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low acyl gellan gum used in plant tissue culture
Low acyl gellan gum used in plant tissue culture

It is a convenient technique to use liquid culture in plant research for generating bulk tissue, liquid culture makes it impossible to the study of root hair development and root system architecture. Plant tissues need to span in three-dimensional space for growth and differentiation. 

The Gel Strength and the transmittance of this product could be adjusted subject to customer’s actual application. 

Low acyl gellan gum is ideal for using in Plant Tissue Culture Medium application and Micro-Organism Tissue Culture. 

It is widely used in tissue culture labs and is a desired alternative to to Agar.

To avoid bubbles during application, the Nitrogen item and Protein item should be carefully controlled. In order to get a clear gel for a better obversation of Plants Growing, the transparency data for this type of gellan gum is very important.

Main advantages are:

Very high transparency (90%) and brittle gels texture; the gels formed by Gellan Gum LA is heat irreversible.

Typical Dosage: 

Regular recommended dosage is 0.015%-0.4% subject to different application.

Dispersion & Dissolution:

To disperse the product without lumps:Premix the powder with the other dry ingredients, and pour the preparation into the liquid under efficient stirring. 

Continue stirring to obtain a complete dispersion.or, disperse it in a non-solvent medium (oil or others). 

The dissolution of the product depends on the medium and the process: it is improved by heat treatment(time, temperature), shear stress(stirrer, homogenizer). A complete dissolution can be rapidly obtained by boiling for 5-10min or at around 85-90℃ for 10-15min with sequestrants.

Plant Tissue Culture Recipe examples:

There are various types of tissues from which a plant can be cultured. Sometimes plant propagation requires multiple stages of tissue culturing, with different media, hormones, or different media consistencies.

Similar to sitting a plant cutting partially submerged in water on a kitchen window sill, sterile liquid media can be used, but most explants should not stay submerged in media or they will be deprived of proper gas exchange and fail to thrive.

Besides keeping all your instruments clean, and properly sterilizing media, the other difficulty is getting your recipe right. We needed to make media, test media, and adjust media to get the correct consistency, ph, and hormone balances.

Experimenting with small batches and making adjustments is the best way to settle in on a good media recipe. Not too firm or you’ll bind up the transportation of nutrients and deprive explants, and not too soft to the point the explant would sink and become deprived of oxygen.

Our base recipe makes approximately 200ml of media. We are able to fill 10 1.5 oz vials about 1/3 full, or sufficiently fill 3 1/2 pint jars. More media can easily be created by multiplying all the values.
· 0.5 – 1.0g gellan gum
· 5.4g Sugar
· 1.5ml Liquid Fertilizer with Macro and Micro Nutrients (Look for a PH adjusted Fertilizer for Hydroponics)
· 18ml All Natural Coconut Water
· 180ml Purified Water H20

Media Preparation: Prepare the autoclavable vials, jars, or other suitable containers that will receive the media, and equipment to be used prior to preparing the media.
1. Measure out the liquid components of purified water, coconut water, liquid nutrients, and any dissolved hormones.
2. Heat and thoroughly dissolve sugar into liquids.
3. Let cool and adjust ph. If you used PH adjusted fertilizers, this step will hopefully be unnecessary. Otherwise the use of ph adjusting chemicals may be necessary
4. Heat the ph adjusted liquid.
5. Thoroughly dissolve agar into liquids.
After all solids have been evenly dissolved, either pipette or carefully pour media into your tissue culture vessels.
Sterilizing Media: This step involves any of three methods, and depends on the tools available. They are Autoclaving, Microwaving, or Pressure Cooking.

Sterilizing Media: Pressure Cooking

The pressure cooker is sometimes abused to make destructive bombs. 
Why? Because some people are sick and deranged. Really though, because containing increasing pressure in any sort of vessel can be dangerous. To avoid, accidents, injury or death, PLEASE READ all of the instructions included with your pressure cooker, then read them again and again until you understand them.

1. Fill the bottom of the pressure cooker with your pressure cookers recommended amount of media for a 3 to 25 minute cook time at 15psi.
2. Place your autoclavable containers with media inside the pressure cooker. Do not seal or cover the containers with anything other than loose foil. A completely closed, or sealed container that cannot breathe will explode.
3. Rapidly heat the pressure cooker to get it up to pressure. Depending on model, the pressure cooker should have some sort of lock that engages as the pressure rises to prevent opening.
4. As soon as the steam pressure regulator starts bobbling, drastically reduce heat until the regulator bobbles gently.
5. Start the stop watch or timer.
6. The regulator should gently rock for the duration of the sterilization time, which can range from just a few minutes to 25 minutes. Time will depend on the volume of media. We manage to get 0% contamination from the volume of media that our base recipe will make, in only 5 minutes of pressure cook time.
7. After cooking, remove from heat, and wait for the lock to disengage.
8. Carefully, remove the pressure cooker lid when it is ready to be released.
9. Without burning yourself, quickly close or tighten media jar lids that have synthetic filter disks. We do this very cautiously as the pressure cooker is still cooling, and the steam is still rising to keep any unwanted pathogens from entering the positive pressure environment of the cooling pressure cooker. Running the hood fan on the stove during this process also helps prevent the settling of any unwanted pathogens.

Improved Recipe:

After some trial and error with the basic recipe, we observed some explant and media browning, and made some adjustments. We added citric acid to the media to prevent and treat phenol exudates from oxidizing.
· 0.5 – 1.0g gellan gum
· 4.0g Sucrose
· 1.0ml Soluble Liquid Fertilizer with Macro and Micro Nutrients ~PH 5.0-5.8
· 18ml All Natural Coconut Water (LESS THAN 10% concentration, greater concentrations caused callus death ~PH 5.2)
· 180ml Purified Water H20 PH 7
· 1.0 mg/l IBA(Indole-3-butyric acid) You may need to dissolve this with a solvent prior to usage
· 50 mg/l Citric Acid Or Less.
High moisture levels in our culture vessels seemed to affect our explants in early trials. In later trials we added autoclavable synthetic filter disks to our jars, and adhered them with high temperature RTV gasket maker/sealer. In other cases we taped over a small holes in the lid of our vials with an adhesive micropore filter.

Conclusion:

Engineering complex tissues for research and clinical applications relies on high-performance biomaterials that are amenable to biofabrication, maintain mechanical integrity, support specific cell behaviours, and, ultimately, biodegrade. In most cases, complex tissues will need to be fabricated from not one, but many biomaterials, which collectively fulfill these demanding requirements. 

Gellan gum is an anionic polysaccharide with potential to fill several key roles in engineered tissues, particularly after modification and blending. This review focuses on the present state of research into gellan gum, from its origins, purification and modification, through processing and biofabrication options, to its performance as a cell scaffold for both soft tissue and load bearing applications. 

Overall, we find gellan gum to be a highly versatile backbone material for tissue engineering research, upon which a broad array of form and functionality can be built.

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ZHENGZHOU CINOGEL BIOTECH CO.,LTD a professional chinese Gellan Gum manufacturer with annual production capacity of 500 tons Gellan Gum...more>>

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