Efficient <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (L.) R. Br. using shoot apices as explant source

A critical step in the development of a reproducible Agrobacterium tumefaciens mediated transformation system for a recalcitrant species, such as pearl millet, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. A multiple shoot regeneration system, without any intervening callus phase, was developed and used as a tissue culture system for Agrobacterium-mediated transformation. Agrobacterium super virulent strain EHA105 harboring the binary vector pCAMBIA 1301 which contains a T-DNA incorporating the hygromycin phosphotransferase (hpt II) and β-glucuronidase (GUS) genes was used to investigate and optimize T-DNA delivery into shoot apices of pearl millet. A number of factors produced significant differences in T-DNA delivery; these included optical density, inoculation duration, co-cultivation time, acetosyringone concentration in co-cultivation medium and vacuum infiltration assisted inoculation. The highest transformation frequency of 5.79% was obtained when the shoot apex explants were infected for 30 min with Agrobacterium O.D.₆₀₀ = 1.2 under a negative pressure of 0.5 × 10⁵ Pa and co-cultivated for 3 days in medium containing 400 μM acetosyringone. Histochemical GUS assay and polymerase chain reaction (PCR) analysis confirmed the presence of the GUS gene in putative transgenic plants, while stable integration of the GUS gene into the plant genome was confirmed by Southern analysis. This is the first report showing reproducible, rapid and efficient Agrobacterium-mediated transformation of shoot apices and the subsequent regeneration of transgenic plants in pearl millet. The developed protocol will facilitate the insertion of desirable genes of useful traits into pearl millet.     

Factors influencing T-DNA transferring into pollen of lily in vitro

Direct pollen transformation method improves the classical transformation procedures because some tissue culture steps and subsequent regeneration can be avoided. A critical step in the development of Agrobacterium-mediated transformation is the establishment of optimum conditions for T-DNA delivery into tissue. The pollen grains of David lily (Lilium davidii Duchartre) are transformable by Agrobacterium during their germination, and extremely high GUS expression frequency of pollen had been achieved (92.7 ± 2.7%), but not for the ungerminated pollen. The culture medium, Agrobacterium cell density, duration of co-cultivation, and the combination of bacterial strains and plasmids should be optimized to get the highest transformation frequency. Thus, a method for pollen monocotyledonous species reproductive tissues transformation by Agrobacterium in monocots has been successfully developed. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 3, pp. 475–480 The text was submitted by the authors in English.     

T-DNA transfer to maize plants

Agrobacterium-mediated transformation is the method of choice to engineer desirable genes into plants. Here we describe a protocol for demonstrating T-DNA transfer from Agrobacterium into the economically important graminaceous plant maize. Expression of the T-DNA-located GUS gene was observed with high efficiency on shoots of young maize seedlings after cocultivation with Agrobacterium.     

CRISPR/Cas9-mediated targeted T-DNA integration in rice

Key message

Combining with a CRISPR/Cas9 system, Agrobacterium-mediated transformation can lead to precise targeted T-DNA integration in the rice genome.

Abstract

Agrobacterium-mediated T-DNA integration into the plant genomes is random, which often causes variable transgene expression and insertional mutagenesis. Because T-DNA preferentially integrates into double-strand DNA breaks, we adapted a CRISPR/Cas9 system to demonstrate that targeted T-DNA integration can be achieved in the rice genome. Using a standard Agrobacterium binary vector, we constructed a T-DNA that contains a CRISPR/Cas9 system using SpCas9 and a gRNA targeting the exon of the rice AP2 domain-containing protein gene Os01g04020. The T-DNA also carried a red fluorescent protein and a hygromycin resistance (hptII) gene. One version of the vector had hptII expression driven by an OsAct2 promoter. In an effort to detect targeted T-DNA insertion events, we built another T-DNA with a promoterless hptII gene adjacent to the T-DNA right border such that integration of T-DNA into the targeted exon sequence in-frame with the hptII gene would allow hptII expression. Our results showed that these constructs could produce targeted T-DNA insertions with frequencies ranging between 4 and 5.3% of transgenic callus events, in addition to generating a high frequency (50?80%) of targeted indel mutations. Sequencing analyses showed that four out of five sequenced T-DNA/gDNA junctions carry a single copy of full-length T-DNA at the target site. Our results indicate that Agrobacterium-mediated transformation combined with a CRISPR/Cas9 system can efficiently generate targeted T-DNA insertions.

