A method for regenerating complete plants from maize tissue cultures has been reported by Green and Phillips (Crop Sci. 15:417-421, 1975). Tissue cultures initiated from immature embryos from 4 of the 5 inbred lines tested subsequently formed plantlets. The proportion of embryos that formed differentiating cultures was as high as 20% in some cases, and approximately 200 complete plants were differentiated. Other efforts to regenerate plants from maize tissue cultures have been much less successful (Gresshoff and Doy, Aust. J. Biol. Sci. 26:505-508, 1973; Rafaill, this News Letter 50:84-86, 1976). Attempts to reproduce the findings described by Green and Phillips have apparently been unsuccessful according to both published (Rafaill, 1976) and unpublished accounts. In this brief note we report the regeneration of plants from maize tissue cultures using methods that reproduce as faithfully as possible the procedures and conditions described by Green and Phillips.
A188 seeds obtained from C. E. Green were grown in a greenhouse during the spring and summer of 1976. Immature embryos 1-2 mm in length were excised 14 to 20 days after self-pollination. The embryos were placed on an agar-solidified culture medium prepared as described by Green and Phillips and containing 2 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). The flat embryonic axis was placed in contact with the medium. Sixty excised embryos were incubated under conditions similar to those described by Green and Phillips with a 16/8 hr photoperiod from fluorescent cool-white VHO tubes (Sylvania) with an intensity of 275-400 ft-c at 26-28 C. Tissue from under the scutellum of greater than 90% of the embryos proliferated and grew upwards eventually surrounding the scutellum. The tissue was pale white initially and was quite organized throughout the culture interval. After 4-5 weeks of incubation approximately 60% of the embryos gave rise to a number of "scutellar-like" structures, both colorless and green, that arched inward toward the scutellum of the excised embryo. As soon as shoot formation occurred, the nascent shoots (as many as 10 per embryo culture) were transferred to a medium containing 0.25 mg/l 2,4-D where they continued to develop. When the leaves were 1-4 cm in length, the shoots were transferred to a medium without 2,4-D, where roots formed within 3-4 days. When the plantlets were about 15 cm high and had 4-6 leaves and an extensive primary root system, they were transplanted to 6 cm peat pots containing a 1:1 peat-perlite mixture. Immediately after transplanting the plants were exposed to continuous light for 24 hr followed by the normal 16/8 hr photoperiod. The plants were maintained at 75% relative humidity at 27-28 C in light and watered with 25% MS salts (Murashige and Skoog, Physiol. Plant. 15:473-497, 1962). More than 85% of the plantlets survived the transfer to soil.
Although we have no data on the effect of subculture on the differentiating clones, this report substantiates many features of the method described by Green and Phillips. The results also emphasize the need for adherence to experimental details. Several modifications of this protocol resulted in tissue cultures that have not differentiated shoots.
Phillip N. Gordon, Masha M. Robarts and Thomas B. Rice
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