Genetic Transformation Approach Research Articles

Agrobacterium rhizogenes-Mediated Genetic Transformation and Establishment of CRISPR/Cas9 Genome-Editing Technology in Limonium bicolor

Limonium bicolor is a perennial herbaceous plant belonging to the Plumbaginaceae family. It can be used as a dried flower or in cut flower arrangements and serves as a model recretohalophyte. Its genome sequencing has been recently completed. However, the research on L. bicolor is limited by the absence of a highly efficient genetic transformation system. In this study, we established a highly efficient Agrobacterium rhizogenes-mediated L. bicolor genetic transformation method. The transgenic hairy roots were induced from the hypocotyl of L. bicolor using A. rhizogenes strain K599 harboring pRdGa4Cas9 plasmid (which carries an expression cassette of 35S::DsRed2). The transgenic shoots were regenerated from hairy root segments (~0.1 cm diameter), and induction efficiency was achieved at 100%. The transgenic shoots with 4–5 rosette leaves were directly planted into the soil to induce the transgenic roots. Therefore, transgenic plantlets were produced. The DsRed2 can be used as a reliable reporter gene in screening transgenic plantlets. Furthermore, we also established a CRISPR/Cas9 system in L. bicolor employing the A. rhizogenes-mediated genetic transformation approach. The highly efficient transformation method and CRIPSP/Cas9 system established will provide a valuable tool for functional genomics investigation and trait improvement in L. bicolor.

Direct leaf-peeling method for areca protoplasts: a simple and efficient system for protoplast isolation and transformation in areca palm (Areca catechu)

BackgroundAreca palm (Areca catechu) is a woody perennial plant of both economical and medicinal importance grown in tropical and subtropical climates. Yet, the molecular biology study of areca palm is extremely impeded by its unavailability of a transformation method. An efficient protoplast isolation and transformation system could be highly desirable to overcome this barrier.ResultsHere, we described a simple and efficient method for protoplast isolation and transformation from the perennial plant areca palm. A high yield of protoplasts (2.5 × 107 protoplasts per gram of fresh leaf tissues) was obtained from the fresh light green leaflet from the newly-emerged leaf digested overnight in the enzyme solution [2% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, 0.7 M mannitol, 10 mM CaCl2, 20 mM KCl, 20 mM MES and 0.1% (w/v) BSA, pH 5.7] by the direct leaf-peeling method. The isolated areca protoplasts maintain viability of 86.6% and have been successfully transformed with a green fluorescent protein (GFP)-tagged plasmid (pGreen0029-GFP, 6.0 kb) via the polyethylene glycol (PEG)-mediated transformation. Moreover, the mannitol concentration (optimal: 0.7 M) was determined as a key factor affecting areca protoplast isolation. We also demonstrated that the optimal density of areca protoplast for efficient transformation was at 1.0–1.5 × 106 cells/ml. With the optimization of transformation parameters, we have achieved a relatively high transformation efficiency of nearly 50%.ConclusionWe have established the first efficient protocol for the high-yield isolation and transformation of areca palm protoplasts. This method shall be applied in various biological studies of areca palm, such as gene function analysis, genome editing, protein trafficking and localization and protein–protein interaction. In addition, the protoplast system offers a great genetic transformation approach for the woody perennial plant-areca palm. Moreover, the established platform may be applied in protoplast isolation and transformation for other important species in the palm family, including oil palm and coconut.

Efficient Transient Expression for Functional Analysis in Fruit Using the Tsukuba System Vector

