Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance

 Transformation of barley and wheat via particle bombardment with a gene derived from Vitis vinifera L. (Vst1 gene) resulted in the expression of the foreign phytoalexin, resveratrol, in the transformed plants. Transgenic barley plants were regenerated from microspores and transgenic wheat plants from immature embryos were both selected on Basta. Stable integration of the gene in the genomes of transgenic barley and wheat plants, as well as their progeny, was analysed by Southern-blot analysis. The induction of the stilbene synthase promoter and the transient expression of stilbene synthase-specific mRNA after induction by wounding and infection were proofed in T₁ and T₂ progeny plants. An enhanced expression of the Vst1 gene under control of the stilbene synthase promoter was observed with enhancer sequences from the cauliflower mosaic virus 35s (CaMV 35s) promoter. The enzyme activity of the stilbene synthase was analysed in T₁ progeny plants. The first pathological results indicated an increased resistance of transgenic barley plants to Botrytis cinerea used as a model experimental system. Received: 5 November 1997 / Accepted: 11 November 1997     

Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower)

We previously established a system of in vitro regeneration and Agrobacterium-mediated transformation for hot pepper plants. The level of protection against cucumber mosaic virus in the progeny of the transgenic hot pepper plants that express cucumber mosaic virus (CMV) satellite RNA was investigated. The transgenic hot pepper plants were self-fertilized, and their progeny were tested for stable inheritance and expression of the cDNA of CMV satellite RNA. Polymerase chain reaction and RNA gel blot analyses showed that the introduced gene was stably transmitted and expressed in the progeny. Symptom attenuation in the offspring was confirmed upon inoculation with CMV-Y or CMV-Korea (CMV-Kor) strains. Received: 30 September 1996 / Revision received: 5 May 1997 / Accepted: 22 May 1997     

Expression of the Commelina yellow mottle virus promoter in transgenic oat

The Commelina yellow mottle virus (CoYMV) infects the monocot weed Commelina diffusa. The objective of this study was to investigate the transgene expression conferred by the CoYMV promoter in a monocot species. Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with the CoYMV promoter fused to the coding region of E. coli β-glucuronidase (uidA, GUS). Developmental and tissue-specific expression of the CoYMV-GUS construct was investigated in regenerated plants and their progeny. Histochemical GUS staining was primarily localized in the vascular tissues of shoots, leaves, floral bracts and in roots. While ovaries stained intensely, no staining was detected in anthers or the endosperm in mature seed. The scutellum of mature and germinating seed exhibited GUS activity. Received: 16 April 1997 / Revision received: 23 July 1997 / Accepted: 2 August 1997     

Inheritance of a bacterial hygromycin phosphotransferase gene in the progeny of primary transgenic pea plants

Summary An analysis of the progeny of primary transgenic pea plants in terms of transmission of the transferred DNA, fertility and morphology is presented. A transformation system developed for pea that allows the regeneration of fertile transgenic pea plants from calli selected for antibiotic resistance was used. Expiants from axenic shoot cultures were co-cultivated with a nononcogenic Agrobacterium tumefaciens strain carrying a gene encoding hygromycin phosphotransferase as selectable marker, and transformed callus could be selected on callus-inducing media containing 15 mg/l hygromycin. After several passages on regeneration medium, shoot organogenesis could be reproducibly induced on the hygromycin resistant calli, and the regenerated shoots could subsequently be rooted and transferred to the greenhouse, where they proceeded to flower and set seed. The transmission of the introduced gene into the progeny of the regenerated transgenic plants was studied over two generations, and stable transmission was shown to take place. The transgenic nature of the calli and regenerated plants and their progeny was confirmed by DNA and RNA analysis. The DNA and ploidy levels of the progeny plants and primary regenerants were studied by chromosome analysis, and the offspring of the primary transformants were evaluated morphologically.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - BA 6-ben-zyladenine - hpt hygromycin phosphotransferase gene - IAA indole acetic acid, kin, kinetin - NAA -naphtalene acetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Transgenic tobaccos transformed with a gene encoding a truncated form of the coat protein of tomato black ring nepovirus are resistant to viral infection

 Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3′ terminal part of the viral RNA2 (positions 2801–4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T₄₀₆₅ so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation. Received: 28 February 1997 / Revision received: 1 August 1997 / Accepted: 24 March 1999     

Indirect effects of an unspecialized endophytic fungus on specialized plant – herbivorous insect interactions

