Factors affecting <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Hybanthus</Emphasis><Emphasis Type="Italic">enneaspermus</Emphasis> (L.) F. Muell.

Agrobacterium tumefaciens-mediated transformation system was established for Hybanthus enneaspermus using leaf explants with the strain LBA4404 harbouring pCAMBIA 2301 carrying the nptII and gusA genes. Sensitivity of leaf explants to kanamycin was standardized (100 mg/l) for screening the transgenic plants. Transformation parameters (OD, virulence inducer, infection time, co-cultivation period, bactericidal antibiotics, etc.) influencing the gene transfer and integration were assessed in the present investigation. Fourteen-day pre-cultured explants were subjected with Agrobacterium strain LBA4404. Optimized parameters such as culture density of 0.5 OD₆₀₀, infection time of 6 min, AS concentration of 150 µM with 3 days co-cultivation revealed maximum transformation efficiency based on GUS expression assay. The presence of gusA in transgenics was confirmed by polymerase chain reaction and Southern blotting analysis. The present transformation experiment yielded 20 shoots/explant with higher transformation efficiency (28 %). The protocol could be used to introduce genes for trait improvement as well as for altering metabolic pathway for secondary metabolites production.     

Genotype independent and efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of the medicinal plant <Emphasis Type="Italic">Withania somnifera</Emphasis> Dunal

Withania somnifera one of the most reputed Indian medicinal plant has been extensively used in traditional and modern medicines as active constituents. A high frequency genotype and chemotype independent Agrobacterium-mediated transformation protocol has been developed for W. somnifera by optimizing several factors which influence T-DNA delivery. Leaf and node explants of Withania chemotype was transformed with A. tumefaciens strain GV3101 harboring pIG121Hm plasmid containing the gusA gene encoding β-glucuronidase (GUS) as a reporter gene and the hptII and the nptII gene as selection markers. Various factors affecting transformation efficiency were optimized; as 2 days preconditioning of explants on MS basal supplemented with TDZ 1 μM, Agrobacterium density at OD₆₀₀ 0.4 with inclusion of 100 μM acetosyringone (As) for 20 min co-inoculation duration with 48 h of co-cultivation period at 22 °C using node explants was found optimal to improved the number of GUS foci per responding explant from 36?±?13.2 to 277.6?±?22.0, as determined by histochemical GUS assay. The PCR and Southern blot results showed the genomic integration of transgene in Withania genome. On average basis 11 T₀ transgenic plants were generated from 100 co-cultivated node explants, representing 10.6 % transformation frequency. Our results demonstrate high frequency, efficient and rapid transformation system for further genetic manipulation in Withania for producing engineered transgenic Withania shoots within very short duration of 3 months.     

Morphological and Molecular Analysis of Isolated Cultures of Tobacco Adventitious Roots Obtained by the Methods of Biolistic Bombardment and <Emphasis Type="Italic">Agrobacterium</Emphasis>-Mediated Transformation

Plant infection with Agrobacterium rhizogenes leads to the development of a hairy root disease notable for the rapid agravitropic growth of roots on hormone-free nutrient media. In order to look into the interaction of A. rhizogenes with plants and assess opportunities of practical application of hairy root culture, new approaches to their production are elaborated. A method of bacterium-free and plasmid-free production of genetically modified roots (hairy roots) by means of biolistic transformation of leaf explants with a DNA fragment (size of 5461 bp) consisting of genes rolA, rolB, rolC, and rolD are proposed. In most cases, such transformation resulted in the emergence of only adventitious roots with transient expression of rol-genes, and the growth of such roots on hormone-free media ceased in 2–3 months in contrast to genuine hairy roots capable of unrestricted growth. Molecular analysis of different systems of target genes’ expression showed an important role of transgene rolC and host gene of cyclin-dependent protein kinase CDKB1-1 in the maintenance of rapid growth of hairy roots in vitro (in isolated cultures).     

