beaker or jar.
    2 or 3 beakers or jars of sterilized water
    An well-lit area away from direct sunlight or full-spectrum gro-lights
    Hormones such as BAP (benzylaminopurine) and NAA (naphthalene acetic acid) to stimulate growth and root development, respectively. (Commercial rooting hormone solutions and powders are also available from hardware stores.)
    Procedure
    Preparation and sterilization of growing medium (when not provided pre-poured)
    These steps will make 1 L of growth medium which is enough to prepare about 65 growing tubes.
    1. Dissolve the MS mixture in about 800 ml of distilled water. Stir the water continuously while adding the salt mixture. Add 30 g sugar and stir to dissolve. Adjust pH to 5.8 using 1M NaOH or 1M HCl as necessary while gently stirring. Add distilled water to make the total volume up to 1 L.
    2. Weigh out 10 grams of agar and add it to the MS solution. Heat the solution gently while stirring until all the agar has dissolved.
    3. Pour the still warm medium into the polycarbonate tubes to a depth of about 4 cm which will use about 15ml of media per tube.
    4. Place the tubes (with lids sitting on the tubes but not tightened) in a pressure cooker and sterilize for 20 minutes. Cool the pressure cooker, then remove the tubes and tighten the lids. Alternatively the tubes can be placed in boiling water for 30 minutes, but make sure that none of the water is able to enter the tubes.
    NOTE: If you wish to use plants other than cauliflower you need to prepare two different media which contain plant hormones necessary to stimulate development of differentiated tissues. The first one should contain a cytokinin such as BAP which promotes shoot formation and the second one a rooting hormone such as NAA or store bought rooting hormone. To do this, prepare the mixture up until the end of step 2. Keeping the mixture warm so that it does not solidify divide it equally into two pre-warmed containers. Each container can be used to prepare 30 or so tubes as above. The first container should have BAP added at the rate of 2.0mg/l. The second container should have the NAA hormone added at the rate of 0.1 mg/L. To do this it is necessary to make concentrated solutions of both BAP (2.0mg/ml) and NAA (1.0mg/ml). Add 1ml of the concentrated BAP stock or 100μl of the NAA concentrated stock to each 1 liter of media that you prepare. If you use rooting hormone that is purchased from your local hardware or nursery supply store instead of NAA then just follow the directions before adding to your media.
    Preparation of a sterile transfer chamber and equipment
    A classroom transfer chamber can be made from a clean glass aquarium turned on its side. Scrub the aquarium thoroughly with a 30% bleach solution, making sure that you wear gloves and do not inhale the fumes. Rinse with sterile distilled water, turn upside down on a clean counter or paper towels and allow to dry. Cut holes in a clean plastic sheet to allow arms to reach into the chamber and reinforce the cut edges with tape if necessary. Tape the clean plastic sheet over the open side of the aquarium making sure that the arm holes are located at a convenient height. Plastic sleeves could also be fitted to these holes if you wish to make it easier to prevent the entry of airborne spores into the chamber. The finished aquarium chamber can be sterilized by spraying with 10% chlorox bleach just prior to each use and drying with sterile paper towel.
    Wrap the forceps, scalpels, razor blades, paper towel and gloves (rubber or surgical) in aluminum foil, seal with tape and sterilize by processing them in a pressure cooker for twenty minutes. These items can also be sterilized by placing in an oven at 350oF for 15 minutes. You can wrap each item separately or put together a "kit" so that each student will have their own sterile equipment to use.
    Alternatively the forceps and blades can be sterilized by dipping in 10% bleach and then rinsing in sterile water, or dipping in alcohol and then placing in a flame, although this is not recommended for use in crowded classrooms. If you choose to dip in bleach and rinse in sterile water, it is best if fresh solutions are available for each 3-4 students since the water can easily be contaminated if care is not used. These liquid containers should only be opened once they are inside of the sterile chamber.
    Plant preparation
    Your plant material must first be surface sterilized to remove any bacteria or fungal spores that are present. We aim to kill all microorganisms, but at the same time not cause any adverse damage to the plant material.
    1. Cauliflower should be cut into small sections of florets about 1 cm across. If using a rose or other cuttings, cut the shoots into about 5 to 7 cm lengths. Whole African violet leaves can also be used.
    2. Wash the prepared plant material in a detergent-water mixture for about 20 minutes. If trying hairy plant material, scrub with a soft brush (toothbrush). This will help remove fungi etc., and the detergent will help wet the material and remove air bubbles that may be trapped between tiny hairs on a plant.
    3. Transfer the washed plant material to the sterilizing chlorox solution. Shake the mixture for 1 minute and then leave to soak for 10-20 minutes. Carefully pour off the bleach solution using the lid to keep the plant tissue from coming out and then carefully cap the container. Note: At this point, the tissue is considered sterile. All subsequent rinses should be done with sterile water, and all manipulations of the tissue performed with sterile instruments and supplies. Open one container at a time and never leave the lid off of any container longer than necessary.
