Non-oncogenic plant vectors for use in the agrobacterium binary system

Summary Agrobacterium strains harbouring the T-region and the virulence-region of the Ti plasmid on separate replicons still display efficient T-DNA transfer to plants. Based on this binary vector strategy we have constructed T-region derived gene vectors for the introduction of foreign DNA into plants. The vectors constructed can replicate in E. coli, thus the genetic manipulations with them can be performed with E. coli as a host. They can be transferred to Agrobacterium as a cointegrate with the wide host range plasmid R772. Their T-regions are transferred to plant cells from Agrobacterium strains conferring virulence functions.The plasmid pRAL 3940 reported here is 11.5 kb large, contains a marker to identify transformed plant cells and unique restriction sites for direct cloning of passenger DNA, flanked by the left- and right-hand border fragments of the T-region (including the 25 bp border repeats). The plasmid is free of onc-genes. Therefore, is does not confer tumorigenic traits on the transformed plant cells and mature, fertile plants can thus be regenerated from them.     

Chromosomal nodulation genes: Sym-plasmid containing Agrobacterium strains need chromosomal virulence genes (chvA and chvB) for nodulation

Summary The chromosomal genes chvA and chvB of Agrobacterium tumefaciens, which mediate attachment to plant cells, were found to be essential not only for tumour induction but also for the formation of root nodules on plants.     

Osa protein encoded by plasmid pSa is located at the inner membrane but does not inhibit membrane association of VirB and VirD virulence proteins in Agrobacterium tumefaciens

Abstract The osa gene of IncW plasmid pSa encodes a 21-kDa protein that completely abolishes the oncogenic activity encoded by virulence genes in Agrobacterium tumefaciens. osa is the last gene of a four-gene operon in pSa, the expression of which appears to be highly regulated since the Osa protein is absent when either pSa or the osa operon is present in the Agrobacterium cell. When the osa gene alone or together with upstream genes within the operon are expressed under the control of a constitutive promoter, Osa protein is produced, enabling us to determine its subcellular location. Immunoblot analyses located Osa protein at the inner membrane of both A. tumefaciens and Escherichia coli . Because Osa inhibits oncogenicity of A. tumefaciens , and because alterations of the products of the virB and virD genes affect oncogenicity, studies were conducted to determine if there are changes in their specific association with the membranes in the presence Osa. Immunoblot analyses of VirB2, VirB3, VirB4, VirB9, and VirD4 in the presence and absence of Osa revealed no differences between the two treatments in these Vir protein associations with the membranes. These results indicate that both virB and virD gene products are produced in the presence of Osa; that they appear unaffected in their association with the membranes; and that Osa is associated with the inner membrane, where VirB2, VirB4, and VirD4 proteins are also located.     

新疆杨高效遗传转化系统的建立

选择新疆杨(Populus alba L.var．pyramidalis Bge．)为遗传转化受体材料,为建立根癌农杆菌介导新疆杨高效遗传转化系统,从预培养时间、侵染时间、共培养时间、添加乙酰丁香酮(AS)的时机、共培养培养基中添加乙酰丁香酮浓度、侵染菌液的制备方法、外植体继代方式等7个方面优化筛选。结果显示较合适的转化系统为：预培养8h,农杆菌菌液(OD600=0．4)侵染15min,共培养5d,侵染菌液的最优制备方法是液体培养活化农杆菌2次加离心收集菌体重悬,共培养培养基中添加乙酰丁香酮80μmol／L。新疆杨叶盘转化频率可达38．10％。     

Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

An Agrobacterium -mediated transformation procedure for aspen ( Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHA101 harboring the binary plasmid pKIW1105 (which carries the uidA and nptII genes, coding for β-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2–3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (1-phenyl-3-[1,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10–14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptII genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptII gene into the plant genome.     

