GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters

Huanglongbing (HLB) is associated with Candidatus Liberibacter spp., endogenous, sieve tube-restricted bacteria that are transmitted by citrus psyllid insect vectors. Transgenic expression in the phloem of specific genes that might affect Ca. Liberibacter spp. growth and development may be an adequate strategy to improve citrus resistance to HLB. To study specific phloem gene expression in citrus, we developed three different binary vector constructs with expression cassettes bearing the beta-glucuronidase (GUS) reporter gene (uidA) under the control of one of the three different promoters: Citrus phloem protein 2 (CsPP2), Arabidopsis thaliana phloem protein 2 (AtPP2), and Arabidopsis thaliana sucrose transporter 2 (AtSUC2). Transgenic lines of 'Hamlin', 'Pera', and 'Valencia' sweet oranges [Citrus sinensis (L.) Osbeck] were produced via Agrobacterium tumefaciens transformation. The epicotyl segments collected from in vitro germinated seedlings were used as explants. The gene nptII, which confers resistance to the antibiotic kanamycin, was used for selection. The transformation efficiency was expressed as the number of GUS-positive shoots over the total number of explants and varied from 1.54 to 6.08 % among the three cultivars and three constructs studied. Several lines of the three sweet orange cultivars analyzed using PCR and Southern blot analysis were genetically transformed with the three constructs evaluated. The histological GUS activity in the leaves indicates that the uidA gene was preferentially expressed in the phloem, which suggests that the use of the three promoters might be adequate for producing HLB-resistant transgenic sweet oranges. The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters. Key message The results reported here conclusively demonstrate the preferential expression of GUS in the phloem driven by two heterologous and one homologous gene promoters.

Funktionelle Untersuchungen von FLORICAULA und KNOX-Genen in Eschscholzia californica Cham.

FLORICAULA (FLO) und KNOTTED1-like Homöobox (KNOX)-Gene übernehmen neben ihren konservierten Funktionen in der Achsenentwicklung in verschiedenen Eudikotylen eine Funktion in der Fiederblattentwicklung. Zur Klärung der Frage nach dem ursprünglichen Regulationsweg der Fiederblattentwicklung in Hinblick auf FLO und KNOX-Gene innerhalb der Eudikotylen wurde hier die Bedeutung dieser Gene für die Fiederblattentwicklung von Eschscholzia californica als Modell für die Ranunculales, die Schwestergruppe aller anderen Eudikotylen untersucht. Es wurde ein Protokoll zur Erzeugung von somatischen Embryonen aus unreifen Samen entwickelt. Wege zur Herstellung von Mutanten durch Agrobacterium-vermittelte Transformation werden vorgeschlagen. Die Bedeutung von Auxin für die Blattentwicklung und die Untersuchung der Interaktion von ESCHSCHOLZIA CALIFORNICA FLORICAULA (EcFLO) und des KNOX- Gens ESCHSCHOLZIA CALIFORNICA SHOOT MERISTEMLESS (EcSTM) mit Auxin wurde durch Hemmung des Auxintransports untersucht. Trotz gravierender Störungen in der Blattpositionierung und -morphologie konnten Expressionsänderungen beider Gene nicht nachgewiesen werden. Ein Funktionsverlust von EcFLO und KNOX-Genen in E. californica wurden mittels Virus induziertem Gen Silencing (VIGS) erzeugt. VIGS von EcFLO rief keinen Phänotypen hervor. VIGS des KNOX-Gens EcSTM erzeugte dagegen in einigen Pflanzen eine Reduktion der Fiederzahl. Auch molekularbiologisch konnte das Silencing von EcSTM, nicht aber das Silencing von EcFLO nachgewiesen werden. Die Ergebnisse belegen die Notwendigkeit des ungestörten Auxintransports für die Blattentwicklung von E. californica und machen die Beteiligung des KNOX-Gens EcSTM an der Blattentwicklung wahrscheinlich. Die Beteiligung von EcFLO an der Fiederbildung konnte nicht nachgewiesen werden.

