A non-destructive screenable marker,OsFAST, for identifying transgenic rice seeds

The production of transgenic plants has contributed greatly to plant research. Previously, an improved method for screening transgenic Arabidopsis thaliana seeds using the FAST (Fluorescence-Accumulating-Seed Technology) method and FAST marker was reported. Arabidopsis seeds containing the FAST marker may be visually screened using a fluorescence stereomicroscope or blue LED handy-type instrument. Although the FAST method was originally designed for Arabidopsis screens, this study endeavors to adapt this method for the screening of other plants. Here, an optimized technology, designated the OsFAST method, is presented as a useful tool for screening transgenic rice seeds. The OsFAST method is based on the expression of the OsFAST-G marker under the control of a seed-embryo-specific promoter, similar to the Arabidopsis FAST-G marker. The OsFAST method provides a simple and non-destructive method for identifying transgenic rice seeds. It is proposed that the FAST method is adaptable to various plant species and will enable a deeper analysis of the floral-dip method.Key words: Oryza sativa, oleosin, seed, green fluorescent protein, transformation, screenable markerThe production of transgenic plants has significantly enhanced many areas of plant science research. Antibiotic/herbicide-resistance genes are traditionally used as screenable markers for the selection of transgenic plants. However, this approach does have disadvantages. First, antibiotics or herbicides occasionally inhibit the growth of transgenic plants, regardless of the incorporation of antibiotic- or herbicide-resistance genes¹ into the transgenic plants. Second, the identification of resistant transgenic plants requires that the seed population be sown onto plates containing antibiotics or herbicides. Third, the selection process is slow and labor intensive, often involving the screening of vast numbers of potentially transgenic seeds on selective plates.To overcome these disadvantages, an improved approach for selecting transgenic Arabidopsis thaliana, designated the FAST (Fluorescence-Accumulating-Seed Technology) method, was developed. This method employs the use of a fluorescent protein that is expressed in seeds and used as a visual screenable marker for the identification of transgenic seeds. The seed-specific protein oleosin, a family of oil-body-membrane proteins,² has an important role as a size regulator of oil bodies.³ AtOLE1, the most abundant oleosin, functions in the freezing tolerance of Arabidopsis seeds.⁴ A plasmid containing an AtOLE1-GFP fusion gene controlled by the AtOLE1 promoter was constructed and designated the FAST-G (Fluorescence-Accumulating-Seed Technology with OLE1-GFP) marker. Interestingly, Arabidopsis seeds containing the FAST-G marker emitted clear fluorescence under a fluorescence stereomicroscope or blue LED handy-type instrument. The transgenic seeds were visually identified by the seed fluorescence without the use of antibiotics or herbicides, thus indicating that the FAST method offers a nondestructive approach. The FAST marker permits the identification of homozygous seeds among the T2 population with a false discovery rate of less than 1% as a co-dominant screenable marker. In contrast to conventional methods using antibiotics or herbicides, the FAST method reduces the amount of time required to acquire homozygous transgenic plants from 7.5 months to 4 months. The fluorescence of the FAST-G marker was limited to a specific organ (i.e., in seeds) and a specific time (i.e., during dormancy), desirable characteristics of selectable and/or screenable markers. Furthermore, the FAST marker does not require sterile seeding and the handling of large numbers of plants.     

Some Physiological Parameters and Seed Lipid Composition of Transgenic Tobacco Plant

To see the effects of foreign gene introduction on the physiological performance and the quality and quantity of seed lipids, we studied transgenic tobacco plant as a model system, as tobacco seeds are oil seeds. Using Agrobacterium Ti plasmid based vectors, tobacco plants cv Petit Havana were transformed by NPT II gene as selectable marker. Transformed T₀ generation plants raised in tissue culture were transferred to pots and selfed. From the seeds, T₁ generation plants were grown in pots and their physiological performance was assessed. The transgenic plants showed slightly slower rates of germination and growth. Total chlorophyll content, chlorophyll a/b ratio and specific leaf weight, however, remained unchanged. The transgenic plants also had delayed flowering. However, total protein, lipid content and fatty acid composition of lipids of seeds in transgenic plants did not show appreciable difference from the seeds from control plants. Thus the physiological cost of transgenic plant for the extra genetic load was only marginal, if any.     

