Introduction of a chimeric gene encoding an oryzacystatin-β-glucuronidase fusion protein into rice protoplasts and regeneration of transformed plants

In order to construct transgenic rice plant with an introduced oryzacystatin (OC)--glucuronidase (GUS) fusion gene, we first introduced it into rice protoplasts by electroporation, together with a marker gene conferring hygromycinresistance (pUC-HPH). In a transient assay using the transfected protoplasts, both OC and GUS activities were detected. The GUS activity was higher when the OC-GUS fusion protein was expressed than when only a single GUS protein was expressed. Next, to isolate stable transformants, hygromycin-resistant calli were selected. Forty one out of 116 hygromycin-resistant calli expressed a 2.2 kb mRNA transcribed from the chimeric gene and their extracts exhibited the activities of both OC and GUS. Finally, the transgenic calli were regenerated into rice plants whose tissues (leaves, roots and seeds) exhibited GUS activity probably derived from the fusion protein.     

Introduction and constitutive expression of a tobacco hornworm (<Emphasis Type="Italic">Manduca sexta</Emphasis>) chitinase gene in soybean

Summary Embryogenic soybean [Glycine max (L.) Merrill] cultures were transformed with a Manduca sexta chitinase (msc) gene using microprojectile bombardment. A 1.7 kb DNA fragment encoding a tobacco hornworm chitinase was cloned into the rice transformation vector pGL2, under the control of the maize ubiquitin promoter and linked to the hpt gene as a selectable marker. After bombardment, hygromycin-resistant tissues were isolated and cultured to give rise to clones of transgenic material. Four hygromycin-resistant clones were converted into plants. Two clones were positive for the msc gene via polymerase chain reaction (PCR) and Southern blot analysis. The integration inheritance, and expression of transgenes were confirmed by molecular analysis of transgenic soybean plants. Progeny analysis showed that the introduced genes were inherited and segregated in a 3:1 Mendelian fashion. DNA blot experiments and progeny inheritance analysis indicated that the plants contained several copies of the msc gene and that the insertion occurred at a single locus. Northern blotting analysis confirmed the expression of the transgenes. Western blot analysis of transgenic plants and their progeny revealed the presence of a protein with a molecular weight of 48kDa that reacted with the Manduca sexta antibody. Progeny from the chitinase-positive plants were tested for their resistance to the soybean cyst nematode. Plants expressing the insect chitinase did not manifest enhanced resistance to the soybean cyst nematode.     

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.     

Oral immunization with transgenic rice seeds expressing VP2 protein of infectious bursal disease virus induces protective immune responses in chickens

The expression of infectious bursal disease virus (IBDV) host-protective immunogen VP2 protein in rice seeds, its immunogenicity and protective capability in chickens were investigated. The VP2 cDNA of IBDV strain ZJ2000 was cloned downstream of the Gt1 promoter of the rice glutelin GluA-2 gene in the binary expression vector, pCambia1301-Gt1. Agrobacterium tumefaciens containing the recombinant vector was used to transform rice embryogenic calli, and 121 transgenic lines were obtained and grown to maturity in a greenhouse. The expression level of VP2 protein in transgenic rice seeds varied from 0.678% to 4.521% µg/mg of the total soluble seed protein. Specific pathogen-free chickens orally vaccinated with transgenic rice seeds expressing VP2 protein produced neutralizing antibodies against IBDV and were protected when challenged with a highly virulent IBDV strain, BC6/85. These results demonstrate that transgenic rice seeds expressing IBDV VP2 can be used as an effective, safe and inexpensive vaccine against IBDV.     

