The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation

This paper describes a so-called ternary transformation system for plant cells. We demonstrate that Agrobacterium tumefaciens strain LBA4404 supplemented with a constitutive virG mutant gene (virGN54D) on a compatible plasmid is capable of very efficient T-DNA transfer to a diverse range of plant species. For the plant species Catharanthus roseus it is shown that increased T-DNA transfer results in increased stable transformation frequencies. Analysis of stably transformed C. roseus cell lines showed that, although the T-DNA transfer frequency is greatly enhanced by addition of virGN54D, only one or a few T-DNA copies are stably integrated into the plant genome. Thus, high transformation frequencies of different plant species can be achieved by introduction of a ternary plasmid carrying a constitutive virG mutant into existing A. tumefaciens strains in combination with standard binary vectors.     

Herbicide-resistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium-mediated transformation

The presence of undesirable plants in sugarcane (Saccharum officinarum L.) plantations reduces crop yields. Using genetic engineering as a complement for traditional breeding methods it is possible to introduce herbicide-resistant traits into Saccharum germplasm. Transgenic sugarcane plants resistant to phosphinothricine (PPT), the active compound of the commercial herbicide BASTA were generated by Agrobacterium tumefaciens-mediated transformation. Meristematic sections of sugarcane were treated with anti-necrotic compounds to minimize oxidative bursts and used as explants. Four transformation protocols were assessed and the transformation frequencies reached 10–35%. The regeneration rate was high and did not appear to be affected by the transformation procedure. Southern blot analysis of several transformed plants indicated the integration per genome of one or two intact copies of the bar gene which encodes PPT acetyltransferase and confers resistance to BASTA. The levels of BASTA resistance were evaluated under greenhouse and small-plot conditions. Received: 8 November 1997 / Accepted: 22 November 1997     

CaMV 35S promoter directs β-glucuronidase expression in the laticiferous system of transgenic Hevea brasiliensis (rubber tree)

Hevea brasiliensis anther calli were genetically transformed using Agrobacterium GV2260 (p35SGUSINT) that harboured the β-glucuronidase (gus) and neomycin phosphotransferase (nptII) genes. β-Glucuronidase protein (GUS) was expressed in the leaves of kanamycin-resistant plants that were regnerated, and the presence of the gene was confirmed by Southern analysis. GUS was also observed to be expressed in the latex and more importantly in the serum fraction. Transverse sections of the leaf petiole from a transformed plant revealed GUS expression to be especially enhanced in the phloem and laticifers. GUS expression was subsequently detected in every one of 194 plants representing three successive vegetative cycles propagated from the original transformant. Transgenic Hevea could thus facilitate the continual production of foreign proteins expressed in the latex. Received: 14 February 1997 / Revision received: 16 August 1997 / Accepted: 20 July 1997     

Bialaphos stimulates shoot regeneration from hairy roots of snapdragon (Antirrhinum majus L.) transformed by Agrobacterium rhizogenes

Hairy roots of snapdragon (Antirrhinum ma-jus L.: Scrophulariaceae) induced by a wild-type strain of Agrobacterium rhizogenes were cultured on media containing various concentrations of a phosphinothricin-based herbicide, bialaphos, or plant growth regulators (PGRs). Adventitious shoot regeneration from hairy roots was observed with a low frequency (10%) on half-strength Murashige and Skoog medium. Addition of α-naphthalene-acetic acid in combination with 6-benzylaminopurine, thidiazuron, or zeatin to the medium had no effect on shoot regeneration from hairy roots. Although bialaphos at 0.9 mg l^–1 or more was toxic to hairy roots, it significantly increased the shoot regeneration frequency up to 56% at 0.5 mg l^–1. In contrast, non-transformed roots and leaves regenerated no shoots on media with or without bialaphos. Regenerated shoots detached from host roots readily developed roots on gellan-gum-solidified medium. Regenerated plants were successfully transferred to the greenhouse, but did not produce seed. Received: 24 February 1997 / Revision received: 10 July 1997 / Accepted: 28 July 1997     

Sonication-assisted Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill] embryogenic suspension culture tissue

Successful transformation of plant tissue using Agrobacterium relies on several factors including bacterial infection, host recognition, and transformation competency of the target tissue. Although soybean [Glycine max (L.) Merrill] embryogenic suspension cultures have been transformed via particle bombardment, Agrobacterium-mediated transformation of this tissue has not been demonstrated. We report here transformation of embryogenic suspension cultures of soybean using “Sonication-Assisted Agrobacterium-mediated Transformation” (SAAT). For SAAT of suspension culture tissue, 10–20 embryogenic clumps (2–4 mm in diameter) were inoculated with 1 ml of diluted (OD_600nm 0.1–0.5) log phase Agrobacterium and sonicated for 0–300 s. After 2 days of co-culture in a maintenance medium containing 100 μM acetosyringone, the medium was removed and replaced with fresh maintenance medium containing 400 mg/l Timentin^?. Two weeks after SAAT, the tissue was placed in maintenance medium containing 20 mg/l hygromycin and 400 mg/l Timentin^?, and the medium was replenished every week thereafter. Transgenic clones were observed and isolated 6–8 weeks following SAAT. When SAAT was not used, hygromycin-resistant clones were not obtained. Southern hybridization analyses of transformed embryogenic tissue confirmed T-DNA integration. Received: 22 August 1997 / Revision received: 22 October 1997 / Accepted: 11 November 1997     

