Plant Tissue Culture Protocols

Gelling Agents

 
Preparation of Alginate, Agargel, Phytagel and Transfergel
Agar Products Description and Use

   Agar has long been used to solidify media for plant tissue culture. The type of agar or gelling agent used can influence the growth of the tissue in culture. Both purity and cost of the gelling agent are important factors in any research or production operation. Sigma has expanded its line of gelling agents to allow greater selection in choosing the plant cell culture tested gelling agent for your particular requirement. Listed in the TABLE below are some criteria which may help you select the appropriate product for your application.

 


 

Preparation of Alginate, Agargel, Phytagel and Transfergel


Alginate Preparation (Product No. A 0682)
    Alginic acid has been used as a physical support similar to agarose and to prepare beads into which immobilized suspension cells and protoplasts are embedded (Adaoha Mbanaso and Roscoe, 1982; Chie and Cantliffe, 1989; Draget et al., 1988; Larkin et al., 1988). Aqueous solutions of alginate form a gel at room temperature in the presence of certain cations, especially calcium. This gel matrix can be reliquified by adding chelating agents (e.g. citrate). Cells entrapped in alginate beads are easily manipulated with less chance of physical damage from handling.
    Alginate should be prepared at 1.75-4.0% (w/v) in a low calcium (e.g. 2 mM) buffered medium containing an appropriate osmoticum. After stirring to completely dissolve, alginate solutions can be filter sterilized (0.45 µm) or autoclaved. However, Larkin et al. (1988) noted a reduction in bead-making capacity with increased autoclave time. Protoplasts are concentrated by centrifugation in low calcium medium and added to alginate at an appropriate density (e.g. 1 x 105). Protoplast-alginate solution is added drop-wise to a solution containing 50 mM CaCl2 and an appropriate osmoticum. Each droplet will form a bead. The beads should remain in the CaCl2 for up to 45 minutes to ensure complete gelation.

Agargel™ Preparation (Product No. A 3301)
    Agargel is a blend of agar and Phytagel™ that was developed to help control vitrification in plant tissue cultures. This product provides the positive attributes of both products and is superior to Phytagel, where vitrification is a problem. It also serves as an economical alternative to agar. Agargel produces a semi-clear gel which allows better detection of contamination. The product should be used at a concentration of 3.5-5.0 g/L, depending upon the desired gel strength.

Phytagel™ Preparation (Product No. P 8169)
    Phytagel is an agar substitute produced from a bacterial substrate composed of glucuronic acid, rhamnose and glucose. It produces a clear, colorless, high-strength gel, which aids in detection of microbial contamination. Phytagel provides an economical alternative to agar as a gelling agent. Use Phytagel at a concentration of 1.5-2.5 g/L. To prevent clumping, Phytagel should be added to rapidly stirring culture medium which is at room temperature.

Transfergel™ Preparation (Product No. T 5660)
    Transfergel is supplied as a white powder and can be used in research and in the micropropagation industry as a carrier gel for tissue culture propagules, such as somatic embryos, microcuttings and shoot tips (Schultheis et al., 1990). Transfergel also is an effective carrier for seed drilling practices. The gel is prepared by mixing a solution containing the desired inorganic salts, vitamins, carbohydrates and growth regulators, with Transfergel, at 1.5-2% (w/v). Transfergel hydrates and thickens immediately upon addition to liquid. For this reason, it requires considerable stirring or mixing to completely dissolve. The resulting gel is of a highly viscous (almost syrupy) nature which does not thicken significantly after autoclaving. Because of its extreme viscosity, Transfergel should be autoclaved in a container approximately four times its volume (e.g. 500 ml in a 2 L flask) to avoid potential boil-over.
    The pH of the gel should be adjusted prior to autoclaving. A pH of 6.5 before autoclaving will lower to 5.5-6.0 after autoclaving for 20 minutes. Tissue culture propagules are placed in the gel under aseptic conditions.

 


 

Agar Products Description and Use

Prod.
No.
 
Desc.
 
Use
Gelling
Temp 0C
pH
at 1.5%
Ash
Content
Typical ICP Analysis (%)
Ca Mg K P Na
A 1296 Agar General purpose agar for most research and production needs.
Use at 6-12 g/L.
32-35 7.0-7.5 2-5% 0.30 0.10 0.01 0.01 0.5
A 6686 Agar Bacteriological Bacteriological grade agar for most microbiological work.
Use at 6-12 g/L.
32-39 6.5-7.5 3-7% 0.17 0.09 0.80 3.1
A 4550 Agar Type A General purpose agar; good bacteriological grade agar.
Use at 6-12 g/L.
26-28 7.2-7.7 5-6% 0.01 0.01 0.1 0.17 1.8
A 4675 Agar Type E General purpose agar;
Use at 5-10 g/L.
26-28 7.5-8.0 3-4% 0.02 0.02 0.07 0.13 1.2
A 4800 Agar Type M General purpose agar;
Use at 5-11 g/L.
34-36 7.0-7.5 3-6% 0.09 0.14 0.07 0.01 1.4
A 9799 Agar High Gel Strength Use when firmer gel is required.
Use at 4-8 g/L.
34-37 6.5-7.0 3-4% 0.03 0.00 0.07 0.09 0.72
A 7921 Agar Purified High purity agar for research and protoplast culture.
Use at 6-12 g/L.
30-35 6.5-7.0 2.0% 0.02 0.01 0.01 0.01 0.35
A 8678 Agar Washed High-purity agar for research and protoplast culture. Prepared through a series of purified water and solvent washes.
Use at 6-10 g/L.
25-27 7.0-7.5 2.2% 0.15 0.08 0.38
A 6560 Agarose A low-gelling temperature agarose with excellent clarity.
Use at 6-10 g/L.
26-30 7.0-8.0 <1%
A 0682 Alginic Acid A polyuronic acid which forms gels. Excellent product for plating out protoplast cultures or cell suspension cultures. 0.17 0.01 2.3 10.1
A 3301 Agargel™ A blend of agar and Phytagel offering the positive aspects of both products. Produces a semi-clear gel.
Use at 3.5-5.0 g/L.
26-28 7.2-7.7 4-5% 0.25 0.06 0.04 0.08 1.05
P 8169 Phytagel™ Produces a clear, colorless high-strength gel.
Use at 1.5-2.5 g/L.
27-31 6.5-7.0 9.5% 0.85 0.35 1.70 0.15 0.45

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