rolABC Genes Research Articles

Enhanced stress tolerance in transformed Ajuga bracteosa Wall. ex Benth. regenerants by upregulated gene expression of metabolic pathways

The rol oncogenes of Agrobacterium rhizogenes enhance the production of medicinally important compounds in plants and provide a first barrier against the overproduction of reactive oxygen species during biotic and abiotic stress. 'this study was designed to evaluate the expression of genes involved in biosynthetic pathways and their impact on metabolic contents and environmental stress tolerance in regenerated Ajuga bracteosa Wall. ex Benth. After successful transformation, real-time quantitative PCR confirmed the increased expression (1.94-6.59-fold) of HMGR, HDS, FDS, PAL, and TTG1 genes in transgenic lines. Furthermore, GC-MS coupled with principal component analysis revealed diverse concentrations of 97 metabolites in A. bracteosa. Transgenic lines showed greater survival under multiple stresses. This was revealed by significant chlorophyll content (8.13-21 mu moles/m(2)), higher quantum efficiency of PSII (F-v/F-m), and the performance index (PIabs) value. Similarly, catalase and peroxidase enzyme activities were enhanced during extreme drought (300-400 mM mannitol) and salinity (150-200 mM NaCl) conditions, compared to untransformed control. Wild type control plant leaves were completely necrotized by Aspergillus fumigatus (FCBP 66) and Fusarium solani (FCBP 0291), whereas transformed leaves had improved antifungal resistance. In conclusion, our data suggest that rolABC genes have a significant impact on the synthesis of metabolites involved in enhancing multistress tolerance in A. bracteosa.

Isolation and identification of Agrobacterium rhizogenes inducing hairy roots of Catharanthus roseus in vivo

Agrobacterium rhizogenes is a common tool for transforming foreign genes into plant cell and inducing hairy root formation. A total of 274 colonies of Agrobacterium species was isolated on selective MG-Te medium from 235 samples of rhizophere soils. There were only thirteen colonies belonged to A. rhizogenes being able to efficiently induce the hairy root formation of Catharanthus roseus in vivo based on the morphological, biochemical, and pathogenicity tests. These thirteen colonies were identified as A. rhizogenes based on 16S rRNA sequence and possessing rolABC genes. The results indicated A. rhizogenes was universal in rhizophere soil in Vietnam and riched transgenic tools for researching C. roseus hairy roots in the future.

Transformation of Lettuce with rol ABC Genes: Extracts Show Enhanced Antioxidant, Analgesic, Anti-Inflammatory, Antidepressant, and Anticoagulant Activities in Rats.

Lettuce is an edible crop that is well known for dietary and antioxidant benefits. The present study was conducted to investigate the effects of rol ABC genes on antioxidant and medicinal potential of lettuce by Agrobacterium-mediated transformation. Transgene integration and expression was confirmed through PCR and real-time RT-PCR, respectively. The transformed plants showed 91-102% increase in total phenolic contents and 53-65% increase in total flavonoid contents compared to untransformed plants. Total antioxidant capacity and total reducing power increased up to 112 and 133% in transformed plants, respectively. Results of DPPH assay showed maximum 51% increase, and lipid peroxidation assay exhibited 20% increase in antioxidant activity of transformed plants compared to controls. Different in vivo assays were carried out in rats. The transgenic plants showed up to 80% inhibition in both hot plate analgesic assay and carrageenan-induced hind paw edema test, while untransformed plants showed only 45% inhibition. Antidepressant and anticoagulant potential of transformed plants was also significantly enhanced compared to untransformed plants. Taken together, the present work highlights the use of rol genes to enhance the secondary metabolite production in lettuce and improve its analgesic, anti-inflammatory, antidepressant, and anticoagulatory properties.

Cellular engineering of Artemisia annua and Artemisia dubia with the rol ABC genes for enhanced production of potent anti-malarial drug artemisinin.

