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

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.     

Irregular patterns of transgene silencing in allohexaploid oat

An irregular pattern of transgene silencing was revealed in expression and inheritance studies conducted over multiple generations following transgene introduction by microprojectile bombardment of allohexaploid cultivated oat (Avena sativa L.). Expression of two transgenes, bar and uidA, delivered on the same plasmid was investigated in 23 transgenic oat lines. Twenty-one transgenic lines, each derived from an independently selected transformed tissue culture, showed expression of both bar and uidA while two lines expressed only bar. The relationship of the transgenic phenotypes to the presence of the transgenes in the study was determined using (1) phenotypic scoring combined with Southern blot analyses of progeny, (2) coexpression of the two transgenic phenotypes since the two transgenes always cosegregated, and (3) reactivation of a transgenic phenotype in self-pollinated progenies of transgenic plants that did not exhibit a transgenic phenotype. Transgene silencing was observed in 19 of the 23 transgenic lines and resulted in distorted segregation of transgenic phenotypes in 10 lines. Silencing and inheritance distortions were irregular and unpredictable. They were often reversible in a subsequent generation of self-pollinated progeny and abnormally segregating progenies were as likely to trace back to parents that exhibited normal segregation in a previous generation as to parents showing segregation distortions. Possible causes of the irregular patterns of transgene silencing are discussed.     

The relationship between homozygous and hemizygous transgene expression levels over generations in populations of transgenic rice plants

Segregating T₁, T₂ and T₃ transgenic rice populations, derived from independent particle-bombardment-mediated transformation events were examined in order to assess the effect of gene dosage on transgene expression levels and stability. The expression level of the unselected β-glucuronidase (gusA) reporter gene was quantified in plants from these populations. The gusA gene dosage was determined by segregation analysis of progeny seedlings at the structural level (by PCR) and at the expression level. For some transformation events a gene dosage effect on transgene expression was observed, leading to higher transgene expression levels in homozygous progeny than in hemizygous progeny or primary transgenic plants. However, in many other transformation events, the homozygous state appears to be disadvantageous, being associated with lower transgene expression levels, gene silencing or counter-selection of homozygous plants across generations. Change of gene dosage is probably one of the key factors influencing transgene expression levels and stability in transgenic rice. This is particularly important when considering molecular genetic studies and crop improvement programmes. The possible influence of matrix attachment regions (MARs) in increasing the likelihood of an additive effect on transgene expression level is discussed. Received: 21 March 2001 / Accepted: 29 June 2001     

Integration and inheritance of transgenes in crop plants and trees

Transgene integration and inheritance have been investigated in a number of crop plants and few tree species. Transgene integration is predominantly a random process, whether mediated by Agrobacterium or particle bombardment. Depending on the genomic position of the integrated transgene and structure of the integration site as well as copy number of the transgene in the genome, its expression may be stable or variable. Therefore, integration patterns would affect the mode of transgene inheritance in plants, regardless of the method of gene transfer. So far, both Mendelian and non-Mendelian inheritance of transgenes has been reported across several generations (T1–T3) of crop plants. In few tree species (apple, poplar, plum, and American chestnut), mostly Mendelian inheritance of the transgenes has been observed in the T1 or BC1 generations. However, detailed studies in the transgenic papaya trees showed Mendelian segregation of the transgene in the T1 generation but non-Mendelian inheritance in the T2 generation. Variation in transgene inheritance was also detected in transgenic apple and plum trees. Long generation cycles in many economically important tree species preclude investigation of inheritance of transgenes in the tree progeny. Production of early flowering trees, either by genetic modification or by environmental modulation, would facilitate the study of transgene inheritance across generations of transgenic trees. In order to overcome problems of randomness of transgene integration, targeted transgene insertions by homologous or site-specific recombination or by designer recombinases or nucleases offer prospects for stable integration of transgenes in predetermined locations in the plant genome. And perhaps, that might provide a platform for stable expression and Mendelian inheritance of transgenes in plants.     

