Identification of IAA-producing endophytic bacteria from micropropagated Echinacea plants using 16S rRNA sequencing

The presence of latent bacteria is a serious problem in plant tissue cultures. While endophytes are generally beneficial to plants in situ, they may affect culture growth under the modified conditions in vitro. The present study was undertaken to identify and characterize endophytic bacteria associated with the medicinal plant Echinacea in tissue culture. Based on classical microbiological tests and 16S rRNA analyses, it was found that endophytic bacteria associated with aseptically micropropagated Echinacea plantlets are representatives of several genera, Acinetobacter, Bacillus, Pseudomonas, Wautersia (Ralstonia) and Stenotrophomonas. Based on TLC and HPLC analyses, we found that Pseudomonas stutzeri P3 strain produces plant hormone, auxin (indole-3-acetic acid, IAA). Antibiotic resistance was also assessed as a virulence factor. The majority of endophytic bacteria were resistant to the antibiotic kanamycin, but susceptible to chloramphenicol. Recommendations for propagating Echinacea in vitro cultures involve the addition of chloramphenicol, tetracycline, and ampicillin, antibiotics that cause no side effects on these plant species.     

Alkamide production from hairy root cultures of <Emphasis Type="Italic">Echinacea</Emphasis>

Hairy root cultures of Echinacea, one of the most important medicinal plants in the US, represent a valuable alternative to field cultivation for the production of bioactive secondary metabolites. In this study, the three most economically important species of Echinacea (Echinacea purpurea, Echinacea pallida, and Echinacea angustifolia) were readily transformed with two strains of Agrobacterium that produce the hairy root phenotype. Transformed roots of all three species exhibited consistent accelerated growth and increased levels of alkamide production. Optimization of the culture of Echinacea hairy roots was implemented to enhance both growth and alkamide production concomitantly. The use of half-strength Gamborg’s B5 medium supplemented with 3.0% sucrose was twice as effective in maintaining hairy root production than any other media tested. The addition of indolebutyric acid increased the growth rate of roots by as much as 14-fold. Alkamide production increased severalfold in response to the addition of the elicitor, jasmonic acid, but did not respond to the addition of indolebutyric acid. Induced accumulation of the important bioactive compounds, alkamides 2 and 8, was observed both in transformed roots and in response to jasmonic acid treatments. The results of this study demonstrate the efficacy of hairy root cultures of Echinacea for the in vitro production of alkamides and establish guidelines for optimum yield.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of some important chinese medicinal plants and their sustainable usage

Summary Medicinal plants are valuable sources of medicinal and many other pharmaceutical products. The conventional propagation method is the principal means of propagation and takes a long time for multiplication because of a low rate of fruit set, and/or poor germination and also sometimes clonal uniformity is not maintained through seeds. The plants used in the phyto-pharmaceutical preparations are obtained mainly from the natural growing areas. With the increase in the demand for the erude drugs, the plants are being overexploited, threatening the survival of many rate species. Also, many medicinal plant species are disappearing at an alarming rate due to rapid agricultural and urban development, uncontrolled deforestation, and indiscriminate collection. Advanced biotechnological methods of culturing plant cells and tissues should provide new means for conserving and rapidly propagating valuable, rare, and endangered medicinal plants. The purpose of the present review is to focus the application of tissue culture technology for in vitro propagation via somatic embryogenesis and organogenesis and the cell suspension culture with suitable examples reported earlier. An overview of tissue culture studies on important Chinese medicinal plants and related species is presented.     

