Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium × hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.     

Role of purine metabolism in thidiazuron-induced somatic embryogenesis of geranium (Pelargonium×hortorum) hypocotyl cultures

Somatic embryogenesis in geranium ( Pelargonium × hortorum Bailey cv. Scarlet Orbit Improved) was achieved by culturing hypocotyl sections on Murashige and Skoog (1962) (MS) medium containing 10 μ M thidiazuron (TDZ) (induction medium) for 3 days and subsequently transferring the sections onto a basal medium lacking any plant growth regulators (expression medium). Addition of the purine analogue 2.6-diaminopurine (DAP) to the somatic embryo induction medium completely inhibited the embryogenic response as well as chlorophyll accumulation without affecting enlargement of the treated tissues. Addition of 20 μ M adenine sulphate to the expression medium, i.e during embryo growth and development phase, completely reversed the DAP-induced inhibition of the embryogenic response while addition during the induction phase caused only a 50% reversal of the inhibition. Analysis of endogenous levels of plant growth substances indicated that TDZ alone elevated the levels of auxins, cy-tokinins and abscisic acid while the presence of DAP during the induction phase caused a further increase in the levels of adenine and adenosine. These findings indicate a possible critical role for purines in embryogenesis from geranium hypocotyl tissues. However, the conversion of cytokinin bases to their corresponding nucleotide forms was not evident as the levels of isopentenyl adenine and zeatin increased during the second day of culture.     

Thidiazuron-induced somatic embryogenesis in intact seedlings of peanut (Arachis hypogaea): Endogenous growth regulator levels and significance of cotyledons

The regulatory role of thidiazuron (TDZ) and explant factors in imparting somatic embryogenic potential was assessed in relation to endogenous growth regulator levels in peanut ( Arachis hypogaea L. cv. Tango). TDZ induced somatic embryogenesis over a range of concentrations (0.5 to 10 μ M ). Culture of seedlings for just 2 days on TDZ-supplemented medium was sufficient to induce somatic embryogenesis. Seedling age and retention or removal of cotyledons during culture influenced morphogenic potential significantly. The younger the seedlings, the better was the embryogenic response to TDZ treatment. The embryogenic potential of seedlings was limited for explants with no cotyledons and they did not respond to increasing levels of TDZ. In contrast, retention of at least one or both the cotyledons resulted in increased response to TDZ. Endogenous levels of cytokinins, auxins and abscisic acid were influenced by TDZ treatment, while TDZ itself was detected only in the cotyledons. The cytokinin N⁶-(2-iso-pentenyl)adenine (2iP) fluctuated on a 10-day cycle in the cotyledons; TDZ treatment suppressed this change and caused an overall decrease in the pool of available 2iP in the tissue. It appeared that TDZ induced somatic embryogenesis in peanut by influencing endogenous levels of both auxin and cytokinins.     

Thidiazuron required for efficient somatic embryogenesis from suspension-cultured cells ofPimpinella brachycarpa

To maintain embryogenic cell lines ofPimpinella brachycarpa, we suspension-cultured friable and rapidly growing yellowish calli in an MS liquid medium containing 0.2 ~ 2,4-D and 0.5pM BAP. Efficient somatic embryogenesis was achieved when selected cells were then transferred to an MS medium (0.2% gelrite) that contained 0.2gM 2,4-D, 0.5 uM BAP, and 10.0 laM TDZ (thidiazuron). These cells were cultured at 27°C under continuous illumination (21.5 I~E m^-2 s^-l). Embryogenic calli expanded about four-fold, and developed into pale yellow calli. Somatic embryogenesis was initiated only from glossy and nodular-type calli. After two more weeks of culture, globular embryos appeared on the surface of calli grown in the MS medium that contained 10.0 /aM TDZ only, or in combination with 0.5 gM NAA. Experimenting with 2,4-D, an auxin, to promote embryogenic calli resulted in excessive browning and death. We overcame this problem by growing glossy embryogenic and nodular calli on media that contained 10.0 gM TDZ. Calli that were not treated with TDZ turned dark brown and were not viable. Up to 74% of the calli showed somatic embryos when the medium was supplemented with 10.0 uM TDZ and 0.5 uM NAA. Embryos from these TDZ-induced, somatic embryogenic calli grew efficiently, forming multiple shoots and developing into normal plants. Therefore, efficient differentiation of suspension-cultured cell clusters into embryogenic calli, along with treatment of subsequent somatic embryos by TDZ, suggests that TDZ probably helps in establishing the optimum cytokinin-auxin ratio required for induction and expression of somatic embryogenesis.     

