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Intrinsic GUS-like activities in seed plants
Abstract
Fifty-two plant species, covering some Gymnosperms and all the key groups of Angiosperms, were chosen for surveying their intrinsic beta-glucuronidase-like activities. Histochemical (overnight incubation) and qualitative fluorometric (24 h incubation) assays indicated that, with few exceptions, such activities were detected in certain part(s) of the fruit walls, seed coats, endosperms or, especially, the embryos of the tested plants. Most of such activities in the excised immature embryos of soybean and string bean disappeared after one to a few days' in vitro culturing. Such activities in the intact mature seeds of these two species diminished also during germination process. The vegetative organs of seedlings/mature plants usually lack such activities. The enzyme(s) responsible for such activities was antigenically dissimilar to E. coli beta-glucuronidase.
... In the last years researchers focused their attention on intrinsic GUS activities in plants. The presence of GUS was evaluated in different species, included pear, corn, pea, tobacco and also in different tissues as fruit walls, seed coats, immature and mature embryos, seedlings and plant at flowering stage [31]. In all of these species intrinsic GUS activity, which was widely distributed in different tissues, was found. ...
... Also in these species, as wild-type crops, the ubiquitous presence of GUS was detected and it was higher in young tissues than the older or mature tissues. Our results can be explained considering the evidence above reported; achenes are younger tissue than seed-derived plants and also because the germination process decreases GUS activity [31,32]. To confirm these hypotheses further investigation must be done, including Southern hybridisation and an evaluation of the ubiquitous presence of GUS in wild-type plants of F. vesca. ...
... First, the donor plant could have not been homozygous, but for this to be true in both of the groups of achenes we would have observe the same percentage for GUS expression. Second, the GUS assay could have been inefficient in detecting expression of the reporter genes when achenes were directly analysed [31,32]. ...
The transmission of transgenes (uid-A and nptII) was studied in crosses between transgenic plants, with Fragaria x ananassa cultivar Teodora as the donor plant and various Fragaria species as recipient plants. GUS expression and kanamycin resistance were evaluated in embryos and in seedlings after aseptical germination, both in intra- and inter-specific crosses as demonstrated by different ploidy level. This expression in most of the T1 seedlings shows that one or more functional transgenes were transferred from T0 plants to T1 seedlings. These results, obtained in a greenhouse, confirm the possibility of genetic exchange both at the intra- and inter-specific level, though the inter-specific hybrids seem to have reduced germination ability. These observations need to be further investigated.
... This enzymatic activity is also reported in maize developing kernels (Muhitch, 1998). Moreover, endogenous GUSB activity is present in many other plants such as fruit wall, seed coat, endosperm, embryos (Hu et al., 1990) and during male gametophyte development of potato, tobacco, and tomato (Plegt and Bino, 1989). ...
... Following research on plant GUSB or "endogenous" GUSB that plays key roles in development of the primary leaves of rye plant (Secale cereale), the same is found to be in the pedicel of maize kernels during its developing and lignin biosynthesis (Schulz and Weissenböck, 1987;Anhalt and Weissenböck, 1992;Muhitch, 1998). Endogenous GUSB activity in plant is reported in fruit wall, seed coat, endosperm, embryos, and during male gametophyte development of many plants (Plegt and Bino, 1989;Hu et al., 1990). Inhibition of endogenous GUSB activity leads to seedling developmental problems such as arresting the growth and inhibiting root-hair development in tobacco (Nicotiana tabacum) and parasitic angiosperm, Cuscuta pentagona (Sudan et al., 2006;Schoenbeck et al., 2007). ...
... three different GUS genes have been identified in Arabidopsis thaliana: AtGUS1, AtGUS2 and AtGUS3 [42]. Further studies have confirmed the wide diffusion of GUSs in plants [35,[43][44][45][46][47] and the presence of enzymatic activity in all the different organs [40,[48][49][50][51][52][53][54][55][56][57][58][59]. ...
Gene duplication played a fundamental role in eukaryote evolution and different copies of a given gene can be present in extant species, often with expressions and functions differentiated during evolution. We assume that, when such differentiation occurs in a gene copy, this may be indicated by its maintenance in all the derived species. To verify this hypothesis, we compared the histological expression domains of the three β-glucuronidase genes (AtGUS) present in Arabidopsis thaliana with the GUS evolutionary tree in angiosperms. We found that AtGUS gene expression overlaps in the shoot apex, the floral bud and the root hairs. In the root apex, AtGUS3 expression differs completely from AtGUS1 and AtGUS2, whose transcripts are present in the root cap meristem and columella, in the staminal cell niche, in the epidermis and in the proximal cortex. Conversely, AtGUS3 transcripts are limited to the old border-like cells of calyptra and those found along the protodermal cell line. The GUS evolutionary tree reveals that the two main clusters (named GUS1 and GUS3) originate from a duplication event predating angiosperm radiation. AtGUS3 belongs to the GUS3 cluster, while AtGUS1 and AtGUS2, which originate from a duplication event that occurred in an ancestor of the Brassicaceae family, are found together in the GUS1 cluster. There is another, previously undescribed cluster, called GUS4, originating from a very ancient duplication event. While the copy of GUS4 has been lost in many species, copies of GUS3 and GUS1 have been conserved in all species examined.
... Un problema similar se ha reportado en callo y foliolos de palma de aceite , y, en callo de trigo (Loeb & Reynolds, 1994). Esta complicación puede deberse a una difusión pobre de los productos de la reacción (Hu et al., 1990;Thomasset et al., 1996). Pero la misma se puede resolver ajustando la solución tampón usada durante el ensayo, manteniendo el pH en 7,0 y disolviendo de manera más eficiente los reactivos, agregando una mayor proporción de metanol (Nishihara et al., 1993;Kosugi et al., 1990). ...
... This protocol resulted in minimal tissue damage at the harvest time point (3 dpi, Figure 6A). Endogenous β-glucuronidase-like (GUS-like) activity can be detected in specific tissues of various plant species (Hu et al., 1990); however, we detected no GUS-like activity in C. roseus seedlings after transformation with A. tumefaciens lacking a GUS reporter (Figure 6B). Transformation of seedlings with A. tumefaciens containing the CaMV2x35s driven GUS reporter at OD 600 = 0.2 led to an even transformation of the cotyledons ( Figure 6C). ...
Catharanthus roseus
produces medicinal
terpenoid indole alkaloids, including the critical anti-cancer compounds vinblastine and vincristine in its leaves. Recently, we developed a highly efficient transient expression method relying on Agrobacterium-mediated transformation
of seedlings to facilitate rapid and high-throughput studies on the regulation of terpenoid indole alkaloid
biosynthesis in C. roseus
. We detail our optimized protocol known as efficient Agrobacterium-mediated seedling infiltration method (EASI), including the development of constructs used in EASI and an example experimental design that includes appropriate controls. We applied our EASI method to rapidly screen and evaluate transcriptional activators and repressors and promoter activity. Our EASI method can be used for promoter transactivation studies or transgene overexpression paired with downstream analyses like quantitative PCR
or metabolite analysis. Our protocol takes about 16 days from sowing seeds to obtaining the results of the experiment.Key words
Catharanthus roseus
Agrobacterium tumefaciens
Agrobacterium-mediated transformationVacuum-infiltrationTransient transformationTerpenoid indole alkaloids
... In most studies, the sGUS activities in plants have been reported under acidic conditions (pH 4.0−6.0; Sood 1980; Plegt and Bino 1989;Hu et al. 1990;Alwen et al. 1992;Hodal et al. 1992;Wozniak and Owens 1994;Sudan et al. 2006;Schoenbeck et al. 2007). However, the sGUS from S. baicalensis is different, because its pH optimum is between 6.0 and 7.0, and the activity completely disappeared at pH values lower than 4.5 (Marimoto et al. 1995). ...
