Willy: "Gary has kindly shared his insights from the 9 years experience with us.
This article will be adjusted for the format."
Dionaea muscipula
Micropropagation Manual
by GARY GIPSON
------------------------------------------------------------------------------------
TABLE OF CONTENTS
FORWARD
I. PREPARING THE MEDIA
Media Preparation Technique, Sterilization and Storage.
Stage 1: Media Recipe for Seed, Leaf and Flower Stalk Initiation
Stage 2: Media Recipe for Multiplication
Stage 3: Media Recipe for Growing
Gelling agents: Agar and Carageenans
Adjusting PH and Care of Your Electronic Meter
II. EXPLANT SELECTION
Seed
leaf
flower stalk
III. CLEAN ROOM
Equipment Sterilization
Clean room Techniques
IV. STERILIZATION OF EXPLANT
Common accepted method using Sodium Hypo-chlorate
Alternative method
Seeds
Leafs
Flower Stalks
V. CARE OF CULTURES; IN VITRO TISSUE REQUIREMENTS
STAGE 1: Initiation
Seeds
Leaf
Flower Stalk
STAGE 2: Multiplication
Seeds
Leaf
Flower Stalk
STAGE 3: Growth
STAGE 4: Ex-vitro Planting and Hardening
VI CONTAMINATION
Types and Treatment
------------------------------------------------------------------------------------
Forward
This Manual will never be finished. Micro-propagation; even with the focused study of a specific Species of Plant, is an ongoing process of experimentation with absolute perfection never achieved. All One can hope for is a compatible or acceptable environment in which to propagate a particular plant species in a temporary environment at accelerated growth in which makes it beneficial to do so in the first place.
Therefore, the purpose of this manual is to be a ‘go to’ source of methods that have been proven or has been evident to be at least successful to a certain degree for the Plant species Dionaea Muscipula.
This manual is geared for Wholesale and Retail environment of Dionaea Muscipula, therefore the methods described are for maximizing productivity for the least amount of time and therefore cost.
------------------------------------------------------------------------------------
Media Preparation Technique
I have only used distilled, RO, or De-ionized Water, as I wanted uniformity. Purity of the water also affects clarity of final product. Clarity is important for early detection of infections BEFORE One multiplys it out over many vessels. For better dissolving properties, add all ingredients (Basal Salts, Vitamins, PGR‘s, etc.) into the distilled water before adding the Sugar.
Add the Gelcarin (Carageenan GP-812 or Hi Clarity) Stir until all lumps are gone in the media. It is important that the media is cool to room temperature for the Gelcarin to dissolve quickly. If the media is even slightly warm, it considerably adds to the time it takes to get rid of the Gelcarin lumps when stirring.
Test the PH with ALL the ingredients added, including the Gelcarin.
High clarity Carageenan requires a pre-autoclave PH of 4.4 to acheive a post autoclave PH of 5.5/6.
Test with an electric Meter only and adjust PH down with White Vinegar, Citric Acid (better as it is an antioxidant), or HCL. Raise PH with Sodium Bicarbonate. A little goes a long way!
Dispense into vessels, stirring the media every now and then. I currently use a magnetic stir plate. Constant stirring prevents gelling agent settling which can cause inconsistant gel firmness from vessel to vessel.
Autoclave at 15psi for 15 minutes for 25 to 30ml. Media vessel content. Add a couple of minutes for 40ml or higher media containing vessels.
Store in baggies or tubs until use; store in refrigerator if sensitive PGR’S are used to extend their life. Storing in Laminar hood is best, but needs to be used as soon as possible as the water slowly evaporates, hardening the media.
------------------------------------------------------------------------------------
MEDIA RECIPE for Dionaea Muscipula
Stage 1: Seed, leaf, and flower stalk initiation
1,000 ml distilled water
1/3 suggested label strength ms basal salts (without vitamins)
1 ml ms vitamins 1000x stock
.50 ml kinetin (1mg/1ml) or 1.0ml bap/ .1ml naa combo.
1 to 1.5 ml ppm (optional)
30 grams sugar
7.2 grams gelcarin gp812 carageenan or high clarity carageenan (hi clarity carageenan is crystal clear).
Adjust ph to 4.6 for gp812 ( 4.4 for high clarity carageenan) with citric acid, or hcl.
