Studies on Auxin Protectors X. Protector Levels and Lignification in Sunflower Crown Gall Tissue

The lignification and differentiation of phloem fibers in sunflower stems is inhibited by growing crown gall tumors. Crown gall tumor tissue has previously been shown to contain large quantities of auxin protectors. Since auxin protectors are antioxidants which inhibit peroxidase-catalyzed reactions, and since the formation of lignin is known to involve a peroxidase-catalyzed reaction, an investigation was undertaken to examine the relationship between auxin protectors and lignification in sunflower crown gall tissue. Sunflower crown gall tissue placed into media low in mineral content, rapidly lignifies. In the low mineral media, protectors appear in the medium within an hour or two, implying that endogenously-synthesized protectors rapidly leak out of the tissue. In control media, the tissue neither lignified appreciably, nor did it exhibit an excessive amount of protector release. The addition of Ca²⁺ to the low mineral medium markedly slowed, but did not entirely prevent lignification; similarly Ca²⁺ markedly slowed the release of protector into the low mineral medium. Auxin protectors added to the low mineral medium did not inhibit lignification apparently because, in the medium, the protectors are rapidly oxidized to quinones. The addition of catechol, a substance which mimics protector, also failed to inhibit lignification and also formed a colored compound in the medium suggesting o-qui none formation. In contrast, dithiothreitol, a strong anti-oxidant which upon oxidation does not form a strong oxidant (such as o-quinone), when added to the low mineral medium does inhibit lignification. It is suggested that in the in vitro situation lignification and senescence occurs in low mineral media because the protectors leak out rapidly causing the cell's metabolism to favor peroxidase-catalyzed oxidations including those leading to lignification, while in the in vivo situation the excess protectors produced by crown gall tumor tissue diffuse into surrounding tissue, maintaining a reduced state in such tissues and thereby inhibiting differentiation and lignification. The synthesis of large quantities of protectors by the tumor tissue therefore could account for the anaplasia of the bundle caps observed in sunflower internodes in the vicinity of growing crown gall tumors.     

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.     

Auxin Synthesis in Crown Gall Tumor Tissue: A Comparison of Three Putative Precursors

Axenic crown gall tumor callus (from Vinca rosea L.) which is known to synthesize its own auxin is able to convert exogenous ¹⁴C-indole or tryptamine to indoleacetic acid [5.4 and 10 × 10⁻⁶μmol × h⁻¹× (g fr wt)⁻¹ respectively], but little or no ³H-tryptophan is converted [less than 6.4 × 10⁻⁸×μmol × h⁻¹× (g fr wt)⁻¹].     

Diversity of Opines and Opine-Catabolizing Bacteria Isolated from Naturally Occurring Crown Gall Tumors

The diversity of opines from 43 naturally occurring crown gall tumors on several plant species was analyzed for the presence of agropine, chrysopine, iminodiacid, an unidentified leucinopine-like iminodiacid (IDA-B), mannopine, octopine, nopaline, DL- and LL-succinamopine, leucinopine and heliopine. Opine utilization patterns of agrobacteria and fluorescent pseudomonads resident in a tumor were then analyzed and compared for agreement with the opine isolated from that tumor. Nopaline was the most common opine found and was detected in tumors from cherry, blackberry, grape, and plum. Octopine was not found, although octopine-catabolizing bacteria were isolated from several tumors. A new, previously undescribed iminodiacid of the succinamopine-leucinopine type (provisionally designated IDA-B) was isolated from tumors of wild blackberry. Field tumors from apple, blueberry and grape yielded no detectable opines, even though opine-utilizing bacteria were present. Bacterial isolates from plum and cherry showed the best correspondence between the opine in tumors (nopaline) and the presence of bacteria that catabolized that opine. However, several unusual opine catabolic combinations were identified, including isolates that catabolized a variety of opines but were nonpathogenic. More variability was observed among isolates from field tumors on the remaining plant species. We isolated novel mannopine-nopaline type agrobacteria from field tumors of cherry, plum and blackberry that induced tumors containing either mannopine (plus agropine) or nopaline, but not both. Epidemiologically, the galled plants from an area were not of clonal origin (same Ti plasmid), indicating that the field tumors from a small area were incited by more than one type of Ti plasmid.     

