The transfer of small shoot apices onto rootstocks (termed micrografting), can be carried out in vivo or in vitro. Navarro (1988) lists four uses which have been found for the technique: • Obtaining plants free from specific virus and virus-like diseases;
• A method for separating virus and virus-like organisms in mixed infections;
• For studying graft incompatibility between scions and rootstocks, and the histological and physiological aspects of grafting;
• A minimum risk method for importing plant material through quarantine.
Micrografting is thus indirectly useful in micropropagation: the necessary techniques are usually too time consuming and the proportion of successful takes is generally too low, for it to be of direct application.
The rootstocks used for micrografting are commonly newly-germinated seedlings, but it is also possible to use rooted cuttings or micropropagated shoots. Before micrografting can be carried out, it is necessary to prepare suitable rootstock material. When seedling rootstocks are used, and all stages of grafting are conducted in vitro, seeds are surface sterilised and germinated aseptically in vessels containing nutrient salts (e.g. those of MS medium). The seedlings may be supported on agar medium or on a porous substrate, such as sterile vermiculite, which allows the growth of a branched root system. Micrografting is then effected by cutting off the tops of the seedling rootstocks and placing small shoot apices onto the exposed surface. When grafts are successful, rootstock and scion grow together to produce a plant. It is usually necessary to examine freshly grafted seedlings on a regular basis and remove any adventitious shoot arising on or below the graft union.
Shoot tips to be grafted onto seedling rootstocks (i.e. the scions) are carefully excised from preferred plant material. In Citrus there has been most success when scions have been placed directly in one of two positions:
• into inverted T-shaped incisions immediately below the cut surface of a decapitated rootstock, or,
• onto the cambium layer or vascular ring of the cut surface (Fig 2.7).
Shoot or meristem tips intended for grafting can be taken from apical or axillary buds of actively growing shoots in the greenhouse or field, or may be removed from shoots growing in vitro. Once transferred, the survival of micrografted apices is partly dependent on their size. Very small apices must be used for virus elimination, making the technique difficult and unreliable. Tips 0.1-0.2 mm in length have been grafted for virus elimination from vines; with peach, slightly larger apices (0.5-1 mm) have been employed. The excision and transfer of very small shoot apices requires precise micromanipulation under a binocular microscope. If large shoot apices are to be grafted, their bases are often cut into a wedge which is then inserted into a vertical cut on the rootstock (Fig 2.8).
Several techniques have been found to increase the proportion of successful graft unions:
• Tissue blackening, which commonly results in the death of very small scions, can be reduced by soaking explants in an anti-oxidant solution, and/or placing a drop of solution onto the severed rootstock immediately before inserting the scion. A solution of 2 g/l sodium diethyldithiocarbamate (DIECA) has been used for this purpose. Navarro (1988) advocates rapid manipulations to prevent phenolic oxidation and says that it is more effective than anti-oxidants.
• Apices to be grafted may be placed either directly onto a decapitated rootstock, or cultured for a short period before being transferred. There is often a better 'take' and more rapid growth if they are pre-cultured for a short while supported on paper above an MS mineral salt medium containing growth regulators. Jonard et al. (1983) found that adding a cytokinin to the medium (e.g. 0.1 mg/l zeatin if the apex is cultured for 48 h; 0.01 mg/l zeatin if the culture is continued for 48-240 h) was particularly effective in encouraging the rapid formation of leafy shoots once the graft has been made. An alternative is to place scion shoot tips into a growth regulator solution for a short period before grafting: a 5-10 minute immersion in either 10 mg/l 2, 4-D or 1 mg/l BA, doubled the number of successful micrografts of Citrus (Edriss and Burger, 1984). Starrantino and Caruso (1988) got a greater percentage of viable grafts when they dipped both shoot tips and the cut apex of young rootstocks in 0.5 mg/l BA for 20 min before the two were united. Yet another method is to place cytokinin (e.g. 2mg/l BA or, for peach, 10 mg/l zeatin) in a drop of water or agar gel between the scion and the rootstock.
• A greater proportion of graft unions may result from growing isolated meristem tips to a larger size before they are implanted. Isolated scion tips of peach have been cultured in vitro by the initial stages of meristem tip culture for a period of about two weeks until they have grown from 0.5-1 mm to ca. 10 mm.
• Desiccation is a major cause of the failure of graft unions. To prevent drying, Pliego-Alfaro and Murashige (1987) applied a layer of moist nutrient agar gel to connect the graft area with the medium. The gel had to be progressively removed from the top downwards during weeks 1-3 after grafting, or poor unions resulted.
When, as has been most common, micrografting has been carried out in vitro, considerable care needs to be taken over transferring grafted plants to the external environment. However, several authors have found that sterility is not necessary and that shoot apices can be united onto rootstocks grown in vivo. The proportion of completed grafts may be less than under aseptic conditions, but problems with eventual transfer are eliminated. A variety of scion material has been utilised for in vivo grafting, including directly-excised 0.1-0.2 mm tips of Citrus limon, and meristem-cultured scion tips of peach. Once again, graft unions may be improved if cut surfaces are anointed with a DIECA (1g/l) plus cytokinin (10 g/l) solution (Jonard et al., 1983). Plants are probably best kept in a growth room for a period after grafting, and desiccation of the graft union prevented by enclosing each plant in a plastic bag or by placing an elastic strip around the graft.
Grafting mature shoots of woody perennials onto juvenile rootstocks is known to induce juvenile characteristics in the resulting shoots, particularly if it is repeated successively (Chapter 11). Half of the plants resulting from micrografting adult lateral buds of avocado onto seedling rootstock were found to have some juvenile symptoms (Pliego-Alfaro and Murashige, 1987). But in Citrus, micrografting does not seem to induce juvenile characteristics, providing shoot tips are taken from an adult source: plants are thornless and come into flower rapidly (Navarro, 1988).
From having been developed as a method of producing virus-tested Citrus, micrografting is now widely used for the improvement of plants of this and related genera. It has also been practised on a wide range of other plants, primarily for virus-elimination (see reviews by Jonard et al., 1983; Burger, 1985 and Navarro, 1988)
General reviews on micropropagation have been written by: Cassells (2005), Harney (1982), Holdgate (1977), Hussey (1978a, 1983), Jain et al. (2006), Lane (1982), Murashige (1974), and Rout and Jain (2004)
Books devoted completely or in great part to in vitro propagation are: Ahuja (1993), Conger (1981), Debergh and Zimmerman (1991), de Fossard (1981), Hall (1999), Hermann (1991-2006), Hvoslef-Eide and Preil (2005), Pierik (1997), Karata and Kozai (1992), Kyte (1983), Lumsden et al. (1994), Pennell (1987), Wetherell (1982), and Razdan (2003). A number of volumes of Acta Horticulturae deals specifically with micropropagation (Volumes 212, 225, 319, 530 and 616).
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