Function of C1A barley peptidases and their inhibitors (cystatins) in barley seed germination : Funcion de las peptidasas C1A de cebada y sus inhibidores (cistatinas) en la germinacion de la semilla

Plant proteolysis is a metabolic process where specific enzymes called peptidases degrade proteins. In plants, this complex process involves broad metabolic networks and different sub-cellular compartments. Several types of peptidases take part in the proteolytic process, mainly cysteine-, serine-, aspartyl- and metallo- peptidases. Among the cysteine-peptidases, the papain-like or C1A peptidases (family C1, clan CA) are extensively present in land plants and are classified into catepsins L-, B-, H- and Flike. The catalytic mechanism of these C1A peptidases is highly conserved and involves the three amino acids Cys, His and Asn in the catalytic triad, and a Gln residue which seems essential for maintaining an active enzyme conformation. These proteins are synthesized as inactive precursors, which comprise an N-terminal signal peptide, a propeptide, and the mature protein. In barley, we have identified 33 cysteine-peptidases from the papain-like family, classifying them into 8 different groups. Five of them corresponded to cathepsins L-like (5 subgroups), 1 cathepsin B-like group, 1 cathepsin F-like group and 1 cathepsin H-like group. Besides, C1A peptidases are the specific targets of the plant proteinaceous inhibitors known as phytocystatins (PhyCys). The cystatin inhibitory mechanism is produced by a tight and reversible interaction with their target enzymes. In barley, the cystatin gene family is comprised by 13 members. In this work we have tried to elucidate the role of the C1A cysteine-peptidases and their specific inhibitors (cystatins) in the germination process of the barley grain. Therefore, we selected a representative member of each group/subgroup of C1A peptidases (1 cathepsin B-like, 1 cathepsin F-like, 1 cathepsin H-like and 5 cathepsins L-like). The molecular characterization of the cysteine-peptidases was done and the peptidase-inhibitor interaction was analyzed in vitro and in vivo. A study in the structural basis for specificity of pro-peptide/enzyme interaction in barley C1A cysteine-peptidases has been also carried out by inhibitory assays and the modeling of the three-dimensional structures. The barley grain maturation produces the accumulation of storage proteins (prolamins) in the endosperm which are mobilized during germination to supply the required nutrients until the photosynthesis is fully established. In this work, we have demonstrated the participation of the cysteine-peptidases and their inhibitors in the degradation of the different storage protein fractions (hordeins, albumins and globulins) present in the barley grain. Besides, transgenic barley plants overexpressing or silencing cysteine-peptidases or cystatins were obtained by Agrobacterium-mediated transformation of barley immature embryos to analyze their physiological function in vivo. Preliminary assays were carried out with the T1 grains of several transgenic lines. Comparing the knock-out and the overexpressing lines with the WT, alterations in the germination process were detected and were correlated with their grain hordein content. These data will be validated with the homozygous grains that are being produced through the double haploid technique by microspore culture. Resumen La proteólisis es un proceso metabólico por el cual se lleva a cabo la degradación de las proteínas de un organismo a través de enzimas específicas llamadas proteasas. En plantas, este complejo proceso comprende un entramado de rutas metabólicas que implican, además, diferentes compartimentos subcelulares. En la proteólisis participan numerosas proteasas, principalmente cisteín-, serín-, aspartil-, y metalo-proteasas. Dentro de las cisteín-proteasas, las proteasas tipo papaína o C1A (familia C1, clan CA) están extensamente representadas en plantas terrestres, y se clasifican en catepsinas tipo L, B, H y F. El mecanismo catalítico de estas proteasas está altamente conservado y la triada catalítica formada por los aminoácidos Cys, His y Asn, y a un aminoácido Gln, que parece esencial para el mantenimiento de la conformación activa de la proteína. Las proteasas C1A se sintetizan como precursores inactivos y comprenden un péptido señal en el extremo N-terminal, un pro-péptido y la proteína madura. En cebada hemos identificado 33 cisteín-proteasas de tipo papaína y las hemos clasificado filogenéticamente en 8 grupos diferentes. Cinco de ellos pertenecen a las catepsinas tipo L (5 subgrupos), un grupo a las catepsinas tipo-B, otro a las catepsinas tipo-F y un último a las catepsinas tipo-H. Las proteasas C1A son además las dianas específicas de los inhibidores protéicos de plantas denominados fitocistatinas. El mecanismo de inhibición de las cistatinas está basado en una fuerte interacción reversible. En cebada, se conoce la familia génica completa de las cistatinas, que está formada por 13 miembros. En el presente trabajo se ha investigado el papel de las cisteín-proteasas de cebada y sus inhibidores específicos en el proceso de la germinación de la semilla. Para ello, se seleccionó una proteasa representante de cada grupo/subgrupo (1 catepsina tipo- B, 1 tipo-F, 1 tipo-H, y 5 tipo-L, una por cada subgrupo). Se ha llevado a cabo su caracterización molecular y se ha analizado la interacción enzima-inhibidor tanto in vivo como in vitro. También se han realizado estudios sobre las bases estructurales que demuestran la especificidad en la interacción enzima/propéptido en las proteasas C1A de cebada, mediante ensayos de inhibición y la predicción de modelos estructurales de la interacción. Finalmente, y dado que durante la maduración de la semilla se almacenan proteínas de reserva (prolaminas) en el endospermo que son movilizadas durante la germinación para suministrar los nutrientes necesarios hasta que la nueva planta pueda realizar la fotosíntesis, en este trabajo se ha demostrado la participación de las cisteínproteasas y sus inhibidores en la degradación de las diferentes tipos de proteínas de reserva (hordeinas, albúmins y globulinas) presentes en el grano de cebada. Además, se han obtenido plantas transgénicas de cebada que sobre-expresan o silencian cistatinas y cisteín-proteasas con el fin de analizar la función fisiológica in vivo. Se han realizado análisis preliminares en las semillas T1 de varias líneas tránsgenicas de cebada y al comparar las líneas knock-out y las líneas de sobre-expresión con las silvestres, se han detectado alteraciones en la germinación que están además correlacionadas con el contenido de hordeinas de las semillas. Estos datos serán validados en las semillas homocigotas que se están generando mediante la técnica de dobles haploides a partir del cultivo de microesporas.

