Particle Bombardment Technique Research Articles

In Planta Genome Editing in Commercial Wheat Varieties: Use of TaQsd1 to Lengthen Seed Dormancy.

Dependency on in vitro culture and regeneration limits the ability to use genome editing on elite wheat (Triticum aestivum L.) varieties. We recently developed an in planta particle bombardment (iPB) technique for gene editing in wheat that utilizes shoot apical meristems (SAMs) as a target tissue. Since the method does not require in vitro culture, it can therefore be used on recalcitrant varieties. In this chapter, we describe in detail the steps used in the iPB method. With this protocol, 3% to 5% of T0 plants grown from bombarded SAMs typically carry mutant alleles and approximately 1% to 2% of the T0 plants inherit mutant alleles in the next generation.

Surface nano-crystallization and its effect on the microstructure evolution and mechanical properties of lightweight Fe–28Mn–8Al–1C steel subjected to supersonic fine particle bombardment

The surface nano-crystallization of lightweight Fe–28Mn–8Al–1C steel was achieved via the supersonic fine particle bombardment (SFPB) technique. The effects of impact time of SFPB and gas pressure on the surface morphology, microstructure, and mechanical properties were systematically investigated. The results indicated that SFPB treatment can form a gradient nanostructure on the surface of the lightweight Fe–28Mn–8Al–1C steel. With a gas pressure of 1.0 MPa and an impact time of 60 s, the surface roughness was minimized, and the corresponding strength parameters reached their highest values. With increases in the impact time and gas pressure, the grain size of the surface nanocrystalline layer gradually decreased, whereas the surface microhardness and the depth of the severe plastic deformation layer increased. However, excessively long impact time or excessively high gas pressure led to the formation of microcracks, resulting in a decrease in the strength. After SFPB treatment, lightweight Fe–28Mn–8Al–1C steel still exhibited good plasticity with elongation above 46 %, but the tensile fracture morphology changed from ductile fracture (before SFPB) to mixed ductile and brittle fracture (after SFPB).

Coffea arabica L. Resistant to Coffee Berry Borer (Hypothenemus hampei) Mediated by Expression of the Bacillus thuringiensis Cry10Aa Protein.

Coffea spp. are tropical plants used for brewing beverages from roasted and grounded seeds, the favorite drink in the world. It is the most important commercial crop plant and the second most valuable international commodity after oil. Global coffee trade relies on two Coffea species: C. arabica L. (arabica coffee) comprising 60% and C. canephora (robusta) comprising the remaining 40%. Arabica coffee has lower productivity and better market price than robusta. Arabica coffee is threatened by disease (i.e., coffee leaf rust), pests [i.e., Hypothenemus hampei or coffee berry borer (CBB) and nematodes], and susceptibility to climate change (i.e., drought and aluminum toxicity). Plant biotechnology by means of tissue culture inducing somatic embryogenesis (SE) process, genetic transformation, and genome editing are tools that can help to solve, at least partially, these problems. This work is the continuation of a protocol developed for stable genetic transformation and successful plant regeneration of arabica coffee trees expressing the Bacillus thuringiensis (Bt) toxin Cry10Aa to induce CBB resistance. A highly SE line with a high rate of cell division and conversion to plants with 8-month plant regeneration period was produced. To validate this capability, gene expression analysis of master regulators of SE, such as BABY BOOM (BBM), FUS3, and LEC1, embryo development, such as EMB2757, and cell cycle progression, such as ETG1 and MCM4, were analyzed during induction and propagation of non-competent and highly competent embryogenic lines. The particle bombardment technique was used to generate stable transgenic lines after 3 months under selection using hygromycin as selectable marker, and 1 month in plant regeneration. Transgenic trees developed fruits after 2 years and demonstrated expression of the Bt toxin ranging from 3.25 to 13.88 μg/g fresh tissue. Bioassays with transgenic fruits on CBB first instar larvae and adults induced mortalities between 85 and 100% after 10 days. In addition, transgenic fruits showed a seed damage lower than 9% compared to 100% of control fruits and adult mortality. This is the first report on stable transformation and expression of the Cry10Aa protein in coffee plants with the potential to control CBB.