(electric meter only) this will make the final ph after autoclaving to be around 5.6 as prior tests have shown.
Dispense.
Autoclave for 18 minutes at 15 psi. Cool in laminar flowhood.
Stage 2: Multiplication
Make 1/3 MS with Full Vitamin and full Sugar (30g/Liter).
1-1 liquid strength = 1ml/L BAP, .1ml/Liter NAA
It is said another excellent Multiplication media is to use Phytotech Labs Dionaea/ Drosera multiplication media. I am currently experimenting with it.
Kinetin is a proven Cytokinin for Dionaea for multiplication, however BAP in My early tests gives better results in individual corms and doesn't have a tendancy to form a tumerous like solid callous.
After experimenting with varying concentrations of Kinetin in multiplication stocks, it is evident that Kinetin is much more powerful than I gave it credit for. I also underestimated the natural inclination of Dionaea to multiply on it’s own even without any Cytokinins. I have gone full circle from .5ml/Liter to 2.5ml/L and finally back to .5ml/Liter.
The reason for finally ‘settling’ on the .5ml/Liter on multiplication stocks is that in Dionaea propagation, it is not necessary to have large amounts of Kinetin to get the desired multiplication rate necessary even for large production. My ex-plants on higher amounts of Kinetin continued to multiply for several platings afterward on the grow rack, and I was turning one Ga-7 Grow Box re-plate to 4 additional vessels with virtually no maturation or significant rooting (vital to growth) evident. This multiplied out to hundreds of vessels with virtually nothing to de-flask. If I continued on this trend, I would eventually have tens of thousands of Plants coming out of vitro at One time, not to mention losses from contamination inevitable from the multiple platings and labor and materials involved.
I am currently experimenting with 1mL BAP and .1mL NAA.
Stage 3: Growing and/or Acclimation for Vivo
Makes 1/3 MS w/ full Vitamin
1,000 ML DISTILLED WATER
1/3 MS BASAL SALTS
1.00 ML MS VITAMINS 1000X STOCK
1 ML PPM (optional)
20 GRAMS SUGAR ( 30 Grams if just Growing to increase size)
7.3 GRAMS CARAGEENAN
Alternatively, use PHYTOTECH LABS VFT/DROSERA Pre-transplant media.
------------------------------------------------------------------------------------
Agar
The staple and proven Gelling media for use on Dionaea ex-plants Use 6-7 Grams per Liter.
Tends to lower PH some depending on purity. Micro-propagation Grade Agar possess’ better clarity and doesn’t affect the PH as much.
Agar is better for shipping in vitro plants.
Gelcarin
Gelcarin, specifically Carageenan GP-812 is used with great success by many Culturists. The clarity (that can vary from batch to batch) is as good or slightly better than Micro-propagational grade Agar, and can have a slight golden hue. Clarity is important for catching phenols, contamination, etc.
Another advantage is that it is partially water soluble, so clean up is easy. It’s recommended usage is between 6 and 8 Grams per Liter in strength. I’m finding that 7-7.5 Grams/Liter is a good happy medium concentration for baby food jars. 7.5 -8 Grams is more suitable for larger volumes of media in larger vessels.
Gelcarin GP812 does not readily clog the ph meter, at least the glass bulb type, as it is water soluble (partially), and the electronic meter could be used to test ph with all ingredients added including the Gelcarin before boiling. I was told by Gary of Phytotech labs that the industry standard is to test and adjust the PH of the media with ALL the ingredients added including the Agar or Gelcarin, but before boiling.
Phytotech introduced a new ‘High Clarity’ Carageenan and has proven to be ultra clear and mixes much easier.
Both Carageenans (GP812 and Hi-clarity) raises the PH of the autoclaved media much Higher. This needs to be compensated for. I have found adjusting ½ MS media to 4.6 for GP812 and 4.4 for the new Hi-Clarity Carageenan before autoclaving will yield the recommended PH of 5.5 to 5.6 after autoclaving.
PH of the Media
PH is important to the health of explants, but to what degree is debatable and depends on what is being cultivated. The proper PH for Dionaea media seems best at 5.6 more or less. I talked to Gary at Phytotech Labs and His take on PH and its importance echoed what a lot of experienced TC’rs that I’ve talked to have said. It can be important, but there tends to be an overemphasis on the importance of getting the PH just right. Don’t over think it because a culture won’t crash if it isn’t spot on, at least with most plants, anyway. The PH drops over time, anyway, as the explant extracts nutrients from the media, yet the explants are thriving?
