Expression of CaMV35S-GUS gene in transgenic rice plants

Summary To understand the properties of the cauliflower mosaic virus (CaMV) 35S promoter in a monocotyledonous plant, rice (Oryza sativa L.), a transgenic plant and its progeny expressing the CaMV35S-GUS gene were examined by histochemical and fluorometric assays. The histochemical study showed that -glucuronidase (GUS) activity was primarily localized at or around the vascular tissue in leaf, root and flower organs. The activity was also detected in the embryo and endosperm of dormant and germinating seeds. The fluorometric assay of various organs showed that GUS activity in transgenic rice plants was comparable to the reported GUS activity in transgenic tobacco plants expressing the CaMV35S-GUS gene. The results indicate that the level of expression of the CaMV 35S promoter in rice is similar to that in tobacco, a dicotyledonous plant, suggesting that it is useful for expression of a variety of foreign genes in rice plants.     

Transgenic plant production mediated by Agrobacterium in Indica rice

Summary A reproducible system has been developed for the production of transgenic plants in indica rice using Agrobacterium-mediated gene transfer. Three-week-old scutella calli served as an excellent starting material. These were infected with an Agrobacterium tumefaciens strain EHA101 carrying a plasmid pIG121Hm containing genes for -glucuronidase (GUS) and hygromycin resistnace (HygR). Hygromycin (50 mg/l) was used as a selectable agent. Inclusion of acetosyringone (50M) in the Agrobacterium suspension and co-culture media proved to be indispensable for successful transformation. Transformation efficiency of Basmati 370 was 22% which was as high as reported in japonica rice and dicots. A large number of morphologically normal, fertile transgenic plants were obtained. Integration of foreign genes into the genome of transgenic plants was confirmed by Southern blot analysis. GUS and HygR genes were inherited and expressed in R1 progeny. Mendelian segregation was observed in some R1 progeny.Abbreviations GUS ß-glucuronidase - HygR hygromycin-resistance - AS acetosyringone     

Field assessments of gene flow from transgenic to cultivated rice (Oryza sativa L.) using a herbicide resistance gene as tracer marker

Development of plant genetic engineering has led to the deployment of transgenic crops and, simultaneously, to the need for a thorough assessment of the risks associated with their environmental release. This study investigated the occurrence of gene flow from transgenic rice to non-transgenic rice plants under agronomic conditions using a herbicide resistance gene as a tracer marker. Two field experiments were established in the paddy fields of two main Mediterranean rice-growing areas of Spain and Italy. In both locations analyses of phenotypic, molecular and segregation data showed that pollination of recipient plants with pollen of the transgenic source occurred at a significant frequency. A gene flow slightly lower than 0.1% was detected in a normal side-by-side plot design. Similar results were found in a circular plot when the plants were placed at 1-m distance from the transgenic central nucleus. A strong asymmetric distribution of the gene flow was detected among this circle and highest values (0.53%) were recorded following the direction of the dominant wind. A significant lowest value (0.01%) was found in the other circle (5 m from the transgenic plants) as was expected according to the characteristics of rice pollen. Such circular-field trial designs could also prove to be very useful in studying the gene flow to other commercial cultivars of rice with the aim of establishing strategies to prevent pollen dispersal from commercial transgenic fields to the neighbouring conventional fields. Received: 23 February 2001 / Accepted: 31 March 2001     

Expression of the rice Osgrp1 promoter-Gus reporter gene is specifically associated with cell elongation/expansion and differentiation

To study the expression and regulation of a rice glycine-rich cell wall protein gene, Osgrpl, transgenic rice plants were regenerated that contain the Osgrpl promoter or its 5 deletions fused with the bacterial -glucuronidase (GUS) reporter gene. We report here a detailed histochemical analysis of the Osgrpl-Gus expression patterns in transgenic rice plants. In roots of transgenic rice plants, GUS expression was specifically located in cell elongation and differentiation regions, and no GUS expression was detectable in the apical meristem and the mature region. In shoots, GUS activity was expressed only in young leaves or in the growing basal parts of developing leaves, and little GUS activity was expressed in mature leaves or mature parts of developing leaves. In shoot apices, GUS activity was detected only in those leaf cells which were starting to expand and differentiate, and GUS expression was not detected in the apical meristem and the young meristematic leaf primordia. GUS activity was highly expressed in the young stem tissue, particularly in the developing vascular bundles and epidermis. Thus, the expression of the Osgrpl gene is closely associated with cell elongation/expansion during the post-mitotic cell differentiation process. The Osgrpl-Gus gene was also expressed in response to wounding and down-regulated by water-stress conditions in the elongation region of roots. Promoter deletion analysis indicates that both positive and negative mechanisms are involved in regulating the specific expression patterns. We propose a simple model for the developmental regulation of the Osgrpl gene expression.     

Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties

Genetic transformation of rice (Oryza sativa L.) mediated by Agrobacterium ttumefaciens has been confirmed for japonica varieties and extended to include the more recalcitrant indica varieties. Immature embryos were inoculated with either A. tumefaciens At656 (pCNL56) or LBA4404 (pTOK233). Experimental conditions were developed initially for immature embryos treated with strain At656, based upon both transient and stable -glucuromdase (GUS) activities. However, plant regeneration following selection on G418 (pCNL56 contained the nptII gene) did not occur. Using the same basic protocol, but inoculating immature embryos of rice with LBA4404 (pTOK233), resulted in efficient (about 27%) production of transgenic plants of the japonica variety, Radon, and an acceptable efficiency (from 1–5%) for the indica varieties IR72 and TCS10. Transformation was based upon resistance to hygromycin (pTOK233 contains the hpt gene), the presence of GUS activity (from the gusA gene), Southern blots for detection of the integrated gusA gene, and transmission of GUS activity to progeny in a Mendelian 3:1 segregation ratio. Southern blots indicated two to three copies of the gene integrated in most transformants. Transgenic plants of both the japonica and indica varieties were self-fertile and comparable in this respect to seed-grown plants. Key factors facilitating the transformation of rice by Agrobacterium tumefaciens appeared to be the use of embryos as the expiant, the use of hygromycin as the selection agent (which does not interfere with rice regeneration), the presence of extra copies of certain vir genes on the binary vector of pTOK233, and maintaining high concentrations of acetosyringone for inducing the vir genes during co-cultivation of embryos with Agrobacterium.Abbreviations AS acetosyringone - DMRT Duncan's Multiple Range Test - GUS -glucuronidase - T-DNA transferred DNA We wish to thank Dr. Toshihiko Komari, Japan Tobacco Inc. for providing Ayrobacterium tumefaciens strain LBA4404 (pTOK322). Support by the Rockefeller Foundation in the form of a fellowship to R.R.A. and a grant to T.K.H. is acknowledged. This is journal paper number 14,914 from the Purdue University Agricultural Experiment Station.     

Characterization of a rice gene family encoding root-specific proteins

Two cDNA clones (RCc2 and RCc3) corresponding to mRNAs highly expressed only in root tissues of rice (Oryza sativa L.) seedlings were characterized. Respectively, they encode polypeptides of 146 (14.5 kDa) and 133 amino acids (13.4 kDa) that share high (<70%) sequence similarity with a polypeptide encoded by a cDNA (ZRP3) encoding an mRNA preferentially expressed in young maize roots. Genomic DNA blot analysis revealed that they are members of a small gene family and RCg2, the gene corresponding to RCc2, was isolated. A 1656 bp 5-upstream sequence of RCg2 was translationally fused to a -glucuronidase (GUS) reporter gene and stable introduction of the chimeric construct into rice was confirmed by PCR and genomic DNA blot analyses. Histochemical analysis of transgenic rice plants containing the full-length chimeric gene showed high levels of GUS activity in mature cells and the elongation and maturation zones of primary and secondary roots, and in the root caps, but no GUS activity was detected in root meristematic regions. Surprisingly, high GUS activity was also detected in leaves of the same plants. This raises the possibility that the RCg2 5-upstream element may not be sufficient for the proper spatial control of root specificity in transgenic rice.     

Analysis of an ABA-responsive rice gene promoter in transgenic tobacco

We have analyzed in transgenic tobacco the expression of a chimeric gene containing 5 sequences of the rice rab-16B gene fused to the -glucuronidase (GUS) reporter gene. This construct, a translational fusion (–482 to +184) including 14 amino acids of the RAB-16B protein, is expressed only in zygotic and pollen-derived embryos. In zygotic embryos, GUS activity begins to accumulate 10 days after flowering (daf), and increases until seed maturation at 25 daf. Immunological measurements of endogenous abscisic acid (ABA) accumulation in these seeds showed a close parallel between hormone levels and GUS activity. However, GUS activity could not be reproducibly induced by treatment of immature embryos with ABA (10 M). Neither GUS activity nor GUS mRNA could be detected in leaves of transgenic tobacco even after ABA treatment. In contrast, GUS activity could be induced to high levels in pollen-derived embryos by treatment with ABA. Our results show that 482 bp of 5 sequences of the rice rab-16B promoter can confer in transgenic tobacco developmentally regulated expression in embryos but not ABA-responsive expression in vegetative tissues.     