Identification and expression of DoCCaMK during Sebacina sp. symbiosis of Dendrobium officinale

Dendrobium officinale Kimura et Migo is a famous precious medicinal plant in China. Seed and seedling were cultivated with the mycorrhizal fungus Sebacina sp. CCaMK was initially cloned from D. officinale based on a SSH cDNA library of symbiotically germinated seeds with Sebacina sp. Phylogenetic analysis was performed among DoCCaMK and other CCaMKs. The particle bombardment technique was used to visualize DoCCaMK-GFP. qRT-PCR and western blot analysis were conducted to determine the tissue expression patterns of DoCCaMK with (SGS) and without (UGS) Sebacina sp. Furthermore, the effect of KN-93 on CCaMK expression was also examined. Using NMT the net Ca2+ fluxes and the CCaMK concentration were measured during D. officinale seed germination. DoCCaMK had the highest homology with Lilium longiflorum CCaMK. The DoCCaMK-GFP protein localized in the nucleus and cell membrane. CCaMK expression was significantly upregulated after symbiosis with Sebacina sp. KN-93 could be used as an inhibitor of CCaMK to inhibit D. officinale seed germination. Ca2+ influx and the concentration of the CCaMK in the SGS group was significantly more than that of the UGS group. The characterization of CCaMK provides certain genetic evidence for the involvement of this gene during seed germination and mycorrhizal cultivation in D. officinale.

A modified transient gene expression protocol for subcellular protein localization analysis in rice

Transient protein expression is a useful technique for investigating the protein localization and functional analysis of stress responses in rice plants. Currently, available methods for in planta transient expression analysis include Agrobacterium-mediated transformation, protoplast transformation using polyethylene glycol or electroporation, and biolistic bombardment expression which have several disadvantages and are not well suited for the rice. Therefore, development of a method for rapid and efficient analysis of protein expression, subcellular localization, pathogen effector screening, and protein–protein interaction in rice is required. We developed a protocol for in planta gene expression analysis in sliced rice sheath cells by modifying and optimizing the biolistic particle bombardment technique. By obtaining thin sections (~400 μm) of rice sheath cells, auto-fluorescence from chlorophyll was eliminated. This system was validated through the localization of marker genes specifically expressed in nuclei, plasma membranes, and tonoplast. In addition, high transformation efficiency of 30% was achieved. Therefore, this protocol provides a new and rapid method for transient gene expression assay in rice. Protein secretion was examined in rice sheath cells using predicted secretory proteins from rice blast fungus, indicating that this method is applicable to plant–microbe interaction studies. The transient expression protocol established here is well optimized for protein localization, secretion, and host–pathogen protein interaction studies in rice. A typical experiment can be completed in three days.

Enhance Maize <i>(Zea mays L.)</i> Tolerance to Abiotic Stress Through the Genetic Transformation with Anthranilate Synthase (ASA2 Gene) Using Particle Bombardment

The agricultural plans in Egypt aim to increase maize production and yield to reduce the importation and to meet the country's need from maize. Currently the crop production is affected by the climatic changes which rise the abiotic and biotic stresses problems. In an attempt to raise the tryptophan level in maize, particle bombardment technique was used with two hybrids maize, namely Sc168 and Sc10. The results of this study showed that both hybrids were transformed efficiently and showed increased levels of tryptophan in the two hybrids. The double shot 1100 psi was higher effective than single shot. It can be concluded that levels of tryptophan increased clearly in the two hybrids which is considered an indicator for ASA2 successful expressing as compared to control. The present study established effective tissue culture protocol for maize hybrids suitable for gene transformation which may used in maize improvement programs in the future.

Transformasi Gen Antisens ACC Oksidase pada Pepaya dengan Teknik Penembakan Partikel

Papaya (Carica papaya L.) is a climacteric fruit that exhibit a<br />very fast ripening rate. Ethylene controls the ripening event<br />in the papaya fruit. 1-aminocyclopropane-1-carbocxylic acid<br />(ACC) oxidase gene encodes a specific enzyme for ethylene<br />biosynthesis. The gene had become a target for manipulation<br />to make a gene construct of an antisense ACC oxidase<br />to regenerate transgenic papaya that has a characteristic of<br />delayed ripening. The objective of the experiment is to engineer transgenic papaya that has a delayed ripening characteristic by transforming papaya with the antisense ACC oxidase gene through particle bombardment technique. The immature embryos of papaya variety Burung<br />were used for the explants. Antisense ACC oxidase and reporter (gus) genes were co-transformed to papaya calli. Four hundreds eighteen calli were bombarded by the antisense ACC oxidase gene. The transformation experiment resulted 25 putatives transgenic plants out of fifty plants<br />acclimatized in a greenhouse. Gus gene expression assay observed at 9 days after bombardment showed that the papaya explants bombarded twice at 9 cm shoot distance had 53.3% transformation rate of gus positive and 5.25 blue spots number in average. The results of PCR analysis showed that four out of 25 transgenic putative papaya plants (TR6, TR9, TR20, dan TR24), indicated a positive PCR of the antisense ACC oxidase gene with the amplified fragment DNA size of 800 base pair.