Organization and characterization of the virCD genes from Agrobacterium rhizogenes

Summary We have precisely localized virulent (vir) genes of the hairy root-inducing plasmid pRiA4b on the basis of sequence similarity with the tumor-inducing plasmid pTiA6NC, and shown that the overall organizations of vir genes in both plasmids are fairly analogous, although sizes and spacer lengths in some genes differ from each other. Among the vir genes thus mapped, the virC and virD loci were characterized in detail. Transposon insertions in virD led to loss of tumorigenicity on Kalanchoe stems and carrot discs, and one within virC exhibited an attenuated pathogenicity. The avirulent phenotype of the virD2 strain among these mutants was due to the lack of ability to recombine T-DNA border repeats in Agrobacterium cells. The nucleotide sequence of most parts of the virCD loci were similar in both plasmids. The virCD genes of these two plasmids, therefore, seem comparable both functionally and structurally. Phylogeny of pRi and pTi has also been discussed from the sequence data.     

Membrane location of the Ti plasmid VirB proteins involved in the biosynthesis of a pilin-like conjugative structure on Agrobacterium tumefaciens

Abstract The virB operon of the Agrobacterium tumefaciens Ti plasmid encodes 11 proteins. Specific antisera to VirB2, VirB3 and VirB9 were used to locate these virulence proteins in the A. tumefaciens cell. Immunoblot analysis located VirB2 protein to the inner and outer membranes; VirB3 and VirB9 were likewise associated with both membranes, but mainly in the outer membrane. VirB2 is processed from a 12.3-kDa protein into a 7.2-kDa polypeptide. Such sized protein results from cleavage at residue Ala⁴⁷, upstream of which two additional alanine residues Ala⁴⁵-Ala⁴⁶ are contained and bearing resemblance to a signal peptide peptidase-I cleavage sequence. VirB2 and VirB3 sequences are strikingly similar to the pilin biosynthetic proteins TraA and TraL encoded by the tra operon of F and R1-19 plasmids. Since traA encodes a propilin that is cleaved into a 7.2-kDa conjugative pilin product and since this cleavage site is present in both TraA and VirB2, we propose that virB2 encodes a pilin-like protein which together with VirB3 and VirB9 as well as other VirB proteins may be used for interkingdom T-DNA transfer between bacteria and plants.     

由根癌农杆菌介导向毛白杨导入激素合成基因建立遗传转化系统

用携带基因1,2的根癌农杆菌AG(84)转化毛白杨外植体,在无激素的MS0培养基上获得转化根。分离单根或切成根段在分化培养基上能分化芽而再生完整植株。由T－DNA上带有基因4的根癌农杆菌C58C1（PBZ6111）转化毛白杨外植体,在MS0培养基上能直接分化不定芽而再生植株．在转化中使用叶柄作外植体比使用叶片的转化率提高一倍以上。基因1,2引入毛白杨后,植株根系发达,生根率达100％。基因4引入毛白杨则使植株节间变短,植株矮化．纸电泳分析表明,带有基因1,2的转化植株能表达特异的农杆碱,带有基因4的转化植株能表达特异的胭脂碱。     

Integration of Agrobacterium T-DNA into a tobacco chromosome: Possible involvement of DNA homology between T-DNA and plant DNA

Summary We established tobacco tumour cell lines from crown galls induced by Agrobacterium. Restriction fragments containing T-DNA/plant DNA junctions were cloned from one of the cell lines, which has a single copy of the T-DNA in a unique region of its genome. We also isolated a DNA fragment that contained the integration target site from nontransformed tobacco cells. Nucleotide sequence analyses showed that the right and left breakpoints of the T-DNA mapped ca. 7.3 kb internal to the right 25 by border and ca. 350 by internal to the left border respectively. When the nucleotide sequences around these breakpoints were compared with the sequence of the target, significant homology was seen between the region adjacent to the integration target site and both external regions of the T-DNA breakpoints. In addition, a short stretch of plant DNA in the vicinity of the integration site was deleted. This deletion seems to have been promoted by homologous recombination between short repeated sequences that were present on both sides of the deleted stretch. Minor rearrangements, which included base substitutions, insertions and deletions, also took place around the integration site in the plant DNA. These results, together with previously reported results showing that in some cases sequences homologous to those in T-DNA are present in plant DNA regions adjacent to left recombinational junctions, indicate that sequence homology between the incoming T-DNA and the plant chromosomal DNA has an important function in T-DNA integration. The homology may promote close association of both termini of a T-DNA molecule on a target sequence; then TDNA may in some cases be integrated by a mechanism at least in part analogous to homologous recombination.Shogo Matsumoto is on leave from Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd, Ogaki, Gifu-ken 503, Japan     