Production of high levels of poly‐3‐hydroxybutyrate in plastids of Camelina sativa seeds

Poly‐3‐hydroxybutyrate (PHB) production in plastids of Camelina sativa seeds was investigated by comparing levels of polymer produced upon transformation of plants with five different binary vectors containing combinations of five seed‐specific promoters for expression of transgenes. Genes encoding PHB biosynthetic enzymes were modified at the N‐terminus to encode a plastid targeting signal. PHB levels of up to 15% of the mature seed weight were measured in single sacrificed T₁ seeds with a genetic construct containing the oleosin and glycinin promoters. A more detailed analysis of the PHB production potential of two of the best performing binary vectors in a Camelina line bred for larger seed size yielded lines containing up to 15% polymer in mature T₂ seeds. Transmission electron microscopy showed the presence of distinct granules of PHB in the seeds. PHB production had varying effects on germination, emergence and survival of seedlings. Once true leaves formed, plants grew normally and were able to set seeds. PHB synthesis lowered the total oil but not the protein content of engineered seeds. A change in the oil fatty acid profile was also observed. High molecular weight polymer was produced with weight‐averaged molecular weights varying between 600 000 and 1 500 000, depending on the line. Select lines were advanced to later generations yielding a line with 13.7% PHB in T₄ seeds. The levels of polymer produced in this study are the highest reported to date in a seed and are an important step forward for commercializing an oilseed‐based platform for PHB production.     

An oleosin-fusion protein driven by the CaMV35S promoter is accumulated in Arabidopsis (Brassicaceae) seeds and correctly targeted to oil bodies

Oleosin-fusion technology is used to express desired proteins. It was developed based on the properties of oleosin; the heterologous protein gene is fused to the oleosin gene and the fusion gene is driven by a seed-specific promoter. We replaced the seed specific promoter with the CaMV35S promoter to dive a gfp-oleosin fusion gene in transformed Arabidopsis. The heterologous oleosin-fusion protein was mainly accumulated in the transgenic Arabidopsis seeds and correctly targeted to oil bodies. This provides an alternate choice of promoter in oleosin-fusion technology.     

Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants

 Barley (Hordeum vulgare L.) hordeins are alcohol-soluble redundant storage proteins that accumulate in protein bodies of the starchy endosperm during seed development. Strong endosperm-specific β-glucuronidase gene-(uidA; gus) expression driven by B₁- and D-hordein promoters was observed in stably transformed barley plants co-transformed with the selectable herbicide resistance gene, bar. PCR analysis using DNA from calli of 22 different lines transformed with B₁- or D-hordein promoter-uidA fusions showed the expected 1.8-kb uidA fragment after PCR amplification. DNA-blot analysis of genomic DNA from T₀ leaf tissue of 13 lines showed that 12 (11 independent) lines produced uidA fragments and that one line was uidA-negative. T₁ progeny from 6 out of 12 independent regenerable transgenic lines tested for uidA expression showed a 3 : 1 segregation pattern. Of the remaining six transgenic lines, one showed a segregation ratio of 15 : 1 for GUS, one expressed bar alone, one lacked transmission of either gene to T₁ progeny, and three were sterile. Stable GUS expression driven by the hordein promoters was observed in T₅ progeny in one line, T₄ progeny in one line, T₃ progeny in three lines and T₂ or T₁ progeny in the remaining two fertile lines tested; homozygous transgenic plants were obtained from three lines. In the homozygous lines the expression of the GUS protein, driven by either the B₁- or D-hordein promoters, was highly expressed in endosperm at early to mid-maturation stages. Expression of bar driven by the maize ubiquitin promoter was also stably transmitted to T₁ progeny in seven out of eight lines tested. However, in most lines PAT expression driven by the maize ubiquitin promoter was gradually lost in T₂ or later generations; one homozygous line was obtained. In contrast, six out of seven lines stably expressed GUS driven by the hordein promoters in T₂ or later generations. We conclude that the B₁- and D-hordein promoters can be used to engineer, and subsequently study, stable endosperm-specific gene expression in barley and potentially to modify barley seeds through genetic engineering. Received: 28 May 1998 / Accepted: 19 December 1998     

Commercial production of aprotinin in transgenic maize seeds

The development of genetic transformation technology for plants has stimulated an interest in using transgenic plants as a novel manufacturing system for producing different classes of proteins of industrial and pharmaceutical value. In this regard, we report the generation and characterization of transgenic maize lines producing recombinant aprotinin. The transgenic aprotinin lines recovered were transformed with the aprotinin gene using the bar gene as a selectable marker. The bar and aprotinin genes were introduced into immature maize embryos via particle bombardment. Aprotinin gene expression was driven by the maize ubiquitin promoter and protein accumulation was targeted to the extracellular matrix. One line that showed a high level of aprotinin expression was characterized in detail. The protein accumulates primarily in the embryo of the seed. Southern blot analysis showed that the line had at least 20 copies of the bar and aprotinin genes. Further genetic analysis revealed that numerous plants derived from this transgenic line had a large range of levels of expression of the aprotinin gene (0–0.069%) of water-soluble protein in T₂ seeds. One plant lineage that showed stable expression after 4 selfing generations was recovered from the parental transgenic line. This line showed an accumulation of the protein in seeds that was comparable to the best T₂ lines, and the recombinant aprotinin could be effectively recovered and purified from seeds. Biochemical analysis of the purified aprotinin from seeds revealed that the recombinant aprotinin had the same molecular weight, N-terminal amino acid sequence, isoelectric point, and trypsin inhibition activity as native aprotinin. The demonstration that the recombinant aprotinin protein purified from transgenic maize seeds has biochemical and functional properties identical to its native counterpart provides a proof-of-concept example for producing new generation products for the pharmaceutical industry.     