转AtNHX1基因玉米的产生及其耐盐性分析

以玉米(ZeamaysL.)骨干自交系DH4866、齐319和鲁原16106的胚性愈伤组织为材料,采用农杆菌介导法将AtNHX1和hpt基因转入玉米培养细胞,经筛选获得了抗潮霉素的愈伤组织并再生植株。经PCR检测和Southernblot验证,确定了22.8%的再生植株为转基因植株。农杆菌液浓度、愈伤组织基因型及共培养时间对转化率均有明显影响。外源基因在转基因植株后代中的分离呈多样性,在部分株系中表现出孟德尔遗传规律。耐盐筛选表明,一些转基因植株及其后代具有很好的耐盐性,部分株系可在0.8%-1.0%NaCl溶液浇灌下萌发和生长。Northern杂交表明,植株耐盐性提高与AtNHX1基因的转录水平相一致。     

Rapid production of fertile transgenic barley (Hordeum vulgare L.) by direct gene transfer to primary callus-derived protoplasts

Protoplasts were isolated from primary calli of barley (Hordeum vulgare L.), and an antibiotic (G418) resistance gene was introduced into these protoplasts using a polyethylene glycol (PEG) DNA uptake method. Sixty-four G418 resistant calli were obtained in nine experiments, and two plants were regenerated from these calli. NPTII ELISA and Southern analysis indicated that the G418 resistance gene was introduced and expressed in two T₀ plants. These plants set seed and the introduced gene was transmitted to T₁ plants. These results suggest that our transformation system using primary callus-derived protoplasts is a useful method for the generation of transgenic barley. Received: 14 November 1997 / Revision received: 12 March 1998 / Accepted: 24 April 1998     

Visualization of somatic deletions mediated by R/<Emphasis Type="Italic">RS</Emphasis> site-specific recombination and induction of germinal deletions caused by callus differentiation and regeneration in rice

A transgenic rice plant expressing the recombinase of Zygosaccharomyces rouxii under the control of the CaMV 35S promoter was crossed with a transgenic plant carrying a cryptic (beta-glucuronidase) GUS reporter gene, which was activated by recombinase-mediated deletions between two specific recombination sites ( RSs). In F(1) plants, GUS activity was observed as blue spots and stripes in vascular bundles in several parts of the leaves. GUS expression was detected in all of the calli induced from F(1) seeds and throughout the regenerated plants. DNA analysis using the polymerase chain reaction and Southern blotting showed that R/ RS-mediated deletions occurred in all of the cells of the regenerated plants. Stable GUS expression was confirmed in the progeny resulting from self-pollination. Thus, the deletions obtained in the regenerated plants were genetically equivalent to the germinal deletions. These results indicate that the induction of callus differentiation and shoot regeneration is an effective manner to activate the R/ RS system and to produce plants with chromosomal deletions.     

Expression of Rice Stripe Virus Coat Protein Gene in Transgenic Rice Plants

Rice stripe virus (RSV) is a pathogen of rice stripe disease causing great damage to rice. The disease is transmitted by Laodelphax striatellus and three other planthoppers. RSV infects as much as 37 cereals including rice, wheat, maize and results in a significant reduction in yield in epidemic year. In order to develop efficient means of controlling the disease, authors have studied the amino acid composition of RSV coat protein (CP), synthesized and cloned the cDNA to CP, sequenced the full-length CP gene. Having inserted the RSV CP gene into plant expression vector pROK Ⅱ, authors transformed rice suspension culture via microprojectile bombardment and obtained transgenic plants expressing the CP gene. The suspension culture was initiated by inoculating yellowish, compact and embryogenic calli derived from seeds into suspension medium containing proline and maltose. After being cultured at 26℃ in the dark for about half a year, finely-dispersed and embryogenic suspension culture was estabolished. Before bombardment the suspension culture was evently applied onto three-layered filter-paper discs in a petri dish. CaCl2 and spermidine was employed to coat tungsten particle with plasmid DNA. 2.5 μl of coated particle was loaded onto bullet and each dish was bombarded three times. Immediately after being bombarded, the suspensions were cultured in modified N6 medium. 2 days later the suspensions were transferred to the same medium but containing G418, which were subcultured weekly. Being subject to G418 selection for two months, white and fast-growing clones were emerged from the brownish cultures. Green plants regenerated when the resistant calli were transferred to differentiation medium. The regenerated plants were firm enough to grow well in the greenhouse. 10 plants regenerated from G418 resistant calli were tested for their transformed nature by Southern blot using 32P-labelled CP gene as a probe. Among the plants tested, 2 plants showed clearly hy bridizing bands with a molecular weight corresponding to RSV CP gene. Western blot further demonstrated that RSV CP gene was expressed in transgenic rice plants. At present tests on the antiviral effects of transgenic plants by feeding plantphoppers infccted with RSV are being underway.     