Evaluating the function of genes expressed in fruit tissues of fruit tree species using a genetic transformation approach is a long process because the trees are generally recalcitrant to genetic transformation and cannot bear fruit during their long juvenile phases. Transient gene expression in fruit enables the functional analysis of genes associated with fruit traits, which may accelerate the study of fruit physiology. Here, by using the recently developed “Tsukuba system”, we successfully established an efficient transient expression system in harvested fruit tissues. The “Tsukuba system” utilizes a combination of the geminiviral replication system and a double terminator, which ensures sufficient levels of transgene expression. We used blueberry fruit as a model to characterize the applicability of this system for transient expression in fruit tissue. The pTKB3-EGFP vector was introduced by agroinfiltration into the fruit tissues of several blueberry cultivars. We found that transient GFP fluorescence in fruit peaked 4–6 days after agroinfiltration. Agrobacterium suspensions were easily injected into soft, mature fruit, and GFP was strongly expressed; however, hard, immature fruit were not penetrable by Agrobacterium suspensions, and GFP was rarely detected. We then tested the applicability of the developed system to other fruit tree species: six families, 17 species, and 26 cultivars. GFP fluorescence was detected in all species, except for Japanese apricot. In blueberry, bilberry, sweet cherry, apricot, and satsuma mandarin, GFP was highly expressed and observed in a large proportion of the flesh. In kiwifruit, hardy kiwifruits, persimmon, peach, apple, European pear, and grape, GFP fluorescence was limited to certain parts of the fruits. Finally, transient VcMYBA1 overexpression in blueberry was tested as a model for gene functional analysis in fruit. Transient VcMYBA1 overexpression induced red pigmentation in the flesh, suggesting that VcMYBA1 expression caused anthocyanin accumulation. This study provides a technical basis for the rapid evaluation of genes expressed in fruit, which will be useful for gene function evaluation studies in fruit crops with long juvenile phases.

An Efficient Agrobacterium-Mediated Genetic Transformation Using Embryonic Axis in Cotton (Gossypium hirsutum L.)

Agrobacterium-mediated genetic transformation approach allows for introducing novel genes in cotton (Gossypium hirsutum L.). Development of efficient regeneration and transformation protocol is very important for recalcitrant plants like cotton. In the present study, five-day-old germinated mature embryo parts especially embryonic axis, hypocotyl and plumule of cotton ‘Lashata’ were wounded and inoculated with A. tumefaciens strain GV2260 harbouring plasmid p35S-GUS-INT. The binary plasmid p35S-GUS-INT contains neomycin phosphotransferase II (NPTII) gene driven by nopaline synthase (NOS) promoter and β-glucuronidase (GUS) gene controlled by cauliflower mosaic virus (CaMV) 35S promoter. After inoculation, explants were co-cultivated in liquid and agar-solidified MS medium for 48 h in dark condition. Agar-solidified co-cultivation medium increased transformation frequency as compared to liquid medium. The putative primary transformants were confirmed with histochemical GUS assay, PCR and RT-PCR. However, comparing the three culture explant, the embryonic axis explants had significant difference with embryonic hypocotyl and plumule explants in terms of number and percentage (%) of GUS, PCR and RT-PCR positive plant. The total transformation efficiency was recorded as 3% with varying GUS expression levels.

Over‐expression of DXS gene enhances terpenoidal secondary metabolite accumulation in rose‐scented geranium and Withania somnifera: active involvement of plastid isoprenogenic pathway in their biosynthesis

Rose-scented geranium (Pelargonium spp.) is one of the most important aromatic plants and is well known for its diverse perfumery uses. Its economic importance is due to presence of fragrance rich essential oil in its foliage. The essential oil is a mixture of various volatile phytochemicals which are mainly terpenes (isoprenoids) in nature. In this study, on the geranium foliage genes related to isoprenoid biosynthesis (DXS, DXR and HMGR) were isolated, cloned and confirmed by sequencing. Further, the first gene of 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway, 1-deoxy-d-xylulose-5-phosphate synthase (GrDXS), was made full length by using rapid amplification of cDNA ends strategy. GrDXS contained a 2157 bp open reading frame that encoded a polypeptide of 792 amino acids having calculated molecular weight 77.5 kDa. This study is first report on heterologous expression and kinetic characterization of any gene from this economically important plant. Expression analysis of these genes was performed in different tissues as well as at different developmental stages of leaves. In response to external elicitors, such as methyl jasmonate, salicylic acid, light and wounding, all the three genes showed differential expression profiles. Further GrDXS was over expressed in the homologous (rose-scented geranium) as well as in heterologous (Withania somnifera) plant systems through genetic transformation approach. The over-expression of GrDXS led to enhanced secondary metabolites production (i.e. essential oil in rose-scented geranium and withanolides in W. somnifera). To the best of our knowledge, this is the first report showing the expression profile of the three genes related to isoprenoid biosynthesis pathways operated in rose-scented geranium as well as functional characterization study of any gene from rose-scented geranium through a genetic transformation system.

