亚硒酸钠对烟草冠瘿组织生长的影响及其与内源激素的关系

低浓度(0.1～1μmol/L)亚硒酸钠(Na_2-ScO_s)对烟草冠瘿组织的生长有促进作用;但浓度较高时(5～20μmol/L)不仅对生长有抑制作用,同时能促使其内源吲哚乙酸(IAA)和玉米素(ZA)含量下降,脱落酸(ABA)含量增加,IAA氧化酶活性增强。适宜浓度的外源2,4-D,IAA,6-BA,ZA都可部分缓解较高浓度Na_2SeO_3对生长的抑制作用,其中2,4-D,6-BA的缓解能力分别大于IAA和ZA。Na_2SeO_3还引起烟草冠瘿组织过氧化物同工酶谱发生显著变化,这种变化可被适宜浓度的外源6-BA逆转,并向正常组织方向转化,缓解较高浓度Na_2SeO_3对生长的抑制作用与逆转其过氧化物同工酶谱变化的6-BA浓度范围完全一致。     

Functional Expression of Agrobacterium tumefaciens T-DNA onc-genes in Asparagus Crown Gall Tissues

We analyzed Asparagus crown gall tissues transformed with A.tumefaciens C58 and C58C1 (pTiB6S3) and selected for hormoneautotrophic growth. No increased IAA levels were observed inthe Asparagus tumor lines notwithstanding the presence of allthree T-DNA onc genes. The endogenous cytokinin levels indicatethat Asparagus crown gall is dependent on enhanced zeatin ribosideequivalent levels for its growth. We conclude that phytohormone autotrophic growth of Asparaguscrown gall tissue seems only to be dependent upon an activegene 4, inducing enhanced cytokinin levels. Moreover, the presenceof an active gene 1 seems to be lethal as was indicated by theabsence of tryptophan-2-mono-oxygenase activity in transformedtissues and the toxicity of exogenously supplied indole-3-acetamide(IAM) or naphthalene-1-acetamide (NAM) as a substitute for anactive gene 1. (Received August 7, 1989; Accepted October 31, 1989)     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Tobacco Crown Gall Tissues of Different Morphologies

Endogenous levels of gibberellin (GA) as well as IAA and cytokininsin teratomas and unorganized crown gall tissues of tobacco wereexamined by GC-SIM (for GA and cytokinin) or HPLC with a fluorescencedetector (for IAA). Two different types of crown gall inducedby octopine-type and nopaline-type Ti-plasmids were used. Inboth types, GA contents were higher in shoot-forming teratomasthan in unorganized calluses, while IAA contents were higherin unorganized calluses. But cytokinin contents in octopine-typecells were higher in unorganized calluses than in teratomas,whereas the contents in nopaline-type cells were higher in teratomas.Our results suggest that there is not always a relationshipbetween the cytokinin/IAA balance and tobacco crown gall morphology,but GA production in tobacco tissues is closely related to itsdifferentiation. ⁴ Present address: Agency for the Assessment and Applicationof Technology (BPPT), Jakarta Pusat, Indonesia. (Received September 1, 1986; Accepted February 16, 1987)     

Endogenous Levels of Gibberellins, IAA and Cytokinins in Catharanthus Crown Gall Tissues of Different Tumor Types

Endogenous levels of gibberellins, auxins and cytokinins incrown gall tissues of Catharanthus roseus G. Don. (Vinca roseaL.) of two different tumor types, induced by a nopaline-typeand an octopine-type Ti-plasmid, were examined. The levels of gibberellins were higher in nopaline-type V208cells than in octopine-type V277 cells. GA₁₂ and GA₂₄ were identifiedfrom V208 cells. The level of IAA was 20-fold higher in V208cells than in V277 cells. The level of trans-ribosylzeatin washigher in V277 cells than in V208 cells, whereas trans-zeatinwas present at higher levels in V208 cells than in V277 cells. Our results revealed that the production of gibberellins, aswell as of IAA and cytokinins, in the crown gall tissues ofCatharanthus roseus differed between the octopine- and nopaline-typetumors, even though the crown galls apparently grow as unorganizedaggregates of cells in both cases. ⁴Present address: Facultad de Ciencias, Pontificia UniversidadJaveriana, Bogota, Colombia. (Received September 29, 1989; Accepted February 2, 1990)     

Studies on Auxin Protectors. VII. Association of Auxin Protectors With Crown Gall Development in Sunflower Stems

Mature sunflower internodes contain only very low concentrations of auxin protectors. Wounding such internodes results in a temporary increase of protector substances. Inoculating the wounds with a virulent strain of Agrobacterium tumefaciens Conn results in a dramatic rise in protector substances, particularly the very high molecular weight protector “A”, which continues as the tumors develop. The levels attained in tumor tissue are comparable to those normally encountered in meristematic tissue. Since the auxin protectors are antioxidants which inhibit the peroxidase-catalyzed oxidation of IAA, and in view of the findings by other workers that the meristematic tissues are maintained in a relatively reduced state, the presence of such large quantities of protector substances in the tumor tissue could explain the physiological and morphological similarity of tumor tissue to young meristematic tissue.     

