Independent integration and seed-transmission of the TR-DNA of the octopine Ti plasmid pTi Ach5 in Nicotiana plumbaginifolia

Summary After co-cultivation of diploid Nicotiana plumbaginifolia protoplasts with an octopine-type Agrobacterium tumefaciens strain (LBA 4013) putative transformants were selected for hormone-independent growth, and were tested for T-DNA markers. The number of transformants expressing only T_L-DNA markers, i.e. phytohormone autotrophy and octopine synthase, was an order of magnitude higher than that of the cell lines which were simultaneously positive for both T_L- and T_R-DNA markers (the latter being mannopine and agropine). In one transformant, line no. 101, only the T_R-DNA markers were found. Not each of the T_L-, or T_R-DNA markers were expressed in each transformant resulting in a variety of phenotypes. It included the unorganized or the shoot-teratoma type of growth combined with the presence or absence of opines; e.g. agropine was absent from some of the transformants containing its precursor, mannopine. 5-Azacytidine did not induce agropine synthesis in these lines. Southern blot analysis showed that the T_R-DNA region coding for agropine synthesis was rearranged or absent in one of these lines. Similar variation in the expression of agropine and mannopine production was observed in transformants obtained with the leucinopine-type strain A281.From line 101 plants could be easily regenerated with the ability to synthesize agropine and mannopine. The segregation in the self-progeny fitted to a 3:1 ratio, indicating that the T_R-DNA was carried by a single chromosome. The Southern blot analysis showed that only opine-positive plants contained T_R-DNA. It also confirmed the absence of the T_L-DNA, demonstrating the independent integration of the T_R-region of the octopine-type Ti plasmid pTi Ach5.     

Expression of GUS in primary transformants and segregation patterns of GUS,TL- and TR-DNA in the T1 generation of hairy root transformants ofLotus corniculatus

Agrobacterium rhizogenes was assessed as a vehicle for transformation ofLotus corniculatus. Plants were co-transformed usingA. rhizogenes strain LBA 9402 harbouring the bacterial plasmid pRi1855 and the binary transformation vector pJit 73. pRi 1855 transfers both T_L and T_R sequences, while pJit 73 encodes β-glucuronidase (GUS) and also two selectable marker genes giving resistance to the antibiotics kanamycin and hygromycin. Three primary transformants (lines 1,6 and 12) were subjected to detailed morphological and biochemical analysis and lines 6 and 12 were also analysed at the molecular level. Tissues of both lines 6 and 12 were resistant to hygromycin and expressed GUS. Analysis of various tissues of each line showed a significantly lower GUS activity in line 6 than in line 12. Genetical analysis of progeny produced between control plants and lines 6 and 12 indicated that line 6 had one dose of theuid gene while line 12 had two or more independently segregating doses of the gene. Both lines 6 and 12 contained multiple copies of T_L-DNA, while only line 6 was T_R positive. In the progeny of lines 6 and 12 there was no evidence for linkage of T_L-DNA withuid, while in the progeny of line 6, T_R-DNA was under-represented. GUS-positive progeny which were free of both T_L and T_R sequences were identified from both lines.     

Overexpression of a soybean gene encoding cytosolic glutamine synthetase in shoots of transgenic Lotus corniculatus L. plants triggers changes in ammonium assimilation and plant development

A soybean cytosolic glutamine synthetase gene (GS15) was fused with the constitutive 35S cauliflower mosaic virus (CaMV) promoter in order to direct overexpression in Lotus corniculatus L. plants. Following transformation with Agrobacterium rhizogenes, eight independent Lotus transformants were obtained which synthesized additional cytosolic glutamine synthetase (GS) in the shoots. To eliminate any interference caused by the T-DNA from the Ri plasmid, three primary transformants were crossed with untransformed plants and progeny devoid of T_L- and T_R-DNA sequences were chosen for further analyses. These plants had a 50–80% increase in total leaf GS activity. Plants were grown under different nitrogen regimes (4 or 12 mM NH₄ ⁺) and aspects of carbon and nitrogen metabolism were examined. In roots, an increase in free amino acids and ammonium was accompanied by a decrease in soluble carbohydrates in the transgenic plants cultivated with 12 mM NH₄ ⁺ in comparison to the wild type grown under the same conditions. Labelling experiments using ¹⁵NH₄ ⁺ were carried out in order to monitor the influx of ammonium and its subsequent incorporation into amino acids. This experiment showed that both ammonium uptake in the roots and the subsequent translocation of amino acids to the shoots was lower in plants overexpressing GS. It was concluded that the build up of ammonium and the increase in amino acid concentration in the roots was the result of shoot protein degradation. Moreover, following three weeks of hydroponic culture early floral development was observed in the transformed plants. As all these properties are characteristic of senescent plants, these findings suggest that expression of cytosolic GS in the shoots may accelerate plant development, leading to early senescence and premature flowering when plants are grown on an ammonium-rich medium. Received: 17 July 1996 / Accepted: 16 October 1996     