Stable nuclear transformation of Pandorina morum.

BackgroundVolvocine green algae like Pandorina morum represent one of the most recent inventions of multicellularity diverged from their unicellular relatives. The 8–16 celled P. morum alga and its close multicellular relatives constitute a model lineage for research into cellular differentiation, morphogenesis and epithelial folding, sexual reproduction and evolution of multicellularity. Pandorina is the largest and most complex organism in the volvocine lineage that still exhibits isogamous sexual reproduction. So far, molecular-biological investigations in P. morum were constricted due to the absence of methods for transformation of this species, which is a prerequisite for introduction of reporter genes and (modified) genes of interest.ResultsStable nuclear transformation of P. morum was achieved using chimeric constructs with a selectable marker, a reporter gene, promoters and upstream and downstream flanking sequences from heterologous sources. DNA was introduced into the cells by particle bombardment with plasmid-coated gold particles. The aminoglycoside 3′-phosphotransferase VIII (aphVIII) gene of Streptomyces rimosus under control of an artificial, heterologous promoter was used as the selectable marker. The artificial promoter contained a tandem arrangement of the promoter of both the heat shock protein 70A (hsp70A) and the ribulose-1,5-bisphosphat-carboxylase/-oxygenase S3 (rbcS3) gene of Volvox carteri. Due to the expression of aphVIII, transformants gained up to 333-fold higher resistance to paromomycin in comparison to the parent wild-type strain.The heterologous luciferase (gluc) gene of Gaussia princeps, which was previously genetically engineered to match the nuclear codon usage of Chlamydomonas reinhardtii, was used as a co-transformed, unselectable reporter gene. The expression of the co-bombarded gluc gene in transformants and the induction of gluc by heat shock were demonstrated through bioluminescence assays.ConclusionStable nuclear transformation of P. morum using the particle bombardment technique is now feasible. Functional expression of heterologous genes is achieved using heterologous flanking sequences from Volvox carteri and Chlamydomonas reinhardtii. The aphVIII gene of the actinobacterium S. rimosus can be used as a selectable marker for transformation experiments in the green alga P. morum. The gluc gene of the marine copepod G. princeps, expressed under control of heterologous promoter elements, represents a suitable reporter gene for monitoring gene expression or for other applications in P. morum.

Transfer of a β-glucuronidase marker gene to triticale (xTriticosecale Wittmack) via particle bombardment (biolistic) method

Abstract This study was carried out to investigte the biotechnology laboratory of Department of Field Crops, Ankara University with the aim of investigating the optimum parameters of gene transfer to mature embryo and leaf segment explants of Triticale (x Triticosecale Wittmack) via particle bombardment (Biolistic) method. Mature embryo and leaf segment explants obtained from Triticale (x Triticosecale Wittmack) were bombardment with accelerated gold or tungsten particles coated with the plasmid pBI221.23 containing the b -glucuronidase (GUS: u idA ) marker gene. Different bombardment distances from the stopping plate (6, 9, 12, cm) and different rupture disk pressures (900, 1100, 1550 psi ) were used as physical parameters. Blue cells expressing the b -glucuronidase gene were detected by using a histochemical assay. The variation in the number of blue spots in bombarded explants was used to determine the gene transfer efficiency. The mature embryos of triticale were found to be more amenable for the direct delivery of foreign DNA by the particle bombardment technique than the leaf segments. Also the gold particles were more suitable than tungsten particles in the gene transfer efficiency. The highest gene transfer results were obtained in mature embryos when 6 cm bombardment distance and 1100 psi rupture disk pressure were used.

