Morphogenetic sequences of the rooting process after in vitro culture or Agrobacterium rhizogenes inoculation

Abstract

After a brief review dealing with the factors inducing adventitious rhizogenesis, the morphogenetic patterns of the rooting process induced by in vitro culture or after Agrobacterium rhizogenes inoculations are described. In vitro cultures of Nicotiana tabacum, Datura innoxia and Crepis capillaris leaf explants on Murashige and Skoog's medium supplemented with sucrose and IAA or NAA have been used in order to identify the target cells for direct and indirect rooting. The structural and functional features of the prerhizogenetic cells are described and the ploidy levels of both direct and indirect root meristems are examined by means of DNA microspectrophotometry. Data on the synergistic effects between auxin, sucrose and amino acids (particularly L-ornithine) for the reactivation of the prerhizogenetic cells are also given.

Transformed roots from carrct root discs or pea epicotyls after Agrobacterium rhizogenes inoculation arise indirectly from cambium-like layers differentiated inside a previously formed callus. Numerous auxin-like symptoms at the cell level are detected after the inoculation, suggesting modifications in the endogenous contents of auxin prior to the rooting process. It is also shown that initially non-susceptible cells (fully differentiated tobacco pith) can be induced to become susceptible by in vitro treatments resulting in cell reactivation before inoculation with the bacteria. Further work is in progress to extend these observations to non-susceptible species in order to check, via the occurrence of transformed roots, their ability to react positively to foreign Ri T-DNA.     

Effect of the rol genes from Agrobacterium rhizogenes on polyamine metabolism in tobacco roots

Plants of the mangrove species Pelliciera rhizophoreae and Avicennia germinans, exhibit pronounced oscillations in stomatal aperture under certain climatic conditions. During these oscillations, changes in transpirational water loss were closely followed by those in leaf water potential (ψ1) as indicated by continuous monitoring with an in situ dewpoint hygrometer. With this instrument, it was possible to measure dynamic changes in ψ1 for several days under constant conditions. Subsequently, the leaf was detached from the shoot and a pressure-volume (PV) curve was established by repeatedly weighing the leaf, still attached to the hygrometer during short interruptions of the water potential recordings. The pressure-volume relationship was then used to derive other water relations parameters from these water potential data. Thus, the procedure described herein allows a continuous analysis of the relevant components of bulk leaf water relations. Oscillations in water potential were also measured with single leaves using a pressure chamber. Water relations data obtained with these two different methods were in good agreement. In addition, osmotic potentials derived from the PV-analysis were well within the range of those determined cryoscopically using extracted cell sap.     

Establishment of new axenic hairy root lines by inoculation with Agrobacterium rhizogenes

Cultured hairy root lines resulting from infection by Agrobacterium rhizogenes are known for approximately thirty plant species. We extend this range by establishing forty original dicotyledonous hairy root lines with A. rhizogenes strain A₄. Hairy roots have been cultured for at least 2–6 years on Murashige & Skoog medium. Some hairy root cultures such as Anagallis arvensis and Antirrhinum majus spontaneously regenerated whole plants.     

The rolB-like part of the Agrobacterium rhizogenes orf8 gene inhibits sucrose export in tobacco

Many Agrobacterium T-DNA genes belong to the highly diverse rolB family. The mode of action of most of these genes is still unknown. rolB-like sequences also are present at the 5' ends of the T-DNA-located iaaM genes and the iaaM homolog orf8, whereas iaaM genes from Pseudomonas and Erwinia spp. lack such sequences. iaaM genes encode tryptophan monooxygenases; these enzymes convert tryptophan into indole-3-acetamide, a precursor of indole-3-acetic acid. Tobacco plants expressing the rolB-like part of the A4 orf8 gene (2x35S-A4-Norf8 plants) accumulate glucose, fructose, sucrose, and starch and resemble sucrose transporter (NtSUT1) antisense plants. Different lines of evidence indicate that 2x35S-A4-Norf8 plants export less sucrose from source leaves. Glucose, fructose, sucrose, and starch accumulate in source leaves during sink-source transition, whereas sink tissues like petioles and midveins contain lower levels than normal. Petiole exudation experiments demonstrate a significant decrease in export of label after 14C-sucrose infiltration and after 14CO2 labeling. Grafting of stunted homozygous 2x35S-A4-Norf8 plants onto wild-type rootstocks restores growth, indicating that unloading is not affected. Growth of 2x35S-A4-Norf8 seedlings is inhibited on naphthalene acetic acid-containing media, suggesting a link between sucrose transport and auxin sensitivity.     

