Obtaining of transgenic French bean plants (Phaseolus vulgaris L.) resistant to the herbicide pursuit by Agrobacterium-mediated transformation

The transgenic plants of French bean (Phaseolus vulgaris) resistant herbicide Pursuit and kanamycin have been obtained. The genetic transformation was carried out with Agrobacterium tumefaciens strain LBA4404 containing binary vector carrying mutant ahas/als and selective nptII genes. Integration of the transgenes into plant genome was confirmed by polymerase chain reaction.     

Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors

A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed.     

Localization of replication origins in pea chloroplast DNA.

The locations of the two replication origins in pea chloroplast DNA (ctDNA) have been mapped by electron microscopic analysis of restriction digests of supercoiled ctDNA cross-linked with trioxalen. Both origins of replication, identified as displacement loops (D-loops), were present in the 44-kilobase-pair (kbp) SalI A fragment. The first D-loop was located at 9.0 kbp from the closest SalI restriction site. The average size of this D-loop was about 0.7 kbp. The second D-loop started 14.2 kbp in from the same restriction site and ended at about 15.5 kbp, giving it a size of about 1.3 kbp. The orientation of these two D-loops on the restriction map of pea ctDNA was determined by analyzing SmaI, PstI, and SalI-SmaI restriction digests of pea ctDNA. One D-loop has been mapped in the spacer region between the 16S and 23S rRNA genes. The second D-loop was located downstream of the 23S rRNA gene. Denaturation mapping of recombinants pCP 12-7 and pCB 1-12, which contain both D-loops, confirmed the location of the D-loops in the restriction map of pea ctDNA. Denaturation-mapping studies also showed that the two D-loops had different base compositions; the one closest to a SalI restriction site denatured readily compared with the other D-loop. The recombinants pCP 12-7 and pCB 1-12 were found to be highly active in DNA synthesis when used as templates in a partially purified replication system from pea chloroplasts. Analysis of in vitro-synthesized DNA with either of these recombinants showed that full-length template DNA was synthesized. Recombinants from other regions of the pea chloroplast genome showed no significant DNA synthesis activity in vitro.     

Biolistic-mediated genetic transformation of cowpea (<Emphasis Type="Italic">Vigna unguiculata</Emphasis>) and stable Mendelian inheritance of transgenes

We describe a novel system of exploiting the biolistic process to generate stable transgenic cowpea (Vigna unguiculata) plants. The system is based on combining the use of the herbicide imazapyr to select transformed meristematic cells after physical introduction of the mutated ahas gene (coding for a mutated acetohydroxyacid synthase, under control of the ahas 5' regulatory sequence) and a simple tissue culture protocol. The gus gene (under control of the act2 promoter) was used as a reporter gene. The transformation frequency (defined as the total number of putative transgenic plants divided by the total number of embryonic axes bombarded) was 0.90%. Southern analyses showed the presence of both ahas and gus expression cassettes in all primary transgenic plants, and demonstrated one to three integrated copies of the transgenes into the genome. The progenies (first and second generations) of all self-fertilized transgenic lines revealed the presence of the transgenes (gus and ahas) co-segregated in a Mendelian fashion. Western blot analysis revealed that the GUS protein expressed in the transgenic plants had the same mass and isoelectric point as the bacterial native protein. This is the first report of biolistic-mediated cowpea transformation in which fertile transgenic plants transferred the foreign genes to next generations following Mendelian laws.     

A minimal DNA cassette as a vector for genetic transformation of soybean (Glycine max)

Currently, the market demands products committed to protecting human health and the environment, known as clean products. We developed a protocol using DNA fragments containing only the gene sequence of interest, to replace the circular vectors containing genes for antibiotic resistance and other undesirable sequences, for obtaining transgenic soybeans for microparticle bombardment. Vector pAC321 was digested with the restriction enzyme PvuII to produce the 6159 bp ahas fragment, which contains the mutated ahas gene from Arabidopsis thaliana (Brassicaceae), under the control of its own promoter and terminator. This gene confers resistance against imazapyr, a herbicidal molecule of the imidazolinone class, capable of systemically translocating and concentrating in the apical meristematic region of the plant, the same region used for the introduction of the transgenes. This fragment was used to generate 10 putative transgenic soybean lines.     

Transformation and Regeneration of Two Cultivars of Pea (Pisum sativum L.)

A reproducible transformation system was developed for pea (Pisum sativum L.) using as explants sections from the embryonic axis of immature seeds. A construct containing two chimeric genes, nopaline synthase-phosphinothricin acetyl transferase (bar) and cauliflower mosaic virus 35S-neomycin phosphotransferase (nptII), was introduced into two pea cultivars using Agrobacterium tumefaciens-mediated transformation procedures. Regeneration was via organogenesis, and transformed plants were selected on medium containing 15 mg/L of phosphinothricin. Transgenic peas were raised in the glasshouse to produce flowers and viable seeds. The bar and nptII genes were expressed in both the primary transgenic pea plants and in the next generation progeny, in which they showed a typical 3:1 Mendelian inheritance pattern. Transformation of regenerated plants was confirmed by assays for neomycin phosphotransferase and phosphinothricin acetyl transferase activity and by northern blot analyses. Transformed plants were resistant to the herbicide Basta when sprayed at rates used in field practice.     

