Addition Of Benzyladenine Research Articles

Flower Development in Cassava Is Feminized by Cytokinin, While Proliferation Is Stimulated by Anti-Ethylene and Pruning: Transcriptome Responses.

Cassava, a tropical storage-root crop, is a major source of food security for millions in the tropics. Cassava breeding, however, is hindered by the poor development of flowers and a low ratio of female flowers to male flowers. To advance the understanding of the mechanistic factors regulating cassava flowering, combinations of plant growth regulators (PGRs) and pruning treatments were examined for their effectiveness in improving flower production and fruit set in field conditions. Pruning the fork-type branches, which arise at the shoot apex immediately below newly formed inflorescences, stimulated inflorescence and floral development. The anti-ethylene PGR silver thiosulfate (STS) also increased flower abundance. Both pruning and STS increased flower numbers while having minimal influence on sex ratios. In contrast, the cytokinin benzyladenine (BA) feminized flowers without increasing flower abundance. Combining pruning and STS treatments led to an additive increase in flower abundance; with the addition of BA, over 80% of flowers were females. This three-way treatment combination of pruning+STS+BA also led to an increase in fruit number. Transcriptomic analysis of gene expression in tissues of the apical region and developing inflorescence revealed that the enhancement of flower development by STS+BA was accompanied by downregulation of several genes associated with repression of flowering, including homologs of TEMPRANILLO1 (TEM1), GA receptor GID1b, and ABA signaling genes ABI1 and PP2CA. We conclude that flower-enhancing treatments with pruning, STS, and BA create widespread changes in the network of hormone signaling and regulatory factors beyond ethylene and cytokinin.

Evaluation of responses embryogenic Cycas revoluta thumb., from callus culture obtained in vitro

Processes cell differentiation and dedifferentiation are included in the development of biotechnology protocols to promote somatic embryogenesis as an alternative to the in vitro propagation of plants, somatic embryos may be an excellent strategy for both propagation and conservation of fossil species such as cycads. They were evaluated in vitro with different strategies, morphogenic responses associated with obtaining somatic embryos of C. revolute. calli of megagametophytes, subsequently subcultured in four combinations of basic salts of Murashige and Skoog (1962) MS, with the addition of benzyladenine (BA), and 2, dichlorophenoxyacetic (2,4-D) were used, and kinetin (K), and picloram. In the results, It was possible to characterize potentially embryogenic callus, evaluating the levels of both cellular differentiation, necrosis, texture and color; and increases mass or weight considered to start differentiation or proembryogenic or globular type. Calli were subcultured in a medium containing MS salts, incorporating abscisic acid (ABA) in 0, 0.38, 1.13, 3.78 and 5.67 uM doses influenced both the production and maturation of somatic embryos. Embryonic structures, presented a pink coloration characteristic strongly associated towards maturity. The effect of combinations of BA, Kin, 2,4-D, GA3 and ANA influenced the development and germination of mature somatic embryos. And the combination of 1.36 mM 2,4-D+4.44 uM BA promoted the appearance of calluses with a compact texture, characteristic related to their embryogenic potential. The purpose of this research in Cycas sp was to contribute to the study of the in vitro morphogenic responses of this group of plants. And somatic embryogenesis, will allow the obtaining and multiplication as well as its preservation of Cycas sp. Gender that is evolutionary very important

PENGARUH ARANG AKTIF, BENZILADENIN, DAN KINETIN TERHADAP PERTUMBUHAN TUNAS JATI SOLOMON (Tectona grandis Linn. f) IN VITRO

Tissue culture techniques can be used for propagation of uniformaly large teak solomon seeds.The purpose of this research was knowing the effect of activated charcoal, the addition of benzyladenine (BA) and combination of BA with 6-furfurylaminopurine (kinetin) to the growth of shoots of solomon teak in vitro. The solomon teak explants used were singlestem cuttings from aseptic shoots obtained from in vitro cultures. This research was conducted in laboratory with complete randomized design with 3 replications. The experimental treatment was a single factor consisting of basic MS medium (Murashige and Skoog, 1962), with 6 treatments: MS without growth regulator (control), MS without growth regulator + 2 g/l activated charcoal, MS + 0,1 m/l BA , MS + 0,2 m/l BA, MS + 0,1 m/l BA + 0,1 m/l kinetin and MS + 0,2 m/l BA + 0,1 m/l kinetin. Observation on the number of books/ shoots, number of leaves/ shoots, shoot/ bud height and visual apperance of culture was taken at 8 weeks after planting. The data were analyzed for variety and continue the separation of the LSD at 5% level. The results showed that in general, all six treatments could be used for propagation of in vitro teak solomon (Tectona grandis Linn. f) and produced at least 6,22 books/ shoots every 8 weeks. The best media were MS medium + 0,1 m/l BA and MS + 0,1 m/1 BA + 0,1 m/l kinetin, because it able to produce 7,78 books/ shoots. The highest number of leaves was obtained at the treatment of MS + 0,1 m/l BA, while the average shoots/ shoots produced were not different for all.

