Evidence for genomic changes in transgenic rice (Oryza sativa L.) recovered from protoplasts

The occurrence of genomic modifications in transgenic rice plants recovered from protoplasts and their transmission to the self-pollination progeny has been verfied with the random amplified polymorphic DNA (RAPD) approach. The plant was the Indica-type rice (Oryza sativa L.) cultivar Chinsurah Boro II. The analysed material was: (1) microspore-derived embryogenic rice cells grown in suspension culture, (2) transgenic plants recovered from protoplasts produced from the cultured cells and (3) the self-pollination progeny (two successive generations) of the transgenic plants. DNA purified from samples of these materials was PCR-amplified with different random oligonucleotide primers and the amplification products were analysed by agarose gel electrophoresis. Band polymorphism was scored and used in band-sharing analyses to produce a similarity matrix. Relationships among the analysed genomes were expressed in a dendrogram.The extensive DNA changes evidenced in cultured cells demonstrate the occurrence of somaclonal variation in the material used to produce protoplasts for gene transfer. Quantitatively reduced DNA changes were also found in the resulting transgenic plants and i their self-pollination progenies.While confirming the stability of the foreign gene in transgenic plants, this work gives molecular evidence for the occurrence of stable genomic changes in transgenic plants and points toin vitro cell culture as the causative agent. RAPDs are shown to be a convenient tool to detect and estimate the phenomenon at the molecular level. The methodology is also proposed as a fast tool to select those transgenic individuals that retain the most balanced genomic structure and to control the result of back-crosses planned to restore the original genome.     

Transformation of Brazilian eliteIndica-type rice (Oryza sativa L.) by electroporation of shoot apex explants

We have generated transgenic plants of a Brazilian elite Indica-type rice by electroporation of shoot apices. This approach avoids a callus phase and produces 0.4–13.8% resistant plants. Transgenic plantlets were transferred to soil a few weeks after explant electroporation. Root segments from plantlets obtained from transformation experiments with pAHC25 plasmid were GUS positive. Integration of the introduced gene into the genome was demonstrated by PPT and antibiotic screening as well as by PCR and Southern blot hybridization of genomic DNA isolated from R₂ plantlets.     

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     

The Mutant Form of Acetolactate Synthase Genomic DNA from Rice is an Efficient Selectable Marker for Genetic Transformation

The proper use of a marker gene in a transformation process is critical for the production of transgenic plants. However, consumer concerns and regulatory requirements raise an objection to the presence of exogenous DNA in transgenic plants, especially antibiotic-resistant genes and promoters derived from viruses. One approach to overcome this problem is the elimination of marker genes from the plant genome by using several site-specific recombination systems. We propose an alternative method to solve this problem using a marker gene exclusively derived from the host plant DNA. We cloned a genomic DNA fragment containing regulatory and coding sequences of acetolactate synthase (ALS) gene from rice, and mutagenized the ALS gene into a herbicide-resistant form. After transfer of this construct to the rice genome, transgenic plants were efficiently selected with a herbicide, bispyribac-sodium salt, which inhibits the activity of wild type ALS. We also analyzed the regulatory feature of the rice ALS gene promoter with the gusA reporter gene and revealed that GUS expression was observed constitutively in aerial parts of rice seedlings and root tips. The marker system consisted exclusively of host plant DNA and enabled efficient selection in a monocot crop plant, rice. The selection system can potentially be applied to generate transgenic plants of other crop species and can be expected to be publicly acceptable.     

Fertile Transgenic Indica Rice Produced by Expression of Maize Ubiquitin Promoter-bar Chimaeric Gene in the Protoplasts

We report production of fertile transgenic Indica rice plants by transferring a chimaeric construct consisting of promoter, first exon and intron of maize ubiquitin gene (Ubi-1) and the coding sequences of the bar gene from Streptomyces hygroscopicus to the rice protoplasts through electroporation. In total, 11 plants were regenerated. All of them were fertile and set seeds on maturity. These plants were resistant to high concentration of PPT (400 mg l^?1) which was otherwise toxic to the untransformed controls. The gene was inherited to the progenies of the five plants in Mendelian ratio.     

Increased baculovirus susceptibility of armyworm larvae feeding on transgenic rice plants expressing an entomopoxvirus gene.