Modification of phenotype in Belgian endive (Cichorium intybus) through genetic transformation byAgrobacterium rhizogenes: conversion from biennial to annual flowering

Belgian endive (Cichorium intybus) was genetically transformed usingAgrobacterium rhizogenes to insert wild type root-inducing, leftward, transferred DNA (Ri T_L-DNA) into the nuclear genome. Transformed root cultures gave rise to plants (R₀ generation) having the transformed phenotype described for other species, including increased branching, sterility, annual flowering and wrinkled leaves. Transformation circumvented the need for vernalization in order to flower, but not the need for inductive day length. Progeny (R₁ generation) were analysed by molecular hybridization and phenotypes were characterized relative to normal controls and to the R₀ generation. The extent of the T _l -DNA varied among siblings, with restriction fragmentEco R1 15, containing open reading frames 10, 11 and 12 (rol A, B and C), segregating as a single insertion. Phenotypic alterations in these plants indicate that the transformed phenotype in endive is at least partially due to the genes carried on theEco R1 fragment 15.     

Enhancement of Naphthaleneacetic Acid-Induced Rhizogenesis in T(L)-DNA-Transformed Brassica napus without Significant Modification of Auxin Levels and Auxin Sensitivity

Determination of the abscisic acid and indoleacetic acid (IAA) contents of floral stem segments of nontransformed and pRi A₄ T_L-DNA-transformed rape (Brassica napus L. var oleifera, cv Brutor) using a high performance liquid chromatography-enzyme-linked immunosorbent assay procedure and mass spectrometry controls showed that IAA levels were not modified. The regeneration abilities of the in vitro cultured explants were compared on media supplemented with several plant growth regulator combinations. No regeneration occurred on hormone-free media, and shoot production was similar in both genotypes when supplemented with benzyladenine. In the presence of naphthaleneacetic acid (NAA), transformed explants were characterized by faster root regeneration and reduced shoot organogenesis. The optimum for root formation was the same in nontransformed and transformed plants, but the sensitivity threshold was slightly lower in the latter. The NAA inductive period was shorter (14 versus 22 h) with transformed tissue. Root neoformation occurred about 72 h earlier on transformed explants. Our results suggest mainly that there is an acceleration of the auxinic signal transduction and/or that the events preliminary to the formation of roots occur faster in the transformed tissues than in the normal ones.     

TL-DNA from Agrobacterium rhizogenes plasmid pRi1855 reduces the osmotic pressure in transformed plants grown in vitro

Growth, water content, osmotic pressure and solute content were examined for normal potato (Solanum tuberosum L. cv. Desiree) and a derivative (line D9X8a), which was genetically transformed with T_L-DNA from Agrobacterium rhizogenes. Plants were grown (i) in vitro, (ii) in a growth chamber and (iii) in the field. In vitro, the transformed potato plants produced more biomass than the untransformed plants, partly because they had a higher water content. Potassium concentration and osmotic pressure were lower in cell sap extracted from the transformed potato shoots. In some cases the difference was as much as 50%. These differences were less clear, absent or reversed in plants from a growth chamber or from the field. In the field, however, transformed potato senesced early. It is suggested that a cellular basis for these observations may be changes induced by Ri T_L-DNA expression products in plant membrane properties.Abbreviations Ri root inducing - Ti tumour inducing - T-DNA transferred DNA     

Progeny study of tobacco regenerant transformed by agrobacterium rhizogenes

The pRi T-DNA markers were followed in the first generation of the progeny ofAgrobacterium tumefaciens strain C58Cl(pRiA4b) transformed tobacco regenerant. Two categories segregating probably in the ratio 15: 1 can be distinguished: those showing a complex of pRi T-DNA transformation markers and those showing no transformation markers. The presence of both T_L and T_R-DNA was demonstrated by Southern blotting. The most prominent of the transformation markers observedIn vitro was rapid growth of detached plagiotropic roots on media without phytohormones. Most of the progeny plants of the transformant showed irregularities of leaf morphology and inflorescence and flower morphology. The last resulted in sterility in most progeny plants.     

