Isolation of cDNA for Pea Phytochrome Using an Expression Vector

Partially purified phytochrome mRNA was obtained from etiolatedpea epicotyls by polyribosome immunoprecipitation or by sizefractionation of total poly(A)⁺RNA, and used for the synthesisof double-stranded complementary DNA (cDNA). cDNA librarieswere constructed using an Escherichia coli expression vector,pUC9, and screened for phytochrome cDNA by colony immunologicalassay. Nine colonies were found to produce a 27 kDa polypeptidethat was reactive to both polyclonal and monoclonal antipeaphytochrome antibodies. The plasmids from these colonies containedcDNA inserts of 1.2 or 2.0 kbp. Hybridization-arrest translationassay verified that the cDNA clones contained a sequence codingfor phytochrome polypeptide. RNA blot hybridization analysisindicated that the cDNA hybridized to a 4.1 kb poly(A)⁺RNA indark-grown pea. (Received March 22, 1986; Accepted June 13, 1986)     

Maintenance and expression of heterologous genes in chloroplast ofChlamydomonas reinhardtii

The chloroplast genome ofChlamydomonas reinhardtii has been transformed with a chimeric gene consisting of the chloroplastatpA promoter and the bacterial gene for aminoglycoside adenine transferase (aadA). TheatpA-aadA cassette has been placed within the chloroplast DNAEcoRI restriction enzyme fragment 14, or within the chloroplastBamH1 fragment 10. The chimeric constructs were introduced into the chloroplast by particle bombardment. Integration of the cassette into chloroplast DNA then occurred via homologous recombination of sequences flanking the cassette with their corresponding chloroplast sequences. We demonstrate that the chloroplastatpA promoter inatpA-aadA routinely recombines with its endogenous counterpart, resulting in heteroplasmic chloroplast DNA populations that may persist for many generations. The heterologous gene does not require a 3 inverted repeat sequence for its expression. TheatpA-aadA gene copy number, which is dictated here by its position in the chloroplast genome, is proportional to the steady state level ofatpA-aadA mRNA. However, neither genomic position, gene copy number, or mRNA level have a significant effect on cellular resistance to spectinomycin, nor activity of theaadA gene productin vitro. These results suggest that, in the case ofaadA, the limiting step for expression of this gene is at the translational or post-translational level. TheatpA-aadA cassette should prove a useful model for future studies on the maintenance and expression of heterologous genes inC. reinhardtii chloroplasts.     

Highly efficient integration of foreign DNA into the genome of the green sulfur bacterium,Chlorobium vibrioforme by homologous recombination

Highly efficient and reproducible transformation ofChlorobium vibrioforme with plasmid DNA has been achieved by electroporation. Specific parameters have been optimized for the electrotransformation procedure. The method was developed using a construct containing a full copy of thepscC gene encoding the cytochromec ₅₅₁ subunit of the photosynthetic reaction center complex and theaadA gene encoding streptomycin resistance as selectable marker. Southern blotting analysis showed that the tested colonies were true transformants with the plasmid integrated into the genome by single homologous recombination. No transformants were obtained using the vector without thepscC gene showing that this vector does not replicate inC. vibrioforme. Thus transformation is possible only by homologous recombination. When using constructs designed to inactivate thepscC gene by insertion no transformants were obtained, indicating that the gene is indispensable for growth. The vector pVS2 carrying genes for erythromycin and chloramphenicol resistance was shown to replicate inC. vibrioforme. The two transformations shown here, provide an important genetical tool in the further analysis of structure and function of the photosynthetic apparatus in green sulfur bacteria.     

Agrobacterium tumefaciens AKE10-mediated transformation of an Asian pea pear, Pyrus betulaefolia Bunge: host specificity of bacterial strains

The Asian pea pear, Pyrus betulaefolia Bunge, is tolerant to several disorders in the fruit bodies caused by high humidity and dryness and is hence widely used as a rootstock for many pear plants suitable for food sources. We have now successfully transformed P. betulaefolia Bunge by an Agrobacterium-mediated gene transfer system. Among several wild-type A. tumefaciens strains examined, only AKE10 induced shoot-forming tumors at a high frequency on excised cotyledons of P. betulaefolia Bunge cultured on phytohormone-free medium. Both the nptII (kanamycin resistance) and GUS (#-glucuronidase) genes were introduced into the cotyledons by infection with AKE10 harboring a binary vector, and regenerated plants were obtained. Southern hybridization and polymerase chain reaction analyses and histochemical GUS assay indicated that morphologically normal transformed plants faithfully contained genes from the vector but not from wild-type oncogenic T-DNA. However, morphologically abnormal plants additionally possessed the 6b gene (AK-6b) of AKE10. These results show that non-disarmed A. tumefaciens is adequate to transfer genes to the Asian pea pear, P. betulaefolia Bunge.     