It is worth mentioning that more important than the accuracy of the PH to a specific target range, is the CONSISTANCY of the PH from batch to batch for the sake of not stressing the cultures that have acclimated to a particular PH in the media.
The only real opportunity to adjust the PH in the media is in the preparation of the media. The standard accepted protocol for adjusting the PH in media in the TC industry is such that One has ALL the ingredients in the media, including the Gelling agent such as Agar or Gelcarin, but have not boiled the media. Adjust the PH with an electronic meter at room temperature and You are done! Now You can boil, dispense, and sterilize.
Gelcarin and Agar are polysaccharides and won’t clog an electronic meter. When finished, rinse probe under warm tap water, and store moist in it’s solution, if applicable. Gelatin will clog a meter as will any other protein based compound.
PH meters will clog over time even in the best of environments. The Supplier may tell You to replace the probe/meter, but all it really needs in most cases is just a cleaning. Meters should be cleaned every 6 months or so, especially if You are noticing ‘drift,’ meaning it is taking several minutes instead of 2 minutes to get a solid reading on the meter. You can buy cleaning solution for soaking, but I was also told that You can simply clean the probe in a jewelers ultrasonic cleaner (You can buy a cheap one at Wal-Mart) in plain water for a minute or two and let the cavitation explode off the particles in the porous glass probe bulb. If You have a powerful industry ultrasonic, care must be given not to break the glass bulb.
PH STRIPS for Testing Prepared Media
The practice of checking PH with PH strips are useless in the testing of the prepared media as the PH strips were not designed for the high content of the various Salts that We have in the media. If You have a plain glass of tap water that is a PH of say, 5.5 and prepared media that is 5.5, drop a PH strip in both and they will be two completely different colors. It is impossible to get an accurate reading in Our application. PH strips also change color under different light. Check the strip under fluorescent lighting and under an incandescent and it is readily apparent. The color code reference chart won’t change color much, but the wet strip will change color dramatically, from muddy brown to bright apple green.
------------------------------------------------------------------------------------
CLEAN ROOM
Equipment Sterilization
Autoclaved vessels, instruments, paper towels, etc. are best cooled and even stored in the Laminar flow hood or the like with a front cover intact. A running flow hood tends to dry out the media quicker, however.
Whenever possible, One should deep sterilize the utensils in an autoclave. Periodically, even the closed laminar flow hood should be wiped down with 70% alcohol and a sterile cloth or paper towel. Start from the top and work down. Do this with the fan running.
Clean Room Techniques
Prior to starting working under the laminar flow hood, ensure All air conditioning vents and any other source of moving air in the room other than the laminar flow hood should be shut or shut off. All doors, windows, etc. should be shut as well. You need 10 minutes or so for all air movement to still and contaminants to drop. Turn on the UV lamp if so equipped and leave that on for 15 minutes while You wait for the air to settle. Leave the room or avoid exposure to the UV lamp.
Next turn on the laminar flow hood fan and let it run about 10 to 15 minutes prior to working. Turn on the bacteriocinator or glass bead sterilizer at this time.
While in the hood area, avoid opening and closing doors, cabinet doors, etc. rapidly. Keep Your own movement slow and deliberate as well. The 100 to 110 fpm airflow out of the unit can be easily overwhelmed by forces greater introducing contaminants into the unit.
Wear short sleeved shirts or lab coat. Wash hands and forearms up to the elbows. Alcohol hands and arms and put latex or similar gloves on. Hairnet should also be worn. If You suspect You will have to communicate while under the hood, then wear a mask as well. Put on a clean, short sleeved lab coat, rubber gloves, and hair net. Spray gloved hands and forearms with 70% alcohol. Anything introduced into the hood should be sprayed with alcohol or bleach solution.
Vessels with plantlets taken off of the shelves for re-plating should be sprayed with bleach solution or at least alcohol. I currently use 70% alcohol and spray vessel liberally.
Bleach solution (1 part 6% Clorox to 10 parts water, and 2 drops Tween 20 per 100ml) should be made fresh each time or used within 24 hours or so. I currently use heat for instrument sterilization. It is best.