The use of a thermostable β-glucanase gene from Clostridium thermocellum as a reporter gene in plants

In order to take advantage of the high thermostability of its product, β-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine T_R-DNA 2′ gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.     

Growth and terpenoid indole alkaloid production in <Emphasis Type="Italic">Catharanthus roseus</Emphasis> hairy root clones in relation to left- and right-termini-linked Ri T-DNA gene integration

Hairy root cultures of Catharanthus roseus var. Prabal were established by infecting the leaves with Agrobacterium rhizogenes agropine-type A4 strain. Two hundred and fifty independent root clones were evaluated for growth, morphology, number of integration of Ri T-DNA genes and alkaloid contents. On the basis of growth pattern, type of branching and number of lateral roots we were able to separate the hairy root clones into four categories. However based on the integration of the Ri T_L-DNA and T_R-DNA genes, there were only three different categories of independent hairy root clones—C1 (rolA&B ⁺/ags ⁺), C2 (rolA&B ^-/ags ⁺) and C3 (rolA&B ⁺/ags ^–). Southern hybridization analysis revealed both single and multiple copies of T-DNA integration in the root clones. The accumulation of considerable amounts of the root-specific alkaloids ajmalicine and serpentine was observed in the presence of both the T_L-DNA and T_R-DNA genes (C1) and the T_L-DNA gene (C3) alone. Two rolA&B ^– but ags ⁺ clones (C2) accumulated much less or only very negligible amounts of ajmalicine. The possible role of the T_L-DNA and T_R-DNA genes on growth and alkaloid accumulation in these root clones is discussed.Abbreviations ags Agropine synthase - Ri Root-inducing - T _L -DNA Left-terminus DNA - T _R -DNA Right-terminus DNA - TIAs Terpenoid indole alkaloids     

Genetic studies on the role of octopine T-DNA border regions in crown gall tumor formation

Summary Crown gall tumors result from transfer and integration of the T-DNA from the Ti plasmid of Agrobacterium tumefaciens into plant nuclear DNA. In the present study, recombinant plasmids containing deletion and rearrangement deriviatives of the T-DNA region of the octopine Ti plasmid pTiA6 were tested in a binary tumorigenesis system (Hoekema et al. 1983) to determine the requirements for T-DNA border regions in tumor formation. Since two defined segments of the T-DNA region of octopine Ti plasmids can be detected in tumor DNA (the left (T_L-) and right (T_R-) DNA), four border regions exist in this Ti plasmid. Agrobacteria harboring plasmid constructs which contain a T-DNA gene capable of inciting tumors (gene 4, the tmr gene, which is involved in cytokinin biosynthesis) and various T-DNA border regions were tested for ability to cause tumors on Nicotiana glauca and other host plants. Such tmr constructs containing as their only border region the right border of either the T_L-DNA or the T_R-DNA are fully tumorigenic. Analogous tmr constructs containing only the T_L-DNa left border region are not tumorigenic. These results do not depend on the orientation or position of the single border with respect to the tmr gene; furthermore, the T_R-DNA right border can confer tumor-forming ability despite the presence of an intervening copy of the T_L-DNA left border.These results for relatively small plasmids are contrasted with previously determined requirements for border regions in tumorigenesis by intact Ti plasmids. A model previously proposed by Wang et al. (1984) for the role of border regions in DNA transfer to plant cells is extended in order to explain the tumor-forming ability of plasmid constructs containing a single border region. The results of this study interpreted according to the model suggest that the octopine T_L-DNA left border is defective in this DNA-transfer process.     