The study examined the relationship between the contents of principal flavones of Scutellaria baicalensis Georgi and the activity of endogenous b-glucuronidase (sGUS) in differentiated and undifferentiated in vitro cultures grown on solid and liquid medium. Using RP-HPLC, it was shown that sGUS activity did not correlate with the increase in biomass of cultures, but depended on tissue differentiation. In hairy roots cultured both on liquid and solid medium, it was ten times higher than in undifferentiated cultures. At the same time, the share of aglycone forms in the total content of flavones during the culture cycle depended on the growing conditions. It was significantly higher on agar medium than on liquid medium. A correlation between sGUS activity and total aglycone content, as well as wogonin content, was observed only in hairy roots grown in the liquid medium.
... pLAP-A1:GUS tomato plants have the GUS gene fused to a copy of the promoter (and part of the 5′ untranslated region) of the endogenous LAP-A1 gene, such that when the endogenous LAP-A1 is expressed, GUS enzyme is produced in parallel(Chao et al., 1999).Because (young) tomato plants have no intrinsic GUS activity(Hu et al., 1990), the amount of GUS activity in pLAP-A1:GUS plants is proportional to the expression of the endogenous LAP-A1 gene. As a glycosidase, GUS catalyzes the breakdown of carbohydrates. ...
Plant defense suppression is an offensive strategy of herbivores, in which they manipulate plant physiological processes to increase their performance. Paradoxically, defense suppression does not always benefit the defense‐suppressing herbivores, because lowered plant defenses can also enhance the performance of competing herbivores and can expose herbivores to increased predation. Suppression of plant defense may therefore entail considerable ecological costs depending on the presence of competitors and natural enemies in a community. Hence, we hypothesize that the optimal magnitude of suppression differs among locations. To investigate this, we studied defense suppression across populations of Tetranychus evansi spider mites, a herbivore from South America that is an invasive pest of solanaceous plants including cultivated tomato, Solanum lycopersicum, in other parts of the world. We measured the level of expression of defense marker genes in tomato plants after infestation with mites from eleven different T. evansi populations. These populations were chosen across a range of native (South American) and non‐native (other continents) environments and from different host plant species. We found significant variation at three out of four defense marker genes, demonstrating that T. evansi populations suppress jasmonic acid‐ and salicylic acid‐dependent plant signaling pathways to varying degrees. While we found no indication that this variation in defense suppression was explained by differences in host plant species, invasive populations tended to suppress plant defense to a smaller extent than native populations. This may reflect either the genetic lineage of T. evansi—as all invasive populations we studied belong to one linage and both native populations to another—or the absence of specialized natural enemies in invasive T. evansi populations.
... The transformants were selected according to the presence of the insert detected by PCR as above and according to the presence of GFP signal under the epifluorescent microscope. The selected plants were screened for GUS signal [78]. Briefly, various tissue types (seedlings, leaves, cauline leaves, inflorescences, flower buds, flowers, pollen grains, siliques) were incubated usually for 2 h (in case of low signal, the duration was prolonged to 24 h) in GUS staining buffer (100 mM sodium phosphate buffer pH 7.0, 10 mM EDTA, 0.1% (v/v) Triton X-100, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, and 1 mM X-Gluc), and then bleached with ethanol series (90%, 70%, 50%). ...
The nascent polypeptide-associated (NAC) complex was described in yeast as a heterodimer composed of two subunits, α and β, and was shown to bind to the nascent polypeptides newly emerging from the ribosomes. NAC function was widely described in yeast and several information are also available about its role in plants. The knock down of individual NAC subunit(s) led usually to a higher sensitivity to stress. In Arabidopsis thaliana genome, there are five genes encoding NACα subunit, and two genes encoding NACβ. Double homozygous mutant in both genes coding for NACβ was acquired, which showed a delayed development compared to the wild type, had abnormal number of flower organs, shorter siliques and greatly reduced seed set. Both NACβ genes were characterized in more detail—the phenotype of the double homozygous mutant was complemented by a functional NACβ copy. Then, both NACβ genes were localized to nuclei and cytoplasm and their promoters were active in many organs (leaves, cauline leaves, flowers, pollen grains, and siliques together with seeds). Since flowers were the most affected organs by nacβ mutation, the flower buds’ transcriptome was identified by RNA sequencing, and their proteome by gel-free approach. The differential expression analyses of transcriptomic and proteomic datasets suggest the involvement of NACβ subunits in stress responses, male gametophyte development, and photosynthesis.
... This protocol resulted in minimal tissue damage at the harvest time point (3 dpi, Figure 6A). Endogenous β-glucuronidase-like (GUS-like) activity can be detected in specific tissues of various plant species (Hu et al., 1990); however, we detected no GUS-like activity in C. roseus seedlings after transformation with A. tumefaciens lacking a GUS reporter (Figure 6B). Transformation of seedlings with A. tumefaciens containing the CaMV2x35s driven GUS reporter at OD 600 = 0.2 led to an even transformation of the cotyledons ( Figure 6C). ...
The Catharanthus roseus plant is the exclusive source of the valuable anticancer terpenoid indole alkaloids, vinblastine (VB) and vincristine (VC). The recent availability of transcriptome and genome resources for C. roseus necessitates a fast and reliable method for studying gene function. In this study, we developed an Agrobacterium-mediated transient expression method to enable the functional study of genes rapidly in planta, conserving the compartmentalization observed in the VB and VC pathway. We focused on (1) improving the transformation method (syringe versus vacuum agroinfiltration) and cultivation conditions (seedling age, Agrobacterium density, and time point of maximum transgene expression), (2) improving transformation efficiency through the constitutive expression of the virulence genes and suppressing RNA silencing mechanisms, and (3) improving the vector design by incorporating introns, quantitative and qualitative reporter genes (luciferase and GUS genes), and accounting for transformation heterogeneity across the tissue using an internal control. Of all the parameters tested, vacuum infiltration of young seedlings (10-day-old, harvested 3 days post-infection) resulted in the strongest increase in transgene expression, at 18 – 57 fold higher than either vacuum or syringe infiltration of other seedling ages. Endowing the A. tumefaciens strain with the mutated VirGN54D or silencing suppressors within the same plasmid as the reporter gene further increased expression by 2 – 10 fold. For accurate measurement of promoter transactivation or activity, we included an internal control to normalize the differences in plant mass and transformation efficiency. Including the normalization gene (Renilla luciferase) on the same plasmid as the reporter gene (firefly luciferase) consistently yielded a high signal and a high correlation between RLUC and FLUC. As proof of principle, we applied this approach to investigate the regulation of the CroSTR1 promoter with the well-known activator ORCA3 and repressor ZCT1. Our method demonstrated the quantitative assessment of both the activation and repression of promoter activity in C. roseus. Our efficient Agrobacterium-mediated seedling infiltration (EASI) protocol allows highly efficient, reproducible, and homogenous transformation of C. roseus cotyledons and provides a timely tool for the community to rapidly assess the function of genes in planta, particularly for investigating how transcription factors regulate terpenoid indole alkaloid biosynthesis.
... However, this transformation-based method is labor intensive and is difficult to quantitatively measure the strength of a candidate CRE [32]. Additionally, GUS staining cannot be performed on living tissue and can introduce false positives or negatives depending on the sampled development/environmental state [33]. GUS is also not suitable for the observation of conditionally or temporally regulated expression patterns due to its low turnover rate, as the half-life time (T 50 protein) of GUS can be as high as several days [34][35][36]. ...
Background
Enhancers are one of the most important classes of cis-regulatory elements (CREs) and play key roles in regulation of transcription in higher eukaryotes. Enhancers are difficult to identify because they lack positional constraints relative to their cognate genes. Excitingly, several recent studies showed that plant enhancers can be predicted based on their distinct features associated with open chromatin. However, experimental validation is necessary to confirm the predicted enhancer function.