Micropropagation protocol for the wild Brazilian greenberry (Rubus erythroclados)

This study presents the first micropropagation protocol for greenberry (Rubus erythroclados), a wild Brazilian species with edible green fruits. In the in vitro multiplication stage, three concentrations of benzyladenine (BA) were tested (0, 5 and 10 μM), combined with three concentrations of indolebutyric acid (IBA) (0, 3 and 6 μM) in two subsequent subcultures. In the rooting stage, in and ex vitro rooting were compared after pulse treatment of the microcutting for 10 seconds in IBA (0, 2.46, 4.92 and 7.38 mM). For the in vitro trial, the microcuttings were maintained in glass bottles with an MS medium under controlled conditions inside a growth room. For the ex vitro trial, the microcuttings were planted in styrofoam containers with vermiculite and maintained inside a greenhouse with an intermittent mist system. R. erythroclados multiplication was obtained with the addition of BA to the culture medium, while IBA reduced the shoot proliferation and increased mortality. The ex vitro rooting showed the best results, reaching 95.8% for rooted and acclimatizated plants without IBA. An efficient and simple protocol can be used for R. erythroclados micropropagation with 5 μM BA for in vitro shoot proliferation and ex vitro rooting of microcuttings with intermittent misting.

English

Spilanthes oleracea Jacq. has been extensively used due to its pharmacological activities, and it has been considered as a promising vegetable crop. Nodal-segment cultures of S. oleracea were established in order to analyze the content of alkamide spilanthol. On average, 11 buds/nodal segment were developed after 30 days on MS basal salts liquid medium containing 2.22 μM benzyladenine (BA). Regenerated shoots formed complete plantlets in medium without growth regulators, and 80.2% of the regenerated plantlets survived in field conditions. Higher production of spilanthol (58.5% relative area) was obtained from the leaves and stems of plants grown in liquid medium up to 90 days, than in field-grown plants. Addition of BA to the culture medium significantly affected spilanthol accumulation. The production of spilanthol was also detected on the shoot basal callus. The spilanthol content varied with the plant organ in micropropagated plants. Ethanolic extract of leaves from field-grown plants showed no acute toxicology in Swiss mice at a dosage of 3 g/kg. Key words: Spilanthes oleracea, toxicity, plant regeneration, calli, spilanthol, gas chromatography.

Biomass accumulation of Panax vietnamensis in cell suspension cultures varies with addition of plant growth regulators and organic additives.

To evaluate the impact of plant growth regulators including kinetin (KN), benzyl adenine and naphthalene acetic acid, yeast extract and casein hydrolyzate on biomass accumulation of Vietnamese ginseng Panax vietnamensis (P.vietnamensis) in cell suspension culture. Cell suspension cultures were established from friable calluses derived from leaves and petioles of 3-year-old in-vitro P. vietnamensis plants. The cell suspension cultures were grown in Murashige and Skoog basal media supplemented with various concentrations of KN, benzyl adenine, naphthalene acetic acid, and yeast extract and casein hydrolyzate. All tested factors generated an increase in the cell biomass of P.vietnamensis in suspension culture, but the impact of each varies depended on the factor type, concentration, and incubation period. Addition of 2.0mg/L KN resulted in the largest biomass increase after 24d, (57.0±0.9) and (3.1±0.1)mg/mL fresh and dry weight, respectively, whereas addition of benzyl adenine or naphthalene acetic acid produced optimum levels of Panax cell biomass at 1.0 and 1.5mg/L, respectively. Addition of the elicitor yeast extract led to a 1.4-2.4 fold increase in biomass of P.vietnamensis, while addition of casein hydrolyzate enhanced biomass accumulation 1.8-2.6 fold. The addition of each factor causes significant changes in biomass accumulation of P.vietnamensis. The largest biomass accumulation is from cultures grown in MS media containing 2.0mg/L KN for 24d. The outcome of the present study provides new insights into the optimal suspension culture conditions for studies on the invitro cell biomass production of P.vietnamensis.