The effects of Acremonium alternatum Gams (Ascomycotina, Clavicipitacea) on the development and nutrition of diamondback moth larvae Plutella xylostella L. (Lepidoptera, Plutellidae) were studied in the laboratory. All experiments were conducted before the endophyte reached the green parts of the plants; thus P. xylostella, a folivore, was not in direct contact with the endophyte. Larvae feeding on leaves of previously inoculated plants suffered from increased mortality, especially during the first 10 days of development. Likewise, during early development surviving larvae had a reduced relative growth rate (RGR), which, however, did not result in reduced pupal or adult weight. We found sexual differences in the food utilization efficiency; female P. xylostella progeny reacted more sensitively to endophytic infection of cabbage than male larvae. Female larvae feeding on leaves of endophyte-infested plants responded to reduced efficiency of conversion of ingested food (ECI) by increasing their relative consumption rate (RCR). The underlying mechanisms for these results are discussed in relation to changes in plant phytosterol metabolism which could account for reduced larval growth on inoculated cabbage plants. Our data suggest that unspecialized, soil-borne endophytic fungi, even when their association with the host plant is weak, can influence aboveground herbivore development and should be considered when investigating plant-insect interactions. Received: 3 November 1997 / Accepted 29 December 1997     

Somaclonal-variation-induced aluminum-sensitive mutant from an aluminum-inbred maize tolerant line

Somaclonal-variation-induced multiple mutations were observed in a progeny of the S1587 plant, regenerated from type I calli of the aluminum-tolerant inbred maize line Cat-100-6. After five generations of self-pollination, 14 progeny families of the S1587 somaclone were found to show aluminum toxicity symptoms with altered root tip morphology and reduced primary root growth. The most sensitive progeny, S1587-17, was crossed to the Cat-100-6 inbred line. The parental lines and the F1 were tested in nutrient solutions containing an aluminum activity gradient of 0–93 ⋅ 10^–6. The heterozygote behaves like the tolerant parent at aluminum activities up to 40 ⋅ 10^–6 and showed an intermediate phenotype at higher aluminum concentrations. Histological sections of aluminum-treated roots from tolerant and sensitive plants stained with hematoxylin, an aluminum marker, showed a progressive destruction of the root tip of the aluminum-sensitive genotype over time and indicated that tolerance in Cat-100-6 could be due to an aluminum exclusion mechanism. Segregation analysis of the F2 and backcross to the sensitive parent based on root morphology of plants subjected to an aluminum activity of 30 ⋅ 10^–6 showed the typical 3:1 and 1:1 tolerant:sensitive segregation ratios, respectively, indicating that tolerance in the Cat-100-6 inbred maize line is controlled by a single nuclear, semidominant gene, named Alm1. Received: 9 May 1996 / Revision received: 24 February 1997 / Accepted: 8 March 1997     

Identification and characterization of photosystem II chlorophyll a/b binding proteins in Marchantia polymorpha L.

The minor chlorophyll a/b-binding (CAB) proteins of the liverwort Marchantia polymorpha L. were investigated in order to compare the antenna organization and the light-acclimation potential in lower and higher plants. Homologues to the minor CAB proteins CP24, CP26 and CP29 were identified by the following criteria: enrichment in photosystem II preparations, immunological cross-reactivities, spectroscopic properties and protein-fragment amino acid sequences. The high violaxanthin content of the minor CAB proteins in M. polymorpha indicates that their role in protection from high light is comparable in lower and higher plants. Considerably more-alkaline isoelectric points are found for the minor CAB proteins of M. polymorpha than for their higher-plant counterparts. This might be due to a higher content of basic amino acids. While the N-terminal sequence of angiosperm CP29 contains a threonine that becomes phosphorylated during cold stress, this amino acid is substituted by valine in M. polymorpha. Therefore, the regulatory properties of this protein could differ in lower and higher plants. Received: 25 March 1997 / Accepted: 21 July 1997     

Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny

Several modifications of a wild-type green fluorescent protein (GFP) gene were combined into a single construct, driven by the ubi-1 promoter and intron region, and transformed into maize. Green fluorescence, indicative of GFP expression, was observed in stably transformed callus as well as in leaves and roots of regenerated plants and their progeny. Cell wall autofluorescence made GFP expression difficult to observe in sections of leaves and roots. However, staining sections with toluidine blue allowed detection of GFP in transgenic tissue. Bright GFP fluorescence was observed in approximately 50% of the pollen of transgenic plants. These results suggest that GFP can be used as a reporter gene in transgenic maize; however, further modification, i.e., to alter the emission spectra, would increase its utility. Received: 17 December 1997 / Revision received: 6 March 1998 / Accepted: 20 March 1998     

Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

An efficient Agrobacterium tumefaciens-mediated transformation and regeneration system for cotyledons of spinach (Spinacia oleracea L.)