Results
We developed a rapid enhancer validation system based on Nicotiana benthamiana. A set of 12 intergenic and intronic enhancers, identified in Arabidopsis thaliana, were cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene, and were then infiltrated into N. benthamiana leaves mediated by agrobacterium. The enhancer activity of each candidate was quantitatively assayed based on bioluminescence measurement. The data from this luciferase-based validation was correlated with previous data derived from transgenic assays in A. thaliana. In addition, the relative strength of different enhancers for driving the reporter gene can be quantitatively compared. We demonstrate that this system can also be used to map the functional activity of a candidate enhancer under different environmental conditions.
Conclusions
In summary, we developed a rapid and efficient plant enhancer validation system based on a luciferase reporter and N. benthamiana-based leaf agroinfiltration. This system can be used for initial screening of leaf-specific enhancers and for validating candidate leaf enhancers from dicot species. It can potentially be used to examine the activity of candidate enhancers under different environmental conditions.
Electronic supplementary material
The online version of this article (10.1186/s13007-019-0407-y) contains supplementary material, which is available to authorized users.
... Whereas, GUSs from GH1 and GH79 degrade steroid β-glucuronides and heparan sulfate proteoglycans, respectively [13,14]. Several studies have been reported endogenous activity of GUS in various regions of plant body including roots, leaves, stems, pollen grains and fruits [15][16][17]. ...
A multimodular hyperthermophilic β‑glucuronidase (TpGUS) from Thermotoga petrophila RKU-1T, belongs to glycoside hydrolase family 2 (GH2), was cloned and overexpressed in Escherichia coli BL21 CodonPlus (DE3)-RIPL. Expression and production of extracellular TpGUS was enhanced through various specific cultivation and induction strategies. Extracellular TpGUS activity was improved by 3.44 and 7 fold in 4 × ZB medium induced with 0.5 mM IPTG and 100 mM lactose, respectively. The enzyme was purified to homogeneity with a single band of 65.6 kDa on SDS-PAGE, using two subsequent steps of anion exchange and hydrophobic interaction chromatography after heat precipitation (70 °C, 1 h). Optimal activity of TpGUS was observed at 95 °C and pH 6.0; and it displayed prodigious thermal stability over a temperature range of 50–85 °C for 12 h at pH 6.0–7.5. Km, Vmax, VmaxKm−1, kcat, and kcatKm−1 were calculated to be 0.7 mM, 227 mmol mg−1 min−1, 324.3 min−1, 164,492.7 s−1 and 234,989.6 mM−1 s−1, respectively using pNPGU as a substrate. Recombinant TpGUS exhibited favorable properties which make this a promising candidate for various biotechnological and pharmacological applications.
... Regulatory genes of the maize anthocyanin biosynthetic pathway, such as C1, B, and R, have also been successfully used in wheat transformation (Ludwig et al., 1990;Kloti et al., 1993;Dhir et al., 1994;McCormac et al., 1998;Chawla et al., 1999aChawla et al., , 1999b. Expression of anthocyanin genes in plant tissues can be useful where false positive results due to intrinsic GUS-like activity (Hu et al., 1990) and GUS production by endophytic micro-organisms have been reported (Tor and Ainsworth, 1992). A related transcription factor from Arabidopsis, AtMYB12, which is a flavonol-specific regulator of phenylpropanoid biosynthesis (Mehrtens et al., 2005), has been used successfully to activate anthocyanin expression in wheat and serve as a scorable marker (Gao et al., 2011). ...
Wheat is one of the most important staple food crops of the world, and continuous genetic improvement is vital to meet the demands of the rapidly growing world population. Conventional breeding has led to the development of current high yielding wheat varieties, and recent achievements in genetic engineering are expected to augment conventional breeding to further increase production. Advances in genome sequencing and molecular breeding have increased the rate of gene discovery, leading to a need for highly efficient and robust transformation systems. Targeted genome editing will require efficient delivery of sequence-specific nucleases, such as zinc fingers (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided engineered nucleases such as CRISPR-Cas9. Since the first report of fertile transgenic wheat in 1992, optimization of plant tissue culture techniques, DNA delivery methods, gene expression cassettes, and marker genes have led to reliable transformation protocols for a range of wheat model cultivars. However, like other cereal crops, wheat transformation has also been hampered by genotype effects. The limited range of transformable tissues in wheat is considered another challenge. Several excellent review papers have described the progress made towards developing robust genetic transformation systems for wheat, so we have focused our attention on a detailed analysis of selectable markers and promoters that have been used. The choice of selectable marker and promoter can dramatically influence the outcome of a transformation project. Both Agrobacterium tumefaciens and microprojectile-mediated transformation systems have been employed successfully for genetic transformation of wheat using genes of agronomic importance. Since improvement in agronomic traits of wheat will affect a sizeable population, we have provided an analysis of the progress made towards developing genetically superior wheat containing gene(s) of agronomic importance. Recent efforts on targeted genome editing in wheat are also discussed.
... According to Jefferson (1987), blue background signals can occur when long incubation times are chosen, as in the present study. Furthermore, intrinsic GUSlike activities have been reported in other plants previously (Hu et al., 1990;Taylor, 1997). However, the faint coloration of the controls allowed them to be distinguished from the deep blue color and sharp localization of transgenic GUS-expressing cells. ...
Cyclamen persicum is an ornamental plant with economic relevance in many parts of the world. Moreover, it can be regarded as an applied model for somatic embryogenesis, since transcriptomic, proteomic, and metabolomic comparisons have revealed insights into this regeneration process on the molecular level. To enable gene function analyses, the aim of this study was to establish an efficient Agrobacterium tumefaciens-mediated genetic transformation protocol for C. persicum. For the first time, embryogenic callus cultures were used as a target material. The advantages of embryogenic callus are the defined and known genotype compared to seedlings, the high regeneration potential and the stability of the regenerated plants. A. tumefaciens strains EHA105 and LBA4404 were most efficient for transformation, resulting in transformation efficiencies of up to 43 and 20%, respectively. In regenerated plants, the presence of the transgenes was verified by PCR, Southern hybridization, and a histochemical GUS assay. The protocol was applied successfully to two C. persicum genotypes. Moreover, it served to transfer two reporter constructs, the auxin-responsive promoter DR5 driving the gus gene and the redox sensor roGFP2_Orp1, to the C. persicum genotypes, allowing the localization of high auxin concentrations and reactive oxygen species in order to study their roles in somatic embryogenesis in the future. For success in transformation, we regard the following factors as important: highly embryogenic cell lines, the use of Silwet® L-77 as a surfactant during co-culture, a genotype-specific appropriate selection schedule with hygromycin, and A. tumefaciens strains EHA105 and LBA4404.
... As expected the uidA-gene was not detected in any of them (Table 3). Hence this is assumed to be an endogenous activity in Petunia seeds, which − to our knowledge − has not yet been reported for Petunia, but for various other seedlings [39][40][41][42][43]. Thus, GUS-staining could not be used as reliable marker for plastid transmission but only for preselection of events. ...
As already demonstrated in greenhouse trials, outcrossing of transgenic plants can be drastically reduced via transgene integration into the plastid. We verified this result in the field with Petunia, for which the highest paternal leakage has been observed. The variety white 115 (W115) served as recipient and Pink Wave (PW) and the transplastomic variant PW T16, encoding the uidA reporter gene, as pollen donor. While manual pollination in the greenhouse led to over 90% hybrids for both crossings, the transgenic donor resulted only in 2% hybrids in the field. Nevertheless paternal leakage was detected in one case which proves that paternal inheritance of plastid-located transgenes is possible under artificial conditions. In the greenhouse, paternal leakage occurred in a frequency comparable to published results. As expected natural pollination reduced the hybrid formation in the field from 90 to 7.6% and the transgenic donor did not result in any hybrid.
