Kohei Irifune

Nucleotide sequence of a highly repeated DNA sequence and its chromosomal localization in Allium fistulosum

A highly repeated DNA sequence with a repeating unit of approximately 380bp was found in EcoRV digests of the total genomic DNA of Allium fistulosum. Three independent clones containing this unit were isolated, and their repeating units sequenced. These units showed more than 94% sequence homology, and the copy number was estimated to be about 2.8×106 per haploid genome. In situ hybridization, with the repeating unit as a probe, and C-banding analyses indicated that the repeated DNA sequence of A. fistulosum is closely associated with the major C-heterochromatin in the terminal regions of all 16 chromosomes at mitotic metaphase. The characters of the repeating unit are similar to those of the A. cepa unit, which is taxonomically closely related to A. fistulosum.

Nucleotide sequence of a gene for nitrite reductase from Arabidopsis thaliana

A nitrite reductase (NiR) gene was recovered from Arabidopsis thaliana genomic library by the homology with a cDNA of spinach NiR and sequenced. Based on the comparison with the spinach cDNA, the Arabidopsis NiR gene was concluded to contain 4 exons [exon 1 of 376 bp (beginning with ATG start codon), exon 2 of 355 bp, exon 3 of 289 bp and exon 4 of 741 bp (ending at TGA stop codon)] and 3 introns (intron 1 of 196 bp, intron 2 of 81 bp and intron 3 of 77 bp). This conclusion was confirmed by the analysis using the RT-PCR method. The deduced amino acid sequence of the coding region of the Arabidopsis NiR gene had high similarities with those of NiR genes of other plants including spinach.

Stable transformation ofArabidopsis with thebar gene using particle bombardment

A plasmid pARK 22 harbouring thebar gene encoding phosphinothricin acetyltransferase (PAT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator was constructed and introduced into root sections ofArabidopsis thaliana using the pneumatic particle gun. The root sections that had been bombarded with this plasmid gave four to eight times higher yield of drug-resistant calluses than those sections bombarded with pCaMVNEO or pCH, which respectively contain the neomycin phosphotransferase and hygromycin phosphotransferase genes. Among a number of primary transformant (T0) plants obtained from independent bialaphos-resistant calluses, three were studied by Southern blot hybridization and PAT enzyme activity analyses, confirming the stable integration of the foreign gene into theArabidopsis genome and its expression in plants. The progeny analysis showed transmission of the foreign gene and its expression in up to the T2 generation. Some of the T1 progeny showed morphological abnormalities. Thus, thebar gene can be used effectively to allow selection of transgenicA. thalianna plants.

Bombardment-mediated transformation of plant cells

Significance of bombardment-mediated transformation in plant research is discussed. Apart from general subjects related to this transformation study, a particular emphasis has been placed on the pollen and organelle transformation and on integration mechanism. Over 70 literatures related to this field are cited here.

Fluorescent banding pattern analysis of eight taxa of Phaseolus and Vigna in relation to their phylogenetic relationships

Phylogenetic relationships among eight taxa of seven species of Phaseolus and Vigna (Phaseolus angularis, P. aureus, P. calcaratus, P. coccineus, P. vulgaris, Vigna sesquipedalis and V. sinensis; 2n = 22 each) were studied by the fluorescent chromosome banding technique. Preparations of somatic metaphase chromosomes of each taxon were sequentially stained with Giemsa, GC-specific fluorochrome chromomycin A3 (CMA) and AT-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI). On the basis of the fluorescent banding patterns of the 22 chromosomes of each taxon, P. angularis, P. coccineus (from China and Korea) and P. vulgaris were grouped into one group (“Phaseolus group”), P. aureus and two Vigna species were grouped into another (“Vigna group”) and P. calcaratus was grouped in an independent group.

