BAP Treatment Research Articles

Cytokinin induces expansin gene expression in Melilotus alba Desr. wild-type and the non-nodulating, non-mycorrhizal (Nod-Myc-) mutant Masym3

We previously showed that applying 10-6 M of the cytokinin 6-benzylaminopurine (BAP) to uninoculated roots of Masym3, a Nod-Myc- Melilotus alba (Desr.) mutant, resulted in the accumulation of MaENOD40 transcripts to levels similar to BAP-treated wild-type roots. In contrast, inoculation with a Nod- S. meliloti mutant expressing the trans-zeatin synthase gene of Agrobacterium tumefaciens (Nod-/pTZS+) did not induce MaENOD40 transcript accumulation in either wild-type or Masym3 roots. However, Masym3 root hairs became swollen at their tips following inoculation with wild-type or Nod-/pTZS+ rhizobia. Because root hair distention and elongation are often correlated with increased expansin activity, we investigated whether BAP treatment or inoculation with Nod-/pTZS+ S. meliloti upregulated the expression of expansion mRNAs. We first determined that treating wild-type roots with 10-5 or 10-6 M BAP resulted in greater MaEXPA1 transcript accumulation than treating roots with comparable concentrations of the auxin NAA. When Masym3 roots were treated with 10-6 M BAP, MaEXPA1 mRNAs accumulated to levels comparable to wild-type roots. We then showed that MaEXPA1 mRNAs accumulated in wild-type M. alba roots in response to the Nod-/pTZS+ S. meliloti strain. Masym3 mutant roots inoculated with Nod-/pTZS+ rhizobia were also upregulated for MaEXPA1 expression.

Effects of Different Hormonal Treatments on the Callus Production and Plantlet Regeneration in Saffron (Crocus sativus L.)

Saffron (Crocus sativus L.) is one of valuable and native plants in the land of Iran. By this investigation the best hormonal compositions for callus production from protoplast and for plantlet regeneration from callus were determined. To isolate protoplasts, the embryogenic calli were used. The embryogenic calli were immersed in enzymatic solution to degrade the cell walls. The treated mixture was filtered and then centrifuged at 100 g for 3-5 min and the resulted pellet was rinsed. After one step of washing and another step of centrifugation, the protoplasts were gently mixed with sterile sodium alginate solution and added to MS broth consisting 1% calcium chloride and 0.3 M manitol to form calcium alginates granules. The protoplast-containing granules were exposed to MS broth including 0.3 M manitol and various treatments of two kinds of auxins (2, 4-D and NAA) and three kinds of cytokinins (2ip, Kin, BAP), respectively in four rates of 0.1, 0.2, 0.5, 1.0 mg L(-1) for auxins and in three rates of 0.1, 0.2 and 0.5 mg L(-1) for cytokinins, incubated in dark at 22 +/- 2 degrees C for a period of 30 days. Out of all the treatment of 2, 4-D (1.0 mg L(-1)) and Kin (0.2 mg L(-1)) was the best in callus induction. In order to regenerate plantlets, the resulted calli were transferred to MS broth amended with different rates of ABA (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 mg L(-1)) so that they could pass the steps of embryonic maturation. The mature embryos were transferred to MS media with different rates of GA3 (0.0, 5.0, 15.0, 20.0, 25.0 and 30.04 mg L(-1)) to initiate germination. The germinated embryos were then placed in solid MS media with various rates of NAA and 2, 4-D auxins (0.0, 0.5, 1.0 and 2.0 mg L(-1)) and different levels of BAP and Kin cytokinins (0.0, 0.5, 1.0 mg L(-1)). Results from statistical analyses indicated the treatment of NAA and BAP (each 1 mg L(-1)) as the best hormonal treatment for the plantlet regeneration from the domestic saffron calli.