Induction by thidiazuron of somatic embryogenesis in intact seedlings of peanut

In planta differentiation of somatic embryos was induced in seedlings of peanut (Arachis hypogaea L.) obtained from mature seeds germinated on a medium supplemented with thidiazuron (TDZ: N-phenyl-N¹- (1,2,3 thiadiazol-yl)urea). At optimum levels of TDZ (10 M), all germinating seeds produced embryogenic seedlings, and somatic embryos developed in the apical region and on the surface of cotyledons and hypocotyls. These somatic embryos matured, germinated, and formed shoots which eventually developed into whole plants. Thidiazuron-induced direct embryogenesis from morphologically intact seedlings may provide an excellent experimental system for investigating somatic embryogenesis and the morphoregulatory role of TDZ.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium - TDZ thidiazuron (N-phenyl-N¹(1,2,3 thiadiazol-yl)urea) This research was supported by an operating grant from the Natural and Engineering Research Council of Canada to P.K.S. We thank Drs. J.A. Qureshi and Judith Strommer for helpful discussions, and Sangeeta Saxena for technical assistance. A gift of technical-grade thidiazuron from Nor-Am Chemical Co., Wilmington, Del., USA is gratefully acknowledged.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.     

Embryogenic cell lines from somatic embryos of grape (Vitis vinifera L.)

Somatic embryo formation occurred on leaf callus of grape (Vitis vinifera cv. Koshusanjaku). An embryogenic callus was induced from somatic embryo clusters cultured on vitamin-, inositol- and glycine-free Nitsch and Nitsch (1969) medium supplemented with 1.0M 2,4-D. This callus has retained a high embryogenic activity after repeated subculture on the same medium for over two years, and has produced numerous embryos after transfer to a hormone-free medium. The effect of cytokinin treatment on somatic embryogenesis from leaf callus was also examined. N-(2-chloro-4-pyridyl)-N-phenylurea (KT-30) and N-(1,2,3-thiadiazol-5-yl)-N-phenylurea (TAG), both synthetic cytokinins, were found to be effective for the induction of somatic embryogenesis. When leaf callus was induced by these cytokinins combined with 2,4-D at either 5.0 or 10.0M, somatic embryos were produced.Abbreviations B₅ Basal medium, Gamborg et al. (1968) - BA 6-benzylaminopurine - 2,4-D 2,4- dichlorophenoxyacetic acid - IAA indole-3-acetic acid - 2iP (2-isopentenyl)adenine - KIN kinetin - KT-30 N-(2-chloro-4-pyridyl)-N'-phenylurea, also called 4PU-30, Kyowa Hakko Kogyo Co., Japan - NAA 1-naphthaleneacetic acid - NN Basal medium, Nitsch and Nitsch (1969) - MS Basal medium, Murashige and Skoog (1962) - TAG N-(1,2,3-thiadiazol-5-yl)-N'-phenylurea, also called thidiazuron or TDZ, Tomono Noyaku Co., Japan - ZEA zeatin     

Molecular fate of thidiazuron and its effects on auxin transport in hypocotyls tissues of Pelargonium × hortorum Bailey