This article will be adjusted for the format."
Dionaea muscipula
Micropropagation Manual
by GARY GIPSON
------------------------------------------------------------------------------------
TABLE OF CONTENTS
FORWARD
I. PREPARING THE MEDIA
Media Preparation Technique, Sterilization and Storage.
Stage 1: Media Recipe for Seed, Leaf and Flower Stalk Initiation
Stage 2: Media Recipe for Multiplication
Stage 3: Media Recipe for Growing
Gelling agents: Agar and Carageenans
Adjusting PH and Care of Your Electronic Meter
II. EXPLANT SELECTION
Seed
leaf
flower stalk
III. CLEAN ROOM
Equipment Sterilization
Clean room Techniques
IV. STERILIZATION OF EXPLANT
Common accepted method using Sodium Hypo-chlorate
Alternative method
Seeds
Leafs
Flower Stalks
V. CARE OF CULTURES; IN VITRO TISSUE REQUIREMENTS
STAGE 1: Initiation
Seeds
Leaf
Flower Stalk
STAGE 2: Multiplication
Seeds
Leaf
Flower Stalk
STAGE 3: Growth
STAGE 4: Ex-vitro Planting and Hardening
VI CONTAMINATION
Types and Treatment
------------------------------------------------------------------------------------
Forward
This Manual will never be finished. Micro-propagation; even with the focused study of a specific Species of Plant, is an ongoing process of experimentation with absolute perfection never achieved. All One can hope for is a compatible or acceptable environment in which to propagate a particular plant species in a temporary environment at accelerated growth in which makes it beneficial to do so in the first place.
Therefore, the purpose of this manual is to be a ‘go to’ source of methods that have been proven or has been evident to be at least successful to a certain degree for the Plant species Dionaea Muscipula.
This manual is geared for Wholesale and Retail environment of Dionaea Muscipula, therefore the methods described are for maximizing productivity for the least amount of time and therefore cost.
------------------------------------------------------------------------------------
Media Preparation Technique
I have only used distilled, RO, or De-ionized Water, as I wanted uniformity. Purity of the water also affects clarity of final product. Clarity is important for early detection of infections BEFORE One multiplys it out over many vessels. For better dissolving properties, add all ingredients (Basal Salts, Vitamins, PGR‘s, etc.) into the distilled water before adding the Sugar.
Add the Gelcarin (Carageenan GP-812 or Hi Clarity) Stir until all lumps are gone in the media. It is important that the media is cool to room temperature for the Gelcarin to dissolve quickly. If the media is even slightly warm, it considerably adds to the time it takes to get rid of the Gelcarin lumps when stirring.
Test the PH with ALL the ingredients added, including the Gelcarin.
High clarity Carageenan requires a pre-autoclave PH of 4.4 to acheive a post autoclave PH of 5.5/6.
Test with an electric Meter only and adjust PH down with White Vinegar, Citric Acid (better as it is an antioxidant), or HCL. Raise PH with Sodium Bicarbonate. A little goes a long way!
Dispense into vessels, stirring the media every now and then. I currently use a magnetic stir plate. Constant stirring prevents gelling agent settling which can cause inconsistant gel firmness from vessel to vessel.
Autoclave at 15psi for 15 minutes for 25 to 30ml. Media vessel content. Add a couple of minutes for 40ml or higher media containing vessels.
Store in baggies or tubs until use; store in refrigerator if sensitive PGR’S are used to extend their life. Storing in Laminar hood is best, but needs to be used as soon as possible as the water slowly evaporates, hardening the media.
------------------------------------------------------------------------------------
MEDIA RECIPE for Dionaea Muscipula
Stage 1: Seed, leaf, and flower stalk initiation
1,000 ml distilled water
1/3 suggested label strength ms basal salts (without vitamins)
1 ml ms vitamins 1000x stock
.50 ml kinetin (1mg/1ml) or 1.0ml bap/ .1ml naa combo.
1 to 1.5 ml ppm (optional)
30 grams sugar
7.2 grams gelcarin gp812 carageenan or high clarity carageenan (hi clarity carageenan is crystal clear).
Adjust ph to 4.6 for gp812 ( 4.4 for high clarity carageenan) with citric acid, or hcl.
