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Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants.

Choi HW, Yu XH, Lemaux PG, Cho MJ - Plant Cell Rep. (2009)

Bottom Line: Both lines stably expressed the two transgenes in the generations prior to the cross.FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F(3) and F(4) generations.Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T(8) plant, having uidA (or gus) driven by the barley endosperm-specific B(1)-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T(4) plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F(1) progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F(2) seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F(2) plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F(3) and F(4) generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.

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FISH of transgenes [uidA and sgfp(S65T)] in F2 plants. a, b. Hemizygous plant with both (a) a single signal of uidA (arrow) inserted on the centromeric region of chromosome 7H and (b) a single signal of sgfp(S65T) (arrow) inserted on the subtelomeric region of chromosome 2H. c, d Homozygous plant with both (c) doublet signals of uidA and (d) sgfp(S65T) on homologous chromosomes (Fig. 4)
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Fig3: FISH of transgenes [uidA and sgfp(S65T)] in F2 plants. a, b. Hemizygous plant with both (a) a single signal of uidA (arrow) inserted on the centromeric region of chromosome 7H and (b) a single signal of sgfp(S65T) (arrow) inserted on the subtelomeric region of chromosome 2H. c, d Homozygous plant with both (c) doublet signals of uidA and (d) sgfp(S65T) on homologous chromosomes (Fig. 4)

Mentions: Screening of homozygous plants using GUS/GFP assay and FISH from crossed transgenic barley plants. F1 seeds were obtained from the cross of two parental plants, a homozygous T8 plant derived from GPBhGN-7 and a homozygous T4 plant derived from GPDhGFP-12; three F1 plants were tested for GUS/GFP activities. GFP expression in F2 seeds was performed using cross-sectioned half-seeds without immature embryos; GUS assay was then performed using the same materials. Expression of sgfp(S65T) is marked by an asterisk. Half-seeds with embryos expressing both GFP and GUS were saved and grown for next generations. Numbers indicate the seed number examined (Table 1). FISH technique was employed to screen the homozygous [uidA and sgfp(S65T)] F2 plants by direct mapping of transgenes on the chromosomes (Table 2; Fig. 3). Inserted uidA and sgfp(S65T) genes were localized on the centromeric region of chromosome 7H and on the subtelomeric region of chromosome 2H, respectively. Homozygous F3 generation seeds were obtained by analyzing segregation ratios of transgenes


Stability and inheritance of endosperm-specific expression of two transgenes in progeny from crossing independently transformed barley plants.

Choi HW, Yu XH, Lemaux PG, Cho MJ - Plant Cell Rep. (2009)

FISH of transgenes [uidA and sgfp(S65T)] in F2 plants. a, b. Hemizygous plant with both (a) a single signal of uidA (arrow) inserted on the centromeric region of chromosome 7H and (b) a single signal of sgfp(S65T) (arrow) inserted on the subtelomeric region of chromosome 2H. c, d Homozygous plant with both (c) doublet signals of uidA and (d) sgfp(S65T) on homologous chromosomes (Fig. 4)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2717377&req=5

Fig3: FISH of transgenes [uidA and sgfp(S65T)] in F2 plants. a, b. Hemizygous plant with both (a) a single signal of uidA (arrow) inserted on the centromeric region of chromosome 7H and (b) a single signal of sgfp(S65T) (arrow) inserted on the subtelomeric region of chromosome 2H. c, d Homozygous plant with both (c) doublet signals of uidA and (d) sgfp(S65T) on homologous chromosomes (Fig. 4)
Mentions: Screening of homozygous plants using GUS/GFP assay and FISH from crossed transgenic barley plants. F1 seeds were obtained from the cross of two parental plants, a homozygous T8 plant derived from GPBhGN-7 and a homozygous T4 plant derived from GPDhGFP-12; three F1 plants were tested for GUS/GFP activities. GFP expression in F2 seeds was performed using cross-sectioned half-seeds without immature embryos; GUS assay was then performed using the same materials. Expression of sgfp(S65T) is marked by an asterisk. Half-seeds with embryos expressing both GFP and GUS were saved and grown for next generations. Numbers indicate the seed number examined (Table 1). FISH technique was employed to screen the homozygous [uidA and sgfp(S65T)] F2 plants by direct mapping of transgenes on the chromosomes (Table 2; Fig. 3). Inserted uidA and sgfp(S65T) genes were localized on the centromeric region of chromosome 7H and on the subtelomeric region of chromosome 2H, respectively. Homozygous F3 generation seeds were obtained by analyzing segregation ratios of transgenes

Bottom Line: Both lines stably expressed the two transgenes in the generations prior to the cross.FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F(3) and F(4) generations.Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720, USA.

ABSTRACT
To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T(8) plant, having uidA (or gus) driven by the barley endosperm-specific B(1)-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T(4) plant, having sgfp(S65T) driven by the barley endosperm-specific D-hordein promoter (localized on the subtelomeric region of chromosome 2H). Both lines stably expressed the two transgenes in the generations prior to the cross. Three independently crossed F(1) progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F(2) seeds followed a 3:1 Mendelian segregation ratio and transgenes were localized by FISH to the same location as in the parental plants. FISH was used to screen F(2) plants for homozygosity of both transgenes; four homozygous plants were identified from the two crossed lines tested. FISH results showing presence of transgenes were consistent with segregation ratios of expression of both transgenes, indicating that the two transgenes were expressed without transgene silencing in homozygous progeny advanced to the F(3) and F(4) generations. Thus, even after crossing independently transformed, homozygous parental plants containing a single, stably expressed transgene, progeny were obtained that continued to express multiple transgenes through generation advance. Such stability of transgenes, following outcrossing, is an important attribute for trait modification and for gene flow studies.

Show MeSH
Related in: MedlinePlus