Enhanced Lung Tumor Development in Tobacco Smoke-Exposed p53 Transgenic and Kras2 Heterozygous Deficient Mice

A/J mice bearing either a mutation in the p53 gene or a Kras2 heterozygous deficiency were investigated for their susceptibility to tobacco smoke-induced lung tumorigenesis. Transgenic mice and their wild-type littermates were exposed to mainstream tobacco smoke (MS) for 5 mo, followed by 4 mo of recovery in filtered air. In sham (filtered air) groups, p53 transgenic mice did not exhibit a higher tumor multiplicity but did exhibit larger tumors, with tumor load increased 3.6-fold, when compared with wild-type mice. With exposure to MS, tumor multiplicity was increased 60% but there was a strikingly increased tumor load (15.9-fold) in p53 transgenic mice. Increased tumor load (5.3-fold) but not tumor multiplicity was seen in MS-exposed Kras2 heterozygous deficient mice. Interestingly, MS exposure did not increase benzo[a]pyrene-induced lung tumorigenesis when MS exposure was initiated after BaP treatment. These results indicate that a p53 mutation or loss of a Kras2 allele increases susceptibility to MS-induced lung tumor development.

Multiplicação in vitro de Eucalyptus grandis sob estímulo com benzilaminopurina

O objetivo deste trabalho foi avaliar os efeitos do estímulo com benzilaminopurina (BAP) na multiplicação in vitro de Eucalyptus grandis. Foram avaliadas as interações entre concentrações de BAP (0, 200, 400 e 600 mg L-1), tempo de exposição (1, 2 e 3 horas), pH (3 e 5,8) da solução e alterações morfológicas dos explantes. Semanalmente, foi determinada a massa da matéria fresca dos explantes. Aos 21 dias de cultura, foram avaliados: o número de brotações por tratamento, o número de brotações obtidas por explante (taxa de multiplicação), e foi realizado o resgate das plântulas para avaliação histológica. O pH não apresentou interação com os demais fatores estudados. Os tratamentos com BAP a 200 mg L-1, durante 1 e 2 horas, apresentaram-se como os mais indicados na multiplicação do E. grandis. Houve intensificação da divisão celular, no parênquima cortical e no procâmbio, representada pelo surgimento de meristemóides, em resposta aos tratamentos com 200 mg L-1 de BAP, durante 1 e 2 horas. O estímulo com BAP na multiplicação in vitro de E. grandis é viável.

Streptozotocin (STZ) diabetes enhances benzo( α)pyrene induced renal injury in Sprague Dawley rats

Information is lacking regarding the biological response to environmental chemicals in the context of pre-existing disease. Benzo( α)pyrene (BaP), a polycyclic aromatic hydrocarbon, is a byproduct of combustion that causes renal injury and elicits a nephropathic response. This study evaluated the nephrotoxicity of BaP in normoglycemic and diabetic rats. Female Sprague Dawley rats were divided into four groups: normoglycemic-vehicle (NV), normoglycemic-BaP (N-BaP), diabetic-vehicle (DV) and diabetic-BaP (D-BaP). Diabetes was induced by intraperitoneal (ip) injection of streptozotocin (60 mg/kg, 1 ml/kg). Rats were injected (ip) with vehicle or 10 mg/kg BaP (1 ml/kg) once per week for 5 weeks. Urinary protein and albumin, plasma creatinine and light microscopy were performed to assess the effects of BaP on kidney function. Diabetes was confirmed by plasma glucose levels >400 mg/dl in the DV and D-BaP groups. BaP increased kidney weight and blood urea nitrogen (BUN) levels in the D-BaP relative to the DV group. No change in BUN was observed following 5 weeks of BaP treatment in the normoglycemic animals, however, kidney weight was increased ( p = 0.013) in the N-BaP relative to the NV animals. STZ diabetes increased susceptibility to BaP mediated renal damage following repeated treatment for 5 weeks when compared to age matched normoglycemic rats.

The two ribosomal proteinL23Agenes are differentially transcribed inArabidopsis thaliana

