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.     

Regeneration of transgenic,microspore-derived,fertile barley

We have developed a system for the biolistic transformation of barley using freshly-isolated microspores as the target tissue. Independent transformation events led, on average, to the recovery of one plant per 1×10⁷ bombarded microspores. Putative transformants have been regenerated using phosphinothricin as a selective agent. R₀ plants have been transferred to soil approximately 2 months after bombardment. Integration of the marker genes bar and uidA has been confirmed by Southern analysis. The marker genes are inherited in all progeny plants confirming the expected homozygous nature of the R₀ plants.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

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     

Analysis of a large number of independent transgenic rice plants produced by the biolistic method

Summary Over 500 independent transgenic rice plants have been obtained by the biolistic method with an average transformation frequency of 9.7% for japonica variety Taipei 309. A tight selection procedure using 50 mg/l of hygromycin B successfully prevented the growth of nontransformed tissues. Analysis of the T0 transgenic rice plants revealed that more than 97% of the transgenic plants were morphologically normal and more than 80% were at least partially fertile. The hyg^r trait was inherited as a dominant trait in a Mendelian manner in 8 out of 11 transgenic events assayed. Thirty-seven out of fifty transgenic plants were estimated to contain no more than five copies of the transgenes. In six out of seven transformation events, unlinked, co-transformed genes co-segregated in the T1 generation. The hyg^r trait has been stably inherited to the T4 generation. No chimerical transgenic plant has been found in an intensive search. Novel phenomena observed in transgenic rice plants are also reported.     

A mannose selection system for production of fertile transgenic maize plants from protoplasts

 Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T₁ and T₂ plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation. Received: 12 July 1999 / Revision received: 11 October 1999 / Accepted: 11 October 1999     

Rapid,efficient production of homozygous transgenic tobacco plants withagrobacterium tumefaciens: A seed-to-seed protocol

An optimized complete protocol was developed forAgrobacterium tumefaciens-mediated transformation of tobacco (Nicotiana tabacum L. cultivar SR1), producing T₁ flowering plants homozygous for the inserted T-DNA as verified by kanamycin resistance in T₂ seedlings in 6 to 7 months from the time of cocultivation withAgrobacterium. Previous protocols require up to 9 to 12 months to obtain similar results. Procedures unique and important to this protocol include; a modified “whole-leaf” transformation coupled with a long duration of cocultivation, resulting in high rates of transformation, high levels of kanamycin in selection media resulting in few escapes, and extensive rooting of regenerants prior to a greenhouse hardening procedure. Once in the greenhouse, primary regenerants were maintained in small containers with long day photoperiod and high light levels, greatly shortening the time to seed set. Flowers from primary transformants were bagged to allow self pollination, and seed capsules harvested and dried prior to normal maturation on the plant. T₁ and T₂ seeds were plated and selected on kanamycin media by an improved seed plating technique which eliminates the need for the placement of individual seeds, saving time and improving selection homogeneity. Using this protocol, over 130 independent tobacco lines from six separate gene constructs have been generated in a very short time period. Of these 130, nearly 60 percent segregated 3∶1 for kanamycin resistance: susceptibility, indicating single transgene insertion events.     

Direct gene delivery into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants

A procedure for direct gene transfer into isolated microspores of rapeseed (Brassica napus L.) and the production of fertile transgenic plants is presented. By modifying the microspore culture method and adopting the firefly luciferase (Luc) gene as a non-destructive marker, we could obtain stably transformed androgenetic embryos from bombarded microspores. Luc-positive embryos were easily isolated from the large non-transformed population using a high-sensitivity bioluminescent image analyzer. PCR and Southern blot analyses confirmed that the introduced transgene was integrated stably into the genome of the selected embryos. Diploidized plants obtained from the haploid embryos were self-pollinated, and all of the offspring tested were Luc-positive, indicating rapid fixation of the transgene which is characteristic of doubled haploids. Received: 14 May 1997 / Revision received: 15 July 1997 / Accepted: 28 July 1997     

Regeneration of fertile transgenic indica (group 1) rice plants following microprojectile transformation of embryogenic suspension culture cells

