Established procedures for maintaining regenerable maize tissue cultures involve visually selecting scutellar-like tissues for subculture and discarding other more abundant tissue types. The latter tissues, if tested on growth regulator-free medium, usually exhibit rhizogenesis and are unable to form embryos or shoots. It was, therefore, unexpected to observe the formation of a somatic embryo on a well-differentiated root in tissue culture.
The culture was initiated from the apical meristem of a seedling of A188. It was totipotent with characteristic scutellar-like tissues. These tissues were subcultured onto solidified medium (macronutrient and micronutrient salts of Murashige and Skoog + 0.25 mg thiamine-HCI/l + 40 9 sucrose/1 + 8 g agar/1) containing 1 mg 2,4-D/l and incubated at 25 C in darkness. After 8 months of subculturing, an embryo was detected 19 days after the ninth subculture on one of the numerous aerial roots. This particular root appeared truncated just past a sharp bend. The epidermis was torn open on the outside of the bend and a ball of friable callus extended from the bend to the end of the root. The embryo arose immediately adjacent to this friable callus on the inside of the bend. The embryo enlarged slowly over the following 45 days and developed a cleft top. Crowding by other roots in the original plate necessitated removing the root bearing the embryo and transferring it to a fresh plate. After 13 days the embryo separated from the supporting root tissue when touched gently. Although the supportive root tissue was now senescing, histological studies comparing this tissue with sections of other roots grown under identical conditions revealed similar cellular structure for both samples. The tissue which produced the embryo also remained white although leaves in the same dish turned green, consistent with its identification as a true root. The free embryo was transferred to a culture room with a 16-hour photoperiod at 25 C, where it rapidly germinated. On auxin-free medium a 10-cm-tall seedling was obtained which did not survive transplantation into the greenhouse.
Although apparently a low frequency event, this observation demonstrates that corn roots can express totipotency in vitro.
Stephen J. Molnar, John L. Grainger and Krystyna Klimaszewska
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