Strain specificity in transformation of alfalfa by Agrobacterium tumefaciens

Production of transgenic alfalfa plants by Agrobacterium-mediated transformation requires Agrobacterium infection and regeneration from tissue culture. Variation in alfalfa (Medicago sativa L.) germplasm for resistance to oncogenic and disarmed strains of A. tumefaciens (Smith & Townsend) Conn was tested in plant populations representing the nine distinct sources of alfalfa germplasm introduced into North America and used to develop modern varieties. For each of the virulent strains there was a positive correlation (p=0.05) of resistance to tumorigenesis with the trait for fall dormancy. There was also a significant correlation between plants selected for ineffective nodulation and resistance to tumorigenesis suggesting that the genetic loci required for successful symbiosis are also involved in tumorigenesis. Tissue explants of seedlings from the nine diversity groups were tested for transformation by three disarmed strains containing a plasmid with the scorable marker -glucuronidase. The strong correlation between dormancy and resistance to oncogenic strains was not observed with disarmed strains. However, there was a strong germplasm-strain interaction or transformation and embryogenesis in a highly embryogenic genotype. Thus, transformation at the whole plant level is germplasm dependent while in tissue culture the bacterial strain used is the critical variable for successful transformation.Abbreviations pTi tumor-inducing plasmid - GUS -glucuronidase     

Development of efficient miniprep transformation methods for Artemisia annua using Agrobacterium tumefaciens and Agrobacterium rhizogenes

Extensive studies have been carried out for the optimization of regeneration and transformation conditions for both Agrobacterium tumefaciens- and Agrobacterium rhizogenes-mediated transformation of the highly medicinal plant Artemisia annua. Most protocols describe laborious transformation procedures requiring no less than 3 mo to obtain transgenic plants. This study reports rapid and efficient protocols for A. tumefaciens- and A. rhizogenes-mediated transformation of A. annua, which were equally effective for transformation of Artemisia dubia. In both transformation procedures, stem explants responded best for maximal production of transformed plants and hairy roots. In the case of A. tumefaciens-mediated transformation, stem explants were pre-cultured for 2 d followed by infection with A. tumefaciens strain LBA4404 for 48 h. A. annua explants showed maximal transformation rate (43.5%) on half-strength Murashige and Skoog medium containing 40 mg/L kanamycin in only 20 d. The same method was tested using a related species A. dubia and resulted in a transformation rate of 41.3%, demonstrating that this protocol is efficient and genotype-independent. In the case of A. rhizogenes-mediated transformation for the production of hairy root cultures, in vitro-grown stem explants were infected with a single colony of A. rhizogenes strain LBA9402 by creating incisions at different places of the stem explants, which resulted in production of hairy roots in only 7 d. The method was tested in both A. annua and A. dubia, which resulted in transformation rates of 90 and 87.5%, respectively. Integration of the transgene and copy number was confirmed by PCR and Southern blot analyses, respectively. The miniprep transformation protocols developed for both A. tumefaciens- and A. rhizogenes-mediated transformation are simple, efficient, and potentially applicable to other species of Artemisia for transfer of pharmaceutically important genes.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens I. Auxin and Cytokinin Contents of Cultured Tissues Transformed with Wild-Type and Mutant Ti Plasmids

Carrot and tobacco plants were transformed with Agrobacteriumtumefaciens harboring wild-type, aux^– or cyt^– Tiplasmids. In tobacco, these wild and mutant Ti plasmids inducedthe formation of non-morphogenic galls, galls with shoots, andgalls with roots, respectively. In carrot, however, transformationwith any of these plasmids resulted in only the formation ofamorphous tumors. Determination of IAA and cytokinin contentsshowed that in tobacco, significantly high amounts of cytokininsor IAA are present in the cells transformed with Ti plasmidspossessing cytokinin or IAA biosynthetic genes, respectively.In carrot, cytokinin contents were also high in the cells transformedwith Ti plasmids having cytokinin biosynthetic genes, whereasIAA contents of the cells were similar regardless of the plasmidsused for transformation. These results suggest that the mechanism regulating IAA metabolismmay be different in tobacco and carrot. (Received June 25, 1987; Accepted February 1, 1988)     

The Endosperm and the Embryo of Arabidopsis thaliana are Independently Transformed through Infiltration by Agrobacterium tumefaciens

Several experiments had indicated that in planta transformation of Arabidopsis thaliana by Agrobacterium involves the female germ line. In order to identify the precise stage at which transformation occurs we have monitored expression of a gusA reporter gene in the two products of the double fertilization of infiltrated plants. The plantlets and the remaining endosperm of seeds were separately tested after germination. It appeared that in the majority of cases only the plantlet or the endosperm were transformed. Based on transformation with two vectors borne by two different Agrobacterium strains, the minority of co-transformed plantlets and endosperm can be explained by simultaneous but independent transformation events. These results indicate that mature female gametes could be the targets of T-DNA.     