Molecular components required for the targeting of PEX7 to peroxisomes in Arabidopsis thaliana

PEX7 is a soluble import receptor that recognizes peroxisomal targeting signal type 2 (PTS2)-containing proteins. In the present study, using a green fluorescent protein (GFP) fusion protein of PEX7 (GFP-PEX7), we analyzed the molecular function and subcellular localization of PEX7 in Arabidopsis thaliana. The overexpression of GFP-PEX7 resulted in defective glyoxysomal fatty acid beta-oxidation, but had no significant effect on leaf peroxisomal function. Analysis of the subcellular localization of GFP-PEX7 in transgenic Arabidopsis showed that GFP-PEX7 localizes primarily to the peroxisome. Transient expression of a C- or N-terminal fusion protein of PEX7 and yellow fluorescent protein (YFP) (PEX7-YFP and YFP-PEX7, respectively) in leek epidermal cells, using the particle bombardment technique, confirmed that fluorescent protein-tagged PEX7 localizes to peroxisomes in Arabidopsis. Immunoblot analysis revealed that GFP-PEX7 accumulates primarily in peroxisomal membrane fractions, whereas endogenous PEX7 was distributed evenly in cytosolic and peroxisomal membrane fractions, which indicated that both endogenous PEX7 and GFP-PEX7 are properly targeted to peroxisomal membranes. The results of bimolecular fluorescence complementation (BiFC) and yeast two-hybrid analyses showed that PEX7 binds directly to PTS2-containing proteins and PEX12 in the peroxisomal membrane. We used red fluorescent protein (tdTomato) fusion protein of PEX7 (tdTomato-PEX7) in several Arabidopsis pex mutants to identify proteins required for the targeting of PEX7 to peroxisomes in planta. The results demonstrated that pex14, pex13 and pex12 mutations disrupt the proper targeting of PEX7 to peroxisomes. Overall, our results suggest that the targeting of PEX7 to peroxisomes requires four proteins: a PTS2-containing protein, PEX14, PEX13 and PEX12.

Studies on genetic transformation of olive (Olea europaea L.) somatic embryos: I. Evaluation of different aminoglycoside antibiotics for nptII selection; II. Transient transformation via particle bombardment

As a first step to the establishment of a genetic transformation protocol for olive somatic embryos obtained from the seeds of cv. 'Picual', the efficiencies of different aminoglycoside antibiotics as selective agents to be used with the nptII marker gene, and the particle bombardment technique for transient transformation have been evaluated. Among the three antibiotics tested, paromomycin and kanamycin showed a similar inhibitory effect and, at 200 mg l -1 , both of them impaired callus growth after 8 weeks of culture. However, when isolated embryos were cultured in the presence of these antibiotics, a 20% of the embryos still remained viable at 400 mg l -1 . Neomycin was discarded as a selective agent since it showed only a mod- erate toxic effect. Contrary to solid medium, when olive callus was cultured in liquid medium supplemented with different paromomycin concentrations for 3 weeks, the callus growth was impaired at the lowest antibiotic concentration, 3 mg l -1 . Best conditions for transient transformation of olive callus using PDS-1000/He system were a 6 cm target distance and a 900 psi bombardment pressure. pCGUD1 plasmid, containing the gus gene under the control of sunflower ubiquitin promoter yielded a sig- nificantly higher number of gus expression areas per bombarded explant than pGUSINT or pJGUS5 plasmids, where the gus gene is driven by CaMV35S promoter or CaMV35S with enhancer, respectively. Almost 45% of bombarded explants showed gus expression 12 weeks after

Biosafety assessment and benefits for co-existence of biological contained plants- regulatory assessment in the EU-project “Transcontainer”