In vitro propagation and cryopreservation of Aerides odorata Lour. (Orchidaceae).

An efficient method for in vitro propagation and cryopreservation of Aerides odorata was established. Leaf segments were cultured on New Dogashima (ND) mediums supplemented with various concentrations of Benzyladenine (BA) (0-5 mg L(-1)) combined with Naphthaleneacetic Acid (NAA) (0-2 mg L(-1)). The optimal treatment for inducing Protocorm-like Bodies (PLBs) from leaf segments was obtained from the combination of 1 or 3 mg L(-1) BA and 0.5 or 1 mg L(-1) NAA; whereas, the addition of BA or NAA alone induced shoot and/or root initiation rather than PLB or callus formation. Shoots rapidly developed on ND mediums containing 5 mg L(-1) BA. Cryopreservation of leaf segment-derived PLBs was successful using the encapsulation-dehydration method. The maximum survival percentage of Cryopreserved (Cryp) PLBs was achieved by encapsulating PLBs with 2% Na-alginate combined with 2 M glycerol and 0.4 M sucrose. The encapsulated PLBs were then precultured in 0.75 M sucrose for 24 h and dehydrated for 6 h before plunging into liquid nitrogen. Genetic stability of Cryp PLBs after regrowth was assessed by flow cytometry. The findings showed no different patterns of ploidy levels and morphology between Cryp and non-cryopreserved (Ncryp) control plantlets.

Influence of growth regulators on callogenesis and somatic embryo development in date palm (Phoenix dactylifera L.) Sahelian cultivars.

This study provides a physiological analysis of somatic embryogenesis in four elite cultivars of date palms: Ahmar, Amsekhsi, Tijib, and Amaside, from the initial callogenesis to establishment and proliferation of embryogenic suspension cultures. Somatic embryos development and in vitro plants rooting were also studied. For each step, auxins and cytokinins concentrations were optimised. The primary callogenesis from leaf explants of seedlings appeared highly dependent on genotype. Ahmar (80%) and Amsekhsi (76%) appeared highly callogenic, whereas Tijib (10%) and Amaside (2%) produced low amounts of calluses. 2,4-Dichlorophenoxyacetic acid appeared favorable to the induction of primary callogenesis and its effect was enhanced by the addition of benzyl adenine or adenine sulfate. Secondary friable calli obtained from chopped granular calli were used to initiate embryogenic cell suspensions in media supplied with 2,4-dichlorophenoxyacetic acid. Suspension cultures showed a growth rate of fourfold after four subcultures in presence of 2,4-dichlorophenoxyacetic acid 2 mg/L. Our results showed that a seven-day transitory treatment with benzyl adenine 0,5 mg/L was necessary to optimize embryos development. Naphthalene acetic acid induced the development of primary orthogravitropic roots during embryos germination. The comparison with cytofluorometry of nuclear DNA amounts showed no significant difference in ploidy level between regenerated plants and seedlings.

Effects of Spectral Composition Conversion Film and Plant Growth Regulators on Proliferation of Cymbidium Protocorm Like Body (PLB) Cultured In Vitro

We examined the effects of the spectral composition conversion red film on the proliferation of protocorm like bodies (PLBs) and formation of shoots and roots in Cymbidium finlaysonianum and C. insigne in the presence and absence of benzyladenine (BA) and 1-naphthaleneacetic acid (NAA). PLBs were cultured under light provided by a cool white fluorescent light and transmitted through the red film with a 16-h photoperiod for 40 days. The addition of NAA (1 mg/L) and BA (2 mg/L) under the cover of the red film increased the number of PLBs significantly; hence, this is the most effective technique for increasing the number of C. finlaysonianum PLBs. Forty days after culture, the order of increase in the number of C. finlaysonianum PLBs was as follows: Red film+NAA+BA>NAA+BA>Red film>Control. In this study, we demonstrated that the spectral composition conversion film can be used as an alternative growth regulator for PLB proliferation in Cymbidium species.

