Electroporation Of Protoplasts Research Articles

Somatic Embryogenesis in the Family Gentianaceae and Its Biotechnological Application.

The family Gentianaceae consists of 1736 species, which play an important role in human being existence due to their pharmacological and horticultural values. Many species accumulate bitter iridoid substances used medicinally and in flavorings, while others are cultivated because of beauty of their flowers showing a wide range of colors and patterns. Out of 99 genera belonging to the gentian family, process of somatic embryogenesis (SE) was reported for 5. The first reports, aimed at micropropagation of ornamental cultivars and production of secondary metabolites, concerned Centaurium erythraea Rafn., Eustoma russellianum Grieseb. and Exacum affine Balf. Somatic embryos were induced on different explants cultured in the liquid Murashige and Skoog medium supplemented with auxins and cytokinins. In the 1990s of the last century, significant progress in the exploration of the phenomenon of SE and its biotechnological application was made for the genus Gentiana. The process was induced on various explants and studied at the structural and ultrastructural levels. Regenerated plants were screened for genetic stability using flow cytometry, chromosome counting, and molecular markers. Besides typical indirect SE, the use of leaf fragments enabled to obtain single-cell origin of somatic embryos. On the other hand, proliferation of embryogenic callus in liquid medium resulted in the establishment of long-term embryogenic cell suspension cultures, paving the way not only to study the formation of somatic embryos and the development of regenerants but also to preserve the morphogenic potential of cell aggregates by cryopreservation. Cell suspensions re-established after storage in liquid nitrogen maintained their embryogenic character and allowed to obtain somatic embryo-derived regenerants that were true-to-type at both genetic and epigenetic levels. Another application of SE was related to genetic manipulation purposes. Efficient protocols of plant regeneration from callus-, cell suspension-, or leaf mesophyll-derived protoplasts allowed engaging procedures of somatic hybridization or protoplast electroporation for gentian genome modifications. Also, high embryogenic potential existing in the numerous gentian species enabled successful Agrobacterium-mediated transformation of G. cruciata L. and G. dahurica Fisch.

Methods for suspension culture, protoplast extraction, and transformation of high-biomass yielding perennial grass Arundo donax.

Arundo donax L. is a promising biofuel feedstock in the Mediterranean region. Despite considerable interest in its genetic improvement, Arundo tissue culture and transformation remains arduous. The authors developed methodologies for cell- and tissue culture and genetic engineering in Arundo. A media screen was conducted, and a suspension culture was established using callus induced from stem axillary bud explants. DBAP medium, containing 9 µM 2,4-D and 4.4 µM BAP, was found to be the most effective medium among those tested for inducing cell suspension cultures, which resulted in a five-fold increase in tissue mass over 14 days. In contrast, CIM medium containing 13 µM 2,4-D, resulted in just a 1.4-fold increase in mass over the same period. Optimized suspension cultures were superior to previously-described solidified medium-based callus culture methods for tissue mass increase. Suspension cultures proved to be very effective for subsequent protoplast isolation. Protoplast electroporation resulted in a 3.3 ± 1.5% transformation efficiency. A dual fluorescent reporter gene vector enabled the direct comparison of the CAMV 35S promoter with the switchgrass ubi2 promoter in single cells of Arundo. The switchgrass ubi2 promoter resulted in noticeably higher reporter gene expression compared with that conferred by the 35S promoter in Arundo.

Transformation of Undomesticated Strains of Bacillus licheniformis by Protoplast Electroporation

Transformation of Undomesticated Strains of Bacillus licheniformis by Protoplast Electroporation

PSiM24 Is a Novel Versatile Gene Expression Vector for Transient Assays As Well As Stable Expression of Foreign Genes in Plants