Effects of down regulation of lignin content in maize (Zea mays L.) plants expressing C4H3 gene in the antisense orientation

ABSTRACTLignin is an aromatic biopolymer, an integral cell wall constituent in all vascular plants including the herbaceous varies. Genetic engineering using an antisense system offers the way to modulate enzymes in the lignin pathway and leads to reduced lignin content. The Hi II, a highly embryogenic genotype of maize, has been extensively used in transformation protocols. The plasmid pZMAS-C4H3 10,907bp has C4H3 antisense (AS) to down regulate O-methyltransferase and reduce lignin level in the maize and bar gene as selectable marker that confers glufosinate herbicide resistance under 35s promoter. Eleven transgenic lines exhibited detectable levels of bar gene activity. Three lines of AS plants, C4H3-AS, displayed a significant reduction in lignin content (14–17%). The results of this study pointed to possible use of the genetic transformation approach to minimize lignin content and consequently enhancing biofuel production from stover.

Conjugative plasmid transfer from Escherichia coli is a versatile approach for genetic transformation of thermophilic Bacillus and Geobacillus species.

We previously demonstrated efficient transformation of the thermophile Geobacillus kaustophilus HTA426 using conjugative plasmid transfer from Escherichia coli BR408. To evaluate the versatility of this approach to thermophile transformation, this study examined genetic transformation of various thermophilic Bacillus and Geobacillus spp. using conjugative plasmid transfer from E. coli strains. E. coli BR408 successfully transferred the E. coli-Geobacillus shuttle plasmid pUCG18T to 16 of 18 thermophiles with transformation efficiencies between 4.1×10(-7) and 3.8×10(-2)/recipient. Other E. coli strains that are different from E. coli BR408 in intracellular DNA methylation also generated transformants from 9 to 15 of the 18 thermophiles, including one that E. coli BR408 could not transform, although the transformation efficiencies of these strains were generally lower than those of E. coli BR408. The conjugation was performed by simple incubation of an E. coli donor and a thermophile recipient without optimization of experimental conditions. Moreover, thermophile transformants were distinguished from abundant E. coli donor only by high temperature incubation. These observations suggest that conjugative plasmid transfer, particularly using E. coli BR408, is a facile and versatile approach for plasmid introduction into thermophilic Bacillus and Geobacillus spp., and potentially a variety of other thermophiles.

Development of a genetic transformation system for Candida tropicalis based on a reusable selection marker of URA3 gene

Candida tropicalis, a diploid asporogenic yeast, is frequently utilized in industrial applications and research studies. However, the low efficiency of genetic transformation limits the strain improvement by metabolic engineering. A reliable transformation and efficient deletion of target gene are prerequisite for molecular improvement of C. tropicalis. In this study, an efficient approach for genetic transformation of C. tropicalis was developed based on the URA3 gene as a reusable selection marker and both of PDC allele genes encoding pyruvate decarboxylase were successfully deleted by this approach. Firstly, an auxotrophic mutant strain of C. tropicalis XZX which is defective in orotidine-5'-phosphate decarboxylase (URA3) was isolated by chemical mutagenesis combined with nystatin enrichment selection and 5-fluoro-orotic acid (5-FOA) resistance selection using C. tropicalis ATCC 20336 as the parent strain. Then, the first PDC deletion cassette PDC1-hisG-URA3-hisG- PDC1 (PHUHP) which contains a 1.6 kb URA3 marker gene, two copies of 1.1 kb Salmonella hisG fragments and homologous arms of target gene was constructed and transformed into C. tropicalis XZX cells. Transformants with a single copy of PDC deleted were isolated and identified by PCR and DNA sequencing, which was designated as C.tropicalis XZX02. The C.tropicalis XZX02 cells were spread on the minimal medium containing 5-FOA to generate mutant C. tropicalis XZX03 in which URA3 marker gene was excised from PHUHP fragment integrated into the PDC gene site. The second PDC gene deletion cassette PDCm-URA3-PDCm (MUM) was constructed and transformed into C. tropicalis XZX03 to generate C.tropicalis XZX04 in which both of PDC allele genes were deleted. All strains were confirmed by PCR and DNA sequencing. This efficient genetic transformation approach laid a foundation for further metabolic engineering of C. tropicalis.

