Gelling Agents – Plant Tissue Culture Protocol

• Preparation of Alginate, Agargel and Phytagel
  
• 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. We have expanded our 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 and Phytagel

Alginate Preparation (Product No. A0682)
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. A3301)
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. P8169)
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.

Agar Products Description and Use

Product No.	Description	Use	Gelling Temp 0C	pH at 1.5%	Ash Content	Typical ICP Analysis (%)
						Ca	Mg	K	P	Na
A1296	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
A6686	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
A4550	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
A4675	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
A4800	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
A9799	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
A7921	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
A8678	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
A9045	Agarose	A low-gelling temperature agarose with excellent clarity. Use at 6-10 g/L.	26-30	7.0-8.0	<1%	—	—	—	—	—
A0682	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
A3301	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
P8169	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