Studying plant genome variation using molecular markers

The authors' studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.     

Phenotypes of Tobacco Plants Expressing Genes for the Synthesis of Growth Regulators

The expression of genes for synthesis of auxin (iaaM and iaaH) and cytokinins (ipt) was studied in tobacco plants transformed by two Agrobacterium tumefaciens strains C 58 and LBA 4404. The strain LBA 4404 carried binary vector plasmid pCB 1334 (ipt gene) and plasmid pCB 1349 (iaaM, iaaH and ila genes). Both plasmids carried reportered gene for npt II. Obtained plants expressed incorporated genes. New proteins with molecular masses of about 74, 40, 26, 25, 21 and 17 kDa for wild plasmid pTi C58; 60, 36, 31.5, 27, 26 and 17 kDa for binary vector plasmid pCB 1334 and 74, 49, 36, 31.5, 26 and 25 kDa for binary vector plasmid pCB 1349 were found in the patterns of soluble proteins. Significant changes in the content of chlorophylls, especially chlorophyll a, were detected in the plants carrying ipt gene and in plants transformed by the wild strain C58 of A. tumefaciens. Tobacco plants expressing ipt gene and genes from T-DNA of pTi C58 plasmid were dwarf, and in comparison to the controls, they had thicker stems, and the surface of the leaf blades was reduced to 20 - 50 %. Adventitious roots, growing from the stem, were typical for transformants overproducing auxins. Regenerants and transformants expressing genes from T-DNA of plasmid pTi C58 differed in the shape of the flowers and their fertility. This revised version was published online in July 2006 with corrections to the Cover Date.     

Transformation of tobacco and birdsfoot trefoil by lupin leghemoglobin I cDNA clone

The full length cDNA clone of leghemoglobin I gene fromLupinus luteus was placed under dual promoter into the plant expression vector pCB1399 and the resulting vector (pCB1415) was transfered into theAgrobacterium strain LBA4404 (pAL4404). The binary system LBA4404 (pAL4404, pCB1415) was then used for transformation ofNicotiana plumbaginifolia andLotus corniculatus. In both species kanamycin-resistant plants have been selected and regenerated. The synthesis of LbI protein in transformed plants has not been shown.     

Nucleotide sequence of the single ribosomal RNA operon of pea chloroplast DNA

The nucleotide sequence of an 8 kbp region of pea ( Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha . The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia . i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene - 5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/ Marchantia and maize/ Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia .     

Conservation of Arabidopsis flowering genes in model legumes

The model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) have provided a wealth of information about genes and genetic pathways controlling the flowering process, but little is known about the corresponding pathways in legumes. The garden pea (Pisum sativum) has been used for several decades as a model system for physiological genetics of flowering, but the lack of molecular information about pea flowering genes has prevented direct comparison with other systems. To address this problem, we have searched expressed sequence tag and genome sequence databases to identify flowering-gene-related sequences from Medicago truncatula, soybean (Glycine max), and Lotus japonicus, and isolated corresponding sequences from pea by degenerate-primer polymerase chain reaction and library screening. We found that the majority of Arabidopsis flowering genes are represented in pea and in legume sequence databases, although several gene families, including the MADS-box, CONSTANS, and FLOWERING LOCUS T/TERMINAL FLOWER1 families, appear to have undergone differential expansion, and several important Arabidopsis genes, including FRIGIDA and members of the FLOWERING LOCUS C clade, are conspicuously absent. In several cases, pea and Medicago orthologs are shown to map to conserved map positions, emphasizing the closely syntenic relationship between these two species. These results demonstrate the potential benefit of parallel model systems for an understanding of flowering phenology in crop and model legume species.     

TheMagnaporthe grisea DNA fingerprinting probe MGR586 contains the 3′ end of an inverted repeat transposon

TheMagnaporthe grisea repeat (MGR) sequence MGR586 has been widely used for population studies of the rice blast fungus, and has enabled classification of the fungal population into hundreds of genetic lineages. While studying the distribution of MGR586 sequences in strains ofM. grisea, we discovered that the plasmid probe pCB586 contains a significant amount of single-copy DNA. To define precisely the boundary of the repetitive DNA in pCB586, this plasmid and four cosmid clones containing MGR586 were sequenced. Only 740 bp of one end of the 2.6-bp insert in the pCB586 plasmid was common to all clones. DNA sequence analysis of cosmid DNA revealed that all the cosmids contained common sequences beyond the cloning site in pCB586, indicating that the repetitive DNA in the fingerprinting clone is part of a larger element. The entire repetitive element was sequenced and found to resemble an inverted repeat transposon. This putative transposon is 1.86 kb in length and has perfect terminal repeats of 42 bp, which themselves contain direct repeats of 16 bp. The internal region of the transposon possesses one open reading frame which shows similarity at the peptide level to the Pot2 transposon fromM. grisea and Fot1 fromFusarium oxysporum. Hybridization studies using the entire element as a probe revealed that some strains ofM. grisea, whose DNA hybridized to the pCB586 probe, entirely lacked MGR586 transposon sequences.     