Heat Stress Responses and Thermotolerance

The rising ambient temperature by plant cells is crucial for the timely activation of various molecular defences before the appearance of heat damage. The heat-threshold level varies considerably at different developmental stages. With a view to survive under heat stress, mechanisms of regulation at the molecular level enable plants to prosper. Traditional breeding contributed for improving heat tolerance meagrely. The genetic transformation approach needs to be accelerated that can mitigate harmful effects by developing improved thermotolerance of crop plants. In this background, a thorough understanding of physiological responses of plants to high temperature, mechanisms of heat tolerance and possible strategies is vital. Temperature changes are sensed through cellular responses due to signal transduction into the cell. A key ingredient for plants temperature sensing ability is a specialized histone protein, dubbed H2A.Z that wraps DNA into a more tightly packed structure. Further, metabolic reactions and internal conductance to CO 2 decrease Rubisco activation, inhibition of Rubisco activase and subsequently slowing down net photosynthesis (Pn). Importance of internal conductance to CO 2 , heat inducible transcription factor for heat shock response (HSR) involve different HSR genes. The mechanisms, which are regulated at the molecular level, facilitate plants to thrive under heat stress. Exogenous applications of protectants such as osmoprotectants, phytohormones, signaling molecules, trace elements etc. have shown beneficial effects on plants growing under high temperature, due to the growth promoting and antioxidant activities of these compounds. There is acute necessity of crop biotechnology research for evolving genetically modified (GM) crops for a high CO 2 world at this juncture. So, attaining thermotolerance is a lively process to escape damages caused by heat stress.

Sugarcane Specific Drought Responsive Candidate Genes Belonging to ABA Dependent Pathway Identified from Basic Species Clones of Saccharum sp. and Erianthus sp.

Development of drought tolerant sugarcane cultivars has long been a major breeding objective in view of depleting water source and longer dry spells. Identification of specific candidate genes would be an efficient approach to the genetic improvement of sugarcane for drought tolerance. As the involvement of abscisic acid (ABA) in mediating drought stress has been well demonstrated, in the present study eleven putative candidate genes from orthologous species belonging to ABA dependent pathway viz ABF 2, ABF 3, ATHB-7, CIPK 14, CDPK 18, LEA 3, MYB 2, RD 28, RGS 1, SNRK 2.5 and TPS 2 were screened on a set of clones belonging to tolerant species viz. Saccharum spontaneum, S. barberi, S. sinense, S. robustum and Erianthus and the susceptible species of S. officinarum. Out of these, ten genes except ATHB-7 were amplified in Saccharum and Erianthus species. The genes ABF 2, CIPK 14, LEA 3, MYB 2, RD 28, RGS1 and SNRK 2.5 were absent in almost all the susceptible clones of S. officinarum and were present in the tolerant species clones implicating their role in imparting drought tolerance in sugarcane. Number of genes detected in S. officinarum ranged from 2 to 6 whereas drought tolerant species clones exhibited 4–10 genes. The clones SES 106 B and Iritty 2 (S. spontaneum), Pathri (S. barberi) and IK 76–99 (Erianthus) were found as the repositories of ten genes to be advantageously used in breeding for drought tolerance in sugarcane. The sequencing of the polymorphic bands and BLAST search would lead to the identification of new drought responsive genes in sugarcane for use as novel and functional candidate genes for improving drought tolerance in sugarcane through marker-assisted introgression breeding and genetic transformation approach.

