Role of bacterial cellulose fibrils in Agrobacterium tumefaciens infection.

During the attachment of Agrobacterium tumefaciens to carrot tissue culture cells, the bacteria synthesize cellulose fibrils. We examined the role of these cellulose fibrils in the attachment process by determining the properties of bacterial mutants unable to synthesize cellulose. Such cellulose-minus bacteria attached to the carrot cell surface, but, in contrast to the parent strain, with which larger clusters of bacteria were seen on the plant cell, cellulose-minus mutant bacteria were attached individually to the plant cell surface. The wild-type bacteria became surrounded by fibrils within 2 h after attachment. No fibrils were seen with the cellulose-minus mutants. Prolonged incubation of wild-type A. tumefaciens with carrot cells resulted in the formation of large aggregates of bacteria, bacterial fibrils, and carrot cells. No such aggregates were formed after the incubation of carrot cells with cellulose-minus A. tumefaciens. The absence of cellulose fibrils also caused an alteration in the kinetics of bacterial attachment to carrot cells. Cellulose synthesis was not required for bacterial virulence; the cellulose-minus mutants were all virulent. However, the ability of the parent bacterial strain to produce tumors was unaffected by washing the inoculation site with water, whereas the ability of the cellulose-minus mutants to form tumors was much reduced by washing the inoculation site with water. Thus, a major role of the cellulose fibrils synthesized by A. tumefaciens appears to be anchoring the bacteria to the host cells, thereby aiding the production of tumors.     

Further observations on the interaction between sugar cane and Gluconacetobacter diazotrophicus under laboratory and greenhouse conditions.

Sugar cane (Saccharum spp.) variety SP 70-1143 was inoculated with Gluconacetobacter diazotrophicus strain PAL5 (ATCC 49037) in two experiments. In experiment 1 the bacteria were inoculated into a modified, low sucrose MS medium within which micropropagated plantlets were rooted. After 10 d there was extensive anatomical evidence of endophytic colonization by G. diazotrophicus, particularly in lower stems, where high numbers of bacteria were visible within some of the xylem vessels. The identity of the bacteria was confirmed by immunogold labelling with an antibody raised against G. diazotrophicus. On the lower stems there were breaks caused by the separation of the plantlets into individuals, and at these 'wounds' bacteria were seen colonizing the xylem and intercellular spaces. Bacteria were also occasionally seen entering leaves via damaged stomata, and subsequently colonizing sub-stomatal cavities and intercellular spaces. A localized host defence response in the form of fibrillar material surrounding the bacteria was associated with both the stem and leaf invasion. In experiment 2, stems of 5-week-old greenhouse-grown plants were inoculated by injection with a suspension of G. diazotrophicus containing 10(8) bacteria ml(-1). No hypersensitive response (HR) was observed, and no symptoms were visible on the leaves and stems for the duration of the experiment (7 d). Close to the point of inoculation, G. diazotrophicus cells were observed within the protoxylem and the xylem parenchyma, where they were surrounded by fibrillar material that stained light-green with toluidine blue. In leaf samples taken up to 4 cm from the inoculation points, G. diazotrophicus cells were mainly found within the metaxylem, where they were surrounded by a light green-staining material. The bacteria were growing in relatively low numbers adjacent to the xylem cell walls, and they were separated from the host-derived material by electron-transparent 'haloes' that contained material that reacted with the G. diazotrophicus antibody.     

The developmental process of xylem embolisms in pine wilt disease monitored by multipoint imaging using compact magnetic resonance imaging

In pine wilt disease (PWD), embolized tracheids arise after virulent pine wood nematodes (PWN), Bursaphelenchus xylophilus, invade the resin canal of pine tree; infected pine trees finally die from significant loss of xylem water conduction. We used a compact magnetic resonance imaging system with a U-shaped radio frequency (rf) probe coil to reveal the developmental process of the xylem dysfunction in PWD. Multiple cross-sectional slices along the stem axis were acquired to periodically monitor the total water distribution in each 1-year-old main stem of two 3-year-old Japanese black pines (Pinus thunbergii) after inoculation of PWN. During the development of PWD, a mass of embolized tracheids around the inoculation site rapidly enlarged in all directions. This phenomenon occurred before the significant decrease of water potential. Some patch-like embolisms were observed at all monitoring positions during the experimental period. Patchy embolisms in a cross-section did not expand, but the number of patches increased as time passed. When the significant decrease of water potential occurred, the xylem dysfunctional rate near the inoculation point exceeded 70%. Finally, almost the whole area of xylem was abruptly embolized in all cross-sections along the stem. This phenomenon occurred just after water conduction was mostly blocked in one of the cross-sections. Thus, it appears that the simultaneous expansion of embolized conduit clusters may be required to induce a large-scale embolism across the functional xylem. Consequently, xylem dysfunction in infected trees may be closely related to both the distribution and the number of PWN in the pine stem.     