... LuPLR1 is responsible for the synthesis of the major (+)secoisolariciresinol enantiomer (87%) while a minor quantity of the (-)-secoisolariciresinol enantiomer (13%) is synthesized by LuPLR2 (Hemmati et al., 2010). The very weak β-glucuronidase (GUS) activity detected in embryos has to be attributed to non-specific GUS that has already been reported in seeds of a number of plants (Hu et al., 1990;Hänsch et al., 1995). GUS activities measured at different developmental stages in the coats and embryos of transgenic flax seeds containing the LuPLR1 gene promoter were in accordance with those of previously published semi-quantitative RT-PCR experiments (Hano et al., 2006), confirming that the SDG biosynthetic pathway is active already at S0 and reaches a maximum during S1, S2, and S3 ( Figure 5). ...
The concentration of secoisolariciresinol diglucoside (SDG) found in flaxseed (Linum usitatissimum L.) is higher than that found in any other plant. It exists in flaxseed coats as an SDG-3-hydroxy-3-methylglutaric acid oligomer complex. A laser microdissection method was applied to harvest material from different cell layers of seed coats of mature and developing flaxseed to detect the cell-layer specific localization of SDG in flaxseed; NMR and HPLC were used to identify and quantify SDG in dissected cell layers after alkaline hydrolysis. The obtained results were further confirmed by a standard molecular method. The promoter of one pinoresinol-lariciresinol reductase gene of L. usitatissimum (LuPLR1), which is a key gene involved in SDG biosynthesis, was fused to a β-glucuronidase (GUS) reporter gene, and the spatio-temporal regulation of LuPLR1 gene expression in flaxseed was determined by histochemical and activity assays of GUS. The result showed that SDG was synthesized and accumulated in the parenchymatous cell layer of the outer integument of flaxseed coats.
... Such a discrepancy between the results/observations of GUS expression in bombarded pollen of lily has been attributed to the presence (Nishihara et al. 1993) or absence van der Leede-Plegt et al. 1992) of 20 % methanol in the X-Gluc solution used for the assay (Jefferson et al. 1987;Kosugi et al. 1990). Discrepancies such as 'background stain' have also been reported in vegetative tissues as well as pollen (Hu et al. 1990;Shi et al. 1995). The modified green fluorescent protein (mgfp) has also been tested in Arabidopsis and Medicago (refs from Chumakov and Moiseeva 2012). ...
In present times, when genetically modified (GM) crops are creating a niche for themselves in the agricultural arena, germline transformation is likely to reduce the time and effort to produce transgenics. In the last three decades of transgenic research, phenomenal success has been achieved but has remained limited to species that lent themselves easily to genetic modification. More than 15 dicot and 11 monocot taxa have been tested for male germline transformation. On the other hand at least 23 three dicot and four monocot taxa have been tested for genetic modification through female germline. Amongst the male germ cells, cellular systems ranging from microspore, immature and mature pollen, pollinaria, pollen protoplasts, pollen tubes, exine detached pollen (EDP) and pollen derived embryos have been tested for transient or stable integration of foreign genes. A variety of methods and variants and combinations of methods such as agrolistics that combines Agrobacterium mediated transformation and biolistics, are available for the introduction of genes into the male germline, as it is accessible to treatments under a variety of conditions. Amongst the methods tested in male germline transformation, particle bombardment remains the most preferred method. The female germline, being largely inaccessible has lent itself to modification mostly via Agrobacterium-mediated methods. The success of in planta vacuum infiltration and floral dip exercise seems to be confined to Crucifers with ovule as the prime target. Applicability of germline transformation methods is being tested on a wider range of crop plants.
... Biochemical and immunological data from our study, and in some others involving higher plant species (Hu et al., 1990;Raineri et al., 1990;Alwen et al., 1992), have indicated a lack of similarity between the plant and microbial glucuronidases. The pH optimum, reaction to inhibitors of GUS, thermal stability, and failure to recognize any common epitopes, demonstrated a disparity between these two activities at the structural level. ...
Accurate detection of a genetic or biochemical marker introduced into sugarbeet (Beta vulgarjs L.) is based on the absence of native sequences or activities in the plant that could confound the analysis of expression of the introduced marke r. During the course of experiments designed to optimize DNA transfer from Agrobacterjum tumef aciens to sugarbeet leaf disc cells, an endogenous enzyme activity was discovered which utilizes all the common substrates recognized by the marker enzyme l3,-glucuronidase (GUS) from E. coli. This native sugarbeet enzyme (SB-GUS) was characterized immunologically and biochemically. GUS and SB-GUS were found to be distinct with regard to pH optima, thermal inactivation, reaction to denaturants and protein modifying reagents, inhibition by metals and saccharo-Iactone, and molecular mass. The two activities are not immunologically related, as judged by Western blot and immunoprecipitation analyses. A protocol was developed to accurately quantitate introduced GUS in the presence of SB-GUS, by utilizing selective inhibition of GUS at pH 7.0 by saccharic acid l,4-lactone. Under these conditions GUS activity is completely eliminated, while SB GUS activity was unaffected.
... At first it was assumed that the β-glucuronidase reaction is restricted to prokaryotes only and that it does not appear in higher plants (Stomp 1992). Hu et al. (1990) demonstrated that a transient "false" or "background" GUS reactions occurs in embryos and vegetative organs of many plant species. They classified cabbage and kohlrabi among plants with the most intensive false GUS reaction. ...
Brassica oleracea L. with its numerous subvarieties is one of the most important vegetable species in the world. Constant improvement of various agronomical traits is a permanent task of Brassica breeders for which methods of genetic engineering have been adopted recently. However transformation in Brassica vegetables is not yet a routine. Development of successful transformation protocols based on common transformation procedures has been presented in many reports. Transformation success depends on many factors including genotype, explant type, gene introduction technique and the construct itself. In this review we present recent data on transformation of Brassica vegetables using both Agrobacterium tumefaciens and A. rhizogenes strains, as well as some other alternative approaches.
... . PCR and endogenous GUS activity analysis demonstrated their expression in all the different organs (Sood, 1980;Schulz and Weissenböck, 1987;Plegt and Bino, 1989;Hu et al., 1990;Alwen et al., 1992;Anhalt and Weissenböck, 1992;Wozniak and Owens, 1994;Morimoto et al., 1995;Morimoto et al., 1998;Muhitch, 1998;Sudan et al., 2006;Schoenbeck et al., 2007;Woo et al., 2007). ...
A new highly sensitive whole-mount in situ hybridization method, based on tyramide signal amplification (TSA-MISH) was developed and a combined GFP detection and TSA-MISH procedure was applied for the first time in plants, to precisely define the spatial pattern of AtGUS1 and AtGUS2 expression in the root apex. β-glucuronidases (GUSs) belonging to the glycosyl hydrolases (GHs) 79 family, are widely distributed in plants, but their functional role has not yet been fully investigated. In the model system Arabidopsis Thaliana, three different AtGUS genes have been identified which encode proteins with putative different fates. Endogenous GUS expression has been detected in different organs and tissues, but the cyto-histological domains of gene expression remain unclear. The results here reported show co-expression of AtGUS1 and AtGUS2 in different functional zones of the root apex (the cap central zone, the root cap meristem, the staminal cell niche and the cortical cell layers of the proximal meristem), while AtGUS2 is exclusively expressed in the cap peripheral layer and in the epidermis in the elongation zone. Interestingly, both genes are not expressed in the stelar portion of the proximal meristem. A spatial (cortex vs. stele) and temporal (proximal meristem vs. transition zone) regulation of AtGUS1 and AtGUS2 expression is therefore active in the root apex. This expression pattern, although globally consistent with the involvement of GUS activity in both cell proliferation and elongation, clearly indicates that AtGUS1 and AtGUS2 could control distinct downstream process depending on the developmental context and the interaction with other players of root growth control. In the future, the newly developed approaches may well be very useful to dissect such interactions.