The EU-project TransContainer deals with the evaluation of environmental impact and benefits for coexistence between GMand non-GM plants. Different containment strategies are applied to a broad spectrum of crops (e.g. oilseed rape, sugar beet, tomato) as well as to perennial plants like trees and grasses. The containment methods focus on three strategies: Chloroplast Transformation, Controllable Flowering and Controllable Fertility. Key issues are (a) safety assessment focussing on three points: molecular characteristics, ecology of the GM species and consequences of a potential break-down of the containment system. (b) Benefit assessment of the containment system for the co-existence of GM and non-GM plants. The environmental impact assessment is performed based on the criteria provided by the EFSA Guidance document (2006) for the placing on the market of GM plants in the EU. Additionally to impact assessment, an economic evaluation is performed. The development of contained GM crop plants is still in an early stage and thus our evaluation is focussed on the safety assessment of the general methodical characteristics. Benefits of Chloroplast Transformed plants: Chloroplast transformation is a promising containment system for plant species with strict maternal plastid inheritance by avoiding the outcrossing of recombinant genes via the pollen. The targeted insertion of a GM sequence in the chloroplast genome by homologous recombination has three advantages: (1) insertional inactivation of unknown functional genes is avoided, (2) endogenic in-situ promoters can be used, and (3) even though plastids harbour relatively small replicons, large insertions are tolerated. However, it has to be verified that the transgene is not inserted unintentionally in the nuclear genome since the transformation (e.g. particle bombardment technique) could be unspecific. Benefits of plants with Controlled Flowering: The suppression of flowering is useful for plants that are cultivated for their vegetative parts, e.g. sugar beet, grasses or trees. For bi-annual sugar beet, the inhibition of undesired bolting and flowering will either prevent out-crossing and introgression into endogenous beet populations as well as facilitate the cultivation of beets for the farmer. In grasses, flower suppression will improve the fodder quality as the shoots have higher lignin content thus hampering the digestibility of the feed. In trees that are intended to be cultivated in plantations for biomass production, the suppression of flower development would offer certain advantages: GM trees with e.g. changed wood properties would neither develop pollen nor seeds containing the inserted DNA, avoiding any unintended hybridisation or spread of the GM seeds by wind. In contrast to trees that can be propagated vegetative, grasses as well as sugar beet need to flower for breeding purposes. Therefore, a molecular switch will be introduced, which will initiate flowering upon an external chemical stimulus. In case of flower induction for breeding, other (physical) measures are requested to avoid unintended out-crossing and spread of hybrids. Benefits for Co-existence: Regarding the legislation on GM plants in the EU, the minimisation of GM escape via pollen into adjacent non-GM fields or wild relatives is an important point in the improvement of co-existence measures. In chloroplast transformed plants, the spread of inserted DNA is limited only at the pollen level, whereas the (transgene) seeds produced by the mother plant could still be lost or spilled unintended during harvest and transport. Upon complete flower suppression, neither pollen nor seeds will be released, thus gaining a very high level of restriction. The containment measures will allow (i) minimising the isolation distances between GM and nonGM fields, (ii) protecting the GM cultivating farmers for liability claims from neighbours, (iii) reduce conflicts with bee keepers, and (iv) avoid unintended mixtures with food products.

Characterization of the S-RNase promoters from sweet cherry (Prunus avium L.).

Genomic DNA fragments containing the S(3)-, S(4)-, and S(6)-RNase genes were isolated from the sweet cherry (Prunus avium L.) and sequenced. Comparison of the 5'-flanking sequences of these three S-RNases indicated that a highly conserved region (designated CR) existed just upstream from the putative TATA boxes. We postulate that CR contains cis-regulatory element(s) involved in pistil expression. To examine the activity of the isolated S-RNase promoters of sweet cherry in the pistil, we transiently introduced approximately 650-bp fragments of the S(4)- and S(6)-RNase promoters fused to beta-glucuronidase (GUS) gene into the pistil of the petunia using a particle bombardment technique. Histochemical analysis showed that the 5'-flanking region of each S-RNase was active in the pistil. This suggests that cis-regulatory element(s) for pistil-specific expression may exist(s) within the 650-bp region upstream from the TATA box in the sweet cherry S-RNase promoter.