Thidiazuron, a synthetic phenylurea-type cytokinin, has previously beenfound to induce somatic embryogenesis and organogenesis in a wide range ofplantspecies and to modulate the metabolism of endogenous auxins and cytokinins. Inspite of these findings, the precise mode of action of TDZ remainsundetermined.The current studies were undertaken to determine the fate of the TDZ moleculeand the effects of TDZ exposure on auxin transport in plants. The fate of tworadiolabelled versions of thidiazuron, [¹⁴C-5-thidiazol]-TDZ and[¹⁴C-U-phenyl]-TDZ, was investigated in sterile hypocotyl culturesofgeranium (Pelargonium×hortorumBailey). Radiolabelled TDZ was recovered from the tissue explants inethanol-insoluble, ethanol-soluble and chloroform fractions as well as inacidic, basic and neutral eluants from Dowex resins. Hypocotyl sections thathadbeen exposed to TDZ were found to accumulate more ¹⁴C-IAA from theculture medium and to translocate the auxin over a greater distance within thetissues. These data provide the first evidence that the TDZ molecule remainsintact in both a free and conjugated form within the plant tissues and providesome indication that TDZ-exposure enhances the accumulation and translocationofauxin within the tissues.     

Identification of thidiazuron-induced ESTs expressed differentially during callus differentiation of alfalfa (Medicago sativa)

Yellowish embryogenic callus, which had been induced from the petioles of alfalfa ( Medicago sativa L. cv. Jinnan) and maintained on kinetin (KN)–containing B5h medium, turned non-embryogenic with the differentiation of tracheary elements when KN in the medium was replaced by thidiazuron (TDZ). To investigate the molecular mechanisms underlying the TDZ-induced morphological competence transition, we cloned the differentially expressed sequence tags from TDZ-treated alfalfa callus using suppression subtractive hybridization. The gene expression patterns induced by TDZ were found to change throughout the period of TDZ treatment. TDZ induced the expression of stress-related genes, including trehalose-6-phosphate phosphatase ( TPP ) gene, 1-aminocyclopropane-1-carboxylate synthase ( ACS ) gene and proline dehydrogenase gene, in both early days (day 2) and late days (day 28) of treatment, which are suggestive of the stress responses from the TDZ-treated callus. The TDZ-induced expression of TPP , which tends to lower the level of trehalose-6-phosphate, and ACS , the gene involved in ethylene production, appears to be the major cause for the termination of somatic embryogenesis.     

Cytokinin induced somatic embryogenesis from immature embryos of Abies nordmanniana Lk.

Summary Abies nordmanniana Lk. is used in short intensive rotations for Christmas tree production. Thus there is a high demand for development of advanced propagation and breeding methods. Somatic embryogenesis was easily induced from immature (precotyledonary) embryos collected in July 1989 with cytokinin as the sole plant growth regulator. The proliferating embryogenic cell masses were characteristic of conifer somatic embryogenesis and could be maintained on a simple basal medium containing 5 M benzylaminopurine. Auxin inhibited induction as well as proliferation. Proliferation was improved by up to 30 % by addition of L-glutamine and/or casein hydrolysate. Neither cytokinin concentration nor culture on 3 different basal media, differing markedly in their nitrogen composition, affected the proliferation rate. Embryos matured using a 4 week subculture on medium containing 10 M abscisic acid and subsequent transfer to medium devoid of plant growth regulators.Abbreviations TDZ thidiazuron (Schering) - BAP benzylaminopurine (Sigma) - KIN kinetin (Sigma) - 2,4-D 2,4-dichlorophenoxyacetic acid (Sigma) - ABA +/2-cis-4-trans-abscisic acid (Sigma) - CH casein hydrolysate (Sigma Type 1, acidic) - L-gln L-glutamine (Sigma) - EDTA ethylene diamine tetra acetic acid (Sigma)     