We have introduced an entomopoxvirus gene encoding a virus enhancing factor (EF) into rice, which resulted in high-level accumulation of the EF in the transgenic plants. The introduced gene was stably inherited in the progeny of the primary transformants, as shown by analysis of their genomic DNA. Bioassays for insect susceptibility to baculovirus infection showed that armyworm larvae feeding on the transgenic rice had increased susceptibility to a Nucleopolyhedrovirus. Thus, introduction of the EF gene into plants can be used as a strategy to increase the effectiveness of baculoviruses in insect pest management.     

Transgenic rice plants produced by electroporation-mediated plasmid uptake into protoplasts

Transgenic rice plants have been regenerated by somatic embryogenesis from cell suspension derived protoplasts electroporated with plasmid carrying the NPTII gene under the control of the 35S promoter from cauliflower mosaic virus. Heat shock of protoplasts prior to electroporation maximised the throughput of kanamycin resistant colonies. Omission of kanamycin from the medium for plant regeneration was essential for the recovery of transgenic rice plants carrying the NPTII gene. This report of the production of kanamycin resistant transgenic rice plants establishes the use of protoplasts for rice genetic engineering.Abbreviations NPTII neomycin phosphotransferase - SDS sodium dodecyl sulphate     

转基因水稻T—DNA侧翼序列的扩增与分析

利用现有的转抗白叶枯病基因Xa21的水稻材料,通过TAIL－PCR技术扩增出携带Xa21基因的T－DNA的侧翼序列,对24个有效扩增片段的序列分析结果表明,其中14个侧翼序列是水稻DNA,9个含载体主干序列,1个是外源基因Xa21片段,14个T－DNA侧翼的水稻DNA序列与直接转化法外源基因整合位点的基因组序列具有不同的特点,这些T－DNA在水稻染色体上整合后其两端序列的特点类似于在转基因双子叶植物中观察到的现象,在含主干序列的侧翼序列（37．5％,9／24）,中,载体主干序列是以不同的类型出现的。     

A protocol for consistent, large-scale production of fertile transgenic rice plants

A protocol for consistent production of fertile transgenic rice plants was established utilizing microparticle bombardment of embryogenic tissues (Oryza sativa L. japonica cv. Taipei 309). This system has been employed to produce several thousand independently transformed plant lines carrying the hygromycin phosphotransferase (hph) gene and various genes of interest. The most efficient target tissue was highly embryogenic callus or suspension cell aggregates, when they were given an osmotic pre- and post-transformation treatment of 0.6 m carbohydrate. By optimizing the age of the tissue at the time of gene transfer and applying an improved selection procedure, transgenic plants were recovered in 8 weeks from the time of gene transfer, at an average of 22.3±9.7 per 100 calli and 22.4±8.0 plant lines per dish of suspension cell aggregates. This system has facilitated a number of studies using rice as a model for genetic transformation and will enable the large-scale production of transgenic rice plants for genomic studies. Received: 12 March 1998 / Revision received: 5 May 1998 / Accepted: 15 May 1998     

Application of novel nanotechnology strategies in plant biotransformation: a contemporary overview

During the past epoch we have gone through the remarkable progress in plant gene transformation technology. The production of transgenic plants is considered as a valuable tool in plant research and the technology is extensively applied in phytomedicines and agricultural research. Gene transformation in plants is normally carried out by Agrobacterium species, application of some chemicals and physical techniques (electroporation, microprojectile, etc.). Now a days with better efficacy and reproducibility, novel technologies for the direct gene transfer like liposome, positively charged liposome (lipofectin) and nanoparticle based delivery systems are used for genetic transformation of plants. In this review, we have enlightened the novel nanotechnologies like liposome, Carbon nano-tube and nanoparticles with their current status and future prospects in transgenic plant development. Moreover, we have also highlighted the limitations of conventional techniques of gene transfer. Furthermore, we have tried to postulate innovative ideas on the footprints of established nanotechnology and chemical based strategy with improved efficacy, reproducibility and accuracy along with less time consumption.     