Evidence for high activity of xylem parenchyma and ray cells in the interface of host stem and Agrobacterium tumefaciens-induced tumours of Ricinus communis

Rapidly developing tumours at hypocotyls of Ricinus communis, induced by Agrobacterium tumefaciens strain C58, were characterized by strong differentiation of vascular bundles and their functional connection to the host bundles. The stem/tumour interface showed increased xylem, with numerous vessels accompanied by multiseriate unlignified rays. To know how nutrients efficiently accumulate in the tumour sink tissue, cell electropotentials (E(m)) in cross-sections were mapped. The measured cells were identified by injected Lucifer Yellow. Xylem and phloem parenchyma cells and stem/tumour-located rays hyperpolarized to E(m) values of about -170 mV, which suggest high plasma membrane proton pump activities. Rapidly dividing cells of cambia or small tumour parenchyma cells had low E(m). The tumour aerenchyma and the stem cortex cells displayed values close to the energy-independent diffusion potential. The lowest values were recorded in stem pith cells. Cell K(+) concentrations largely matched the respective E(m). The pattern of individual cell electropotentials was supplemented by whole organ voltage measurements. The voltage differences between the tumour surface and the xylem perfusion solution in stems attached to the tumours, the trans-tumour electropotentials (TTP), confirm the findings of respiration-dependent and phytohormone-stimulated high plasma membrane proton pump activity in intact tumours, mainly in the xylem and phloem parenchyma and ray cells. TTPs were inhibited by addition of NaN(3), CN(-) plus SHAM or N(2) gas in the xylem perfusion solution and by external N(2) flushing. The data provide functional evidence for the structural basis of priority over the host shoot in nutrient flow from the stem to the tumour.     

Tumor infiltrating leukocytes (tils) during progressive tumor growth and BCG-mediated tumor regression

Tumor regression was induced by intralesional injection with BCG, 7 days after inoculation of line 10 hepatocellular carcinoma cells into strain 2 guinea pigs. Tumor-infiltrating leukocytes (TILS) were characterized immunohistochemically with 11 monoclonal antibodies (MoAbs) during the induction phase of line 10-immunity, and during immune-mediated regression of the tumor, at days 12 and 28 after tumor cell inoculation, respectively. At day 5 after BCG-injection (day 12 after tumor cell inoculation), there were no major differences between the TIL subpopulations of the BCG-treated and untreated tumors. The TILS were mainly T-cells, as identified by MoAbs against Pan T-cells (CT5), T-cytotoxic/suppressor cells (CT6) and T-helper/inducer cells (H155). A limited number of macrophages was also present. However, at day 21 after BCG-treatment (28 days after tumor cell inoculation), the fibrous stroma was increased dramatically in most of the BCG-treated tumors, and as a result, the tumor cell islets were smaller than in control tumors. In the BCG treated tumors, the numbers of T-cells and macrophages were increased. In growing and regressing tumors, MHC class I and II antigens were strongly expressed in TILS and in the tumor stroma. Line 10 tumor cells prior to inoculation expressed no MHC class I or II antigens. In the centers of the tumor islets at days 12 and 28, expression of these antigens was not found. However, MHC class I and II antigens were expressed on tumor cells at sites where they lay close to the fibrous stroma or TILS. This observation was made in progressively growing tumors and was most apparent in BCG-treated tumors.     

THE ANATOMY OF TUMORS INDUCED ON CITRUS BY CITRUS VEIN-ENATION VIRUS

Citrus vein-enation virus-induced tumors on leaves, thorns, stems, and roots of Citrus auranti-folia (Christm.) Swingle and C. jambhiri Lush. were investigated. Leaf vein tumors are initiated as cytological abnormalities of phloem fiber primordial cells adjacent to protophloem sieve tubes. The tumors then enlarge through hyperplasia of the affected fiber primordia. The mesophyll and epidermal tissues on the abaxial side of affected veins divide less prolifically and contribute to the tumor mass. Leaf vein tumors are determinate, like the protophloem where they originate, and cease enlarging as the leaf matures. Most thorn tumors are initiated in fiber primordia and develop similarly to vein tumors. Stem tumors and some thorn tumors develop from affected cells of the procambial tissue that lies between the metaxylem and metaphloem of vascular bundles. The cambium which differentiates in these areas is composed of affected cells and produces large amounts of abnormal xylem tissue. The resulting woody tumors are indeterminate like the cambium and secondary xylem.     