Transient Gene Expression in Plant Cells Mediated by Agrobacterium tumefaciens: Application for the Analysis of Virulence Loci

A simple system is described for detection of the transfer ofT-DNA from Agrobacterium cells to suspension-cultured tobaccoBY-2 cells. A modified reporter gene for rß-glucuronidase(GUS) that contained an intron sequence was introduced intothe T-DNA region such that the GUS protein could be synthesizedin plant cells only after transfer of the T-DNA to plant nuclei.When BY-2 cells were co-cultured with Agrobacterium cells thatcontained the modified reporter gene, transient synthesis ofGUS protein was observed between 36 and 48 h after the onsetof co-culture. The level of GUS activity reached a plateau withinas little as 48 h. This temporal profile of GUS activation suggeststhat the transient activity might have been due to expressionof the GUS gene in the T-DNA that had been transferred to theplant nuclei but had not yet been integrated into the plantchromosomes. Levels of transient GUS activity were also examinedwith various vir mutants of Agrobacterium and in a mutant withan altered chromosomal acvB gene, the gene for a protein thathas been postulated to function outside bacterial cells. Duringco-culture with virB, virD2, virD4 and acvB mutants, GUS activityremained at background levels, and the GUS activity in the caseof the virE2 mutant was thirty-fold lower than with the wildtype. On the basis of these results, we discuss the roles ofthese genes during infection by Agrobacterium of plant cells. ⁴Present address: Biochemistry Laboratory, Kanebo Ltd., 5-3-28Kotobuki-cho, Odawara, Kanagawa, 250 Japan     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens III. Formation of IAA Conjugates in Cultured Carrot Tissues Transformed with Wild-type and Mutant Ti Plasmids

Inactivation of IAA was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens that harbored wild-type,aux^– or cyt^– Ti plasmids. IAA oxidase activities were similar among the three types oftransformed tissue. Metabolites of IAA were examined by followingthe fate of 3-indolyl-[l-^l4C]IAA. IAA conjugates were detectedin all transformed tissues as well as in hypocotyl segmentsof non-transformed carrot seedlings. The rate of formation ofIAA conjugates was ten times higher in the tissues transformedwith wild-type or cyt^– Ti plasmids than in the tissuestransformed with aux^– Ti plasmids. When the tissues transformedwith aux^– Ti plasmids were cultured on medium that containedIAA for 6 h, the rate of formation of IAA conjugates in thesetissues became as high as that in tissues transformed with wild-typeor cyt^– Ti plasmids. The tissues transformed with wild-type or cyt^– Ti plasmidsnot only synthesize larger amounts of IAA but also convert alarger amount of free IAA to conjugated IAA than do non-transformedand aux^– transformed tissues. Presumably, in carrot, theformation of IAA conjugates decreases the amount of free IAAin the transformed tissues that synthesize large amounts ofIAA and, consequently, the level of free IAA can be maintainedfairly constant. (Received June 2, 1989; Accepted May 23, 1990)     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

Evidence of Biological Control of Agrobacterium tumefaciens Strains Sensitive and Resistant to Agrocin 84 by Different Agrobacterium radiobacter Strains on Stone Fruit Trees

The effectiveness of Agrobacterium radiobacter K84, 0341, and a K84 non-agrocin-producing mutant (K84 Agr^-) in biological control of crown gall on rootstocks of stone fruit trees was determined in three experiments. In experiment 1, K84 and 0341 controlled crown gall on plum plants in soil inoculated with two strains of Agrobacterium tumefaciens resistant to agrocin 84. In experiment 2, K84 controlled crown gall on peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84 or with a mixture of both. However, the effectiveness of K84 was higher against the sensitive strain than against the resistant strain. There was a residual effect of K84 from one year to another in soil inoculated with the sensitive strains. In experiment 3, K84 and K84 Agr^- controlled crown gall on plum and peach plants in soils inoculated with strains of A. tumefaciens sensitive or resistant to agrocin 84. The control afforded by K84 was higher than that provided by K84 Agr^- against the sensitive strain but was similar against the resistant strain.     

Isolation and localization by histoimmunology of isoperoxidases specific for male flowers of the dioecious species Mercurialis annua L.

Specific anodic isoperoxidases of male flowers of the dioecious plant Mercurialis annua were extracted, partially purified, and injected into rabbits. The serum against these specific peroxidases was used after absorption to localize these male isoenzymes during flower development by means of histoimmunology. (Indirect immunoperoxidase method). The peroxidases were characteristic of microsporogenesis and tapetum differentiation. Their synthesis started at an early stage of male organogenesis. They were not observed in other sites of peroxidase activity of male flowers (tunica, endothecium, filament, vascular anatomy) or in female flowers (embryo sac or nucellus). I propose that these isoenzymes constitute an early and specific marker of male organogenesis in higher plants.     