In vitro regeneration of Echinacea purpurea L.: Direct somatic embryogenesis and indirect shoot organogenesis in petiole culture

Summary An in vitro propagation system was developed for Echinacea purpurea L. (purple coneflower), a medicinal plant commonly used in the treatment of colds, flu and related ailments. Echinacea seeds were found to be contaminated with systemic fungi and therefore an optimized minimal concentration of Plant Preservation Mixture (PPM) was incorporated in the seed germination medium to recover sterile seedlings. Regeneration was induced on petiole explants from 2-month-old sterile seedlings cultured on medium supplemented with benzylaminopurine (BAP) or thidiazuron (TDZ) in combination with indoleacetic acid (IAA). Two distinct forms of regeneration were identified in cultured petiole explants with histological and morphological observations, viz. the direct formation of somatic embryos on the epidermis and the de novo development of shoots from callus tissues formed in subepidermal cell layers. the results of this study have established a micropropagation system for E. purpurea that will provide sterile plant material for further investigations into medicinally active biochemicals and may facilitate mass production of high-quality E. purpurea plants for the commercial market.     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

Conservation Strategy for African Medicinal Species: <i>In Vitro</i> Biotechnological Approach

The use of medicinal plants for different therapeutic values is well documented in African continent. African diverse biodiversity hotspots provide a wide range of endemic species, which ensures a potential medicinal value. The feasible conservation approach and sustainable harvesting for the medicinal species remains a huge challenge. However, conservation approach through different biotechnological tools such as micropropagation, somatic embryogenesis, synthetic seed production, hairy root culture, molecular markers based study and cryopreservation of endemic African medicinal species is much crucial. In this review, an attempt has been made to provide different in vitro biotechnological approaches for the conservation of African medicinal species. The present review will be helpful in further technology development and deciding the priorities at decision-making levels for in vitro conservation and sustainable use of African medicinal species.     

<Emphasis Type="Italic">In vitro</Emphasis> micropropagation of four lupin species

The complete protocols for long-term micropropagation of some cultivars of four lupin species: Lupinus luteus, L. albus, L. angustifolius and L. mutabilis were elaborated. The shoots were regenerated in vitro via induction of axillary buds development. Plantlets were multiplicated on lowered salts MS-derived media containing BAP in diverse and generally low concentrations. Significant differences in regeneration capacity between species and cultivars were observed. The highest multiplication ratio revealed L. mutabilis and L. luteus. Regenerated shoots were rooted in vitro on low-salts MS-derived media with B5 vitamins. Media were supplemented with different auxins that affected roots formation of particular species and cultivars. Rooting ability of regenerated shoots decreased rapidly through in vitro culture. For that reason, grafting was applied as an alternative method of transfer of shoots to in vivo conditions. This method turned out to be successful for the majority of studied species and cultivars. Complete rooted or grafted plantlets were cultivated in pots with perlit in greenhouse. An erratum to this article is available at .     

Mitochondrial function plasticity in <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> during growth in batch culture

The alterations in mitochondrial bioenergetics during growth in a batch culture of Acanthamoeba castellanii were studied. The capacity of cytochrome pathway-dependent respiration measured in vitro decreased from the intermediary phase, when cell division slowed down. The pattern of the cytochrome pathway capacity changes was paralleled from the intermediary phase by alterations in the amount of total (and reducible) membranous ubiquinone. These changes were accompanied by a decrease in mitochondrial reactive oxygen species production in vitro (when no energy-dissipating system was active), and almost no change in superoxide dismutase activity and protein level, thus indicating an equivalent need for this enzyme in oxidative stress defence in A. castellanii culture. On the other hand, a decrease in the activity and protein level of alternative oxidase and uncoupling protein was observed in vitro, when cells shifted from the exponential growth phase to the stationary phase. It turned out that the contribution of both energy-dissipating systems in the prevention of mitochondrial reactive oxygen species generation in vivo could lead to its constant level throughout the growth cycle of A. castellanii batch culture. Hence, the observed functional plasticity insures survival of high quality cysts of A. castellanii cells.     