Bacterial Attachment to a Specific Wound Site as an Essential Stage in Tumor Initiation by Agrobacterium tumefaciens

The number of tumors initiated by Agrobacterium tumefaciens strain B6 on primary pinto bean leaves was decreased when cells of an avirulent strain (IIBNV6) were included in the inoculum. With sufficient B6 cells to initiate ca. 50% of the maximal number of tumors per leaf, inhibition was detected at a 1:1 ratio of B6 to IIBNV6 cells and increased linearly with the logarithm of the number of IIBNV6. Varying the number of B6 in the presence of a constant number of IIBNV6 or varying the number of both, while maintaining a constant ratio of B6 to IIBNV6, showed that the inhibition was a function of the absolute concentration of each cell type. The data fit a one-particle dose response curve, which indicates that a single IIBNV6 cell can prevent tumor initiation by a single B6 cell. Inhibition was obtained with mixed inocula and when the addition of IIBNV6 preceded B6, but not when B6 preceded IIBNV6. Heat-inactivated IIBNV6 inhibited, as did ultraviolet or heat-inactivated B6. Several unrelated bacteria and certain strains of Agrobacterium failed to inhibit, whereas other related strains gave inhibition. Attachment of IIBNV6 to a specific would site, thus excluding B6 from the site, is proposed to account for these data. A specific complementary binding of a virulent bacterium to a host wound site exposed by the inoculation procedure is suggested as an essential early event in the crown-gall tumor initiation process.     

Genetic transformation of Populus deltoides and P.x euramericana clones using Agrobacterium tumefaciens

The susceptibility of different Populus euramericana (Neva, PE68-022 x P. nigra, 71-060 x P. nigra) and P. deltoides (PE68-022 x P. deltoides) clones to wild-type Agrobacterium tumefaciens strains (A281 and 82.139) was evaluated in an inoculation experiment, and differences in the frequency of tumor formation (0-48) were found. Co-cultivation experiments demonstrated high transformation ability of oncogenic binary A. tumefaciens strains as compared to disarmed strains. Using oncogenic binary strains, transgenic calluses were obtained from all tested clones. The presence of acetosyringone did not influence the transformation frequency of the disarmed strains. Co-inoculation experiments were performed using leaf discs and a bacterial suspension containing both wild-type and disarmed strains. No positive effects on transformation efficiency were noticed in these conditions either. The transformation of tumors and kanamycin resistant calluses was confirmed by DNA analysis.     

Involvement of a vitronectin-like protein in attachment of Agrobacterium tumefaciens to carrot suspension culture cells.

Infections of dicotyledonous plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. Attachment of the bacteria to plant host cells is required for tumor formation. Human vitronectin and antivitronectin antibodies both inhibited the binding of A. tumefaciens to carrot cells. Wild-type bacteria are able to bind radioactive vitronectin; nonattaching mutants showed a reduction in the ability to bind vitronectin. The binding of biotype 1 A. tumefaciens to carrot cells or to radioactive vitronectin was not affected by high ionic strength. Detergent extraction of carrot cells removed the receptor to which the bacteria bind. The extract was found to contain a vitronectin-like protein. These results suggest that A. tumefaciens utilizes a vitronectin-like protein on the plant cell surface as the receptor for its initial attachment to host cells.     