We have constructed a small and highly efficient binary Ti vector pSiM24 for plant transformation with maximum efficacy. In the pSiM24 vector, the size of the backbone of the early binary vector pKYLXM24 (GenBank Accession No. HM036220; a derivative of pKYLX71) was reduced from 12.8 kb to 7.1 kb. The binary vector pSiM24 is composed of the following genetic elements: left and right T-DNA borders, a modified full-length transcript promoter (M24) of Mirabilis mosaic virus with duplicated enhancer domains, three multiple cloning sites, a 3′rbcsE9 terminator, replication functions for Escherichia coli (ColE1) and Agrobacterium tumefaciens (pRK2-OriV) and the replicase trfA gene, selectable marker genes for kanamycin resistance (nptII) and ampicillin resistance (bla). The pSiM24 plasmid offers a wide selection of cloning sites, high copy numbers in E. coli and a high cloning capacity for easily manipulating different genetic elements. It has been fully tested in transferring transgenes such as green fluorescent protein (GFP) and β-glucuronidase (GUS) both transiently (agro-infiltration, protoplast electroporation and biolistic) and stably in plant systems (Arabidopsis and tobacco) using both agrobacterium-mediated transformation and biolistic procedures. Not only reporter genes, several other introduced genes were also effectively expressed using pSiM24 expression vector. Hence, the pSiM24 vector would be useful for various plant biotechnological applications. In addition, the pSiM24 plasmid can act as a platform for other applications, such as gene expression studies and different promoter expressional analyses.

GENETICALLY MODIFIED BANANAS: PAST, PRESENT AND FUTURE

Edible bananas comprise several characteristics that make them an ideal target for improvement through genetic engineering: (i) they constitute the N° 1 fresh fruit crop in the world, (ii) they are highly sterile which makes classical breeding extremely difficult but at the same time prevents transgene drift via pollen into the environment, (iii) their cultivation is monoclonal, and (iv) they are susceptible to a wide range of pests and diseases. The latest outbreak of Fusarium wilt disease caused by Fusarium oxysporum f. sp. cubense Race 4 or TR4, which is seriously threatening the international banana trade, strengthens the need for developing resistant bananas. Despite these facts, the number of publications and research groups dealing with genetic engineering of banana remains low compared to other crops. Possible reasons are among others the difficulties in obtaining transformation competent tissues (in case of banana embryogenic cell suspensions or ECS), the duration to obtain fully transformed plants, the cost and effort of maintaining transgenic lines, the lack of a proper and functional legal biosafety system for testing and culturing GMOs in many banana producing countries and the size of individual banana plants rendering large scale testing of transgenes extremely costly. In this review, we discuss the 19 years history of banana genetic modification. An overview of the different transformation methodologies will be given, from the first reports on protoplast electroporation to Agrobacterium-mediated transformation of ECS. Also the applications of this technology for banana improvement (agronomic treats, quality treats, etc.) will be discussed along with examples of confined field trials of genetically modified bananas. Finally, a brief overview of the research topics on banana genetic engineering at the Laboratory of Tropical Crop Improvement (LTCI, KU Leuven, Belgium) is discussed. Topics include selectable marker genes, promoter and gene characterization as well as molecular breeding for fungus and drought resistance.

Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation

Wang J., Jiang P., Cui Y., Guan X. and Qin S. 2010. Gene transfer into conchospores of Porphyra haitanensis (Bangiales, Rhodophyta) by glass bead agitation. Phycologia 49: 355–360. DOI: 10.2216/09-91.1Genetic transformation by electroporation of protoplasts is a standard procedure for many plants. However, for the genus Porphyra, the method is not effective because of low viability of protoplasts and is a time-consuming and expensive procedure. Based on the life history of Porphyra, a spore-targeted strategy of genetic transformation was developed, that is, using fresh conchospores of Porphyra haitanensis Chang & Zheng transformed by agitation with glass beads. A SV40 promoter-driven lacZ reporter gene was expressed in conchospores 48 h after the agitation. More transformants were obtained by increasing the agitation time from 10 to 25 s. The maximum number of transformants was more than six out of 1 million agitated conchospores. Transfer of a SV40 promoter-driven egfp gene into conchospores resulted in significant green GFP fluorescence. The expression of lacZ and egfp revealed that this strategy of spore-targeted transformation using glass bead agitation is feasible in P. haitanensis and that the SV40 promoter is effective for monitoring expression of foreign genes in this red algal species.

Improvement of bananas for black sigatoka and panama disease resistance through genetic manipulation

Bananas are a staple food in Eastern Africa, with 25.3% of the total world production. The production is, however, threatened by the presence of several diseases, of which the fungal diseases black sigatoka and Panama disease are the most important. With the development of embryogenic cell suspension cultures, the isolation of protoplasts therefrom and their successful regeneration, an invaluable vegetative material for genetic manipulation of bananas became available. The discovery of new types of antifungal proteins (AFP’s) and the cloning of their encoding genes provide a source of resistance to fungal diseases that can be introduced into plant cells by genetic engineering. Transformation techniques are currently under investigation. Electroporation of banana protoplasts have resulted in transient expression frequencies of more than 1% as visualized by the action of the β-glucuronidase (GUS) marker gene. Experiments with particle bombardment on cell suspensions have resulted in transient expression frequencies up to 3300 blue spots/0.5 ml cells, and stable transformants have also been selected. Optimisation of the cell suspension culture technique and transformation methodology is in progress.

