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

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Inheritance and effectiveness of two transgenes determining PVY resistance in progeny from crossing independently transformed tobacco lines

Genetic transformation of plants allows us to obtain improved genotypes enriched with the desired traits. However, if transgenic lines were to be used in breeding programs the stability of inserted transgenes is essential. In the present study, we followed the inheritance of transgenes in hybrids originated from crossing two transgenic tobacco lines resistant to Potato virus Y (PVY): MN 944 LMV with the transgene containing Lettuce mosaic virus coat protein gene (LMV CP) and AC Gayed ROKY2 with PVY replicase gene (ROKY2). Progeny populations generated by successive self-pollination were analyzed with respect to the transgene segregation ratio and resistance to Potato virus Y in tests carried out under greenhouse conditions. The presence of the virus in inoculated plants was detected by DAS-ELISA method. The results demonstrated the Mendelian fashion of inheritance of transgenes which were segregated independently and stably. As a result, we obtained T₄ generation of hybrid with both transgenes stacked and which was highly resistant to PVY.     

染色体重组对转基因大麦中gfp基因表达的作用

利用5种转绿色荧光蛋白基因(gfp)大麦不同株系及野生型大麦为材料,对不同转基因株系间以及转基因株系与野生型植株间进行杂交,分别对不同世代植株的根尖、花粉中gfp基因的表达量进行测定.结果表明,不同转基因株系间的根尖、花粉的gfp基因在表达量上存在差异,同一转基因材料的gfp基因表达存在组织差异;gfp基因在杂交后代中作为一个显性基因以孟德尔方式稳定遗传,不同染色体上的gfp基因重组有利于提高持基因表达.     

Expression and inheritance of foreign genes in transgenic peanut plants generated byAgrobacterium-mediated transformation

To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T₂ plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T₁ plants. Fluorometric GUS assay in T₁ and T₂ generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.Abbreviations GUS -Glucuronidase - MS Murashige and Skoog - MU 4-Methylumbelliferone - NPTII Neomycin phosphotransferase II     

Integration,expression and inheritance of transgenes in hexaploid oat (Avena sativa L.)

Two oat varieties, Melys (spring variety) and Bulwark (winter variety) were transformed by particle bombardment of primary embryogenic callus using either a ubi-bar-ubi-gus co-integration vector or co-transformed (Melys) with a ubi-bar plasmid together with one of three plasmids containing the beta-glucuronidase (gus) gene under the control of either a rice actin promoter, a CaMV35S promoter or a wheat high molecular weight glutenin promoter. Morphologically normal and fertile transgenic plants were regenerated following callus selection with glufosinate ammonium. Evidence for the integration and functioning of the selectable (bar) and reporter (gus) genes in T0 and T1 plants was confirmed by PCR, Southern hybridisation, fluorescence in situ hybridisation (FISH), histochemical assays, and by progeny analysis. Transformation rates varied from 0.2 to 5.0 lines/plate of callus bombarded, with co-transformation frequencies of 83 to 100%, and co-expression frequencies of 60 to 100%. Copy numbers for the bar and gus gene varied from 3 to 17 and from 2 to 20 respectively. Cell and tissue specific expression of the gus gene was evident from the different promoters, with the HMW glutenin promoter showing endosperm specific expression in T1 seed. No expression of the gus gene under the CaMV35S promoter was detected in any tissues. Progeny analysis provided evidence of Mendelian inheritance of the introduced genes suggesting either one or two unlinked integration sites. This was confirmed by fluorescence in situ hybridisation to chromosome spread preparations. No segregation of the gus gene from the bar gene was observed in any of the progeny derived from co-transformation.     

Assessment of expression and inheritance patterns of three transgenes with the aid of techniques for promoting rapid flowering of transgenic apple trees

Rapid flowering of transgenic Royal Gala apple (Malus×domestica) trees was achieved by growing trees under controlled greenhouse conditions. The expression and inheritance of three transgenes were confirmed in the seedling progeny. Grown as single stems on their own roots, the transgenic apple trees produced 80–110 nodes and were 2 m high on average at the end of the 1st year's growth. In the 2nd year, approximately 20% of these trees flowered around node 80. However, when scions collected from the top of 1-year-old trees were grafted onto the dwarfing rootstock Malling 9, 85% produced flowers and fruit within the next year. The grafted trees continued to produce fruit in the following years. Expression of the transgene uidA was monitored by assaying β-glucuronidase (GUS) activity in leaves, flowers and fruit. Inheritance of three transgenes, uidA, neomycin phototransferase II and acetolactate synthase, were demonstrated through the recovery of GUS-positive, kanamycin-resistant and chlorsulfuron-resistant progeny. Segregation patterns fitted a 1:1 ratio in most lines. However, a detailed analysis in one progeny line revealed a complex T-DNA integration pattern. Received: 8 June 1998 / Revision received: 19 November 1998 / Accepted: 26 November 1998     

Molecular characterization of the fate of transgenes in transformed wheat (Triticum aestivum L.)