... Thus, as described earlier activity of reporter enzyme GUS was determined to study the expression of this marker gene in transformed chilli leaves and hypocotyls. However, in some plants, intrinsic gus like activity might interfere with reporter enzyme assay (Hu et al., 1990). genes toPlant Cells. ...
... Thus, as described earlier activity of reporter enzyme GUS was determined to study the expression of this marker gene in transformed chilli leaves and hypocotyls. However, in some plants, intrinsic gus like activity might interfere with reporter enzyme assay (Hu et al., 1990). ...
... Positive GUS activity could be solely attributed to the expression and correct splicing of the inserted uidA in eukaryotic plant cells. P. andrieuxii does not present endogenous GUS activity as reported for carrot, celery, parsley, rice [13], Ricinus communis [14], Agave fourcroydes [15] and Capsicum chinense [16]. Older roots displayed a higher GUS activity than younger root explants and leaf sections. ...
Sections of hypocotyls, roots and leaves from Penta-linon andrieuxii plantlets were transiently transform-ed with Agrobacterium tumefaciens LBA4404 bearing the binary plasmid pCAMBIA2301 with an inter-rupted β-glucuronidase (GUS) gene. Histochemical GUS assays showed transient gene expression in all infected tissues, being older roots those which dis-played the most intense GUS staining. To our knowl-edge, this is the first report of Pentalinon andrieuxii susceptibility to Agrobacterium tumefaciens-mediated genetic transformation.
... GUS reaction was negative in the untransformed control roots (Figure 5). Positive GUS reaction is not reliable evidence of β-glukuronidase activity considering that endogenous enzyme in plant tissue can use same substrate and that most plant species express that enzyme at least in some tissues under certain conditions [40,41]. As a consequence this result is acceptable only as an indicator of the genetic transformation and further confirmed using other techniques or methods (e.g. by PCR analysis). ...
... 10 Some reporters were observed intrinsic GUS like activity in rice. 11 It was controlled by treating tissues at alkaline pH 8.0 or by including 20% methanol in the assay. 12 Transformation screening had increased by selectable marker such as antibiotic resistance. ...
An efficient and reproducible protocol for Agrobacterium- mediated transformation of an agronomically useful Oryza sativa variety, Pusa Basmati- 1 (IET- 10364) has been standardized. Although several reporters have been claimed the Agrobacterium- mediated transformation but here we studied the effect of acetosyringone and age of callus on transformation frequency for scutellum-derived callus of Pusa basmati- 1. By optimizing these two parameters for Agrobaterium- mediated transformation, we can produce an efficient and reproducible protocol. The callus derived from mature rice seeds were used as explants for transformation studies. Callus were co-cultured with Agrobacterium tumefaciens EHA 105 harboring a binary vector p35SGUSINT that carried Beta-glucuronidase (uid A) and It contains gene npt II, determining resistance to kanamycin, under the control of the constitutive (nos) promoter. Maximum Agrobacterium- mediated transformation frequency was observed at 350µM/l acetosyringone with 35 days old callus.
... GUS reaction was negative in the untransformed control roots ( Figure 5). Positive GUS reaction is not reliable evidence of β-glukuronidase activity considering that endogenous enzyme in plant tissue can use same substrate and that most plant species express that enzyme at least in some tissues under certain conditions [40,41]. As a consequence this result is acceptable only as an indicator of the genetic transformation and further confirmed using other techniques or methods (e.g. by PCR analysis). ...
Hairy root cultures of lettuce (Lactuca sativa L.) were obtained by inoculation of cotyledonary leaves of in vitro lettuce seedlings (cvs. Nansen and Ljubljanska ledenka) with Agrobacterium rhizogenes A4M70GUS. Approximately in 96.7% cvs. Nansen and in 91.2% Ljubljanska ledenka inoculated explants produced hairy root when they were incubated on Murashige and Skoog (MS) half-strength medium without plant growth regulators. A total of 54% of all hairy root cultures expressed GUS activity. Every hairy root represented an independent transformation event. Line Ljubljanska ledenka 18 showed the highest biomass (5.5 times the biomass of control root). A PCR analysis of the genomic DNA confirmed the presence of marker and target genes in 15 hairy roots examined.
Numerous classes of endogenous and xenobiotic compounds are conjugated to uridine-5′-diphospho (UDP)-alpha-D-glucuronic acid which is catalyzed by human UDP-Glucuronosyltransferases (UGTs). The resulting beta-D-glucuronides can be hydrolyzed to ß-D-glucuronic acid and the corresponding aglycone in a configuration retaining manner by beta-glucuronidases (GUSBs), which are widely distributed in mammalians, microbiota, insects, molluscs, nematodes, fishes and plants. This study investigates GUSBs’ activity in the presence of ethanol (0–70% by volume) using different ß-D-glucuronides (phenolphthalein-ß-D-glucuronide, 4-nitrophenol-ß-D-glucuronide, morphine-3-O-ß-D-glucuronide, quercetin-3-O-ß-D-glucuronide and 1-/2-propyl-ß-D-glucuronide) as substrates. It was found that ß-D-ethyl glucuronide (EtG), which is a minor UGT-derived metabolite of ethanol in man and one of the most frequently used biomarkers of alcohol consumption today, builds up from all investigated ß-D-glucuronides by means of GUSBs in the presence of ethanol. The glucuronyl transfer reaction, which was neither detected in the absence of ethanol nor in absence of GUSBs, is minor at ethanol concentrations which are commonly observed in blood and tißues after consumption of alcoholic beverages, but predominant at higher concentrations of ethanol. In spite of in vitro characteristics, our observations point to an additional biochemical path and another source of EtG, which should be further evaluated in the context of alcohol biomarker applications. The detection of EtG in several settings independent from of human UGT-metabolism (e.g. EtG post post-collection synthesis in E.coli coli-contaminated urine samples, EtG in wine and ethanolic herbal preparations) can be explained by the described mechanism.
Currently, gene exploration and molecular breeding in pumpkin (Cucurbita moschata Duch.) is a challenging process. There are limited efficient stable transformation methods for pumpkin. Transient transformation is a promising tool for the study of gene function. Here, an efficient Agrobacterium-mediated transient transformation system was developed for gene function studies in pumpkin. In this system, the function of salt-tolerant gene StNHX1 was confirmed by over-expression in germinated seeds. The CmPHD1-EGFP fusion protein was detected in the nucleus of cotyledon epidermal cells. This system can be used to analyze gene function and protein subcellular localization in pumpkin. Its advantages are highly efficient, cost-effective and time-saving (∼14 days). It may play a key role in gene exploration and molecular breeding in pumpkin, especially in large-scale analyses.
Parthenocarpy is the development of an ovary into a seedless fruit without pollination. The ubiquitous downregulation of SlIAA9 induces not only parthenocarpic fruit formation but also an abnormal vegetative phenotype. To make parthenocarpic transgenic tomato plants without unwanted phenotypes, we found two genes, namely, Solyc03g007780 and Solyc02g067760, expressed in ovary tissue but not in vegetative tissues. Solyc03g007780 was expressed in developing ovaries and anthers. Solyc02g067760 mRNA was detected in whole-flower tissues. The promoters of Solyc03g007780 (Psol80) and Solyc02g067760 (Psol60) predominantly induced the expression of genes in the ovule, placenta, endocarp and pollen and in whole-flower tissues, respectively. Psol80/60-SlIAA9i lines, created for SlIAA9-RNA interference controlled by two promoters, successfully formed parthenocarpic fruits without pleiotropic effects in vegetative tissues. Downregulation of SlIAA9, responsible for parthenocarpic fruit formation, was observed in ovules rather than ovaries in the Psol80/60-SlIAA9i lines. Although the weight of parthenocarpic fruits of the Psol80/60-SlIAA9i lines was lower than the weight of pollinated fruits of the wild type (WT), the parthenocarpic fruits presented redder and more saturated colors and higher levels of total soluble solids and titratable acidity than the WT fruits.