Protocol: a rapid and economical procedure for purification of plasmid or plant DNA with diverse applications in plant biology

Research in plant molecular biology involves DNA purification on a daily basis. Although different commercial kits enable convenient extraction of high-quality DNA from E. coli cells, PCR and agarose gel samples as well as plant tissues, each kit is designed for a particular type of DNA extraction work, and the cost of purchasing these kits over a long run can be considerable. Furthermore, a simple method for the isolation of binary plasmid from Agrobacterium tumefaciens cells with satisfactory yield is lacking. Here we describe an easy protocol using homemade silicon dioxide matrix and seven simple solutions for DNA extraction from E. coli and A. tumefaciens cells, PCR and restriction digests, agarose gel slices, and plant tissues. Compared with the commercial kits, this protocol allows rapid DNA purification from diverse sources with comparable yield and purity at negligible cost. Following this protocol, we have demonstrated: (1) DNA fragments as small as a MYC-epitope tag coding sequence can be successfully recovered from an agarose gel slice; (2) Miniprep DNA from E. coli can be eluted with as little as 5 μl water, leading to high DNA concentrations (>1 μg/μl) for efficient biolistic bombardment of Arabidopsis seedlings, polyethylene glycol (PEG)-mediated Arabidopsis protoplast transfection and maize protoplast electroporation; (3) Binary plasmid DNA prepared from A. tumefaciens is suitable for verification by restriction analysis without the need for large scale propagation; (4) High-quality genomic DNA is readily isolated from several plant species including Arabidopsis, tobacco and maize. Thus, the silicon dioxide matrix-based DNA purification protocol offers an easy, efficient and economical way to extract DNA for various purposes in plant research.

English

The ever-increasing world population has created two major problems: more mouths to feed and less land to farm. While classical breeding has enormously helped in providing more food, a lot still needs to be done. Transformation biotechnology can also help especially where classical breeding lacks solution (e.g. limited availability of stable and durable genetic source of resistance). However, plant transformation technologies require selectable marker genes to produce transgenic plants but such genes are of no value thereafter; in fact, marker genes in transgenic plants are perceived to pose potential bio-safety problems. In all genetic transformation technologies based on direct gene transfer (electroporation of protoplasts, particle bombardment etc) the selectable marker genes generally co-integrate with the gene of interest(s) in one Mendelian locus in the plant genome; hence, their removal is highly desirable. This may also help in the acceptability of transgenic plants by society. Transgenic plants that contain the desired gene of interest but lack the selection marker gene used in its production are termed “clean” and the methods utilized in their production are referred to as “clean gene” technology. There are several proved methods of eliminating selectable marker genes and these include: (A) Marker gene excision consisting of (1) intra-genomic relocation of transgenes via transposable elements, (2) site-specific recombination systems and (3) intra-chromosomal recombination. (B) Gene replacement or Targeted gene replacement; and (C) transformation with multiple T-DNAs, which could result in linked and/or unlinked co-integration of transgenes. Unlinked transgenes are then segregated out during meiosis.   Key words: Clean gene, selection-marker free, transposable elements, site-specific recombination systems, intra-chromosomal recombination, multiple T-DNAs co-cultivation, linked / unlinked transgenes.

Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells

Transient expression of fluorescent fusion proteins in plant cells has dramatically facilitated our study of newly identified genes and proteins. This protocol details an in vivo transient expression system to study the subcellular localization and dynamic associations of plant proteins using protoplasts freshly prepared from Arabidopsis or tobacco BY-2 suspension cultured cells. The method relies on the transformation of DNA constructs into protoplasts via electroporation. The whole protocol is comprised of three major stages: protoplast generation and purification, transformation of DNA into protoplasts via electroporation and incubation of protoplasts for protein analysis. Similar to stably transformed cell lines, transformed protoplasts are compatible with protein localization studies, pharmaceutical drug treatment and western blot analysis. This protocol can be completed within 11-24 h from protoplast production to protein detection.