An efficient transformation and regeneration system was established for the production of transgenic spinach (Spinacia oleracea L.) plants. Cotyledon explants were infected with Agrobacterium tumefaciens strain LBA4404 carrying the selectable marker gene, neomycin phosphotransferase II (nptII), and the reporter gene smgfp, encoding soluble-modified green-fluorescent protein, driven by the cauliflower mosaic virus 35S promoter. The infected explants were cultured on Murashige and Skoog medium, containing 1 mg/l benzyladenine and 0.4 mg/l naphthaleneacetic acid. Shoots were regenerated on selection medium containing 50 mg/l kanamycin. Regenerated kanamycin-resistant shoots were rooted on medium containing 1 mg/l indolebutyric acid and subsequently grown in soil in the greenhouse. Southern blot analysis indicated that the smgfp gene had been integrated into the spinach genome. Northern and Western blots showed that the smgfp gene was expressed in progeny plants. Received: 31 March 1998 / Revision received: 27 September 1998 / Accepted: 10 Ocotber 1998     

Transformed Potato Plants Carrying the Gene of the Antifungal Peptide of Amaranthus caudatus

Potato plants (Solanum tuberosumL., var. Desire) were transformed with a pH22Kneo vector harboring the gene ac2, which encodes the fungicidal peptide (defensin) from the seed of amaranth (Amaranthus caudatusL.). The transformation involved cocultivation on a solid MS medium of potato stem explants (excised from aseptically grown plants) andAgrobacterium tumefaciens. The factors affecting in vitroregeneration of the explants and the transformation efficiency were optimized. Regenerated potato plants carrying the amaranth defensin gene were selected by two traits, growth and ability to form roots on a kanamycin-supplemented MS medium. The transgenic state was confirmed by PCR analysis of ac2in tissues of the kanamycin-resistant plants. The transgenic organisms thus obtained differed from the original plants in their patterns of Ambiol-induced growth and proton translocation across the plasma membrane of the tuber cells.     

Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass (Tripsacum dactyloides L.)

Eastern gamagrass, (Tripsacum dactyloides L.) is a perennial, warm-season grass that is being developed as a forage plant. Shoots were derived from callus initiated from immature embryos and immature inflorescences of diploid (2n=2x=36) gynomonoecious eastern gamagrass. These shoots were induced to microtiller in the presence of 3 mg/l benzyladenine. Amiprophosmethyl (10, 15, or 20 μm) was applied to 27 microtillers for 3–5 days to induce chromosome doubling. All 14 surviving plants were tetraploid, (2n=4x=72), as determined by flow cytometry or chromosome counts. These plants were morphologically normal and produced seed. Test crosses were made with a known diploid. Flow cytometry and chromosome counts showed that the progeny were triploid, proving that the induced tetraploids reproduce sexually. Received: 12 February 1997 / Revision received: 18 February 1998 / Accepted: 13 March 1998     

Identification of minor DNA variations in rice somaclonal variants

Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases in previous analysis carried out in our laboratory. An attempt was made to assess the effect of culturing and regeneration of rice plants on DNA variation at microsatellite loci in R₂ progeny of callus-derived rice plants. Different somaclones of the rice line Indrayani differing in yield and disease response (high, low and no change in yield, as compared to the original genotype) were used as genetic material for these analyses. Analysis of microsatellite loci was accomplished by digesting DNA from regenerated rice somaclones and assaying for polymorphisms at microsatellite loci by in-gel hybridization with synthetic oligonucleotide probes such as (GATA)₄, (CAC)₅ and (TG)₁₀. Specific variation at a PCR-amplified locus containing three internal microsatellite repeats (1E₆) using restriction site fingerprinting was also investigated. The locus-specific amplification of a sequence-tagged microsatellite marker followed by digestion with HinfI and Sau3AI restriction endonucleases showed differences in some somaclonal variants. The technique used in this study enables monitoring of DNA changes in successive generations of somaclonal variants as a measure of DNA variability and possibly to identify the regions which are responsible for specific traits. Received: 7 November 1997 / Revision received: 22 April 1997 / Accepted: 5 June 1998     