잡초성벼의 superoxide dismutase cDNA cloning과 재배벼로의 형질전환

냉해나 한발등의 환경스트레스에 대해 저항성을 유발하는 유전자를 환경스트레스에 강한 잡초성벼로부터 선발하고 이들 유전자를 재배벼에 도입하여 도입유전자 산물의 과량 발현을 통해 냉해나 한발 등에 대한 저항성이 향상된 벼를 선발하고자 하였다. 잡초성벼인 Bhutan 14Ad로부터 냉해 및 한발 저항성 유전자로 알려진 superoxide dismutase (SOD) cDNA를 분리하고자 mRNA를 분리하고 이 분리된 mRNA를 이용해 reverse transcriptase PCR방법으로 SOD cDNA를 cloning 하였다. 그 결과 2종의 SOD cDNA가 cloning되어 SOD-A, SOD-B로 명명하였다. 이들 cDNA의 염기서열을 결정한 결과 이들은 아미노산 서열 상동성이 88.4%를 나타내었으며, SOD-A는 Oryza sativa 계열의 Cu/Zn SOD유전자인 GenBank accession No. L36320와 99.3% 동일하였으며, SOD-B는 accession No. D01000과 100% 동일하였다. 이들 SOD-A와 SOD-B cDNA를 재배벼인 낙동벼에 형질전환하여 형질전환체 벼를 선발하였으며, 이들 형질전환체 벼의 냉해저항성및 한발저항성 검정을 통해 저항성이 향상된 형질전환체 벼를 선발하고 있다. Two different cDNA clones for superoxide dismutase (SOD) were isolated from an weedy rice variety (Oryza sativa, cv. Bhutan14Ad) and were introduced into a cultivated rice variety (Oryza sativa, cv. Nakdong) in order to develop the environmental stress-resistant rice plants. Sequence analysis of the cloned cDNAS indicated that the deduced amino acid sequence of SOD-A is 88.4% identical to that of SOD-B. Furthermore, the nucleotide sequence of SOD-A is 99.3% identical to that of a Cu/Zn SOD gene of Oryza sativa (GenBank accession No. L36320). The nueleotide sequence of SOD-B was identical to that of the previously published SOD gene (Accession No. D01000). A cultivated rice variety, Nakdong-byeo, was transformed with chimeric SOD genes containing a actin promoter of rice and pin2 terminator using a particle bombardment technique. Transformed calli were selected on an selection medium containing phosphinothricin (PPT). Transgenic rice plants were regenerated from the PPT-resistant calli. PCR analysis with genomic DNAs from transgenic plants revealed that transgenes are introduced into rice genome.

Delivery of Genes and Fluorescent Dyes into Cells of the Intact Lens by Particle Bombardment

The authors report the use of a particle bombardment technique to deliver exogenous genes and fluorescent dyes into living fibre cells in the intact lens. Gold particles were coated with plasmid DNA encoding green fluorescent protein (GFP) or the lipophilic fluorescent probes DiI and DiO. The particles were introduced into embryonic chicken or neonatal mouse lenses using a pressurized helium charge. A significant fraction of particles penetrated the capsule and came to rest in the superficial lens cortex. Over the range tested, varying the particle size or the pressure of the helium charge had little effect on the final distribution of particles within the lens. Particle bombardment was used initially to deliver DiI and DiO into the lens. Within a few hours, bombarded lenses exhibited multicolored membranous labelling of individual, elongating fibre cells. The particle bombardment technique was also used to introduce a plasmid encoding GFP. After overnight incubation, many fibre cells in the bow region expressed GFP. On close examination by confocal reflectance microscopy, each expressing cell was found to contain a gold particle lodged in its nucleus. The authors examined the fate of GFP-expressing fibre cells over a period of 1 week in organ culture. In the embryonic chicken lens, transfected fibres showed modest (approximately two-fold) elongation. In contrast, GFP-expressing mouse lens fibres underwent dramatic elongation, reaching the anterior and posterior sutures after 7 days in culture. These species differences may reflect the fact that mitosis continued at a near normal rate in the cultured mouse lens but declined precipitously in the cultured chicken lens. These results suggest that particle bombardment, in conjunction with appropriate cell culture conditions, may prove useful in visualizing the behaviour of differentiating fibre cells in the living intact lens in vitro.

Somatic embryogenesis of groundnut (Arachis hypogaea L.): A comparison of four South African genotypes

Genetic engineering is an important tool which can be used together with conventional breeding methods in transferring certain economically important traits into cultivated varieties of almost any crop species. One of the methods for gene transfer is the particle bombardment technique. Several primary explants can be used with this technique for the transformation of groundnut. This study explored the use of mature embryo axes as explants for somatic embryogenesis, and determined the effect of medium composition on four South African genotypes. A modified Murashige and Skoog-medium was used with various concentrations of picloram. Mature embryo axes were found to be a suitable explant for induction of somatic embryogenesis. A significant difference was found in the response of the different genotypes to medium composition.