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.     

Efficient plantlet regeneration from protoplasts isolated from suspension cultures of poplar (Populus alba L.)

We developed an efficient plant regeneration system from protoplasts for poplar (Populus alba L.). Protoplasts were isolated from 4-day-old suspension cultures derived from seed-induced calli with a yield of 6.96× 10⁶ cells/g fresh weight cells and then cultured at a concentration of 2.5×10⁵ cells/ml in NH₄NO₃-free Murashige and Skoog (MS) medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 0.05 μM thidiazuron (TDZ) and 0.5 M glucose as a osmoticum. The plating efficiency of the cultured protoplasts was calculated at 26.5% at day 7 and 31.7% at day 14. Cell colonies were observed after culturing for 4 weeks. Regenerated colonies were propagated through subculture in liquid MS medium supplemented with 5 μM 2,4-D. Buds were induced from regenerated calli on MS medium containing 10 μM kinetin or 1 μM TDZ. Regenerated shoots were rooted on half-strength MS medium, and the plantlets were transplanted in soil. Randomly amplified polymorphic DNA analysis did not detect any DNA polymorphism among the regenerated plants. Received: 7 March 1997 / Revision received: 16 June 1997 / Accepted: 5 July 1997     

rol-Gene expression in transgenic aspen (Populus tremula) plants results in accelerated growth and improved stem production index

Stem and trunk growth, axillary bud break and branching habits are extremely important parameters of wood production in forest trees. The possibility of altering tree form by transformation with genes responsible for hormone biosynthesis and/or activity is most attractive. We examined four different phenotypically selected transgenic clones of a model tree –Populus tremula– expressing rol genes from Agrobacterium rhizogenes under their native promoters. Several of the observed phenotypic modifications were correlated with rol-gene expression, including breaking of stem apical dominance which resulted in the development and branching of up to four axillary buds per explant, as compared to a lack of axillary bud break in a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. rol-Transgenic plants also exhibited a higher cumulative stem length and enhanced growth rate, and hence a higher stem production index. During their first and second years in the greenhouse, rol-transgenic aspen plants exhibited enhanced growth and delayed winter dormancy relative to non-transformed plants. Although initially rol-transgenic plants had smaller, wrinkled leaves, these changes were not observed in the 2-year-old plants, which exhibited a phenotypically true-to-type leaf shape. Received: 13 September 1998 / Accepted: 15 April 1999     

Characterization of a new Agrobacterium tumefaciens strain from alfalfa (Medicago sativa L.)

Agrobacterium tumefaciens strain 1D1609 is reported here as the first field isolate from alfalfa (Medicago sativa L.). Unlike well-characterized A. tumefaciens strains such as C58 and Ach5, strain 1D1609 is highly virulent on alfalfa and has a distinctive host range. Interestingly, strain 1D1609 is naturally resistant to kanamycin and spectinomycin. The Ti plasmid in strain 1D1609 is an octopine-type; thus, tumors formed by strain 1D1609 synthesize octopine, which is utilized by the bacterium as a sole carbon source. Reciprocal exchange of Ti plasmids between strains 1D1609 and C58 showed that both chromosomal and Ti plasmid genes in strain 1D1609 contribute specifically to tumor formation on alfalfa. In addition, the nondormant CUF101 alfalfa cultivar from which strain 1D1609 was isolated was significantly more susceptible to all Agrobacterium strains tested than was the dormant Agate cultivar. Received: 17 October 1997 / Accepted: 8 December 1997     

