William L. Petersen

AgNO3 increases type II callus production from immature embryos of maize inbred B73 and its derivatives

Incorporating 10 to 100 μM AgNO3 into Phytagel™ (0.2%) solidified N6 medium containing 1 mg/L 2,4-D, 100 mg/L casamino acids and 25 mM praline (N6 1-100-25) promoted type II callus production from cultured Zea mays L. immature embryos of FRB73, B73 X A188 and a proprietary B73 BC6 genotype. Under these conditions, approximately 15, 80 and 80% of the respective FRB73, B73 X A188 and B73 BC6 explants produced type II calli after 2 to 3 weeks incubation in the dark at 28 C. In the absence of AgNO3, the type II culture response from B73BC6 immature embryos was 25% on N6 1 100-25 solidified with Phytagel™ (0.2%) as compared to 0% for that solidified with 0.8% agar. Duncans medium was tested using 10 to 100 μm AgNO3 and generally promoted type I callus initiation, although up to 6% of the explants produced type II cultures in the presence of 0.2% Phytagel™. Ethylene emanation rates of up to 370 and 115 nL g-1 h-1 were detected from B73 X A188 immature embryos and calli, respectively, cultured on N6 1-100-25.

Production of transgenic maize plants and progeny by bombardment of hi-II immature embryos

SummaryProduction of transgenic maize (Zea mays L.) callus, plants, and progeny from microprojectile bombardment of 2–5-d cultured Hi-II immature embryos is described. Histological evidence indicates that these tissues are amenable to transformation due to surface layer cell division of the scutellum. Two out of every 100 bombarded embryos produced transgenic callus and R0 transgenic plants were both male and female fertile. Expected segregation of transgenes was observed in progeny. The primary advantage of bombarding these tissues is increased male and female fertility of transgenic plants compared with those produced using long-term callus or suspension cultures.

Factors affecting PEG-mediated stable transformation of maize protoplasts.

Factors influencing the frequency of stable transformation and co-transformation of maize protoplasts utilizing a polyethylene glycol (PEG) mediated DNA uptake procedure have been investigated. Protoplast plating conditions, pre-treatment buffer composition, PEG concentration, and DNA concentration were all found to be important. Carrier DNA was not beneficial when transforming with circular plasmid DNA. The effect of linearizing plasmid DNA was inconsistent across experiments, and may be dependent on the presence of carrier DNA. Functional co-transformation of an unlinked marker gene (hygromycin phosphotransferase) was increased by increasing the ratio of nonselected:selected DNA, and varied from 39% at a 1∶1 ratio to 65% at a 100∶1 ratio. Under optimum conditions, up to 300 transformed calli were recovered per million input protoplasts. The protocol is simple, inexpensive, and effective, and is useful for studies in maize requiring large numbers of stably transformed or co-transformed cell lines.

Effect of nurse cultures on the production of macro-calli and fertile plants from maize embryogenic suspension culture protoplasts.

Fertile plants have been obtained from maize (Zea mays L.) embryogenic suspension culture protoplasts. Friable, embryogenic callus initiated from an immature embryo from a cross involving the genotypes A188, B73, and Black Mexican sweetcorn was used to establish a rapidly growing embryogenic suspension culture. After nine months in culture, high yields of viable protoplasts (30×106/ gram fresh weight) were obtained following a 1.5 hour enzymatic digestion. Protoplasts cultured with feeder cells divided and formed embryogenic callus, from which male and female fertile plants were regenerated. Protoplast-derived R1 plants were self-pollinated and immature R2 embryos isolated for callus initiation. Female fertile plants have also been produced from protoplasts isolated from an R2-derived suspension culture. Significant interactions between protoplast and feeder-cell lines were observed.