Arabidopsis thaliana ribosomal protein (r-protein) L23A (RPL23A) is a member of the conserved L23/L25 family of primary ribosomal RNA (rRNA) binding proteins. The 2 AtRPL23A isoforms, RPL23A-1 and RPL23A-2, are 94% identical at the amino acid level, yet RPL23A-1 and RPL23A-2 share only approximately 40-50% primary sequence identity within the 5' regulatory regions. While the RPL23A-1 and -2 5' regulatory regions share many similar predicted motifs, the arrangement and number of these motifs differs between the 2 genes. Differences in regulation between RPL23A-1 and -2 have been investigated via reverse transcription-PCR (RT-PCR) expression profiles. Overall, transcript abundance for RPL23A-1 and -2 varied slightly in specific tissues and under some abiotic stresses. The highest transcript abundance for both RPL23A genes was detected in mitotically active tissues such as bud, flower and elongating carpel, as well as in root and stem while the lowest transcript levels were found in mature leaf and bract. Hormone-treated seedlings showed increased RPL23A-1 and -2 transcript levels following IAA and BAP treatment while ABA treatment resulted in a transient lowering of transcript levels. Expression patterns differed between RPL23A-1 and -2 in cold-, wound-, and copper-stressed seedlings. In all tissues examined, RPL23A-2 transcript levels were consistently lower than those of RPL23A-1. This report shows differential transcriptional regulation of the 2 RPL23A genes, which should no longer be identified as "housekeeping" genes.

Analysis of Promoter Activity of the Early Nodulin Enod40 in Lotus japonicus

Our comparative studies on the promoter (pr) activity of Enod40 in the model legume Lotus japonicus in stably transformed GusA reporter lines and in hairy roots of L. japonicus demonstrate a stringent regulation of the Enod40 promoter in the root cortex and root hairs in response to Nod factors. Interestingly, the L. japonicus Enod40-2 promoter fragment also shows symbiotic activity in the reverse orientation. Deletion analyses of the Glycine max (Gm) Enod40 promoter revealed the presence of a minimal region -185 bp upstream of the transcription start. Stable transgenic L. japonicus reporter lines were used in bioassays to test the effect of different compounds on early symbiotic signaling. The responses of prGmEnod40 reporter lines were compared with the responses of L. japonicus (Lj) reporter lines based on the LjNin promoter. Both reporter lines show very early activity postinoculation in root hairs of the responsive zone of the root and later in the dividing cells of nodule primordia. The LjNin promoter was found to be more responsive than the GmEnod40 promoter to Nod factors and related compounds. The use of prGmEnod40 reporter lines to analyze the effect of nodulin genes on the GmEnod40 promoter activity indicates that LJNIN has a positive effect on the regulation of the Enod40 promoter, whereas the latter is not influenced by ectopic overexpression of its own gene product. In addition to pointing to a difference in the regulation of the two nodulin genes Enod40 and Nin during early time points of symbiosis, the bioassays revealed a difference in the response to the synthetic cytokinin 6-benzylaminopurine (BAP) between alfalfa and clover and L. japonicus. In alfalfa and clover, Enod40 expression was induced upon BAP treatment, whereas this seems not to be the case in L. japonicus; these results correlate with effects at the cellular level because BAP can induce pseudonodules in alfalfa and clover but not in L. japonicus. In conclusion, we demonstrate the applicability of the described L. japonicus reporter lines in analyses of the specificity of compounds related to nodulation as well as for the dissection of the interplay between different nodulin genes.

Effect of ethephon and 6-benzylaminopurine on chlorophyll degrading enzymes and a peroxidase-linked chlorophyll bleaching during post-harvest senescence of broccoli (Brassica oleracea L.) at 20°C

Effects of ethephon and 6-benzylaminopurine (BAP) treatments on enzyme degrading enzymes and a peroxidase-linked chlorophyll bleaching were investigated in broccoli (Brassica oleracea L.) florets. The florets were dipped in solutions containing either BAP or ethephon and then incubated in darkness at 20 °C. The hue angle values and chlorophyll contents of ethephon-treated florets declined the most of the five-day experiment. In contrast, hue angle and chlorophyll content declined the least in BAP-treated florets. Pheophytin levels increased the most in ethephon-treated florets and the least in BAP-treated florets. Chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching increased over the five days in control (untreated) florets. Ethephon treatment enhanced chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching levels as compared with control. BAP treatment reduced chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching levels. It was concluded that the activities of chlorophyllase, Mg-dechelatase, and peroxidase-linked chlorophyll bleaching could be regulated by external application of either ethylene (applied as ethephon) or a cytokinin (applied as BAP). In the case of ethephon treatment, accelerated rates of chlorophyll degradation were found and in the case of BAP-treatment, reduced rates of chlorophyll degradation were found.