Glucosyl Zeatin and Glucosyl Ribosylzeatin from Vinca rosea L. Crown Gall Tumor Tissue

Recently detected but unidentified cytokinin activity in crown gall tumor tissue from Vinca rosea L. grown on media containing sources of reduced nitrogen has now been attributed to two adenine-type cytokinins. These compounds are glucopyranosyl derivatives of zeatin and ribosylzeatin. The substitution in each case is on the isopentenyl chain of the parent compound. Neither of these compounds had activity in the soybean callus bioassay at concentrations lower than 1 nm whereas zeatin had activity at 0.1 nm.     

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。     

ent-Kaurene Synthesis and Endogenous Levels of Gibberellins in a Shoot Forming Tobacco Crown Gall Tissue

The in vitro ent-Mcaurene synthesizing capacity, as well asthe endogenous GA content of shoot-forming tobacco crown gallsinduced by a nopaline-type Ti plasmid, was studied. For determinationof the ent-kaurene synthesizing capacity, an HPLC procedurepreceded by sample clean-up was used and the GA content wasexamined by GC-SIM. Kaurene synthesis reached a maximum at thebeginning of the logarithmic phase of growth. There was a clearcorrelation between the ent-kaurene synthesizing capacity andthe content of C₂₀-GAs. It seems that gibberellin synthesisis related to growth and development of the tissue. The natureof the GAs identified suggests, that the GA metabolism mightbe an unusual one. (Received October 12, 1987; Accepted April 11, 1988)     

Revised Methods for Purification of Ribosyl-trans-zeatin from Vinca rosea L. Crown Gall Tumor Tissue

Ribosyl-trans-zeatin has been purified from Vinca rosea L. crown gall tumor tissue by using two new sequences of isolation procedures. Identification of the compound has been established by mass spectrometry, ultraviolet absorbancy spectra, chromatographic values, and growth activity. The isolation sequences eliminate the exposures to pH extremes and the strong cation-exchange resin used in the purification reported earlier. The initial extraction procedures have been designed so as to avoid enzymatic alteration or production of active material and to prevent the inclusion in the extracts of nucleic acids which might serve as soures of the small active compounds. The production of ribosylzeatin by the tumor tissue is confirmed and the validity of isolation steps such as the use of cation exchangers is supported.     

A Transformation-induced Alteration of Cellular Membranes in Crown Gall Tumor Cells

Phospholipids were utilized as a membrane marker to test for transformation-induced alteration of cellular membranes of cultured crown gall cells of Vinca rosea L. Fully transformed cells contained less than half the amount of phospholipids (7.8 micrograms lipid P per gram fresh weight) of normal V. rosea cells (21.4 micrograms lipid P per gram fresh weight). The normal V. rosea callus cells were not significantly different (P > 0.05) in phospholipid content from partially transformed crown gall cells (20.7 micrograms lipid P per gram fresh weight). Stimulation to rapid growth of the partially transformed cells by adding higher concentrations of inorganic salts and auxin did not significantly alter their phospholipid content (23.1 micrograms lipid P per gram fresh weight). These findings suggest that the transformation process is directly responsible for an alteration of the cellular membranes and that the membrane alteration cannot be attributed to secondary effects associated with the rapid growth of these neoplastic cells.     

Quantification of Intact Cytokinin Glucosides in Datura innoxia Crown Gall Tissue by Desorption Chemical Ionisation Mass Spectrometry

Cytokinin glucosides are routinely quantified as their aglycones produced by enzymic or chemical hydrolysis. It is, however, important to be able to measure their levels per se. The present paper illustrates the use of desorption chemical ionisation mass spectrometry coupled with stable isotope dilution for the determination of intact, underivatized N- and O- glucosyl conjugates of cytokinins in Datura innoxia crown gall tissue. A total of six glucosyl conjugates were determined; the two N-glucosides, zeatin-7-glucoside and zeatin-9-glucoside, were present in higher quantities than the O-glucosyl derivatives of zeatin, dihydrozeatin and their ribosides.     

Biological Control of Crown Gall: Seed and Root Inoculation

S ummary . Experiments on the control of crown gall by inoculating susceptible plants with a non-pathogenic strain of Agrobacterium radiobacter have continued. In all experiments, highly significant disease control was achieved. In one experiment, 42% of untreated plants growing in soil heavily infested with A. radiobacter var. tumefaciens died; inoculation of seed with the non-pathogenic strain reduced this to nil. Combined seed and root inoculation was more efficient than seed inoculation alone. In naturally infested soil, combined seed and root inoculation at transplanting gave 99% control of gall formation (as dry weight). A significant increase in plant growth resulted from combined seed and root inoculation. At transplanting, roots should probably be inoculated within 2 h of lifting. This method of biological control is now widely practised by commercial growers in South Australia.     

