Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

Cho, M.-J.; Choi, Hyung-Jin; Buchanan, Bob B.; Lemaux, Peggy G.

doi:10.1007/s001220051191

Cited by 47 publications

(48 citation statements)

References 21 publications

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“…DNA blot-hybridization analysis showed that transgenic lines had at least three copies of transgenes, ranging from approximately 3 to 15 copies (Cho et al 1999(Cho et al , 2002. As expected, a more intense FISH signal was observed in transgenic barley lines with higher copy numbers.…”

Section: Discussionmentioning

confidence: 55%

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Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

2002

Theor Appl Genet

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Introduced transgenes, uidA, sgfp (S65T) and/or bar, were localized using fluorescence in situ hybridization (FISH) on metaphase chromosomes of transgenic barley produced by microparticle bombardment of immature embryos. Of the 19 independent transgenic lines (eight diploid and 11 tetraploid), nine had uidA and ten had s gfp (S65T). All lines tested had three or more copies of the transgenes and 18 out of 19 lines had visibly different integration sites. At a gross level, it appeared that no preferential integration sites of foreign DNA among chromosomes were present in the lines tested; however, a distal preference for transgene integration was observed within the chromosome. In diploid T0 plants that gave a 3:1 segregation ratio of transgene expression in the T1, only single integration sites were detected on one of the homologous chromosomes. Homozygous diploid plants had doublet signals on a pair of homologous chromosomes. All tetraploid T0 plants that gave a 3:1 segregation ratio in the T1 generation had only a single integration site on one of the homologous chromosomes. In contrast, the single tetraploid T0 plant with a 35:1 segregation ratio in the T1 generation had doublet signals on a pair of homologous chromosomes. In the one tetraploid T0 line, which had a homozygote-like segregation ratio (45:0), there were doublet signals at two loci on separate chromosomes. We conclude that the application of FISH for analysis of transgenic plants is useful for the gross localization of transgene(s) and for early screening of homozygous plants.

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Section: Discussionmentioning

confidence: 55%

“…Golden Promise, 2n = 2x = 14) plants were produced via microprojectile bombardment of immature embryos as described by Cho et al (1999). Nineteen transgenic barley lines were generated; eight lines were diploid and 11 were tetraploid (see Table 1).…”

Section: Plant Materialsmentioning

confidence: 99%

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

2002

Theor Appl Genet

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“…Likewise, the expression of a cloned fragment from the seed-specifi c β-amylase gene from barley was confi rmed using the β-glucuronidase gene ( gus ) in a homologous system, and GUS activity was found in the subaleurone endosperm during seed maturation . Endospermspecifi c expression during grain maturation in transgenic barley was detected analysing the barley B1 hordein ( Hor2-4 ) and D-hordein ( Hor3-1 ) promoters (Cho et al 1999b(Cho et al , 2002Furtado et al 2009 ), a rice glutelin B1 ( GluB-1 ) promoter (Patel et al 2000 ) and two high-molecular-weight glutenin ( HMW-Glu ) promoters (Schünmann et al 2002 ;Zhang et al 2003 ;Furtado et al 2009 ). Besides that, in a study with the green fl uorescent protein gene ( gfp ) as a reporter, the wheat early methionine (Em) promoter was evaluated which maintained endosperm-specifi c expression in barley (Furtado and Henry 2005 ) suggesting its ability as a strong promoter to direct transgenes in specifi c tissues of barley.…”

Section: Targeted Expression Of Alien Genesmentioning

confidence: 97%

“…Direct DNA delivery frequently results in multicopy integration, and rearrangement of the transgene and gene silencing often has been linked to this phenomenon (Vaucheret et al 1998 ;Cho et al 1999bCho et al , 2002Bregitzer and Tonks 2003 ). Convincing evidences were provided by Travella et al ( 2005 ) comparing transgenic barley lines generated by biolistics and Agrobacterium -mediated method.…”

