Skip to main content
ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Publications at this Location » Publication #324249
Title: The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat

Author
item HAN, YONGHUN - Chinese Academy Of Sciences
item Blechl, Ann
item WANG, DAOWEN - Chinese Academy Of Sciences

Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/19/2015
Publication Date: 9/24/2015
Citation: Han, Y., Blechl, A.E., Wang, D. 2015. The distribution of cotransformed transgenes in particle bombardment-mediated transformed wheat. Transgenic Research. doi: 10.1007/s11248-015-9906-4.

Interpretive Summary: New genes can be added to wheat by genetic transformation methods that employ physical means to transfer DNA. In previous research, we used the particle bombardment method to add extra copies of wheat genes encoding high-molecular-weight-(HMW-) glutenins. The glutenin genes were carried on plasmid DNA’s and the transformations also included separate plasmids carrying a selection marker gene for herbicide resistance. We found that increasing the levels of HMW-glutenin genes and proteins resulted in transgenic wheat flours with higher dough strength and better tolerance to over-mixing. In this manuscript, we determine the locations of the added genes in the genomes of each of 45 such transgenic wheat lines. Insertion sites are found in all but one of the 21 wheat chromosomes and in various locations along the chromosomes, demonstrating that DNA integration was random. Six wheat lines contain two insertion sites and one line has three insertion sites. Even though the transformations included two or three separate plasmid DNA vectors, all the genes integrated into the same location(s), providing support for a model in which the separate incoming DNA’s are joined together before the whole array is integrated into a chromosome. One practical implication of the results is that it will be very difficult to use crossing and segregation to separate the herbicide resistance marker gene from the HMW-glutenin transgenes because they are so close together on the chromosomes. We also detect chromosome rearrangements in 3 of the 45 lines, suggesting an association between such changes and transformation by direct DNA transfer. This study contributes valuable insights into the strengths and limitations of the particle bombardment transformation method.

Technical Abstract: Although particle bombardment is the predominant method of foreign DNA direct transfer, whether transgene is integrated randomly into the genome has not been determined. In this study, we identified the distribution of transgene loci in 45 transgenic wheat (Triticum aestivum L.) lines containing co-transformed high molecular weight glutenin subunit genes and the selectable marker bar using fluorescence in situ hybridization. Transgene loci were shown to distribute unevenly throughout the genome and incorporate into different locations along individual chromosomes. There was only a slight tendency toward the localization of transgenes in distal chromosome regions. High proportions of transgenes in separate plasmid integrated at the same site and only 7 lines had 2 or 3 loci. Such loci may not segregate frequently in subsequent generations so it is difficult to remove selectable markers from transgenic lines after regeneration. We also found that three transgene lines were associated with rearranged chromosomes, suggesting a close relationship between particle bombardment-mediated transgene integration and chromosomal rearrangements.