Scutella of two diploid hybrids (77E-2, 77D-1) and one diploid inbred (sul/sul), and sections of apical meristems of one diploid inbred (77-01) were grown in vitro on modified MS (Murashige and Skoog, 1962) medium with 2 mg/l 2,4-D. After 10 days of culturing, more than 50% of the 252 explants grew into callus, irrespective of being illuminated or not. One month after initial growth of explants, callus pieces were excised and reinoculated into freshly prepared medium consisting of the same nutrients for continued culturing. One to two weeks after transfer, many calli developed into root-like structures. Particularly those which were originally kept in light manifested profuse growth from primitive organ differentiations to teratomous expansions. These organ differentiations (root-like) soon sent out small branches with hairy appearance. It was also observed that some of the organ differentiations later cracked and a greening area exposed from inside. In about six weeks of subculturing, approximately 50 pieces of these developing calli were again cut off and reinoculated into regeneration medium prepared by substituting NAA for 2,4-D (2 mg/l). The rest of the calli were again subcultured on the same maintenance medium. Being on the regeneration medium for one week, these callus pieces began changing color from creamy-white to light-brown. In contrast to the other maintenance culture, their rate of growth was strikingly slow and their organ, developments ceased. The original greening areas gradually became brown with compact instead of friable texture. Four to five weeks later, fine root-like organs appeared in some cultures and grew deep into the agar medium. But plantlets were not found. Even though regular transfers to newly prepared media for regeneration at four to six-week intervals were made for more than a half year, no regenerated plantlets were obtained.
Callus pieces from the maintenance cultures were excised and fixed for ordinary light microscopic examinations. It was found that cells in the friable and creamy-white calli were monstrous and extremely long, about 20 times longer than those in the normal tissues, while those in the hard and brown calli were round or nearly so, with enormous relative increase in size. However, due to limited amount of material available, the study in karyotype stability in various calli was still incomplete. Furthermore, controlled regeneration from maize calli into shoots and plantlets remains a problem to be solved. Maintenance of long-term tissue cultures of maize, nevertheless, can be carried out successfully.
Y. C. Ting, Anne Boyer and Mary J. Plante
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