抗除草剂转基因大豆后代遗传分析

分析了来源于农杆菌介导的4个独立的大豆转化系的后代遗传特性。分别采用种子切片GUS染色方法和除草剂涂抹以及喷洒方法检测gus报告基因和抗除草剂bar基因在后代的表达。其中3个转化系T1代gus基因和bar基因能够以孟德尔方式3：1连锁遗传,说明这2个基因整合在大豆基因组的同一位点。这3个转化系在T2代获得了纯合的转化系,并能够稳定遗传至T5代。有一个转化系在T1代GUS和抗除草剂检测都为阴性,但通过Southern杂交证明转基因存在于后代基因组,显示发生了转基因沉默。为了证明转基因沉默是转录水平还是转录后水平,T1代植物叶片接种大豆花叶病毒（SMV）并不能抑制转基因沉默,说明该转化系基因沉默可能不是发生在转录后水平。     

A robust genetic transformation protocol to obtain transgenic shoots of Solanum tuberosum L. cultivar ‘Kufri Chipsona 1’

The genetic transformation of plants is an important biotechnological tool used for crop improvement for many decades. The present study was focussed to investigate various factors affecting genetic transformation of potato cultivar ‘Kufri Chipsona 1’. It was observed that explants pre-cultured for 2 days on MS2 medium (MS medium containing 10 µM silver nitrate, 10 µM BA, 15 µM GA3), injured with a surgical blade and co-cultivated with Agrobacterium tumefaciens strain EHA105 [O.D600 (0.6)] for 2 days results in maximum transient β-glucuronidase (GUS) expression. The addition of 100 µM acetosyringone in MS2 medium also increased rate of transient GUS expression in both the explants. Clumps of putative transgenic shoots were regenerated using the optimised culture conditions from leaf and internodal explants. The stable integration of T-DNA was established using histochemical staining for GUS and amplification of DNA fragment specific to nptII and uidA genes. Within the clumps, around 67.85% of shoots showed uniform GUS expression in all the tissues and about 32.15% shoots show intermittent GUS expression establishing chimeric nature. Uniform GUS staining of the tissue was used as initial marker of non-chimeric transgenic shoots. Quantitative expression of nptII transgene was found to be directly proportional to uniformity of GUS staining in transgenic shoots. The present investigation indicated that manipulation of culture conditions and the medium composition may help to get transgenic shoots with uniform expression of transgene in all the tissues of potato cultivar ‘Kufri Chipsona 1’.     

Evaluation of tyrosinase minigene co-injection as a marker for genetic manipulations in transgenic mice.

The utility of tyrosinase minigene co-injection was evaluated as a visual marker for the generation and breeding of transgenic mice. In an evaluation of 39 transgenic founder animals and 44 transgenic lines five phenotypic patterns of pigmentation were consistently observed, including albino, dark, light, mottled and himalayan. In these studies co-injection of the tyrosinase minigene along with the transgene of interest (TOI) resulted in genomic integration of the two transgenes in 95% of the F0 generation. Co-segregation of transgenes occurred in 94% of doubly transgenic mice in the F1 generation, without dissociation in subsequent generations. All pigmented phenotypes proved useful for distinguishing homozygous from heterozygous F2 animals via backcross trials, while the light, mottled and himalayan phenotypes proved useful in visually discriminating between homozygous and heterozygous F2 animals. In addition, the light, mottled and himalayan phenotypes proved useful in determining segregation patterns of transgenes in the progeny of crosses between separate transgenic lines. Moreover, there appears to be a correlation between intensity of pigmentation and degree of expression of the co-injected TOI. These studies confirm that tyrosinase co-injection is a useful adjunct in transgenic mouse studies and can serve to reduce routine genetic validation of transgenic lines.     

Stable genetic transformation of<Emphasis Type="Italic"> Vigna mungo</Emphasis> L. Hepper via<Emphasis Type="Italic"> Agrobacterium tumefaciens</Emphasis>

Vigna mungo is one of the large-seeded grain legumes that has not yet been transformed. We report here for the first time the production of morphologically normal and fertile transgenic plants from cotyledonary-node explants inoculated with Agrobacterium tumefaciens carrying binary vector pCAMBIA2301, the latter of which contains a neomycin phosphotransferase ( nptII) gene and a beta-glucuronidase (GUS) gene ( uidA) interrupted with an intron. The transformed green shoots, selected and rooted on medium containing kanamycin, tested positive for nptII and uidA genes by polymerase chain reaction (PCR) analysis. These shoots were established in soil and grown to maturity to collect the seeds. Mechanical wounding of the explants prior to inoculation with Agrobacterium, time lag in regeneration due to removal of the cotyledons from explants and a second round of selection at the rooting stage were found to be critical for transformation. Analysis of T(0) plants showed the expression and integration of uidA into the plant genome. GUS activity in leaves, roots, flowers, anthers and pollen grains was detected by histochemical assay. PCR analysis of T(1) progeny revealed a Mendelian transgene inheritance pattern. The transformation frequency was 1%, and 6-8 weeks were required for the generation of transgenics.     