Pollen-mediated transgene flow in the wind-pollinated grass species tall fescue (Festuca arundinacea Schreb.)

Information regarding gene flow in wind-pollinated, outcrossing forage grasses is essential for any future releases of value-added transgenic cultivars. Experiments on pollen dispersal was carried out by growing transgenic tall fescue (Festuca arundinacea) in a central plot, surrounded by exclosures containing recipient plants up to a distance of 200 m from the central source plants in eight directions. The central transgenic tall fescue plants carried a chimeric hygromycin phosphotransferase gene (hph) and a chimeric -glucuronidase gene (gusA). Seeds were collected from the recipient plants and germinated seedlings were used for high throughput DNA isolation and polymerase chain reaction (PCR) analysis. More than 21,000 seedlings were PCR analyzed for the experiments conducted in three years. Transgenes were detected in recipient plants at up to 150 m from the central transgenic plot. The highest transgene frequencies, 5% at 50 m, 4.12% at 100 m and 0.96% at 150 m, were observed north of the central plot, the prevailing wind direction. Lower transgene frequencies were detected in other directions, particularly at 100 m and 150 m distances. No transgene was detected at 200 m distance in any direction. Transgene flow was less effective or ineffective when recipient plants were further away from the central donor plants. Southern blot hybridization analysis confirmed the transgenic nature of the PCR positive plants. A supplementary experiment demonstrated that transgene flow can be controlled by placing transgenic plantings downwind and long distances from non-transgenic seed increases, thus allowing tall fescue breeding and transgene development programs to be conducted concurrently at the same research station.     

Inheritance of gusA and neo genes in transgenic rice

Inheritance of foreign genes neo and gusA in rice (Oryza sativa L. cv. IR54 and Radon) has been investigated in three different primary (T₀) transformants and their progeny plants. T₀ plants were obtained by co-transforming protoplasts from two different rice suspension cultures with the neomycin phosphotransferase II gene [neo or aph (3) II] and the -glucuronidase gene (uidA or gusA) residing on separate chimeric plasmid constructs. The suspension cultures were derived from callus of immature embryos of indica variety IR54 and japonica variety Radon. One transgenic line of Radon (AR2) contained neo driven by the CaMV 35S promoter and gusA driven by the rice actin promoter. A second Radon line (R3) contained neo driven by the CaMV 35S promoter and gusA driven by a promoter of the rice tungro bacilliform virus. The third transgenic line, IR54-1, contained neo driven by the CaMV 35S promoter and gusA driven by the CaMV 35S.Inheritance of the transgenes in progeny of the transgenic rice was investigated by Southern blot analysis and enzyme assays. Southern blot analysis of genomic DNA showed that, regardless of copy numbers of the transgenes in the plant genome and the fact that the two transgenes resided on two different plasmids before transformation, the introduced gusA and neo genes were stably transmitted from one generation to another and co-inherited together in transgenic rice progeny plants derived from self-pollination. Analysis of GUS and NPT II activities in T₁ to T₂ plants provided evidence that inheritance of the gusA and neo genes was in a Mendelian fashion in one plant line (AR2), and in an irregular fashion in the two other plant lines (R3 and IR54-1). Homozygous progeny plants expressing the gusA and neo genes were obtained in the T₂ generation of AR2, but the homozygous state was not found in the other two lines of transgenic rice.     

The rice metallothionein gene promoter does not direct foreign gene expression in seed endosperm

We generated transgenic tobacco and rice plants harboring a chimeric gene consisting of the 5-upstream sequence of the rice metallothionein gene (ricMT) fused to the -glucuronidase (GUS) gene. The activity and tissue-specific expression of the ricMT promoter were demonstrated in these transgenic plants. In the transgenic rice plants, despite substantial levels of GUS activity in the shoot and root, almost no GUS signal was detected in the endosperm. Thus, the ricMT promoter could be useful in avoiding accumulation of undesired proteins in the seed endosperm.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment

