C.N.R. - Istituto Biosintesi Vegetali

Embryogenic cultures: release and development in liquid medium of proembryonic structures --L.A. Manzocchi, G. Giovinazzo, S. Castelli
Considerable progress has been made recently in induction of embryogenic suspension cultures, from which it is now possible to obtain protoplasts capable of regenerating plants (reviewed in Shillito et al., Bio/Technology 7: 581, 1989). Maize does not seem therefore as reluctant to in vitro manipulation as previously believed, and it is reasonable to expect the possibility to develop for this plant in vitro cell systems able to carry on a developmental pattern in controlled conditions, analogous to the ones existing for better studied species such as carrot (Sung et al., Plant Mol. Biol. Rep. 2:216,1984).

With this aim, we have induced in summer 1989 highly embryogenic friable calli by plating immature embryos of A188 xW64A cross (from Ist. Sper. Cereal., Sez. Bergamo) on MS (Murashige and Skoog, Physiol. Plant. 15:473, 1962) agar medium as modified by Armstrong and Green (Planta 164:207, 1985); calli were subcultured every 15 days on N6 medium (Chu et al., Scientia Sinica 18:659, 1975) containing 2 mg/l 2,4-D. Incubation of calli in liquid media on a rotatory shaker resulted in a rapid release of cells, cell aggregates and proembryonic structures; although the study of their proliferation and differentiation is still in progress, we think it worthwhile to report some preliminary data.

Several media (B5, N6, MS) were tested to optimize cell release from embryogenic calli; the best results were obtained with a medium composed of MS salts (3/4 strength), 30 g/l sucrose, 1 g/l proline, 100 mg/l asparagine, 200 mg/l inositol, 1mg/l niacin, 0.5 mg/l thiamine, 0.2 mg/l pyridoxine, 0.2 mg/l Ca panthothenate, 1.5 mg/l 2,4-D (L medium). Medium with released cells can be taken from the flasks every five days and replaced with fresh medium, allowing proliferation and further release for long periods.

Three cell populations were separated by differential sieving through stainless steel filters, and cultivated in medium L (containing 1.5 mg/l 2,4-D), N6(+) with 2 mg/l 2,4-D, and N6(-) without auxin.

A first fraction, which passed through a 63 um sieve, was composed essentially of single elongated cells, and a few small clusters of dense cells; they divide slowly, only in media with 2,4-D, and no further differentiation is observed.

A second population was separated between 63 and 125 um: it still contains single elongated cells, but is predominantly composed of small round-shaped dense cells (as described by Shillito et al. for rapidly growing suspension cultures), originating compact clusters especially when cultivated in N6(+).

The fraction comprised between 125 and 500 um is represented by a heterogeneous population of compact cell clusters and larger proembryonic structures, either round-shaped or characteristically elongated or resembling the torpedo stage of carrot embryonic development. Cultivation of this fraction in N6(-) does not stimulate either cellular division or increase in the number of proembryonic structures, a few of which develop not to complete embryos but to structures similar to roots. In N6(+) and L media there is apparently an evolution from cell clusters to large proembryonic structures: these, in turn, do not differentiate further, but, after some days, begin to proliferate cells from their surface evolving to small calli.

Although very preliminary, our data seem to indicate that compact cell clusters larger than 125 um released from embryogenic calli are possibly analogous to the proembryo bodies of better described embryogenic systems such as carrot; it is possibly feasible to separate them from other cell populations and to obtain their evolution in culture to embryonic structures; further studies on hormonal balances are necessary to allow in vitro expression of a complete embryonic developmental pattern.

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