Organogênese de explante foliar de clones de Eucalyptus grandis x E. urophylla

O objetivo deste trabalho foi avaliar os efeitos dos reguladores de crescimento TDZ [1-fenil-3-(1,2,3-tia-diazol-5-il)uréia], BAP (6-benzilaminopurina) e ANA (ácido naftalenoacético) no desempenho da propagação in vitro por organogênese de explante foliar de três clones híbridos de Eucalyptus grandis x Eucalyptus urophylla. Houve resposta diferenciada dos clones quanto a intensidade, textura e coloração dos calos, em razão dos tratamentos com os reguladores de crescimento. Os melhores resultados de calejamento dos três genótipos foram observados nos tratamentos com a combinação dos reguladores de crescimento TDZ (0,5 mg L-1) e ANA (0,1 mg L-1), obtendo-se 100% de calejamento no explante foliar. Os piores resultados de calejamento foram observados nos tratamentos com a combinação dos reguladores de crescimento BAP (0,1 mg L-1) e ANA (0,1 mg L-1). Em relação à regeneração, a melhor resposta foi obtida com 1,0 mg L-1 BAP em que 8% dos calos formados a partir de explantes foliares regeneraram gemas, com número médio destas formadas por calo igual a 4,2.

In vitro regeneration of lychee (Litchi chinensis Sonn.)

In vitro plantlet regeneration in the main commercial variety of lychee ( Litchi chinensis Sonn. cv Tai So) in Mauritius was achieved from callus cultures derived from young, tender leaf explants on Murashige and Skoog (MS) medium. Callogenesis was obtained in all media supplemented with auxin, but was most prominent in media supplemented with 2,4 – D ( 1.5 mgL -1 ) with or without benzylamino purine and kinetin. Nodular compact callus obtained in the 2,4 – D and BAP treatment proliferated and differentiated into shoots .When transferred to MS medium supplemented with BAP (2.0 mgL -1 ) and IAA (3.0 mgL -1 ). Regenerated shoots produced prominent roots when transferred to MS medium supplemented with IBA (2.0 mgL -1 ). Regeneration was predominantly through organogenesis. Somatic embryogenesis was observed when callus growing on MS medium supplemented with 2,4–D (1.5 mgL -1 ) was transferred to medium devoid of 2,4-D. Key Words: Litchi chinensis , micropropagation, somatic embyogenesis. African Journal of Biotechnology Vol.3(11) 2004: 576-584

In vitro regeneration of lychee (Litchi chinensis Sonn.)

In vitro plantlet regeneration in the main commercial variety of lychee ( Litchi chinensis Sonn. cv Tai So) in Mauritius was achieved from callus cultures derived from young, tender leaf explants on Murashige and Skoog (MS) medium. Callogenesis was obtained in all media supplemented with auxin, but was most prominent in media supplemented with 2,4 – D ( 1.5 mgL -1 ) with or without benzylamino purine and kinetin. Nodular compact callus obtained in the 2,4 – D and BAP treatment proliferated and differentiated into shoots .When transferred to MS medium supplemented with BAP (2.0 mgL -1 ) and IAA (3.0 mgL -1 ). Regenerated shoots produced prominent roots when transferred to MS medium supplemented with IBA (2.0 mgL -1 ). Regeneration was predominantly through organogenesis. Somatic embryogenesis was observed when callus growing on MS medium supplemented with 2,4–D (1.5 mgL -1 ) was transferred to medium devoid of 2,4-D. Key Words: Litchi chinensis , micropropagation, somatic embyogenesis. African Journal of Biotechnology Vol.3(11) 2004: 576-584

Micropropagation of mature wych elm (Ulmus glabra Huds.).

Explants of mature vigorous donor trees of wych elm ( Ulmus glabra Huds.) that had not been previously exposed to Dutch elm disease were investigated for the influence of phytohormones and media on shoot multiplication rates and organogenic capacity. The regenerates were micropropagated from cultures that originated from 15-year-old progeny of plus trees. Two plus trees aged over 70 years showed recalcitrant responses. Thidiazuron in combination with 6-benzylaminopurine (BAP) induced a significantly higher number of shoots per explant than the most optimal BAP treatment (5.88 vs. 3.05 shoots). Woody plant medium and Dubovský minimal medium had no significant effects on shoot formation and multiplication rates. All plantlets raised in vitro were phenotypically normal and successfully hardened to ex vitro conditions. Two experimental field plots with 3-year-old in vitro-propagated trees were established.