根癌农杆菌介导转化马铃薯与抗病毒基因工程

病毒侵染一直是导致马铃薯品种退化的主要因素,严重影响马铃薯的产量和品质。近年来,随着基因工程的迅速发展和转基因技术体系的日益完善,基因工程技术在提高马铃薯抗病性（尤其是抗病毒）方面显示了极大的潜力,必将成为马铃薯抗病毒育种的主要手段。对其进展进行了综述,并讨论了根癌农杆菌介导马铃薯遗传转化及其体系优化因素。最后提出存在问题及发展趋势,以供广大马铃薯抗病毒育种工作者参考。     

Reconciliation of Sequence Data and Updated Annotation of the Genome of Agrobacterium tumefaciens C58, and Distribution of a Linear Chromosome in the Genus Agrobacterium

Two groups independently sequenced the Agrobacterium tumefaciens C58 genome in 2001. We report here consolidation of these sequences, updated annotation, and additional analysis of the evolutionary history of the linear chromosome, which is apparently limited to the biovar I group of Agrobacterium.     

Involvement of the Acr3 and DctA anti‐porters in arsenite oxidation in Agrobacterium tumefaciens 5A

Microbial arsenite (AsIII) oxidation forms a critical piece of the arsenic cycle in nature, though our understanding of how and why microorganisms oxidize AsIII remains rudimentary. Our model organism Agrobacterium tumefaciens 5A contains two distinct ars operons (ars1 and ars2) that are similar in their coding region content. The ars1 operon is located nearby the aio operon that is essential for AsIII oxidation. The AsIII/H⁺ anti‐porters encoded by acr3‐1 and acr3‐2 are required for maximal AsIII and antimonite (SbIII) resistance, but acr3‐1 (negatively regulated by ArsR‐1) appears more active in this regard and also required for AsIII oxidation and expression of aioBA. A malate‐phosphate anti‐porter DctA is regulated by RpoN and AsIII, and is required for normal growth with malate as a sole carbon source. Qualitatively, a ΔdctA mutant was normal for AsIII oxidation and AsIII/SbIII resistance at metalloid concentrations inhibitory to the Δacr3‐1 mutant; however, aioBA induction kinetics was significantly phase‐shift delayed. Acr3 involvement in AsIII/SbIII resistance is reasonably well understood, but the role of Acr3 and DctA anti‐porters in AsIII oxidation and its regulation is unexpected, and suggests that controlled AsIII trafficking across the cytoplasmic membrane is important to a process understood to occur in the periplasm.     

Host-Bacteriophage Interaction in Agrobacterium tumefaciens I. Characterization of Bacteriophage R4 and Physiological Changes in the Infected Host Cell

Infection of Agrobacterium tumefaciens B6, a tumor-producing plant pathogen, by bacteriophage R4, does not immediately shut off host deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein synthesis. Viral DNA synthesis begins soon after infection, but the host DNA is not shut off until after 35 min; net RNA and protein synthesis are not inhibited until 30 min after infection. The pattern of synthesis of phage particles was confirmed by electron microscopy of thin sections during the infection cycle. The phage particle consists of a polyhedral head, 65 nm in diameter, and a long flexible tail 210 nm long and 10 nm wide with helically arranged subunits. By gel electrophoresis, four major protein components with the following molecular weights were found in the capsid: 72,000, 45,000, 28,000, and 14,500. The phage DNA has a molecular weight of 30 million and a guanine-cytosine content of 59%.     

Genetic transformation of 9 in vitro clones of Alnus and Betula by Agrobacterium tumefaciens

Crown gall tumorigenesis, integration and expression of T-DNA encoded genes from Agrobacterium tumefaciens were investigated in 9 clones of Alnus glutinosa, A. incana and Betula papyrifera. Tumor formation on in vitro shoots was frequent in all clones with strain Ach5 and present in 8 clones with strain C58. Tumors excised from shoots were selected for autotrophic growth in vitro and axenic cultures were established. Octopine or nopaline, respective of the strain type used for inoculation, was detected in tumorous cultures. Southern blot analyses demonstrated T-DNA integration by hybridization of DNA from tumors with tmr and nos gene probes. One clone of B. papyrifera produced tumors with a morphogenic character, unusual in calli of this species, generating viable shoots which did not synthesize opine.Abbreviations Cb Carbenicillin - Cf Cefotaxime - 2,4-D 2,4-Dichloro-phenoxyacetic acid     

成花光敏感的雌雄性不育苎麻活性氧代谢研究

为研究成花光敏感雌雄不育苎麻(Floral initiation photo-sensitive male and female sertile ramie,SMSFS)的败育与活性氧代谢的关系,对SMSFS及其回复突变株SMSFS(R)花器官的活性氧(ROS)清除酶活性和丙二醛(MDA)含量测定．结果表明：SMSFS蕾的过氧化物酶(POD)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性明显高于可育株SMSFS(R),而膜脂过氧化产物MDA的含量低于SMSFS(R);与蕾期相比,败育期SMSFS雌雄花除POD活性有所上升,而SOD和CAT活性则显著下降,MDA含量升高并高于开花期可育株．可见活性氧代谢的失衡引起了的膜脂过氧化,与SMSFS雌雄性败育明显相关．