Goldenseal (<Emphasis Type="Italic">Hydrastis canadensis</Emphasis> L.): <Emphasis Type="Italic">In vitro</Emphasis> regeneration for germplasm conservation and elimination of heavy metal contamination

Summary Hydrastis canadensis L. (Goldenseal) is an endangered medicinal plant used in the treatment of many ailments, such as gastrointestinal disturbances, urinary disorders, hemorrhage, skin, mouth and eye infections, and inflammation. Commercial preparations of wild-harvested goldenseal were found to contain heavy metal contaminants including aluminum (848 μgg⁻¹), cadmium (0.4μgg⁻¹), lead (18.7μgg⁻¹), and mercury (0.1 μgg⁻¹). As well, goldenseal is an endangered species listed in the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES) Appendix II. Therefore, the practice of wild-harvest is actually decimating natural populations of goldenseal and endangering its genetic diversity. In vitro propagation protocol by tissue culture was developed for producing high-quality tissues of goldenseal. Significantly more de novo regeneration was induced on stem explants of 3-mo.-old plants cultured on a medium containing 10 μM 6-benzylaminopurine (BA) (22 regenerants per explant) than any other treatment. Subculture of the regenerants on a medium devoid of growth regulators resulted in the development of complete plants that were acclimatized and thrived in standard greenhouse conditions. The plants regenerated in vitro contained the lowest levels of heavy metals. The findings of this study provide the first evidence that heavy metal contaminants bioaccumulate in goldenseal tissues and also provide a method for germplasm conservation, mass multiplication, and production of goldenseal tissues free from abiotic contamination.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

Propagation and Conservation of Picrorhiza kurrooa Royle ex Benth.: An Endangered Himalayan Medicinal Herb of High Commercial Value

Picrorhiza kurrooa Royle ex Benth., a high value medicinal herb of alpine Himalaya and a source of hepatoprotective picrosides, is listed as ‘endangered’ due to heavy collection from its natural habitat. The present report deals with successful propagation of this species using both conventional and in vitro techniques. Vegetative propagation was achieved by rooting runner cuttings with indole-3-butyric acid (IBA) or α-naphtheleneacetic acid (NAA) treatment before planting. Nearly 87% rooting success was achieved by treatment of cuttings with 50.0 μM IBA. Seeds were given a presoaking treatment with gibberellic acid (GA₃), 6-benzylaminopurine (BAP) or a combination of both to influence germination. More than 11-fold improvement in germination was recorded in seeds treated with 250.0 μM GA₃. In vitro shoot multiplication was achieved through sprouting of axillary buds using nodal segment. Multiple shoots were formed following culture for 3 weeks on Murashige and Skoog (MS; 1962. Physiologia Plantarum 15: 473–497) medium containing 1.0 μM BAP. Cent percent rooting success, without basal callus formation, was observed when individual microshoots were placed in MS medium supplemented with IBA. The plantlets raised using conventional as well as tissue culture methods were hardened and successfully established in the experimental field located at 2450 m elevation. In addition, strategies have been discussed to encourage cultivation and in situ conservation of this highly valued medicinal herb so as to reduce pressure on its natural populations.     

Acmella oppositifolia micropropagation by single-node culture

Acmella oppositifolia plantlet formation was achieved by subculturing single-node explants on Murashige and Skoog medium without growth regulators. The explants from 1-month-old in vitro plantlets produced shoots over a 7-day culture period. From these in vitro cultured nodes readily rooted shoots elongated on auxin-free MS medium. Plants produced were easily acclimatized and subsequently flowered in a greenhouse. This species is of medicinal value in tropical America from Mexico to Colombia.     

<Emphasis Type="Italic">In vitro</Emphasis> biosynthesis of antioxidants from <Emphasis Type="Italic">Hemidesmus indicus</Emphasis> R. Br. cultures

Summary Shoot cultures and callus cultures from roots and leaves of Hemidesmus indicus R. Br (Asclepiadaceae) were established on Murashige and Skoog medium with various hormonal combinations. The production of antioxidants (lupeol, vanillin, and rutin) in shoot cultures, callus cultures derived from leaf cells and root cells, was compared with root and aerial portions of the parent plant. Shoot cultures and leaf callus cultures produced more antioxidants than root callus cultures. In vitro culture of this species might ofter an alternative method for production of these important pharmaccuticals, which would reduce the collection pressure on this rare plant.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of a high value medicinal plant: <Emphasis Type="Italic">Asparagus racemosus</Emphasis> Willd.