Octopine Ti-plasmid deletion mutants of agrobacterium tumefaciens with emphasis on the right side of the T-region

One hundred and twelve Agrobacterium tumefaciens mutants with a deleted octopine Ti plasmid were isolated. They originated from four insertion mutants, each of which carried the transposon Tn904 at a different position in the Ti plasmid. The deletion mutants were selected on the basis of loss of the capacity encoded by the Ti plasmid to degrade octopine. They were tested for the expression of other Ti-plasmid coded functions: tumor induction, presence of lysopine dehydrogenase activity in the tumor, and exclusion of phage Ap-1. For 21 mutants affected in at least one of these functions, the map position of the deletions was determined. It was found that deletions at two separated loci give rise to an Occ^? phenotype. Genes for Ap-1 exclusion were mapped on a small region just outside and to the right of the T_L + T_R region. Most of the T_R region, present as T_R-DNA in a limited number of crown gall tissues only, was shown to be unnecessary for tumor formation, since it could be deleted without affecting virulence (tested on various plant species) of the mutants. However, if the T_R region together with a small part of the adjacent T_L region, which is always present as T_L-DNA in normal crown gall tissues, was deleted the mutants became weakly virulent on Kalanchoë and Nicotiana rustica and avirulent on tomato. We hypothesize that in this case a region necessary for T-DNA integration has been deleted. The same region was found to be essential for lysopine dehydrogenase activity in the tumors.     

Potato transformation byAgrobacterium rhizogenes Ri plasmid

Potato root cultures transformed by_pRiA4 T-DNA do not regenerate plants in vitro spontaneously, but regeneration can be induced by a sequence of callogenesis and regeneration media. Both T_l and T_r-DNA were found by Southern blotting. Regenerated transformants showed characteristic morphological deviations in the in_vitro cultures. Plants in the soil showed differences in the inflorescence and tuber development.     

In planta transformation of Arabidopsis thaliana

Transformants of Arabidopsis thaliana can be generated without using tissue culture techniques by cutting primary and secondary inflorescence shoots at their bases and inoculating the wound sites with Agrobacterium tumefaciens suspensions. After three successive inoculations, treated plants are grown to maturity, harvested and the progeny screened for transformants on a selective medium. We have investigated the reproducibility and the overall efficiency of this simple in planta transformation procedure. In addition, we determined the T-DNA copy number and inheritance in the transformants and examined whether transformed progeny recovered from the same Agrobacterium-treated plant represent one or several independent transformation events. Our results indicate that in planta transformation is very reproducible and yields stably transformed seeds in 7–8 weeks. Since it does not employ tissue culture, the in planta procedure may be particularly valuable for transformation of A. thaliana ecotypes and mutants recalcitrant to in vitro regeneration. The transformation frequency was variable and was not affected by lower growth temperature, shorter photoperiod or transformation vector. The majority of treated plants gave rise to only one transformant, but up to nine siblings were obtained from a single parental plant. Molecular analysis suggested that some of the siblings originated from a single transformed cell, while others were descended from multiple, independently transformed germ-line cells. More than 90% of the transformed progeny exhibited Mendelian segregation patterns of NPTII and GUS reporter genes. Of those, 60% contained one functional insert, 16% had two T-DNA inserts and 15% segregated for T-DNA inserts at more than two unlinked loci. The remaining transformants displayed non-Mendelian segregation ratios with a very high proportion of sensitive plants among the progeny. The small numbers of transformants recovered from individual T₁ plants and the fact that none of the T₂ progeny were homozygous for a specific T-DNA insert suggest that transformation occurs late in floral development.     

T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of nicotiana tabacum

Octopine-type tumor tissue was obtained both by infection of plants or isolated protoplasts with Agrobacterium tumefaciens and by somatic hybridization of normal and crown gall tobacco cells. Analysis of T-DNA by Southern blotting of clones and uncloned tissue reveals that, whereas tumors induced on plants are heterogeneous mixtures of cells differing in T-DNA organization, each tissue derived from transformed protoplasts or from somatic hybridization is homogeneous. Detailed analysis of T-DNA organization showed that T_L- or “core” T-DNA was always present at one or two copies per diploid genome. However, sometimes it was present in a modified form, either deleted, extended, tandemly duplicated or probably methylated. T_R-DNA was not detected. The observed variation in the organization of T-DNA in octopine crown gall tissue did not appear to be a characteristic of the way the tissue was derived.     