Studying plant genome variation using molecular markers

The authors studies on the organization and variation of plant genome with the use of molecular markers are briefly reviewed with special emphasis on random amplified polymorphic DNA (RAPD), inter simple sequence repeat (ISSR), sequence characterized amplified region (SCAR), and cleaved amplified polymorphic sequence (CAPS) markers detected with the use of polymerase chain reaction (PCR). These markers have been demonstrated to be promising for identifying cultivars and determining the purity of genetic strains of pea. Genetic relationships between strains, cultivars, and mutants of pea have been studied. The role of molecular markers in molecular genetic mapping and localizing the genes of commercially important characters of pea has been shown. The possibility of the use of molecular markers for studying somaclonal variation and detecting mutagenic factors in plants during long-term spaceflights is considered. The prospects of using DNA markers for understanding the organization and variability of higher plant genomes are discussed.__________Translated from Genetika, Vol. 41, No. 4, 2005, pp. 480–492.Original Russian Text Copyright © 2005 by Gostimsky, Kokaeva, Konovalov.     

固氮相关的两个植物基因转化烟草及其表达

豆科植物凝集和血红蛋白分别在植物识别其相应的根瘤菌和在根瘤内降低氧分压保护固氮酶的共生固氮作用中起重要作用。将豌豆（Ｐｉｓｕｍ ｓａｔｉｖａ Ｌ．）凝集素基因（ｐｌ）和Ｐａｒａｑｓｐｏｎｉａ ａｎｄｅｒｓｏｎｉｉ血红蛋白基因（ｐｈｂ）构建到同一植物表达载体上,通过根癌土壤杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍ ｔｕｍｅｆａｃｉｅｎｓ（Ｓｍｉｔｈ ｅｔ Ｔｏｗｎｓｅｎｄ）Ｃｏｎｎ）介导法转化烟草（Ｎｉｃｓ     

Transformation of Two Nitrogen-fixation-related Plant Genes into Tobacco and Their Expressions

Lectins and leghemoglobins in legumes play the important roles, respectively, in recognition of host plants to their own rhizobia, and lowering the oxygen partial pressure surround the bacteroids and protecting nitrogenase from oxygen in symbiotic nitrogen-fixing nodules.In order to investigate the non-leguminous recognition of rhizobial bacteria relating to nitrogen fixation, plant expression vectors containing pea lectin gene (pl) and Parasponia hemoglobin gene (phb) have been, respectively, constructed in a plasmid and the plasmid has been introduced into tobacco (Nicotiana tabacum L.) using Agrobacterium tumefaciens (Smith et Townsend) Conn as a vehicle for transformation. PCR and Southern blot demonstrated that the two genes were integrated into the genome of the tobacco plants. Histochemical staining for GUS activity, Western blotting,and in situ hybridization of pea lectin showed that they were expressed at translational level in the plants. These results may provide a clue for exploring whether Rhizobium leguminosarum bv. viciae could extend its host range and make the transgenic tobacco plants have the possibility of being symbiotic, or associative to nitrogen fixation.     

The hyperhydricity ofin vitroregenerants of grass pea (Lathyrus sativus L.) is linked with an abnormal DNA content

The grass pea (Lathyrus sativus) is a wild relative of the protein pea which may be a useful genetic resource for the acquisition of interesting stress resistance traits. However, grass pea is cross incompatible with pea, leaving protoplast fusion as the only alternative to produce interspecific hybrids of grass pea and pea. In addition, as all grass pea seeds contain a toxic aminoacid, low-toxin containing genotypes will have to be produced by gene transfer. In this context, it is therefore essential that regenerated plants are fertile, true-to-type and not chimaeric in nature when they have been obtained in absence of any selection treatment. In the present study, shoot buds were regenerated from hypocotyls of three grass pea genotypes, and flow cytometry permitted us to characterise them in terms of nuclear DNA content. Plant regeneration competence was genotype-dependent and strongly also was correlated with a normal DNA content. The auxin/cytokinin balance of regeneration media affected the DNA level of regenerants. In turn, an abnormal DNA content was systematically associated with severe hyperhydricity symptoms, which hampered the regeneration of rooted, fertile plants.     

Genetic transformation of Nicotiana africana Merxm. with plasmids containing lox recombination

An efficient genetic transformation method for african tobacco Nicotiana africana Merxm. has been established. African tobacco is a valuable source for cytoplasmic male sterility (CMS) and nuclear encoded resistance to potato virus Y (PVY). N. africana transgenic plants have been obtained using both Agrobacterium-mediated and direct transformation of leaf explants with gold particle bombardment using particle inflow gun. Plasmid vectors containing phosphinothricin resistance gene (bar gene) coding region without promoter and independent 35S promoter between lox sites (lox-bar-35S-lox) and nptII gene were used. Transgenic plants were selected according to growth capacity on the selective medium containing 50 mg/l kanamycin. PCR analyses of kanamycin-resistant plants confirmed the presence of nptII and bar genes in their genome. Agrobacterium-mediated transformation of root explants has proved to be the most efficient transformation method for N. africana.