BackgroundMalaria is causing more than half of a million deaths and 214 million clinical cases annually. Despite tremendous efforts for the control of malaria, the global morbidity and mortality have not been significantly changed in the last 50 years. Artemisinin, extracted from the medicinal plant Artemisia sp. is an effective anti-malarial drug. In 2015, elucidation of the effectiveness of artemisinin as a potent anti-malarial drug was acknowledged with a Nobel prize. Owing to the tight market and low yield of artemisinin, an economical way to increase its production is to increase its content in Artemisia sp. through different biotechnological approaches including genetic transformation.MethodsArtemisia annua and Artemisia dubia were transformed with rol ABC genes through Agrobacterium tumefacienes and Agrobacterium rhizogenes methods. The artemisinin content was analysed and compared between transformed and untransformed plants with the help of LC–MS/MS. Expression of key genes [Cytochrome P450 (CYP71AV1), aldehyde dehydrogenase 1 (ALDH1), amorpha-4, 11 diene synthase (ADS)] in the biosynthetic pathway of artemisinin and gene for trichome development and sesquiterpenoid biosynthetic (TFAR1) were measured using Quantitative real time PCR (qRT-PCR). Trichome density was analysed using confocal microscope.ResultsArtemisinin content was significantly increased in transformed material of both Artemisia species when compared to un-transformed plants. The artemisinin content within leaves of transformed lines was increased by a factor of nine, indicating that the plant is capable of synthesizing much higher amounts than has been achieved so far through traditional breeding. Expression of all artemisinin biosynthesis genes was significantly increased, although variation between the genes was observed. CYP71AV1 and ALDH1 expression levels were higher than that of ADS. Levels of the TFAR1 expression were also increased in all transgenic lines. Trichome density was also significantly increased in the leaves of transformed plants, but no trichomes were found in control roots or transformed roots. The detection of significantly raised levels of expression of the genes involved in artemisinin biosynthesis in transformed roots correlated with the production of significant amounts of artemisinin in these tissues. This suggests that synthesis is occurring in tissues other than the trichomes, which contradicts previous theories.ConclusionTransformation of Artemisia sp. with rol ABC genes can lead to the increased production of artemisinin, which will help to meet the increasing demand of artemisinin because of its diverse pharmacological and anti-malarial importance.

Bioinformatics and Expression Analysis of CPMAX2 in Citrange

Transgenic citrange lines with rolABC genes behave rosette branching and extreme dwarfing. To explore the regulatory mechanism of plant hormones in axillary shoot growth of transgenic citrange, a full length cDNA of CPMAX2 was cloned from citrange [Citrus sinensis (L.) Osb × Poncirus trifoliate (L.) Raf.] by RT-PCR in this study. The expression of CPMAX2 was detected in axillary tender leaves of 3 transgenic citrange lines and the wild type. Additionally, we constructed an over-expression vector CPMAX2-pCAMBIA1301 for further study. The results showed that the cDNA sequence and its putative peptide sequence shared 99.66% and 99.14% of identity with its Citrus sinensis ortholog MAX2. The deduced amino acid sequence contained putatively one F-box and two LRR repeat domains that are highly conserved in MAX2 genes. CPMAX2 was obviously down-expressed in tender leaves of 3 transgenic citrange lines compared with the wild type. The results suggest CPMAX2 play an important role in regulating the rosette shoot growth in transgenic citrange with rolABC genes.

Transgenic Artemisia dubia WALL showed altered phytochemistry and pharmacology

The rol genes have been shown to enhance the production of secondary metabolites in plants. This report examines the effect of trans-genes (rol ABC) on possible high production of biologically important phytochemicals and enhanced pharmacological activities. Three transgenic lines (1, 2 and 3) of Artemisia dubia WALL (transformed with Agrobacterium tumefaciens harboring rol ABC genes) were subjected to phytochemical analysis and pharmacological studies. A great variation in phytochemistry and the pharmacological activities was observed not only between the transgenic and non-transgenic control plants but also among the transgenic lines itself. Comparative chemical profile obtained via HPLC, TLC and spectrophotometry showed high degree of variations in the quantity of phytochemicals. An increased production of total flavonoids (71.1% in transgenic line 2) and total phenolics (110.8% in transgenic line 1), increase in caffeic acid and catechin and a decrease in gallic acid content in the extracts of transformed plants compared to the untransformed control plants was decreased. In case of pharmacological activities, moderate to high level increase in antimicrobial (antibacterial and antifungal) activities, cytotoxicity (14.1%), antitumor (29%) and antioxidant activities (23.9%) was observed (in transgenic line 2). In general all the three transgenic lines under study showed improvement in their pharmacological activities in the order of transgenic line 2>1>3>control. The implication of these findings will help to meet the increasing demand of pharmacologically important compounds.