In the mid 1980s, the first transgenic plants were produced via Agrobacterium-mediated transformation of Nicotiana tabacum (tobacco) protoplasts (Horsch et al., 1984; DeBlock et al., 1984). Since then, over 30 different plant species have been genetically engineered by a variety of techniques (Gasser and Fraley, 1989). The three major breakthroughs in the transformation of dicotyledonous plants were: (1) the development of shuttle vectors for harnessing the natural gene transfer capability of Agrobacterium (Fraley et al., 1986), (2) the methods to use these vectors for the direct transformation of regenerable explants obtained from plant organs (Horsch et al., 1985), and (3) the development of selectable markers. Genetic modification of many field crops, as well as horticultural and tree species, has been achieved with Agrobacterium vectors.
Background
In many plants, the amino acid proline is strongly accumulated in pollen and disruption of proline synthesis caused abortion of microspore development in Arabidopsis. So far, it was unclear whether local biosynthesis or transport of proline determines the success of fertile pollen development.
Results
We analyzed the expression pattern of the proline biosynthetic genes PYRROLINE-5-CARBOXYLATE SYNTHETASE 1 & 2 (P5CS1 & 2) in Arabidopsis anthers and both isoforms were strongly expressed in developing microspores and pollen grains but only inconsistently in surrounding sporophytic tissues. We introduced in a p5cs1/p5cs1 p5cs2/P5CS2 mutant background an additional copy of P5CS2 under the control of the Cauliflower Mosaic Virus (CaMV) 35S promoter, the tapetum-specific LIPID TRANSFER PROTEIN 12 (Ltp12) promoter or the pollen-specific At5g17340 promoter to determine in which site proline biosynthesis can restore the fertility of proline-deficient microspores. The specificity of these promoters was confirmed by β-glucuronidase (GUS) analysis, and by direct proline measurement in pollen grains and stage-9/10 anthers. Expression of P5CS2 under control of the At5g17340 promoter fully rescued proline content and normal morphology and fertility of mutant pollen. In contrast, expression of P5CS2 driven by either the Ltp12 or CaMV35S promoter caused only partial restoration of pollen development with little effect on pollen fertility.
Conclusions
Overall, our results indicate that proline transport is not able to fulfill the demand of the cells of the male germ line. Pollen development and fertility depend on local proline biosynthesis during late stages of microspore development and in mature pollen grains.
Electronic supplementary material
The online version of this article (10.1186/s12870-018-1571-3) contains supplementary material, which is available to authorized users.
Although dozens of stress inducible promoters have been identified in rice, detailed and comparative investigations under uniform condition are still limited. In this study, we selected eight previously reported drought-inducible genes [Oshox24, hypothetical protein (hp), OsLEA3-1, OsNAC5, OsNCED3, Rab21, RRJ1 and Wsi18] and analyzed their transcriptional responses to drought and osmotic stresses in rice. Based on their transcription patterns and the required standards for inducible promoters, Oshox24, Rab21 and OsLEA3-1 were chosen for cloning of their promoters for detailed analyses at both the RNA and protein/activity levels, with rice oxalate oxidase gene (OsOxO3) as a reporter gene. By generating transgenic plants and determining oxalate oxidase activity that is activated under different stresses, we defined more quantitatively that the promoters of Oshox24, OsLEA3-1 and Rab21 are ideally applicable to transgene expression for inducibly controlling drought resistance genes or other functional genes in rice. The results provided a quantitative assessment of the strength, sensitivity, stress specificity and time course of the three promoters. Such detailed information is essential for the selection of promoters for use in improving stress resistance.
Verticordia grandis is a native Australian plant of the Myrtaceae family. The genus Verticordia (feather flower) consists of 79 species, most of which are endemic to the southwest of Western Australia. Members of this genus are perennial woody shrubs adapted to sandy soils and low rainfall areas; they occur mainly in heathland and shrubland communities. Their floral display is impressive with loose to tight clusters of showy flowers and feathery calices. Flower color is diverse, ranging from red, orange, yellow, mauve, or white. All have great horticultural value, but some species, including V. grandis, are difficult to propagate.
Any European directive can be implemented in different ways in different countries. Directive EC/90/220, dealing with introducing genetically modified organisms (GMOs) into the environment, is no exception in this respect. The international consensus underlying legislation regarding GMOs is intended to result in world-wide harmonization. Consensus in this context of course only refers to common opinions among competent authorities and national experts. Public acceptance of and appreciation for scientific work within the broad field of biotechnology are far less unanimous (e.g. Zechendorf 1994). It is public opinion, or better still, public opinions, that will determine whether or not genetically modified novel foods, medicines and other products will reach the market successfully in the foreseeable future. Not less important, the public will have its say about continuing the funding of biotechnology research carried out at present at universities and public institutes. The words “out in the open” in the title therefore literally mean “for everyone to see and judge”. Field trial activity reports, like the example presented in Table 1, should be open to public discussion. When comparing field trials in six different countries, it is difficult not to over-emphasize differences. This is especially appealing, when in all of these countries but the United Kingdom the same crop and trait, maize harbouring glufosinate ammonium resistance, were field tested.
Transient expression following agroinfiltration of plant tissue was investigated as a system for producing recombinant protein. As a model system, Agrobacterium tumefaciens containing the beta-glucuronidase (GUS) gene was vacuum infiltrated into lettuce leaf disks. Infiltration with a suspension of 109 colony forming units/mL followed by incubation for 72 h at 22 degrees C in continuous darkness produced a maximum of 0.16% GUS protein based on dry tissue or 1.1% GUS protein based on total soluble protein. This compares favorably to expression levels for commercially manufactured GUS protein from transgenic corn seeds. A. tumefaciens culture medium pH between 5.6 and 7.0 and surfactant concentrations <= 100 ppm in the vacuum infiltration did not affect GUS expression, while infiltration with an A. tumefaciens density of 107 and 108 colony forming units/mL, incubation at 29 degrees C, and a surfactant concentration of 1,000 ppm significantly reduced expression. Incubation in continuous light caused lettuce to produce GUS protein more rapidly, but final levels did not exceed the GUS production in leaves incubated in continuous darkness after 72 h at 22 degrees C. The kinetics of GUS expression during incubation in continuous light and dark were represented well using a logistic model, with rate constants of 0.30 and 0.29/h, respectively. To semi-quantitatively measure the GUS expression in large numbers of leaf disks, a photometric enhancement of the standard histochemical staining method was developed. A linear relationship with an R-2 value of 0.90 was determined between log(10) (% leaf darkness) versus log(10) (GUS activity). Although variability in expression level was observed, agroinfiltration appears to be a promising technology that could potentially be scaled up to produce high-value recombinant proteins in planta. (c) 2005 Wiley Periodicals, Inc.
As an alternative to the traditional histochemical GUS visualization substrate, 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc), the fluorescence substrates ImaGene Green™ (C12FDGLcU) and ImaGene Red™ (C12RGlcU) were used to visualize GUS activity in transformed cassava cells. These fatty acyl-containing GUS substrates become anchored to the cell membrane, by means of hydrophobic interactions with the plasma membrane. Once anchored to the cell membrane, the fluorogenic glucosidic moiety is transferred to the cytosolic side of the membrane, thereby introducing the fluorogenic substrate into the cell without the need to solubilize the cell membranes. Using these two fluorogenic and lipophilic substrates, many of the practical difficulties often experienced in the visualization of GUS activity in transformed cells with X-Gluc can be readily overcome.