High Sensitivity to Auxin is a Common Feature of Hairy Root

The responses to auxin of Lycopersicon esculentum roots transformed by (T_l+T_r)-DNA of the Ri plasmid of agropine-type Agrobacterium rhizogenes strain 15834 and Catharanthus trichophyllus roots transformed by the (T_l+T_r)-DNA, and by T_l- or T_r- DNA alone of the same bacterial strain were compared to that of their normal counterparts. The transmembrane electrical potential difference of root protoplasts was measured as a function of the concentration of exogenous naphthalene acetic acid. The sensitivity to auxin expressed by this response was shown to be independent of the measurement conditions and of the basal polarization of isolated protoplasts. According to this electrical response, as well as to the modulation by auxin of proton excretion by root tips and root tip elongation, roots transformed by (T_l+T_r) DNA are 100 to 1000 times more sensitive to exogenous auxin than normal roots, as is the case with normal and transformed roots from Lotus corniculatus (WH Shen, A Petit, J Guern, J Tempé [1988] Proc Natl Acad Sci USA 85: 3417-3421). Further-more, transformed roots of C. trichophyllus are not modified in their sensitivity to fusicoccin, illustrating the specificity of the modification of the auxin sensitivity. Roots transformed by the T_r-DNA alone showed the same sensitivity to auxin as normal roots, whereas the roots transformed by the T_l-DNA alone exhibited an auxin sensitivity as high as the roots transformed by (T_l+T_r)-DNA. It was concluded that the high sensitivity to auxin is controlled by the T_l-DNA in agropine type Ri plasmids.     

Segregation in the progeny of transformed rapeseed (Brassica napus)

The primary transformant of spring rapeseed cv. HM-81 contained TL- and TR-DNA of agropine plasmid pRi ofAgrobacterium rhizogenes 15834. The presence of TL-DNA corresponds to visible transformed phenotype in its progeny; the leaves are wrinkled and the plants are shorter than normal plants. R₁ R₂ and R₃ generations have mostly transformed phenotype. The normal phenotype appears in a low frequency in F₁ generation. Autogamised F₁ plants segregate in F₂ transformed and normal phenotype in 3:1 ratio. It is possible to suppose that TL-DNA is present in two differentloci of one pair of homologic chromosomes. The recombination frequency is 12 % (microsporogenesis) or 6 % (microsporogenesis and macrosporogenesis). In some crosses the transformed phenotype has a maternal type of inheritance. Maternal inheritance influences also several growth characteristics,e.g. length of plants and number of seeds/pods.     

Efficient Genetic Transformation of Lotus corniculatus L. and Growth of Transformed Plants in Field

An efficient protocol for shoot regeneration and genetic transformation was applied to root segments of a new Lotus corniculatus L. cultivar Bokor. The shoots, that regenerated on root segments, were inoculated with Agrobacterium rhizogenes A4M70GUS, and produced hairy roots, which on media with 0.2 mg dm⁻³ benzylaminopurine, regenerated shoots. After rooting and acclimation, the transformed plants were planted in the experimental field. Their morphological traits were compared to controls. No signs of the rol genes phenotype were present. The transformants were significantly taller than controls, while there were no significant differences in the leaf area. The glucuronidase activity and the presence of uidA gene was demonstrated in transformed plants of T₀ and in seedlings of T₁ generations. It is concluded that A. rhizogenes could be a vector of choice for the transfer of desirable genes into the bird's foot trefoil genome. This revised version was published online in July 2006 with corrections to the Cover Date.     

The Production of Marker-Free Genetically Engineered Broccoli with Sense and Antisense ACC synthase 1 and ACC oxidases 1 and 2 to Extend Shelf-Life

The production of transgenic broccoli (Brassica oleracea) with increased shelf-life using an Agrobacterium rhizogenes-mediated co-transformation protocol is reported. An Agrobacterium rhizogenes Ri vector, pRi1855:GFP was constructed to allow expression of the green fluorescent protein to identify insertion of Ri T_L-DNA into plant cells. The Brassica oleracea ACC synthase 1 and ACC oxidase 1 and 2 cDNAs in sense and antisense orientations were co-transformed into GDDH33, a doubled haploid calabrese-broccoli cultivar. Transformation efficiency was 3.26%, producing 150 transgenic root lines, of which 18 were regenerated into mature plants. The floral buds from T₀ broccoli heads were assayed for post-harvest production of ethylene and chlorophyll levels. Buds from T₀ lines transformed with ACC oxidase 1 and 2 constructs produced significantly less post-harvest ethylene at 20 °C than the untransformed plants and chlorophyll loss was significantly reduced over a 96 h post-harvest period. The T₀ plants transformed with sense and antisense ACC synthase 1 had a significantly reduced 24 h post-harvest ethylene peak and delayed chlorophyll loss. A positive correlation between post-harvest bud ethylene production and chlorophyll loss was described by a regression. This demonstrates that the shelf-life of a very perishable vegetable may be increased up to 2 days at 20 °C by reducing post-harvest ethylene production.