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The Components of Plant Tissue Culture Media II
1986). Chauvin et al. (1999) found that regeneration from cultures of tulip, gladiolus and tobacco shoots was possible in the presence of 200 mg l–1 kanamycin, whereas in several other gelling agents a concentration of 100 mg l–1 inhibited regeneration. 6.2.6. Pectins
A mixture of pectin and agar can be a less expensive substitute for agar. For example, a semisolid medium consisting of 0.2% agar plus 0.8-1.0% pectin, was employed for shoot culture of strawberry and some other plants (Zimmerman, 1979). 6.2.7. Other gelling agents
Battachary et al. (1994) found sago (from Metroxylon sagu) and isubgol (from Plantago ovata) were satisfactory substitutes for agar at, respectively, one eighth and one tenth of the cost of Sigma purified agar A7921. 6.3. POROUS SUPPORTS
Aeration of the tissues on a porous substrate is usually better than it would be in agar or static liquid. Chin et al. (1988) used a buoyant polypropylene membrane floated on top of a liquid medium to culture cells and protoplasts of Asparagus. The membrane (Celgard 3500, Questar Corp., Charlotte, N.C.) has a pore size of 0.04 mm and is autoclavable. Conner and Meredith (1984) found that cells grew more rapidly on filter papers laid over polyurethane foam pads saturated with medium than on agar. Young et al. (1991) supported shoots of tomato over liquid culture medium on a floating microporous polypropylene membrane and entrained the growing shoots through polypropylene netting. They reported opportunities for the development of this method for mechanisation by mass handling. Membrane rafts were also used by Teng (1997) and Watad et al. (1995,1996). Cheng and Voqui (1977) and Cheng (1978) used polyester fleece to support cultures of Douglas fir that were irrigated with liquid media in Petri dishes. Plantlets that were regenerated from cotyledon explants were cultured on 3 mm-thick fabric. When a protoplast suspension was dispersed over 0.5 mmthick fabric, numerous colonies were produced in 12 days, whereas in the absence of the support, cell colonies failed to proliferate beyond the 20 cell stage. A major advantage in using this type of fabric support is that media can be changed simply and quickly without disturbing the tissues. The system has also been used for protoplast culture of other plants (e.g. by Russell and McCown, 1986).
Heller and Gautheret (1949) found that tissues could be satisfactorily cultured on pieces of ashless filter paper moistened by contact with liquid medium. Very small explants, such as meristem tips, that might be lost if placed in a rotated or agitated liquid medium, can be successfully cultured if placed on an M-shaped strip of filter paper (sometimes called a ‘Heller’ support). When the folded paper is placed in a tube of liquid medium, the side arms act as wicks (Goodwin, 1966). This method of support ensures excellent tissue aeration but the extra time required for preparation and insertion has meant that paper wicks are only used for special purposes such as the initial cultural stages of single small explants which are otherwise difficult to establish. Whether explants grow best on agar or on filter paper supports, varies from one species of plant to another. Davis et al. (1977) found that carnation shoot tips grew less well on filter paper bridges than on 0.6% agar but axillary bud explants of Leucospermum survived on bridges but not on agar. Paper was also used in the construction of plugs (marketed by Ilacon Ltd, Tonbridge, UK TN9 1NR) known as Sorbarods (Roberts and Smith, 1990). These are cylindrical (20 mm in length and 18 mm in diameter) and consist of cold-crimped cellulose, longitudinally folded, wrapped in cellulose paper. The plug has a porosity (total volume – volume of cellulose) of 94.2% and high capillarity, so that the culture medium is efficiently drawn up into the plug, leaving the sides of the plug in direct contact with air. Roots permeate the plugs and are protected by the cellulose during transfer to soil. Plantlets of chrysanthemum in Sorbarods formed longer stems, larger leaves, more roots, and developed greater fresh mass, dry weights and fresh to dry mass ratios than plantlets in agar-solidified medium (Roberts and Smith, 1990). The greater fresh to dry mass ratio indicates that contact with liquid medium led to greater hydration of tissues. This was subsequently controlled by the inclusion of a growth retardant, paclobutrazol (1 mg l–1), in the culture medium (Smith et al., 1990a). Other porous materials that have been used to support plant growth include rockwool (Woodward et al., 1991; Tanaka et al., 1991), polyurethane foam (Gutman and Shiryaeva, 1980; Scherer et al., 1988), vermiculite (Kirdmanee et al., 1995), a mixture of vermiculite and Gelrite (Jay-Allemand et al., 1992). Afreen-Zobayed et al. (2000) cultured sweet potato, on sugar-free medium in autotrophic conditions, on mixtures of paper pulp and vermiculite in various proportions. Optimal growth was obtained on a mixture containing 70% paper pulp. On this