Production and identification of somatic hybrids between Solanum tuberosum and S. papita by using the rolC gene as a morphological selectable marker

A successful hybridization of a diploid clone of Solanum tuberosum with a rolC-transgenic, diploid S. papita clone is reported. By using leaf expiants of this S. papita clone, which after transformation expressed kanamycin resistance, intact protoplasts were obtained, but these protoplasts did not develop to microcalli or regenerate to mature plants. However, protoplasts of the S. tuberosum clone showed a high capacity to regenerate plants from isolated protoplasts. On a medium containing Kanamycin only calli regenerated to plants, which revealed a rolC phenotype (reduced apical dominance with a large number of adventitious shoots and a pale green color of leaves) and later on turned out to be true hybrids. Self fusions of S. papita never developed to microcalli and those of S. tuberosum ceased to develop on the kanamycin-containing medium. Identification of somatic hybrids was done by RFLP and RAPD analysis. In the greenhouse, out of four selected hybrids only FK3.1 was successfully crossed with two standard S. tuberosum varieties (Datura, Desirée). Out of all the seeds germinated, only rolC-negative F¹ seedlings were further characterized. Within the seedling population obvious differences were evident in respect of the S. papita and S. tuberosum characteristics.     

Stable haploid poplar callus lines from immature pollen culture

Embryogenesis and plant regeneration have been obtained from isolated immature pollen of two poplar hybrids ( Populus nigra L. × hybrid 'Aue1' and 'Aue2'). In total, 1487 calli or embryos, respectively, larger than 1 mm were generated in a 2-year study. By using a cytokinin containing induction medium, on average 19 calli per responsive immature catkin were formed. Additional supplementation with auxin in 2002 increased the frequency to 72 calli per catkin. Microsatellite marker analyses confirmed haploid origin in most regenerants studied. So far six out of eight obtained regenerative callus lines have maintained their haploid level up to 24 months of development. A number of haploid and doubled haploid plants of different lines are available and have been transferred to soil.     

A full saturated linkage map of<Emphasis Type="Italic"> Picea abies</Emphasis> including AFLP,SSR, ESTP, 5S rDNA and morphological markers

Based on an F₁ progeny of 73 individuals, two parental maps were constructed according to the double pseudo-test cross strategy. The paternal map contained 16 linkage groups for a total genetic length of 1,792 cM. The maternal map covered 1,920 cM, and consisted of 12 linkage groups. These parental maps were then integrated using 66 intercross markers. The resulting consensus map covered 2,035 cM and included 755 markers (661 AFLPs, 74 SSRs, 18 ESTPs, the 5S rDNA and the early cone formation trait) on 12 linkage groups, reflecting the haploid number of chromosomes of Picea abies. The average spacing between two adjacent markers was 2.6 cM. The presence of 39 of the SSR and/or ESTP markers from this consensus map on other published maps of different Picea and Pinus species allowed us to establish partial linkage group homologies across three P. abies maps (up to five common markers per linkage group). This first saturated linkage map of P. abies could be therefore used as a support for developing comparative genome mapping in conifers.Communicated by O. Savolainen     

T-DNA and transposon tagging in aspen

Abstract: We have investigated the somatic activity of the maize Activator (Ac) element in haploid and diploid aspen with the objective of developing an efficient transposon-based system for gene isolation in the model tree species Populus. It was shown that Ac is reinserted, frequently into or near coding regions in aspen, and therefore can be used for gene tagging studies. A number of phenotypic variants were also found following transformation of constructs harbouring the rolC gene. Comparative analyses of T-DNA flanking regions of variants and wild type lines indicate that T-DNA insertion has occurred in or near coding regions. However, the frequency of T-DNA insertion into genes is about one half of the frequency of Ac insertion hitting coding sequences. The results obtained give a proof-of-concept for transposon tagging in a tree system. Given the long generation cycles in tree species, gene tagging strategies are practical only to obtain dominant gain-of-function mutants that do not require selfing or test crossing. In order to obtain recessive loss-of-function mutants, we have regenerated haploid lines from immature pollen. These lines were successfully transformed with a construct containing the rolC transgene from Agrobacterium rhizogenes and Ac element from maize. The results indicate that Ac is also active in haploid aspen and hence can be used in general for gene tagging in trees.     