Hairy root culture optimization and resveratrol production from <Emphasis Type="Italic">Vitis vinifera</Emphasis> subsp. <Emphasis Type="Italic">sylvesteris</Emphasis>

Resveratrol is a polyphenolic compound produced in very low levels in grapes. To achieve high yield of resveratrol in wild grape, three Agrobacterium rhizogenes strains, Ar318, ArA4 and LBA9402, were used to induce hairy roots following infection of internodes, nodes or petioles of in vitro grown Vitis vinifera subsp. sylvesteris accessions W2 and W16, and cultivar Rasha. The effects of inoculation time, age of explants, bacterial concentration and co-cultivation times were examined on the efficiency of the production of hairy roots. Strains Ar318, ArA4 and LBA9402 all induced hairy roots in the tested genotypes, but the efficiency of ArA4 strain was higher than the other strains. The highest hairy root production was with using internodes as explants. The transformation of hairy roots lines was confirmed by PCR detection of rolB gene. Half Murashige and Skoog (MS) medium was better for biomass production compared with MS medium. HPLC analysis of resveratrol production in the hairy root cultures showed that all the genotypes produced higher amounts of resveratrol than control roots. The highest amount of resveratrol was produced from W16 internode cultures, which was 31-fold higher than that of control root. Furthermore, TLC analysis showed that treatments of hairy roots with sodium acetate and jasmonate elevated resveratrol levels both in hairy root tissue and excreted into the half MS medium. These results demonstrate that endogenous and exogenous factors can affect resveratrol production in hairy root culture of grape, and this strategy could be used to increase low resveratrol production in grapes.     

Efficient genetic transformation and regeneration system from hairy root of <Emphasis Type="Italic">Origanum vulgare</Emphasis>

Origanum vulgare L is commonly known as a wild marjoram and winter sweet which has been used in the traditional medicine due to its therapeutic effects as stimulant, anticancer, antioxidant, antibacterial, anti-inflammatory and many other diseases. A reliable gene transfer system via Agrobacterium rhizogenes and plant regeneration via hairy roots was established in O. vulgare for the first time. The frequency of induced hairy roots was different by modification of the co-cultivation medium elements after infection by Agrobacterium rhizogenes strains K599 and ATCC15834. High transformation frequency (91.3 %) was achieved by co-cultivation of explants with A. rhizogenes on modified (MS) medium. The frequency of calli induction with an 81.5 % was achieved from hairy roots on MS medium with 0.25 mg/L^?1 2,4-D. For shoot induction, initiated calli was transferred into a medium containing various concentrations of BA (0.1, 0.25, 0.5, 0.75 and 1 mg/L^?1). The frequency of shoot generation (85.18 %) was achieved in medium fortified with 0.25 mg/L^?1 of BA. Shoots were placed on MS medium with 0.25 mg/l IBA for root induction. Roots appeared and induction rate was achieved after 15 days.     

Alterations in the Antioxidant Status of Transgenic Roots of <Emphasis Type="Italic">Artemisia</Emphasis> spp. Representatives after <Emphasis Type="Italic">A. rhizogenes</Emphasis>-Mediated Genetic Transformation

The effect of A. rhizogenes-mediated genetic transformation on the antioxidant status of Artemisia tilesii, A. vulgaris, A. dracunculus, and A. annua transgenic roots has been studied. Antioxidant activity (AOA) of aqueous extracts was determined using methods based on the ability to reduce DPPH⁺ and ABTS⁺-radicals. The level of AOA (DPPH) in 50% of extracts obtained from transgenic roots was higher than the level of activity possessed by extracts from untransformed roots. An increased ability to reduce the ABTS+ radical was observed in 80% of the extracts. Extracts of A. annua and A. tilesii transgenic roots were the most active, while the lowest antioxidant activity was shown in A. dracunculus extracts. Thus, A. rhizogenes-mediated transformation has led to a change in the antioxidant status of the “hairy” roots of several Artemisia spp. plants (except A. vulgaris). It can be used as a method for the enhancement of the natural antiradical properties of plants belonging to the Artemisia genus.     