Analysis of transgene integration and expression following biolistic transfer of different quantities of minimal expression cassette into sugarcane (Saccharum spp. hybrids)

Sugarcane (Saccharum spp. hybrids) is an interspecific hybrid with a highly polyploid and frequently aneuploid genome. This C4 grass accounts for nearly 70% of the global sugar production and more recently has become an important biofuel feedstock. Biolistic gene transfer of plasmid DNA is the most frequently used approach for genetic transformation of sugarcane. Minimal expression cassettes lacking vector backbone sequences (MC) have been reported to support simple transgene integration in other species. In this study, we introduced a MC of nptII into embryogenic callus derived from immature leaf whorl cross-sections by biolistic gene transfer. The precipitation equivalents of 12.5, 25 or 50 ng of the nptII MC were delivered per shot to the target tissue with 1.0 μm gold particles. A total of 203 independent putative transgenic plants were regenerated following 80 bombardments and selection on geneticin or paromomycin containing media and 176 transgenic lines were confirmed with PCR. Twenty independent transgenic lines were selected for Southern blot analysis and expression analysis by NPTII ELISA from each of the three treatments. Genomic DNA from transgenic sugarcane plants displayed two to 13 nptII hybridization signals on Southern blots. There was a trend toward reduced transgene integration complexity and reduced transgene expression levels when lower (12.5 ng) MC was used per shot. These results demonstrate that backbone free MCs can be efficiently integrated and expressed in sugarcane.

Somatic embryogenesis from the axillary meristems of peanut (Arachis hypogaea L.)

Developmental anomalies in the plumule meristem of peanut (Arachis hypogaea L.) somatic embryos resulted in poor shoot differentiation and reduced plant recovery. Existing meristems with caulogenic potential have never been tested for embryogenesis in peanut. The present experiment was designed to test the mature zygotic embryo axis derived plumule with three meristems for somatic embryogenesis. Embryogenic masses and embryos developed from the caulogenic meristems in the axils. Exposure of 2 weeks in primary medium with 90.5 µM 2,4-D suppressed the shoot tip differentiation temporarily which then regained the ability to form the shoot on withdrawal of 2,4-D. Exposure of 4 weeks in primary medium with 90.5 µM 2,4-D suppressed the shoot tip differentiation irreversibly. No shoot formation was noted from the tips in any of the cultures which were in secondary medium with 13.6 µM 2,4-D. Development of somatic embryos directly from axillary meristems was confirmed histologically. Conversion frequency of these embryos was 11%. Thus, in this report, we describe a method to obtain somatic embryos from the determined organogenic buds of the axillary meristem, by culturing the nodal explant vertically on embryo induction medium. It also displays the possibility of obtaining both embryogenic and organogenic potential in two parts of the same explant simultaneously. The possibility of extending this approach for genetic transformation in in vivo system through direct DNA delivery or Agrobacterium injection in meristems can also be explored. Using Agrobacterium rhizogenes, we have demonstrated the possibility of gene transfer in the axillary meristems of seed-derived plumule explant.

Decreasing unpalatable flavonoid components in Citrus: the effect of transformation construct

Citrus species accumulate large quantities of flavanone glycosides in their leaves and fruit. The physiological role(s) of these compounds in citrus plants are unknown, but they have been documented to benefit human health upon consumption. Flavanone rutinosides are tasteless, whereas flavanone neohesperidosides, such as naringin, give a bitter taste to fruit and fruit juice products, reducing their palatability. In an effort to alter the types and levels of flavanone neohesperidosides in citrus, an Agrobacterium-mediated genetic transformation approach was employed. Citrus paradisi Macf. (grapefruit) epicotyl stem segments were transformed with sense (S) and antisense (AS) constructs of the target genes chalcone synthase (CHS) and chalcone isomerase (CHI), whose products catalyze the first two steps in the flavonoid biosynthetic pathway. Transformation with each of the individual constructs led to a different and unpredictable combination of viability, phenotypic change, transgene steady-state expression and alteration in flavonoid content in the resulting transgenic plants. These qualities were consistent within the transgenic plants obtained using any particular construct. Transgenic plants with decreased leaf naringin levels were obtained, particularly when the CHS-AS constructs were employed.