    Transfer of plant material to tissue culture medium
    Use the sterile gloves and equipment for all of these steps.
    1. Place the plant material still in the chlorox bleach sterilizing container, the containers of sterile water, the sterilized forceps and blades, some
    sterile paper towel to use as a cutting surface and enough tubes containing sterile medium into the sterile aquarium. The outside surfaces of the containers, the capped tubes and the aluminum wrapped supplies should be briefly sprayed with 70% alcohol before moving them into the chamber.
    2. The gloves can be sprayed with a 70% alcohol solution and hands rubbed together to spread the alcohol just prior to placing hands into the chamber. Once students have gloves on and sprayed they must not touch anything that is outside of the sterile chamber.
    3. Carefully open the container with the plant material and pour in enough sterile water to half fill the container. Replace the lid and gently shake the container to wash tissue pieces (explants) thoroughly for 2-3 minutes to remove the bleach. Pour off the water and repeat the washing process 3 more times.
    4. Remove the sterilized plant material from the sterile water, place on the paper towel or sterile petri dish. Cut the cauliflower into smaller pieces about 2 to 3 mm across. If using rose cut a piece of stem about 10 mm in length with an attached bud. The African violet leaf can be cut into small squares about 1-1.5 cm across. Be sure to avoid any tissue that has been damaged by the bleach, which is apparent by its' pale color.
    5. Take a prepared section of plant material in sterile forceps and place into the medium in the polycarbonate tube. Cauliflower pieces should be partly submerged in the medium, flower bud facing up. Rose or other cuttings should be placed so that the shoots are level with the medium surface. The African violet leaf pieces should be laid directly onto the medium surface.
    6. Replace the cap tightly on the tube.
    A cutting or a plant clone grown in a tissue culture
    (Tissue culture)
    Growing the plants
    1. The tubes containing plant sections may be placed in a well-lit area of the classroom although not in direct sunlight. The shoots will probably grow more quickly if the explants are placed under fluorescent or grow-lights to provide at least 12 hours of light per day. The aquarium can be used as a growth chamber with the lighting about 8-10" overhead. This will also help maintain a more regular and warm temperature. Ensure that the temperature does not go over 28oC. New shoots should develop within 2 weeks, and should be well advanced in 3 to 4 weeks. Check the tubes daily and discard any that show signs of infection (before discarding first sterilize in the pressure cooker or add bleach into the tube).
    2. Roots can appear within 6 weeks on cauliflowers. The roses, African violet and other cuttings will need to be moved into rooting media for roots to properly develop. This transfer to the second, rooting media must be conducted under the same sterile conditions as at the initiation of the culture. All necessary equipment and the aquarium should be set up as before and properly sterilized.
    3. Working inside the sterile aquarium chamber remove, the cap from the culture tube. There will usually be several shoots that have arisen from each explant. These shoots should be carefully separated by gently removing the whole explant from the media with sterile forceps and then separating the shoots by gently pulling them apart using two pairs of forceps. Each shoot should then be placed into a tube of rooting media and the bottom of the shoot pushed into the media so that good contact is made. The cap is replaced and the shoots are then allowed to grow as in step 1 until roots are formed, usually within 2-3 weeks.
    Potting the clones
    Once roots are well formed the plants are ready to be transferred into soil.
    (plant tissue culture, including multiplication and elongation of nodules, meristem or shoot clumps, microtubers, bulbs and hairy root culture)
    1. Each plant should be carefully removed from its tube of media and planted into a small pot containing a clean light potting mix. Gently wash off all of the agar medium prior to planting. The plants will still need to be protected at this stage since they are not accustomed to the drier air of the classroom when compared to the moist environment of the tube of media.
    2. Place all of the pots onto a tray and cover lightly with a plastic dome or tent. Place the plants in an area with 12-16 hours of light (either natural or artificial) but not direct sunlight.
    3. After a week the cover can be gradually removed and the plants acclimated to stronger light and drier atmospheric conditions.
    4. You now have a collection of plants in your classroom that are genetically exactly the same. You could use these plants to carry out other experimental tests knowing that one of the main variables in the experiment has been eliminated. Some of these tests could include looking at plant responses to low light levels, to drought or to saline soil conditions. (see activity 7)
    just tryin to live the good life with my friend and wife now its time to and stay and be a
    http://boards.cannabis.com/basic-gro...-dankness.html
    complete guide to mothers clones and trimming roots http://www.cannabase.com/cl/index.html
    these last 2 links have most grow books to read http://greenmanspage.com/guides/ growmoreweed.com