Effect of thidiazuron on somatic embryogenesis of Cayratia japonica

Unpollinated ovary explants of Cayratia japonica (Thump.) Gagnep, were cultured on the revised Murashige & Skoog's medium supplemented with different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-d) alone, or in combination with 0.009 M thidiazuron (TDZ) or 0.23 M kinetin for induction of embryogenic callus. The best results were obtained on medium containing 2.3 – 4.6 M 2,4-d and TDZ. When the calluses were subcultured on the basal medium (BM), somatic embryogenesis took place spontaneously at surfaces of the calluses, but only about 5% of the somatic embryos could develop to cotyledonary stage and most of the rest remained at the globular stage of development. If the calluses were transferred onto medium containing TDZ or TDZ combined with 0.27 M -napthaleneacetic acid, the number of cotyledonary somatic embryos increased up to 25%. When the somatic embryos of different stages were transferred onto fresh BM, only the cotyledonary embryos could convert into the plantlets. The results revealed that for the induction of embryogenic callus and somatic embryogenesis of Cayratia japonica, both cytokinin and auxin are required in the medium and the cytokinin activity of TDZ is much stronger than that of kinetin even when the concentration of TDZ used was only 4% of kinetin.Abbreviations BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid - NAA -naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron (N-phenyl-1,2,3,-thi-diazol-5-ylurea)     

Somatic embryogenesis and plant regeneration of neem (Azadirachta indica A. Juss.)

Somatic embryos were initiated with mature seeds of neem (Azadirachta indica A. Juss.) when cultured on Murashige and Skoog's medium supplemented with thidiazuron (TDZ). Regeneration occurred via somatic embryogenesis: direct embryo formation and through an intermediary callus phase. TDZ was very effective and induced somatic embryogenesis across a wide range of concentrations (1–50 μm). However, somatic embryogenesis was accompanied by callus formation at concentrations of 20 μm and above. Cell suspension cultures were established with the TDZ-induced callus and groups of large cell clumps were formed within 2–3 weeks. Plants were regenerated from both directly formed somatic embryos and somatic embryos derived from cell suspensions plated on semisolid medium devoid of growth regulators. Regenerated plantlets continued to grow after transfer to a greenhouse environment and were similar phenotypically to zygotic seedlings. This simple regeneration system may be beneficial for mass propagation of selected elite clones of neem. Received: 13 May 1997 / Revision received: 13 November 1997 / Accepted: 2 December 1997     

Effects of ethylene and cytokinins on vase life of cut Eucalyptus parvifolia Cambage branches

The role of ethylene and cytokinins was investigated during postharvestsenescence of cut Eucalyptus parvifolia Cambage branches.The effect of endogenous and exogenous ethylene on the vase life of the cutbranches was studied using 2 mM 1-aminocyclopropane-1-carboxylicacid (ACC) as a continuous treatment or pulse treatment for 48h with 20, 40 and 80 l l^–1ethylene. Both endogenous and exogenous ethylene reduced the vase life of thebranches; however, the effect of the endogenous hormone was stronger than theexogenous applications. Ethylene biosynthesis was inhibited by pulse treatmentfor 24 h using 1 mM AOA or 2 mMCoCl₂. The latter treatment significantly extended the vase life ofthe branches by delaying senescence. The effect of cytokinins was evaluated onthe vase life of cut E. parvifolia branches by pulsetreatment for 24 h with 10, 50 and 100 mM thidiazuron(TDZ) or 85, 130 and 260 mM N6-benzyladenine (BA). The resultsobtained showed that no response was observed following pulse treatment with BAwhile, although TDZ had little effect on vase life, it was a good inhibitor ofchlorophyll degradation.     

Somatic embryogenesis and organogenesis in peanut: The role of thidiazuron and N6-benzylaminopurine in the induction of plant morphogenesis

Intact peanut (Arachis hypogaea L.) seeds, incubated on media containing N⁶-benzylaminopurine (BAP) or thidiazuron (TDZ) exhibited de novo regeneration at the hypocotyledonary notch region. Regeneration was observed when seeds were cultured on either TDZ or BAP but the optimal level of media supplementation was 10 mol·L^–1 for TDZ and 50 mol–L^–1 for BAP. Light microscopic observations revealed that the regenerants induced by TDZ were somatic embryos while those induced by BAP were shoots. An alternative approach of exposing the seeds to TDZ was through vacuum infiltration followed by culture on basal media but BAP did not induce regeneration by this method. Although TDZ has often been classified as a synthetic cytokinin, our results clearly demonstrate that seedlings treated with TDZ undergo a different morphological route of development than that induced by purine cytokinins.     