Construction of a barley (Hordeum vulgare L.) YAC library and isolation of a Hor1-specific clone

We have constructed an EcoRI-based YAC (yeast artificial chromosome) library from barley (Hordeum vulgare L. cv. Franka) using the vector pYAC4. The library consists of approximately 18 000 recombinant YACs with insert sizes ranging between 100 and 1000 kb (average of 160 kb) corresponding to 50% of the barley genome. Size fractionation after ligation resulted in an increased average insert size (av. 370 kb) but also in a substantial decrease in cloning efficiency. Less than 1% of the colonies showed homology to a plastome-specific probe; approximately 50% of the colonies displayed a signal with a dispersed, highly repetitive barley-specific probe. Using a primer combination deduced from the sequence of a member of the small Hor1 gene family coding for the C-hordein storage proteins, the library was screened by polymerase chain reaction and subsequently by the colony hybridization technique. A single YAC, designated Y66C11, with a 120 kb insert was isolated. This DNA fragment represents a coherent stretch from the terminal part of the Hor1 gene region as judged from the correspondence of the restriction patterns between Y66C11 DNA and barley DNA after hybridization with the Hor1-specific probe. Restriction with the isoschizomeric enzymes HpaII/MspI suggests a high degree of methylation of the Hor1 region in mesophyll cells but not in YAC-derived (yeast) DNA.     

Genetic transformation ofPelargonium X hortorum

Summary TransgenicPelargonium X hortorum have been producedvia Agrobacterium tumefaciens-mediated transformation. The regeneration protocol used provided a regeneration frequency approximately to 95 percent. Clumps of regenerants, from cotyledons and hypocotyls ofPelargonium X hortorum seedlings, were inoculated with the disarmed strain EHA101 ofAgrobacterium tumefaciens. This strain contains a binary vector carrying neomycin phosphotransferase II, hygromycin B phosphotransferase and ß-glucuronidase genes. Selection on the regeneration medium supplemented with hygromycin allowed production of transgenic plants in up to 20% of the inoculated explants. The insertion of foreign DNA was demonstrated by Southern and polymerase chain reaction analysis: these experiments indicated that the inserted T-DNA is not full length for most of the plants. All RO transgenic plants exhibited a normal phenotype and are fertile.Abbreviations GUS ß-glucuronidase coding sequence - PCR polymerase chain reaction - CaMV cauliflower mosaic virus - NPTII neomycin phosphotransferase coding sequence - NOS nopaline synthase gene promoter and terminator - HPH hygromycin B phosphotransferase coding sequence - SDS sodium dodecyl sulphate - EDTA (ethylenedinitro trilo)tetra-acetic acid disodium salt     

Transgenic papaya plants from Agrobacterium-mediated transformation of somatic embryos

Summary Transgenic papaya (Carica papaya L.) plants were regenerated from embryogenic cultures that were cocultivated with a disarmed C58 strain of Agrobacterium tumefaciens containing one of the following binary cosmid vectors: pGA482GG or pGA482GG/cpPRV-4. The T-DNA region of both binary vectors includes the chimeric genes for neomycin phosphotransferase II (NPTII) and ß-glucuronidase (GUS). In addition, the plant expressible coat protein (cp) gene of papaya ringspot virus (PRV) is flanked by the NPTII and GUS genes in pGA482GG/cpPRV-4. Putative transformed embryogenic papaya tissues were obtained by selection on 150 g·ml^–1 kanamycin. Four putative transgenic plant lines were obtained from the cp gene^– vector and two from the cp gene⁺ vector. GUS and NPTII expression were detected in leaves of all putative transformed plants tested, while PRV coat protein expression was detected in leaves of the PRV cp gene⁺ plant. The transformed status of these papaya plants was analyzed using both polymerase chain reaction amplification and genomic blot hybridization of the NPTII and PRV cp genes. Integration of these genes into the papaya genome was demonstrated by genomic blot hybridizations. Thus, like numerous other dicotyledonous plant species, papayas can be transformed with A. tumefaciens and regenerated into phenotypically normal-appearing plants that express foreign genes.Journal Series no. 3757 of the Hawaii Institute of Tropical Agriculture and Human Resources     