Stacking resistance to crown gall and nematodes in walnut rootstocks

BackgroundCrown gall (CG) (Agrobacterium tumefaciens) and the root lesion nematodes (RLNs) (Pratylenchus vulnus) are major challenges faced by the California walnut industry, reducing productivity and increasing the cost of establishing and maintaining orchards. Current nematode control strategies include nematicides, crop rotation, and tolerant cultivars, but these methods have limits. Developing genetic resistance through novel approaches like RNA interference (RNAi) can address these problems. RNAi-mediated silencing of CG disease in walnut (Juglans regia L.) has been achieved previously. We sought to place both CG and nematode resistance into a single walnut rootstock genotype using co-transformation to stack the resistance genes. A. tumefaciens, carrying self-complimentary iaaM and ipt transgenes, and Agrobacterium rhizogenes, carrying a self-complimentary Pv010 gene from P. vulnus, were used as co-transformation vectors. RolABC genes were introduced by the resident T-DNA in the A. rhizogenes Ri-plasmid used as a vector for plant transformation. Pv010 and Pv194 (transgenic control) genes were also transferred separately using A. tumefaciens. To test for resistance, transformed walnut roots were challenged with P. vulnus and microshoots were challenged with a virulent strain of A. tumefaciens.ResultsCombining the two bacterial strains at a 1:1 rather than 1:3 ratio increased the co-transformation efficiency. Although complete immunity to nematode infection was not observed, transgenic lines yielded up to 79% fewer nematodes per root following in vitro co-culture than untransformed controls. Transgenic line 33-3-1 exhibited complete crown gall control and 32% fewer nematodes. The transgenic plants had thicker, longer roots than untransformed controls possibly due to insertion of rolABC genes. When the Pv010 gene was present in roots with or without rolABC genes there was partial or complete control of RLNs. Transformation using only one vector showed 100% control in some lines.ConclusionsCG and nematode resistance gene stacking controlled CG and RLNs simultaneously in walnuts. Silencing genes encoding iaaM, ipt, and Pv010 decrease CG formation and RLNs populations in walnut. Beneficial plant genotype and phenotype changes are caused by co-transformation using A. tumefaciens and A. rhizogenes strains. Viable resistance against root lesion nematodes in walnut plants may be accomplished in the future using this gene stacking technology.

Establishment of fluorescence quantitative RT-PCR assay on <I>rolABC</I> genes in transgenic Troyer citrange

B,D,E transgenic clones with rolABC genes and wildtype plants were used as materials to construct binary standard curves of rol genes and β-actin gene with SYBR Green I fluorescent dye.After normalizing the influence of amplification efficiency with different primers in qRT-PCR,comparative Delta-delta Ct method was applied to develop suitable qRT-PCR method for analysising the expression of rolA,rolB,rolC genes in 5 tested tissues of B,D,E clones.The primary result showed that rolC expressed highest among 3 rol genes in tender stem,tender leaf,functional leaf and bark,rolA gene's expression in the middle level and mainly in tender stem,and rolB expressed lowest and mainly in root.

Is Laparoscopic donor nephrectomy really the way ahead?

Transgenic citrange lines with rolABC genes behave rosette branching and extreme dwarfing. To explore the regulatory mechanism of plant hormones in axillary shoot growth of transgenic citrange, a full length cDNA of CPMAX2 was cloned from citrange [Citrus sinensis (L.) Osb × Poncirus trifoliate (L.) Raf.] by RT-PCR in this study. The expression of CPMAX2 was detected in axillary tender leaves of 3 transgenic citrange lines and the wild type. Additionally, we constructed an over-expression vector CPMAX2-pCAMBIA1301 for further study. The results showed that the cDNA sequence and its putative peptide sequence shared 99.66% and 99.14% of identity with its Citrus sinensis ortholog MAX2. The deduced amino acid sequence contained putatively one F-box and two LRR repeat domains that are highly conserved in MAX2 genes. CPMAX2 was obviously down-expressed in tender leaves of 3 transgenic citrange lines compared with the wild type. The results suggest CPMAX2 play an important role in regulating the rosette shoot growth in transgenic citrange with rolABC genes.