We compared rice transgenic plants obtained by Agrobacterium-mediated and particle bombardment transformation by carrying out molecular analyses of the T₀, T₁ and T₂ transgenic plants. Oryza sativa japonica rice (c.v. Taipei 309) was transformed with a construct (pWNHG) that carried genes coding for neomycin phosphotransferase (nptII), hygromycin phosphotransferase (Hyg^r), and -glucuronidase (GUS). Thirteen and fourteen transgenic lines produced via either method were selected and subjected to molecular analysis. Based on our data, we could draw the following conclusions. Average gene copy numbers of the three transgenes were 1.8 and 2.7 for transgenic plants obtained by Agrobacterium and by particle bombardment, respectively. The percentage of transgenic plants containing intact copies of foreign genes, especially non-selection genes, was higher for Agrobacterium-mediated transformation. GUS gene expression level in transgenic plants obtained from Agrobacterium-mediated transformation was more stable overall the transgenic plant lines obtained by particle bombardment. Most of the transgenic plants obtained from the two transformation systems gave a Mendelian segregation pattern of foreign genes in T₁ and T₂ generations. Co-segregation was observed for lines obtained from particle bombardment, however, that was not always the case for T₁ lines obtained from Agrobacterium-mediated transformation. Fertility of transgenic plants obtained from Agrobacterium-mediated transformation was better. In summary, the Agrobacterium-mediated transformation is a good system to obtain transgenic plants with lower copy number, intact foreign gene and stable gene expression, while particle bombardment is a high efficiency system to produce large number of transgenic plants with a wide range of gene expression.     

Inheritance of a co-transferred foreign gene in the progenies of transgenic rice plants

The integration pattern and the inheritance of exogenous DNA in transgenic rice plants were analysed. Plasmid pCH (4.8 kb), that contains chimaeric cauliflower mosaic virus 35S promoter-hygromycin phosphotransferase structural gene, and plasmid pGP400 (7.2 kb), possessing oat phytochrome promoter and structural gene of bacterial -glucuronidase, were co-transferred into protoplasts of rice (Oryza sativa L.) plants via electroporation. Primary transformants (T₀ generation) and their progenies (T₁, T₂ and T₃) were selected by hygromycin B. Southern blot analysis of inserted genes in transgenic rice plants suggests the integration of an intact hygromycin phosphotransferase gene and non-functional DNA fragments into host genome. Co-inheritance of the hygromycin phosphotransferase gene and -glucuronidase gene was also observed. There were no significant differences in terms of the morphology and size of seeds between untransformed and transgenic plants (T₃ generation).     

Intron position affects expression from thetpi promoter in rice

A series of promoter-GUS fusion constructs containing a portion of the rice triosephosphate isomerase (tpi) promoter, the firsttpi intron, and the gene encoding bacterial -glucuronidase (GUS) were made. These constructs were electroporated into rice protoplasts and transient expression was monitored. Inclusion of the first intron from the ricetpi gene enhanced expression of the GUS gene from thetpi promoter when it was placed 5 of the GUS gene. When thetpi intron was placed in the 3-untranslated region no enhancement of GUS gene expression was observed, indicating the importance of position in intron-mediated enhancement of gene expression.     

Investigation of rice transgene flow in compass sectors by using male sterile line as a pollen detector

Rice is the most important staple food in the world. The rapid development of transgenic rice and its future commercialization have raised concerns regarding transgene flow and its potential environmental risk. It is known that rice is a self-pollinated crop; the outcrossing rate between common cultivars is generally less than 1%. In order to improve the detection sensitivity of rice transgene flow, a male sterile (ms) line BoA with a high outcrossing rate was used as a pollen detector in this study. A concentric circle design was adopted, in which the transgenic rice B2 containing bar gene as a pollen donor was planted in the center circle and the recipient BoA was planted in eight compass sectors. The frequency of transgene flow in compass sectors was analyzed by continuous sampling to generate cumulative data. The results of two years with sound reproducibility demonstrated that the rice gene flow was closely associated with the wind direction. According to the mean frequency of transgene flow, the eight sectors can be divided into two groups: a higher frequency group downstream of the prevailing wind (DPW) with a mean frequency ranging from 6.47 to 26.24%, and a lower frequency group lateral to or upstream of the prevailing wind (UPW) with a mean frequency of 0.39 to 3.03%. On the basis of the cumulative data, 90–96% of the cumulative gene flow events occurred in the four DPW sectors, while it was 4–10% in the four UPW sectors. By using these systematic data, simulation models and isograms of transgene flow in the eight compass sectors were calculated and drawn, respectively. Q. H. Yuan, L. Shi and S. R. Jia contributed equally to the work.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.     

Bt水稻中crylAb基因的遗传分析

用PCR、GUS染色和Western点杂交技术检测了Bt水稻杂交后代群体,在394株GUS阳性株中,有392株表达Bt蛋白,协同表达株率达99.49%。由此表明,在杂交后代中报告基因gus和目的基因crylAb紧密连锁遗传与表达。GUS组织染色和Southern杂交检测表明Bt水稻中的crylAb基因呈单位点显性基因遗传,且在有性世代中能稳定传递,还发现,在BC1、BC1F2和粳粳交F2群体中crylAb基因呈单位点显性基因遗传,而在籼粳交F2群体中偏离3：1分离。     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%–30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.