Preparation and Electroporation of Oat Protoplasts from Cell Suspension Culture

Protoplasts provide a convenient system for introduction of nucleic acids into plant cells. Protoplasts allow rapid assays of gene expression and virus replication, with advantages for plant biology similar to those of cultured animal cells for the study of animal systems. Traditionally, preparation and handling of protoplasts has been as much art as science, requiring a special touch by the user. The purpose of this unit is to lay out in clear detail all the methods and nuances involved in protoplast preparation using a robust, reliable system that does not require skills beyond those expected of an unspecialized molecular biologist. Because dicots and monocots differ in many biological properties, and because different procedures may work better for different plants, separate units in this chapter are devoted to protoplast preparation from dicots (Arabidopsis, tobacco; refer to UNITS 16D.1 & 16D.4) and from a monocot (oat). This unit describes methods for preparation and transfection by electroporation of protoplasts derived from an oat suspension culture.

Transformation of undomesticated strains of Bacillus subtilis by protoplast electroporation

A rapid method combining the use of protoplasts and electroporation was developed to transform recalcitrant wild strains of Bacillus subtilis. The method described here allows transformation with both replicative and integrative plasmids, as well as with chromosomal DNA, and provides a valuable tool for molecular genetic analysis of interesting Bacillus strains, which are hard to transform by conventional methods.

Transgenic grapevine rootstock clones expressing the coat protein or movement protein genes of Grapevine fanleaf virus: Characterization and reaction to virus infection upon protoplast electroporation

The reaction to Grapevine fanleaf virus (GFLV) infection in 42 independent transgenic grapevine rootstock 41B clones expressing the coat protein (CP) or movement protein (MP) gene of GFLV was assayed by protoplast electroporation. Two of the 26 transgenic clones expressing the CP gene did not support the accumulation of GFLV MP to detectable levels, 12 accumulated substantially lower levels of MP, and 12 accumulated equivalent levels of MP relative to protoplasts of nontransformed controls at 72 h post-electroporation, as shown by Western blots with anti-MP γ-globulins. Interestingly, inhibition of MP accumulation was achieved against virions but not viral RNAs, and was dependent on the inoculum dose. No interference was observed with the multiplication of Arabis mosaic virus, which is closely related to GFLV, likely due to low nucleotide identity between the CP genes. Also, one of the 16 transgenic clones expressing the MP gene significantly reduced the accumulation level of GFLV CP at 72 h post-electroporation, as shown by DAS-ELISA with anti-GFLV γ-globulins. The potential of protoplast electroporation as rapid identification of GFLV-resistant grapevine clones at the cell level will be discussed relative to field screening for resistance at the plant level by nematode-mediated GFLV transmission.

Improvement of transformation and electroduction in avermectin high-producer,Streptomyces avermitilis

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.

Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene.

Creeping bentgrass (Agrostis palustris Huds.) is a cool season grass widely used on putting greens in golf courses. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. The objective of our study is to develop an alternative and more efficient approach in transforming the grass using Agrobacterium (strain EHA 101). This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose. Dozens of transgenic plants have been produced from two independent transformed calli. Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings. Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the GFP gene were clearly shown in transgenic plants. These results indicated that Agrobacterium transformation can successfully be applied to creeping bentgrass.

Chimeric RNA/DNA oligonucleotide-based site-specific modification of the tobacco acetolactate syntase gene.

Single amino acid substitutions at either of two crucial positions in acetolactate synthase (ALS) result in a chlorsulfuron-insensitive form of this enzyme and, as a consequence, a herbicide-resistant phenotype. Here, we describe the successful in vivo targeting of endogenous tobacco (Nicotiana tabacum) ALS genes using chimeric RNA/DNA and all-DNA oligonucleotides at two different locations. Similar number of conversion events with two different chimeras indicates the absence of restricting influence of genomic target sequence on the gene repair in tobacco. Chlorsulfuron-resistant plants were regenerated from calli after mesophyll protoplast electroporation or leaf tissue particle bombardment with these specifically constructed chimeras. Sequence analysis and enzyme assays proved the resulting alterations to ALS at both DNA and protein levels. Furthermore, foliar application of chlorsulfuron confirmed the development of resistant phenotypes. Lines with proline-196-alanine, threonine, glutamine, or serine substitutions or with tryptophan-573-leucine substitutions were highly resistant at both cellular and whole plant levels, whereas lines with proline-196-leucine substitutions were less resistant. The stability of these modifications was demonstrated by the continuous growth of calli on chlorsulfuron-containing medium and by the transmission of herbicide resistance to progeny in a Mendelian manner. Ability of haploid state to promote chimera-mediated conversions is discussed.

