Transformation of endosperm cell suspension cultures and immature seeds by high-velocity microprojectile bombardment
--A. Genga, L. A. Manzocchi, S. Faranda and A. Viotti
The analysis concerning the expression and regulation of tissue-specific sequences from maize has in the majority of cases been performed in heterologous systems, due to difficulties in setting up easy and routine methods of maize transformation. However, high-velocity microprojectile bombardment has recently proved to be a successful method in obtaining both transient and stable transformation of this species.
In order to develop an experimental protocol for the study of zein gene expression, a particle gun apparatus, based on an electric device that allows controlled flow from a gas cylinder, has been constructed in our laboratory, and transformation experiments have been carried out using two different types of targets: endosperm cell suspension cultures and immature seeds.
Cell suspension cultures, established from immature endosperms of an A69Y maize line, appear to be a valuable homologous system, since these cells synthesize and accumulate zein polypeptides during their growth cycle (Manzocchi, LA, Plant Cell Reports 9:555-558, 1991).
Immature seeds of the W22 maize line were harvested at 20 DAP and immediately subjected to bombardment. The target was represented either by the pericarp (intact seeds) or by the endosperm tissue directly exposed, after removal of the pericarp, to DNA-coated gold particles.
The target surface area was about 5 square centimeters. Cells from suspension cultures were layered on sterilized filter paper and maize seeds were placed on agar plates.
In order to optimize the conditions of the bombardment the two plasmid constructs DP74 and DP33 were used. Both plasmids contained the promoter-leader region of the 35S-CaMV fused to the 5' end of the GUS gene, and differed by the presence (DP74) or the absence (DP33) of the maize AdhI intron (Pierce, DA et al., Plant Gene Systems and Their Biology, Alan R. Liss, Inc., pp. 301-310, 1987). For the functional analysis of endosperm specific sequences we employed the entire promoter region (1450bp upstream to the ATG start codon) or derived fragments containing different regulatory elements of the zE19 clone, a light chain zein gene (Spena, A et al., J. Mol. Biol. 169:799-811, 1983).
Transient expression of the GUS activity was determined by the histochemical method (Jefferson et al., EMBO J. 6:3901-3907, 1987), usually 24h after bombardment.
The 35S-GUS constructs gave a higher number of blue spots (GUS expression unit, GEU) on entire seeds (pericarp tissue subjected to bombardment) than on the exposed endosperm tissue. The average GEU values for pericarp and endosperm tissue were about 90 and 15 per shot, respectively. The endosperm cell suspension cultures showed with the same plasmids an average GEU value of 302±111 spots per shot.
Using the zE19 construct, suspension cultured cells did not show any GUS expression. However, preliminary results of seeds bombarded with the zein E19 promoter indicate a low, but significant, GEU value (up to 20 spots) only on the exposed endosperm tissue, but not on seeds with pericarp.
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