Vacuum infiltration of Petunia hybrida pollen with Agrobacterium tumefaciens to achieve plant transformation

 Genetic transformation of Petunia hybrida with a reporter gene and selectable marker gene (35S-bar) was achieved in similar frequencies by pollinating flowers with pollen vacuum-infiltrated with Agrobacterium tumefaciens or applying a drop of Agrobacterium suspension to the stigma immediately prior to pollination. Nine percent of the T₁, and 5% of the T₂ progeny germinated in nutrient medium with 3 mgl/l Basta^R. Polymerase chain reaction assays indicated that of the Basta^R-resistant plants, 66% of the T₁ plants, and 61% of the T₂ plants harboured the GUS gene. Histochemical assays showed that 10% of the putatively transformed T₁ plants and 5% of their progeny expressed GUS in leaf tissue, pistils and young anthers. Southern hybridization confirmed genomic integration of the bar gene in one to three places in selected T₁ and T₂ progeny. Received: 12 March 1999 / Revision received: 1 October 1999 / Accepted: 20 October 1999     

Co-transformation with one Agrobacterium tumefaciens strain containing two binary plasmids as a method for producing marker-free transgenic plants

Co-transformation was investigated as a method that would allow the use of a selectable marker during plant regeneration followed by recovery of progeny which contain the desired gene(s) but lack a marker gene. Rapeseed (Brassica napus cv `212/86') and tobacco (Nicotiana tabacum cv `Xanthi NC') were co-cultivated with a single Agrobacterium tumefaciens strain containing two binary plasmids. Genes from both plasmids were expressed in approximately 50% of the primary transformants. Progeny expressing only one of the transgenes were observed in about 50% of the co-transformed lines, indicating that the genes were inserted at different loci. This single-strain co-transformation method allowed the use of a selectable marker during plant regeneration and subsequent recovery of marker-free progeny. Received: 23 December 1996 / Revision received: 23 September 1997 / Accepted: 11 October 1997     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Factors influencing Agrobacterium tumefaciens-mediated transformation of Artemisia annua L.

Following our previously described Agrobacterium tumefaciens-mediated transformation procedure for Artemisia annua L., we have undertaken several additional experiments to establish the importance of some parameters such as explant type, age of explant source, A. tumefaciens strain and type of binary vector. Several binary vectors were useful for the production of transgenic callus on explants of different ages. In transformed calli, a good correlation between integration and expression of foreign DNA was observed: different assays showed expression of β-Glucuronidase, neomycin phosphotransferase II, superoxide dismutase and bleomycin acetyl transferase. The regeneration of transgenic plants required more restricted conditions. Only with the pTJK136 vector could transgenic plants be obtained from leaf and stem explants from 12- to 18-week-old plants. Co-cultivation for 48 h seemed favorable for the regeneration of transgenic plants. Stable integration and expression of the transgenes was also shown in the progeny. Received: 18 August 1997 / Revision received: 3 December 1997 / Accepted: 3 July 1998     

Control of Lepidopteran insect pests in transgenic Chinese cabbage (Brassica rapa ssp. pekinensis) transformed with a synthetic Bacillus thuringiensis cry1C gene

 A synthetic Bacillus thuringiensis cry1C gene was transferred to three Korean cultivars of Chinese cabbage via Agrobacterium tumefaciens-mediated transformation of hypocotyl explants. Hygromycin resistance served as an efficient selective marker. The transformation efficiency ranged from 5% to 9%. Transformation was confirmed by Southern blot analysis, PCR, Northern analysis, and progeny tests. Many transgenic plants of the closed-head types (lines Olympic and Samjin) flowered in vitro. Over 50 hygromycin-resistant plants were successfully transferred to soil. The transgenic plants and their progeny were resistant to diamondback moths (DBM, Plutella xylostella), the major insect pest of crucifers world-wide, as well as to cabbage loopers (Trichoplusia ni) and imported cabbage worms (Pieris rapae). Both susceptible Geneva DBM and a DBM population resistant to Cry1A protein were controlled by the Cry1C-transgenic plants. The efficient and reproducible transformation system described may be useful for the transfer of other agriculturally important genes into Chinese cabbage. Received: 12 June 2000 / Revision received: 21 August 2000 / Accepted: 22 August 2000