Correlation between Auxin Resistance and the Lack of a Membrane-Bound Auxin Binding Protein and a Root-Specific Peroxidase in Nicotiana tabacum

The levels of a membrane-bound auxin binding protein (MABP) and a root-specific peroxidase (RSP) were studied in several tobacco (Nicotiana tabacum L.) cell lines including an auxin-resistant variant. Groups of cell lines were distinguished which behaved differentially with respect to MABP and RSP depending on the hormonal composition of the medium. In cell lines in which there existed a correlation between the presence or absence of MABP and that of RSP both phenotypes were expressed if kinetin (1-2 micromolar) was supplied. In contrast, neither MABP nor RSP could be detected under any hormonal conditions tested in the auxin-resistant variant which retains the ability to differentiate shoots but lacks the ability to differentiate roots. About an eightfold increase in the concentration of MABP and a dramatic increase in the activity of RSP occurred in a transformant by a mutant strain of Agrobacterium tumefaciens lacking an intact cytokinin gene when it was grown on medium containing 1 to 2 micromolar kinetin. A correlation between auxin resistance and the lack of MABP and RSP suggests that MABP might be involved in auxin-mediated root differentiation in tobacco.     

Changes in Endogenous Ribosyl-trans-zeatin and IAA Levels in Relation to the Proliferation of Tobacco Crown Gall Cells

Changes in the contents of major endogenous plant hormones intobacco crown gall cells, namely IAA and ribosyl-trans-zeatin,during cell growth were examined using HPLC and ¹⁴C-labeledplant hormones. The content of IAA was high at the early logarithmicstage, while that of ribosyl-trans-zeatin was high at the middlelogarithmic stage. This suggests that cell growth is affectedfirst by IAA, then by ribosyl-trans-zeatin. ³ Present address: Department of Agricultural Chemistry, TottoriUniversity, Koyama, Tottori 680, Japan (Received July 13, 1981; Accepted September 11, 1981)     

烟草毛状根诱导及其茄尼醇含量初探

茄尼醇是合成泛醌类药物的重要中间体.以发根农杆菌（Agrobacterium rhizogenes）W.T15834感染烟草叶片诱导产生毛状根,探讨其茄尼醇含量变化.结果显示,获得的毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长,但在液体培养基中培养的毛状根生长更迅速,也不会形成愈伤组织.甘露碱检测及PCR结果证实,发根农杆菌Ri质粒的rolB基因已在烟草（Nicotiana tabacum）毛状根基因组中整合并得到表达.用改进的HPLC法测定烟草毛状根中的茄尼醇含量,其结果为对照根（种子萌发产生的幼苗根）的1.12倍,但仍比废弃烟叶中茄尼醇含量低43.2%.     

烟草毛状根诱导及其茄尼醇含量初探

茄尼醇是合成泛醌类药物的重要中间体。以发根农杆菌(Agrobacterium rhizogenes)W.T15834感染烟草叶片诱导产生毛状根, 探讨其茄尼醇含量变化。结果显示, 获得的毛状根能在无外源生长调节剂的MS固体和液体培养基上自主生长, 但在液体培养基中培养的毛状根生长更迅速, 也不会形成愈伤组织。甘露碱检测及PCR结果证实, 发根农杆菌Ri质粒的rolB基因已在烟草(Nicotiana tabacum)毛状根基因组中整合并得到表达。用改进的HPLC法测定烟草毛状根中的茄尼醇含量, 其结果为对照根(种子萌发产生的幼苗根)的1.12倍, 但仍比废弃烟叶中茄尼醇含量低43.2％。     

The Initiation of Auxin Autonomy in Tissue from Tobacco Plants Carrying the Auxin Biosynthesizing Genes from the T-DNA of Agrobacterium tumefaciens

