Modulation of berberine bridge enzyme levels in transgenic root cultures of California poppy alters the accumulation of benzophenanthridine alkaloids

California poppy (Eschscholzia californica Cham.) root cultures produce a variety of benzophenanthridine alkaloids, such as sanguinarine, chelirubine and macarpine, with potent biological activity. Sense and antisense constructs of genes encoding the berberine bridge enzyme (BBE) were introduced into California poppy root cultures. Transgenic roots expressing BBE from opium poppy (Papaver somniferum L.) displayed higher levels of BBE mRNA, protein and enzyme activity, and increased accumulation of benzophenanthridine alkaloids compared to control roots transformed with a -glucuronidase gene. In contrast, roots transformed with an antisense-BBE construct from California poppy had lower levels of BBE mRNA and enzyme activity, and reduced benzophenanthridine alkaloid accumulation, relative to controls. Pathway intermediates were not detected in any transgenic root lines. Suppression of benzophenanthridine alkaloid biosynthesis using antisense-BBE also reduced the growth rate of the root cultures. Two-dimensional ¹H-NMR spectroscopy showed no difference in the abundance of carbohydrate metabolites in the various transgenic roots lines. However, transformed roots with low levels of benzophenanthridine alkaloids contained larger cellular pools of certain amino acids compared to controls. In contrast, cellular pools of several amino acids were reduced in transgenic roots with elevated benzophenanthridine alkaloid levels relative to controls. The relative abundance of tyrosine, from which benzophenanthridine alkaloids are derived, was only marginally altered in all transgenic root lines; thus, altering metabolic flux through benzophenanthridine alkaloid pathways can affect cellular pools of specific amino acids. Consideration of such interactions is important for the design of metabolic engineering strategies that target benzophenanthridine alkaloid biosynthesis.     

Cell type-specific protoberberine alkaloid accumulation inThalictrum flavum

Berberine is a common benzylisoquinoline alkaloid with potent antimicrobial properties, which suggest it functions to protect some plants from pathogen challenge. Berberine was identified as the major alkaloid in meadow rue (Thalictrum flavum ssp. glaucum), a medicinal member of the Ranunculaceae, and was detected in seeds and all organs of the plant. The high level of berberine in roots, rhizomes, and older petioles is mainly responsible for the intense yellow color of these organs. In rhizomes, protoberberine alkaloids were detected throughout the pith and, to a lesser extent, the cortex, but were absent from the vascular tissues. Similarly, protoberberine alkaloids were detected in the rib parenchyma of older petioles. In roots, alkaloid accumulation was detected only in the endodermis at the onset of secondary growth. Rather than being sloughed off, the endodermis was found to undergo extensive anticlinal division leading to an expanding cellular cylinder that ultimately displaced all external tissues. Endodermal-specific protoberberine alkaloid accumulation continued throughout root development, but was extended to include 3 to 4 layers of smaller pericycle cells in the oldest roots near the base of the stem. The cell type-specific accumulation of antimicrobial alkaloids and the unusual development of the endodermis and pericycle in T. flavum roots support the putative role of berberine in plant defense.     

Tropane Alkaloids from Callus Cultures, Differentiated and Shoots of Hyoscyamus muticus L.

Alkaloid production has been observed in cotyledonary leaf derived callus tissues, and also in in vitro differentiated shoots, and roots of Hyoscyamus muticus. The callus tissue was developed form cotyledonary leaf explants on Murashige and Skoog medium enriched with 2 mg 1^-1 2, 4-D and 0.5 mg 1^-1 BAP. Cotyledonary leaf derived callus was proliferated in the same medium for 2 passages (1 passage 28-30 days). Green and compact callus was used for alkaloid extraction. Shoots and roots formed on MS medium containing 0.05 mg 1^-1 NAA and 0.5 mg 1^-1 BAP, and also compact, nodular and embryogenic calli from which these shoots and roots differentiated, were used for alkaloid extraction. Chromatographic studies performed with TLC showed the presence of hyoscyamine as the major alkaloid present in the callus tissues, differentiated shoots and roots. However, alkaloid content varied in different tissues. Differentiated roots were found to contain maximum amount of hyoscyamine.     