Review of Techniques to Inoculate Grapevines with Grapevine Fanleaf Virus: Lessons and Perspectives

Grapevine fanleaf virus (GFLV) occurs in all major grape-growing regions of the world, causing a severe degeneration of vegetation, reductions in grape yield, and poor-quality harvest. GFLV is transmitted through the soil by the ectoparasitic nematode <i>Xiphinema index</i>. Control of GFLV with agrochemicals to eradicate nematode vectors is not feasible in established vineyards. Several laboratories are examining the development of GFLV-resistant rootstocks as a control strategy. In such programs, screening of large plant populations is a critical step in the identification of GFLV-resistant individuals. Different approaches to inoculate grapevine with GFLV have been used with varying degrees of success. We compared four innoculation techniques: nematode-mediated virus transmission, in vitro micrografting, leaf bombardment with gold particles coated with purified virus or viral RNA, and electroporation of mesophyll protoplasts. This review evaluates the efficacy of these techniques and offers insights on the most appropriate approach to inoculate grapevine plants with GFLV according to specific research goals.

Efficient preparation of maritime pine (Pinus pinaster) protoplasts suitable for transgene expression analysis

Establishment of protoplast techniques for conifers is of crucial importance to forest biotechnology. Protoplasts can be used to regenerate whole trees and to study transcriptional regulation in woody plants by promoter analysis. Here we describe a technique for isolating protoplasts fromPinus pinaster shoots. Protoplast viability was determined by fluorescein diacetate staining. Transient gene expression was studied after electroporation of protoplasts with plasmid DNA containing a β-glucuronidase (GUS) reporter gene. The 35S promoter and a pine (cytosolic glutamine synthetase) promoter both were able to driveGUS expression, indicating applicability of conifer protoplasts as a model to study gene promoters using transient expression.

Transient gene expression in electroporated intact tissues of Stylosanthes guianensis (Aubl.) Sw.

Genetic transformation though protoplast electroporation has been established for commercially important plant species. In this work, explant sources, electric field strengths, electroporation buffers, DNA forms and osmotic pretreatment were assayed in order to optimize transient reporter gene expression in electroporated tissues of Stylosanthes guianensis, a tropical forage legume. Higher transformation rates were obtained employing cotyledonary explants and an electric field strength of 250 V cm-1. Linear plasmid DNA, chloridefree electroporation buffer and osmotic pretreatment with 1.6 mol L-1 mannitol also improved transient transformation but non-significantly. Transgene specific PCR amplification was employed to prove the transformed status of the tissues.

Transformation of sweet potato tissues with green-fluorescent protein gene

The expression of the green-fluorescent protein (GFP) gene from Aequorea victoria (jellyfish) was analyzed by transient and stable expression in sweet potato Ipomoea batatas L. (Lam.) ev. Beauregard tissues by electroporation and particle bombardment. Leaf and petiole segments from in vitro-raised young plantlets were used for protoplast isolation and electroporation. Embyrogenic callus was also produced from leaf segments for particle bombardment experiments. A buffer solution containing 1×106 protoplasts ml−1 was mixed with plasmid DNA containing the GFP gene, and electroporated at 375 V cm−1. Approximately 25–30% of electroporated mesophyll cell protoplasts subsequently cultured in KM8P medium regenerated cell walls after 48 h. Of these, 3% emitted bright green fluorescence when exposed to UV-blue light at 395 nm. Transformed cells continued to grow after embedding in KM8P medium solidifed with 1.2% SeaPlaque agarose. Stable expression of GFP was observed after 4 wk of culture in approximately 1.0% of the initial GFP positive cells (27.5 GFP positive micro callases out of 3024 cells which transiently expressed GFP 48 h after electroporation). In a separate experiment, 600–700 bright green spots were observed per plate 48 h after bombarding leaf segments or embryogenic cellus. In bombarded cultures, several stable GEP-expressing sectors were observed in leafderived embryogenic callus grown without selection for 4 wk. These results show that GFP gene expression can occur in various sweet potato tissues, and that it may be a useful sereenable marker to improve transformation efficiency and obtain transgenic sweet potato plants.