Metabolism of Indole-3-Acetic Acid: III. Identification of Metabolites Isolated from Crown Gall Callus Tissue

The metabolism of labeled indole-3-acetic acid (IAA-2-¹⁴C) was investigated in Parthenocissus tricuspidata crown gall callus tissue. After 48 hours incubation, 85 to 90% of the supplied IAA was taken up by the tissue, and of that taken up, about 45% was conjugated with five amino acids. The conjugates found were aspartic and glutamic acid (minor ones) as well as glycine, alanine, and valine (major ones). The last four are being reported for the first time as metabolites of IAA. These conjugates were identified through their chromatographic properties, hydrolysis products, and their mass spectra. The possible significance of these amino acid conjugates is discussed.     

Biological Control of Crown Gall: Field Measurements and Glasshouse Experiments

S ummary : An ecological study showed that the pathogeniec form of Agrobacterium radiobacter (A.r. var. tumefaciens ), biotype 2 was present in soil near galled almond seedlings, where it formed a significant proportion of the total biotype 2 population; the pathogen was not detected near healthy plants. One nonpathogenic biotype 2 isolate was able, when present in numbers approximately equal to the pathogen, to prevent crown gall induction in tomato and peach seedlings. This isolate gave complete biological control of crown gall in a glasshouse test. Six other nonpathogenic isolates did not significantly inhibit gall induction by equal numbers of the pathogen. In the field, gall induction probably depends on the size of the population of the pathogen (both absolutely and relative to that of the nonpathogen) at a wound site and on the inhibitory efficiency of the nonpathogen.     

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)     

Effect of Selected Polysaccharide-Producing Soil Bacteria on Hyperhydricity Control in Oregano Tissue Cultures

Hyperhydricity, or vitrification, is a physiological malformation affecting tissue culture-generated plants. This malformation is associated with excessive hydration and poor lignification and results in poor regeneration of plants. We have tested hyperhydricity prevention in oregano by several nonspecific polysaccharide-producing rhizosphere bacteria. Among these bacteria, Pseudomonas mucidolens and another Pseudomonas sp. prevented hyperhydricity and improved acclimation of oregano clones. These two bacteria have more advantages for commercial applications than Pseudomonas strains isolated previously from oregano.     

A Simple Technique for Purifying Fungal Cultures Contaminated with Bacteria and Mites

A simple technique was developed for purifying fungal cultures contaminated with bacteria and mites. It was based on the observation that the growth of bacteria and movement of mites were confined to the upper surface of the agar. A culture contaminated with bacteria and mites was transferred to a piece of clean paper with the upper surface facing down. Small thin pieces (approximately 3 mm × 3 mm × 0.5 mm) of agar were removed from the exposed surface and transferred to a V-8 agar plate. Colonies that developed from these agar pieces were free from bacteria and mites.     

Suppression of the Tumorous State in Crown Gall Teratomas of Tobacco A Clonal Analysis

Two types of experimental approaches designed to test the possible involvement of chimeras in the persistent but readily reversible suppression of the tumorous state and in a recovery from that state in crown gall teratomas of tobacco were investigated. These studies showed that the tumorous state was suppressed in specialized cells present in the different tissues derived from each of the three layers of the apical meristem of teratoma-derived shoots of clonal origin. Although differences existed in the exogenous requirements needed for the reestablishment of the tumorous state in the different specialized cell types present in the different tissues, all again acquired a capacity for autonomous growth when appropriately stimulated. No evidence was found for the existence of periclinal or sectoral chimeras in teratoma-derived shoots of a single cell origin. Chimerism was, however, found to exist in one noncloned teratoma-derived shoot. Analysis of more than 500 clones derived from leaf mesophyll cells of cloned teratoma shoots showed that the vast majority of them (> 99%) were tumorous, while all of the 144 seed-grown plants from these shoots had completely recovered from the tumorous state. The results of these studies suggest that (a) a teratoma shoot does not depend for its development on the presence of significant numbers, if any, of cells that had recovered from the tumorous state; (b) the tumorous state may be suppressed in even the most highly specialized cells; (c) some special mechanism exists in the meiotic process for the elimination of all or most of the foreign DNA from transformed cells.