Regulatory genes of the maize anthocyanin biosynthetic pathway have proven useful as scorable markers for transformation because of their high sensitivity, ease of visualization, cell-autonomous expression and lack of requirement for exogenous substrates. This is particularly advantageous when using particle bombardment as a DNA delivery system, since cells expressing genes can be counted easily and unambiguously (Bowen 1992). Furthermore, the cells that have been damaged by microprojectile bombardment can still express GUS (giving false positive), but they will not accumulate anthocyanin since the latter requires an intact vacuole and coordinate expression of many genes. Expression of anthocyanin genes in plant tissues will be useful where false positive results have been reported due to intrinsic GUS-like activity in several plant parts (Hu et al. 1990) and GUS production by endophytic microorganisms (Tor et al. 1992). The anthocyanin markers permit visualization of transgenic tissue from the beginning and throughout development without sacrificing the tissues. In addition, expression of anthocyanin genes in transformed tissues can provide a system for investigating the regulation of gene expression in plants, which is difficult with GUS (Ben-fey et al. 1990). Transient and stable expression of anthocyanin genes in various corn intact tissues (Klein et al. 1988; Ludwig et al. 1990; Wong et al. 1991; Bowen 1992; Dunder et al. 1993) and transient expression in wheat seedling tissues (Wong et al. 1991) has been reported previously. We have used anthocyanin as a visual marker for optimization of bombardment conditions using different target tissues (transiently) and evaluation of different selectable markers (stably) for wheat transformation by selecting the transformants visually in the early stage of development.
Today a number of cell culture and molecular techniques are used or are under development to manipulate sexual reproduction. Protoplast fusion is used not only to create somatic hybrids but also to produce cybrids that regenerate into cytoplasmic male sterile plants. Unreduced gametophytes are selected for polyploidization or distant hybridization. In vitro pollination and in vitro fertilization after isolation of egg and sperm cells are used to overcome pre- and postfertilization crossing barriers (Shivanna and Johri, 1985).
Modern commercial sugarcane varieties are highly heterozygous, complex polyploid and aneuploid hybrids, often with four different species of Saccharum in their ancestry. Agronomically superior sugarcane cultivars are obtained through a multistage selection scheme over a period of approximately 10 years to identify a few elite clones in very large populations of seedlings. The costs of selection approach $1 million per cultivar from advanced breeding programs. In practice, elite clones may be completely abandoned because of a single fault such as disease susceptibility. Traditional breeding approaches to correct such faults in an existing cultivar are impractical in sugarcane, because of the genetic complexity of cultivars and the long periods needed for each round of selection for some agronomically important traits such as ratooning ability. The capacity to introduce specific genes by genetic engineering, without the major genetic reassortment following crossing, could save the sugarcane industry millions of dollars by rescuing flawed cultivars.
A study on expiant inoculation method using promoterless tagging vector, pCAMDIN, on local tomato (MT1 variety) for the purpose of T-DNA insertional mutagenesis by agro-infiltration have been carried out. The injection of young leaves with a solution containing Agrobacterium tumefaciens, 100 mgll Str, 50 mgll Kan, 10mM MES and 100μM Acetosyringone. The infiltration was carried out with the use of LB medium by gently wounding the surface of the leaves and every procedures were carried out in the glass house. Infiltrated tissues were not damaged and remained green even after 5 days. Advantages of the technique included ease of infiltration and the observed transient expression on the sample, gus gene fragments were successfully amplified. Successful integration of gus gene into the tomato can also be observed in the results of Southern hybridization. Thus, it can be suggested that transgene insertion from recombinant vector pCAMDIN has occurred in tomato genome.
“Transformation of soybean has been far from routine.... Unfortunately, all of the procedures described in the literature suffer from problems of low efficiency, poor reproducibility, and limited cultivar specificity.” This is the assessment of the field recently provided by The Soybean Center for Tissue Culture and Genetic Engineering (Trick et al. 1997). As a major force in promoting and developing soybean transformation technology, the Center has compiled worldwide data on soybean biotechnology. Readers may obtain updated soybean transformation information and protocols from their homepage: http:// mars. cropsoil. uga. edu/ homesoybean/ >.
A number of transformation systems have been developed to insert foreign DNA into the appropriate plant genome (nuclear or plastid) (discussed in Chap. 3). However, only a small fraction of the treated cells become transgenic, while the majority of the cells remain untransformed using any of these methods. Thus, effective selection and screening strategies are needed to pick up the rare transgenic lines from a pool of nontransformed cells or plants. To date, more than 50 marker genes and a few molecular techniques have been developed to serve this essential purpose.
Marker genes (MGs) are essential tools for plant research and biotechnology. Positive selectable marker genes (SMGs) are used in genetic transformation to allow only transgenic cells to grow and develop and are necessary for efficient transformation. Negative SMGs confer a selective disadvantage to the cells that express them, and have several uses in both basic and applied research. Reporter genes (RGs) make it possible to easily screen cells or tissues for their expression. Several tens of different genes from bacteria, fungi, plants, and animals have been demonstrated to function as SMGs. Here, SMGs are classified based on the mechanism of action of the gene products. To provide the readers with practically useful information, details on transformation and selection efficiency are given. RGs are the object of intense research. Refinement of existing RGs and development of new ones is constant, and has provided powerful aids for fine studies on cell biology and more efficient genetic engineering. They are classified as vital and non vital, depending on the possibility to screen their expression in living cells. The effect of MG expression on the phenotype and their safety in crops is briefly discussed. The picture emerging from this literature review is that a plentiful array of powerful and versatile tools for basic and applied research is available.
A transient ß-glucuronidase (GUS)-assay was performed to evaluate electroporation parameters and optimize DNA delivery conditions into strawberry protoplasts. Optimal GUS-activity was obtained when protoplasts were subjected to 400 V/cm for 20 ms. GUS-activity could be further increased by the addition of carrier DNA to the electroporation mixture. Callus selected on 10 μg/ml hygromycin produced shoots which exhibited GUS-activity. The transformed nature of the shoots obtained after selection was confirmed by DNA-analysis.
False positive transformants obtained during plant transformation experiments on species of the monocotyledonous genus Dioscorea (yam) are described. The false positive results were found to be due to endophytic bacteria which exist within aseptically micropropagated shoot cultures and which express β-glucuronidase (GUS). The bacteria were isolated and identified as two species of Curtobacterium. The expression of GUS in these organisms was found to be induced by a variety of glucuronide substrates. The induction of GUS activity in the bacteria can be inhibited by chloramphenicol, tetracycline, ticarcillin and sodium azide. Implications of these results for use of the gus gene in plant transformation work are discussed.
The cauliflower mosaic virus 35S (35S) and the enhanced 35S (E35S) promoters fused with maize alcohol dehydrogenase (Adh1) intron1 or maize shrunken locus (sh1) intronl along with maize Adh1 and rice actin (Act1) promoters fused to their respective first introns were tested for transient expression of the E.coli β-glucuronidase (gus) reporter gene in cultured barley (Hordeum vulgare L) cells. The plasmids, carrying the respective promoterintron combinations to drive the gus fused to nopaline synthase (nos) terminator, were introduced into cultured barley cells using a particle gun. The rice Act1 promoter with its first intron gave the highest expression of all promoter intron combinations studied. This was followed by the E35S promoter and no significant differences were observed between the other two promoters tested. The rice actin promoter is now being used to drive selectable marker genes to obtain stably transformed cereal cells.
In this paper we demonstrate that RNA sequences present upstream and downstream of a reporter gene coding region play an important role in determining the amount of protein produced from an mRNA. A translational enhancer, omega, derived from tobacco mosaic virus, when present at the 5'-end of beta-glucuronidase mRNA increased the efficiency of translation 16-fold to 18-fold in electroporated tobacco or carrot protoplasts, and threefold to 11-fold in maize or rice protoplasts. The presence of omega did not alter the half-life of the mRNA in vivo. We also demonstrate for the first time that a minimum polyadenylated tail length of 25 adenylate residues is sufficient to substantially increase the expression and half-life of the reporter mRNA in plants. When in vitro-produced mRNAs were synthesized such that extra sequence was added to the 3'-end of the poly(A) tail, however, the final level of expression was decreased up to 80%. Omega, the translational enhancer, and a poly(A) tail function independently of each other; their combined effect on translation, when both are present in an mRNA, is the multiplication of their individual effects. Histochemical analysis for the presence of beta-glucuronidase in tobacco established that virtually all viable cells receive mRNA during electroporation. Video image analysis of tobacco protoplasts electroporated with luciferase mRNA demonstrated that there is a wide range in the level of expression of this marker. Carrier RNA, when present during electroporation, had only a modest effect on increasing mRNA uptake. Reporter mRNA expression in electroporated protoplasts was directly proportional to the input mRNA up to at least 30 micrograms/ml.