转AtNHX1基因玉米的产生及其耐盐性分析

以玉米(Zea mays L.)骨干自交系DH4866、齐319和鲁原16106的胚性愈伤组织为材料,采用农杆菌介导法将AtNHX1和hpt因转入玉米培养细胞,经筛选获得了抗潮霉素的愈伤组织并再生植株.经PCR检测和Southernblot验证,确定了22.8%的再生植株为转基因植株.农杆菌液浓度、愈伤组织基因型及共培养时间对转化率均有明显影响.外源基因在转基因植株后代中的分离呈多样性,在部分株系中表现出孟德尔遗传规律.耐盐筛选表明,一些转基因植株及其后代具有很好的耐盐性,部分株系可在0.8%-1.0%NaCl溶液浇灌下萌发和生长.Northern杂交表明,植株耐盐性提高与AtNHX1基因的转录水平相一致.     

Improvement of rice transformation using bombardment of scutellum-derived calli

Although several reports on rice transformation have been published, producing transgenic plants ofIndica rice varieties is still problematic. We report an improved protocol for transformingIndica rice genotypes. An important agronomic MexicanIndica rice variety, Morelos A-92, was used. Calli derived from scutellum seeds were produced by using auxins and bombarded with 2 vectors, one harboring the reporteruidA gene and the other with thehptII gene conferring hygromycin resistance. The influence of the molar relation of these vectors (uidA-hptIII) in generating callus and plants expressing theuidA reporter gene was analyzed. Selection of bombarded calli was performed under 2 conditions: 50 mg/L of hygromycin with 3 subcultures and 80 mg/L of hygromycin with no subcultures. The best conditions were a 20∶1uidA-hptII molar vector relationship and selection at 80 mg/L of hygromycin, producing 14% of calli expressing GUS. The minimal callus size in regenerating plants was 3 mm. Transformed rice plants were generated with 4.6% efficiency, considering the initial number of bombarded calli. Heredity of theuidA gene behaved as a single locus in transformed rice plants. Homozygous plants were identified in the T₁ generation by means of pollen staining.     

A polyethylene glycol-mediated protoplast transformation system for production of fertile transgenic rice plants

We have established an efficient procedure for protoplast transformation and regeneration of fertile transgenic plants of rice (Oryza sativa L.) cultivars Nipponbare and Taipei 309. Protoplasts were mixed with a plant-expressible hygromycin resistance gene and treated with 25% (w/v) polyethylene glycol. Stringent selection of transformed colonies was applied to 14-day-old regenerated protoplasts in the presence of 95 micromolar of hygromycin B for 12 days. After selection, 450 and 200 resistant colonies were recovered per million treated Taipei 309 and Nipponbare protoplasts, respectively. Southern hybridization analysis of hygromycin-resistant cell lines and regenerated plants indicated that 1 to 10 copies of transferred DNA were integrated at 1 to 4 loci of the rice genome. Southern DNA analysis suggests that the introduced plasmid DNA may form concatemers by intermolecular recombination prior to integration. Four Taipei 309 and 39 Nipponbare transgenic rice plants were regenerated and grown to maturity in the greenhouse. Two Taipei 309 and 35 Nipponbare plants set viable seeds. Agronomic traits of Taipei 309 transgenic plants and inheritance of the hygromycin resistance trait by progeny of the selfed transgenic plants were analyzed.     