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    texas grass,

    Thanks for the above info, but as you can see they where dealing with cauliflower.
    The technique itself I am familiar with; I have seen and helped with african violets and it is truly amazing. While it requires a sterile enviorment, I can do a small laminar flow hood myself (my groom room is almost sterile anyway) and I have a electric pressure cooker to serve as an atuoclave.

    But I don't want to do research here.....I want to apply this technology to my grow.
    I am willing to experiment tho....
    but I need to know out of the many grow mediums, which works best with MJ.
    Also, what part of the plant is it best to take cell material.
    Which hormones and chems at what stage.
    etc, etc.

    I need info specific to the plant. Lucky for me hemp is legal for use in many areas and is commerically viable. Currently I'm sifting thru various tech papers and writings from Universties trying to find the info I crave.

    I would love to be able to grow REAL CLONES not cuttings, in test tubes, petri dishes and baby food jars on a few feet of counter space in my work area, then having to keep 6 mature and 2-3 up and comming mothers for each strain I want to run. I currently switch between two strains....so I keep 16 or so plants, just for cuttings.

    I know there's a better way, just gotta get the info.

  7. #6
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    Just as IBA rooting hormone works on a wide range of plants, the hormones for culturing various dicotyledonous plants are very similar, it is often just the ratios that you'll see researchers tweaking. I'd suggest starting with the violet medium.
    Take a piece of leaf and press it with a sterile loop, TOP DOWN, onto the medium. You want the stomata under the leaf to be exposed so the cells do not suffocate.

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    OK, I'm going to order the kitchen culture kit with the stock violet set up.
    Maybe I'll do better with all of this if I don't overthink it.

    Worst I could do is create a "franken-canabis".

    Thanks stinky, tho I'm not sure why I'm taking encourgement from someone who can't sprout a chia pet.

  9. #8
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    Quote Originally Posted by oldmac View Post
    Thanks stinky, tho I'm not sure why I'm taking encourgement from someone who can't sprout a chia pet.
    lol, don't worry. Chia pets are surprisingly difficult- after all, it IS a hydroponic medium akin to hydtroton... I wonder if they pre-soak the critters at the factory and set them at 6.0 before sale?