Modified nicotine metabolism in transgenic tobacco plants expressing the human cytochrome P450 2A6 cDNA

In this study, the human cytochrome P450 (CYP) 2A6 was used in order to modify the alkaloid production of tobacco plants. The cDNA for human CYP2A6 was placed under the control of the constitutive 35S promoter and transferred into Nicotiana tabacum via Agrobacterium-mediated transformation. Transgenic plants showed formation of the recombinant CYP2A6 enzyme but no obvious phenotypic changes. Unlike wild-type tobacco, the transgenic plants accumulated cotinine, a metabolite which is usually formed from nicotine in humans. This result substantiates that metabolic engineering of the plant secondary metabolism via mammalian P450 enzymes is possible in vivo. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

The Introduction of Transgenes to Control Blackheart in Pineapple: Biolistics Vs Agrobacterium Transformation, in 'Contributing to a Sustainable Future'

Techniques for the introduction of transgenes to control blackheart by particle bombardment and Agrobacterium co-transformation have been developed for pineapple cv. Smooth Cayenne. Polyphenol oxidase (PPO) is the enzyme responsible for blackheart development in pineapple fruit following chilling injury. Sense, anti-sense and hairpin constructs were used as a means to suppress PPO expression in plants. Average transformation efficiency for biolistics was approximately 1% and for Agrobacterium was approximately 1.5%. These results were considered acceptable given the high regeneration potential of between 80-90% from callus cultures. Southern blot analysis revealed stable integration of transgenes with lower copy number found in plants transformed with Agrobacterium compared to those transformed by biolistics. Over 5000 plants from 55 transgenic lines are now undergoing field evaluation in Australia