Molecular analysis of the transgenes bar and gus was carried out over successive generations in six independent transgenic lines of wheat, until the plants attained homozygosity. Data on expression and integration of the transgenes is presented. Five of the lines were found to be stably transformed, duly transferring the transgenes to the next generation. The copy number of the transgenes varied from one to five in the different lines. One line was unstable, first losing expression of and then eliminating both the transgenes in R3 plants. Although the gus gene was detected in all the lines, GUS expression had been lost in R2 plants of all but one line. Rearrangement of transgene sequences was observed, but it had no effect on gene expression. All the stable lines were found to segregate for transgene activity in a Mendelian fashion.     

A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques

Two barley transformation systems, Agrobacterium-mediated and particle bombardment, were compared in terms of transformation efficiency, transgene copy number, expression, inheritance and physical structure of the transgenic loci using fluorescence in situ hybridisation (FISH). The efficiency of Agrobacterium-mediated transformation was double that obtained with particle bombardment. While 100% of the Agrobacterium-derived lines integrated between one and three copies of the transgene, 60% of the transgenic lines derived by particle bombardment integrated more than eight copies of the transgene. In most of the Agrobacterium-derived lines, the integrated T-DNA was stable and inherited as a simple Mendelian trait. Transgene silencing was frequently observed in the T₁ populations of the bombardment-derived lines. The FISH technique was able to reveal additional details of the transgene integration site. For the efficient production of transgenic barley plants, with stable transgene expression and reduced silencing, the Agrobacterium-mediated method appears to offer significant advantages over particle bombardment.     

The inheritance of genetic markers in microspore-derived plants of barley Hordeum vulgare L.

Summary Biochemical, molecular and morphological markers have been used to monitor the segregation of alleles at major gene loci in microspore-derived lines of four spring barley crosses and their parents. Significant deviations from the expected Mendelian ratios were observed for four of the ten markers studied in the cross. Distorted ratios were associated with loci located on chromosomes 4H and 6H. The differential transmission of alleles was in favour of the responsive parent (Blenheim) used in the anther culture studies. For the -Amy-1 locus on chromosome 6H, the preferential transmission of Blenheim alleles was most pronounced in the haploid regenerants that were colchicine treated. These results are discussed in relation to the genetic control of androgenetic response in barley and with respect to the exploitation of another culture in barley improvement.     

Overlap of gametophytic and sporophytic gene expression in barley

Summary The overlap of gametophytic and sporophytic gene expression in barley was studied by means of enzyme electrophoresis. Of the isozymes found, 60% were expressed in both gametophyte and sporophyte, 30% were sporophyte specific, and 10% were gametophyte specific. A considerable amount of the barley genome is thus potentially amenable to gametophytic selection. The estimated sizes of the common, sporophytic and gametophytic domains in barley gene expression correspond with the estimates obtained in other plant species.     

Transgenic rice plants: Characterization of two generations of seed progeny

Transgenic rice plants have been regenerated from kanamycin-resistant callus of Oryza sativa (cv. Taipei 309) derived from protoplasts electroporated with pCaMVNEO carrying the neomycin phosphotransferase II ( nptII ) gene. Of 6 randomly selected plants, all contained the nptll gene, but only 2 plants expressed NPTII activity. The transgenic plants were significantly shorter, produced fewer tillers, took longer to flower and had reduced fertility compared to non-transformed protoplastderived plants. Fifty-six seeds collected from one transgenic plant expressing NPTII activity germinated on medium containing kanamycin sulphate to give 16 green, first seed generation (R₁) plants. The latter could be divided into 3 groups: (i) Plants which set seed, had normal floret morphology and produced a total of 76 seeds; (ii) Plants which flowered, but which failed to set seed; (iii) Plants which failed to flower, were shorter and had significantly fewer tillers than plants of groups (i) and (ii). The nptII gene was present in all transgenic R₁ plants, but only 8 plants expressed the gene. Phenotypic characteristics, observed in transgenic R₁ plants were also seen in the transforned R₂ plants. These included reduced stature, a longer vegetative phase and reduced fertility compared to non-transformed plants.     