Plastid marker gene excision by the phiC31 phage site-specific recombinase

Marker genes are essential for selective amplification of rare transformed plastid genome copies to obtain genetically stable transplastomic plants. However, the marker gene becomes dispensable when homoplastomic plants are obtained. Here we report excision of plastid marker genes by the phiC31 phage site-specific integrase (Int) that mediates recombination between bacterial (attB) and phage (attP) attachment sites. We tested marker gene excision in a two-step process. First we transformed the tobacco plastid genome with the pCK2 vector in which the spectinomycin resistance (aadA) marker gene is flanked with suitably oriented attB and attP sites. The transformed plastid genomes were stable in the absence of Int. We then transformed the nucleus with a gene encoding a plastid-targeted Int that led to efficient marker gene excision. The aadA marker free Nt-pCK2-Int plants were resistant to phosphinothricin herbicides since the pCK2 plastid vector also carried a bar herbicide resistance gene that, due to the choice of its promoter, causes a yellowish-golden (aurea) phenotype. Int-mediated marker excision reported here is an alternative to the currently used CRE/loxP plastid marker excision system and expands the repertoire of the tools available for the manipulation of the plastid genome.     

Transient and stable expression of marker genes in cotransformedPetunia protoplasts in relation to X-ray and UV-irradiation

Irradiation of protoplasts with X-rays or ultraviolet light does not seem to influence the level of transient expression of foreign DNA inPetunia protoplasts, whereas the number of stably transformed colonies is significantly raised. This may indicate that irradiation influences integration and/or the expression of marker genes and does not result in enhanced uptake rates of plasmids into protoplasts and cell nuclei. Co-transformation with plasmids carrying a gene for kanamycin resistance (neomycin phosphotransferase II) and a gene for hygromycin resistance (hygromycin phosphotransferase) revealed that the cotransformation rates were not stimulated by irradiation when measuring expression. Twenty-five kanamycin resistant but hygromycin sensitive colonies were examined with Southern or slot blotting and all were found to contain the coding sequence for the hygromycinphosphotransferase gene in their genomes. No obvious differences regarding copy number of integrated genes were observed when comparing transformed colonies derived from irradiated and non-irradiated protoplasts.     

Genetic transformation of Brassica nigra by agrobacterium based vector and direct plasmid uptake

Summary Genetic transformation systems have been established for Brassica nigra (cv. IC 257) by using an Agrobacterium binary vector as well as by direct DNA uptake of a plasmid vector. Both the type of vectors carried nptII gene and gus gene. For Agrobacterium mediated transformation, hypocotyl tissue explants were used, and up to 33% of the explants produced calli on selection medium. All of these expressed B-glucuronidase gene on histochemical staining. Protoplasts isolated from hypocotyl tissues of seedlings could be transformed with a plasmid vector by FEG mediated uptake of vector DNA. A number of fertile kanamycin resistant plants were obtained using both the methods, and their transformed nature was confirmed by Southern blot analysis and histochemical staining for GUS. Backcrossed and selfed progenies of these transformed plants showed the presence of npt and gus genes.     

Transformation of alfalfa chloroplasts and expression of green fluorescent protein in a forage crop

The ability to transform chloroplasts in multiple species is important for improving agricultural traits. Chloroplast transformation of alfalfa (Medicago sativa L.), a useful forage plant with high market value, was achieved using a vector carrying aadA and gfp genes being introduced into the chloroplasts of alfalfa via particle bombardment using leaves and calli as explants. Resistant somatic embryos were generated and developed into plantlets from explants. The transformation efficiency was 1.3% for callus explants and 2.7% for leaf explants. PCR and Southern blotting analyses revealed that the foreign genes were integrated into the transformed chloroplast genome. The occurrence of GFP was further confirmed by fluorescence microscopy. Expression of foreign genes in alfalfa chloroplasts is therefore possible, and provides a novel means for genetic improvement of agronomically important traits and production of value-added proteins.     

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     

An estimate of the physical distance between two linked markers inHaemophilus influenzae

Using DNA clones, the physical distance between the linked genesnov andstr inHaemophilus influenzae was estimated. Although none of the cloned inserts contained both the markers, pJ1-8Str^R 13 (insert of 18·7 kb) includedstr gene at one end and part ofnov gene at the other end of the insert. By EcoRI restriction analysis and by Southern hybridization, the distance between the two EcoRI sites, cutting at which inactivates the two genes, was estimated to be 17·7 kb. A single continuous EcoRI fragment (containing 4EcoRI sites within it) carrying both the genes intact would need to be 20·4 kb in size. These estimates were confirmed independently using different clones ofnov ^r andstr ^r alleles as probes for hybridization with BamHI-digested chromosomal DNA.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of embryogenic cell suspension cultures of <Emphasis Type="Italic">Santalum album</Emphasis> L.