The Tailorability of Lightweight Wool and Wool-blend Fabrics

By using the FAST system, a range of lightweight wool and wool-blend fabrics, including three wool fabrics treated with Synthappret BAP shrinkproofing resin to obtain machine-washable properties, was investigated in terms of tailorability. The main problems with these fabrics were found to be their low warp extensibility, low bending rigidity, low shear rigidity, and low formability, which would lead to tailoring problems during laying, cutting, sewing, and fabric manipulation. A commercial Synthappret BAP treatment was found to improve the tailorability of the lightweight wool fabrics by increasing fabric bending rigidity and formability.

A PROPOSED METHODOLOGY OF CANCER RISK ASSESSMENT MODELING USING BIOMARKERS

A methodology for cancer risk assessment modeling was developed using a biomarker of DNA adduct, exposure dose, and tumor response. DNA adducts in the blood and lung were measured after single or multiple administration of [3H]benzo[a]pyrene (1 2 BaP) in ICR mice. Making the assumption that DNA adducts are formed in a dose-dependent manner as observed in 1 2 BaP treatment, kinetics patterns of DNA adducts were predicted at two other hypothetical BaP doses (2 2 BaP, 1/2 2 BaP) for single and continuous BaP treatments because the difference between the simulated and the experimental kinetic responses only amounted to 5.49-5.86% in terms of the integrated area under the curve. Correlations between the formation of DNA adducts and exposure doses or between blood DNA and lung DNA adducts were determined to be linear. The doseresponse relationship between biomarker and exposure dose was further incorporated into a dose-tumor response equation, obtained from 2-yr bioassay, to predict cancer risk. The interrelationships between exposure dose, biomarker, and tumor response allowed the prediction of cancer risk in animals, once the information on biomarker levels was obtained. Moreover, this methodology could be further applied to human cancer risk assessment after appropriate safety factors were employed.

Interactive effect of cytokinin and potassium on sink-source relationships in Lupinus angustifolius

This study examined whether increased K supply in conjunction with BAPcould increase lupin seed yield and harvest index by enlarging sink volume (podnumber), increasing assimilate and improving assimilate partitioning to filltheadditional pods induced by BAP treatment. Narrow-leafed lupin(Lupinusangustifolius, cv. Danja abs− mutant) was grown inaglasshouse, in pots containing sandy soil with four K treatments (0, 15, 60 and120 mg K/kg soil). BAP (2 mM) was applied daily toallmain stem flowers throughout the life of each flower from opening to senesced.BAP application did not affect assimilate production (as measured by totalabove-ground biomass), but changed assimilate partitioning. On BAP-treatedplants, there were greater proportions of seed to pod wall dry weight on themain stem but smaller proportions on the branches, and an increased weightratioof seed to pod wall overall which meant more assimilate was used for seedgrowthrather than pod wall growth. BAP increased the number of pods per plant by35% and this more than compensated for the decreases in seeds per podandseed weight. Therefore, there was an increased harvest index (+11%)and seed yield per plant (+13%) in BAP-treated plants. BAP alsoincreased the number of pods with filled seeds (146%) on the main stemand main stem seed K+ concentration (from 0.81% to0.87%). Added K increased biomass but only slightly affected assimilatepartitioning. As applied K increased, relatively more assimilate was used forpod wall growth rather than seed growth. Added K increased seed yield per plantby about 14% due to increases in seed weight and the number of pods onthe main stem. Moreover, K+ concentration in seeds and shootsincreased with increasing level of applied K. Seed yield was enhanced more byBAP when K was supplied at high levels. Increasing K supply interactedpositively with added BAP by increasing narrow-leaf lupin seed yield andharvestindex through increases in assimilate supply and its partitioning into seeds.