High-efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation in black gram (<Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper)

In vitro culture and genetic transformation of black gram are difficult due to its recalcitrant nature. Establishment of gene transfer procedure is a prerequisite to develop transgenic plants of black gram in a shorter period. Therefore, genetic transformation was performed to optimize the factors influencing transformation efficiency through Agrobacterium tumefaciens-mediated in planta transformation using EHA 105 strain harbouring reporter gene, bar, and selectable marker, gfp-gus, in sprouted half-seed explants of black gram. Several parameters, such as co-cultivation, acetosyringone concentration, exposure time to sonication, and vacuum infiltration influencing in planta transformation, have been evaluated in this study. The half-seed explants when sonicated for 3 min and vacuum infiltered for 2 min at 100 mm of Hg in the presence of A. tumefaciens (pCAMBIA1304– bar) suspensions and incubated for 3 days co-cultivation in MS medium with 100 µM acetosyringone showed maximum transformation efficiency (46 %). The putative transformants were selected by inoculating co-cultivated seeds in BASTA^® (4 mg l^?1) containing MS medium followed by BASTA^® foliar spray on 15-day-old black gram plants (35 mg l^?1) in green house, and the transgene integration was confirmed by biochemical assay (GUS), Polymerase chain reaction, Dot-blot, and Southern hybridisation analyses.     

Improved <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation and direct plant regeneration in four cultivars of finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.)

We have developed an improved Agrobacterium-mediated transformation and rapid regeneration system for four cultivars (‘CO(Ra)-14’, ‘PR-202’, ‘Try-1’ and ‘Paiyur-2’) of finger millet using optimized transformation and direct plant regeneration conditions. The shoot apical meristems (SAMs) were used as explants in this study. Agrobacterium strain EHA105 carrying binary vector pCAMBIA1301 was used to optimize the transformation conditions. Concentration of hygromycin, the optical density of the culture, infection time, age of the explants, co-cultivation period, the concentrations of acetosyringone and antibiotics were optimized to improve the transformation frequency. The highest frequency of mean transient gus expression (85.1%) was achieved in cultivar ‘CO(Ra)-14’. The entire transformation procedure, from initiating SAMs to planting putative transgenic plantlets in the greenhouse, was completed within 45 days with the highest stable transformation frequency of 11.8% for ‘CO(Ra)-14’. PCR, gus staining and Southern blot analyses were performed in T₀ and T₁ generations to confirm the gene integration. Six events from T₀ had a single copy of the transgene and showed a normal Mendelian pattern of segregation. To our knowledge, this is the first report on the high frequency transformation of finger millet by Agrobacterium and subsequent recovery of transgenic plants via direct plant regeneration without a callus phase, in short duration (45 days). The proposed protocol could be supportive in breaking through the bottleneck in transformation and regeneration of finger millet cultivars.     

Expression of <Emphasis Type="Italic">Withania somnifera</Emphasis> Steroidal Glucosyltransferase gene Enhances Withanolide Content in Hairy Roots

In Withania somnifera, sterol molecules of immense medicinal value are diversified by means of glycosylation. Identifying sterol glycosyltransferases provides an imperative insight of diverse sterol modifications, thereby helping to comprehend the underlying plant mechanisms. In the present study, one of the W. somnifera sterol glycosyltransferase-4 (Ws-Sgtl4) gene was transformed into the W. somnifera leaf explant through Agrobacterium rhizogene. Transformed W. Somnifera Ws-Sgtl4 leaf explants were subjected to hairy root induction and analyzed for biomass accumulation. The analysis of Ws-Sgtl4 gene expression was performed at different time exposures with the application of salicylic acid and methyl jasmonate. The elicitation of W. somnifera hairy root expressing the Ws-Sgtl4 gene was also evaluated for the enhancement if any, in the total withanolide yield as well as the withanolides-A contents. The results suggested that Ws-Sgtl4 gene expression enhanced the production of total withanolide yield and withanolides-A in the hairy root culture of W. somnifera in the response to the elicitors.     