<Emphasis Type="Italic">In vitro</Emphasis> propagation of an endangered plant species, <Emphasis Type="Italic">Thermopsis turcica</Emphasis> (Fabaceae)

This report deals with micropropagation of the critically endangered and endemic Turkish shrub, Thermopsis turcica using callus, root and cotyledonary explants. Callus cultures were initiated from root and cotyledon explants on MS medium supplemented with 0.5–20 μM NAA or 2,4-D. The root explants were found to be better in terms of quick responding and callusing percentages as compared to the cotyledons. Organogenic callus production with adventitious roots and shoots were obtained on MS medium with only NAA. The calli obtained with NAA, root and cotyledonary explants were cultured with BA and kinetin (2–8 μM) alone or in combination with a low level (0.5 μM) of 2,4-D or NAA. The best regeneration of shoots from root explants was observed on hormone-free MS medium. NAA with BA or kinetin in the medium improved shoot induction from the calli obtained with NAA. Maximum percentage of shoots (93.3%), maximum number of shoots (6.2) and maximun length of shoots (8.22 cm) were achieved from cotyledonary explants at 4 μM BA and 0.5 μM NAA. The presence of 0.5 μM or higher levels of 2,4-D in shoot induction medium inhibited the regeneration in T. turcica explants. 83% of in vitro rooting was attained on pulsed-IBA treated shoots. The regenerated plants with well developed shoots and roots were successfully acclimatized. Application of this study’s results has the potential to conserve T. turcica from extinction.     

In Vitro Organogenetic Responses of Gloriosa superba

An efficient protocol was developed for regeneration of healthy plant derived from six categories of explants from both in vivo and in vitro raised plants, viz. roots, corm buds (dormant and nondormant), young leaves, stems, pedicels, and shoot tips from aerial shoots. MS medium supplemented with various concentrations and combinations of auxin, cytokinin, and organic acids was used. 98% of callus induction occurred in nondormant corm bud explants. The greatest number of multiple shoots (57) was observed in corm-derived calluses. Vigorous root formation occurred in all cases when multiple shoots were derived. Histomorphogenetic studies revealed that not only the origin of shoot and root buds in in vitro systems, but the morphology and structure of leaves resemble those of in vivo plants too.     

Adventitious rooting of Jatropha curcas L. is stimulated by phloroglucinol and by red LED light

An efficient root induction system has been established for in vitro-regenerated Jatropha curcas L. shoots. Callus formation on shoots transferred to auxin containing medium was found to be a prominent and recurrent problem for rooting of in vitro-cultivated J. curcas. In particular, the type of auxins and cytokinins applied in the culture media were shown to strongly influence the severity of callus formation. Shoots cultivated on meta-methoxytopolin riboside (MemTR) were free of callus and produced elongated stems and well-developed leaves in comparison to the cytokinins benzyl adenine, zeatin, and thidiazuron. Subsequent root induction experiments were performed with shoots precultured on MemTR-containing medium. Shoots were excised and transferred to Murashige and Skoog (MS) medium supplemented with different concentrations of indole-3-butyric acid (IBA), indole-3-acetic acid (IAA), and α-naphtaleneacetic acid (NAA). The induction of excessive callus formation was avoided only on IBA-containing medium. The optimum rooting medium with good root induction (35%) and 1.2 roots per shoot contained half-strength MS salts supplemented with 2.5 μM IBA. The same medium supplemented with 0.25% (w/v) activated charcoal produced 46% rooted shoots. Further improvement of rooting was obtained by transferring in vitro grown shoots to woody plant medium containing phloroglucinol (PG). In the presence of 2.5 μM IBA and 238 μM PG, 83% of the shoots rooted with on average 3.1 roots per shoot. We also analyzed the impact of light quality on the rooting capacity of Jatropha in vitro grown shoots. In general, light-emitting diodes (LEDs) light sources were less efficient for root induction. Red LED light provided the most favorable growth conditions, inducing a rooting response in 65% of the shoots, which produced on average 5.5 roots per shoot. These results indicate that adventitious rooting in J. curcas is under control of photoreceptors and that optimal rooting requires fine-tuning of the salt concentration, auxin, and cytokinin balance and application of synergistic compounds.     