cDNA cloning,characterization and expression of an endosperm-specific barley peroxidase

A barley peroxidase (BP 1) of pI ca. 8.5 and M _r 37000 has been purified from mature barley grains. Using antibodies towards peroxidase BP 1, a cDNA clone (pcR7) was isolated from a cDNA expression library. The nucleotide sequence of pcR7 gave a derived amino acid sequence identical to the 158 C-terminal amino acid residues of mature BP 1. The clone pcR7 encodes an additional C-terminal sequence of 22 residues, which apparently are removed during processing. BP 1 is less than 50% identical to other sequenced plant peroxidases. Analyses of RNA and protein from aleurone, endosperm and embryo tissue showed maximal expression 15 days after flowering, and high levels were found only in the endosperm. BP 1 was not expressed in the leaves.     

Variable T-DNA linkage configuration affects inheritance of carotenogenic transgenes and carotenoid accumulation in transgenic indica rice

Transgenics for the expression of β-carotene biosynthetic pathway in the endosperm were developed in indica rice background by introducing phytoene synthase (psy) and phytoene desaturase (crtI) genes through Agrobacterium-mediated transformation, employing non-antibiotic positive selectable marker phosphomannose isomerase (pmi). Twenty-seven transgenic lines were characterized for the structural organization of T-DNA inserts and the expression of transgenes in terms of total carotenoid and β-carotene accumulation in the endosperm. Ten lines were also studied for the inheritance of transgenic loci to the T₁ progenies. Copy number and sites of integration of the transgenes ranged from one to four. Almost 50% of the transgenic lines showed rearrangement of T-DNA inserts. However, most of the rearrangements occurred in the crtI expression cassette which is adjacent to the right T-DNA border. Differences in copy numbers of psy and crtI were also observed indicating partial T-DNA integration. Beyond T-DNA border transfer was also detected in 25% of the lines. Fifty percent of the lines studied showed single Mendelian locus inheritance, while two lines showed bi-locus inheritance in the T₁ progenies. Some of the lines segregating in 3:1 ratio showed two sites of integration on restriction digestion analysis indicating that the T-DNA insertion sites were tightly linked. Three transgenic lines showed nonparental types in the segregating progenies, indicating unstable transgenic locus. Evidences from the HPLC analysis showed that multiple copies of transgenes had a cumulative effect on the accumulation of carotenoid in the endosperm. T₁ progenies, in general, accumulated more carotenoids than their respective parents, the highest being 6.77 μg/g of polished seeds. High variation in the carotenoid accumulation was observed within the T₁ progenies which could be attributed to the variation in the structural organization and expression of transgenes, minor variations in the genetic background within the progeny plants, or differences in the plant microenvironments. The study identified lines worthy of further multiplication and breeding based on transgene structural integrity in the segregating progeny and high expression levels in terms of the β-carotene accumulation.     

Inheritance and expression of Bt cry1Ba3 gene in progeny from transformed cabbage plants

Molecular Biology Reports - Stable inheritance and expression of transgene are important parameters for successful use of a transgenic crop. We previously transformed a Bt cry1Ba3 gene into cabbage...     

Inheritance and Expression of Copies of Transgenes 1Dx5 and 1Ax1 in Elite Wheat （Triticum aestivum L.） Varieties Transferred from Transgenic Wheat through Conventional Crossing

To study the inheritance and expression of multiple copies of transgenes from transgenic wheat lines, three crosses between transgenic wheat lines B72-8-11b and B102-1-2 and Chinese elite wheat varieties Chuan89-107 and Email 8 were carried out. Chuan89-107×B72-8-11b, Chuan89-107×B102-1-2 and Email 8×B72-8-11b, and F_1 plants were selfed or backcrossed to obtain different generation populations. Protein analysis in grains of F_1 and F_2 and backcross progenies of BC_1F_1, BC_1F_2, BC_1F_3, BC_2F_1, BC_2F_2 and BC_2F_3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the transgenes lDx5 and lAx1 were expressed and segregated in the target wheat according to Mendelian laws. A range of lDx5 expression levels were observed in the progenies of Chuan89-107×B72-8-11b and Emai 18×B72-8-11b, but the expression levels of lAx1 in progenies of Chuan89-107×B102-1-2 rarely changed. It suggested that the two foreign genes had different mechanisms of expression in the cross progeny, even though they were produced in the same way and the foreign lDx5 gene of 5-10 copies had the more complicated expression mechanism than the lAx1 gene of 4-5 copies.