Agrobacterium Rhizogenes-mediated Transformation Research Articles

A protocol for sonication-assisted Agrobacterium rhizogenes-mediated transformation of haploid and diploid sugar beet (Beta vulgaris L.) explants.

Hairy root cultures obtained after Agrobacterium rhizogenes-mediated genetic transformation can serve as a model system for studying plant metabolism and physiology, or can be utilized for the production of secondary metabolites. So far no efficient protocol of hairy root development in sugar beet has been publically released. In this work, two A. rhizogenes strains (A4T and LBA1334) carrying a binary vector pBIN-m-gfp5-ER or pCAMBIA1301 possessing gfp and uidA reporter genes were used to transform petiole explants of haploid and diploid sugar beet genotypes. Five treatment combinations of sonicated-assisted Agrobacterium-mediated transformation were compared. Hairy roots appeared on 0% to 54% of explants depending on the treatment combination used. The highest frequency was achieved when explants of a diploid genotype were sonicated for 15 s in the inoculum containing A. rhizogenes of OD600=0.5 and then co-cultured for three days. Using the same treatment combinations the explants of haploid genotypes developed hairy roots with the frequency ranging from 10% to 36%. Transformation efficiency was independent on the bacterial strain used. The results indicate that haploid sugar beet explants are amenable to transformation using A. rhizogenes, and that the efficiency of that process can be increased by applying short ultrasound treatment.

Development of efficient miniprep transformation methods for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium rhizogenes

Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes.

Identification of a STOP1-like protein in Eucalyptus that regulates transcription of Al tolerance genes

Tolerance to soil acidity is an important trait for eucalyptus clones that are introduced to commercial forestry plantations in pacific Asian countries, where acidic soil is dominant in many locations. A conserved transcription factor regulating aluminum (Al) and proton (H+) tolerance in land-plant species, STOP1 (SENSITIVE TOPROTON RHIZOTOXICITY 1)-like protein, was isolated by polymerase chain reaction-based cloning, and then suppressed by RNA interference in hairy roots produced by Agrobacterium rhizogenes-mediated transformation. Eucalyptus STOP1-like protein complemented proton tolerance in an Arabidopsis thaliana stop1-mutant, and localized to the nucleus in a transient assay of a green fluorescent protein fusion protein expressed in tobacco leaves by Agrobacterium tumefaciens-mediated transformation. Genes encoding a citrate transporting MULTIDRUGS AND TOXIC COMPOUND EXTRUSION protein and an orthologue of ALUMINUM SENSITIVE 3 were suppressed in transgenic hairy roots in which the STOP1 orthologue was knocked down. In summary, we identified a series of genes for Al-tolerance in eucalyptus, including a gene for STOP1-like protein and the Al-tolerance genes it regulates. These genes may be useful for molecular breeding and genomic selection of elite clones to introduce into acid soil regions.

Enhanced triterpene accumulation in Panax ginseng hairy roots overexpressing mevalonate-5-pyrophosphate decarboxylase and farnesyl pyrophosphate synthase.

To elucidate the function of mevalonate-5-pyrophosphate decarboxylase (MVD) and farnesyl pyrophosphate synthase (FPS) in triterpene biosynthesis, the genes governing the expression of these enzymes were transformed into Panax ginseng hairy roots. All the transgenic lines showed higher expression levels of PgMVD and PgFPS than that by the wild-type control. Among the hairy root lines transformed with PgMVD, M18 showed the highest level of transcription compared to the control (14.5-fold higher). Transcriptions of F11 and F20 transformed with PgFPS showed 11.1-fold higher level compared with control. In triterpene analysis, M25 of PgMVD produced 4.4-fold higher stigmasterol content (138.95 μg/100 mg, dry weight [DW]) than that by the control; F17 of PgFPS showed the highest total ginsenoside (36.42 mg/g DW) content, which was 2.4-fold higher compared with control. Our results indicate that metabolic engineering in P. ginseng was successfully achieved through Agrobacterium rhizogenes-mediated transformation and that the accumulation of phytosterols and ginsenosides was enhanced by introducing the PgMVD and PgFPS genes into the hairy roots of the plant. Our results suggest that PgMVD and PgFPS play an important role in the triterpene biosynthesis of P. ginseng.

Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi.