     

Caspase-resistant VirD2 protein provides enhanced gene delivery and expression in plants

Agrobacterium tumefaciens VirD2 protein is one of the key elements of Agrobacterium-mediated plant transformation, a process of transfer of T-DNA sequence from the Agrobacterium tumour inducing plasmid into the nucleus of infected plant cells and its integration into the host genome. The VirD2 protein has been shown to be a substrate for a plant caspase-like protease activity (PCLP) in tobacco. We demonstrate here that mutagenesis of the VirD2 protein to prevent cleavage by PCLP increases the efficiency of reporter gene transfer and expression. These results indicate that PCLP cleavage of the Agrobacterium VirD2 protein acts to limit the effectiveness of T-DNA transfer and is a novel resistance mechanism that plants utilise to combat Agrobacterium infection. Brian Reavy and Svetlana Bagirova contributed equally to this work.     

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     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated <Emphasis Type="Italic">in planta</Emphasis> transformation of maize germ cells

A transfer DNA (T-DNA) carrying the marker gene nptII was detected in the genomes of diploid and haploid maize plants obtained after the treatment of pistil filaments with a suspension of Agrobacterium during artificial pollination. PCR analysis of total DNA isolated from 155 canamycin-resistant diploid F1 seedlings revealed T-DNA insertions in the genomes of 111 plants (32.7% of the total number of analyzed seeds). The example of matroclinal haploids was used to demonstrate that T-DNA may be transported to the egg cell by the growing pollen tube (PT). Twelve out of 16 analyzed haploid plants contained the T-DNA insertion. The possible mechanism of the transfer of the Agrobacterium T-DNA to the maize genome during pollination is discussed.     

Rice <Emphasis Type="Italic">Importin β1</Emphasis> gene affects pollen tube elongation

Importin β1 interacts with nuclear transport factors and mediates the import of nuclear proteins. We isolated a pollen-expressed gene, rice Importin β1 (OsImpβ1), from a T-DNA insertional population that was trapped by a promoterless β-glucuronidase (GUS) gene. The GUS reporter was expressed in the anthers and ovaries from early through mature developmental stages. Its expression was also observed in all floral organs. However, these patterns changed as the spikelet developed. T-DNA was inserted into the OsImpβ1 gene at 339 bp downstream from the translation initiation site. We obtained another T-DNA insertional allele by searching the flanking sequence tag database. In both lines, the wild-type and T-DNA-carrying progeny segregated at a ratio close to 1:1. The latter genotype was heterozygous (OsImpβ1/osimpβ1). Reciprocal crosses between WT and heterozygous plants demonstrated that the mutant alleles could not be transmitted through the male gametophyte. Close examination of the heterozygous anthers revealed that the mutant pollen matured normally. However, in vitro assays showed that tube elongation was hampered in the mutant grains. These results indicate that OsImpβ1 is specifically required for pollen tube elongation.     

Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of citrus tristeza virus

Factors influencing transformation frequencies using the Agrobacterium-mediated protocol developed for Citrus seedling internodal stem segments in this laboratory were evaluated, with particular emphasis on decreasing the numbers of ``escape' shoots produced. Although the use of a wild-type ``shooty' Agrobacterium strain allowed relatively high frequencies of β-glucuronidase positive (GUS+) shoots to be produced, none of the shoots were free of wild-type T-DNA and would not root. Both use of a liquid medium/kanamycin overlay and horizontal placement of stem segments increased the efficiency of kanamycin selection. Wounding via particle bombardment prior to Agrobacterium inoculation did not increase transformation frequencies. The concentration of benzyladenine (BA) in the regeneration/selection medium inversely influenced the numbers of shoots that regenerated and the subsequent ability of the shoots to root. Regeneration in the presence of kanamycin also influenced the ability of shoots to root. Many of the shoots that regenerated on selection medium were chimeric for GUS expression, and plants established from such shoots ranged from non-staining to solidly staining for GUS. However, solidly transformed plants with integrated T-DNA were obtained, and these plants have maintained the expression of transgenes over several years. The transgenic plants include ones of sour orange (C. aurantium L.) and Key lime (C. aurantifolia (Christm.) Swing.), two species not previously transformed, and have integrated and express the coat protein gene of citrus tristeza virus. This is the first report of a potentially agriculturally important transgene being expressed in Citrus. Received: 8 October 1996 / Revision received: 1 April 1997 / Accepted: 18 April 1997     