不同理化因子对发根农杆菌Ri质粒转化骆驼刺的影响

骆驼刺苗茎切段有很强的离体培养再生能力,用野生型发根农杆菌Ａ,菌株转化骆驼刺度轩茎段,并建立了发根转化体的培养体系。高压纸电泳显示转化组织可合成冠瘿。试验不同试验经条件及理化因子对提高转化率和加快发根生长的影响。结果表明,经过３天预培养及０．３ｍｏｌ／Ｌ甘露醇１２ｈ高渗处理后培养于ＭＳ培养基中可有效提高转化率。而２％～３％蔗糖,３ｍｇ／Ｌ,ＧＡ３,１０μｍｏｌ／ＬＣｕ＾２＋,１０ｍｇ／ＬＡｇ＾＋,     

Recovery of morphologically normal transgenic tobacco from hairy roots co-transformed with Agrobacterium rhizogenes and a binary vector plasmid

Summary Co-transformation of tobacco (Nicotiana tabacum) leaf explants with Agrobacterium rhizogenes harbouring pRi1855 and the binary vector pBin19 was achieved at a frequency of 67%. The kanamycin resistant hairy roots were cultured via a callusing phase to regenerate plants which were partially fertile when outcrossed with wild-type pollen. Phenotypic and molecular analysis of the F₁ progeny demonstrated the efficient segregation of the hairy root marker from the kanamycin resistance marker, enabling morphologically normal plants to be recovered which retained the binary vector marker gene. This co-transformation strategy provides a means of introducing non-selectable genes into plants in cases where antibiotic resistance markers are undesirable.     

Differential effects of ethylene on pith peroxidase of intact tobacco plants and excised tissue

Ethylene increases the pith peroxidase activity of intact tobacco plants (Nicotiana tabacum) but not of excised pith, either at atmospheric or reduced pressures. In the intact plant, the increased activity involves augmentation of the two constitutive anodic isoperoxidases. In the excised pith, ethylene strongly represses one injury-induced isoperoxidase, while not markedly affecting other isozymes known to be repressed by auxin. Thus, the previously described auxin-induced repression of peroxidase is not due mainly to auxin-induced ethylene formation.     

Factors influencing the incidence of habituation for cytokinin of tobacco pith tissue in culture

Pith tissue of Nicotiana tabacum L. cv. Havana 425 exhibits a gradient in its tendency to habituate for cytokinin on an auxin-containing medium at 35° C, about 10° C above the standard culture temperature. Explants of pith from below the 8th to 11th internode, counting from the bottom of the plant, rarely habituate for cytokinin; explants from above this threshold habituate rapidly. The explants must also be above a critical size, about 20–30 mg, to habituate. There was a pronounced interaction between size and position effects; the threshold position for cytokinin habituation shifted upward with decreasing explant size.     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5′ end of a reporter gene, β-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

Promoter of the rolC gene of Agrobacterium rhizogenes can be strongly regulated in glandular cell of transgenic tobacco

A 577-bp promoter segment of Agrobacterium rhizogenes rolC, previously known as the phloem-specific gene expression promoter, was fused to the 5' end of a reporter gene, beta-glucuronidase (GUS), uidA. This rolC-promoter-driven expression of the GUS gene was found to be significantly strong in glandular cells in transgenic tobacco plants. Analysis of this segment of the promoter sequence revealed a myb response element.     