PHA production, from bacteria to plants.

The genes encoding the polyhydroxyalkanoate (PHA) biosynthetic pathway in Ralstonia eutropha (3-ketothiolase, phaA or bktB; acetoacetyl-CoA reductase, phaB; and PHA synthase, phaC) were engineered for plant plastid targeting and expressed using leaf (e35S) or seed-specific (7s or lesquerella hydroxylase) promoters in Arabidopsis and Brassica. PHA yields in homozygous transformants were 12-13% of the dry mass in homozygous Arabidopsis plants and approximately 7% of the seed weight in seeds from heterozygous canola plants. When a threonine deaminase was expressed in addition to bktB, phaB and phaC, a copolyester of 3-hydroxybutyrate and 3-hydroxyvalerate was produced in both Arabidopsis and Brassica.     

Feasibility of the seed specific cruciferin C promoter in the self excision Cre/<Emphasis Type="Italic">lox</Emphasis>P strategy focused on generation of marker-free transgenic plants

This work is focused on the generation of selectable marker-free transgenic tobacco plants using the self excision Cre/loxP system that is controlled by a strong seed specific Arabidopsis cruciferin C (CRUC) promoter. It involves Agrobacterium-mediated transformation using a binary vector containing the gus reporter gene and one pair of the loxP sites flanking the cre recombinase and selectable nptII marker genes (floxed DNA). Surprisingly, an ectopic activation of CRUC resulting in partial excision of floxed DNA was observed during regeneration of transformed cells already in calli. The regenerated T₀ plants were chimeric, but no ongoing ectopic expression was observed in these one-year-long invitro maintained plants. The process of the nptII removal was expected in the seeds; however, none of the analysed T₀ transgenic lines generated whole progeny sensitive to kanamycin. Detailed analyses of progeny of selected T₀-30 line showed that 10.2% GUS positive plants had completely removed nptII gene while the remaining 86.4% were still chimeras. Repeated activation of the cre gene in T₂ seeds resulted in increased rate of marker-free plants, whereas four out of ten analysed chimeric T₁ plants generated completely marker-free progenies. This work points out the feasibility as well as limits of the CRUC promoter in the Cre/loxP strategy. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2–4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was varified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T_o plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T₁ and T₂ progenies. Enzyme analyses on T₁ progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T_o plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.     

A biotechnology‐based male‐sterility system for hybrid seed production in tomato

Incorporating male sterility into hybrid seed production reduces its cost and ensures high varietal purity. Despite these advantages, male‐sterile lines have not been widely used to produce tomato (Solanum lycopersicum) hybrid seeds. We describe the development of a biotechnology‐based breeding platform that utilized genic male sterility to produce hybrid seeds. In this platform, we generated a novel male‐sterile tomato line by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9)‐mediated mutagenesis of a stamen‐specific gene SlSTR1 and devised a transgenic maintainer by transforming male‐sterile plants with a fertility‐restoration gene linked to a seedling‐colour gene. Offspring of crosses between a hemizygous maintainer and the homozygous male‐sterile plant segregated into 50% non‐transgenic male‐sterile plants and 50% male‐fertile maintainer plants, which could be easily distinguished by seedling colour. This system has great practical potential for hybrid seed breeding and production as it overcomes the problems intrinsic to other male‐sterility systems and can be easily adapted for a range of tomato cultivars and diverse vegetable crops.     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing

The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T₂ and T₃ generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T₃ and T₄ generation Camelina seeds was associated with a combination of germ‐line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.     

Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T₂ generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T₃ lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.     

亲环素AtCYP1 RNA干涉载体的构建及对拟南芥发育的影响

根据拟南芥AtCYP1基因序列设计特异引物,以拟南芥总DNA为模板,扩增AtCYP1基因中344 bp转录本,插入表达载体PTCK303,构建目的基因RNA干扰载体Ubi::AtCYP1i。利用改良的农杆菌浸染技术获得拟南芥RNAi转基因株系,RT-PCR分析结果表明转基因株系中AtCYP1基因的表达量低于野生型,表型观察结果表明RNAi转基因纯合株系抽苔时间比野生型晚3.32 d,抽苔叶片数较野生型多2.49片,其开花时间、结出第一个种荚的时间、株高等方面也与野生型存在明显差异。此结果说明AtCYP1可能参与了拟南芥的早花发育过程,为进一步研究其在植物生长发育中的功能奠定了基础。