Ribozyme-mediated high resistance against potato spindle tuber viroid in transgenic potatoes

A hammerhead ribozyme [R(-)] targeting the minus strand RNA of potato spindle tuber viroid (PSTVd) and a mutated nonfunctional ribozyme [mR(-)] were designed, cloned, and transcribed. As predicted, both monomer and dimer transcripts of the active R(-) ribozyme gene could cleave the PSTVd minus strand dimer RNA into three fragments of 77, 338, and 359 bases in vitro at 25 and 50°C. The tandem dimer genes of R(-) and mR(-) were subcloned separately into the plant expression vector pROK2. Transgenic potato plants (cultivar Desirée) were generated by Agrobacterium tumefaciens-mediated transformation. Twenty-three of 34 independent transgenic plant lines expressing the active ribozyme R(-) resulted in having high levels of resistance to PSTVd, being free of PSTVd accumulation after challenge inoculation with PSTVd, but the remaining lines showed weaker levels of resistance to PSTVd with low levels of PSTVd accumulation. In contrast, 59 of 60 independent transgenic lines expressing the mutated ribozyme mR(-) were susceptible to PSTVd inoculation and had levels of PSTVd accumulation similar to that of the control plants transformed with the empty vector. The resistance against PSTVd replication was stably inherited to the vegetative progenies.

Complementation of plant mutants with large genomic DNA fragments by a transformation-competent artificial chromosome vector accelerates positional cloning

To accelerate gene isolation from plants by positional cloning, vector systems suitable for both chromosome walking and genetic complementation are highly desirable. Therefore, we developed a transformation-competent artificial chromosome (TAC) vector, pYLTAC7, that can accept and maintain large genomic DNA fragments stably in both Escherichia coli and Agrobacterium tumefaciens. Furthermore, it has the cis sequences required for Agrobacterium-mediated gene transfer into plants. We cloned large genomic DNA fragments of Arabidopsis thaliana into the vector and showed that most of the DNA fragments were maintained stably. Several TAC clones carrying 40- to 80-kb genomic DNA fragments were transferred back into Arabidopsis with high efficiency and shown to be inherited faithfully among the progeny. Furthermore, we demonstrated the practical utility of this vector system for positional cloning in Arabidopsis. A TAC contig was constructed in the region of the SGR1 locus, and individual clones with ca. 80-kb inserts were tested for their ability to complement the gravitropic defects of a homozygous mutant line. Successful complementation enabled the physical location of SGR1 to be delimited with high precision and confidence.

Gene transfer to plants by diverse species of bacteria

Agrobacterium is widely considered to be the only bacterial genus capable of transferring genes to plants. When suitably modified, Agrobacterium has become the most effective vector for gene transfer in plant biotechnology1. However, the complexity of the patent landscape2 has created both real and perceived obstacles to the effective use of this technology for agricultural improvements by many public and private organizations worldwide. Here we show that several species of bacteria outside the Agrobacterium genus can be modified to mediate gene transfer to a number of diverse plants. These plant-associated symbiotic bacteria were made competent for gene transfer by acquisition of both a disarmed Ti plasmid and a suitable binary vector. This alternative to Agrobacterium-mediated technology for crop improvement, in addition to affording a versatile ‘open source’ platform for plant biotechnology, may lead to new uses of natural bacteria– plant interactions to achieve plant transformation.