A putative rolB gene homologue of the Agrobacterium rhizogenes TR-DNA has different morphogenetic activity in tobacco than rolB

Agrobacterium rhizogenes strains of the agropine type harbor on their Ri-plasmid two T-DNAs, a left TL-DNA and a right TR-DNA. The rolB gene of the TL-DNA is the major factor in the pathogenesis of the hairy-root disease and its constitutive expression interferes profoundly with plant morphogenesis. We have tested whether the expression of its sequence related putative homologue from the TR-DNA (rolB_TR) may cause also bacterial virulence or affect plant development. Unlike rolB, rolB_TR is unable to induce root formation on tobacco leaf discs. Tobacco plants expressing a chimeric 35S::rolB_TR gene have reduced stature, off-shoots at the stem base and bent and wrinkled leaves with epinastic growth. 14 N-terminal amino acids which are absent in the rolB protein are indispensable to rolB_TR protein activity. The characteristic tyrosine phosphatase super family motif CX₅R is absent in the rolB_TR protein. For rolB this motif is possibly functionally relevant. We conclude that the rolB_TR gene product has morphogenic activity but is not a functional homologue of the rolB protein.     

Expression of nitrous oxide reductase from Pseudomonas stutzeri in transgenic tobacco roots using the root-specific rolD promoter from Agrobacterium rhizogenes

The nitrous oxide (N(2)O) reduction pathway from a soil bacterium, Pseudomonas stutzeri, was engineered in plants to reduce N(2)O emissions. As a proof of principle, transgenic plants expressing nitrous oxide reductase (N(2)OR) from P. stutzeri, encoded by the nosZ gene, and other transgenic plants expressing N(2)OR along with the more complete operon from P. stutzeri, encoded by nosFLZDY, were generated. Gene constructs were engineered under the control of a root-specific promoter and with a secretion signal peptide. Expression and rhizosecretion of the transgene protein were achieved, and N(2)OR from transgenic Nicotiana tabacum proved functional using the methyl viologen assay. Transgenic plant line 1.10 showed the highest specific activity of 16.7 μmol N(2)O reduced min(-1) g(-1) root protein. Another event, plant line 1.9, also demonstrated high specific activity of N(2)OR, 13.2 μmol N(2)O reduced min(-1) g(-1) root protein. The availability now of these transgenic seed stocks may enable canopy studies in field test plots to monitor whole rhizosphere N flux. By incorporating one bacterial gene into genetically modified organism (GMO) crops (e.g., cotton, corn, and soybean) in this way, it may be possible to reduce the atmospheric concentration of N(2)O that has continued to increase linearly (about 0.26% year(-1)) over the past half-century.     

Treatment of Agrobacterium or leaf disks with 5-azacytidine increases transgene expression in tobacco

We have studied the effect of the demethylating agent azacytidine (azaC) on expression of a -glucuronidase (GUS) gene transferred to tobacco leaf disks by Agrobacterium-mediated transformation. In a system where no selection was performed, where shoot formation was partially repressed, and where Agrobacterium does not express the GUS gene, we were able to follow the early events of transient and stable expression. Two days after inoculation, 8% of the cells expressed GUS but this proportion rapidly decreased to near zero in the following week. Treatment of leaf disks with azaC just after transformation retarded this inactivation to some extent, while treatment of Agrobacterium prior to transformation increased the frequency of transient expression. Three weeks after inoculation the number of GUS-expressing cells increased 4- to 6-fold in the leaf disks treated with azaC and in the leaf disks transformed with azaC-treated bacteria, while the control remained low. These data suggest that DNA methylation is involved in transgene inactivation and that a large number of silent but potentially active transgenes become integrated.     

Transfer of a 4.3-kb fragment of the TL-DNA of Agrobacterium rhizogenes strain A4 confers the pRi transformed phenotype to regenerated tobacco plants

Two strategies were used to transfer into tobacco a 4.3-kb fragment of the TL-DNA of the Ri plasmid of Agrobacterium rhizogenes strain A4. In the liposome-mediated procedure a plasmid containing a neomycin phosphotransferase II (NPT II) gene conferring kanamycin resistance and another plasmid containing the 4.3-kb Eco RI fragment (pRiA4 Eco RI-15) were co-transferred into the tobacco genome. In the Agrobacterium transformation procedure, a micro-Ri vector containing a kanamycin resistance gene and the same pRiA4 fragment was used to transform tobacco leaf fragments. Kanamycin resistant plants were regenerated in both cases. They present a phenotype similar to that of plants regenerated from hairy roots induced by A. rhizogenes, that is wrinkled leaves, reduced apical dominance and ability to form hairy root on leaf fragments. In one plant (Ka158), the organization, expression and transmission to the progency of the inserted foreign DNA were analyzed more precisely.