Somatic embryogenesis in cultured immature kernels of Pistachio,Pistacia vera L.

Embryogenic tissue was produced from kernels of immature fruits of Pistachio (Pistacia vera L.) cultured in liquid Murashige and Skoog media, supplemented with 200 mgl^–1 casein hydrolysate, 114 M 1-ascorbic acid, and benzylaminopurine. Compact embryogenic masses differentiated directly from the fruit explants after culture for 2 weeks in liquid medium with 8.9 M benzylaminopurine. After transfer of the embryogenic masses into the same medium, but with 4.4 M benzylaminopurine, somatic embryos appeared. Several stages of embryogenesis were present in the cultures. Adventive embryos were readily separated from the friable embryogenic masses by shaking. Separated somatic embryos, germinated on solidified Murashige & Skoog medium without growth regulators, developed into plantlets.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP benzylaminopurine (N⁶-benzyladenine) - EMS embryogenic mass - MS Murashige and Skoog medium (Sigma M-0404) - NAA -naphthalene acetic acid - PGR plant growth regulator - TDZ thidiazuron (1-phenyl-3-(1,2,3, thiadiazol-5-yl)urea) - WP McCown's Woody plant medium (Sigma M6774) - ABA abscisic acid     

Effect of abscisic acid and cytokinins on the development of somatic embryos in Hevea brasiliensis

Addition of liquid medium, conditioned by an embryogenic suspension, to MH1 solid medium (3,4-dichlorophenoxyacetic acid 9 M, 6-benzyladenine 9 M) permitted the frequent induction of highly embryogenic calli from slices of internal integument of immature seeds of Hevea brasiliensis Müll. Arg. The proliferation of embryogenic cell clusters was achieved in MH1 liquid medium. Abscisic acid (ABA), cytokinins and adenine were tested for their ability to affect development of somatic embryos to plantlets. The transfer of embryogenic cell clusters on auxin-free solid medium with 10^-5M ABA for 2 months stimulated embryo development. When torpedo-shaped embryos were transferred to medium with adenine or cytokinins they turned green in 1 month. Green embryos produced secondary embryos when they were collected and placed on medium without growth regulators.Abbreviations ABA abscisic acid - BA 6-benzyladenine - IBA indole-3-butyric acid - NOA -naphthoxyacetic acid - 2iP 2-iso-pentenyladenine - 3,4-D 3,4-dichlorophenoxyacetic acid     

The effect of in vitro pretreatments on subsequent shoot organogenesis from excised Rubus and Malus leaves

Shoot regeneration from Rubus leaves was obtained on a medium containing MS salts, vitamins and sugars, Staba vitamins, casein hydrolysate (100 mg l^–1) and 10 M thidiazuron. Shoot regeneration from Malus leaves was obtained on N⁶ rice anther medium with 5 M thidiazuron. In vitro pretreatment of source shoots with either colchicine or thidiazuron enhanced the organogenic potential of detached leaves of two Rubus hybrids. The response to colchicine was quadratic and occurred at non-mutagenic concentrations (75–250 M). The response to thidiazuron was exponential between 0 and 5 M. When applied as a pretreatment, the effectiveness of several different cytokinins (benzyladenine, thidiazuron, zeatin) at enhancing Malus and Rubus organogenesis was related to the shoot proliferation activity of the cytokinin and to treatment-induced variation in leaf and petiole size.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IBA indolebutyric acid - MS Murashige & Skoog basal medium devoid of plant growth regulators - OI organogenesis-initiating subculture - PTI colchicine pretreatment subculture - PTII cytokinin pretreatment subculture - NAA naphthaleneacetic acid - TDZ thidiazuron - zeatin trans-zeatin     

