Dietary micronutrient deficiencies, such as the lack of vitamin A, are a major source of morbidity and mortality worldwide. Carotenoids in food can function as provitamin A in humans, while grains of Chinese elite wheat cultivars generally have low carotenoid contents. To increase the carotenoid contents in common wheat endosperm, transgenic wheat has been generated by expressing the maize y1 gene encoding phytoene synthase driven by a endosperm-specific 1Dx5 promoter in the elite wheat (Triticum aestivum L.) variety EM12, together with the bacterial phytoene desaturase crtI gene from Erwinia uredovora under the constitutive CaMV 35S promoter control. A clear increase of the carotenoid content was detected in the endosperms of transgenic wheat that visually showed a light yellow color. The total carotenoids content was increased up to 10.8-fold as compared with the nontransgenic EM12 cultivar. To test whether the variability of total carotenoid content in different transgenic lines was due to differences in the transgene copy number or expression pattern, Southern hybridization and semiquantitative reverse transcriptase polymerase chain reaction analyses were curried out. The results showed that transgene copy numbers and transcript levels did not associate well with carotenoid contents. The expression patterns of endogenous carotenoid genes, such as the phytoene synthases and carotene desaturases, were also investigated in wild-type and transgenic wheat lines. No significant changes in expression levels of these genes were detected in the transgenic endosperms, indicating that the increase in carotenoid transgenic wheat endosperms resulted from the expression of transgenes.

Two groups of linear gene constructs (gus and bar, and 1Ax1 and bar) lacking vector backbone sequences were independently transferred into the elite wheat (Triticum aestivum L.) variety EM12, and genetically stable transgenic plants with low copy number transgene integration were recovered. Co-transformation experiments were carried out in parallel using either circular whole plasmid(s) or linear gene cassettes which were purified from the same plasmid by restrictive digestion, each cassette consisting of a promoter, an open reading frame, and a terminator. Six transgenic wheat lines transformed with 1Ax1 plus bar gene cassettes, five lines with gus plus bar gene cassettes, three lines with p1Ax1 plus pAHC20, and two lines with pAHC25 were regenerated with transformation frequencies of 0.6, 0.5, 0.3, and 0.2%, respectively. Southern blotting analysis showed that there were 1-4 hybridizing bands in transgenic lines carrying gene cassettes, of which most lines displayed single-copy transgene insertion. Expression analyses showed that 50.5% of the T1 lines carrying gus plus bar gene cassettes have the expression signals of two genes. SDS-PAGE analysis of the T1 generation revealed that 71% of herbicide-resistant plants carrying 1Ax1 plus bar gene cassettes expressed the high molecular weight subunit 1Ax1 in the endosperm. Gene cassettes were transmitted and segregated in the subsequent generations, in simple Mendelian ratios. In addition, reverse transcription-polymerase chain reaction (RT-PCR) results confirmed that 1Ax1 gene cassettes were expressed specifically in the endosperm of the transgenic wheat plant. It is proposed that gene transfer using multiple gene cassettes offers an efficient and rapid method to obtain the single-copy transgenic wheat.

The promoter of a pollen-specific gene TaPSG719 was isolated from wheat (Triticum aestivum L.) by inverse-PCR (IPCR). Sequence analysis revealed that the promoter contains two cis-acting elements (AGAAA and GTGA) known to confer anther/pollen-specific gene expression which suggests that the promoter of TaPSG719 gene is a pollen-specific one. To ascertain the regulatory function of TaPSG719 promoter, two deleted fragments (-1,776 to -1 bp and -1,019 to -1 bp) were fused to the beta-glucuronidase (GUS) gene and transformed into tobacco plants. Similar GUS expression patterns were observed in all transformed plants and its activity was detected exclusively in pollen. No GUS activity in any other floral or vegetative tissue was observed. The results confirm that TaPSG719 promoter is pollen-specific and active during the middle stages of pollen development till anther matured, and it can drive pollen-specific gene expression across the species.

Genetic manipulation using gene cassettes was applied to the elite wheat variety EM12 via particle bombardment, which allows an improvement in transformation frequency. We simultaneously transferred to wheat immature embryos with two non-linked genes, gus and bar, on either separate gene cassettes or one plasmid. The linear gene cassettes were excised and purified by restriction digestion of the plasmid, and consisted of promoters, open reading frames and terminators. No difference was observed in GUS transient expression of between gene cassettes and single whole plasmid. However, the stable transformation frequency was significantly increased to 1.1% using gene cassettes, compared with 0.4% when using single plasmid. Procedures of the efficient co-transformation with gene cassettes were developed. Factors influencing on the transformation frequency were also studied in order to optimize the procedure. These were acceleration pressure, target distance, gold particle size, the quantity ratio of gene cassettes and the age of target explants. Based on the transient and stable expression of the gus gene cassettes, optimization of transformation parameters improved the reproducibility of transformation in the elite wheat variety.