Expression of Kanamycin resistance introduced byAgrobacteriutn binary vector intoNicotiana tabacum andAtropa belladonna

Kanamycin resistance gene was introduced into tobacco and Atropa belladonna cells by binary vectors, based on Agrobacterium, by means of inoculation of seedlings. The plasmid pGA472, which carries chimaeric kanamycin resistance gene expressed in plants was introduced by transformation into A. tumefaciens Bo542, harbouring pTiBo542 plasmid and A. rhizogenes 8196, carrying pRi8196 plasmids and the resulting two strains were used as binary vectors. Tobacco tumors induced by A. tumefaciens Bo542(pGA472) grew as undifferentiated, kanamycin resistant tissues. Those induced by A. rhizogenes 8196(pGA472) differentiated into transformed plants. When cultivated in vitro on 200 μg ml^-1 kanamycin medium, they showed yellow green sectoring, which was not selected out during vegetative propagation. Atropa belladonna tissues transformed by both A. tumefaciens Bo542(pGA472) and A. rhizogenes 8196(pGA472) differentiated plants which grew well on 200 μg ml^-1 kanamycin as green, non-sectoring plants; sensitive cells obviously did not divide at all. Selection of Atropa belladonna transformed tissues on kanamycin medium is much more efficient than selection of transformed tobacco tissues with introduced kanamycin resistance gene.     

ELECTRON MICROSCOPY OF PLASMA-CELL TUMORS OF THE MOUSE : I. MPC-1 and X5563 Tumors

An electron microscope study was made of a series of transplanted MPC-1 plasma-cell tumors carried by BALB/c mice. Large numbers of particles similar in morphology to virus particles were present inside the endoplasmic reticulum of tumor plasma cells. Very few particles were seen outside the cells or in ultracentrifuged preparations of the plasma or ascites fluid. In very early tumors particles were occasionally seen free in the cytoplasm adjacent to finely granular material. In general, the distribution of these particles inside endoplasmic reticulum is similar in early and late tumors. A few transplanted X5563 tumors of C3H mice were also examined. Large numbers of particles were found in the region of the Golgi apparatus in late X5663 tumors. A newly described cytoplasmic structure of plasma cells, here called a "granular body," appears to be associated with the formation of the particles. Particles present in MPC-1 tumors are exclusively of a doughnut form, whereas some of those in the inclusions of the late X5563 tumors show a dense center. Normal plasma cells, produced by inoculation of a modified Freund adjuvant into BALB/c mice. have been compared morphologically with tumor plasma cells of both tumor lines.     

高寒草地甘肃臭草茎-叶性状的坡度差异性

茎与叶的生长形态决定植物与外界环境的物质交换能力, 茎叶的异速生长模式对认识植物表型可塑性及其调节机理具有重要意义。在祁连山高寒退化草地, 利用ArcGIS建立研究区域的数字高程模型(DEM), 并提取样地坡度数据, 采用标准化主轴估计(SMA)方法, 研究了不同坡度甘肃臭草(Melica przewalskyi)种群茎与叶的生长。结果表明: 随着坡度增大, 甘肃臭草茎干质量、叶干质量、叶面积均呈逐渐减小趋势, 叶片数呈增加趋势; 甘肃臭草叶干质量的增长速度显著大于茎干质量的增长速度, 叶面积与茎干质量近等速增长; 不同坡度间的比较显示, 随着坡度变陡甘肃臭草茎干质量与叶干质量异速斜率显著减小(p < 0.05), 陡坡上的甘肃臭草若要生成与缓坡样地中相同的叶生物量需要投入更多的茎生物量, 茎干质量与叶面积的y轴截距显著减小(p < 0.05), 即相同的茎干质量投入下, 较大坡度的甘肃臭草叶面积投入显著降低, 趋向于减小叶面积增加叶数量。坡度梯度上甘肃臭草加快了茎的相对生长速率而减小了在叶面积上的投入, 体现了不同坡度甘肃臭草茎-叶生物量分配机制及资源利用策略, 同时说明高寒草地中小叶更具生境适应性。     

Polymyxin resistance in Agrobacterium tumefaciens and its effect on crown gall tumor induction.

Polymyxin-resistant (PBLr) mutants of Agrobacterium tumefaciens A6, B6, and B6M were isolated from polymyxin-sensitive (PBLs) parent strains in a defined medium containing 600 microgram of polymyxin B sulfate per millilitre. The weight and number of tumors induced by PBLr mutants on a variety of host plants such as carrot, potato, and pinto bean were 45--75% less than those induced by PBLs wild types. The crude cell envelopes (CCE) prepared from both PBLs and PBLr bacteria were inhibitory for tumor initiation when they were applied before or during the inoculation of viable tumorigenic bacteria, but not when they were applied 30 min after the inoculation of infectious bacteria. The potency to inhibit the tumor initiation by the CCE prepared from PBLs cells was approximately 50% higher than that by the equal amount of the CCE prepared from PBLr cells. The concentration of CCE preparations required to reduce tumor induction 50% in carrot and pinto bean was determined to be 2.6 mg/mL and 4.0--6.2 mg/mL for the CCE derived from PBLs and PBLr cells, respectively. These data suggest that the envelope structure or composition of PBLs and PBLr cells is distinct, and that the acquisition of resistance to polymyxin by agrobacteria modifies envelope structure or components which are essential for tumor initiation.     