Activity of the CaMV 35S promoter in various parts of transgenic early flowering birch clones

Early flowering together with small size would be useful for various biotechnical or genetic studies on trees. We report here the selection and micropropagation of early flowering birch (Betula pendula) clones (BPM1–12) obtained from seeds of birches bred elsewhere for early flowering. Under conditions that accelerate flowering (a high CO₂ level, strong and continuous illumination), the first male inflorescences emerged in 3–5 months, the trees then being 20–80 cm high. Transgenic lines (CaMV 35S-GUS INT) were produced through Agrobacterium-mediated gene transfer from BPM2, BPM5 and JR1/4 (a normally flowering birch). β-Glucuronidase (GUS) activities in the different lines, assayed 1–1.5 years after transformation, varied greatly. During further in vitro culture for 10 months, the activities decreased to 0.3–7% of the original values. GUS activities were detected in all organs studied, including the developing male inflorescences; the highest activity was in the roots. Received: 28 April 1997 / Revision received: 5 September 1997 / Accepted: 30 November 1997     

Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation

The effects of timentin on shoot regeneration of tobacco (Nicotiana tabaccum) and Siberian elm (Ulmus pumila L.) and its use for the suppression of Agrobacterium tumefaciens in Agrobacterium-mediated genetic transformation were determined. Timentin is a mixture of ticarcillin and clavulanic acid, and at concentrations of 200–500 mg/l with ratios of ticarcillin:clavulanic acid of 50:1 and 100:1, it had little effect on shoot regeneration of tobacco or Siberian elm. Timentin was as effective in suppressing A. tumefaciens as carbenicillin and cefatoxime at concentrations commonly used in transformation. The disarmed A. tumefaciens strain LBA4404 in infected tobacco leaf tissues was visually undetectable after three subcultures on medium with 500 mg/l of timentin and 250 mg/l carbenicillin. Timentin was stable in solid agar medium and remained effective for at least 70 days, but was unstable when stored as a mixed stock solution or as separate ticarcillin and clavulanic acid stock solutions at –20°C or –80°C freezer for 4 weeks. Timentin may be an alternative antibiotic for the effective suppression of A. tumefaciens in genetic transformation. Received: 8 September 1997 / Revision received: 19 November 1997 / Accepted: 2 December 1997     

Agrobacterium-mediated genetic transformation of groundnut (arachis hypogaea L.): An assessment of factors affecting regeneration of transgenic plants

Transgenic groundnut (Arachis hypogaea L.) plants were produced efficiently by inoculating different explants withAgrobacterium tumefaciens strain LBA4404 harbouring a binary vector pBM21 containinguidA (GUS) andnptll (neomycin phosphotransferase) genes. Genetic transformation frequency was found to be high with cotyledonary node explants followed by 4 d cocultivation. This method required 3 days of precultivation period before cocultivation withAgrobacterium. A concentration of 75 mg/l kanamycin sulfate was added to regeneration medium in order to select transformed shoots. Shoot regeneration occurred within 4 weeks; excised shoots were rooted on MS medium containing 50 mg/I kanamycin sulfate before transferring to soil. The expression of GUS gene (uidA gene) in the regenerated plants was verified by histochemical and fluorimetric assays. The presence ofuidA andnptll genes in the putative transgenic lines was confirmed by PCR analysis. Insertion of thenptll gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis. Factors affecting transformation efficiency are discussed.     

A 42 bp fragment of the pmas1′ promoter containing an ocs-like element confers a developmental,wound- and chemically inducible expression pattern

Synthesis of mannopine in plant tissues infected with Agrobacterium tumefaciens is controlled by a divergent promoter (pmas2 and pmas1) that in 479 bp contains all the cis-acting elements necessary to direct tissue-specific and wound-inducible expression. In this report, using transgenic tobacco plants harboring a pmas1--glucuronidase (GUS) gene fusion, we investigated the developmental expression pattern directed by pmas1 in the early stages of development and the responses of pmas1 to different chemical inducers. It was found that this promoter can respond to auxins, cytokinins, methyl jasmonate (MJ), salicylic acid (SA) and its analogue 2,6-dichloroisonicotinic acid (iNA). Treatment with chemical inducers also showed that the effects of iNA are organ-dependent, that wound-induction is a complex response mediated by at least two different chemical signals, and that MJ stimulates changes in the tissue-specific and developmental expression pattern directed by the pmas1 promoter. Using chimeric promoters we demonstrate that an ocs-like element (ocs+1) directs MJ responses in an orientation-dependent manner and that sequences around the ocs+1 are important to maintain the inducible and developmental properties of this cis-regulatory element.     

利用农杆菌系统将超甜定基因导入烟草

由pUR528构建中间载体pEHT9,再构建超甜定植物表达载体pBIT7。通过直接转化法将pBIT7导入农杆菌,然后利用农杆菌系统转化烟草。经PCR、PC R-S outhern和Southern杂交证实,超甜定基因已整合到烟草基因组中。 Abstract:pEHT9 was constructed from pUR528,and used for the construction of plant thaumatin expression vector pBIT7.Then pBIT7 was introduced to Agrobacterium through direct transformation.Subsequently tobacco was transformed through Agrobacterium-mediated system.It has been confirmed that thaumatin gene has integrated into the genome of transgenic tobacco by PCR,PCR-Southern and Southern blot analyses.