Stable transformation of mature zygotic embryos and regeneration of transgenic plants of chir pine (Pinus roxbughii Sarg.).

A particle inflow gun was used to transfer the plasmid pAHC25 containing the bar gene conferring resistance to glufosinate and the gusA reporter gene, each driven by the maize ubiquitin promoter, to mature embryos of Pinus roxburghii (chir pine). High levels of transient expression were obtained when embryos were cultured for 6 days on 10 microM benzyl adenine-containing medium and then exposed to high osmoticum (0.5 M sucrose) before and after bombardment. Selection on medium containing Basta enabled recovery of stably transformed shoots, both from the epicotyl and from adventitious buds. The primary transformed shoots from the epicotyl were multiplied via axillary shoots. Transformation was confirmed by histochemical staining for beta-glucuronidase (GUS) activity, by polymerase chain reaction (PCR) amplification of fragments of gusA and nos terminator, and by the resistance of needles to Basta.

Indications of limited altered gene expression in Pinus sylvestris trees from the Chernobyl region.

To evaluate the consequences of irradiation on the vegetation of the Chernobyl region, gene expression was compared in morphologically normal and dwarf needles from the same Pinus sylvestris trees in a region where the absorbed dose was 3-5 Gy. To compare the levels of gene expression, arrays consisting of 373 Pinus taeda cDNAs were hybridized with labeled cDNA derived from normal and dwarf needles of P. sylvestris. Twelve genes were significantly (P<0.01) up- or down-regulated between normal and dwarf needles for all five trees taken together. Five of these, related to stress or development, were up- or down-regulated 1.25-1.7-fold in the dwarf needles. There were no significant differences in (137)Cs content in the normal and dwarf needles, or in elongation growth rate of seedlings raised under controlled conditions from seed derived from trees in the region that had received a radiation dose over the range 2-12 Gy.

Phytochrome types in Picea and Pinus. Expression patterns of PHYA-Related types.

Knowledge of the genes in gymnosperms encoding the apoproteins of the plant photoreceptor phytochrome is currently scanty as for gymnosperm nuclear protein coding sequences in general. Here we report two complete cDNA-derived sequences which code for two different types of gymnosperm phytochrome. One sequence stems from Norway spruce (Picea abies) and the other from Scots pine (Pinus sylvestris). More detailed studies have shown that both types of phytochrome gene are present in Norway spruce. From phylogenetic analyses, these types appear to branch off from progenitors that are also the common ancestors of the angiosperm PHYA/PHYC and PHYB/PHYD/PHYE lineages. Partial phytochrome sequences of other gymnosperms cluster with either the one type or the other of the gymnosperm phytochrome genes characterized here. Southern blot analysis of Picea DNA using probes derived from the full-length Picea gene indicated a family of at least five members. Whether they code for new types may be doubted since only two phylogenetic clusters were found. Studies using RNA-PCR of Picea RNA extracted from either light- or dark-grown seedlings indicated that the steady-state levels of the transcripts of two PHYA/C-related genes were hardly affected by light.

Expression of an abscisic acid responsive promoter in Picea abies (L.) Karst. following bombardment from an electric discharge particle accelerator.

The 1.5 kilobase promoter sequence upstream of Dc8, a late embryo abundant gene of Daucus, fused to the reporter ß-glucuronidase gene was introduced into several tissues of Picea abies via a custom-made electric-discharge particle accelerator. Transient expression was measured histochemically as spot number 2 d after bombardment. Embryogenic suspensions gave higher levels of expression depending upon cell line than embryogenic callus or zygotic embryos. Expression was enhanced when cultures were treated with abscisic acid for 3 d before bombardment. A mean and maximum of 17 and 34 spots/disk, respectively, were observed with the best cell line, which was comparable with the level of expression driven by an enhanced 35S promoter.

T-DNA presence and opine production in tumors of Picea abies (L.) Karst induced by Agrobacterium tumefaciens A281.

The hypervirulent Agrobacterium tumefaciens strain A281 formed frequent tumors (31%) on Picea abies (Norway spruce), an economically important tree species in Swedish forests. Three-month-old seedlings were inoculated and tumors were established that grew hormone-independently in culture. Tumors contained agropine and mannopine/mannopinic acid as determined by acid pH paper electrophoresis. In addition, DNA hybridization studies showed that the DNA from these tumor lines contained sequences homologous to Ti plasmid T-DNA, whereas wild-type spruce seedling DNA did not. These results suggest that Agrobacterium vectors can be used for gene transfer into this important forest species.

Display of mitochondria in root tip cells.

A method for displaying mitochondria and proplastids in root tip sections of Tradescantia paludosa and cereals was modified from Altmann and Volkonsky. Root tips were fixed in 3% glutaraldehyde in phosphate buffer, pH 7.1, or acetate buffer, pH 4.8, for 3 hr, rinsed and postchromed overnight in 3% potassium dichromate, all at room temperature (20 C), dehydrated through a tertiary butanol series and embedded in ester wax. Four-micrometer sections were stained in hot acid fuchsin in aniline water, rinsed, treated with 1% sodium phosphomolybdate for 30 sec, rinsed and stained progressively with azure B for 3-10 min before being made permanent. Mitochondria and proplastids were stained brilliant crimson against a light blue cytoplasm with deep blue chromosomes. Previously reported difficulties with Altmann staining techniques are attributed to the erratic action of the classical fixatives used.