Development of transgenic rice pure lines by particle bombardment‐mediated transformation and genetic analysis‐based selection

Mature seed‐derived callus from an elite Chinese japonica rice cv. Eyl 105 was transformed with a plasmid containing the selectable marker hygromycin phosphotransferase (hpt) and the reporter β‐glucuronidase (gusA) genes via particle bombardment. After two rounds of selection on hygromycin (30 mg/l)‐containing medium, resistant callus was transferred to hygromycin (30 mg/l)‐containing regeneration medium for plant regeneration. Twenty‐three independent transgenic rice plants were regenerated from 127 bombarded callus with a transformation frequency of 18.1%. All the transgenic plants contained both gusA and hpt genes, revealed by PCR/Southern blot analysis. GUS assay revealed 18 out of 23 plants (78.3%) proliferated on hygromycin‐containing medium had GUS expression at various levels. Genetic analysis confirmed Mendelian segregation of transgenes in progeny. From R2 generations with their R1 parent plants showing 3:1 Mendelian segregation, we identified three independent homozygous transgenic rice lines. The homozygous lines were phenotypically normal and fertile compared to the control plants. We demonstrate that homozygous transgenic rice lines can be obtained via particle bombardment‐mediated transformation and through genetic analysis‐based selection.     

Agrobacterium-Mediated Transformation of Switchgrass and Inheritance of the Transgenes

Switchgrass (Panicum virgatum L.) has been developed into an important biofuel crop. Embryogenic calli induced from caryopses or inflorescences of the lowland switchgrass cultivar Alamo were used for Agrobacterium-mediated transformation. A chimeric hygromycin phosphotransferase gene (hph) was used as the selectable marker and hygromycin as the selection agent. Embryogenic calli were infected with Agrobacterium tumefaciens strain EHA105. Calli resistant to hygromycin were obtained after 5 to 8 weeks of selection. Soil-grown transgenic switchgrass plants were obtained 4 to 5 months after Agrobacterium infection. The transgenic nature of the regenerated plants was demonstrated by PCR, Southern blot hybridization analysis, and GUS staining. T1 progeny were obtained after reciprocal crosses between transgenic and untransformed control plants. Molecular analyses of the T1 progeny revealed various patterns of segregation. Transgene silencing was observed in the progeny with multiple inserts. Interestingly, reversal of the expression of the silenced transgene was found in segregating progeny with a single insert.     

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

To study stability and inheritance of two different transgenes in barley, we crossed a homozygous T₈ plant, having uidA (or gus) driven by the barley endosperm-specific B₁-hordein promoter (localized in the near centromeric region of chromosome 7H) with a second homozygous T₄ 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 F₁ progeny were analyzed by PCR for both uidA and sgfp(S65T) in each plant and functional expression of GUS and GFP in F₂ 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 F₂ 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 F₃ and F₄ 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.     

一种快速鉴定转基因植物纯合体的新方法

植物转化中鉴定转基因植物的整合性是一个很重要的步骤,常规方法是对独立分离的转基因T1代植株产生的T2代进行转基因分离比率研究,以检测T1代的转基因整合状态,不仅费时费力,而且浪费了T1代资源。本介绍一种应用双重定量实时PCR技术鉴定转基因植物纯合子的新方法：以T1代植物DNA为模板,根据转基因后代的Ct表型值鉴定其转基因整合状态,Ct值接近2的为转基因纯合型,Ct值接近1的为转基因杂合型。用这种方法,可以同时对数十个T1代转基因幼苗的整合状态进行快速鉴定,准确率为100％。     

Central cell nuclear-cytoplasmic incongruity:a mechanism for segregation distortion in advanced backcross and selfed generations of (Allium cepa L. x Allium fistulosum L.) x A. cepa interspecific hybrid derivatives

A model is presented as an explanation for an anomaly observed in germination and establishment and isozyme segregation patterns in Allium cepa x A. fistulosum F2BC3 populations generated in an introgression-breeding program. The F1BC3 parent of these populations was selected for its heterozygous PGI phenotype, Pgi-1(2/3); Pgi-1(2) was inherited from an A. cepa (Ac) seed parent and Pgi-1(3) from an A. fistulosum (Af) pollen parent. Germination and establishment was recorded for the F2BC3 progeny population. Segregation of Ac and Af Pgi-1 alleles was investigated in F2BC3 seeds and embryo and endosperm tissue was isolated and tested for isozyme expression. A pooled goodness-of-fit test of the segregation of Pgi-1 alleles in the populations to the expected Mendelian 1:2:1 ratio using the chi-square statistic gave a chi2 = 185.9, well beyond the accepted limits at 2 degrees of freedom. The 1:2:1 ratio expected for simple Mendelian inheritance was rejected, while a pooled chi-square goodness-of-fit test of the segregation of Pgi-1 alleles in the populations fit a 1:1 ratio with a chi2 = 0.203, based on the incongruity model. We present here the central cell nuclear-cytoplasmic incongruity hypothesis to explain the observed anomalies.     