An efficient hairy root induction system for an important endangered medicinal plant, Dracocephalum kotschyi, was developed through Agrobacterium rhizogenes-mediated transformation by modifying the co-cultivation medium using five bacterial strains, A4, ATCC15834, LBA9402, MSU440, and A13 (MAFF-02-10266). A drastic increase in transformation frequency was observed when a Murashige and Skoog medium lacking NH4NO3 KH2PO4, KNO3 and CaCl2 was used, resulting in hairy root induction frequencies of 52.3%, 69.6%, 48.6%, 89.0%, and 80.0% by A4, A13, LBA9402, MSU440, and ATCC15834 strains, respectively. For shoot induction, hairy roots and unorganized tumors induced by strain ATCC15834 were placed on an MS media supplemented with 0.1, 0.25, 0.5, and 1mg/l BA plus 0.1mg/l NAA. The high frequency of shoot regeneration and number of shoot were obtained in the medium containing 0.25mg/l BA and 0.1mg/l NAA. Root induction occurred from the base of regenerated shoots on the MS medium supplemented with 0.5mg/l IBA after 10 days.

Agrobacterium rhizogenes-mediated transformation of a multi-medicinal herb, Boerhaavia diffusa L.: optimization of the process and anti-microbial activity against bacterial pathogens causing urinary tract infections

Hairy root cultures (HRCs) of Boerhaavia diffusa L., an ethno-medicinally important herb, were established via explant co-cultivation with Agrobacterium rhizogenes. Several key factors influencing transformation efficiency were optimized. Rhizoclones showing prolific growth were maintained through successive subcultures in solidified Murashige & Skoog's medium without growth regulators (MS0) under diffused illumination. PCR analysis of the rhizoclones demonstrated positive amplification of rolB and rolC genes of pRi TL-DNA. Reverse transcription-PCR (RT-PCR) and opine assay revealed the presence and expression of the pRi TR-DNA borne ags gene. The methanol and dichloromethane (M:DM) extracts of hairy roots (HRs) and natural roots (NRs) were screened against a wide range of bacterial pathogens causing human urinary tract infections (UTIs). The MIC and MBC values of HRs were markedly lower when compared to NRs against microorganisms causing UTIs. Extracts of HRs were more effective than those of NRs not only against wild type strains but also, and more importantly, against multi-drug resistant strains. Establishment of fast-proliferating renewable HRCs of B. diffusa would provide a direct source of anti-microbial phytocompounds against several pathogenic bacteria, including resistant strains, causing UTIs.

A novel method based on combination of semi-in vitro and in vivo conditions in Agrobacterium rhizogenes-mediated hairy root transformation of Glycine species

Despite numerous advantages of the many tissue culture-independent hairy root transformation protocols, the process is often compromised in the initial in vitro culture stage where inability to maintain high humidity and the delivery of nourishing culture medium decrease cellular morphogenesis and organ formation efficiency. Ultimately, this influences the effective transfer of produced plantlets during transfer from in vitro to in vivo conditions, where low survival rates occur during the acclimation period. We have developed an intermediate protocol for Agrobacterium rhizogenes transformation in Glycine species by combining a two-step in vitro and in vivo process that greatly enhances the efficiency of hairy root formation and which simplifies the maintenance of the transformed roots. In this protocol, cotyledonary nodes of Glycine max and Glycine canescens seedlings were infected by A. rhizogenes K599 carrying a reporter gene construct constitutively expressing green fluorescent protein (GFP). Glass containers containing sand and nutrient solution were employed to provide a moist clean microenvironment for the generation of hairy roots from inoculated seedlings. Transgenic roots were then noninvasively identified from nontransgenic roots based on the detection of GFP. Main roots and nontransgenic roots were removed leaving transgenic hairy roots to support seedling development, all within 1 mo of beginning the experiment. Overall, this protocol increased the transformation efficiency by more than twofold over traditional methods. Approximately 88% and 100% of infected plants developed hairy roots from G. max and G. canescens, respectively. On average, each infected plant produced 10.9 transformed hairy roots in G. max and 13–20 in G. canescens. Introduction of this simple protocol is a significant advance that eliminates the long and genotype-dependent tissue culture procedure while taking advantage of its optimum in vitro qualities to enhance the micropropagation rate. This research will support the increasing use of transient transgenic hairy roots for the study of plant root biology and symbiotic interactions with Rhizobium spp.

