Research Article

Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos

Published: February 06, 2015
Genet. Mol. Res. 14 (1) : 1096-1105 DOI: https://doi.org/10.4238/2015.February.6.13
Cite this Article:
(2015). Expression and genomic integration of transgenes after Agrobacterium-mediated transformation of mature barley embryos. Genet. Mol. Res. 14(1): gmr4716. https://doi.org/10.4238/2015.February.6.13
1,126 views

Abstract

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

Mature embryos in tissue cultures are advantageous because of their abundance and rapid germination, which reduces genomic instability problems. In this study, 2-day-old isolated mature barley embryos were infected with 2 Agrobacterium hypervirulent strains (AGL1 and EHA105), followed by a 3-day period of co-cultivation in the presence of L-cystein amino acid. Chimeric expression of the b-glucuronidase gene (gusA) directed by a viral promoter of strawberry vein banding virus was observed in coleoptile epidermal cells and seminal roots in 5-day-old germinated seedlings. In addition to varying infectivity patterns in different strains, there was a higher ratio of transient b-glucuronidase expression in developing coleoptiles than in embryonic roots, indicating the high competency of shoot apical meristem cells in the mature embryo. A total of 548 explants were transformed and 156 plants developed to maturity on G418 media after 18-25 days. We detected transgenes in 74% of the screened plant leaves by polymerase chain reaction, and 49% of these expressed neomycin phosphotransferase II gene following AGL1 transformation. Ten randomly selected T0 transformants were analyzed using thermal asymmetric interlaced polymerase chain reaction and 24 fragments ranged between 200-600 base pairs were sequenced. Three of the sequences flanked with transferred-DNA showed high similarity to coding regions of the barley genome, including alpha tubulin5, homeobox 1, and mitochondrial 16S genes. We observed 70-200-base pair filler sequences only in the coding regions of barley in this study.

About the Authors