Impact of hypoxia on the growth and alkaloid accumulation in Catharanthus roseus cell suspension

The Madagascar periwinkle (Catharanthus roseus) produces numerous indole alkaloids, several of which have an important pharmaceutical uses such as ajmalicine, vinblastine and vincristine. The relationship between hypoxia and ajmalicine production in a cell suspension culture of C. roseus were investigated during the cycle of cell culture, in correlation with the effects on growth. The results show that the lack of oxygenation in C20D cells provokes a very strong inhibition in accumulation of the alkaloids and of other possible substances. Moreover, the addition of loganin, a metabolic intermediate of the biosynthetic pathway, in the culture medium of cells subjected to hypoxia restored the alkaloid production. Also, the results showed that the addition of benzyladenine (BA) to the culture medium increased the ajmalicine production and that the inhibitory effect of hypoxia was almost absent in these conditions. Therefore, it could be suggested that BA can without doubt decrease the effects of the hypoxia and increase the ajmalicine production in periwinkle cell suspensions.

Light cytokinin interactions in shoot formation in epicotyl cuttings of Troyer citrange cultured in vitro

Ilumination did not affect the pathway of shoot regeneration at the cut edges of epicotyl explants of Troyer citrange (Moreira-Dias et al. 2000, 2001), but signigficantly affected the number of developed shoots and the response to exogenous cytokinins. Shoot regeneration at the apical end occurred through a direct organogenic pathway without callus formation. For explants incubated in the light, this regeneration did not require cytokinin addendum, but the number of shoots formed was significantly increased by benzyl adenine, but not by zeatin or kinetin. Incubation in the dark almost suppressed shoot formation at the apical end. The addition of benzyl adenine or kinetin, but not of zeatin, restored shoot formation in the dark to the value obtained in the light. At the basal end of the explants shoot regeneration occurred through an indirect organogenic pathway after the formation of a primary callus. In explants incubated in the light, callus formation and shoot growth was supported by a low (0.5–1 mg l−1) benzyl adenine concentration and by zeatin. Kinetin did not support callus growth. Shoot formation was higher in the presence of benzyl adenine (0.5–1 mg l−1) than of zeatin, but was inhibited by a high (5 mg l−1) benzyl adenine concentration. Incubation in the dark increased callus growth and shoot formation at the basal cut as compared to explants incubated in the light. The three cytokinins tested supported callus growth and shoot formation in the dark, zeatin being the most effective and kinetin the least. In terms of number of shoots developed, the optimum cytokinin addendum depended on the pathway of organogenesis and the conditions of incubation. The maximum number of shoots developed at the apical end was obtained when the incubation was performed in the light in the presence of benzyl adenine. At the basal end, the optimal conditions were incubation in the dark in the presence of zeatin. It was not always possible to define an optimal cytokinin concentration as the curve concentration/response varied from experiment to experiment, which seemed unrelated to the endogenous cytokinin concentration in the explants.

Improved plant regeneration and in vitro somatic embryogenesis of St Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze

AbstractSt Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is an important warm season turf and pasture grass. In vitro tissue culture of St Augustinegrass could serve as an important mean for its improvement through genetic transformation as well as induced somaclonal variation. To optimize tissue culture conditions for plant regeneration of St Augustinegrass, tissue culture responses of 11 explant tissues and four callus induction/subculture media have been examined. Embryogenic calli with regeneration potential were observed on cultures of early immature embryo [3 days after pollination (DAP)], immature embryo (7–14 DAP), and shoot base of young seedlings. The addition of benzyladenine (BA) in the callus induction/subculture medium enhances callus regeneration ability and does not harm callus induction for immature embryos. The best response came from 7 to 14 DAP immature embryo on MS medium containing 1 mg/l 2,4‐dichlorophenoxyacetic acid and 0.5 mg/l BA. The callus induction and regeneration rates were 97.7% and 47.6% respectively. However, BA supplement reduced callus formation and failed to enhance regeneration for young leaf bases. Scanning electron microscopy revealed that plant regeneration of St Augustinegrass is via somatic embryogenesis.