Overexpression of a Catharanthus tryptophan decarboxylase (tdc) gene leads to enhanced terpenoid indole alkaloid (TIA) production in transgenic hairy root lines of Rauwolfia serpentina

To enhance the production of terpenoid indole alkaloids in Rauwolfia serpentina, Catharanthus tryptophan decarboxylase (Crtdc) gene was over-expressed in transgenic hairy root cultures using Agrobacterium rhizogenes-mediated transformation. Among six transgenic hairy root lines, line RT4 accumulated the highest alkaloid content, with 0.1202 % dry weight (DW) reserpine and 0.0064 % DW ajmalicine, after 10 weeks of culture. Whereas, wild-type roots accumulated 0.0596 ± 0.003 % DW reserpine and 0.0011 ± 0.001 % DW ajmalicine. Transgenic hairy root line RT7 produced the lowest alkaloid content (reserpine: 0.0896 ± 0.002 % DW; ajmalicine: 0.002 ± 0.0 % DW). On the basis of alkaloid content the six hairy root lines were grouped as RT4/RT2 > RT3/RT5 > RT7/RT8. Analysis of gene expression profile indicated that Crtdc was expressed at a higher level in transgenic lines, which could be correlated with enhanced metabolite accumulation in roots. This study confirms that over-expression of Crtdc is a superlative method to improve the biosynthetic potential of Rauwolfia hairy root cultures. Enhanced reserpine and ajmalicine production can serve as an alternative choice to provide resources for relative pharmaceutical industries.

A chimeric cry8Ea1 gene flanked by MARs efficiently controls Holotrichia parallela

Peanuts transformed with the synthetic cry8Ea1 gene flanked by MARs are a potentially effective control strategy against white grubs. Cry8Ea1 protein levels of the construct containing MARs were increased by 2.5 times. White grubs are now recognized as the most important pests of peanut worldwide. A synthetic cry8Ea1 gene, which was toxic to Holotrichia parallela larvae, was expressed in chimeric peanut roots using an Agrobacterium rhizogenes-mediated transformation system. The relative mRNA and protein levels of the cry8Ea1 gene were confirmed by quantitative real-time PCR and ELISA, respectively. The effects of matrix attachment regions (MARs) on the expression and activity of the cry8Ea1 gene were analyzed. The average expression level of cry8Ea1 in peanut roots was higher for the plants harboring constructs flanked by MARs from tobacco. Moreover, differing from previous studies, the synthetic cry8Ea1 gene flanked by MARs showed more variation in protein levels than mRNA levels. These composite plants containing cry8Ea1 gene flanked by MARs exhibited a high toxicity against Holotrichia parallela larvae as shown by bioassay analysis, thus offering a potential effective combination to control subterranean insects in peanuts.

Agrobacterium rhizogenes transformed soybean roots differ in their nodulation and nitrogen fixation response to genistein and salt stress

We evaluated response differences of normal and transformed (so-called 'hairy') roots of soybean (Glycine max L. (Merr.), cv L17) to the Nod-factor inducing isoflavone genistein and salinity by quantifying growth, nodulation, nitrogen fixation and biochemical changes. Composite soybean plants were generated using Agrobacterium rhizogenes-mediated transformation of non-nodulating mutant nod139 (GmNFR5α minus) with complementing A. rhizogenes K599 carrying the wild-type GmNFR5α gene under control of the constitutive CaMV 35S promoter. We used genetic complementation for nodulation ability as only nodulated roots were scored. After hairy root emergence, primary roots were removed and composite plants were inoculated with Bradyrhizobium japonicum (strain CB1809) pre-induced with 10 μM genistein and watered with NaCl (0, 25, 50 and 100 mM). There were significant differences between hairy roots and natural roots in their responses to salt stress and genistein application. In addition, there were noticeable nodulation and nitrogen fixation differences. Composite plants had better growth, more root volume and chlorophyll as well as more nodules and higher nitrogenase activity (acetylene reduction) compared with natural roots. Decreased lipid peroxidation, proline accumulation and catalase/peroxidase activities were found in 'hairy' roots under salinity stress. Genistein significantly increased nodulation and nitrogen fixation and improved roots and shoot growth. Although genistein alleviated lipid peroxidation under salinity stress, it had no significant effect on the activity of antioxidant enzymes. In general, composite plants were more competitive in growth, nodulation and nitrogen fixation than normal non-transgenic even under salinity stress conditions.