Regenerable embryogenic suspensions of elite Indica (group 1) rice varieties IR24, IR64, IR72 and an advanced Indica rice breeding line IR57311-95-2-3 were established within 6–8 weeks from 3–4 week old calli derived from mature seeds. Transgenic rice plants were obtained by introducing a plasmid carrying genes encoding hygromycin phosphotransferase (hph, conferring resistance to hygromycin B) and ß-glucuronidase (uidA), both driven by the CaMV 35S promoter, via particle bombardment of embryogenic suspensions. The effect of osmotic conditioning on transformation was evaluated. Regenerated plants were resistant to hygromycin B and expressed the uidA (GUS) gene. The growth of mother plants (R₀) was normal and seeds were produced. Southern blot analysis of R₀ and R₁ plants showed that hygromycin resistant plants contained intact hph genes that were inherited in a Mendelian fashion. A protocol for a simple, efficient, repeatable, genotype- and environment-independent Indica rice transformation system is described.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA -naphthalene acetic acid - kb kilobase - GUS ß-glucuronidase - hph hygromycin B phosphotransferase     

Glyphosate as a selective agent for the production of fertile transgenic maize (Zea mays L.) plants

Efficient and reproducible selection of transgenic cells is an essential component of a good transformation system. In this paper, we describe the development of glyphosate as a selective agent for the recovery of transgenic embryogenic corn callus and the production of plants tolerant to Roundup^® herbicide. Glyphosate, the active ingredient in Roundup^® herbicide inhibits the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and thus prevents the synthesis of chorismate-derived aromatic amino acids and secondary metabolites in plants. A maize EPSPS gene has been cloned, mutated to produce a modified enzyme resistant to inhibition by glyphosate, and engineered into a monocot expression vector. In addition, a bacterial gene which degrades glyphosate (glyphosate oxidoreductase, or GOX) was also cloned into a similar expression vector. Stably transformed callus has been reproducibly recovered following introduction of mutant maize EPSPS and GOX genes into tissue culture cells by particle bombardment and selection on glyphosate-containing medium. Plants have been regenerated both on and off glyphosate selection medium, and are tolerant to normally lethal levels of Roundup^®. Excellent seed set has been obtained from both self and outcross pollinations from both sprayed and unsprayed regenerated plants. Progeny tests have demonstrated normal Mendelian transmission and tolerance to the herbicide for some of the transgenic events.     

A fluorescent antibiotic resistance marker for rapid production of transgenic rice plants

Blasticidin S (BS) is an aminoacylnucleoside antibiotic used for the control of rice blast disease. To establish a new cereal transformation system, we constructed a visual marker gene designated gfbsd, encoding an enhanced green fluorescent protein (EGFP) fused to the N-terminus of BS deaminase (BSD). It was cloned into a monocot expression vector and introduced into rice (Oryza sativa L. cv. Nipponbare) calluses by microprojectile bombardment. Three to five weeks after the bombardment, multicellular clusters emitting bright-green EGFP fluorescence were obtained with 10 microg/ml BS, which is not sufficient to completely inhibit the growth of non-transformed tissues. Fluorescent sectors (approximately 2mm in diameter) excised from the calluses regenerated into transgenic plantlets (approximately 10 cm in height) as early as 51 (average 77+/-11) days after the bombardment. The visual antibiotic selection was more efficient and required less time than the bialaphos selection with bar. In addition, the small size (1.1 kb) of gfbsd is preferable for construction of transformation vectors. This new marker gene will make a significant contribution in molecular genetic studies of rice plants.     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Production of fertile transgenic lentil (Lens culinaris Medik) plants using particle bombardment

Summary A reproducible system for gene transfer in lentil through particle bombardment is presented. Lentil cotyledonary nodes excised from germinated seedlings were bombarded with a plasmid containing a mutant acetolactate synthase gene (ALS) from tobacco conferring resistance to sulfonylurea herbicides. Putative transgenic shoots regenerated on Murashige and Skoog medium supplemented with 6-benzylaminopurine (BA) and chlorsulfuron (5 nM for first 4 wk followed by 2.5 nM for the remainder of the culture period) were micrografted and successfully transferred to soil. T₀ and selfed progeny plants were screened using metsulfuron herbicide leaflet painting. The non-transformed escapes died and transformed plants survived the test. The surviving plants were phenotypically normal and produced viable seeds. The presence and stable transmission of the transgene into genomic DNA of screened T₁ transformants was confirmed by PCR and Southern hybridization. This method for producing transformed plants will allow new opportunities for lentil breeding to produce improved cultivars.     