Establishment of Camptotheca acuminata regeneration from leaf explants

Plantlet regeneration through shoot formation from young leaf explant-derived callus of Camptotheca acuminata is described. Calli were obtained by placing leaf explants on Woody plant medium (WPM) supplemented with various concentrations of 6-benzyladenine (BA) and naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). Callus induction was observed in all media evaluated. On the shoot induction medium, the callus induced on the WPM medium containing 19.8 μM BA and 5.8 μM NAA was the most effective, providing high shoot regeneration frequency (70.3 %) as well as the highest number of shoots (11.2 shoots explant⁻¹). The good rooting percentage and root quality (98 %, 5.9 roots shoot⁻¹) were achieved on WPM medium supplemented with 9.6 μM indole-3-butyric acid (IBA). 96 % of the in vitro rooted plantlets with well developed shoots and roots survived transfer to soil.     

The role of auxins in differentiation of rice tissues culturein Vitro

Effects of various auxins on callus induction (dedifferentiation) and organ redifferentiation from the callus were studied by using various tissues of rice,Oryza sativa L. cv. Kyoto Asahi. 2,4-D, NAA and IAA were used as auxins for the test of their ability to induce callus. All of these were active. This callus induction by auxin was successful in all tissues used; seed, root, shoot nodule, anther and ovary. In all of the calluses induced by various auxins such as 2,4-D, NAA and IAA and derived from various tissues such as seed, root, shoot nodule, anther and ovary, organ redifferentiation, i.e., formation of shoots and roots was achieved by removing the auxins from the medium used for the callus calture. Cytokinins were not necessary for the organ redifferentiation in these calluses. These results suggest that auxin is the only exogenous factor that determines dedifferentiation and redifferentiation in rice plant tissues culturedin vitro.     

In vitro regeneration of Solanum nigrum with enhanced solasodine production

High frequency of shoot formation was achieved from Solanum nigrum L. leaves on Murashige and Skoog (MS) medium without any callusing stage. Shoot forming ability was more pronounced on leaves positioned dorsally. For shoot induction, 2.0 mg dm⁻³ benzylaminopurine and 1.5 mg dm⁻³ kinetin were observed to be the most effective plant growth regulators (PGRs). The present paper also describes first successful induction of in vitro flowering in S. nigrum. The leaf derived shoots were excised and treated with various root promoting PGRs and 0.25 mg dm⁻³ indole-3-butyric acid produced maximum number of roots (15.2 per plant). Plants were later transplanted in field with 100 % survival. Solasodine content was higher in in vitro raised shoots and leaf derived callus, compared to ex vitro grown shoots.     

Genetic transformation of Tylophora indica with Agrobacterium rhizogenes A4: growth and tylophorine productivity in different transformed root clones

We have developed an efficient transformation system for Tylophora indica, an important medicinal plant in India, using Agrobacterium rhizogenes strains LBA9402 and A4 to infect excised leaf and stem explants and intact shoots at different sites. The induction of callus and transformed roots was dependent on the bacterial strain, explant type and inoculation site used. Transformed roots were induced only in explants infected with A. rhizogenes strain A4, while an optimal transformation frequency of up to 60% was obtained with intact shoots inoculated at the nodes. The presence of the left-hand transferred DNA (T_L-DNA) in the genome of T. indica roots induced by A. rhizogenes was confirmed by PCR amplification of the rooting locus genes of A. rhizogenes. Root growth and the production of tylophorine, the major alkaloid of the plant, varied substantially among the nine root clones studied. Both parameters increased over time in liquid cultures, with maximum biomass and tylophorine accumulation occurring within 4–6 weeks of growth in fresh medium. Interestingly, in liquid culture, the culture medium also accumulated tylophorine up to concentrations of 9.78±0.21 mg l^–1.     