Efficient transformation of wheat by using a mutated rice acetolactate synthase gene as a selectable marker

Acetolactate synthase (ALS) is a target enzyme for many herbicides, including sulfonylurea and imidazolinone. We investigated the usefulness of a mutated ALS gene of rice, which had double point mutations and encoded an herbicide-resistant form of the enzyme, as a selectable marker for wheat transformation. After the genomic DNA fragment from rice containing the mutated ALS gene was introduced into immature embryos by means of particle bombardment, transgenic plants were efficiently selected with the herbicide bispyribac sodium (BS). Southern blot analysis confirmed that transgenic plants had one to more than ten copies of the transgene in their chromosomes. Adjustment of the BS concentration combined with repeated selection effectively prevented nontransgenic plants from escaping herbicide selection. Measurement of ALS activity indicated that transgenic plants produced an herbicide-resistant form of ALS and therefore had acquired the resistance to BS. This report is the first to describe a selection system for wheat transformation that uses a selectable marker gene of plant origin.     

Ac as a tool for the functional genomics of rice

To examine whether the maize autonomous transposable element Ac can be used for the functional analysis of the rice genome, we used Southern blot analysis to analyze the behaviour of Ac in 559 rice plants of four transgenic families through three successive generations. All families showed highly active transposition of Ac, and 103 plants (18.4%) contained newly transposed Ac insertions. In nine of the 12 independent transpositions analyzed, their germinal transmission was detected. Partial sequencing of 99 Ac-flanking sequences revealed that 21 clones exhibited significant similarities with protein-coding genes in databases and four of them matched rice cDNA sequences. These results indicate preferential Ac transposition into protein-coding rice genes. To examine the feasibility of PCR-based screening of gene knockouts in rice Ac plants, we prepared bulked genomic DNA from the leaves of approximately 6000 rice Ac plants and pooled the DNA according to a three-dimensional matrix. Of 14 randomly selected genes, two gene knockouts were identified, and one encoding a rice cytochrome P450 (CYP86) gene was shown to be stably inherited to the progeny. Together, these results suggest that Ac can be efficiently used for the functional analysis of the rice genome.     

Fertile transgenic Indica rice plants obtained by electroporation of the seed embryo cells

We have obtained fertile transgenic plants of Indica rice variety IR36, by using electroporation to transfer the neomycin phosphotransferase II (nptII) gene into cells of mature embryos. Resistant calli were selected in the presence of 30 g/ml G418. Nearly thirty transgenic plants were regenerated within three months after transformation. Many of them yielded seeds following self-pollination. Data from molecular analysis and enzyme assay proved that the foreign gene was stably integrated into the genome of resistant calli, R0 and R1 plants, and also expressed. Mendelian segregation of the nptII gene was observed in R1 progeny plants.Abbreviations NOS nopaline synthase - NPTII and nptII neomycin phosphotransferase II - OCS octopine synthase - Km kanamycin     

转水稻NibT基因玉米植株的获得及抗病性研究

以玉米(Zea mays L.)自交系'金黄96B'为受体材料,供体为质粒pWM101并携带有水稻矮缩病病毒复制酶基因Nib的提前终止突变体基因NibT,采用超声波处理花粉介导植物基因转化方法将NibT基因导入受体,经PCR检测和Southern杂交分析证实获得转基因植株,进而对T1～T3代转基因植株(株系)进行分子分析、田间抗病鉴定和农艺性状调查.逐代分子检测分析结果证明,目的基因可稳定遗传.抗病鉴定结果证明转基因植株(株系)各代抗病水平基本一致,抗病性比对照提高3级.农艺性状调查分析表明,与对照比较,转基因植株株高增加7～18 cm、穗位高增高0～13 cm、穗长增加0.7～2.1 cm、穗粒数多8～35粒、百粒重增加1.1～2.6 g,转基因株系与阴性对照间、各代转基因株系相互间都差异显著(P＜0.05);调查还发现转基因植株的株高和穗位高随着世代的增加,与对照间的差异逐代减少.研究也说明,超声波处理花粉介导植物基因转化方法是一种简捷、快速和有效的植物转化工具.     

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).     