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    I was talking to my hydro man the other day who mentioned that while it's a great idea.......practical application at this stage (everyone who owns a good pressure cooker raise your hand......) most likely is NOT near at this point.
    Last edited by Weedhound; Apr-12-2008 at 12:35.
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    Hey Weedhound,

    Really won't know if your hydro man is right, if I don't give it a try.
    I was really hoping someone was doing it and I could get an insight into what media, hormones, chems etc..
    I'll try to give it a shot like Stinkyattic said. Also whatever I can learn from the kit instructions themselves.

    As to the pressure cooker, it reminded me of autoclaving petri dishes for setting mushroom spores on algar. Looked thru my old collection of crap and came up with 2-glassine envelopes with 'shroom spores from 1974! 34 years old...bet they are still viable.

    As to tissue culture being a good idea, I really don't know how someone does large scale commerical grows without doing it. I only do small scale hobby grow, but maintaining two set of mom's, plus the work involved with doing cuttings; well it's just work.

    Certianly someone is into micropropagation of MJ, maybe I can find someone.

  12. #11
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    Quote Originally Posted by stinkyattic View Post
    lol, don't worry. Chia pets are surprisingly difficult- after all, it IS a hydroponic medium akin to hydtroton... I wonder if they pre-soak the critters at the factory and set them at 6.0 before sale?
    Stinkyattic,

    Glad you have a sence of humor, guess it helps if your a mod.
    I am new here and have spent more time reading then posting, and have figured out in all that reading that you are one smart person. I don't think I have seen you give an answer or advice that I could disagree with. Sticky kudos to ya. Oh, and belated thanks in helping a old man get his thread where it belonged.

    In my reading I cam across a Jan 2, 08 post of yours indicating you where going to buy a tissue culture kit. Also seems you expressed interest in this before. So......
    did you get a kit?
    progress with it?

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    You know what, I never ended up getting one. I'm still interested, but gearing up for the spring vegetable season kinda got me sidetracked. I need to get past this crazy busy summer season first, lol! Also, I switched to rapidrooter plugs for my cloning and had such good success that I didn't feel the need to learn another complicated procedure quite yet. Probably in fall when the farmin' quiets down and I have some time to devote to learning a new method.
    Thanks for the kind words, too.

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    Well I ordered one, so we'll get to see if it can be done.
    I promise to keep you posted.

    I know on one hand it's complicated, but seems if it can be made to work it would save; space, time and tedious work. I get less and less pleasure out of doing cuttings and it don't help with my aching fingers.

    The big plus it will force me to learn something new,
    probably have to read a few books on the subject too.

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    Yep, that looks like something I could certainly zip right through as well.
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    Quote Originally Posted by oldmac View Post
    texas grass,

    Thanks for the above info, but as you can see they where dealing with cauliflower.
    The technique itself I am familiar with; I have seen and helped with african violets and it is truly amazing. While it requires a sterile enviorment, I can do a small laminar flow hood myself (my groom room is almost sterile anyway) and I have a electric pressure cooker to serve as an atuoclave.

    But I don't want to do research here.....I want to apply this technology to my grow.
    I am willing to experiment tho....
    but I need to know out of the many grow mediums, which works best with MJ.
    Also, what part of the plant is it best to take cell material.
    Which hormones and chems at what stage.
    etc, etc.

    I need info specific to the plant. Lucky for me hemp is legal for use in many areas and is commerically viable. Currently I'm sifting thru various tech papers and writings from Universties trying to find the info I crave.

    I would love to be able to grow REAL CLONES not cuttings, in test tubes, petri dishes and baby food jars on a few feet of counter space in my work area, then having to keep 6 mature and 2-3 up and comming mothers for each strain I want to run. I currently switch between two strains....so I keep 16 or so plants, just for cuttings.