Hairy Root Cultures and Plant Regeneration in <i>Solidago nemoralis</i> Transformed with <i>Agrobacterium rhizogenes</i>

By screening a native plant extract library we identified Solidago nemoralis as a novel source of agonists for alpha7 nicotinic receptors for acetylcholine with therapeutic potential. The next phase of our drug discovery strategy is to increase the yields of active compounds in the plant species by gain of function mutations in hairy root cultures [1]. Here we report a protocol for Agrobacterium rhizogenes-mediated genetic transformation of hairy root cultures of Solidago nemoralis which will enable this. Leaf explants of this species were successfully transformed with a frequency of 30%-35% using A. rhizogenes strain R1000 harboring the binary vector pCambia 1301. Transformation was confirmed using the β-glucuronidase (GUS) histochemical assay. Transformed hairy roots showed spontaneous regeneration of adventitious shoots in media without the addition of cytokines, albeit at very low frequency. However, media supplementation with auxin (α-naphthaleneacetic acid, NAA) increased shoot regeneration frequency to 35% and resulted in viable adventitious shoots. Transformation was confirmed at all phases of plant regeneration by GUS staining. Hairy root transformation of Solidago altissima has been previously reported, but this is the first report of genetic transformation of S. nemoralis. The protocol will allow for a large population of activation tagged mutants of S. nemoralis to be generated which will be then screened for the presence of stable mutants which are over-producing metabolites with activity at alpha7 nicotinic receptors. These over-producing mutant cultures will then be regenerated into intact mutant plants.

IN VITRO MASS PRODUCTION OF STEVIA REBAUDIANA BERTONI (AN ANTIDIABETIC ENDANGERED HERB) AND ASSESSMENT OF PHYTOTOXICITY OF DIFFERENT HEAVY METALS ON STEVIOSIDE SYNTHESIS

Diabetes is a major global epidemic currently affecting 50 million people across the world and is expected to rise to 300 million by 2025. However, the present pharmacological approach for the management of this serious disease is far from ideal, which behooves the exploration of newer and safer agents. Currently, there is an enormous increase in the use of herbal and other alternative medicines for the treatment of diabetes. Stevia rebaudiana, a member of Asteraceae has emerged as a natural sweetener against synthetic sugars and an alternative herbal cure for diabetes. This property of Stevia is attributed mainly due to the presence of major steviol glycosides e.g., steviosides, rebaudioside A, rebaudioside C and dulcoside A, besides, several other bioactive compounds. These compounds are known to stimulate the pancreatic cells to produce insulin in diabetic patients. It has, therefore, become imperative to undertake different biotechnological approaches such as its ex situ conservation and Agrobacterium rhizogenes-mediated genetic transformation for improved production of stevioside and related bioactive compounds. In light of the above, an in vitro technology has been developed for mass production of Stevia rebaudiana and optimization of stevioside production using various elicitors e.g., heavy metals, osmolytes, etc. Multiple shoots have been induced in the seedling derived node and leaf explants on MS medium augmented with different cytokinins. Of the various combinations tried, MS+1 µM BA proved optimum for differentiating an average of 12.05+/-0.31 and 10.40+/-1.02 shoots from nodal and leaf explants respectively, in 100% cultures after 30 days. Half-strength MS basal proved helpful in inducing rhizogenesis in 100% cultures. The plants were gradually hardened and subsequently transferred to garden soil where 97% plants survived and induced floral buds after 60 days. The impact of heavy metal stress on morphogenesis of nodal explants has also been studied.

RNAi suppression and overexpression studies of hydroxyphenylpyruvate reductase (HPPR) and rosmarinic acid synthase (RAS) genes related to rosmarinic acid biosynthesis in hairy root cultures of Coleus blumei

Hydroxyphenylpyruvate reductase (HPPR) and rosmarinic acid synthase (RAS) have been described as enzymes involved in rosmarinic acid (RA) biosynthesis. In this study, elucidating the role of HPPR and RAS in the formation of RA was pursued. For this purpose, hairy root lines of Coleus blumei carrying HPPR or RAS RNAi suppression and overexpression constructs were established using Agrobacterium rhizogenes-mediated transformation. Transgenic lines were evaluated for RA accumulation. Hairy root lines showing undetectable HPPR or RAS mRNA levels displayed strongly reduced RA contents with reduction of up to 92 % compared to control hairy root lines. An increase of RA levels up to 176 % was shown in overexpression hairy root lines with equally increased HPPR transcript levels while RAS overexpression resulted in co-suppression effects with reduced RA levels. These observations support the participation of HPPR and RAS in RA biosynthesis.

