For the obtaining of plant somaclonal variants the application of long-term embryogenic callus culture is preferable, because large numbers of genetic mutations during cultivation are accumulated. In this report the obtaining, stabilization and several peculiarities of five-year old embryogenic callus culture of maize inbred DK675 are described. In the spring of 1993 the culture was induced from immature embryos (length 1.0-2.0 mm) on the medium containing the inorganic nutrients of N6 medium, vitamins of D medium (Duncan D.R. et al., Planta, 165: 322-332, 1985), L-proline (690 mg/l), myo-inositol (100 mg/l), casein hydrolisate (100 mg/l), silver nitrate (10 mg/l), sucrose (20 mg/l), 2,4-D (1 mg/l), agar (7 g/l). The explants were grown in the dark at 25-27 C and were transferred on fresh medium every 12-20 days.

The frequency of embryogenic callus formation was 63.0- 77.5%. The embryoids were observed on the callus surface at 10-14 days after induction as globular structures, situated either one by one or in groups. The first 60 days calli were grown on medium with initial content of 2,4-D. Under such conditions the signs of differentiation and regeneration were observed on the tissue surface. Therefore after 2 months of cultivation the level of 2,4-D was elevated to 1.25 mg/l, and after the next 2 months it was increased to 1.45 mg/l. Under these conditions the callus has been growing all the following period. The callus produced plantlets very intensively on media with 0.1 mg/l 2,4-D and without hormone.

The callus proliferated more actively at the spring-summer period of growth than at the autumn-winter one. In addition, it showed better growth when callus pieces were placed very densely, near each other.

To the end of the growing cycle (20 days) the callus included white compact scutellum-like bodies, embryoids, primordia and numerous sectors of friable undifferentiated nonmucilagenous tissue. It collapsed easily into small pieces by slight mechanical influence or self-voluntarily. These pieces were used for propagation of the callus. In the spring of 1997 we twice determined the rate of growth of culture. The fresh weight of the callus increased about 2 times for the 20 days of growing.

As the result of propagation and growing we obtained a large amount of callus material which has been growing about five years without loss of regeneration capacity. The material is unique because it resulted from one embryo and is of interest for the researching of somaclonal variability, cell selection and genetic engineering.
 
 
 

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