Determination of transgene repeat formation and promoter methylation in transgenic plants

The integration of transgenes into a plant host genome following Agrobacterium tumefaciens-mediated or direct transformation may occur as a single copy or in the form of tandem repeats. The latter has been associated with promoter methylation and silencing of transgenes. Thus, the early screening of such transgenic plants is desirable for ruling out future repeat-dependent transgene instability. We developed a simple PCR-based method in which primer pairs were specifically designed so that amplifications could only be obtained if the transgene was present in the form of multiple inserts in a transgenic line. The method was established using 35S-rolC transgenic aspen lines showing morphologically visible transgenic silencing. Later, it was possible to screen independent transgenic lines showing no visible marker gene expression. Furthermore, a method was developed in which positive PCR amplification was indicative of promoter methylation. The results were consistent and reproducible across different independent transgenic lines. The methods were quick, reliable, consistent and reproducible, and can be useful for routine screening of transgene silencing in lines derived from many different systems.     

Morphology, wood structure and cell wall composition of rolC transgenic and non-transformed aspen trees

Morphology, wood structure and cell wall composition of 35S-rolC transgenic hybrid aspen (P. tremula2tremuloides) were compared with non-transformed control trees. The transgenics are characterised by stunted growth, altered physiological parameters and light green leaves of reduced size. Histometric measurements revealed thinner fibre walls as compared to the controls. UV microspectrophotometry of individual wall layers did not reveal distinctive differences in the lignification of xylem cells, but in the extremely thin-walled fibres of the transgenics the secondary walls were less lignified as revealed by KMnO₄ staining in transmission electron microscopy. In the transgenics the formation of xylem cells was delayed and the differentiation zone reduced to only a few rows. Immunocytochemical analyses revealed the deposition of lignins in less differentiated xylem cells as compared to the controls. The first labelling of condensed lignin appeared in cell corners and of non-condensed lignin in secondary walls near cell corners during the deposition of S1 polysaccharides. Because of alterations in the formation and differentiation of xylem cells, 35S-rolC transgenic aspen may be useful for studies on molecular factors controlling the differentiation continuum.     

Successful crossings with early flowering transgenic poplar: interspecific crossings,but not transgenesis,promoted aberrant phenotypes in offspring

In forest tree species, the reproductive phase is reached only after many years or even decades of juvenile growth. Different early flowering systems based on the genetic transfer of heat‐shock promoter driven flowering‐time genes have been proposed for poplar; however, no fertile flowers were reported until now. Here, we studied flower and pollen development in both HSP::AtFT and wild‐type male poplar in detail and developed an optimized heat treatment protocol to obtain fertile HSP::AtFT flowers. Anthers from HSP::AtFT poplar flowers containing fertile pollen grains showed arrested development in stage 12 instead of reaching phase 13 as do wild‐type flowers. Pollen grains could be isolated under the binocular microscope and were used for intra‐ and interspecific crossings with wild‐type poplar. F1‐seedlings segregating the HSP::AtFT gene construct according to Mendelian laws were obtained. A comparison between intra‐ and interspecific crossings revealed that genetic transformation had no detrimental effects on F1‐seedlings. However, interspecific crossings, a broadly accepted breeding method, produced 47% seedlings with an aberrant phenotype. The early flowering system presented in this study opens new possibilities for accelerating breeding of poplar and other forest tree species. Fast breeding and the selection of transgene‐free plants, once the breeding process is concluded, can represent an attractive alternative even under very restrictive regulations.     

Variation in the psbC gene region of gymnosperms and angiosperms as detected by a single restriction site polymorphism

 This paper reports on a PCR-RFLP analysis in a chloroplast DNA region consisting of coding and intergenic spacer sequences of trnS and the adjacent psbC gene. This region was PCR-amplified in 62 woody plant species, predominantly tree species, that represent a broad systematic range in both gymnosperms and dicotyledonous angiosperms. The amplification products were digested by the restriction endonuclease HaeIII (GG↓CC). Fourteen different restriction patterns occurred, 5 of which characterised representatives of the gymnosperms, and 9 angiosperm representatives. A single restriction site polymorphism revealed most of the species to share restriction patterns. Groups formed which showed relationships to plant systematic units. This phenomenon is discussed with regard to the psbC gene and the GGCC motif for tracing species’ relationships on a high taxonomic level of gymnosperms and angiosperms. Received: 5 June 1996/Accepted: 20 September 1996     

Excision of the maize transposable element Ac in periclinal chimeric leaves of 35S-Ac-rolC transgenic aspen-Populus

The transposable element Ac from maize, in combination with the phenotypic selectable marker rolC, was employed in transformation experiments of a hybrid aspen clone. A number of transgenic clones exhibited light-green sectors on green leaves. In vitro regeneration from leaves showing a high number of light-green spots resulted in R2 plants, which also showed light-green sectored leaves. However, only one out of 385 regenerated plants obtained showed green leaves. Both PCR and northern analysis indicated Ac excision and restoration of rolC expression. In Southern blot analysis of this green plant additional bands were observed as compared to the original R1 plant. The occurrence of these bands and a suggested Ac excision in the non-green L1-epidermal layer leading to periclinal chimerism of this plant is discussed.