Ti质粒介导的磷酸烯醇式丙酮酸羧化酶cDNA转化烟草植株

将玉米Ｃ４－磷酸烯醇式丙酮酸羧化酶（ＰＥＰ羧化酶）ｃＤＮＡ亚克隆至穿梭质粒ｐＢｉｎ１９,通过在杆菌Ｔｉ质粒（ＬＢＡ４４０４）介导的时圆片共培养法将其转入Ｃ３植物烟草中。在获得的抗性转化植株中,８０％具有较强的ＮＰＴⅡ报道基因表达。Ｓｏｕｔｈｅｒｎ杂交表明Ｃ４－ＰＥＰ羧化酶ｃＤＮＡ已被整合到了烟草核基因组中。     

rol-Transgenic Populus tremula: root development, root-borne bud regeneration and in vitro propagation efficiency

 The potential use of the rol genes from Agrobacterium rhizogenes to improve the root system horticultural characteristics was evaluated in transgenic aspen (Populus tremula) plants, harboring the rol genes under their native promoters. Southern blot and RT-PCR analyses confirmed the presence and expression of A. rhizogenes rolC and rolB genes in four different phenotypically selected transgenic clones. Several of the observed phenotypic modifications were related to rol-gene expression and included, in particular, modified root systems. All in vitro-cultured rol-transgenic plants exhibited extensive root formation in a hormone-free medium, as well as a larger root surface area and mass, as compared to a uidA (β-glucuronidase-encoding)-transgenic aspen line and control (non-transformed) plants. Adventitious root formation in stem segments of rol-transgenic plants exhibited very rapid kinetics, resulting in a much shorter rooting time for rol-transgenic stem segments (e.g. 10 days for 80% rooting in rol-transgenic lines T-26 and T-27, as compared to more than 18 days for control non-transformed or uidA-transgenic aspen plants). rol-Transgenic plants maintained the capacity for 100% rooting throughout the year, versus 70–80% rooting in non-transformed plants during the winter. The four rol-transgenic lines exhibited differences in root development; in two of them enhanced root development was accompanied by increased shoot fresh weight. The root:shoot fresh weight ratio was always higher in rol-transgenic lines than in non-transformed plants. In the T-27 rol-transgenic line, the propagation coefficient of shoot-bud regeneration in liquid root culture was almost three times higher than in non-transformed plants. To the best of our knowledge this is the first report on quantitative phenotypic alterations in rol-transgenic woody plants. Received: 1 December 1997 / Accepted: 8 March 1998     

Genetic transformation of Populus genotypes with different chimaeric gene constructs: transformation efficiency and molecular analysis

Aspen (Populus tremula) and hybrid aspen (P. tremula × P. tremuloides) were transformed with different gene constructs using two types of promoter. The aim was to determine the influence of the reporter gene rolC, controlled by promoters of viral or plant origin, on genetic and morphologic expression of different transgenic aspen clones. An improved transformation method using leaf discs was developed, by which putative transgenic plantlets were regenerated at high efficiencies (up to 34%) on kanamycin-containing medium. Transgenic aspen carrying the rolC gene from Agrobacterium rhizogenes under control of the cauliflower-35S-promoter are reduced in size with smaller leaves, whereas aspen transgenic for the same rolC gene, but under control of the light inducible rbcS promoter from potato, are only slightly reduced in size compared to untransformed controls. However, all clones carrying 35S-rolC and rbcS-rolC genes revealed light-green colouration of leaves when compared to untransformed aspen. Owing to this special feature, constructs were used in which expression of the rolC gene was inhibited by insertion of a transposable element, Ac, from maize. Transgenic aspen transformed with the 35S-Ac-rolC and rbcS-Ac-rolC genes were morphologically similar to untransformed aspen, but out of 54 independently regenerated 35S-Ac-rolC transgenic aspen clones, 30 clones showed light-green/dark green variegated leaves. In contrast, out of 19 independently transformed rbcS-Ac-rolC aspen clones, only two clones revealed light-green/dark green variegated leaves. The role of bacterial strains in transformation, and molecular genetics of transgenic aspen plants (including the function of the transposable element, Ac, in the aspen genome) are discussed     