Transgenic haploid plants ofNicotiana rustica produced by bombardment-mediated transformation of pollen

Immature pollen fromNicotiana rustica was bombarded with gold particles coated with plasmid DNA encoding neomycin phosphotransferase II (NPTII) and β-glucuronidase (GUS) genes which, respectively, are under the control of the cauliflower mosaic virus (CaMV) 35S promoter and nopaline synthase (NOS) terminator in the plasmid. Kanamycin-resistant pollen embryoids were selected from the bombarded pollen cells and two independent lines of transgenic plants were regenerated. Enzyme assays showed that one has both NPTII and GUS activities and the other only weak NPTII activity. Southern blot analyses indicated that the former has a DNA fragment corresponding to the intact expression cassettes for both genes in its genome; whereas the latter lacks intact expression cassettes for both genes and has only the intactnptII coding sequence in its genome. The transgenic plants of both lines have 24 chromosomes, confirming haploidy, and they are infertile. These results indicate that transgenic haploid plants can be produced directly by the bombardment-mediated transformation of immature pollen.

Stable transformation of Eustoma grandiflorum by particle bombardment

Abstract Explants (7.5±2.5 mm) cut from stems and roots of 3-week-old Eustoma grandiflorum Grise, (lisianthus) cv. Glory White seedlings were bombarded with plasmid pBI221, which harbors the uidA gene encoding β-glucuronidase (GUS) driven by the cauliflower mosaic virus (CaMV) 35S promoter. More than 800 blue spots of GUS-expressing cells were observed per 90 explants. Explants bombarded with pARK22 harboring the bar gene encoding phosphinothricin acetyltransferase driven by the CaMV 35S promoter were selected for bialaphos resistance. Putative transgenic plants were obtained about 3 months after bombardment. Southern blot analysis of putative transgenic plants revealed the presence of the bar gene in their genome.

Transgenic roots produced by introducing Ri-rol genes into cucumber cotyledons by particle bombardment

Transformed roots ofCucumis sativus were obtained from cotyledon tissues that had been bombarded with gold particles coated with plasmid pE7.4 using a pneumatic particle gun. This plasmid containsrolA, rolB, rolC genes and ORF 13 of the 7.4 kbEco RI fragment of T-DNA of pRi 1724 isolated fromAgrobacterium rhizogenes MAF 03-01724. The nature of the tissue and the composition of the culture media used greatly influenced the recovery of transformed roots. The transgenic nature of the derived roots was confirmed by the vigorous. highly-branched growth seen on a phytohormone-free medium. The stable integration ofrol genes into the cucumber genome was confirmed by Southern blot analysis.

Successful expression in pollen of various plant species of in vitro synthesized mRNA introduced by particle bombardment

Gold particles coated with β-glucuronidase (GUS) mRNA with a 5′ cap structure that had been synthesized in vitro were introduced, by use of a pneumatic particle gun, into pollen grains of lily (Lilium longiflorum), freesia (Freesia refracta) and tulip (Tulipa gesneriana). A fluorometric assay for the GUS activity indicated that in vitro synthesized GUS mRNA introduced into these pollen cells by particle bombardment was successfully expressed. GUS activity in extracts of the bombarded lily pollen became detectable fluorometrically within 30 min after bombardment, peaked at 6 h, then gradually decreased. This activity changed as a function of the developmental stage of the pollen cell of lily.

Stable transformation of cultured cells of the liverwortMarchantia polymorpha by particle bombardment

Suspension-cultured cells (A-18 line) of the liverwortMarchanta polymorpha were bombarded by a pneumatic particle gun with plasmid pCH harbouring the hygromycin phosphotransferase (HPT) gene (hpt) under the control of the cauliflower mosaic virus (CaMV) 35 S promoter and the nopaline synthase polyadenylation region. Nine weeks after bombardments, 128 hygromycin-resistant calluses were obtained from an approximate total of 7×106 cells. Ten cell lines chosen randomly were analysed further. Southern blot analysis showed that all of the ten lines contain thehpt gene in the genome, demonstrating that these lines are transformants. An HPT enzyme activity assay confirmed the expression of the gene in all of the transformant lines.