The Physiology of Rooting Populus Cuttings

The changing levels of carbohydrates and the rates of photosynthesis in rooting Populus cuttings are described. The cuttings were planted in a sand medium, in controlled environment conditions. There was no evidence to support the view that failure to root in P. tremula was caused by insufficient carbohydrate reserves. In leafless hardwood cuttings, the carbohydrate levels were initially very high (14–19%) but fell rapidly (to 5–10%) as roots, callus or shoots developed. In leafy softwood cuttings, a steady accumulation of carbohydrate reserves was observed, rising from 5–10% on planting to 15–25% when roots had developed. One of the differences between the easy-to-root P. × euramericana and the hard-to-root P. tremula was the apparent downward transport of assimilates in P. × euramericana softwood cuttings.     

An in vitro approach to investigate medicinal chemical synthesis by three herbal plants

Ever since regulatory changes introduced herbals into mainstream supermarkets and pharmacies, there has been an explosion of demand for herbal plants and extracts which can be used to improve human health and well being. Science still lacks a basic mechanistic understanding of how environmental triggers regulate phytochemical accumulation, but this gap can be bridged using in vitro models to examine herbal species responses. For St. John's wort (Hypericum perforatum), uniform in vitro shoot cultures were set up as a parallel to a previously established sand culture system for investigation of physical and chemical environmental factors that control hypericin accumulation. Cytokinin supplementation of shoot culture medium resulted in a proliferation of abundant leaf glands with enhanced levels of hypericin, as compared to controls. Cell cultures of echinacea (Echinacea angustifolia) were established, and hydrophilic pharmacological components (caffeic acid derivatives) were detected. A protocol of rigorous explant pretreatment, and use of newly emerging vegetative shoots permitted establishment of axenic kava (Piper methysticum) callus, which was used to regenerate roots (organogenesis). Kavapyrone synthesis was achieved in both undifferentiated cell cultures and in cultured roots, although at lower levels than found in in vivo root systems. The predominance of kavain and methysticin in both forms of the in vitro cultures was parallel to the relative proportions from kava roots in vivo. The cell and organ cultures of all three herbal medicinals provide advantageous, easily-manipulated models to decipher environmental controls of phytochemical biosynthesis.     

Highly efficient regeneration and medicinal component determination of Phellodendron chinense Schneid

Phellodendron chinense Schneid is an important Chinese herb with berberine and phellodendrine in stems and leaves, but with little information available on in vitro culture of this species. Disinfection of explants in 75% alcohol for 45 s, sterilization in 0.1% HgCl₂ for 20 min, and submersion in 1.0 mol L⁻¹ gibberellin3 (GA₃) solution for 24 h was the optimal condition for seed germination. Murashige and Skoog’s (MS) medium supplemented with 2.0 mg L⁻¹ 6-benzylaminopurine (6-BA) in combination with 1.5 mg L⁻¹ 1-naphthylacetic acid (NAA) was optimal for callus induction. MS medium supplemented with 2.0 mg L⁻¹ 6-BA was the appropriate medium for induction of adventitious shoots, and 1/2MS medium supplemented with 2.0 mg L⁻¹ indole-3-butytric acid (IBA) and 0.5% active carbon was the optimal medium for root induction. The 15-d survival rate of regenerated plantlets after transplanting to basins containing perlite and peat moss (1:4) was greater than 80%, and the berberine and phellodendrine accumulation was lower in callus compared with regenerated plantlets. The establishment of highly efficient regeneration system provides technical support for genetic breeding of Phellodendron chinense Schneid.