An efficient method for Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. is described. Embryogenic cell suspension cultures derived from stem internode callus were transformed with Agrobacterium tumefaciens harbouring pCAMBIA 1301 plant expression vector. Transformed colonies were selected on medium supplemented with hygromycin (5 mg/l). Continuously growing transformed cell suspension cultures were initiated from these colonies. Expression of β-glucuronidase in the suspension cultures was analysed by RT-PCR and GUS histochemical staining. GUS specific activity in the transformed suspension cultures was quantified using a MUG-based fluorometric assay. Expression levels of up to 105,870 pmol 4-MU/min/mg of total protein were noted in the transformed suspension cultures and 67,248 pmol 4-MU/min/mg of total protein in the spent media. Stability of GUS expression over a period of 7 months was studied. Plantlets were regenerated from the transformed embryogenic cells. Stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. This is the first report showing stable high-level expression of a foreign protein using embryogenic cell suspension cultures in S. album. U. K. S. Shekhawat and T. R. Ganapathi contributed equally to this work.     

Cassettes for seed-specific expression tested in transformed embryogenic cultures of soybean

Soybean (Glycine max [L.] Merrill) lectin is a seed protein that accumulates in protein bodies of cotyledons during seed development. We have constructed two expression cassettes containing the 5′ and 3′ regions of the soybean lectin gene connected by aNot I restriction site. One vector also contains the 32 amino acid signal sequence. Using polymerase chain reaction (PCR), the coding region of the β-glucuronidase (uidA) gene was inserted into theNot I site of each vector. We tested the function of the expression cassettes in transformed embryogenic cultures of soybean. Development-specific GUS expression was observed in developing somatic embryos transformed with the chimeric lectin promoter-GUS constructs as determined by histochemical assays. Our data indicate that these cassettes could be used to drive expression of foreign genes to modify embryo-specific traits of soybean as protein quality or quantity in the seed.     

Introduction of Foreign Gene into Small Cell Groups Digested Partial-Enzymically in Rice and Regeneration of Transgenic Plants

Suspension cultures of small cell groups (SCG; ca. 50–100 cells per group) were established from calli of Japonica rice Fang 7 and Hl24. The SCG were partially digested and transformed by plasmid pBll21 harboring the NPT-II (neomycin phosphotransferase) and GUS (betaglucuronidase) genes. Plasmid DNA was introduced into cells' by PEG, electroporation and PEG plus electroporation. NTP-II and GUS activity assay showed that the report genes were expressed in transformed cells. Transgenic plants were regeneiated possessing GUS activity due to the integration of intact foreign DNA into their genome as evidanced by hybridization. The results prove that the partially digested SCG is a potential, feasible system as receptor for gene transfer, especially for plants which are difficult for protoplast culture and plant regeneration from protoplasts.     

Cloning and characterization of thewhite andtopaz eye color genes from the sheep blowflylucilia cuprina

Summary Clones carrying thewhite andtopaz eye color genes have been isolated from genomic DNA libraries of the blowflyLucilia cuprina using cloned DNA from the homologouswhite andscarlet genes. respectively, ofDrosophila melanogaster as probes. On the basis of hybridization studies using adjacent restriction fragments, homologous fragments were found to be colinear between the genes from the two species. The nucleotide sequence of a short region of thewhite gene ofL. cuprina has been determined, and the homology to the corresponding region ofD. melanogaster is 72%; at the derived amino acid level the homology is greater (84%) due to a marked difference in codon usage between the species. A major difference in genome organization between the two species is that whereas the DNA encompassing theD. melanogaster genes is free of repeated sequences. that encompassing theirL. cuprina counterparts contains substantial amounts of repeated sequences. This suggests that the genome ofL. cuprina is organized on the short period interspersion pattern. Repeated sequence DNA elements, which appear generally to be short (less than 1 kb) and which vary in repetitive frequency in the genome from greater than 10⁴ copies to less than 10² copies, are found in at least two different locations in the clones carrying these genes. One type of repeat structure, found by sequencing, consists of tandemly repeating short sequences. Restriction site and restriction fragment length polymorphisms involving both thewhite andtopaz gene regions are found within and between populations ofL. cuprina.