Evaluation of a cotyledonary node regeneration system forAgrobacterium-mediated transformation of pea (Pisum sativum L.)

Summary A rapid regeneration system was used for studies ofAgrobacterium-mediated transformation inPisum sativum L. Cotyledonary node explants were inoculated withAgrobacterium tumefaciens strains containing binary vectors carrying genes for nopaline synthase (NOS),β-glucuronidase (GUS), and neomycin phosphotransferase (NPTII) and placed on selection medium containing either 75 or 150 mg/liter kanamycin. A GUS encoding gene (uidA) containing an intron was used to monitor gene expression from 6 to 21 days postinoculation. GUS activity could be observed 6 days after inoculation in the area of the explant in which regeneration-occurred. Regenerating tissue containing transformed cells was observed in explants on selection medium 21 days postinoculation. Using this system, a single transgenic plant was obtained. Progeny of this plant, which contained two T-DNA inserts, demonstrated segregation for the inserts and for expression of the NOS gene in the selfed R₁ progeny. NPTII activity was observed in the R₂ generation, indicating inheritance and expression of the foreign DNA over at least two generations. Attempts to repeat this procedure were unsuccessful.     

Analyses in transgenic tobacco of the promoter from a Brassica napus gene highly expressed in the stigma

A genomic clone, Pis G363, containing the Brassica napus stigma-expressed gene Pis 63-2 was isolated and sequenced. The coding region of Pis G363 does not possess introns and shows 82% identity to the nucleotide sequence of a gene from Arabidopsis BAC clone T01B08. A 2-kb promoter fragment from Pis G363 was fused to the coding sequence of the marker enzyme β-glucuronidase (GUS) and introduced into tobacco via Agrobacterium-mediated transformation. The promoter fragment directed expression of the GUS gene in the stigma of transgenic tobacco. Some transformants also showed relatively low GUS activity in the pollen. Received: 25 May 1998 / Revision received: 30 July 1998 / Accepted: 21 August 1998     

Analysis of proliferation and survival of agrobacteria after inoculation of maize pistil filaments

While the authors have previously developed a method of pistil filament treatment with Agrobacterium cells during blossoming for the transformation of maize generative cells, the mechanism for bacterial T-DNA penetration into the embryo sac remained unknown. This article analyzes the possibility of agrobacterial penetration into the maize embryo via pollen tubes. Microbiological, PCR, and GUS techniques were used to confirm that agrobacteria could spread for up to 20 cm from the site of inoculation and were detected in maize embryo tissues as early as 24 h after inoculation, while they were not revealed after 5–13 days.     

Tumor Transformation of Petunia hybrida via Pollen Co-Cultured with Agrobacterium tumefaciens

Pollen preparations usually show a high nuclease activity. Therefore, to avoid DNA degradation, co-cultures of pollen and Agrobacterium tumefaciens were evaluated as a new tool in gene transfer experiments. As a model system, Petunia pollen was co-cultured with an A. tumefaciens wild strain. The co-cultured pollen was used for pollination of Petunia flowers. The seeds obtained were germinated and one cotyledon per seedling was removed and put stalk upwards on nutrient agar. In 80% of the cotyledons a callus developed from the cut surface of the stalks which was screened for tumor transformation on hormone-free medium. In repeated subculturing some calli maintained growth on hormone-free medium. Two of these calli were habituated. One callus, the best growing one, showed on Southern blot analysis a distinct hybridization signal at 3.2 kb when probed with Hind III fragment 22 DNA, covering two genes responsible for hormone free growth. This is the exact size that could be expected when plant material has been transformed with T-DNA. Another callus gave a hybridization signal at 2 kb which could only be explained with chromosomal rearrangement. In these two calli there was no co-transformation of the nos-gene: nopaline synthase activity could be detected from none of the calli, and none of the calli DNAs hybridized when probed with the nos-gene. Bacterial contamination could be excluded by probing the DNAs with virfragments.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Characterisation of T-DNA loci and vector backbone sequences in transgenic wheat produced by Agrobacterium-mediated transformation

