PROLIFERATION AND PLANT REGENERATION FROM THE NODAL REGION OF ZEA MAYS L. (MAIZE, GRAMINEAE) EMBRYOS

Immature embryos of Zea mays L. (maize), cultured on Murashige and Skoog's medium containing 2,4-dichlorophenoxyacetic acid (2,4-D, 0.25-1.0 mg/i) and sucrose (3, 6 and 12%), produced a narrow ridge of tissue in the nodal region of the embryo axis, opposite the scutellum. Only those embryos which were placed with the scutellum in contact with medium showed such a response. The ridge of tissue further proliferated and formed a compact and opaque callus that appeared similar to the scutellar callus. Morphogenesis took place on media containing low concentrations of both 2,4-D and sucrose. Plant regeneration was achieved and is interpreted to take place by the precocious germination of somatic embryos. It is suggested that the ridge of tissue formed at the node may represent an evolutionarily extinct epiblast of the maize embryo and may be cotyledonary in nature. IMMATURE EMBRYOS of Zea mays L. (maize) produce a morphogenic callus when cultured on nutrient media containing 2,4-dichlorophenoxyacetic acid (2,4-D). The callus is formed by proliferation of the scutellum at the coleorhizal end. Such cultures give rise to plants either via the organization of shoot buds (Green and Phillips, 1975; Springer, Green and Kohn, 1979) or by the formation of somatic embryos (Lu, Vasil and Ozias-Akins, 1982). The shootroot axis of the cultured embryos may also proliferate but is not known to produce shootforming or embryogenic callus. In this report we describe the regeneration of plants from a morphogenic callus tissue derived from proliferation at the scutellar node region of immature embryos of maize. MATERIALS AND METHODS-Immature embryos (1-1.5 mm) were removed aseptically from sterilized cobs of several cultivars (Asgrow Rx 1 2, Coker 16, Coker 22, Dekalb XL 80, Dekalb XL 82, Florida Staysweet, Funks G 4864, Funks G 4507 A, Pioneer 3030, Pioneer 3320, Silver Queen). The dehusked cobs were sterilized by treatment with 70% ethanol (1 min), followed by 20% Clorox (10-15 min), and then rinsed several times with sterile water. In the first set of experiments almost all of the 6,000 embryos cultured were placed with the embryo axis in contact with the medium (for further details see Lu, Vasil and Vasil, 1983). Only in a few cases was the scutellum in contact I Received for publication 12 October 1982; accepted 23 February 1983. Florida Agriculture Experiment Station Journal Series No. 4186. with the medium. In the next series of experiments one out of every six embryos (150/ 900) in each Petri dish was oriented deliberately in such a fashion that the embryo axis was exposed. Each Petri dish contained 5 ml of Murashige and Skoog's (1962) mineral salts and vitamins supplemented with thiamine hydrochloride (0.4 mg/l), inositol (100 mg/l), coconut milk (5%), and 2,4-D (0.25, 0.5, 1, and 2 mg/l). Three concentrations of sucrose (3, 6, and 12%) were used with each 2,4-D concentration. Bacto-agar (0.8%) was added after adjusting the pH to 5.8. The medium was sterilized by autoclaving at 15 psi, 121 C for 17 min. The cultures were incubated in a growth chamber in the dark at 27 C. Tissues for histological examination were fixed in formalin-acetic acid-ethanol, dehydrated in a tertiary-butanol series and embedded in Paraplast. Sections (7 gm) were stained with hematoxylin and safranin. For scanning electron microscopy the samples were fixed in 2.50% glutaraldehyde, post-fixed in 1% osmium tetroxide, and dehydrated in ethanol. After critical point drying, the samples were coated with gold and photographed in a Hitachi S-450 scanning electron microscope. RESULTS-The immature embryos at the time of culture possessed a shoot-root axis that was approximately half the length of the scutellum (Fig. 1, 2). The coleoptile and the scutellum were attached to the embryo axis at the same level and a mesocotyl was not developed (Fig. 2). Within 4 days after culture, a swollen ridge of tissue developed on the shoot-root axis at the scutellar node, opposite the scutellar attachment, but only in embryos that were