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

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.

Fig. 1

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

Fig. 2

PCR analysis of genomic DNA from nontransgenic control and from three F₁ lines from crosses. a 1.8-kb uidA fragment. b 0.72-kb sgfp(S65T) fragment. Plasmids, p16 (a) and pDhsGFP-1 (b) were used in positive control reactions; water was used in negative control reactions (Control). Molecular weights in kb are indicated on left

Fig. 3

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)

Fig. 4

GUS activities in mature transgenic seeds. GUS activity was determined by fluorometric assays on protein extracts from ten, single mature seeds derived from each homozygous plant. T₉ seeds derived from a parental GUS homozygote, GPBhGN-7, and F₃ seeds from two homozygotes for both GUS and GFP, GPBhGN/DhGFP-2-7 and GPBhGN/DhGFP-5-11, were used for GUS activity measurements

Fig. 5

GFP expression levels in mature transgenic seeds. GFP quantification was determined with western blot hybridization analysis using protein extracts from five mature seeds derived from each line. T₅ seeds derived from a parental GFP homozygote, GPDhGFP-12, and F₃ seeds from two homozygotes for both GUS and GFP, GPBhGN/DhGFP-2-7 and GPBhGN/DhGFP-5-11, were used for GFP expression level measurements. Lane 1 BenchMark™ prestained protein ladder, lane 2 Golden Promise, lane 3 GFP homozygotes, lane 4 GPBhGN/DhGFP-2-7, lane 5: GPBhGN/DhGFP-5-11, lane 6 GFP hemizygotes, lane 7 20 ng GFP protein