Detailed molecular characterisation of transgene loci is a requirement for gaining regulatory approval for environmental release of genetically modified crops. In cereals, it is generally accepted that Agrobacterium-mediated transformation generates cleaner transgene loci with lower copy number and fewer rearrangements than those generated by biolistics. However, in wheat there has been little detailed analysis of T-DNA insertions at genetic and molecular level. Wheat lines transformed using Agrobacterium tumefaciens with bar and gusA (GUS) genes were subjected to genetic and molecular analysis. Unlike previous studies of transgene loci in wheat, we used functional assays for PAT and GUS proteins, combined with PCR and Southern analysis to detect the presence, copy number, linkage and transmission of two transgenes inserted in the same T-DNA. Thirty-four independent transgenic lines were categorised into three types: type I events (38% of total) where the gusA and bar genes displayed complete genetic linkage, segregating together as a single functional locus at the expected ratio of 3:1; type II events (18%), which possessed two or more transgene loci each containing gusA and bar; and type III events (44%), containing an incomplete T-DNA in which either the gusA or bar gene was lost. Most lines in this last category had lost the bar gene situated near the left T-DNA border. Southern analysis indicated that 30% of all lines possessed a single T-DNA copy containing gusA and bar. However, when data on expression and molecular analysis are combined, only 23% of all lines have single copy T-DNAs in which both gene cassettes are functioning. We also report on the presence of plasmid backbone DNA sequence in transgene loci detected using primer pairs outside the left and right T-DNA borders and within the plasmid selectable marker (NptI) gene. Approximately two thirds of the lines contained some vector backbone DNA, more frequently adjacent to the left border. Taken together, these data imply unstable left border function causing premature T-strand termination or read-through into vector backbone. As far as we are aware, this is the first report revealing near border T-DNA truncation and vector backbone integration in wheat transgenic lines produced by Agrobacterium-mediated transformation.     

A seed coat-specific promoter for canola

The canola industry generates more than $11 billion of yearly income to the Canadian economy. One problem of meal quality is the dark polyphenolic pigments that accumulate in the seed coat. Seed coat-specific promoters are a pre-requisite to regulate the genes involved in seed coat development and metabolism. The β-glucuronidase (GUS) reporter gene was used to test an Arabidopsis promoter in developing and mature seeds of canola (Brassica napus). The promoter tested is the regulatory region of the laccase gene (AtLAC15) from Arabidopsis thaliana. The AtLAC15 promoter::GUS construct was inserted into canola double haploid line DH12075 using Agrobacterium-mediated transformation. Southern blot analysis using a 536 bp GUS probe showed variation among the transformed plants in the T-DNA copy numbers and the position of the insertion in their genomes. Histochemical assay of the GUS enzyme in different tissues (roots, leaves, stem, pollen grains, flowers, siliques, embryos and seed coats) showed ascending GUS activity only in the seed coat from 10 days after pollination (DAP) to the fully mature stage (35 DAP). GUS stain was observed in the mucilage cell layer, in the outer integument layer of the seed coat but not in the inner integument. The AtLAC15 promoter exhibited a specificity and expression level that is useful as a seed coat-specific promoter for canola.     