Asparagus racemosus Willd. is an important medicinal plant of tropical and subtropical India. Its medicinal usage has been reported in the Indian and British Pharmacopoeias and in traditional systems of medicine such as Ayurveda, Unani, and Siddha. The multiple uses of this species have increased its commercial demand, resulting in over-exploitation. Because of destructive harvesting, the natural population of A. racemosus is rapidly disappearing, and it is recognized as ‘vulnerable’ (Warner et al., Some important medicinal plants of the Western Ghats, India: a profile. International Development Research Centre, Artstock, New Delhi, India, 15 pp, 2001). The development of an efficient micropropagation protocol will play a significant role in meeting the requirements for commercial cultivation, thereby conserving the species in its natural habitat. In the present study, in vitro shoot proliferation was obtained by culturing single node segments in Murashige and Skoog’s (MS) medium supplemented with 3.69 μM 2-isopentyl adenine and 3% sucrose with a multiplication rate of 3.5. For proper root formation, the in vitro-formed shoot clusters were cultured on half strength (major salts reduced to half) MS medium with 1.61 μM 1-naphthalene acetic acid, 0.46 μM kinetin, 98.91 μM adenine sulfate, 500 mg/l malt extract, 198.25 μM phloroglucinol, and 3% sucrose. On this medium, 85% rooting was observed within 20 d. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%.     

Diversity of the <Emphasis Type="Italic">Fusarium</Emphasis> species associated with head and seedling blight on wheat in Slovakia

Species associated with Fusarium head blight are depending on the production and edaphic conditions. The differences are found in the representation of various Fusarium spp. in the diseases, which sporadically occur all over the territory of Slovakia, in all agricultural production types. We identified fifteen Fusarium species during ten years of investigation. Most of the mentioned species F. culmorum (W.G. Smith) Sacc., F. graminearum Schwabe, recently F. cerealis (Cooke) Sacc. (crookwellense Burgess, Nelson & Tousson) and F. sambucinum Fuckel in diseased caryopsis are seed transmitted. The significant differences among species and intra species in cultural and pathogenicity assays in vitro and in vivo were correlated. Some of them are able to produce toxic metabolites — deoxynivalenone, which probably play a role in the aggressiveness of the pathogen and promote disease development and pathogen colonization.     

<Emphasis Type="Italic">Exiguobacterium acetylicum</Emphasis> strain 1P (MTCC 8707) a novel bacterial antagonist from the North Western Indian Himalayas

Exiguobacterium acetylicum strain 1P (MTCC 8707) is a rhizospheric, Gram positive, rod shaped, yellow pigmented bacterium isolated from an apple orchard rhizospheric soil, on nutrient agar plates incubated at 4°C. The species level identification was arrived on the basis of 16S rRNA gene sequencing. The sequence showed 98% similarity with sequences of E. acetylicum available in the public domain. The strain was positive for siderophore and HCN production. In separate invitro assays it was found to inhibit the growth and development of Rhizoctonia solani, Sclerotium rolfsii, Pythium and Fusarium oxysporum. The volatile compound produced by the bacterium was found to be the most potent in inhibiting the hyphal development of R. solani, S. rolfsii, Pythium and F. oxysporum by 45.55, 41.38, 28.92 and 39.74% respectively. Commonly observed deformities caused by the diffusible and volatile compounds produced by the bacterium included hyphal inhibition, constriction and deformation. Under pot culture conditions the bacterium improved the germination and early growth parameters of pea (Pisum sativum) in the presence of R. solani and S. rolfsii.     

Interspecific somatic hybrids <Emphasis Type="Italic">Solanum bulbocastanum</Emphasis> (+) <Emphasis Type="Italic">S. tuberosum</Emphasis> H-8105

Interspecific somatic hybrids between a dihaploid potato clone H-8105 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuberizing species Solanum bulbocastanum were generated and analysed. Only ten regenerants displaying the intermediate morphology with dominating characteristics of the wild parent (simple leaves, anthocyanin pigmentation) were produced in 15 weeks after a single PEG-mediated fusion event. The RAPD patterns confirmed the hybridity of all of them. The hybrids rooted poorly and grew slowly in vitro. The cytological analysis revealed a high degree of aneuploidy in the hybrids with morphological and growth anomalies in vitro, while the morphologically normal hybrids were tetraploids. All the S. bulbocastanum (+) H-8105 hybrids were unstable in culture and three of them were consequently lost during three years of propagation in vitro. The possible reasons for instability of somatic hybrids between the distantly related species are discussed.     