The role of Pentadienoic growth regulator acid by number of growth regulators in the initiation, growth and differentiation of Nigella sativa Callus

The effect of Pentadienoic acid (PDA) as a synthetic growth regulator, on the induction, growth and differentiation of callus from stem segments of seedlings of black seed Nigella sativa L. was carried out. PDA was used at the concentrations 10"4, 10 and 10"8 molar alone, or with the optimal concentration of Benzyl Adenine (BA) which is 10"3 molar,and that for Dichlorophenoxyacetic acid (2,4-D) 1 0 molar. The results showed that the addition of PDA alone to the nutritional medium disencourage the induction of callus , However when PDA was added along with the optimal concentrations of the standard growth regulators, the best growth of callus obtained on the medium supplied wHh(10" ) molar of each of 2,4-D and PDA.Thu; fresh weight of callus reached (25.6) gm at 100 days age .it was found also that the addition of BA with PDA encourages growth of shoots since the first stage of the growth, with the stimulation of the callus growth also ,but the presence of 2,4-D with PDA does not encourage any differentiation except the induction and growth of the callus.

Spatially quantitative control of the number of cotyledons in a clonal population of somatic embryos of hybrid larch Larix x leptoeuropaea.

Many conifer embryos, both in natural seeds and in clonal populations of somatic embryos, display variability in the number of cotyledons. In hybrid larch, Larix x leptoeuropaea (synonymous with L. x marschlinsii Coaz), such variability has previously been reported in somatic embryos, together with a decrease in the average cotyledon number when benzyladenine (BA) is applied exogenously. Described here is a spatially quantitative study with the aim of throwing some light on the way cotyledon number is determined, and hence the mechanism of cotyledon formation. Stock cultures of embryogenic tissue were maintained and later made embryogenically active by standard methods. Development through cotyledon formation was followed by optical microscopy with quantitative measurement of embryo diameter and number of cotyledons. SEMs of representative stages and cotyledon numbers were done for purposes of illustration in this account. Existing mathematics of waveforms on a disc were cast into a form suitable to compare with the quantitative data. The number of cotyledons is linearly related to the diameter of the apical surface of the embryo (which approximates a circular disc) at the time of first appearance of the cotyledon primordia. This linearity is a constant-spacing phenomenon between adjacent primordia. Addition of BA to the medium restricts the range of apical diameters without changing inter-cotyledon spacing. Slope/intercept ratio of the linear plot matches expectation for initiation of cotyledon pattern as a harmonic waveform on a circular disc. The entire pattern of cotyledon primordia arises as a single entity coordinated by a mechanism with wave-forming properties. This is explicable by diverse mechanisms, especially either mechanical buckling ('biophysical') or reaction-diffusion kinetics ('physicochemical').

Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins.

The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.

Isolation and expression of a novel starch-storing cell-specific gene containing the KH RNA binding domain from tobacco-cultured cells BY-2.

In cultured Bright Yellow-2 tobacco (Nicotiana tabacum) cells, the depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) in the culture medium induces amyloplast development. This differentiation also includes a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and accelerated by the addition of benzyladenine (BA). Three cDNAs were identified whose transcript levels are specifically increased during differentiation of starch-storing cells using the differential display method, and designated as starch-storing cell induced genes (SCI genes). One of these cDNAs, SCI2 encodes a 285 amino acids long protein with a KH RNA-binding domain. A database search revealed that this protein showed similarity to respective domains of mammalian quaking proteins. 2,4-D addition, which can convert starch-storing cells into dividing cells, to starch-storing BY-2 cells, immediately decreases the SCI2 transcript level, suggesting that SCI2 may have some role in starch-storing cell differentiation in BY-2 cells.

Micropropagation of Echinacea angustifolia, E. pallida, and E. purpurea from Stem and Seed Explants

Micropropagation of three Echinacea species, E. angustifolia DC., E. pallida Nutt., and E. purpurea Moench., was investigated as a potential means of germplasm preservation of species faced with overcollection in the wild and rapid clonal propagation of elite individuals with unique medicinal or ornamental properties. Very high contamination rates occurred with shoot-tip explants but not with nodal segments. Contamination rates for seed explants were inversely related to the number of seedcoat layers removed, ranging from 100% contamination from intact seeds to near 0% contamination from excised embryos. Dormancy of seed explants was also eliminated when the pericarp and integument were removed. Addition of benzyladenine (BA) to the culture medium induced shoot multiplication and inhibited root formation in all three species. Shoot multiplication rates were low (1-3 shoots per culture) when seed explants were placed on a medium with BA levels low enough to avoid adventitious shoot formation (0.45 μm). Shoot count was higher on half-strength Murashige and Skoog (MS) minerals, while leaf size was greater on full-strength MS minerals. Cultures did not perform well in Woody Plant Medium. Reducing subculture frequency from 4 to 2 weeks increased shoot multiplication from 1.4 to 1.8 shoots per subculture and total shoots produced per subculture after 12 weeks from 2.8 to 23.9. Rooting occurred readily on shoots isolated from E. purpurea cultures and was not promoted by addition of IBA to the rooting medium. Rooting was low and nil on shoots from cultures of E. angustifolia and E. pallida, respectively. Methods described in this study allow rapid multiplication of three Echinacea species and subsequent rooting of E. purpurea. Future improvements in root induction treatments will allow these methods to be used effectively for micropropagation and maintenance of disease-free germplasm of Echinacea species. Chemical names used: N-(phenylmethyl)-1H-purine-6-amine (BA); 1H-indole-3-butyric acid (IBA).