Transformation system of rice suspension-cultured microcolonies by Electroporation

For establishing a transformation system of rice (Oryza sativa), after three days of culture embryogenic suspension-cultured cell clusters were enzymatically macerated for 2 hours in electroporation buffer containing 2% cellulase and filtered through 550, 400, 250 and 100 μm stainless mesh. Filtered embryogenic microcolonies of 100–250 μm with pBI121 were electroporated at 400 V/cm for 1.2 ms. Four weeks after the electroporation, stable transformed calli were obtained at a frequency of 72% on the selection medium containing 100 mg/L kanamycin. GUS gene in the genomic DNA among 20 out of 22 putative transformed calli lines were detected by PCR analysis. The expression of GUS gene into the kanamycin-resistance calli was confirmed by spectrophotometric assay and histochemical assay of GUS activity. In a histochemical study of the transgenic rice regenerants, it was shown that the GUS activity directed by the CaMV 35S promoter was localized mainly in leaf vein and root apex.     

以基因枪介导获转ps1—barnase基因的工程雄性不育水稻植株

以ps1-barnase(brn)为目的基因,pHcintG(PG)为选择/标记基因进行共转化,以PDS-1000-氦气基因枪介导,将brn及PG基因转化到水稻台北309及秋光的核DNA中,得到了转ps1-barnase基因的工程雄性不育植株。以悬浮细胞作为基因枪轰击的靶材料,转化植株再生频率较初级愈伤组织的为高。转brn基因植株的其他主要性状与供体亲本无显著差异,但却表现不育。其不育的程度在不同的植株之间表现不同。在转brn基因植株中观察到全不育(占全部brn阳性植株的40.6％)、高不育(占15.6％)及半不育的个体(占43.7％)。全不育的转基因植株自交完全不能结实(结实率为零),除个别植株外,花粉完全不被I-KI染色;而人工授以正常的花粉则可以获得杂交种子。而brn基因的阴性植株及未进行转化的对照植株则完全可育,表明转基因植株之雄性不育乃brn基因所致。结果表明,brn基因在水稻中是完全可以正常表达的,其表达的时期推测在花粉母细胞减数分裂前至花粉形成之间的整个时期。     

Genomic stability in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transgenic plants obtained by floral dip

The occurrence of DNA modification is an undesired phenomenon accompanying plant cell transformation. The event has been correlated with the stress imposed by the presently utilised transformation procedures, all depending on plant differentiation from in vitro cell culture, but other causes have not been excluded. In this work, transgenic Arabidopsis thaliana plants have been produced by an approach that does not require cell dedifferentiation, being based on in planta Agrobacterium-mediated gene transfer by flower infiltration, which is followed by recovery and selection of transgenic progeny. Genomic DNA changes in transgenic and control plants have been investigated by AFLP and RAMP analysis. Results show no statistically relevant genomic modifications in transgenic plants, as compared with control untreated plants. Variations were observed in callus-derived A. thaliana plants, thus supporting the conclusion that somaclonal variation is essentially correlated with the stress imposed by the in vitro cell culture, rather than with the integration of a foreign gene.     

几种转基因植物体细胞克隆变异的多样性研究

分析了杨树、水稻和甘蔗转基因植株体细胞克隆的表型变异和基因组DNA多态性。探讨了以下问题:转基因植株体细胞克隆的1)表型多样性, 2)基因组DNA多样性, 3)二者的相关性, 4)表型变异和DNA变异的可遗传特性, 5)产生的可能原因,以及6)在农业生产上的应用。     

Rice transformation: bombardment

Bombardment-based methodology is responsible for the effective genetic manipulation of major cereals including rice. Many groups reported significant advances on various aspects of rice molecular biology and genetic engineering using procedures based on bombardment technology. Molecular and genetic characterization of large numbers of these plants (more than 500 independent transgenic plants) provided information on structure, expression and stability of integrated DNA through multiple generations. Such evaluations were carried out in the greenhouse and in the field. Stability of expression was found to be dependent on the nature of the promoter and the transgene, and in specific cases on gene copy number. Direct DNA transfer utilizing particle bombardment for the delivery of foreign DNA into rice tissue results in the recovery of large numbers of independently derived transgenic plants in a variety-independent fashion. Gene copy number, level and stability of expression of transgenes can be compared to other DNA delivery methods, direct or indirect, including Agrobacterium-mediated gene transfer. In this paper, the technology is summarized and discussed in terms of present and future applications, including field trials and potential commercialization of transgenic rice expressing a number of genes of agronomic interest such as pest and herbicide resistance.