Plant regeneration from tissue culture

Methodology for regeneration of complete plants from maize tissue cultures of immature embryos was established by Green & Phillips (Crop Sci. 15:417-421, 1975). A report by Harms, Lorz & Potrykus (Z. Pflanzenzucht. 77:237-259, 1976) describes regeneration from tissue culture using 4-day seedlings. We wish to report regeneration of plants from maize tissue cultures obtained from 14-day seedlings.

Cultures were initiated from 14-day seedlings of the A188 inbred line. Seed was germinated and plants were grown in test tubes (2.5 x 10 cm) on White's medium for 4 days in the dark at 27 C, then in daylight for 10 days more. About 2-3 mm cross sections of stem, between and including the mesocotyl and the apical meristem, were placed basal side down on an agar-solidified culture medium in plastic petri dishes (100 x 25 mm) prepared as described by Green & Phillips (1975) and containing 2 mg/I of 2,4-dichlorophenoxyacetic acid (2,4-D). The explants were incubated in the dark at 27 C for one month, during which time callus formed. The colorless callus was subcultured on the same medium in the dark for 5 days and then in the light as previously described (Gordon, Robarts & Rice, MGCNL 51:79-80) for regeneration of maize tissue cultures. In a few days, areas of the callus greened, and within 6 days leaf primordia could be observed. Subcultures were made onto the same medium for 14 days, then onto a medium with 0.5 mg/l 2,4-D. Shoots began to appear after 9 days. The shoots were transferred to plates without 2,4-D until roots were observed (after approximately 14 days). Plantlets (1 cm high) were placed onto the same medium in storage jars (100 x 80 mm) for 3 weeks or until the plantlets were 8-10 cm high. They were then transferred to Jiffy peat pellets and subsequently to soil in the greenhouse.

The tissue culture "clone" is similar morphologically to the "clones" obtained from immature embryo culture and is maintained on the basic medium with 1 mg/l 2,4-D. Twenty plantlets and 5 plants have been obtained from this particular "clone" (Work done at Pfizer Central Research, Eastern Point Road, Groton, CT 06340).

Phillip N. Gordon


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