Improvement of cotton fiber quality by transforming the<Emphasis Type="Italic"> acsA</Emphasis> and<Emphasis Type="Italic"> acsB</Emphasis> genes into<Emphasis Type="Italic"> Gossypium hirsutum</Emphasis> L. by means of vacuum infiltration

A novel method for the genetic transformation of cotton pollen by means of vacuum infiltration and Agrobacterium-mediated transformation is reported. The acsA and acsB genes, which are involved in cellulose synthesis in Acetobacter xylinum, were transferred into pollen grains of brown cotton with the aim of improving its fiber quality by incorporating useful prokaryotic features into the colored cotton plants. Transformation was carried out in cotton pollen-germinating medium, and transformation was mediated by vector pCAMBIA1301, which contains a reporter gene -glucuronidase (GUS), a selectable marker gene, hpt, for hygromycin resistance and the genes of interest, acsA and acsB. The integration and expression of acsA, acsB and GUS in the genome of transgenic plants were analyzed with Southern blot hybridization, PCR, histochemical GUS assay and Northern blot hybridization. We found that following pollination on the cotton stigma transformed pollen retained its capability of double-fertilization and that normal cotton seeds were produced in the cotton ovary. Of 1,039 seeds from 312 bolls pollinated with transformed pollen grains, 17 were able to germinate and grow into seedlings for more than 3 weeks in a nutrient medium containing 50 mg/l hygromycin; eight of these were transgenic plants integrated with acsA and acsB, yielding a 0.77% transformation rate. Fiber strength and length from the most positive transformants was 15% greater than those of the control (non-transformed), a significant difference, as was cellulose content between the transformed and control plants. Our study suggests that transformation through vacuum infiltration and Agrobacterium mediated transformation can be an efficient way to introduce foreign genes into the cotton pollen grain and that cotton fiber quality can be improved with the incorporation of the prokaryotic genes acsA and acsB.Communicated by D. Bartels     

Microprojectile bombardment of plant tissues increases transformation frequency by Agrobacterium tumefaciens

Bombardment of plant tissues with microprojectiles in an effective method of wounding to promote Agrobacterium-mediated transformation. Tobacco cv. Xanthi leaves and sunflower apical meristems were wounded by microprojectile bombardment prior to application of Agrobacterium tumefaciens strains containing genes within the T-DNA encoding GUS or NPTII. Stable kanamycin-resistant tobacco transformants were obtained using an NPTII construct from particle/plasmid, particle-wounded/Agrobacterium-treated or scalpel-wounded/Agrobacterium-treated potato leaves. Those leaves bombarded with particles suspended in TE buffer prior to Agrobacterium treatment produced at least 100 times more kanamycin-resistant colonies than leaves treated by the standard particle gun transformation protocol. In addition, large sectors of GUS expression, indicative of meristem cell transformation, were observed in plants recovered from sunflower apical explants only when the meristems were wounded first by particle bombardment prior to Agrobacterium treatment. Similar results in two different tissue types suggest that (1) particles may be used as a wounding mechanism to enhance Agrobacterium transformation frequencies, and (2) Agrobacterium mediation of stable transformation is more efficient than the analogous particle/plasmid protocol.     

Agrobacterium-mediated transformation of peanut (Arachis hypogaea L.) embryo axes and the development of transgenic plants

Transgenic peanut (Arachis hypogaea L.) plants have been produced using an Agrobacterium-mediated transformation system. Zygotic embryo axes from mature seed were cocultured with Agrobacterium tumefaciens strain EHA101 harboring a binary vector that contained the genes for the scorable marker B-glucuronidase (GUS) and the selectable marker neomycin phosphotransferase II. Nine percent of the germinated seedlings were GUS+. Polymerase chain reaction analysis confirmed that GUS+ shoots and T₁ progeny contained T-DNA. Molecular characterization of one primary transformant and its T₁ and T₂ progeny plants established that T-DNA was integrated into the host genome.     

Radiation-sensitive Arabidopsis mutants are proficient for T-DNA transformation.

Stable transformation of plants by Agrobacterium T-DNAs requires that the transgene insert into the host chromosome. Although most of the Agrobacterium Ti plasmid genes required for this process have been studied in depth, few plant-encoded factors have been identified, although such factors, presumably DNA repair proteins, are widely presumed to exist. It has previously been suggested that the UVH1 gene product is required for stable T-DNA integration in Arabidopsis. Here we present evidence suggesting that uvh1 mutants are essentially wild type for T-DNA integration following inoculation via the vacuum-infiltration procedure. Received: 23 June 1998 / Accepted: 21 February 1999