Identification of highly transformable wheat genotypes for mass production of fertile transgenic plants.

The efficiency of wheat biolistic transformation systems strongly depends on the bombardment parameters, the condition of the donor plant, and the plant genotype chosen for the transformation process. This paper analyzes the transformation efficiency of the 129 wheat sister lines generically called 'Bobwhite', originally obtained from the cross 'Aurora'//'Kalyan'/'Bluebird 3'/'Woodpecker'. A number of factors influencing the transformation were examined, such as the ability to produce embryogenic callus, regeneration in selection medium, and overall transformation performance. Of the 129 genotypes evaluated, eight demonstrated transformation efficiencies above 60% (60 independent transgenic events per 100 immature embryos bombarded). Among the eight genotypes identified, we studied agronomic characteristics such as earliness to identify the most adaptable line(s) for different lab conditions. 'Bobwhite' SH 98 26 was identified as a super-transformable wheat line.     

Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

The microprojectile bombardment of immature embryos has proven to be effective in transforming many indica rice varieties. One of the drawbacks of using immature embryos is the requirement of a large number of high quality immature embryos, which itself is a tedious and laborious process. To circumvent these problems, we have developed a procedure, using indica variety TN1 as a model that generates highly homogenous populations of embryogenic subcultured calli by selectively propagating a small number of regeneration-proficient calli derived from seeds. Thousands of embryogenic calli were produced from 50 seeds within 10 weeks. Ten to 20 independent R0 transgenic lines were regenerated per 500 embryogenic calli bombarded. The convenience and reliability offered by this transformation system has made transformation of indica rice a routine procedure.Abbreviations 2,4-D 2,4-dichlorophenoxy acetic acid - NAA naphthalene acetic acid - BAP 6-benzylaminopurine - kb kilobase - GUS -glucuronidase - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - HPH hygromycin B phosphotransferase     

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa)

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed. Received: 26 November 1998 / Revision received: 15 March 1999 / Accepted: 21 March 1999     

Transformation of maize embryogenic calli mediated by ultrasonication and regeneration of fertile transgenic plants

An insecticidal protein gene isolated fromBacillus thuringiensis was transferred into maize by using ultrasonication. The fertile transgenic plants and their progeny were obtained. The Southern hybridization results indicated that the foreign gene had integrated into the maize genome. It has been found that the acoustic intensity and the duration of treatment are the important parameters influencing transformation efficiency by ultrasonication. The maximum relative transformation frequency of 34.1 % was achieved after 30 min of sonication at 0. 5 W/cm² acoustic intensity. With appropriate parameters the ultrasonication can make a number of micropores formed on the cell surface and minimize the treatment damage to the foreign DNA molecules, thus facilitating the DNA molecules to enter the cells. Project supported by “863” State High Technology Development Program.     

转基因技术在兰花上的应用(综述)

本文综述兰花转基因技术的应用、转入的目的基因和转基因组织筛选与检测的研究进展。     

Optimization of biolistic method for transient gene expression and production of agronomically useful transgenic Basmati rice plants

We have developed a reproducible biolistic procedure for the efficient transformation of embryogenic suspension cells of an improved aromatic Indica rice variety, Pusa Basmati 1. The -glucuronidase gene was used to assay transient transformation; other plasmids carrying either a potato protease inhibitor 2 (Pin2) gene, or a late embryogenesis-abundant protein (LEA3) gene from barley, were used for the optimization of biolistic process and transgenic plant production. After optimization of the procedure, over 600 transient transformants and at least five fertile plants showing integrative transformation were obtained per bombarded filter. At least 30% of the plants were derived from independent transformation events. The new improved procedure involves the use of a reporter gene or other useful genes driven by the strong rice actin 1 gene (Act1) promoter, osmotic pre-conditioning of cells for 24 h on medium supplemented with 0.25 M mannitol prior to bombardment, use of gold particles for DNA delivery, and use of plant regeneration medium with high (1.0%) agarose concentration.