(electric meter only) this will make the final ph after autoclaving to be around 5.6 as prior tests have shown.
Dispense.
Autoclave for 18 minutes at 15 psi. Cool in laminar flowhood.
Stage 2: Multiplication
Make 1/3 MS with Full Vitamin and full Sugar (30g/Liter).
1-1 liquid strength = 1ml/L BAP, .1ml/Liter NAA
It is said another excellent Multiplication media is to use Phytotech Labs Dionaea/ Drosera multiplication media. I am currently experimenting with it.
Kinetin is a proven Cytokinin for Dionaea for multiplication, however BAP in My early tests gives better results in individual corms and doesn't have a tendancy to form a tumerous like solid callous.
After experimenting with varying concentrations of Kinetin in multiplication stocks, it is evident that Kinetin is much more powerful than I gave it credit for. I also underestimated the natural inclination of Dionaea to multiply on it’s own even without any Cytokinins. I have gone full circle from .5ml/Liter to 2.5ml/L and finally back to .5ml/Liter.
The reason for finally ‘settling’ on the .5ml/Liter on multiplication stocks is that in Dionaea propagation, it is not necessary to have large amounts of Kinetin to get the desired multiplication rate necessary even for large production. My ex-plants on higher amounts of Kinetin continued to multiply for several platings afterward on the grow rack, and I was turning one Ga-7 Grow Box re-plate to 4 additional vessels with virtually no maturation or significant rooting (vital to growth) evident. This multiplied out to hundreds of vessels with virtually nothing to de-flask. If I continued on this trend, I would eventually have tens of thousands of Plants coming out of vitro at One time, not to mention losses from contamination inevitable from the multiple platings and labor and materials involved.
I am currently experimenting with 1mL BAP and .1mL NAA.
Stage 3: Growing and/or Acclimation for Vivo
Makes 1/3 MS w/ full Vitamin
1,000 ML DISTILLED WATER
1/3 MS BASAL SALTS
1.00 ML MS VITAMINS 1000X STOCK
1 ML PPM (optional)
20 GRAMS SUGAR ( 30 Grams if just Growing to increase size)
7.3 GRAMS CARAGEENAN
Alternatively, use PHYTOTECH LABS VFT/DROSERA Pre-transplant media.
------------------------------------------------------------------------------------
Agar
The staple and proven Gelling media for use on Dionaea ex-plants Use 6-7 Grams per Liter.
Tends to lower PH some depending on purity. Micro-propagation Grade Agar possess’ better clarity and doesn’t affect the PH as much.
Agar is better for shipping in vitro plants.
Gelcarin
Gelcarin, specifically Carageenan GP-812 is used with great success by many Culturists. The clarity (that can vary from batch to batch) is as good or slightly better than Micro-propagational grade Agar, and can have a slight golden hue. Clarity is important for catching phenols, contamination, etc.
Another advantage is that it is partially water soluble, so clean up is easy. It’s recommended usage is between 6 and 8 Grams per Liter in strength. I’m finding that 7-7.5 Grams/Liter is a good happy medium concentration for baby food jars. 7.5 -8 Grams is more suitable for larger volumes of media in larger vessels.
Gelcarin GP812 does not readily clog the ph meter, at least the glass bulb type, as it is water soluble (partially), and the electronic meter could be used to test ph with all ingredients added including the Gelcarin before boiling. I was told by Gary of Phytotech labs that the industry standard is to test and adjust the PH of the media with ALL the ingredients added including the Agar or Gelcarin, but before boiling.
Phytotech introduced a new ‘High Clarity’ Carageenan and has proven to be ultra clear and mixes much easier.
Both Carageenans (GP812 and Hi-clarity) raises the PH of the autoclaved media much Higher. This needs to be compensated for. I have found adjusting ½ MS media to 4.6 for GP812 and 4.4 for the new Hi-Clarity Carageenan before autoclaving will yield the recommended PH of 5.5 to 5.6 after autoclaving.
PH of the Media
PH is important to the health of explants, but to what degree is debatable and depends on what is being cultivated. The proper PH for Dionaea media seems best at 5.6 more or less. I talked to Gary at Phytotech Labs and His take on PH and its importance echoed what a lot of experienced TC’rs that I’ve talked to have said. It can be important, but there tends to be an overemphasis on the importance of getting the PH just right. Don’t over think it because a culture won’t crash if it isn’t spot on, at least with most plants, anyway. The PH drops over time, anyway, as the explant extracts nutrients from the media, yet the explants are thriving?
