Istituto Biosintesi Vegetali-CNR

Transformation of maize endosperm cells by electroporation
--Locatelli, F; Castelli, S; Genga, A; Viotti, A and Manzocchi, LA

In vitro endosperm cell cultures represent a valid system to investigate cereal seed maturation, in that they maintain some physiological features of the native tissue (Felker and Goodwin, Physiol. Plant. 88:1235-1239, 1988). In this laboratory, long term endosperm cell cultures have been established from A69Y wildtype and opaque-2 maize. Cultured cells synthesize (though at low levels) zeins with the typical pattern of the native endosperms (Manzocchi, Bianchi and Viotti, Plant Cell Rep. 7:639-643. 1989; Manzocchi, Plant Cell Rep. 9:555-558, 1991). Cells have been used to isolate protoplasts, which have been transformed by polyethylene glycol in experiments of transient expression and stable integration (Giovinazzo, Manzocchi, Bianchi, Coraggio and Viotti, Plant Mol. Biol. 19:257-263, 1992; Faranda, Genga, Viotti and Manzocchi, Plant Cell, Tissue Organ Cult. 37:39-46, 1994).

We present preliminary data here on the transactivation of a 21Kd zein promoter by the transcriptional activator OPAQUE2, through transformation by direct delivery of DNA to endosperm cells by electroporation, according to the method of D'Halluin et al. (K. D'Halluin, Bonne, Bossut, De Beukeleer, and Leemans, Plant Cell 4:1495-1505, 1992).

The DNA constructs used in the experiment were: p472-GUS, containing the uidA (beta-glucuronidase) gene under the control of the 800bp promoter of a 21 kd zein gene, fused to a zein enhancer-like element; and p501, containing the 1550 bp coding region of maize Opaque2 gene, under a CaMV 35S promoter (Quattrocchio, personal communication). Aliquots of approximately 200 mg of A69Y endosperm cultured cells were electroporated, in the conditions described by D'Halluin et al., in the presence of 20 ug DNA of each construct. After electroporation, they were plated on standard agar growth medium (Manzocchi, 1991). The expression of the GUS reporter gene was detected on cell extracts at different times after electroporation, with the spectrofluorimetric method (Jefferson, Kavanagh and Bevan, EMBO J. 6:391-397, 1987).

Data reported in Table 1 show that, in the presence of the sequence coding for the transcriptional activator O2, the expression of GUS under the control of a zein 21 Kd promoter is 6 fold enhanced. Enhancement can be detected both at short times after cell transformation, and several months later. These data are in agreement with a possible stable integration of the constructs in the DNA of a number of cells; this would confirm, in a stably transformed homologous cell system, the transactivation of a zein promoter by the transcriptional activator O2, which had been observed by Ueda et al. (Plant Cell 4:701-709, 1992) in experiments of transient expression. In this experiment no selectable marker was employed, but experiments of co-transformation with NPTII and bar constructs are in progress, in the aim to select stable transformants.

Table 1. GUS expression (pmoles MU/min/mg protein) in cultured A69Y maize endosperm cells transformed through electroporation.
Weeks after electroporation --- p472 p472+p501
2 0.6 7.9 33.2
16 0.44 1.06 6.48

We can conclude that the method of intact cell electroporation can be successfully employed to transform maize endosperm cell cultures; with a suitable selection system allowing the isolation of transformed cell lines, and their molecular characterization, it will provide a useful tool in the study of gene regulation in maize endosperm. 

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