Section: Transgene Insertion and Stabilitymentioning

confidence: 99%

Barley

Schulze¹

2013

Alien Gene Transfer in Crop Plants, Volume 2

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Barley ( Hordeum vulgare L.) is a widely adapted cereal crop with an extremely wide geographic distribution throughout the world. It fi nds great use for animals as a feed and for humans as a grain, especially as the source for malt for the brewing industry. In recent times, there is considerable interest in the nutritional properties of barley due to the discovery of the cholesterol-lowering effect of β-glucan, a cell wall polysaccharide. Exploitation of genetic diversity in the primary and secondary gene pool of barley using DNA-based technologies has yielded interspecifi c crosses with improved grain properties, malting quality and resistance to biotic and abiotic stresses. The signifi cant achievements regarding introgression of alien genes include the genes Rym14(Hb) , Rym16(Hb) and Ryd4(Hb) which were introgressed from Hordeum bulbosum conferring resistance to BaMMV, BaYMV and BYDV in barley. Signifi cant advances in genetic engineering of barley have been obtained, and strategies for establishment of regenerative cell and tissue culture systems as well as for development of DNA delivery techniques have been formulated. Lately, a huge potential has been realised in barley grains to produce pharmaceutical proteins like oral vaccines, growth supplements and food additives which are being exploited in a commercial scale. Nevertheless, several problems still remain like the strong genotype dependency of barley transformation protocols, transformation effi ciency, transgene stability and public acceptance. The review focuses on all these issues and elaborates achievements made in the last two decades in genetic enhancement of barley using different alien gene transfer approaches.

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“…Transgenic barley plants have been obtained by PEG-mediated transfer, electroporation, microprojectile bombardment and Agrobacterium-mediated transformation (Carlson et al 2001;Cho et al 1998Cho et al , 1999aCho et al , b, 2002Funatsuki et al 1995;Hagio et al 1995;Jähne et al 1994;Koprek et al 1996;Ritala et al 1994;Tingay et al 1997;Wan and Lemaux 1994;Zhang et al 1999). For commercial application of transformation technologies, it is imperative that agronomically useful transgenes be stably expressed during generation advance.…”

Section: Introductionmentioning

confidence: 99%

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

2003

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In order to evaluate the long-term stability of transgene expression driven by the B(1)- and D-hordein promoters in transgenic barley ( Hordeum vulgare L., 2 n=2 x=14), we analyzed plants from 15 independent transgenic barley lines [6 for uidA and 9 for sgfp(S65T)] produced via microprojectile bombardment of immature embryos; 4 were diploid and 11 were tetraploid. The expression and inheritance of transgenes were determined by analysis of functional transgene expression, polymerase chain reaction and fluorescence in situ hybridization (FISH). Ability to express transgenes driven by either B(1)- or D-hordein promoter was inherited in T(4) and later generations: T(4) (2 lines), T(5) (8 lines), T(6) (3 lines), T(8) (1 line) and T(9) (1 line). Homozygous transgenic plants were obtained from 12 lines [5 for uidA and 7 for sgfp(S65T)]; the remaining lines are currently being analyzed. The application of the FISH technique for physical mapping of chromosomes was useful for early screening of homozygous plants by examining for presence of the transgene. For example, one line expressing uidA, and shown to have doublet fluorescence signals on a pair of homologous chromosomes was confirmed as a homozygous line by its segregation ratio; additionally this line showed stable inheritance of the transgene to T(9) progeny. The expression of transgenes in most lines (14 out of 15 lines) driven by hordein promoters was stably transmitted to T(4) or later generations, although there was a skewed segregation pattern (1:1) from the T(1) generation onward in the remaining line. In contrast, transgene silencing or transgene loss under the control of the maize ubiquitin promoter was observed in progeny of only 6 out of 15 lines.

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Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

Cited by 47 publications

References 21 publications

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

Use of fluorescence in situ hybridization for gross mapping of transgenes and screening for homozygous plants in transgenic barley (Hordeum vulgare L.)

Barley

Long-term stability of transgene expression driven by barley endosperm-specific hordein promoters in transgenic barley

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