It is worth mentioning that more important than the accuracy of the PH to a specific target range, is the CONSISTANCY of the PH from batch to batch for the sake of not stressing the cultures that have acclimated to a particular PH in the media.
The only real opportunity to adjust the PH in the media is in the preparation of the media. The standard accepted protocol for adjusting the PH in media in the TC industry is such that One has ALL the ingredients in the media, including the Gelling agent such as Agar or Gelcarin, but have not boiled the media. Adjust the PH with an electronic meter at room temperature and You are done! Now You can boil, dispense, and sterilize.
Gelcarin and Agar are polysaccharides and won’t clog an electronic meter. When finished, rinse probe under warm tap water, and store moist in it’s solution, if applicable. Gelatin will clog a meter as will any other protein based compound.
PH meters will clog over time even in the best of environments. The Supplier may tell You to replace the probe/meter, but all it really needs in most cases is just a cleaning. Meters should be cleaned every 6 months or so, especially if You are noticing ‘drift,’ meaning it is taking several minutes instead of 2 minutes to get a solid reading on the meter. You can buy cleaning solution for soaking, but I was also told that You can simply clean the probe in a jewelers ultrasonic cleaner (You can buy a cheap one at Wal-Mart) in plain water for a minute or two and let the cavitation explode off the particles in the porous glass probe bulb. If You have a powerful industry ultrasonic, care must be given not to break the glass bulb.
PH STRIPS for Testing Prepared Media
The practice of checking PH with PH strips are useless in the testing of the prepared media as the PH strips were not designed for the high content of the various Salts that We have in the media. If You have a plain glass of tap water that is a PH of say, 5.5 and prepared media that is 5.5, drop a PH strip in both and they will be two completely different colors. It is impossible to get an accurate reading in Our application. PH strips also change color under different light. Check the strip under fluorescent lighting and under an incandescent and it is readily apparent. The color code reference chart won’t change color much, but the wet strip will change color dramatically, from muddy brown to bright apple green.
------------------------------------------------------------------------------------
CLEAN ROOM
Equipment Sterilization
Autoclaved vessels, instruments, paper towels, etc. are best cooled and even stored in the Laminar flow hood or the like with a front cover intact. A running flow hood tends to dry out the media quicker, however.
Whenever possible, One should deep sterilize the utensils in an autoclave. Periodically, even the closed laminar flow hood should be wiped down with 70% alcohol and a sterile cloth or paper towel. Start from the top and work down. Do this with the fan running.
Clean Room Techniques
Prior to starting working under the laminar flow hood, ensure All air conditioning vents and any other source of moving air in the room other than the laminar flow hood should be shut or shut off. All doors, windows, etc. should be shut as well. You need 10 minutes or so for all air movement to still and contaminants to drop. Turn on the UV lamp if so equipped and leave that on for 15 minutes while You wait for the air to settle. Leave the room or avoid exposure to the UV lamp.
Next turn on the laminar flow hood fan and let it run about 10 to 15 minutes prior to working. Turn on the bacteriocinator or glass bead sterilizer at this time.
While in the hood area, avoid opening and closing doors, cabinet doors, etc. rapidly. Keep Your own movement slow and deliberate as well. The 100 to 110 fpm airflow out of the unit can be easily overwhelmed by forces greater introducing contaminants into the unit.
Wear short sleeved shirts or lab coat. Wash hands and forearms up to the elbows. Alcohol hands and arms and put latex or similar gloves on. Hairnet should also be worn. If You suspect You will have to communicate while under the hood, then wear a mask as well. Put on a clean, short sleeved lab coat, rubber gloves, and hair net. Spray gloved hands and forearms with 70% alcohol. Anything introduced into the hood should be sprayed with alcohol or bleach solution.
Vessels with plantlets taken off of the shelves for re-plating should be sprayed with bleach solution or at least alcohol. I currently use 70% alcohol and spray vessel liberally.
Bleach solution (1 part 6% Clorox to 10 parts water, and 2 drops Tween 20 per 100ml) should be made fresh each time or used within 24 hours or so. I currently use heat for instrument sterilization. It is best.