Shoot regeneration process and optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Sinningia speciosa</Emphasis>

The florist’s Gloxinia, Sinningia speciosa, which bears considerable flower trait variations, is an emerging model plants to study floral traits development. However, the investigation of the genetic information linking these floral traits is limited due to a lack of a reliable and efficient transformation system for functional studies. This study aims to optimize a stable genetic transformation system for S. speciosa. Detailed regeneration process and tissue culture parameters are also elucidated. The results show that the plant regeneration, initiated from a single perivascular parenchyma cell, can be induced from leaf and petiole explants in the presence of 1 mg/mL 6-benzylaminopurine (BA) and 0.1 mg/mL naphthalene-acetic acid (NAA) through embryogenesis. In the presence of 0.1 mg/mL NAA only, the adventitious roots form prior to the re-differentiation of shoot tissues in leaf explants. When the proximal end of the petiole is orientated upright with the distal end to the medium, it results in higher success of regeneration, suggesting that hormone supplies must follow endogenous basipetal auxin polarity. Using a glucuronidase (GUS) reporter gene construct, maximum transformation (3.13%) was obtained after a 3 day pre-culture and 5 day co-culture from cotyledons and leaves of 3-week-old seedlings inoculating Agrobacterium strain EHA105. The putative transgenic lines were validated by RT-PCR, Southern blotting and GUS activity. Our result demonstrates that young seedlings are the best material for transformation, probably because young leaves are only a few cell layers thick allowing inner perivascular cell (the origin of regeneration) to be more accessible for Agrobacterium infiltration.     

Overexpression of <Emphasis Type="Italic">AtGRDP2</Emphasis> gene in common bean hairy roots generates vigorous plants with enhanced salt tolerance

Proteins with glycine-rich repeats have been identified in plants, mammalians, fungi, and bacteria. Plant glycine-rich proteins have been associated to stress response. Previously, we reported that the Arabidopsis thaliana AtGRDP2 gene, which encodes a protein with a glycine-rich domain, plays a role in growth and development of A. thaliana and Lactuca sativa. In this study, we generated composite Phaseolus vulgaris plants that overexpress the AtGRDP2 gene in hairy roots generated by Agrobacterium rhizogenes. We observed that hairy roots harboring the AtGRDP2 gene developed more abundant and faster-growing roots than control hairy roots generated with the wild type A. rhizogenes. In addition, composite common bean plants overexpressing the AtGRDP2 gene in roots were more tolerant to salt stress showing increments in their fresh and dry weight. Our data further support the role of plant GRDP genes in development and stress response.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Thidiazuron: an efficient plant growth regulator for enhancing <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Efficient shoot regeneration and Agrobacterium-mediated genetic transformation systems were developed for Petunia hybrida cv. Mitchell. Leaf explants of petunia were cultured on Murashige and Skoog (MS) medium with different concentrations of thidiazuron (TDZ) without auxin. The highest frequency of shoot regeneration (52.1%) and mean number of shoots per explant (4.1) were obtained on medium containing 2 mg l^?1 TDZ. Leaf explants inoculated with Agrobacterium tumefaciens strain EHA101/pIG121Hm harboring ß-glucuronidase (uidA) and hygromycin resistance genes developed putative transformant shoots. The highest frequency of shoot regeneration (22.5%) and mean number of transformant shoots per explant (2.4) were obtained on a selection medium consisting of the above described regeneration medium and containing 25 mg l^?1 hygromycin as the selection agent. Approximately 95% of putative transformant shoots expressed the uidA gene following histochemical ß-glucuronidase (GUS) assay. These were confirmed to be transgenic by PCR analysis and Southern blot hybridization.     

A modified <Emphasis Type="Italic">in planta</Emphasis> method of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in safflower (<Emphasis Type="Italic">Carthamus tinctorius</Emphasis> L.)

Plant transformation has emerged as an important tool to integrate foreign genes in the plant genome to modify the plants for desired traits. Though many techniques of plant transformation are available; getting single copy transgenic events and cost associated remains a big challenge. Thus Agrobacterium-mediated transformation remains the method of choice due to multiple advantages. In the present work a tissue culture free protocol of Agrobacterium-mediated transformation was optimized in safflower, an oil seed crop recalcitrant to transformation. As a proof of concept we selected pCAMBIA2300 gene cassette containing Arabidopsis specific delta 15 desaturase (FAD3) downstream to truncated seed specific promoter beta-conglycinin and optimized tissue culture free protocol of Agrobacterium-mediated transformation using embryos as explants. Addition of silwet L-77, sonication treatment, vacuum infiltration in infection medium and use of paper wicks in co-cultivation period increased the transformation efficiency to 19.3%. Further, success in transformation was confirmed via product accumulation in 21 independent transgenic events wherein oil in transformed seeds showed significant accumulation of alpha-linolenic acid (ALA; 18:3; n3) which is generated from linoleic acid (LA; 18:2; n3) in a FAD3 catalyzed reaction. The present protocol can be utilized to produce transgenic safflower with different desired characters.     

A simple <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation method for rapid transgene expression in <Emphasis Type="Italic">Medicago truncatula</Emphasis> root hairs

Medicago truncatula is widely used as a model legume for symbiotic and pathogenic microbial interaction studies. Although a number of Agrobacterium-mediated transformation methods have been developed for M. truncatula, a rapid root transformation system was not yet available for this model plant. Here, we describe an easy method for rapid transgene expression in root hairs of M. truncatula, using young seedlings co-cultivated with the disarmed hypervirulent A. tumefaciens strain AGL1. This method leads to efficient expression of various GUS and fluorescent reporters in M. truncatula root hairs. We showed that transgene expression is detected as soon as 2 days following co-culture, in root hairs of a particular responsive zone lying 0.5–2 cm behind the root tip. This method can be used with a variety of M. truncatula genotypes, and is particularly useful for rapid investigation of the sub-cellular localization of fluorescent fusion proteins. Moreover, combining distinct Agrobacterium strains during the initial co-culture step efficiently generates co-transformed root hairs, suitable for co-localization of different fluorescent fusion proteins in the same cell.     

Enhanced multiplication and improved <Emphasis Type="Italic">ex vitro</Emphasis> acclimatization of <Emphasis Type="Italic">Decalepis arayalpathra</Emphasis>

The proposed work describes a protocol for high-frequency in vitro regeneration through nodal segments and shoot tips in Decalepsis arayalpathra, a critically endangered medicinal liana of the Western Ghats. Nodal segments were more responsive than shoot tips in terms of shoot proliferation. Murashige and Skoog’s (MS) basal medium supplemented with 5.0 μM 6-benzyladenine (BA) was optimum for shoot initiation through both the explants. Among different combinations of plant growth regulators and growth additive screened, MS medium added with 5.0 μM BA + 0.5 μM indole-3-acetic acid + 20.0 μM adenine sulphate effectuated the highest response: 11.8 shoots per nodal segment and 5.5 shoots per shoot tip with mean shoot length of 9.2 and 4.8 cm, respectively. Half-strength MS medium with 2.5 μM α-naphthalene acetic acid was optimum for in vitro root induction. The plantlets with the well developed shoot and root were acclimatized in Soilrite? with 92 % survival rate in the field conditions. During acclimatization, chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate were gradually changed in dependence of formation of new leaves. Further, the changes in activities of antioxidant enzymes, i.e., superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) as well as activity of carbonic anhydrase were also observed: a continuous rise in SOD activity, but a rise and fall in the activities of CAT, APX, and GR were also noticed. Maximum fresh mass (3.1 g plant^-1), dry mass (0.35 g plant^-1) of roots and 2-hydroxy-4-methoxybenzaldehyde content of 9.22 μg cm^-3(root extract) were recorded after 8 weeks of acclimatization.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of oleaginous yeast <Emphasis Type="Italic">Lipomyces</Emphasis> species

Interest in using renewable sources of carbon, especially lignocellulosic biomass, for the production of hydrocarbon fuels and chemicals has fueled interest in exploring various organisms capable of producing hydrocarbon biofuels and chemicals or their precursors. The oleaginous (oil-producing) yeast Lipomyces starkeyi is the subject of active research regarding the production of triacylglycerides as hydrocarbon fuel precursors using a variety of carbohydrate and nutrient sources. The genome of L. starkeyi has been published, which opens the door to production strain improvements through the development and use of the tools of synthetic biology for this oleaginous species. The first step in establishment of synthetic biology tools for an organism is the development of effective and reliable transformation methods with suitable selectable marker genes and demonstration of the utility of the genetic elements needed for expression of introduced genes or deletion of endogenous genes. Chemical-based methods of transformation have been published but suffer from low efficiency. To address these problems, Agrobacterium-mediated transformation was investigated as an alternative method for L. starkeyi and other Lipomyces species. In this study, Agrobacterium-mediated transformation was demonstrated to be effective in the transformation of both L. starkeyi and other Lipomyces species. The deletion of the peroxisomal biogenesis factor 10 gene was also demonstrated in L. starkeyi. In addition to the bacterial antibiotic selection marker gene hygromycin B phosphotransferase, the bacterial β-glucuronidase reporter gene under the control of L. starkeyi translation elongation factor 1α promoter was also stably expressed in six different Lipomyces species. The results from this study demonstrate that Agrobacterium-mediated transformation is a reliable and effective genetic tool for homologous recombination and expression of heterologous genes in L. starkeyi and other Lipomyces species.     

In vitro regeneration, <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation,and genetic assay of chalcone synthase in the medicinal plant <Emphasis Type="Italic">Echinacea pallida</Emphasis>

In vitro plant regeneration was established in Echinacea pallida, a plant that is commonly used as a folk medicine to treat the common cold, fevers, inflammation and so on. Conditions for callus induction, lateral root and shoot regeneration were determined. Subsequently, two vectors pCHS and pOSAG78, carrying different selection marker genes resistant to kanamycin and hygromycin, respectively, were independently used to transform leaf explants of E. pallida using an Agrobacterium-mediated method. Genomic PCR analysis confirmed the presence of the transgene and selection marker gene in obtained transgenic lines. Southern hybridization indicated that the T-DNA insertion in some transgenic E. pallida was single copy. Among them, transformants carrying Petunia chalcone synthase (CHS) were selected for further study. CHS is a key enzyme in the biosynthesis of diverse flavonoids including anthocyanin pigmentation. Here, we analyzed the roles and compared the gene expression of two clusters of CHSs, EpaCHS-A and EpaCHS-B (EpaCHS-B1 and EpaCHS-B2), isolated from E. pallida. Two of the genes, EpaCHS-A and EpaCHS-B1, were abundantly expressed in petals, whereas EpaCHS-B2 was expressed at high levels in leaves. The expression of EpaCHSs remained constant in leaves and roots of Petunia CHS transformants, while EpaCHS-B2 expression was changed in flowers of transgenic plants. The biosynthesis of caffeic acid derivatives, cichoric acid and caftaric acid, was increased in leaves and roots of CHS transformants, respectively, while the amount of echinacoside in roots of transgenic plants was decreased. This is the first report on genetic engineering of E. pallida. The information contained herein can be used as a tool for further study of the biological pathways and secondary metabolism of specific compounds from medicinal Echinacea species.