Development of successive cambia and formation of flat stems in Rhynchosia pyramidalis (Lam.) Urb. (Fabaceae)

Stem flattening in Rhynchosia pyramidalis (Fabaceae) is achieved by the development of crescent-shaped successive cambia on two opposite sides of the stem (referred hereafter as distal side). Other lateral sides of the stem (adjacent to supporting host and its opposite side, referred as proximal sides) usually possess single cambium. In the young stems, parenchymatous cells located outside to protophloem of distal side dedifferentiate and develop small segments of cambium. Concomitant to bidirectional differentiation of the secondary xylem and phloem, these newly developed cambial segments also extend in tangential directions. Differential activity of newly developed crescent-shaped cambial segments deposits more secondary xylem at median position as compared to their terminal ends of the stem on distal side; consequently, it pushes the cambial segment outside, thus resulting in crescent-shaped arcs of the cambia only on two opposite sides. After the production of 1–2 mm of secondary xylem, they cease to divide and new segments of cambial arc develop on the same side in a similar fashion. Such repeated behaviour of successive cambia development consequently leads to the formation of tangentially flat stems. The secondary xylem is diffusely porous with indistinct growth rings and is composed of vessels (wide and narrow), fibres, axial ray parenchyma cells, while phloem consisted of sieve elements, companion cells, axial and ray parenchyma. Rays in both xylem and phloem are uni- to multiseriate and heterocellular. The structure of secondary xylem and development of successive cambia is correlated with climbing habit.     

Wood Anatomy and the Development of Interxylary Phloem of Ipomoea hederifolia Linn. (Convolvulaceae)

In Ipomoea hederifolia Linn., stems increase in thickness by forming successive rings of cambia. With the increase in stem diameter, the first ring of cambium also gives rise to thin-walled parenchymatous islands along with thick-walled xylem derivatives to its inner side. The size of these islands increases (both radially and tangentially) gradually with the increase in stem diameter. In pencil-thick stems, that is, before the differentiation of a second ring of cambium, some of the parenchyma cells within these islands differentiate into interxylary phloem. Although all successive cambia forms secondary phloem continuously, simultaneous development of interxylary phloem was observed in the innermost successive ring of xylem. In the mature stems, thick-walled parenchyma cells formed at the beginning of secondary growth underwent dedifferentiation and led to the formation of phloem derivatives. Structurally, sieve tube elements showed both simple sieve plates on transverse to slightly oblique end walls and compound sieve plates on the oblique end walls with poorly developed lateral sieve areas. Isolated or groups of two to three sieve elements were noticed in the rays of secondary phloem. They possessed simple sieve plates with distinct companion cells at their corners. The length of these elements was more or less similar to that of ray parenchyma cells but their diameter was slightly less. Similarly, in the secondary xylem, perforated ray cells were noticed in the innermost xylem ring. They were larger than the adjacent ray cells and possessed oval to circular simple perforation plates. The structures of interxylary phloem, perforated ray cells, and ray sieve elements are described in detail.     

Tumor Induction by Agrobacterium tumefaciens: Specific Transfer of Bacterial Deoxyribonucleic Acid to Plant Tissue

When Agrobacterium tumefaciens cells grown in the presence of tritiated thymidine to label specifically the bacterial deoxyribonucleic acid (DNA) are incubated with carrot root tissue for short periods of time, an appreciable fraction of the label becomes firmly associated with the root tissue. Such association is not observed in identical experiments when A. tumefaciens cell ribonucleic acid or protein are labeled. The extent of the retention of thymidine-derived label from bacterial cells by the root tissue in experiments with A. radiobacter and poorly tumorigenic strains of A. tumefaciens is significantly less than that afforded by tumorigenic strains of A. tumefaciens but greater than the level afforded by Escherichia coli. Transfer of DNA-specific label from A. tumefaciens to carrot root discs is not enhanced by treatments designed to provoke lysis of the bacterial cells, nor is it decreased by addition of deoxyribonuclease or excess unlabeled thymidine to the incubation medium. Bacterial cell-to-plant cell contact is necessary for transfer. Unlabeled A. radiobacter cells decrease in a competitive manner transfer of label when mixed with labeled A. tumefaciens cells. These findings suggest that transfer of DNA from A. tumefaciens to plant tissue after binding of the bacterial cells to specific plant tissue site(s) is a necessary feature of the mechanism by which A. tumefaciens provokes tumors in plants and provides an experimental technique of potentially great value in study of the early steps in the process of tumor induction by A. tumefaciens.     