     

Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.Key words: In vitro propagation, Pyridone alkaloid, Cerpegin, Callus, Ceropegia juncea     

Callus,shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants

We analyzed the impact of ethylene and auxin disturbances on callus, shoots and Agrobacterium rhizogenes-induced hairy root formation in tomato (Solanum lycopersicum L.). The auxin low-sensitivity dgt mutation showed little hairy root initiation, whereas the ethylene low-sensitivity Nr mutation did not differ from the control Micro-Tom cultivar. Micro-Tom and dgt hairy roots containing auxin sensitivity/biosynthesis rol and aux genes formed prominent callus onto media supplemented with cytokinin. Under the same conditions, Nr hairy roots did not form callus. Double mutants combining Rg1, a mutation conferring elevated shoot formation capacity, with either dgt or Nr produced explants that formed shoots with little callus proliferation. The presence of rol + aux genes in Rg1 hairy roots prevented shoot formation. Taken together, the results suggest that although ethylene does not affect hairy root induction, as auxin does, it may be necessary for auxin-induced callus formation in tomato. Moreover, excess auxin prevents shoot formation in Rg1.     

Efficient callus-mediated regeneration and <Emphasis Type="Italic">in vitro</Emphasis> root tuberization in <Emphasis Type="Italic">Trichosanthes kirilowii</Emphasis> Maxim., a medicinal plant

Trichosanthes kirilowii Maxim. is a climbing herb with considerable medicinal value. In this study, efficient protocols for callus-mediated regeneration and in vitro tuberization of this plant were developed. Sterilized stem and leaf tissues were cultured on Murashige and Skoog (MS) medium with plant growth regulators (PGRs), and additives that promoted callus induction and regeneration. Both stem and leaf tissues showed the best response (100%) for callus initiation on MS medium supplemented with 4.5-μM 2,4-dichlorophenoxyacetic acid (2,4-D). Efficient shoot organogenesis was obtained by exposing the callus tissue to 4.6-μM kinetin, 2.2-μM 6-benzylaminopurine, and 2.7-μM 1-naphthylacetic acid (NAA) along with 12.6-μM copper sulfate, which yielded a shoot regeneration rate of 85.5% and 28 shoots derived from each callus. In vitro shoots were best rooted on half-strength (1/2) MS medium with 2.7-μM NAA. Tuberous roots were efficiently induced on rooting medium with 5% (w/v) sucrose under short illumination conditions (8 h photoperiod). Rooted plantlets were successfully acclimatized in pots with a >?90% survival rate. This protocol provides an effective method for callus-mediated regeneration and in vitro root tuberization.     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration and plant establishment of <Emphasis Type="Italic">Tylophora indica</Emphasis> (Burm. F.) Merrill: Petiole callus culture

Summary A protocol has been developed for high-frequency shoot regeneration and plant establishment of Tylophora indica from petiole-derived callus. Optimal callus was developed from petiole explants on Murashige and Skoog basal medium supplemented with 10μM2,4-dichlorophenoxyacetic acid +2,5μM thidiazuron (TDZ). Adventitious shoot induction was achieved from the surface of the callus after transferring onto shoot induction medium. The highest rate (90%) of shoot multiplication was achieved on MS medium containing 2.5μM TDZ. Individual elongated shoots were rooted best on halfstrength MS medium containing 0.5μM indole-3-butyric acid (IBA). When the basal cut ends of the in vitro-regenerated shoots were dipped in 150μM IBA for 30 min followed by transplantation in plastic pots containing sterile vermiculite, a mean of 4.1 roots per shoot developed. The in vitro-raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in a greenhouse with 100% survival. Four months after transfer to pots, the performance of in vitro-propagated plants of T. indica was evaluated on the basis of selected physiological parameters and compared with ex vitro plants of the same age.