Impact of 2,4-DP and BAP upon pod set and seed yield in soybean treated at reproductive stages

A large number of soybean (Glycine max L. Merr.)flowers and young pods abscise rather than develop into mature pods. Flower andpod drop or abortion accounts for the majority of total reproductive abscissionand influences potential soybean yield. The objectives of this study were todetermine the patterns of flower, pod and seed development under treatmentswiththe growth regulators, 2-(2,4-dichlorophenoxy) propanoic acid (2,4-DP) and6-benzylaminopurine (BAP), applied at the early reproductive stages, and toexamine the association of reproductive abscission with growth characteristicsand agronomic traits, including seed yield and seed weight. Small seeded [cvPungsan (11.1 ± 0.4 g100-seed−1)] and large seeded [cv Manlee(21.0 ± 0.5 g 100-seed−1)]genotypes were separately planted in the greenhouse and field, and treated witheither 2,4-DP or BAP. 2,4-DP (a synthetic auxin) and BAP (a syntheticcytokinin)were each applied at three concentrations (i.e. high, intermediate or low):0.12mM, 0.08 mM, 0.04 mM, and 1.5mM, 1 mM, 0.5 mM respectively. High andlow concentrations were employed for greenhouse experiments to examine thenumber of flowers per plant in pots. With the exception of low BAP (0.5mM) treatment in Pungsan, all treatments increased total podnumberwith various numbers of seeds per pod. Low 2,4-DP (0.04 mM) inbothgenotypes or BAP (0.5 mM) in Manlee significantly reduced flowerabortion and delayed abscission of pods in both genotypes, resulting inincreased pod setting rates. Under field conditions using intermediateconcentrations, 1 mM BAP significantly increased 100-seed weightto22.3 g at R1 in Manlee and 11.9 g at R3 in Pungsan.BAP (1 mM) at R3 in Pungsan significantly improved seed yield(40.1g plant−1). Maturity was not significantlyaffected by either application in Manlee, but was significantly affected by BAPin Pungsan. In Pungsan, 2,4-DP increased pod number, plant height and nodenumber, but decreased 100-seed weight in Pungsan treated at R1, causing nosignificant change of seed yield. This study suggested that exogenousregulatorssignificantly influenced reproductive and growth characteristics, andconsequently seed yield, but increase of pod number was not always beneficialfor seed yield.

Differential RNA expression of α-expansin gene family members in the parasitic angiosperm Triphysariaversicolor (Scrophulariaceae)

Haustoria are parasitic plant specific organs that locate, attach to, and invade host plant tissues. Parasitic species of the Scrophulariaceae develop haustoria on their roots in response to chemical signals released by host plant roots. Haustorium development was induced invitro in roots of the parasitic Scrophulariaceae Triphysariaversicolor by treating them with exudates obtained from maize roots, the chemical 2,6-dimethoxybenzoquinone (DMBQ) or the cytokinin 6-benzylaminopurine (BAP). Morphological responses of T.versicolor roots to these haustoria inducing factors (HIFs) included localized swelling and epidermal hair proliferation near the root tips. These responses were not observed when roots of the non-parasitic Scrophulariaceae Lindenbergiamuraria were similarly treated. Because expansin proteins are closely associated with plant cell wall expansion and growth, we examined the expression of expansin genes in response to HIFs. We isolated cDNAs homologous to transcripts encoding three distinct α-expansin proteins in T.versicolor. Northern-blot analyses indicated that these transcripts were differentially abundant in different tissues. Steady-state levels of two expansin transcripts increased in T.versicolor roots exposed to BAP, but not DMBQ or maize root exudates. Expansin transcript abundance also increased in L.muraria in response to BAP treatment. These results suggest that the expansins examined fulfill functions distinct from haustorium development.

Benzyladenine-induced inhibition of flowering in Chenopodium rubrum in vitro is not related to the levels of isoprenoid cytokinins