The effect of the Agrobacterium tumefaciens attR mutation on attachment and root colonization differs between legumes and other dicots

Infections of wound sites on dicot plants by Agrobacterium tumefaciens result in the formation of crown gall tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus, A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.     

Fine structure and distribution of extracellular polymer surrounding selected aerobic bacteria.

The structure and distribution of extracellular polymer surrounding Bacillus circulans, Diplococcus (Streptococcus) pneumoniae, Streptococcus salivarius, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Herella vaginacola (Acinetobacter calcoaceticus), and Agrobacterium tumefaciens were studied by electron microscopy. A modified ruthenium red staining procedure was used to examine the fine structure of capsule and slime. Freeze-etching and critical-point drying were used to examine the quantity of unaltered exocellular material. Comparative data demonstrate that fibrillar extracellular polymer surrounding B. circulans, D. pneumoniae, and K. pneumoniae is capsule (cell wall attached) which is characteristic of the producing organism. Capsular polymer generally appeared fibrillar, although globular polymer consisted of capsular subunits bound to S. salivarius and H. vaginacola. Exocellular slime was present about S. aureus, P. aeruginosa, and A. tumefaciens.     

Wound-induced traumatic resin duct development in stems of Norway spruce (Pinaceae): anatomy and cytochemical traits

Wounding of Norway spruce by inoculation with sterile agar, or agar containing the pathogenic fungus Ceratocystis polonica, induced traumatic resin duct formation in the stem. Visible anatomical responses occurred in the cambium 6-9 d post-inoculation. Near the inoculation site cellular proliferation, polyphenolic accumulation, and lignification were induced as a wound reaction to seal the damaged area. Five centimetres from the inoculation site cells in the cambial zone swelled and divided to form clusters. By 18 d post-inoculation, these cells began to differentiate into resin duct epithelial cells surrounding incipient schizogenous lumens. Mature axial traumatic ducts appeared by 36 d as a row of ducts in the xylem centripetal to the cambium. The ducts formed an interconnected network continuous with radial resin ducts. Parenchyma cells surrounding the ducts accumulated polyphenols that disappeared as the cells differentiated into tracheids. These polyphenols appeared to contain fewer sugar residues compared to those accumulating in the secondary phloem, as indicated by the periodic acid-Schiff's staining. The epithelial cells did not accumulate polyphenols but contained immunologically detectable phenylalanine ammonia lyase (EC 4.3.1.5), indicating synthesis of phenolics as a possible resin component. These findings may represent a defense mechanism in Norway spruce against the pathogenic fungus Ceratocystis polonica.     

THE INDUCTION OF LEAF TUMORS BY AGROBACTERIUM TUMEFACIENS

Using carborundum as an abrasive and light rubbing with a culture of Agrobacterium tumefaciens, leaves of various species of bean and tobacco develop tumors on the leaf lamina. The induction of these tumors requires wounding, the presence of a virulent strain of the bacterium and is due to the bacterium, not substances released into the bacterial culture medium during growth. Observations of the histology and cytology of these tumors on the primary leaves of pinto bean show no significant differences from the more commonly studied stem tumors. The tumors on pinto beans first appear as chlorotic nests of dividing cells which gradually accumulate chlorophyll, eventually becoming dark green in color as opposed to the surrounding leaf tissue which is completely chlorotic at this stage. Tumor development is enhanced by a dark period following inoculation while growth of the leaf is essentially stopped. The tumors thus exhibit a pattern of growth and development independent of that of the normal leaf. The number of tumors obtained on pinto bean leaves was found to depend on the concentration of bacteria in the inoculum and on the age of the plants. A sharp peak in response was observed at about 7 days from planting. Best results were obtained by adding the bacterium at the time of wounding. The tumors were shown to differ from IAA-induced leaf proliferations with respect to their point of origin on the leaf, morphology, physiology and development.