A protocol for <Emphasis Type="Italic">in vitro</Emphasis> germination and sustainable growth of two tropical mistletoes

We report a protocol for in vitro germination and sustainable growth of two tropical mistletoes, Dendrophthoe pentandra and Macrosolen cochinchinensis . Normal mistletoe shoots with haustoria have been produced using Murashige and Skoog (MS) media supplemented with coconut water (15–20% v/v) in ventilated culture vessels. Germination of mistletoes took place on hormone-free basal MS media, and the time needed for germination was much shorter for Macrosolen than for Dendrophthoe . After germination, further development of the mistletoes required a hormonal input from the medium. We found that 20 M 6-benzylaminopurine (BA) did allow normal development of only Dendrophthoe , while coconut water (15–20% v/v) gave complete development of both species in ventilated culture vessels. Multiplication of these mistletoes is also possible via the callus stage using half-strength MS media supplemented with BA at various concentrations in ventilated culture vessels. This is probably the first report of a successful culture for the mistletoe genus of Macrosolen . Compared to Macrosolen , the nutritional and hormonal requirements for Dendrophthoe are less demanding, making the latter a useful model for further mistletoe research.     

Effect of carbohydrates on the production of thaxtomin A by <Emphasis Type="Italic">Streptomyces acidiscabies</Emphasis>

Several Streptomyces species cause plant diseases, including S. scabies, S. acidiscabies and S. turgidiscabies, which produce common scab of potato and similar diseases of root crops. These species produce thaxtomins, dipeptide phytotoxins that are responsible for disease symptoms. Thaxtomins are produced in vivo on diseased potato tissue and in vitro in oat-based culture media, but the regulation of thaxtomin biosynthesis is not understood. S. acidiscabies was grown in a variety of media to assess the impact of medium components on thaxtomin A (ThxA) production. ThxA biosynthesis was not correlated with bacterial biomass, nor was it stimulated by α-solanine or α-chaconine, the two most prevalent potato glycoalkaloids. ThxA production was stimulated by oat bran broth, even after exhaustive extraction, suggesting that specific carbohydrates may influence ThxA biosynthesis. Oat bran contains high levels of xylans and glucans, and both of these carbohydrates, as well as xylans from wheat and tamarind, stimulated ThxA production, but not to the same extent as oat bran. Starches and simple sugars did not induce ThxA production. The data indicate that complex carbohydrates may act as environmental signals to plant pathogenic Streptomyces, allowing production of thaxtomin and enabling bacteria to colonize its host.     

Fusarium root rot of coneflower seedlings and integrated control using <Emphasis Type="Italic">Trichoderma</Emphasis> and fungicides

Fusarium root rot (Fusarium spp.) is one of the most important seedling diseases of coneflower (Echinacea spp.) in Alberta greenhouses. Effects of microbial antagonists (Trichoderma spp.) and fungicides, including difenoconazole, fludioxonil, and a mixture of fludioxonil, metalaxyl and difenoconazole, on the management of this disease, were investigated in Alberta. Twenty Trichoderma isolates demonstrated antagonistic activity to Fusarium in agar plate bioassays, with inhibition rates ranging from 44 to 65%. Some Trichoderma isolates significantly ( p < 0.05) reduced disease incidence and severity on seedlings in greenhouse experiments. An in vitro bioassay indicated that difenoconazole and the mixture equally inhibited the growth of both Fusarium and Trichoderma, but, while fludioxonil strongly inhibited the growth of Fusarium, it had little effect on Trichoderma, according to the dose--response models developed ( p < 0.01, R²= 0.902-0.998). Two Trichoderma isolates, T1 and T13 were applied singly or in combination with a low rate of fludioxonil in greenhouse evaluations. The results suggested that fludioxonil and Trichoderma could be integrated into a disease management program for fusarium root rot in coneflower.