Effect of Benzyladenine and Indolebutyric Acid on Ultrastructure, Glands Formation, and Essential Oil Accumulation in Lavandula Dentata Plantlets

Lavandin (Lavandula dentata) axillary buds were grown in Linsmaier-Skoog (LS) medium solidified with 10 % bactoagar (control) and supplemented with 0.1 mg dm-3 benzyladenine (BA), 0.1 mg dm-3 indolebutyric acid (IBA) or both plant growth regulators. In the studied conditions the axillary buds developed into plantlets. The addition of BA inhibited the formation of glands by 44 % as compared with the control plantlets and also inhibited their development: these plantlets had the highest number of unbroken glands (in pre-secretory state) when compared with plantlets grown in the other conditions. The presence of BA stimulated chloroplast formation, and increased the content of essential oils by 150 % with respect to the control plantlets. It also increased their secretion, and the number of lipid droplets in the chloroplasts, cytosol and plasmalemma. On the contrary, the presence of IBA decreased the essential oil concentration in plantlets by 31 % when compared with the control ones and inhibited their secretion capacity.

Direct and Indirect Shoot Organogenic Pathways in Epicotyl Cuttings of Troyer Citrange Differ in Hormone Requirements and in their Response to Light

Abstract Bud differentiation by direct organogenesis at the apical end of Troyer citrange (Citrus sinensis[L]. Osbeck×Poncirus trifoliata[L]. Raf.) epicotyl cuttings inserted vertically in a semi-solid culture medium did not require hormone additions. The number of buds regenerated was slightly, but significantly, increased when the incubation was performed in the light as compared to the dark, and by the addition of benzyladenine (BA; 2.2 to 22 μ m ) to the medium. Bud sprouting and subsequent shoot formation required the addition of BA and was increased by light to a higher extent than bud formation. The best response was obtained with the highest BA concentration tested (22 μ m ). Regeneration through the indirect organogenic pathway at the two edges of the epicotyl cuttings when in contact with the culture medium did not occur in the absence of benzyladenine, which was an absolute requirement for callus development. The best regeneration response was obtained when the explants were incubated in the light in the presence of 4.4 μ m BA and an auxin. Indole-3-acetic acid (IAA; 5.8 μ m ) was more effective in increasing shoot formation than naphthaleneacetic acid (NAA; 0.54 μ m ). Higher NAA concentrations inhibited shoot formation. Incubation in the dark or increasing the BA concentration (22 μ m ) increased markedly callus growth, but inhibited both bud differentiation and sprouting, almost completely suppressing shoot formation. The conditions during regeneration affected the rooting of the regenerated shoots. Rooting of 86% of the shoots was achieved in a medium with 2.7 μ m NAA and 2.6 μ m indole-3-butyric acid. All the rooted explants acclimated and survived transplanting. Under the optimal conditions tested, the proliferation rate obtained through the indirect regeneration pathway ranged from 60 to 86 plants per seedling.

Secondary Metabolites in Transformed Root Cultures of Campanula glomerata

Summary The transformed roots of Campanula glomerata L. var. dahurica Fish., induced by infection with Agrobacterium rhizogenes MAFF 03-01724, produced the anthocyanins cyanidin 3-glucoside and cyanidin 3-rutinoside, a flavonoid rutin and a phenylpropanoid glucoside coniferin, together with the polyacetylene constituents lobetyol, lobetyolin and lobetyolinin. Although illumination was inhibiting for root proliferation, it was an essential factor for the accumulation of anthocyanin and flavonoid in the tissues. Addition of benzyladenine (BA) to the medium also promoted the accumulation of phenolics (anthocyanins, rutin and coniferin) in the roots. Polyacetylene production in the roots was not greatly affected by either illumination or BA treatment.