Isolation and expression of an elongation‐dependent gene of mung bean (Vigna radiata) hypocotyl

A cDNA clone of an auxin up‐regulated gene, ARG8, was isolated from hypocotyl sections of etiolated mung bean [Vigna radiata (L.) Wilczek] seedlings by differential screening. The deduced amino acid sequence suggested that ARG8 may encode a cell wall protein. The steady state mRNA level of ARG8 increased by treatment of hypocotyl sections not only with indole‐3‐acetic acid (IAA) but also with fusicoccin, and the auxin inducibility was inhibited by the addition of 0.3 M mannitol in the incubation medium. This indicated that it was not auxin but elongation that regulated the expression of ARG8. The promoter activity of the 5′‐flanking region of ARG8 was determined by assaying the transient expression of a luciferase fusion gene that was introduced into mung bean hypocotyl sections by the particle bombardment technique. The basal activity of the ARG8 upstream region was about a few tenths of that of a modified cauliflower mosaic virus 35S promoter, and it was increased a few fold by treatment with IAA. The auxin inducibility was completely suppressed by the addition of mannitol. A 5′‐deletion analysis showed that a 53‐bp region in the ARG8 promoter was important for the basal and elongation‐dependent promoter activities.

Genetic engineering of an ectomycorrhizal fungus Laccaria bicolor for use as a biological control agent

We are developing genetically engineered ectomycorrhizal fungi for use as biological control agents. Using the gold particle bombardment technique, we transformed Laccaria bicolor, an ectomycorrhizal fungus capable of forming ectomycorrhizae on a wide range of plant hosts. Structural gene sequences of the hygromycin B phosphotransferase (hph) gene (hygromycin resistance for selection of transformants), β-glucuronidase (GUS) gene (reporter gene for tracking transformants) and Bacillus thuringiensis δ-endotoxin (BtCryIIIA) gene (Coleopteran specific insecticidal gene from Bacillus thuringiensis var tenebrionis) were introduced into L. bicolor simultaneously. Southern and western blot analyses showed that the introduced sequences were inserted into the fungal genome and transcribed and translated. One of the transformants produced the insecticidal toxin; however, the levels were not sufficient to perform toxicity assays. The technique developed can be employed to generate superior strains of L. bicolor suitable for use as biological control agents in various soil and plant conditions.

Transient and stable transfection of Leishmania by particle bombardment

Transfection of trypanosomatids, particularly the transfection of their mitochondrion and different developmental stages, is still a limiting step in many experiments using recombinant DNA technology. Two types of trypanosomatid transfection protocols have been described so far. The standard protocol is based on electroporation first described by Bellofatto and Cross [1]. The second protocol is based on transfection using a homemade particle gun [2]. The biolistic method was first applied to plants [3], but is now routinely used for transfection of a wide variety of targets ranging from bacterial to mammalian cells [4–10] and it is the only method described so far to transfect organelles [10,11]. In trypanosomatids, the genetic organization of the mitochondrion shows several unusual features like a complex DNA network composed of thousands of catenated circular DNA molecules [12], the lack of encoded tRNAs [13] and the still enigmatic process of RNA editing [14]. However, the trypanosomatid mitochondrion is not yet accessible to recombinant DNA technology, and therefore the experimental in vivo analysis of the mentioned features is severely restricted. It is intended that the particle bombardment technique should be applied to transfect mitochondria of trypanosomatids. Since the optimal bombarding conditions for nuclear and organellar transfection of a given organism generally are identical [6,10,15,16] and as a first step, the factors influencing the nuclear transfection efficiency have been investigated. The ability of microprojectiles to deliver DNA into Leishmania cells was demonstrated either by the presence of transient b-glucuronidase (GUS) activity or by selection of stable transformants on plates following bombardment with the plasmid pX63NEO-GUS [17] using the DuPont Biolistic Abbre6iations: GUS, b-glucuronidase. * Corresponding author. Tel.: +41 31 6314342; fax: +41 31 6313383; e-mail beat.blum@ibc.unibe.ch

Advances in cereal gene transfer

Over the past five years, transgenic strains of various major cereals have been produced, with transformation of rice and maize being most common. A majority of the cereal transformants obtained to date has been generated by the particle bombardment technique, but Agrobacterium-mediated transformation is rapidly becoming the method of choice. Rice, the plant in which transformation-related technology is most advanced, appears to be the model monocotyledon for basic and applied studies.