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

Recovery of transgenic rice plants expressing the rice dwarf virus outer coat protein gene (S8)

 The coding region of the eighth largest segment (S8) of the rice dwarf virus (RDV) was obtained from a RDV Fujian isolate. It was then cloned into pTrcHisA for expression in E. coli and into vector pE3 for plant transformation. By using callus derived from mature rice embryos as the target tissue, we obtained regenerated rice plants after bombardment of the former with plasmid pE3R8 containing the RDV S8 gene and the marker gene neomycin phosphotransferase (NPT II). Southern blotting confirmed the integration of the RDV S8 gene into the rice genome. The expression of the outer coat protein in both E. coli and rice plants was confirmed by western blotting. The recovery of transgenic rice plants expressing S8 gene is an important step towards studying the function of the RDV genes and obtaining RDV-resistant rice plants. Received: 1 March 1996 / Accepted: 2 August 1996     

Transfer of a grapevine stilbene synthase gene to rice (Oryza sativa L.)

A gene derived from grapevine (Vitis vinifera) coding for stilbene synthase has been transferred into protoplasts of the commercially important japonica rice cultivar Nipponbare using PEG-mediated direct gene transfer. Transgenic plants were regenerated from calli selected on kanamycin. Southern blot analysis of genomic DNA isolated from regenerants and progeny plants demonstrated that the stilbene synthase gene is stably integrated in the genome of transgenic rice plants and inherited in the offspring. The transient formation of stilbene-synthase-specific mRNA shortly after inoculation with the fungus of the rice blast Pyricularia oryzae has demonstrated that the grapevine stilbene synthase promoter is also active in monocotyledonous plants. Preliminary results indicate an enhanced resistance of transgenic rice to P. oryzae. Received: 1 July 1996 / Revision received: 5 November 1996 / Accepted: 30 November 1996     

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.     

In Planta visual monitoring of green fluorescent protein in transgenic rice plants

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a widely used reporter that can be directly visualized in the living cells in both animals and plants. We inserted a synthetic gene (sgfp) encoding a modified form of the GFP into expression vector, Act1-sgfp for the direct expression of GFP which is easily detectable in rice plants. Green fluorescence emitted from GFP could be visualized in calli, dry seeds, roots and seedlings with green shoots of transgenic rice plants. In our visualization system with a charge-coupled device camera, band-pass filters and a light source, the presence of red chlorophyll autofluorescence from chloroplasts did not alter the green fluorescence of GFP. These results demonstrate that GFP could be used as a non-destructive visual selection marker for examining gene expression in transformed calli, dry seeds and young plants.     

高赖氨酸蛋白基因导入水稻及可育转基因植株的获得

构建了一个植物高效表达质粒,使来源于四棱豆（Psophocarpus tetragonolobus(L.)DC)的高赖氨酸蛋白基因（lys）受控于单子叶植物ubiqutin强启动子下表达。用基因枪法将其导入水稻(Oryza sativa L.)幼胚诱导的愈伤组织,经潮霉素抗性筛选,得到可育的再生植株。经PCR和Southem blotting检测,表明该基因已整合到水稻的基因组织。GUS组织化学染色表明转基因水稻植株的叶、茎和根中均有gus基因的表达。测定112株转基因水稻叶片中赖氨酸叶量,大部分植株有不同程度的提高,最高幅度为16．04％。     

Fertile transgenic barley generated by direct DNA transfer to protoplasts

We report the generation of transgenic barley plants via PEG-mediated direct DNA uptake to protoplasts. Protoplasts isolated from embryogenic cell suspensions of barley (Hordeum vulgare L. cv Igri) were PEG-treated in a solution containing a plasmid which contained the neomycin phosphotransferase (NPT II) gene under the control of the rice actin promoter and the nos terminator. Colonies developing from the treated protoplasts were incubated in liquid medium containing the selective antibiotic G418. Surviving calli were subsequently transferred to solid media containing G418, on which embryogenic calli developed. These calli gave rise to albino and green shoots on antibiotic-free regeneration medium. NPT II ELISA revealed that approximately half of the morphogenic calli expressed the foreign gene. In total, 12 plantlets derived from NPT-positive calli survived transfer to soil. Southern hybridization analysis confirmed the stable transformation of these plants. However, the foreign gene seemed to be inactivated in plants from one transgenic line. Most of the transgenic plants set seed, and the foreign gene was transmitted and expressed in their progenies, which was ascertained by Southern hybridization and NPT II ELISA.