Until recently, immature embryos have been a choice tissue for manipulation in culture for regeneration and production of transgenic maize plants. The utility of this explant has been compromised by low output, genotype dependence and time-consuming incubation in tissue culture. We have developed a new explant, the split-seed, which addresses these limitations by formally treating each seed as though it were a "dicot". By splitting maize seed longitudinally, three different tissues: the scutellum, the coleoptilar-ring and the shoot apical meristems are simultaneously exposed. The cells of these tissues can be made competent to enhance the regeneration, given that the molecular networks resulting from exposure of the split-seed to hormones is likely to be different from whole seed and, in turn, affects the in vitro response. Using this explant, callus induction frequency exceeded 92% and the regeneration frequency was 76%. The mean number of shoots regenerated via callus was 11 shoots per callus clump and 28 shoots per explant at first sub-culture. All of the regenerated plants survived and were 95% fertile. The large numbers of fertile plants produced were regenerated in 6-8 weeks. Finally, the incidence of regenerated plants varies as a function of growth regulator profile.

We report on a rapid high-frequency somatic embryogenesis and plant regeneration protocol for Zea mays. Maize plants were regenerated from complete shoot meristem (3-4 mm) explants via organogenesis and somatic embryogenesis. In organogenesis, the shoot meristems were directly cultured on a high-cytokinin medium comprising 5-10 mg x L(-1) 6-benzylaminopurine (BAP). The number of multiple shoots produced per meristem varied from six to eight Plantlet regeneration through organogenesis resulted in just four weeks. Callus was induced in five days of incubation on an auxin-modified Murashige and Skoog (MS) medium. Prolific callus, with numerous somatic embryos, developed within 3-4 weeks when cultured on an auxin medium containing 5 mg 2,4-dichlorophenoxyacetic acid x L(-1). The number of multiple shoots varied from three to six per callus. Using R23 (Pioneer, Hi-Bred, Johnston, Iowa), the frequency of callus induction was consistently in excess of 80% and plant regeneration ranged between 47 and 64%. All regenerated plantlets survived in the greenhouse and produced normal plants. Each transgenic plant produced leaves, glumes, and anthers that uniformly expressed green fluorescent protein (GFP). The GFP gene segregated in the pollen. Based on this data it is concluded that the transgenics arose from single-cell somatic embryos. The rate of transfer DNA (T-DNA) transfer to complete shoot meristems of Zea mays was high on the auxin medium and was independent of using super-virulent strains of Agrobacterium.

No abstract

No abstract

Transgene stacking in trait development process through genetic engineering is becoming complex with increased number of desired traits and multiple modes of action for each trait. We demonstrate here a novel gene stacking strategy by combining bidirectional promoter (BDP) and bicistronic approaches to drive coordinated expression of multi-genes in corn. A unidirectional promoter, Ubiquitin-1 (ZMUbi1), from Zea mays was first converted into a synthetic BDP, such that a single promoter can direct the expression of two genes from each end of the promoter. The BDP system was then combined with a bicistronic organization of genes at both ends of the promoter by using a Thosea asigna virus 2A auto-cleaving domain. With this gene stacking configuration, we have successfully obtained expression in transgenic corn of four transgenes; three transgenes conferring insect (cry34Ab1 and cry35Ab1) and herbicide (aad1) resistance, and a phiyfp reporter gene using a single ZMUbi1 bidirectional promoter. Gene expression analyses of transgenic corn plants confirmed better coordinated expression of the four genes compared to constructs driving each gene by independent unidirectional ZmUbi1 promoter. To our knowledge, this is the first report that demonstrates application of a single promoter for co-regulation of multiple genes in a crop plant. This stacking technology would be useful for engineering metabolic pathways both for basic and applied research.

Transformation of maize (Zea mays L.) split‐seed explants from inbred line R23 was performed following particle bombardment with a construct carrying the Arabidopsis transcriptional factor CBF3 under the control of the inducible promoter rd29A and the selectable marker hygromycin phosphotransferase. Overexpressing CBF3 has been shown to enhance cold, drought, and salt tolerance in Arabidopsis, tobacco (Nicotiana tabacum L.), and wheat (Triticum aestivum L.). The CBF3 gene was detected in 18 lines by polymerase chain reaction (PCR), and stable integration of multiple copies of CBF3 was confirmed by Southern blot analysis in three selected lines. Reverse transcription PCR detected expression of CBF3 in the transgenic lines under unstressed conditions despite the use of the stress‐inducible rd29A promoter. This constitutive expression was associated with growth retardation and sterility in most of the transgenic lines. Transmission of the gene in a Mendelian fashion to T1 and T2 generations was confirmed in one line by Southern blot analysis. Plants of this line showed stress‐inducible expression of the CBF3 gene and hardly detectable expression under unstressed conditions along with significant tolerance to cold, drought, and salinity compared with wild‐type plants. These results demonstrate that stress‐inducible overexpression of CBF3 has the potential to enhance abiotic stress tolerance in corn.

No abstract

No abstract