Production of transgenic rice with rice ragged stunt virus synthetic resistance genes

Rice ragged stunt virus (RRSV) is an important pathogen of rice affecting its cultivation in South and South East Asia. An approach based on pathogen derived resistance (PDR) was used to produce RRSV resistant rice cultivars. Sequences from the coding region of RRSV genome segments 7 and 10 (non-structural genes), and 5, 8 and 9 (structural genes) were placed in sense or antisense orientation behind the plant expression promoters CaMV35S, RolC, Ubil, Actl and RBTV. Rice cultivars Taipei 309 and Chinsurah Boro II were transformed by biolistic and/or Agrobacterium-mediated delivery of one or more of these PDR gene constructs. A large number of transgenic lines were produced from calli derived from mature or immature embryos, co-bombarded with the marker gene hph encoding hygromycin resistance and RRSV PDR genes or co-cultivated with strains having the binary vector containing these two genes. Both Mendelian and non-Mendelian segregations were observed in transgenic progeny, especially with transgenic lines produced by biolistics. Preliminary tests conducted in China on selected transgenic lines indicate that plants with RRSV segment 5 antisense PDR gene confer RRSV resistance.

Ectopic overexpression of a mungbean vacuolar Na+/H+ antiporter gene (VrNHX1) leads to increased salinity stress tolerance in transgenic Vigna unguiculata L. Walp

Salinity is a major threat to sustainable agriculture worldwide. Plant NHX exchangers play an important role in conferring salt tolerance under salinity stress. In this study, a vacuolar Na+/H+ antiporter gene VrNHX1 (Genbank Accession No. JN656211.1) from mungbean (Vigna radiata) was introduced into cowpea (Vigna unguiculata) by the Agrobacterium tumefaciens-mediated transformation method. Polymerase chain reaction and Southern blot hybridization confirmed the stable integration of VrNHX1 into the cowpea genome. Comparative expression analysis by semi-quantitative RT-PCR revealed higher expression of VrNHX1 in transgenic cowpea plants than wild-type. Under salt stress conditions, T2 transgenic 35S:VrNHX1 cowpea lines exhibited higher tolerance to 200 mM NaCl treatment than wild-type. Furthermore, T2 transgenic 35S:VrNHX1 lines maintained a higher K+/Na+ ratio in the aerial parts under salt stress and accumulated higher [Na+] in roots than wild-type. Physiological analysis revealed lower levels of lipid peroxidation, hydrogen peroxide and oxygen radical production but higher levels of relative water content and proline, ascorbate and chlorophyll contents in T2 transgenic 35S:VrNHX1 lines.

Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells

Early detection of plant transformation events is necessary for the rapid establishment and optimization of plant transformation protocols. We have assessed modified versions of the green fluorescent protein (GFP) from Aequorea victoria as early reporters of plant transformation using a dissecting fluorescence microscope with appropriate filters. Gfp-expressing cells from four different plant species (sugarcane, maize, lettuce, and tobacco) were readily distinguished, following either Agrobacterium-mediated or particle bombardment-mediated transformation. The identification of gfp-expressing sugarcane cells allowed for the elimination of a high proportion of non-expressing explants and also enabled visual selection of dividing transgenic cells, an early step in the generation of transgenic organisms. The recovery of transgenic cell clusters was streamlined by the ability to visualize gfp-expressing tissues in vitro.

Morphological Characteristics of Genetically-Modified Pineapple Fruit

Genetic engineering is an attractive method for changing a single characteristic of ‘Smooth Cayenne’ pineapple, without altering its other desirable attributes. Techniques used in pineapple transformation, however, such as tissue culture and biolistic-mediated or Agrobacterium-mediated gene insertion are prone to somaclonal variation, resulting in the production of several morphological mutations (Smith et al., 2002). Fruit mutations can include distortion in fruit shape (round ball, conical, fan-shaped), reduced fruit size, multiple crowns, crownless fruit, fruitless crowns, and spiny crown leaves (Dalldorf, 1975; Sanewski et al., 1992). The present paper describes the variability in fruit-shape mutations between transgenic and non-transgenic fruit, and its subsequent impact on organoleptic characteristics.