Tobacco (Nicotiana tabacum cv Havana 425) plants containing the indole-3-acetic acid biosynthesizing genes (1 and 2) from the T-DNA of Agrobacterium tumefaciens strain T37-ADH₂ (mutated at the cytokinin biosynthesis gene 4) were used to study the physiological basis of the suppression and reinitiation of the auxin autonomous phenotype. The plants, though normal in appearance and cross-fertile with nontransformed, wild type tobacco, are shown to contain multiple copies of genes 1 and 2. Plants carrying these genes respond to inoculation by Agrobacterium strains mutated at genes 1 and 2 in a virulent fashion. Despite the presence and potential in planta activity of these genes, pith explants from such plants require auxin or tryptophan for growth in vitro, as does wild type tobacco. In both cases the indole-3-acetic acid levels increase rapidly in pith explants cultured on tryptophan-containing medium. However, only the tissues containing genes 1 and 2 grow subsequently on auxin-free medium and accumulate indole-3-acetic acid to levels that support growth. The capacity of such tissues to utilize naphthalene acetamide as an auxin suggests that gene 2 is rapidly activated during the reinitiation process.     

Effect of Succinic Acid-2,2-Dimethylhydrazide on Endogenous Auxin Level in Apple Shoots

Endogenous auxin levels in the shoots of apple cv. Ingrid Marie (red mutant) treated with SADH (succinic acid-2,2-dimethylhydrazide) were measured. SADH was applied in aqueous solution at 12.5, 25 or 50 mM concentrations in June, 3 weeks after flowering. The defoliated shoot tips were analysed just before SADH spray and 7, 15, 30 and 60 days after spraying. Similar treatments had been made in earlier years. When analysed before the treatment of the year started, shoot extracts of control plants contained higher auxin level than those shoots which were to receive SADH treatment, indicating a residual effect of SADH treatmens during previous years. There was almost no auxin activity in the shoots analysed after 7, 15 and 30 days of SADH treatment. The control plants maintained a high level of auxin activity during this period. However, after 60 days of treatment, shoots treated with 25 or 50 mM exhibited higher auxin activity than control shoots. There appears to be no relationship between the SADH concentration and reduction in auxin activity in the shoots at early stages after treatment.     

Single rol Genes from the Agrobacterium rhizogenes T(L)-DNA Alter Some of the Cellular Responses to Auxin in Nicotiana tabacum

Two kinds of cellular responses to auxin, the hyperpolarization of protoplasts and the division of protoplast-derived cells, were compared in Nicotiana tabacum plants transformed by different T-DNA fragments of Agrobacterium rhizogenes strain A4. Using transmembrane potential difference measurements to characterize hormonal sensitivity of mesophyll protoplasts, we found a 30-fold increase in sensitivity to auxin in protoplasts transformed by the whole Ri A4 T-DNA. Furthermore, the rol genes of the Ri A4 T_L-DNA, together or as single genes, were able to increase the sensitivity to auxin by factors up to 10⁴. The different effects of the single rol genes on the sensitivity of mesophyll protoplasts to auxin, rolB being the most powerful, were consistent with their respective rhizogenic effects on leaf fragments (A Spena, T Schmülling, C Koncz, J Schell [1987] EMBO J 6: 3891-3899). No difference was seen concerning the effects of auxin on division of cells derived from normal or transformed protoplasts. These results suggest that only some cellular responses to auxin could be selectively altered by rol genes. They also show that rol-transformed tobaccos can be a model system to study auxin action in plants.     

蜜环菌诱导子对丹参冠瘿组织积累丹参酮的影响

Using a computer software `uniform design, regression analysis and optimization system' (UROS), the effects of Armillaria mellea Karst elicitor, with respect to its concentration, time of supplement and harvest time, on the accumulation of tanshinones in the crown gall cultures of Salvia miltiorrhiza Bunge in 67-V liquid medium were studied. The result showed that the maximum tanshinone yield was 147 mg/L (P＜0.05）, 62 mg/L (P＜0.05）, 94 mg/L (P＜0.05） in the liquid medium and the cultures 29 day after addition of A. mellea (119 mL/L) at day 0, in the medium 26 day after the addition of 113 mL/L of A. mellea, 28 day after addition of 87 mL/L of A. mellea, respectively. The results confirmed that the computer software UROS was a good tool in the multi-factor study.     