Characterization of hairy root-phenotype in transgenic Hypericum perforatum L. clones

Hairy root-regenerated clones of Hypericum perforatum L. grown in vitro similarly to those successfully adapted to ex vitro conditions showed phenotype features typical for plants transformed with Agrobacterium rhizogenes T-DNA. These included reduced apical dominance, increased branching, dwarfing and reduced fertility. Transgenic clones differed in ability to develop root system as a necessary condition for transfer to the soil. One of the profiling characters, capability of hypericin biosynthesis was altered as well. Dark glands as the sites of hypericin accumulation and/or synthesis exhibited significantly higher densities on both, leaves and petals of transgenic clones comparing to controls. In the genome of transgenic clones, rolABC genes were detected. Both clones harboured similar copy number of individual rol genes. However, copy numbers descended from rolA to rolC gene in both clones.

ASSOCIATION OF FUSARIUM SOLANI WITH ROLABC AND WILD TYPE TROYER CITRANGE

SUMMARY To determine if rolABC genes of Agrobacterium rhizogenes could modify the susceptibility of Troyer citrange to dry root rot, conidial suspensions of a Fusarium solani strain isolated from citrus roots (PVCT1A) were used in cutting, leaf midrib and root inoculation assays of wild type (wt) and rolABC Troyer citrange. In all assays, the pathogen was reisolated equally well from wt and transformant. Interveinal chlorosis, wilt and defoliation of inoculated cuttings and leaf midribs, observed both in rolABC and wt, were more severe in transgenic lines. Two months after root inoculation with the pathogen, root weight was significantly reduced in transgenic lines. No root rot symptoms were observed on rolABC or wt lines, but wilting was more severe on wt. Cell-free fungal culture filtrate induced leaf wilt and defoliation within 10 days from immersion in the filtrate, without any significant difference between rolABC and wt lines. Scanning electron microscopy observations of infected roots showed that fungal penetration and colonization was similar in both lines. Evidence is presented that F. solani colonizes Troyer citrange tissues even in absence of visible symptoms and without differences between wt and rolABC lines.

Terpenoid indole alkaloid production by Catharanthus roseus hairy roots induced by Agrobacterium tumefaciens harboring rol ABC genes

We have established Catharanthus roseus hairy root cultures transgenic for the rol ABC genes from T(L)-DNA of the agropine-type Agrobacterium rhizogenes strain A4. The rol ABC hairy root lines exhibit a wild-type hairy root syndrome in terms of growth and morphology on solid medium. However, they differ from wild-type hairy root lines in that they more frequently have excellent adaptability to liquid medium and do not appear to form calli during cultivation. Moreover, they do not produce detectable levels of mannopine and agropine which, in contrast, are often synthesized abundantly in wild-type hairy root lines. The absence of these opines does not appear to cause the rol ABC lines to have higher levels of terpenoid indole alkaloids than wild-type hairy root lines. Unlike wild-type lines, rol ABC lines produce very similar levels of total alkaloids despite wide variations in individual alkaloid contents. This work demonstrates that the three genes rol ABC are sufficient to induce high-quality hairy roots in Catharanthus roseus.

(286) Hairy Root Culture as a Source of Unique Secondary Metabolites from Echinacea Species

Echinacea is one of the best-selling medicinal plants in the United States. It was historically harvested from wild populations, but its demand has increased so significantly that commercial production has become a necessity to supply the increasing demand and to protect wild populations. The medicinal properties of echinacea are associated with secondary metabolites that are produced mainly in the roots. Hairy roots, induced by the Ri plasmid of Agrobacterium rhizogenes, have been produced in other crops as alternative sources of secondary metabolites that commonly are produced and synthesized in the roots of mature plants. This method of production offers some advantages over traditional agricultural systems, such as the possibility of producing novel compounds year-round. The overall goal of this project is to explore the utility of hairy root cultures (mediated by A. rhizogenes) as an efficient, alternative, and enriched source of secondary compounds with medicinal properties, such as alkamides, flavonoids, and caffeic acid derivatives. We have been successful in transforming roots from E. angustifolia, E. pallida, and E. purpurea plants, and confirming the presence of rol ABC genes in hairy roots using molecular techniques. Roots from control plants show no active growth under dark conditions, whereas transformed roots from E. pallida and E. purpurea show a low degree of branching with a slow growth rate on solid media under darkness. However, transformed E. angustifolia show a faster growth rate and higher degree of branching under the same conditions. Currently, we are working on the optimization of the growing conditions of the transformed roots and will proceed to the biochemical analysis phase of the project.