Cytokinins induce direc somatic embryogenesis of <Emphasis Type="Italic">Dendrobium</Emphasis> chiengmai pink and subsequent plant regeneration

Summary Four auxins (2,4-dichlorophenoxyacetic acid [2,4-D], indole-3-acetic acid [IAA], indole-3-butyric acid [IBA], and naphthaleneacetic acid [NAA]), and five cytokinins (N ⁶-[2-isopentenyl]-adenine [2iP], N ⁶-benzyladenine [BA], 6-furfurylaminopurine [kinetin], 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea [TDZ], and 6-[4-hydroxy-3-methylbut-2-enylamino]purine [zeatin]) were examined for their effects on direct embryo induction from leaf explants of Dendrobium cv. Chiengmai Pink cultured on 1/2 Murashige and Skoog (MS) medium. Whether in light or darkness, explants easily became necrotic and no embryos were obtained on growth regulator-free or auxin-containing media after 60 d of culture. By contrast, five cytokinins tested induced direct embryo formation from leaf explants, and explants cultured in light had a higher embryogenic response compared with those cultured in darkness. The best condition for direct embryo induction was at 18.16 μM TDZ cultured in light for 60 d, where 33% of explants formed a mean number of 33.6 embryos per explant. During subculture on growth regulator-free 1/2 MS medium, embryos gradually developed into plantlets. Secondary embryogenesis was occasionally found on sheath leaves of embryos. Regenerated plantlets were successfully transplanted and grown in a greenhouse environment.     

Morphoregulatory role of thidiazuron : substitution of auxin and cytokinin requirement for the induction of somatic embryogenesis in geranium hypocotyl cultures

Somatic embryogenesis was induced in hypocotyl explants of geranium (Pelargonium × hortorum) cultured on media supplemented with various concentrations of N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (thidiazuron). In less than 2 weeks, somatic embryos were observed in treatments containing levels of thidiazuron (TDZ) ranging from 0.2 to 1.0 micromolar. The use of N⁶-benzylaminopurine in combination with indole-3-acetic acid also evoked embryogenesis, but the efficiency of somatic embryo production was significantly lower than that obtained with TDZ. Hypocotyl culture for only 2 days on TDZ-supplemented medium before transfer to a basal medium was sufficient for inducing somatic embryogenesis. This distinction between the induction and expression of embryogenesis may provide an experimental system for studying the developmental biology of somatic embryogenesis. Substitution of the auxin-cytokinin requirement for the induction of somatic embryogenesis by TDZ suggests the possibility of a novel mode of its action by modulation of endogenous growth regulators.     

Partial auxin deprivation affects endogenous cytokinins in an auxin-dependent,cytokinin-independent tobacco cell strain

The dynamics of individual endogenous cytokinins within the growth cycle (subculture interval) of an auxin-dependent and cytokinin-independent cell suspension culture ofNicotiana tabacum L. (strain VBI-0) were determined using high performance liquid chromatography and radioimmunoassay. In cells grown at an optimum auxin concentration the transient maxima of N⁶-(²-isopentenyl)adenine and N⁶-(²-isopentenyl)-adenosine correlated with the onset of cell division. Cultivation of the cells in a partially auxin-deprived medium resulted in ca. tenfold increase of all endogenous cytokinins. A very distinct maximum of N⁶-(²-sopentenyl) adenine appeared at the beginning of subculture. This indicates that a lack of auxin induced an accumulation of cytokinins predominantly in the form of the free bases, which are physiologically more active than the corresponding ribosides.Abbreviations iP N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - t-Z trans-zeatin - t-[9R]Z trans-zeatin riboside - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - f.w. fresh weight - SBI subculture interval - C complete medium - PAD partially auxin-deprived medium - RP-HPLC reverse phase high performance liquid chromatography - RIA radioimmunoassay - PAL L-phenylalanine ammonia lyase