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Fig. 1.6 Ovule and embryo culture.

The discovery that roots could be grown apart from shoot tissue was one of the first significant developments of modern tissue culture science. Root culture initially attracted a great deal of attention from research workers and the roots of many different species of plants were cultured successfully (see the comprehensive reviews of Street, 1954, 1957, 1969; and Butcher and Street, 1964).

Plants fall generally into three categories with regard to the ease with which their roots can be cultured. There are some species such as clover, Datura, tomato and Citrus, where isolated roots can be grown for long periods of time, some seemingly, indefinitely (Said and Murashige, 1979) providing regular subcultures are made. In many woody species, roots have not been grown at all successfully in isolated cultures. In other species such as pea, flax and wheat, roots can be cultured for long periods but ultimately growth declines or insufficient lateral roots are produced to provide explants for subculture.

The inability to maintain isolated root cultures is due to an induced meristematic dormancy or 'senescence', related to the length of time that the roots have been growing in vitro. Transferring dormant meristems to fresh medium does not promote regrowth, possibly due to the accumulation of naturally-occurring auxinic growth substances at the root apex. The addition of so-called anti-auxin, or cytokinin growth regulators can often prolong active growth of root cultures, whereas placing auxins or gibberellic acid in the growth medium, causes it to cease more rapidly. Cultures, which cannot be maintained by transferring root apices, can sometimes be continued if newly-initiated lateral root meristems are used as secondary explants instead.

Fig. 1.7 Methods of root culture.

Isolated plant roots can usually be cultured on relatively simple media such as White (1954) containing 2% sucrose. Liquid media are preferable, as growth in or on a solid medium is slower. This is presumably because salts are less readily available to the roots from a solidified medium and oxygen availability may be restricted. Although roots will accept a mixed nitrate/ammonium source, they will not usually grow on ammonium nitrogen alone. Species, and even varieties or strains, of plants, are found to differ in their requirement for growth regulators, particularly for auxins, in the root culture medium.

Isolated root cultures have been employed for a number of different research purposes. They have been particularly valuable in the study of nematode infections and provide a method by which these parasites can be cultured in aseptic conditions. Root cultures may also be used to grow beneficial mycorrhizal fungi, and to study the process of root nodulation with nitrogen-fixing Rhizobium bacteria in leguminous plants. For the latter purpose, various special adaptations of standard techniques have been adopted to allow roots to become established in a nitrate-free medium (Raggio et al, 1957; Torrey, 1963).

Unlike some other cultured tissues, root cultures exhibit a high degree of genetic stability (see Chapter 10). It has therefore been suggested that root cultures could afford one means of storing the germplasm of certain species (see Volume 2). For suitable species, root cultures can provide a convenient source of explant material for the micropropagation of plants, but they will only be useful in micropropagation if shoots can be regenerated from roots. There are however, several ways in which this can be done, although they are likely to be effective in only a small number of plant genera which have a natural tendency to produce suckers, or new shoots from whole or severed roots:

• From direct adventitious shoots;

• From shoots or embryos originating indirectly on root callus;

• By conversion of the apical root meristem to a shoot meristem.

Adventitious shoots form readily on the severed roots of some plant species, and root cuttings are employed by horticulturists to increase plants in vivo (see, for example, the review by Hodge, 1986). Shoot regeneration from roots has not been widely used as a method of micropropagation, even though direct shoot regeneration from roots has been observed in vitro on many plants. Sections of fleshy roots used as primary explants are especially likely to form new shoots. Adventitious shoots always develop at the proximal end of a root section while, as a rule, new roots are produced from the distal end. Isolated root cultures would be useful in micropropagation if shoots could be induced to form directly upon them. Unfortunately plants seem to have a high degree of genetic specificity in their capacity to produce shoots directly on isolated root cultures. Shoot induction often occurs after the addition of a cytokinin to the medium. Seeliger (1956) obtained shoot buds on cultured roots of Robinia pseudoacacia and Torrey (1958), shoot buds on root cultures of Convolvulus. Direct shoot formation was induced in three species of Nicotiana and on Solanum melongena by Zelcer et al. (1983) but in N. tabacum and N. petunoides shoots were only obtained after callus formed on the roots. The most optimistic report we have seen comes from Mudge et al. (1986), who thought that the shoot formation, which they could induce in raspberry root cultures would provide a convenient and labour-saving method of multiplying this plant in vitro.

Plants may also be regenerated from root-derived callus of some species e.g. tomato (Norton and Boll, 1954); Isatis tinctoria (Danckwardt-Lilliestrom, 1957); Atropa belladonna (Thomas and Street, 1972). Embryogenesis, leading to the formation of protocorm-like bodies, occurs in the callus derived from the root tips of certain orchids e.g. Catasetum trulla x Catasetum (Kerbauy, 1984a); Epidendrum obrienianum (Stewart and Button, 1978); Oncidium varicosum (Kerbauy, 1984b).

Changing the determined nature of a root meristem, so that it is induced to produce a shoot instead of a root, is a very rare event but has been noted to occur in vitro in the orchid Vanilla planifolia. The quiescent centre of cultured root tip meristems was changed into a shoot meristem so that cultured root tips grew to produce plantlets or multiple shoots (Philip and Nainar, 1986). Ballade (1971) maintained that newly initiated root initials, arising from single nodes of Nasturtium officinale, could be made to develop into shoot meristems by placing a crystal of kinetin on each explant which was then transferred to a medium containing 0.05% glucose.

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