Kinetics of Endogenous Cytokinin, IAA and ABA Levels in Relation to the Growth and Morphology of Tobacco Crown Gall Tissue

The kinetics of endogenous cytokinin, IAA and ABA levels duringthe growth cycle of a wild-type tobacco crown gall (W₃₈-B₆S₃)were compared with that of a shoot-inducing (Shi^-) mutant. Inboth tumor types, high IAA and cytokinin (essentially ribosyl-trans-zeatinand its corresponding glucoside) levels were built up by theend of the linear growth phase and maintained during the greaterpart of the exponential growth period. The stationary phasewas preceded by a very drastic decrease in the endogenous levelof both hormones. Quantitatively, the wild-type tumour showed a higher IAA leveland a reduced cytokinin level compared with the Shi^- mutant.No significantly different endogenous ABA pattern was observed.The reduced cytokinin level might correspond to the ratio oftransformed/untransformed cells in the wild-type tumour whereasthe reduced IAA level in the Shi^- mutant may be correlated withthe deletion of gene 2 in the T-DNA of the pGV 2206 Ti plasmid. The elevated cytokinin/IAA ratio induced shooting mainly ofthe untransformed cells in the Shi^- mutant tissue whereas inthe wild-type, the shoot suppression was compatible with thereduced cytokinin/IAA ratio. ⁴Senior Research Associate Nationaal Fonds WetenschappelijkOnderzoek (N.F.W.O.). ⁵Research Associate N.F.W.O. ⁶Recipient of an Instituut voor Wetenschappelijk Onderzoek inNijverheid en Landbouw grant. (Received February 23, 1984; Accepted June 19, 1984)     

蜜环菌诱导子对丹参冠瘿组织积累丹参酮的影响

以 6 7_V液体培养基为基本培养基 ,利用计算机软件“均匀设计、回归分析及优化系统” ,研究了蜜环菌(ArmillariamelleaKarst)诱导子浓度、诱导子的加入时间与处理的收获时间对丹参 (SalviamiltiorrhizaBunge)冠瘿组织积累丹参酮的影响。结果表明 ,蜜环菌诱导子浓度为 119mL/L、第 0天加入诱导子、第 2 9天收获培养物与培养液时可获得最高的丹参酮生产量 (147mg/L ,P <0 .0 5 ) ,蜜环菌诱导子浓度为 113mL/L、第 0天加入诱导子、第 2 6天收获培养液时可获得最高的丹参酮生产量 (6 2mg/L ,P <0 .0 5 ) ,蜜环菌诱导子浓度为 87mL/L、第 0天加入诱导子、第2 8天收获培养物时可获得最高的丹参酮生产量 (94mg/L ,P <0 .0 5 )。结果证实 ,计算机软件“均匀设计、回归分析及优化系统”对于观察值受多因素影响的研究非常有效和方便。     

Effects of the Introduction of Agrobacterium tumefaciens T-DNA ipt Gene in Nicotiana tabacum L. cv. Petit Havana SR1 Plant Cells

Transformation of tobacco leaf discs with the ‘cytokinin’ipt gene yielded several transgenic callus tissue lines, respectiveto the kind of ipt construction present in the A. tumefacienscointegrates. Those calli containing an active ipt gene wereable to grow hormone-autotrophically and showed an increasedendogenous cytokinin level in comparison with controls. Analysisof endogenous IAA level did not allow any quantitative correlationwith the cytokinin content. However, a minimal level of auxinseems to be necessary to obtain hormone-autotrophic growth.Exogenously supplied NAA significantly reduced the endogenouscytokinin content without modifying growth characteristics. The varying chlorophyll content in the different callus lineselicited the study of the ultrastructure of the plastids. Thecontrols contained small plastids, often filled with starchor accumulated vesicles that did not allow observation of theinternal membrane system. The ‘Pssu-ipt’ line, havinga higher cytokinin content, showed plastids with an internalmembrane system consisting of stroma and grana thylakoids, butthis structure was lost during subculture. Swollen thylakoidsappeared, the amount of starch was reduced and vesicles wereaccumulating. (Received November 15, 1990; Accepted March 4, 1991)     

Effect of pol III dependent elements on CAT reporter gene transcription in Nicotiana tabacum protoplasts

ABSTRACT

The effect of RNA polymerase III promoter elements on the expression of RNA polymerase II dependent genes was evaluated. The influence of tRNA(Tyr) gene, derived from Nicotiana rustica, on the expression of CAT gene, driven by CaMV35S, was tested in transformation experiments using Nicotiana tabacum protoplasts. Five “tRNA(Tyr) gene-CaMV35S?d combinations, differing in the position and orientation of the RNA polymerase III dependent gene, were utilised. Transient gene expression was evaluated by HPLC analysis. CAT expression increased with only two plasmidic constructs compared with control. Our results suggest that RNA polymerase III promoter elements exert a position- and orientation-dependent enhancer effect in Nicotiana tabacum protoplasts.