Antioxidant potential of Agrobacterium-transformed and non-transformed Physalis ixocarpa plants grown in vitro and ex vitro

Oxidative stress is involved in pathogenesis of a number of chronic diseases hence is an increasing interest in plant-derived natural antioxidants with respect to their potential health benefits. Plants from the genus Physalis are particularly rich in secondary metabolites and show significant antioxidant potential. Recent development in transgenic research has opened new possibilities for enhanced production of secondary metabolites with plant cell and organ cultures. The hairy root-regenerated Physalis ixocarpa plants grown in vitro and ex vitro were compared to the non-transformed plants with respect to their antioxidant potential. The total antioxidant capacity (TAC), the contents of total phenols and ascorbate were evaluated in fruits, flowers, leaves and roots of P. ixocarpa using the ferric reducing antioxidant power assay (FRAP), the Folin-Ciocalteu method and the 2,2'-dipyridyl method, respectively. The antioxidant profiles, in terms of TAC, ascorbate and phenols were organ-specific and depended on the culture conditions. Neither the total phenol content nor the ascorbate level appeared to determine the TAC of the studied plant extracts. The aqueous extracts exhibited lower antioxidant activities than the acetone ones indicating that lipophilic antioxidants made a major contribution to TAC of the plant tissues. Agrobacterium rhizogenes-mediated transformation changed the antioxidant status with respect to TAC, phenols and ascorbate and this effect was observed in the plants grown in vitro and ex vitro.

Isolation and Characterization of Medicago truncatula U6 Promoters for the Construction of Small Hairpin RNA-Mediated Gene Silencing Vectors

RNA silencing using vector-based double-stranded RNA is an attractive strategy to suppress gene expression in plants. Especially, a short hairpin RNA (shRNA)-mediated approach is potent for specific target gene silencing. Here, shRNA expression vectors were constructed based on U6 small nuclear RNA (snRNA) gene promoters in Medicago truncatula. Ten U6 snRNA gene loci identified showed highly homologous sequences with those of other eukaryotes. Their RNA polymerase III-specific promoters had a conserved upstream sequence element and TATA box that were located approximately three helical DNA turns apart. The U6 promoters were also insensitive to α-amanitin, actively guiding transcription of fused GUS reporter gene fragments. When the U6 fusion constructs were introduced into M. truncatula via Agrobacterium rhizogenes-mediated transformation, GUS fragment transcripts were detected in nearly every transformed root albeit in varying amounts. Consistently, under the control of U6 promoters, constructs of 21-nucleotide-stem shRNAs directed silencing of GUS expression in transformed roots. Moreover, a phytoene desaturase gene (MtPDS3)-targeting 27-nucleotide-stem U6-shRNA constructs, when introduced into hairy roots, decreased the MtPDS3 transcript levels by approximately 80 % as a result of endogenous gene silencing, as verified by the presence of shPDS-derived siRNAs detected by stem-loop PCR. The U6 promoter-directed shRNA expression plasmids constructed herein can be a useful tool for functional gene analyses in this model legume.

14-3-3 Proteins SGF14c and SGF14l Play Critical Roles during Soybean Nodulation

The soybean (Glycine max) genome contains 18 members of the 14-3-3 protein family, but little is known about their association with specific phenotypes. Here, we report that the Glyma0529080 Soybean G-box Factor 14-3-3c (SGF14c) and Glyma08g12220 (SGF14l) genes, encoding 14-3-3 proteins, appear to play essential roles in soybean nodulation. Quantitative reverse transcription-polymerase chain reaction and western-immunoblot analyses showed that SGF14c mRNA and protein levels were specifically increased in abundance in nodulated soybean roots 10, 12, 16, and 20 d after inoculation with Bradyrhizobium japonicum. To investigate the role of SGF14c during soybean nodulation, RNA interference was employed to silence SGF14c expression in soybean roots using Agrobacterium rhizogenes-mediated root transformation. Due to the paleopolyploid nature of soybean, designing a specific RNA interference sequence that exclusively targeted SGF14c was not possible. Therefore, two highly similar paralogs (SGF14c and SGF14l) that have been shown to function as dimers were silenced. Transcriptomic and proteomic analyses showed that mRNA and protein levels were significantly reduced in the SGF14c/SGF14l-silenced roots, and these roots exhibited reduced numbers of mature nodules. In addition, SGF14c/SGF14l-silenced roots contained large numbers of arrested nodule primordia following B. japonicum inoculation. Transmission electron microscopy further revealed that the host cytoplasm and membranes, except the symbiosome membrane, were severely degraded in the failed nodules. Altogether, transcriptomic, proteomic, and cytological data suggest a critical role of one or both of these 14-3-3 proteins in early development stages of soybean nodules.