The 5' region of the wound-inducible gene wun1, derived from potato, has been sequenced and analyzed for cis-acting elements important in controlling gene expression in transgenic tobacco plants. Different 5' deletion fragments were linked to the reporter gene beta-glucuronidase (GUS) as transcriptional fusions, and the expression of these chimeric genes was analyzed in leaf tissue. Sequences 111 base pairs upstream of the transcriptional start site were not able to drive the GUS expression over background levels, whereas sequences between -111 and -571 showed a slightly higher activity with equal levels of transcription in wounded and nonwounded tissue. The addition of further upstream sequences (-571 to -1022) enhanced the level of expression by a factor between 13 and 370. The expression driven by this fragment was inducible by a factor of twofold to ninefold by wounding. Histochemical analysis of different tissue from transgenic plants that contain wun1-GUS fusions demonstrates wound-inducible and cell-specific wun1 promoter activity in plants containing the -1022-base pair fragment. The location of GUS activity appears to be cell-specific, being highest in epidermal cells of leaves and stems and lower in vascular cells. Activity was reduced to levels that could not be detected by histochemical staining in leaves, stems, and roots of plants containing the deleted promoter fragments. Plants that contain the different deletion constructs and plants that carry the -1022-base pair fragment show high expression in anthers and pollen grains that could not be stimulated by wounding.
We have developed a gene-fusion system based on the Escherichia coli beta-glucuronidase gene (uidA). The uidA gene has been cloned from E. coli K-12 and its entire nucleotide sequence has been determined. beta-Glucuronidase has been purified to homogeneity and characterized. The enzyme has a subunit molecular weight of 68,200, is very stable, and is easily and sensitively assayed using commercially available substrates. We have constructed gene fusions of the E. coli lacZ promoter and coding region with the coding region of the uidA gene that show beta-glucuronidase activity under lac control. Plasmid vectors have been constructed to facilitate the transfer of the beta-glucuronidase coding region to heterologous control regions, using many different restriction endonuclease cleavage sites. There are several biological systems in which uidA-encoded beta-glucuronidase may be an attractive alternative or complement to previously described gene-fusion markers such as beta-galactosidase or chloramphenicol acetyltransferase.
The development of a transposon mutagenesis system in soybean would aid in the isolation of unknown genes. The maize controlling element (Ac) has, therefore, been introduced into the soybean (Glycine max (L.) Merr.) genome byAgrobacterium-mediated transformation.Ac was inserted into the untranslated leader region of the bacterial ß-glucuronidase gene (GUS) such that the excision ofAc resulted in restoration of the GUS gene activity. Excision events of theAc element were monitored by detecting blue cells or sectors in transgenic soybean tissues. Using the GUS gene assay and with hybridization data, we have demonstrated that theAc element transposes in transgenic soybean calli, leaves, stems, and roots.
Patatin is a family of lipid acyl hydrolases that accounts for 30 to 40% of the total soluble protein in potato tubers. Class-I patatin genes encode 98 to 99% of the patatin mRNA in tubers, but are not normally expressed in other tissues. They are not totally 'tuber-specific'; however, since they can be induced to express at high levels in other tissues under conditions of sink limitation or in explants cultured on medium containing elevated levels of sucrose. To examine the evolution of the mechanisms that regulate patatin gene expression, we introduced a chimeric patatin-beta-glucuronidase (GUS) gene containing 2.5 kb of 5' flanking sequence from the Class-I potato patatin gene PS20 into tobacco plants. The construct was not expressed at significant levels in leaves of juvenile plants or plantlets cultured in vitro, but was expressed at high levels in explants cultured on medium containing 0.3 to 0.4 M sucrose. While there were differences in the expression of the chimeric gene between transgenic tobacco and potato plants, the pattern of sucrose induction was very similar. These results suggest that the mechanism that controls patatin gene expression in potato tubers evolved from a widely distributed mechanism in which gene expression is regulated by the level of available photosynthate.
Transposition of the Anthirrinum majus Tam3 element and the Zea mays Ac element has been monitored in petunia and tobacco plants. Plant vectors were constructed with the transposable elements cloned into the leader sequence of a marker gene. Agrobacterium tumefaciens-mediated leaf disc transformation was used to introduce the transposable element constructs into plant cells. In transgenic plants, excision of the transposable element restores gene expression and results in a clearly distinguishable phenotype. Based on restored expression of the hygromycin phosphotransferase II (HPTII) gene, we established that Tam3 excises in 30% of the transformed petunia plants and in 60% of the transformed tobacco plants. Ac excises from the HPTII gene with comparable frequencies (30%) in both plant species. When the beta-glucuronidase (GUS) gene was used to detect transposition of Tam3, a significantly lower excision frequency (13%) was found in both plant species. It could be shown that deletion of parts of the transposable elements Tam3 and Ac, removing either one of the terminal inverted repeats (TIR) or part of the presumptive transposase coding region, abolished the excision from the marker genes. This demonstrates that excision of the transposable element Tam3 in heterologous plant species, as documented for the autonomous element Ac, also depends on both properties. Southern blot hybridization shows the expected excision pattern and the reintegration of Tam3 and Ac elements into the genome of tobacco plants.
We have used the Escherichia coli beta-glucuronidase gene (GUS) as a gene fusion marker for analysis of gene expression in transformed plants. Higher plants tested lack intrinsic beta-glucuronidase activity, thus enhancing the sensitivity with which measurements can be made. We have constructed gene fusions using the cauliflower mosaic virus (CaMV) 35S promoter or the promoter from a gene encoding the small subunit of ribulose bisphosphate carboxylase (rbcS) to direct the expression of beta-glucuronidase in transformed plants. Expression of GUS can be measured accurately using fluorometric assays of very small amounts of transformed plant tissue. Plants expressing GUS are normal, healthy and fertile. GUS is very stable, and tissue extracts continue to show high levels of GUS activity after prolonged storage. Histochemical analysis has been used to demonstrate the localization of gene activity in cells and tissues of transformed plants.
The nutrient requirements of suspension cultures from soybean root have been investigated, and a simple medium consisting of mineral salts, sucrose, vitamins and 2,4-dichlorophenoxyacetic acid (2,4-d) has been designed.The cells required thiamine, 2,4-d and ammonium in addition to the usual mineral salts and sucrose.Optimum concentrations of nitrate and ammonium were 25 and 2 mM respectively. The highest yield of cells was achieved at an initial pH of 4.5–5.5. During the growth cycle the pH gradually increased to 6.0–6.2.
Transgenic plants are an effective system for the study of regulated gene expression. Developmental control of expression
can be monitored by assaying different tissues or by assaying a plant at different developmental stages. Analysis of the petunia
5-enolpyruvylshikimate-3-phosphate synthase gene, which is highly expressed in flowers, allowed identification of an upstream
region that confers tissue-specific and developmentally regulated expression. The cell specificity of expression in floral
tissues has been defined by histochemical localization. This expression is contrasted to that of the 35S promoter of cauliflower mosaic virus, a nominally constitutive promoter that shows a definite specificity of expression in
floral tissues. Moreover, this expression differs in transgenic hosts of different species.
VI Int'l Congr Plant Cell & Tissue Cult
- Cy Hu
- Im Sussex