Cytological Observations on Nodules,Nodule-Like Structures and Crown Galls on the Roots of Phaseolus vulgaris

The transconjugant of Agrobacterium tumefaciens with C58 genetic background containing plasmid pSym3622 of Rhizobium leguminosarum by. phaseoli was capable of infecting the roots of Phaseolus vulgaris of the wounding sites and inducing the formation of nodule-like structures near the sites of inoculation. These structures were different from the nodule and crown galls induced respectively by R. leguminosarumbv, phaseoli 3622--15 and a virulant strain A208 (pTiT37) of A. tumefaciens. The vascular bundles of the nodule-like structures were located at the central region of the structures and were surrounded by highly vacuolated thin-walled cells rich in starch grains. There was no bacterium neither in cell nor in the intercellular space of these structures evidenced by light and electron microscopic observations. In contrast, many metabolically active bacteroids were observed within the cells and in the intercellular spaces of the effective nodules, in the case of crown galls, there was also no bacterium in any part of the tumours. The internal structures of tumour on the root of Phaseolus vuigaris were visualized as distinguishable coherent areas of cells partitioned by disorganized cell zones. These areas of cells were traced back to be derived from different meristematic zones. At some instance, a number of root-like protrusions which were likely to develop into new roots were observed. In other parts, host cells were visible as a result of cell degeneration.     

Vital Parameters Assessments of Starvation Tolerance of <i>in vitro Populus alba</i> Culture

Populus alba is a large woody deciduous plant. The plant has been introduced to shooting, then multiplication of rooting on Murashige and Skoog (MS) medium. This work was designed to estimate the effect of two factors (low levels of 1-Naphthaleneacetic acid NAA and sucrose) on P. alba response resulting in 6 treatments compared to the control, with twelve measured responses. There was a significant difference in some measurements in morphology, like plantlets fresh-weight, shoot-, root-length, and leaf number. In the physiological measurements, there were significant differences in all the measured parameters. The low concentrations of sucrose and media composition/power (MS grams/L) led to starvation in plants; however, these conditions led to enhancement in some morphological and physiological parameters to overcome the starvation effect, compared to the control. The RAPD-PCR molecular marker (four decamers) was used to evaluate the new individuals’ genetic variation (instability), resulting in a total polymorphism percentage of 50.83%. It was formerly known that the plantlets were identical to each other and to the mother plant. In this study, however, the use of distinct media power, hormonal and sucrose levels resulted in molecular variation reflected in P. alba’s morphological and physiological responses.     

Occurrence of crown gall disease on Ficus benjamina in Fars and Isfahan provinces of Iran

Ficus benjamina, commonly known as weeping fig, Benjamin’s fig or Ficus tree is a species of flowering plant in the family of Moraceae. It is native to south and south-east Asia and Australia. Crown gall tumours were collected from branches of one-year-old weeping fig (F. benjamina L.) trees. A total of 50 strains of Agrobacterium tumefaciens were isolated from diseased Ficus plants and their morphological, molecular and biochemical characteristics were studied; pathogenicity tests on tomato, F. benjamina and Bryophyllum daigremontianum were also conducted. Based on the biochemical characteristics, pathogenicity test and PCR amplification of 730?bp fragment using VCR\VCF primers, the tested bacterial strains were identified as A. tumefaciens. This is the first report of crown gall on F. benjamina in Isfahan and Fars provinces of Iran.