The effect of 6-benzylaminopurine (BAP) on floweringand on endogenous levels of isoprenoid cytokinins wasinvestigated in explanted terminal shoots of Chenopodium rubrum cultivated in vitro. Themother plants were grown under continuous light andexplants were cut off when the 6th leaf primordiumoriginated at the shoot apex. The explants wereexposed to one dark period of 13 hours inductive forflowering or to continuous light on medium with orwithout BAP (0.05;0.2;0.4 mg.l-1). Undernon-inductive conditions no flowering was observedeither in the control or after BAP treatment. Afterreceiving one inductive dark period, the controlexplants flowered. However, BAP application either atthe beginning of the inductive dark period and/orduring the following light cultivation inhibitedflowering and stimulated initiation and growth of leafprimordia. In the case of the most efficient BAPconcentration (0.05 mg.l-1) flowering wasinhibited by 80% and the number of leaf primordia wasincreased by 3. Explantation caused a significantincrease in the total amount of endogenous cytokininsin the explants within first 13 h, provided they werekept in light. When explants were kept in darkness,only a slight increase in cytokinin levels wasobserved. BAP treatment had no influence on the levelsof endogenous cytokinins either in light or indarkness. We may thus conclude, that BAP applicationinhibited flowering of photoperiodically inducedterminal shoot explants and stimulated leaf primordiaformation with no significant effect on changes inlevels of endogenous isoprenoid cytokinins. This maysuggest the direct ability of BAP to regulate morphogenesis.

DNA-damaging Aryl Hydrocarbons Induce Mdm2 Expression via p53-independent Post-transcriptional Mechanisms

During previous studies, we found that mdm2 mRNA levels were elevated in benzo[a]pyrene (BaP, a polycyclic aryl hydrocarbon)-treated cells under conditions of DNA damage-induced cell cycle arrest (Vaziri, C., and Faller, D. V. (1997) J. Biol. Chem. 272, 2762-2769). We have identified potential aryl-hydrocarbon receptor-binding sites in the mdm2 promoter. However, we show that induction of mdm2 mRNA by BaP is entirely dependent upon aryl-hydrocarbon-induced genotoxicity and does not involve direct aryl-hydrocarbon receptor-mediated transcriptional activation of the mdm2 gene. Heterologous mdm2 promoter-reporter constructs containing p53-response elements were not responsive to BaP treatment. Therefore the p53-response elements in the mdm2 promoter are insufficient to confer DNA damage-dependent expression of mdm2. Furthermore, mdm2 transcripts were induced by BaP in p53 null cells from transgenic mice (although both basal and BaP-induced mdm2 expression levels were reduced in these cells relative to p53(+/+) cultures). These data show that p53-mediated mechanisms cannot account for BaP/DNA damage-induced mdm2 expression. Mdm2 promoter-reporter gene assays and nuclear run-off analyses of nascent mdm2 transcripts showed that transcriptional induction was unable to account for the large changes in mdm2 transcript levels following BaP treatment. However, mdm2 mRNA half-life measurements showed stabilization of the mdm2 transcript (from approximately 1 h to >4 h) in response to BaP. To our knowledge, this is the first report of control of mdm2 at the post-transcriptional level and in a p53-independent manner. Transient ectopic expression of mdm2 strongly augmented aryl-hydrocarbon-induced apoptosis, demonstrating that mdm2 levels can have a profound effect on the cellular response to DNA damage. Overall, our results suggest a potentially important link between DNA damage signaling and RNA stability that may be relevant to cell cycle regulation, tumor suppression, and environmental carcinogenesis.

Grafting micropropagated tea [ Camellia sinensis (L.) O. Kuntze] shoots on tea seedlings - a new approach to tea propagation

Tea microshoots excised from well-established multiple shoot cultures grown in vitro and 8-week-old, three- to five-leaved seedlings from a local chinery stock (Banuri-96) and UPASI-9 (from southern India) were selected as scions and root stocks, respectively, for grafting. In addition, 4-month- and 12-month-old seedlings of Banuri-96 were also used as root stocks. Cut ends of root stocks and scions were pretreated with varying concentrations of BAP and NAA for 10 min. A treatment of BAP (5 mg/l) and NAA (5 mg/l) to both scion and stocks in water renewed foliar development at a relatively early stage (40–60 days). The grafted plants were kept in hardening chambers with CO2-enriched air. No significant difference was observed between autograft (scion and root stock of Banuri clone) and heterograft (scion of the Banuri clone and root stock of UPASI-9). Of the three types (in terms of age) of seedling-raised root stocks employed, grafts on young tea (4-month-old) performed the best (88.33%). Grafts made in early summer established relatively faster and at a high rate of success. The percentage survival of plants transferred to the field was 88.33%.