Effect of ploidy and homozygosity on transgene expression in primary tobacco transformants and their androgenetic progenies

Expression of a transgene is rarely analysed in the androgenetic progenies of the transgenic plants. Here, we report differential transgene expression in androgenetic haploid and doubled haploid (DH) tobacco plants as compared to the diploid parental lines, thus demonstrating a gene dosage effect. Using Agrobacterium-mediated transformation, and bacterial reporter genes encoding neomycin phosphotransferase (nptII) and β-glucuronidase (uidA/ GUS), driven respectively by the mas 1′ and mas 2′ promoters, we have generated more than 150 independent transgenic (R₀) Nicotiana tabacum plants containing one or more T-DNA copies. Transgene analyses of these R₀, their selfed R₁ lines and their corresponding haploid progenies showed an obvious position effect (site of T-DNA insertion on chromosome) on uidA expression. However, transgene (GUS) expression levels were not proportional to transgene copy number. More than 150 haploids and doubled haploids, induced by treatment with colchicine, were produced from 20 independent transgenic R₀ plants containing single and multiple copies of the uidA gene. We observed that homozygous DH plants expressed GUS at approximately 2.9-fold the level of the corresponding parental haploid plants. This increase in transgene expression may be attributed mainly to the increase (2-fold) in chromosome number. Based on this observation, we suggest a strong link between chromosome number (ploidy dosage effect) and transgene expression. In particular, we demonstrate the effect on its expression level of converting the transgene from the heterozygous (in R₀ plants) to the homozygous (DH) state: e.g. an increase of 50% was observed in the homozygous DH as compared to the original heterozygous diploid plants. We propose that ploidy coupled with homozygosity can result in a new type of gene activation, creating differences in gene expression patterns. Received: 27 April 1998 / Accepted: 12 August 1998     

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

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.     

The Influence of Matrix Attachment Regions on Transgene Expression in Transgenic Tobaccos

To investigate the effect of matrix attachment regions (MARs) on levels of transgene expression in transgenic plants, β-glucuronidase (GUS) gene ( uidA ) was flanked by the MARs isolated from the genome of pea ( Pisum sativum L.) to form plant expression vector. The plant expression vectors with and without MARs were transferred into tobaccos ( Nicotiana tabacum L.) via Agrobacterium -mediated transformation procedure. The results of GUS activity showed that MARs could increase levels of uidA gene expression, the mean GUS activity could be increased two fold compared with that of transformants without MARs, the highest GUS activity of transformant could arrive at five fold.     

Self-fertile transgenic wheat plants regenerated from isolated scutellar tissues following microprojectile bombardment with two distinct gene constructs†

A system for enhanced induction of somatic embryo-genesis and regeneration of plants from isolated scutellar tissue of wheat has been developed. This system has been successfully used in the development of a simple and reproducible protocol for the production of self-fertile transgenic wheat plants. The procedure is rapid resulting in the production of transgenic plantlets within 12 weeks from initiation of cultures and it avoids the need for establishing long-term callus, cell suspension or protoplast cultures. Somatic embryos regenerated from scutella bombarded with plasmid pBARGUS were selected on L-phosphinothricin (L-PPT) to obtain herbicide-resistant self-fertile transgenic plants. Phosphinothricin acetyltransferase (PAT) activity was observed at varying levels in 50% of the plants selected on L-PPT whereas none of the plants showed β-glucuronidase (GUS) activity. Molecular analysis of PAT-positive plants confirmed stable integration of both bar and gus genes in R₀ and R₁ progeny plants. Segregation of the PAT activity and herbicide resistance in R₁ progeny plants confirmed the Mendelian inheritance of the bar gene. Additionally, isolated scutella bombarded with plasmid DNA containing a gus::nptII fusion gene driven by a rice actin promoter and its first intron were selected in the presence of geneticin to obtain fully fertile transgenic plants. Functional expression of the fusion gene was demonstrated in transgenic plants by GUS and neomycin phospho-transferase (NPTII) enzyme assays. Southern blot analysis confirmed the integration of transgenes into the wheat genome. Histochemical GUS staining showed transmission of the fusion gene to floral organs of primary transformants and confirmed Mendelian segregation of the transgene in R₁ progeny.