MORPHOLOGICAL AND PHYSIOLOGICAL EFFECTS OF ROLABC GENES INTO CITRUS GENOME

ISHS XXVI International Horticultural Congress: Citrus and Other Subtropical and Tropical Fruit Crops: Issues, Advances and Opportunities MORPHOLOGICAL AND PHYSIOLOGICAL EFFECTS OF ROLABC GENES INTO CITRUS GENOME

Rooting and Other Characteristics of a Transgenic Walnut Hybrid (Juglans hindsii × J. regia) Rootstock Expressing rolABC

Walnuts (Juglans spp.) are difficult-to-root woody plants. The rolABC genes (rolA + rolB + rolC), derived from the bacteria Agrobacterium rhizogenes, have been shown to increase the rooting potential of other difficult-to-root woody plants. We inserted the rolABC genes into somatic embryos of a `Paradox' hybrid (J. hindsii × J. regia) clone PX1 using the A. tumefaciens gene transfer system. A transgenic sub-clone, designated PX1 rolABC 2-2 was selected and compared to the untransformed clone for a variety of phenotypic characteristics, including rooting potential. Transformed and untransformed shoots were budded onto seedling J. regia rootstock in the greenhouse and established in the field. Transformed trees displayed reduced internode length, an increase in lateral branching, and wrinkled leaves. In another test, a commercial persian walnut cultivar J. regia `Chandler' was grafted onto rooted cuttings of both the untransformed and transformed plants. The presence of the rolABC genes in the rootstock had no visible effects on the grafted scion. Several of these trees were excavated from the field and the root systems of each genotype were examined for root number, diameter, and biomass. Trees with the rolABC rootstock had significantly more small diameter roots compared to the controls and less recovered biomass. Tests of the rooting potential of leafy semi-hardwood cuttings for two years resulted in 14% to 59% rooting of the transformed cuttings compared to 51% to 81% rooting of the control. Both transformed hardwood cuttings and microshoots in tissue culture also rooted significantly less (52% and 29% respectively) than untransformed hardwood cuttings and tissue cultured shoots (82% and 54% respectively). Thus, although the rolABC genes induced a shorter internode length and a more fibrous root system (typical of rol-tranformed plants), they were not useful for increasing the rooting potential, and as rootstock they did not affect the phenotype of the scion.

Increased Susceptibility to Pseudomonas syringae pv. syringae and Pseudomonas viridiflava of Kiwi Plants having Transgenic rolABC genes and its Inheritance in the T1 Offspring

The staminate kiwi (Actinidia deliciosa A. Chev) cv. GTH (Late Yellow Hayward) showed high resistance to the pathogens Pseudomonas syringae pv. syringae and Pseudomonas viridiflava, contrary to the pistillate cv. Hayward which was highly susceptible to both bacteria. The resistant staminate GTH acquired susceptibility when transformed with rolABC of Agrobacterium rhizogenes. This susceptibility was inherited by the T1 offspring containing rolABC genes, derived by crossing pollen of transgenic rolABC GTH with the pistillate cv. Hayward. The susceptibility was probably correlated to high nitrogen content in the leaves of the rolABC plants. The higher susceptibility to P. s. pv. syringae than to P. viridiflava could be correlated with the high degree of auxin production by first bacterium. The high resistance to both bacteria of the staminate GTH was inherited by all the offspring lacking rolABC genes. The possibility of reducing susceptibility of rolABC plants to the pathogens by applying fewer fertilizers in the orchards, which would reduce both costs and environmental impact, is discussed.

Effects of the rol C gene on hairy root: induction development and tropane alkaloid production by Atropa belladonna.

