Excised immature embryos placed on tissue culture initiation medium A (see previous article) showed, after one month of culture, an extensive callus proliferation from the scutellum. In previous observations we have noticed that when such cultures were placed on medium with 0.5 mg 2,4-D/liter or without 2,4-D, some morphogenic events occur. In order to examine such cultures, the calli were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and then rinsed with buffer. After acetone dehydration, the samples were critical point dried and coated with gold-palladium, and scanned at 15 KL on a Jeol JSM-U3 Scanning Electron Microscope (SEM).
Although tissue cultures appeared to the naked eye to grow as true callus, SEM examination showed this growth to be a mix of isodiametric and elongated cells (Fig. 1--see opposite page). Both unorganized and organized callus cells appeared to be the first step of culture initiation observed in our material. As Springer et al. (Protoplasma 101:269-281, 1979) and Mott and Cure (Physiol. Plant 43:139-145, 1978) pointed out, maize callus cultures obtained through either immature embryos or excised mature embryos in basal media with 2,4-D are not composed of uniform or undifferentiated cells. We have observed in our cultures variations in cell size, shape and cell wall characteristics (Fig. 2). Extensive regions of the callus surface also showed a cuticular wax-like surface (Fig. 1).
On the 20th day of culture, large regions of callus surface exhibited morphological modifications. The occurrence of specialized growing points appeared to be the second developmental step in our primary cultures. Numerous primordia-like protuberances appeared which could eventually develop into roots or shoots (Fig. 3). Numerous growing points continued to appear at the end of one month of culture.
Random differentiation of xylem occurs in primary cultures (Fig. 4). Also, distinct xylem development and secondary wall growth occur in the primordia-like protuberances (Fig. 5). We have not observed embryogenesis in the callus cultures obtained in medium A. All the morphogenic events observed appeared to be organogenic in origin.
The observations made here indicate a multicellular origin of these phenomena (Fig. 6). The regeneration of plants showing higher somaclonal variation (Larkin and Scowcroft, TAG, 60:197-214, 1981) could be explained in part by a multicellular origin of such plants. This fact would be very important for increasing the genetic variability necessary to any plant breeding enterprise.
Miguel Angel Rapela and Jorge Herkovits*
*Instituto de Biologia de la Reproduccion y Desarrollo
Embrionario, UNLZ, Lomas de Zamora, Argentina
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