Bacterial transformation using micro-shock waves

Shock waves are one of the most competent mechanisms of energy dissipation observed in nature. We have developed a novel device to generate controlled micro-shock waves using an explosive-coated polymer tube. In this study, we harnessed these controlled micro-shock waves to develop a unique bacterial transformation method. The conditions were optimized for the maximum transformation efficiency in Escherichia coli. The maximum transformation efficiency was obtained when we used a 30 cm length polymer tube, 100 mu m thick metal foil, 200 mM CaCl(2), 1 ng/mu l plasmid DNA concentration, and 1 x 10(9) cell density. The highest transformation efficiency achieved (1 x 10(-5) transformants/cell) was at least 10 times greater than the previously reported ultrasound-mediated transformation (1 x 10(-6) transformants/cell). This method was also successfully employed for the efficient and reproducible transformation of Pseudomonas aeruginosa and Salmonella typhimurium. This novel method of transformation was shown to be as efficient as electroporation with the added advantage of better recovery of cells, reduced cost (40 times cheaper than a commercial electroporator), and growth phase independent transformation. (C) 2011 Elsevier Inc. All rights reserved.

Heterologous expression of P5CS gene in chickpea enhances salt tolerance without affecting yield

Vigna Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA was transferred to chickpea (Cicer arietinum L.) cultivar Annigeri via Agrobacterium tumefaciens mediated transformation. Following selection on hygromycin and regeneration, 60 hygromycin-resistant plants were recovered. Southern blot analysis of five fertile independent lines of T0 and T1 generation revealed single and multiple insertions of the transgene. RT-PCR and Western blot analysis of T0 and T1 progeny demonstrated that the P5CS gene is expressed and produced functional protein in chickpea. T1 transgenic lines accumulated higher amount of proline under 250 mM NaCl compared to untransformed controls. Higher accumulation of Na(+) was noticed in the older leaves but negligible accumulation in seeds of T1 transgenic lines as compared to the controls. Chlorophyll stability and electrolyte leakage indicated that proline overproduction helps in alleviating salt stress in transgenic chickpea plants. The T1 transgenics lines were grown to maturity and set normal viable seeds under continuous salinity stress (250 mM) without any reduction in plant yield in terms of seed mass.

由农杆菌介导将葡萄糖氧化酶基因转入水稻

过氧化氢（Hydrogen peroxide,H2O2）是植物和病原微生物互作中快速合成的一种早期活性氧类（reactive oxygen species, ROS ），它在植物受到病原微生物侵染后引发的一系列防御反应中起着非常重要的作用，因此通过外源基因导入提高植物体内过氧化氢的含量，可以增强植物的广谱抗病性。葡萄糖氧化酶（glucose oxidase, GO）可以催化β-D-葡萄糖氧化生成过氧化氢和葡萄糖酸，此酶已在数种细菌和真菌中检测到，但在植物和动物中仍未发现。为了尝试将此酶应用于水稻广谱抗病基因工程，本研究将葡萄糖氧化酶基因插入具有潮霉素抗性选择标记的双元载体pCAMBIA1301，新构建为水稻高效表达载体pCAG1301。将此质粒导入根癌农杆菌（Agrobacterium tumefaciens ）菌株LBA4404后,转化粳稻(Oryza sativa )品种日本晴(Nipponbare)成熟胚来源的愈伤组织和幼胚，并由筛选出的潮霉素抗性愈伤组织分化再生植株。对所得到的潮霉素抗性植株的Southern杂交分析表明GO基因已整合到受体基因组,为单拷贝或双拷贝插入。利用过氧化氢与淀粉-碘化钾反应显蓝色的特性检测到了转基因植株产生的过氧化氢，证实GO基因表达产生的葡萄糖氧化酶已经在水稻中发挥功能，这是将GO基因转入单子叶植物的首例报道。 基于过氧化氢诱导的植物防御反应没有种属专一性的优点，可以预期所得转基因水稻植株很可能对水稻的多种病原菌具有良好的抗性。已完成的抗病性鉴定表明，所得转基因水稻植株对稻瘟病具有良好的抗性。