    I know there's a better way, just gotta get the info.
    ok so i was reading this and found this chinese study about what they used.
    http://www.pakbs.org/pjbot/PDFs/41%2...%282%29603.pdf
    i hope this is whatur looking for seemd tdz is best but im a big noob @ this just started

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    thats quiet a wash they gave those seeds

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    Question

    update? have you had time to run experiments?

    can you now discuss:
    1 jars,
    2 media,
    3 hormones,
    4 chems
    5 kits

    or maybe a simple cannabis "kitchen culture kit" review?

    found this via google... nice topic
    Thanks m8

    p.s. i am looking for the perfect jars and gel for the jars... ive seen things on youtube that dont discuss what the chemical gel is.

    also... are there <3 organic <3 alternatives to this?

  19. #18
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    Hey there Thrive,

    I got the African Violet micro-propagation down but never had any luck working out the formula for mj. Plus was busy this past summer with medical problems.

    In fact I did not see the post from jon6942 this past August....just saw it now. I'm going to go over the paper and see if it makes sence.

    This is still a fasinating area but I haven't found anyone doing it with mmj.
    Keep it civil please, gentlemen. -StinkyAttic

  20. #19
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    photos of my tissue culture

    yes it is possible - after much reading, building etc etc. I gave it a try

    Some photos of success and failure. If contaminated, you will know in a min of 3 days and by 7 days you will see the fog/mold starting.

    or you will see growth like in the Jack Herer attached.

    I am trying a tissue culture on my best Blueberry plant I have seen in a while. about 20 different seeds but there was one with the best smell, bud definition and the buzz that made you see colors.

    of course, i did not get a clone of this one and hope the Tissue culture can save the day

    Much Respect
    Attached Thumbnails Attached Thumbnails Tissue Culture-01-tissue-culture.jpg  

  21. #20
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    cool pic Glazed!
    Can you tell us about the jars your using, and the medium ? etc?
    thanks!

    also, what plant bodypart are you using?

  22. #21
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    Quote Originally Posted by oldmac View Post
    Has anyone here done successful tissue culture with marijuana, or knows of someone who has.

    I am told by experts that mj should be no problem to culture, but I have not been able to find any research or studies into mj tissue culture.

    Anybodies help here would be appreciated
    I have a patented cannabis tissue culture media send me an email and lets talk

    bill@bbforganicsolutions.com

    bill
    theberry

  23. #22
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    We are just getting into it. Here are some plantlets growing from plant stem cells in a media. We haven't raised any full term yet.



    Tissue Culture-cannabis-plantlets-tissue-culture.jpg
    oldmac likes this.

  24. #23
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    Graywolf,

    Very nice start there. I'm curious how you came up with and how you did work with stem cells. Everything I came across indicated the best cells to use were from the center of the leaf, vein area.

    I gave up long ago, just like stinkyattic, I had too much else going on. But my scientific curiosity still finds this process interesting.

    Keep up the great work, and please keep us posted as you procede.

    OM
    Keep it civil please, gentlemen. -StinkyAttic

  25. #24
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    Quote Originally Posted by oldmac View Post
    Graywolf,

    Very nice start there. I'm curious how you came up with and how you did work with stem cells. Everything I came across indicated the best cells to use were from the center of the leaf, vein area.

    I gave up long ago, just like stinkyattic, I had too much else going on. But my scientific curiosity still finds this process interesting.

    Keep up the great work, and please keep us posted as you procede.

    OM
    Our red head is using stem cells from stem nodes.

    I will ask him to post a process after he gets it shaken out. Tissue Culture-plantlet-2-1.jpgThis is his first run, which so far has been successful on a couple of dozen strains, but he hasn't grown any to the point of roots or subsequent adulthood.

  26. #25
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    Thanks Graywolf,

    Since yesterday I kept running thru my head....stem, where would I get cells from? Was thinking maybe cross cut a stem and use pith cells?

    This AM I saw your post and eureka.....
    NODES, of course, the light bulb went on over my head.

    These would be meristem cells or meristematic tissue.

    BTW, I had managed to get green growth plantlets started (not as nice as what you showed) but could never round the corner and get roots to grow...the point I finnally gave up at.

    Thanks again
    OM
    Keep it civil please, gentlemen. -StinkyAttic

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