Pollen‐mediated transformation of Sorghum bicolor plants

Pollen from sorghum (Sorghum bicolor L. Moench) were transformed by a novel genetic transformation approach using mild ultrasonication. The plasmid pBI121 contained the NPT II gene encoding NPT II (neomycin phosphotransferase II) and a GUS (beta-glucuronidase) reporter gene. Plasmid DNA and pollen from sorghum A(2)V4B were submerged in a 0.3 mol/l sucrose solution and then subjected to ultrasonication. The treated pollen was then used for pollination of stigmas from the male sterile line A(2)V4A. Detection of transient and stable GUS activity confirmed that the GUS gene was expressed in transgenic plants. PCR amplification and Southern-blot analysis of transgenic plants confirmed that the NPT II gene had been integrated into the sorghum genome. These results indicate that direct gene transfer to pollen can be mediated by mild ultrasonication.

Production of a Biodegradable Plastic Polymer, Poly‐β‐Hydroxybutyrate, in Transgenic Alfalfa

Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate the potential of alfalfa as a source of industrial materials, we employed a genetic transformation approach to produce a biodegradable plastic, poly‐β‐hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha (formerly Alcaligenes eutrophus) encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrate‐co‐hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium‐mediated transformation. DNA and RNA blot analyses of transgenic plants indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants ranged from ≈0.025 to 1.8 g kg−1 dry weight (DW). Agglomerations of PHB granules 0.2 to 0.4 μm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants, demonstrating that phb gene products were targeted into the plastids of transgenic alfalfa. Transgenic plants exhibited growth similar to untransformed plants, suggesting that expression of PHB biosynthetic pathway genes at current levels and accumulation of product in the plastids had no deleterious effect on growth and fertility. F1 hybrid progeny, obtained from crosses of PHB transgenic plants with elite alfalfa germplasm, exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.

Production of a Biodegradable Plastic Polymer, Poly-β-Hydroxybutyrate, in Transgenic Alfalfa

Alfalfa (Medicago sativa L.) is primarily used as a source of forage and animal feed. Improving the economic value of alfalfa could be achieved by developing new uses of this perennial crop. To investigate the potential of alfalfa as a source of industrial materials, we employed a genetic transformation approach to produce a biodegradable plastic, poly-β-hydroxybutyrate (PHB), in the leaves of alfalfa plants. Three genes from Ralstonia eutropha (formerly Alcaligenes eutrophus) encoding the enzymes for synthesis of PHB (phbA, phbB, phbC) and a copolymer of PHB and polyhydroxyvalerate (polyhydroxybutyrate-co-hydroxyvalerate, or PHB/V) (bktB, phbB, phbC) engineered for plastid targeting were introduced into alfalfa by Agrobacterium-mediated transformation. DNA and RNA blot analyses of transgenic plants indicated integration and expression of the PHB biosynthetic pathway genes. Polyhydroxybutyrate content in the leaves of transgenic plants ranged from ≈0.025 to 1.8 g kg−1 dry weight (DW). Agglomerations of PHB granules 0.2 to 0.4 μm in diameter, similar to bacterial PHB, were located in the chloroplasts of transgenic plants, demonstrating that phb gene products were targeted into the plastids of transgenic alfalfa. Transgenic plants exhibited growth similar to untransformed plants, suggesting that expression of PHB biosynthetic pathway genes at current levels and accumulation of product in the plastids had no deleterious effect on growth and fertility. F1 hybrid progeny, obtained from crosses of PHB transgenic plants with elite alfalfa germplasm, exhibited leaf PHB levels similar to the transgenic parental line, demonstrating that PHB production in alfalfa is a stable and dominantly inherited trait.