Two series of Atropa belladonna hairy root lines were obtained, the first one transformed via Agrobacterium tumefaciens harboring rol C and npt II genes, and the other transformed with rol ABC and npt II genes. Thirteen hairy root lines were obtained and selected on hormone-free medium. The transformation was confirmed by PCR analysis, and these root lines were first examinated for their growth rate. Then hyoscyamine and scopolamine production was measured after 3 and 4 weeks of culture to evaluate the possible role of rol C gene in tropane alkaloid formation. The rol C gene alone played a significant role in the hairy root growth rate (17-fold increase). However this effect was much lower than that induced by the rol ABC genes together (75-fold increase). In contrast, the rol C gene alone was as efficient as the rol ABC genes together (mean value of total alkaloids: 0.36% dry weight, i.e., 12-fold times more than in untransformed roots) to stimulate the biosynthesis of tropane alkaloids in A. belladonna hairy root cultures.

Tropane alkaloid production by hairy roots of Atropa belladonna obtained after transformation with Agrobacteriumrhizogenes 15834 and Agrobacterium tumefaciens containing rolA, B, C genes only

Atropa belladonna leaf disks were infected by a wild strain Agrobacterium rhizogenes 15834 harboring the Ri-TL-DNA and by a disarmed Agrobacterium tumefaciens strain harboring a construction with only rolABC and npt II genes. Thirteen root lines were established and examined for their growth rate and alkaloid productivity to evaluate the possible role of rol genes in morphological differentiation and in tropane alkaloid formation. A great diversity has been observed in the growth rate of these 13 root lines. The root biomass increased up to 75 times. The total alkaloid contents were similar in the root lines obtained by infection with A. rhizogenes 15834 and A. tumefaciensrolABC. The last ones accumulated between 4 (1.1 mg g−1 DW) and 27 (8 mg g−1 DW) times more alkaloids than the intact roots (0.3 mg g−1 DW). This work has shown that the rolABC genes were sufficient to increase tropane alkaloid production in A. belladonna hairy root cultures.

Characterisation of morphology and root formation in the model woody perennial shrub Solanum aviculare Forst. expressing rolABC genes of Agrobacterium rhizogenes

Transgenic plants of Solanum aviculare Forst. expressing the rolABC genes of Agrobacterium rhizogenes exhibited altered shoot morphologies in comparison to untransformed plants. Altered growth and development characteristics included retarded growth, suppressed apical dominance, shortened internodes, smaller highly lanceolated leaves and smaller, sterile flowers. Pronounced leaf wrinkling was a common feature of the transgenic plants. Shoot slices and leaf fragments of transgenic plants when cultured in vitro exhibited significantly higher root formation capacities than explants derived from non-transformed plants. Explants from transgenic plants were able to produce roots in limited manner without exogenous auxin and presented higher sensitivity to promoting effect of IBA. The suppressing effects of light, kinetin, the inhibitor of putrescine synthesis α-difluoromethyl-ornithine and gibberellin (GA 3) on root formation were significantly lower than in control explants. Vanadate (an inhibitor of phosphatases/kinases) strongly blocked root differentiation in control and in transformant lines. The antigibberellin paclobutrazol increased the numbers of roots in transformant shoot segments and in segments of control plants, whereas the antiauxin p-chlorophenoxyisobutyric acid, the polyamines putrescine and spermidine, the conjugated auxin indoxyl-β-glucoside and the conjugated cytokinin BAP-9-glucoside had no marked effects on root development. Differences in root formation abilities between transformed lines in response to the different treatments were found.

Nucleotide sequence of the rol region of the mikimopine-type root-inducing plasmid pRi1724.

The rol genes of root-inducing plasmids (pRi) are essential for tumorigenesis on various dicotyledonous plants. The nucleotide sequences of the rol genes together with their flanking regions from the mikimopine-type pRi1724 were analyzed and compared with the corresponding portions of the agropine-type pRiA4 and the mannopine-type pRi8196 that were previously reported [Slightom et al., J. Biol. Chem., 261, 108-121 (1986); Hansen et al., Proc. Natl. Acad. Sci. U.S.A., 88, 7763-7767 (1991)]. The sequenced region of 8967 bp contained seven ORFs, five on the upper strand and two on the lower one. These ORFs resembled the ORFs 8, 10 (rolA), 11 (rolB), 12 (rolC), 13, 13a, and 14 of pRiA4 and pRi8196. However, the similarity of the rolABC genes, particularly rolA, among the three plasmids, was considerably lower than that of other ORFs. The spacer regions between the ORFs were also conserved, but to a smaller extent than ORFs, among the three plasmids.