High-efficiency Agrobacterium rhizogenes-mediated genetic transformation in Artemisia vulgaris: Hairy root production and essential oil analysis

Agrobacterium rhizogenes-mediated genetic transformation of Artemisia vulgaris was investigated using four bacterial strains: A4GUS, R1000, R1601and ATCC15834 and three explant types: shoot tip, leaf and node. The A4GUS proved to be more competent than other strains and the highest transformation rates were observed in leaf explant (92.6%). Among 92 hairy root clones acquired, clones AV1 and AV2 exhibited the highest growth. Growth of hairy roots was assessed on the basis of total root elongation, lateral root density and biomass accumulation. Out of four media composition, the ½ MS medium was found to be most suitable for biomass production. Biomass accumulation was the highest when the medium was supplemented with 40gL−1 sucrose. PCR and Southern hybridization analysis revealed both the TL- and TR-DNA integration in the root clones AV1 and AV2. Hairy roots showed higher growth rate and accumulated substantial amount of essential oils than non-transformed roots. Eighty-seven compounds were identified from transformed root essential oils, whereas 77 compounds were identified from non-transformed root essential oils. The main compounds identified were camphor, camphene, α-thujone, germacrene D, 1,8-cineole and β-caryophyllene.

Overexpression of phenylalanine ammonia-lyase improves flavones production in transgenic hairy root cultures of Scutellaria baicalensis

Transgenic hairy root lines from Scutellaria baicalensis overexpressing phenylalanine ammonia-lyase (PAL) (SbPAL1, SbPAL2, and SbAPL3) were established using an Agrobacterium rhizogenes-mediated transformation system. Stable genetic transformation with the SbPAL genes was confirmed by real-time PCR. These transgenic hairy root lines produced higher quantities flavones (i.e., baicalin, baicalein, and wogonin) than the control hairy root line. In particular, the wogonin content was increased by 4–11 times in the SbPAL1-, SbPAL2-, and SbPAL3-overexpressing hairy roots than the S. baicalensis wild-type roots. This research showed the importance of PAL in flavone biosynthesis and demonstrated the efficiency of metabolic engineering in S. baicalensis hairy roots.

Composite Cucurbita pepo plants with transgenic roots as a tool to study root development

In most plant species, initiation of lateral root primordia occurs above the elongation zone. However, in cucurbits and some other species, lateral root primordia initiation and development takes place in the apical meristem of the parental root. Composite transgenic plants obtained by Agrobacterium rhizogenes-mediated transformation are known as a suitable model to study root development. The aim of the present study was to establish this transformation technique for squash. The auxin-responsive promoter DR5 was cloned into the binary vectors pKGW-RR-MGW and pMDC162-GFP. Incorporation of 5-ethynyl-2'-deoxyuridine (EdU) was used to evaluate the presence of DNA-synthesizing cells in the hypocotyl of squash seedlings to find out whether they were suitable for infection. Two A. rhizogenes strains, R1000 and MSU440, were used. Roots containing the respective constructs were selected based on DsRED1 or green fluorescent protein (GFP) fluorescence, and DR5::Egfp-gusA or DR5::gusA insertion, respectively, was verified by PCR. Distribution of the response to auxin was visualized by GFP fluorescence or β-glucuronidase (GUS) activity staining and confirmed by immunolocalization of GFP and GUS proteins, respectively. Based on the distribution of EdU-labelled cells, it was determined that 6-day-old squash seedlings were suited for inoculation by A. rhizogenes since their root pericycle and the adjacent layers contain enough proliferating cells. Agrobacterium rhizogenes R1000 proved to be the most virulent strain on squash seedlings. Squash roots containing the respective constructs did not exhibit the hairy root phenotype and were morphologically and structurally similar to wild-type roots. The auxin response pattern in the root apex of squash resembled that in arabidopsis roots. Composite squash plants obtained by A. rhizogenes-mediated transformation are a good tool for the investigation of root apical meristem development and root branching.

Features of lettuce transgenic plants with the ifn-α2b gene regenerated after Agrobacterium rhizogenes-mediated transformation

"Hairy" roots of lettuce Lactuca sativa and regenerated plants with interferon-alpha2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-alpha2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions.