Flavones in genetically transformed <Emphasis Type="Italic">Scutellaria baicalensis</Emphasis> roots and induction of their synthesis by elicitation with methyl jasmonate

The composition and content of flavones were estimated in pRi T-DNA-transformed skullcap (Scutellaria baicalensis Georgi) roots obtained by the inoculation of axenically grown seedlings with a wild A4 strain of the soil bacterium Agrobacterium rhizogenes. It was elucidated by analytical and preparative HPLC of phenolic compounds in the extracts from the pRi T-DNA-transformed roots and also by ultraviolet spectroscopy and ¹H and ¹³C NMR that cultured skullcap roots contained similar basic flavones as intact roots of this plant species, i.e., baicalein and wogonin and corresponding glucuronides, baicalin and wogonoside. The content of these flavones in cultured roots was threefold lower than in the roots of intact five-year-old plants. When skullcap roots were cultured on B5 or Murashige and Skoog medium, the ratios between major flavones changed but their total content remained unchanged. The treatment of three-week-old cultured roots with methyl ether of jasmonic acid (MeJa) doubled the total concentration of major flavones in roots; the content of aglycons, baicalein and wogonin, increased to a greater degree, e.g., by 2.3 and 3.3 times, respectively. The induction of flavone production by elicitors indicates that flavones behave as phytoanticipins because major flavones of skullcap manifest a distinct antimicrobial activity. The results of the short-term treatment of skullcap roots with MeJa show that stress biotic factors can considerably increase the content of physiologically active flavones.Translated from Fiziologiya Rastenii, Vol. 52, No. 1, 2005, pp. 90–96.Original Russian Text Copyright © 2005 by Kuzovkina, Guseva, Kovács, Szöke, Vdovitchenko.     

Fusicoccin-Like Ligands in Higher Plants

Fusicoccin-like ligands in higher plants were sought by combining high performance liquid chromatography with radioreceptor analysis and radioimmunoassay. Such substances were found in genetically transformed cultured roots of horseradish, alteus and lupine as well as in native horseradish, cucumber, horse chestnut, and maize plants. In root crops such as carrot and sugar beet or in potato tubers, only traces of fusicoccin-like ligands were detected. Fusicoccin A was detected in genetically transformed cultured roots of horseradish by gas chromatography/mass spectrometry. Received October 15, 1996; accepted February 14, 1997     

Artificial seed preparation as the efficient method for storage and production of healthy cultured roots of medicinal plants

The technique for the refinement of pRi T-DNA-transformed root cultivation by the root fragment encapsulation in the gel coat, i.e., so-called “artificial seed” (AS) production, was studied. AS were produced from genetically transformed roots of Baikal skullcap (Scutellaria baicalensis Georgi) and common rue (Ruta graveolens L.). The effects of duration of AS storage at 4°C on their subsequent growth activity and a capability for resumption of actively growing root cultures were analyzed. Encapsulation of contaminated Baikal skullcap root culture with the addition of antibiotic and storage during 2–5 weeks at low above-zero temperature resulted in a complete elimination of infection, i.e., obtaining the healthy root culture. Growth activity and total flavone concentration were markedly increased in this culture, so that total productivity of this renewed root culture increased substantially. Using AS produced from the root fragments of common rue, it was shown that, after long-term storage at low above-zero temperature, they are capable of not only root growth resumption but also active shoot formation, which is of interest for plant micropropagation. Long-term retaining growth activity of AS produced from root cultures of valuable medicinal plants permits their usage as a reserve and also, in the case of necessity, for long-distance transport as compact axenic root inocula. The storage of viable root fragments within AS also helps to optimize intervals between numerous subculturings of root cultures required for the maintenance of IPPRAS collection in the active state.     

Genetically transformed roots as a model system for studying physiological and biochemical processes in intact roots

The sequence of operations during plant genetic transformation using wild strains of the soil bacterium Agrobacterium rhizogenes and subsequent root production capable of long-term growth on the relatively simple media free of growth compounds are described. The basic morphological, physiological, and biochemical characteristics of transformed roots are reported, and a technology of their in vitro cultivation is described. The modes of growth optimization of long-term cultivated roots of valuable plants and of the increase in the concentration of secondary metabolites synthesized by these roots are enumerated. The efficiency of the method of so-called “artificial seed” application for preservation of valuable lines of cultivated roots and a possibility of their healthy state restoration are briefly appraised. On the basis of literature and our own experiments, the main directions for the usage of genetically transformed roots in applied and fundamental studies are outlined. The terminology used for designation of genetically transformed roots by Russian researches is briefly discussed. Main materials and equipment required for plant transformation with the soil bacterium and for the maintenance of long-term growth of obtained roots are listed.     

Catharanthus roseus: micropropagation and in vitro techniques

Different methods of in vitro culture of Catharanthus roseus provide new sources of plant material for the production of secondary metabolites such as indole alkaloids. Callus, cell suspension, plantlets, and transgenic roots cultured in the bioreactor are used in those experiments. The most promising outcomes include the production of the following indole alkaloids: ajmalicine in unorganised tissue, catharanthine in the leaf and cell culture in the shake flask and airlift bioreactor, and vinblastine in shoots and transformed roots. What is very important, enzymatic coupling of monomeric indole alkaloids, vindoline and catharanthine, is possible to form vinblastine in cell cultures. The method of catharanthine and ajmalicine production in the suspension culture in bioreactors has been successful. In this method, elicitation may be used acting on different metabolic pathways. Also of interest is the method of obtaining arbutin from the callus culture of C. roseus conducted with hydroquinone. The transformed root culture seems to be the most promising for alkaloid production. The genetically transformed roots, obtained by the infection with Agrobacterium rhizogenes, produce higher levels of secondary metabolites than intact plants. Also, whole plants can be regenerated from hairy roots. The content of indole alkaloids in the transformed roots was similar or even higher when compared to the amounts measured in studies of natural roots. The predominant alkaloids in transformed roots are ajmalicine, serpentine, vindoline and catharanthine, found in higher amounts than in untransformed roots. Transformed hairy roots have been also used for encapsulation in calcium alginate to form artificial seeds.     

Agrobacterium-mediated transformation of Artemisia absinthium L. (wormwood) and production of secondary metabolites

Hairy roots were obtained after infection of Artemisia absinthium shoots with Agrobacterium rhizogenes strains 1855 and LBA 9402. The susceptibility to hairy root transformation varied between plant genotypes and bacterial strains. Hairy roots showed macroscopic differences from control root cultures. Southern blot hybridization confirmed the integration of T-DNA from both p1855 and pBin19, while polymerase chain reaction analysis indicated the presence of the neomycin phosphotransferase gene in the hairy root genome. Subcultured transformed root lines grew well in selective B5 agar-solidified medium containing kanamycin or rifampicin and without hormones. Shake-flask experiments with fast-growing root lines showed that 40 g l^–1 was the best sucrose concentration for biomass production, yielding a 463-fold increase in dry weight after 28 days of culture. Great differences were found in the profiles of the essential oils isolated from normal and hairy roots. Gas chromatography/mass spectrometry analysis showed the oil produced by transformed cultures to be a mixture of 50 compounds with only one major component representing 37% of the oil content. Received: 19 March 1996 / Revision received: 15 July 1996 / Accepted: 13 December 1996     

Formation of phenolic compounds in the roots of <Emphasis Type="Italic">Hedysarum theinum</Emphasis> cultured in vitro

A steadily growing culture of genetically transformed roots of a valuable medicinal Altaic plant Hedysarum theinum Krasnob. was established using a Ri-plasmid (pRi) T-DNA of A4 wild strain of Agrobacterium rhizogenes. The composition of secondary substances accumulated in the in vitro roots and the roots of H. theinum intact seedlings was investigated. Isoflavonoids were found to represent by their main low-molecular metabolites. Preparative HPLC made it possible to isolate four substances from the methanolic extract of H. theinum cultured roots. Using ¹H- and ¹³C-NMR-spectrometry, these substances were identified as formononetin, ononin (formononetin glycoside), malonyl ononin, and texasin glucoside. The qualitative composition of secondary metabolites of the genetically transformed roots and the roots of H. theinum seedlings was essentially the same, except that malonyl ononin was not found in the latter. The technique of producing artificial seeds on the basis of H. theinum roots cultured in vitro was tested, and the possibility of their use as a rhizogenic inoculum was substantiated. The culture of H. theinum roots is considered as a potential source of ecologically pure raw material for medicinal preparations, and the artificial seeds with root inoculum are a promising vehicle for propagation and conservation of this valuable plant.     

A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots

Summary Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.     

Glycyrrhizin production by in vitro cultured <Emphasis Type="Italic">Glycyrrhiza glabra</Emphasis> elicited by methyl Jasmonate and salicylic acid

Licorice (Glycyrrhiza glabra L. var. glabra, Fabaceae) is considered as a model plant synthesizing triterpenoid secondary compounds. It is known that glycyrrhizin is accumulated in thickened intact licorice roots. The effects of methyl jasmonate (MeJa) and salicylic acid (SA) on plant growth and production of glycyrrhizin in the roots of in vitro cultured 65-day-old plants were studied. Increasing amounts of glycyrrhizin in the roots treated with MeJa inhibited root growth, while SA increased the amount of glycyrrhizin without negative effects on growth. Treatment of plantlets with 0.1–2 mM MeJa and 0.1 and 1 mM SA enhanced the production of glycyrrhizin by 3.8 and 4.1 times, respectively, as compared to the controls. Results support the hypothesis that production of glycyrrhizin is related to a defense response system of the licorice.     

New terpenoids in cultivated and wild chamomile (in vivo and in vitro)

The effect of Chamomilla recutita (L.) Rauschert is made up by several groups of active substances, among which terpenoids in the inflorescences are of greatest importance. Among cultivated species, the Hungarian BK-2 contains more chamazulene in its essential oil than the German Degumil type, which is mainly cultivated for its (-)-alpha-bisabolol. Both components have important antiinflammatory activities. Among wild chamomile populations in Hungary, a population was found in the area of Szabadkigyós containing significant amounts-on average 48%-of (-)-alpha-bisabolol in its inflorescence oil. In vitro cultures were made from this population to obtain propagation material containing a high number of active substances. The intact roots contained no (-)-alpha-bisabolol but the sesquiterpene alcohol beta-eudesmol as new compound was identified by our group. Sterile plantlets, cultured in vitro, were multiplied for phytochemical investigations. Pharmacologically important compounds of the essential oils were followed in great detail. The amount of in vitro cultured terpenoids and polyin compounds was compared with that of in vivo plants. These volatile compounds were identified by comparing their retention times with those of authentic standards, essential oils of known composition and peak enrichment. The confirmation of identity was done by comparison of their mass spectra with those reported in the literature and reference compounds. The percentage evaluation of each component was made by area normalisation. Gas chromatography (GC) and mass spectrometry (MS) showed that sterile chamomile cultures generated the most important terpenoid and polyin compounds characteristic of the parent plant. We identified germacrene-D, berkheyaradulene, 4-(2', 4', 4'-trimethyl-bicyclo[4.1.0]hept-2'-en-3'-yl)-3-buten-2-one, geranyl-isovalerate and cedrol as new components in these sterile cultures.     

Hairy Root Culture for Mass-Production of High-Value Secondary Metabolites

ABSTRACT

Plant cell cultivations are being considered as an alternative to agricultural processes for producing valuable phytochemicals. Since many of these products (secondary metabolites) are obtained by direct extraction from plants grown in natural habitat, several factors can alter their yield. The use of plant cell cultures has overcome several inconveniences for the production of these secondary metabolites. Organized cultures, and especially root cultures, can make a significant contribution in the production of secondary metabolites. Most of the research efforts that use differentiated cultures instead of cell suspension cultures have focused on transformed (hairy) roots. Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic (cancerous) roots produced by A. rhizogenes infection are characterized by high growth rate, genetic stability and growth in hormone free media. These genetically transformed root cultures can produce levels of secondary metabolites comparable to that of intact plants. Hairy root cultures offer promise for high production and productivity of valuable secondary metabolites (used as pharmaceuticals, pigments and flavors) in many plants. The main constraint for commercial exploitation of hairy root cultivations is the development and scaling up of appropriate reactor vessels (bioreactors) that permit the growth of interconnected tissues normally unevenly distributed throughout the vessel. Emphasis has focused on designing appropriate bioreactors suitable to culture the delicate and sensitive plant hairy roots. Recent reactors used for mass production of hairy roots can roughly be divided as liquid-phase, gas-phase, or hybrid reactors. The present review highlights the nature, applications, perspectives and scale up of hairy root cultures for the production of valuable secondary metabolites.     

Transgenic hairy roots. recent trends and applications

Agrobacterium rhizogenes causes hairy root disease in plants. The neoplastic roots produced by A. rhizogenes infection is characterized by high growth rate and genetic stability. These genetically transformed root cultures can produce higher levels of secondary metabolites or amounts comparable to that of intact plants. Hairy root cultures offer promise for production of valuable secondary metabolites in many plants. The main constraint for commercial exploitation of hairy root cultures is their scaling up, as there is a need for developing a specially designed bioreactor that permits the growth of interconnected tissues unevenly distributed throughout the vessel. Rheological characteristics of heterogeneous system should also be taken into consideration during mass scale culturing of hairy roots. Development of bioreactor models for hairy root cultures is still a recent phenomenon. It is also necessary to develop computer-aided models for different parameters such as oxygen consumption and excretion of product to the medium. Further, transformed roots are able to regenerate genetically stable plants as transgenics or clones. This property of rapid growth and high plantlet regeneration frequency allows clonal propagation of elite plants. In addition, the altered phenotype of hairy root regenerants (hairy root syndrome) is useful in plant breeding programs with plants of ornamental interest. In vitro transformation and regeneration from hairy roots facilitates application of biotechnology to tree species. The ability to manipulate trees at a cellular and molecular level shows great potential for clonal propagation and genetic improvement. Transgenic root system offers tremendous potential for introducing additional genes along with the Ri T-DNA genes for alteration of metabolic pathways and production of useful metabolites or compounds of interest. This article discusses various applications and perspectives of hairy root cultures and the recent progress achieved with respect to transformation of plants using A. rhizogenes.     

Genetic transformation of oilseed rape (Brassica napus) by the Ri T-DNA of Agrobacterium rhizogenes and analysis of inheritance of the transformed phenotype

Summary Genetically transformed repeseed (Brassica napus) roots were obtained by in vitro inoculation of excised stem segments with Agrobacterium rhizogenes. Axenic root organ clones were established and they exhibited a phenotype characteristic of transformed roots: rapid growth, reduced apical dominance and root plagiotropism. Stem regeneration was induced by exposing root fragments to 2,4-dichloroacetic acid (2,4-D) in liquid medium, followed by transfer to solid regeneration medium. The resulting plants exhibited the transformed phenotype observed in other species where similar experiments have been performed. Direct evidence for genetic transformation was obtained from opine assays and molecular hybridization. Sexual transmission of the transformed phenotype was Mendelian, and a probable case of T-DNA insertion into two independent loci within the same plant was detected. The estimated optimal time necessary to obtain transformed oilseed rape plants using this approach is 2 months.     

Agrobacterium infection of hemp (Cannabis sativa L.): establishment of hairy root cultures

Abstract

Experimental conditions were optimized for hemp, a difficult to transform plant, to be effectively infected with either Ri or Ti plasmid-bearing agrobacteria and to establish stably transformed tissues. Hypocotyl of intact seedlings was the most responsive material and the response depended on both bacterial strain and plant variety. Transformed tissues, hairy roots and tumors, were cultured and stabilized in vitro and showed the characteristic traits of fast and phytohormone-independent growth as well as high incidence of lateral branching and abundance of root hairs in the case of roots. They all contained T-DNA of the corresponding Ri or Ti plasmid as revealed by PCR analysis with specific primers and further hairy roots induced by AR10GUS strain showed normal pattern of β-glucuronidase positive staining. To our knowledge, this represents the first reported protocol for the establishment of Cannabis sativa hairy root cultures.     

Effect of plant growth regulators on growth and biosynthesis of phenolic compounds in genetically transformed hairy roots of<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer

In this study, morphological alterations, biomass growth, and secondary metabolite production of genetically transformed hairy roots ofPanax ginseng C. A. Meyer, were evaluated after administration of plant growth regulators. The addition of benzylamino purine and kinetin to the culture media increased biomass formation and phenolic compound biosynthesis in the hairy roots. α-Naphthaleneacetic acid and indole-3-butyric acid inhibited hairy root growth, however, low concentrations of indole-3-acetic acid slightly increased hairy root growth. Low concentrations of 2,4-Dichlorophenoxyacetic acid profoundly inhibited growth of hairy roots. The addition of plant growth regulators, such as auxin, did not increase total phenolic compounds in hairy roots that did not contain gibberellic acid and cytokinins. Callus formation was induced in cultures suspended in liquid medium amended with benzylamino purine and kinetin. Hairy roots regenerated from these calluses exhibited an active growth pattern with extensive lateral branching in non-amended medium, similar to the growth pattern of the original hairy roots.     

Chemical Diversity and Antimicrobial Activity of Volatile Compounds from Zanthoxylum zanthoxyloides Lam. according to Compound Classes,Plant Organs and Senegalese Sample Locations

The chemical diversity of Zanthoxylum zanthoxyloides growing wild in Senegal was studied according to volatile compound classes, plant organs and sample locations. The composition of fruit essential oil was investigated using an original targeted approach based on the combination of gas chromatography (GC) and liquid chromatography (LC) both coupled with mass spectrometry (MS). The volatile composition of Z. zanthoxyloides fruits exhibited relative high amounts of hydrocarbon monoterpenes (24.3 – 55.8%) and non‐terpenic oxygenated compounds (34.5 – 63.1%). The main components were (E)‐β‐ocimene (12.1 – 39%), octyl acetate (11.6 – 21.8%) and decanol (9.7 – 15.4%). The GC and GC/MS profiling of fruit essential oils showed a chemical variability according to geographical locations of plant material. The LC/MS/MS analysis of fruit oils allowed the detection of seven coumarins in trace content. The chemical composition of fruit essential oils was compared with volatile fractions of leaves and barks (root and trunk) from the same plant station. Hexadecanoic acid, germacrene D and decanal were identified as the major constituents of leaves whereas the barks (root and trunk) were dominated by pellitorine (85.8% and 57%, respectively), an atypic linear compound with amide group. The fruit essential oil exhibited interesting antimicrobial activities against Staphylococcus aureus and Candida albicans, particularly the alcohol fraction of the oil.     

Flavonoid Production in Transformed Scutellaria baicalensis Roots and Ways of Its Regulation

A root culture of skullcap (Scutellaria baicalensisGeorgi) transformed with pRi T-DNA was initiated by the inoculation of sterile seedlings with Agrobacterium rhizogenes(wild-type strain A-4). The flavonoid concentration in cultured roots comprised 5% of the root dry weight and was maintained essentially constant during a subculture. For four weeks of culturing, the weight of the roots increased by 20–30 times; when the roots were cultured for a longer time and with periodic enrichment of the nutrient medium, their weight increased 50-fold. Skullcap roots were shown to synthesize flavones characteristic of intact roots (wogonin, baicalein, and baicalin). The addition of 0.01–1 mM L-phenylalanine (a precursor of flavonoids) to the nutrient medium affected neither root growth, nor their flavonoid concentration. Root elicitation with 100 M methyl jasmonate for 72 h increased the flavonoid content per flask and per root dry weight by 1.8 and 2.3 times, respectively.     

Root damage to apple plants by cockchafer larvae induces a change in volatile signals below‐ and above‐ground

Volatile organic compounds (VOCs) mediate communication between plants and insects. Plants under insect herbivore attack release VOCs either at the site of attack or systemically, indicating within‐plant communication. Some of these VOCs, which may be induced only upon herbivore attack, recruit parasitoids and predatory insects to feed on the attacking insects. Moreover, some plants are able to ‘eavesdrop’ on herbivore‐induced plant volatiles (HIPVs) to prime themselves against impending attack; such eavesdropping exemplifies plant–plant communication. In apple orchards, the beetle Melolontha melolontha L. (Coleoptera: Scarabaeidae) is an important insect pest whose larvae live and feed on roots for about 4 years. In this study, we investigated whether the feeding activity of M. melolontha larvae (1) alters the volatile profile of apple roots, (2) induces the release of HIPVs systemically in the leaves, and (3) whether infested plants communicate to neighbouring non‐infested conspecifics through HIPVs. To answer these questions, we collected constitutive VOCs from intact M9 roots as well as M. melolontha larvae‐damaged roots using a newly designed ‘rhizobox’, to collect root‐released volatiles in situ, without damaging the plant root system. We also collected VOCs from the leaf‐bearing shoots of M9 whose roots were under attack by M. melolontha larvae and from shoots of neighbouring non‐infested conspecifics. Gas chromatography‐mass spectrometry analysis showed that feeding activity of M. melolontha larvae induces the release of specific HIPVs; for instance, camphor was found in the roots only after larvae caused root damage. Melolontha melolontha also induced the systemic release of methyl salicylate and (E,E)‐α‐farnesene from the leaf‐bearing shoots. Methyl salicylate and (E,E)‐α‐farnesene were also released by the shoots of non‐infested neighbouring conspecifics. These phenomena indicate the induction of specific VOCs below‐ and above‐ground upon M. melolontha larvae feeding on apple roots as well as plant–plant communication in apple plants.     

Secoiridoids and xanthones in the shoots and roots of <Emphasis Type="Italic">Centaurium pulchellum</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

Summary The qualitative and quantitative composition of secondary metabolites was studied in the shoots and roots of Centaurium pulchellum cultured in vitro. Secoiridoids (gentiopierin, swertiamarin, and sweroside) and xanthones (methylbellidifolin, demethyleustomin, and deccussatin) were isolated. Sweroside was found to be the major secoiridoid compound in the aerial parts of plants growing in nature. while swertiamarin dominated in plants cultured in vitro. In roots of all plants, genciopicrin was the major compound. Xanthone demethyleustomin was the major compound both in the shoots and roots of plants growing in nature and cultured in vitro. Different sugars (glucose, fructose, and sucrose) added in different concentrations in the medium affected the production of secondary compounds.     

Chemical compounds and essential oil release through decomposition process from Lavandula stoechas in Mediterranean region

All aromatic plants emit volatile substances into the environment either during their life or during the decomposition process. During decomposition, these volatile constituents affect the top soil microflora and the nutrients recycle process. Lavandula stoechas is a perennial aromatic shrub, rich in aromatic substances which can be found abundant across the Mediterranean. The aim of the present study was to investigate the essential oil degradation during decomposition process of L. stoechas and how the essential oil major compounds are released during this process. Fresh leaves and flowers of lavender were used for this study using the litter bag technique. The essential oil content of the initial plant was 1.46% dw and after seventeen months period dropped at 0.06%. There were no differences in oil degradation among the different experimental sites. Most of the essential oil compounds had disappeared after a year. However, the most toxic compounds in the plant material still remained after seventeen months.