Prevention of the flowering of a tree,silver birch

Genetic modification of trees presents great advantages but it is hampered by the possible spread of introduced genes to native populations. However, the spread would be prevented if the modified trees would be sterile. We have previously shown that the induction of sterility by the prevention of flowering is possible in tobacco and Arabidopsis by introducing a gene construct composed of the ribonuclease gene BARNASE ligated to the flower-specific promoter of the birch gene BpMADS1. In the present study, we test this gene construct in silver birch (Betula pendula Roth). When this gene construct was introduced into very early-flowering birch clones, 81 kanamycin resistant lines were obtained. In 38 lines, the vegetative development was disturbed, e.g., the leaves were small and the plants were short and bushy or the growth of plants was weak. More importantly, in 7 other lines no male inflorescences formed or they aborted early. If male inflorescences were formed, they did not contain any stamens. The initial growth of these lines was similar to the non-transgenic control lines. Later, however, the growth of the non-flowering lines differed from that of the controls in showing some dichotomic branching and a reduced number of branches. Preliminary results showed that the gene construct can prevent the development of female inflorescences as well. The results show clearly that BpMADS1::BARNASE can prevent the flowering in a tree but the prevention of flowering may cause some side effects. Studies with ordinary birch clones will show whether the side effects are a property of the early flowering clones or all birches.     

Overexpression of BpAP1 induces early flowering and produces dwarfism in Betula platyphylla × Betula pendula

The involvement of APETALA1 (AP1) in the flowering transition has been the focus of much research. Here, we produced Betula platyphylla × Betula pendula (birch) lines that overexpressed BpAP1 using Agrobacterium‐mediated transformation; we obtained five independent 35S::BpAP1 transgenic lines. Polymerase chain reaction (PCR), Southern, northern and western analyses were used to identify the transformants. As determined by quantitative real‐time PCR (qRT‐PCR), BpAP1 expression in roots, shoots, leaves and terminal buds of 35S::BpAP1 transgenic lines was significantly higher than that in the wild type (WT, P < 0.01). The average height of 2‐year‐old 35S::BpAP1 plants was significantly lower (41.17%) than that of non‐transgenic plants. In the 35S::BpAP1 lines, inflorescences emerged successively beginning 2 months after transplanting. In addition, the length–diameter ratio of fully developed male and female inflorescences were both significantly less than those of the WT (P < 0.05), i.e. the morphological characteristic was stubby. The male inflorescences emerged early, with empty, draped anthers, and pollen was rarely produced, whereas the female floret structure was not different from WT. The pistils developed normally and could accept pollen, leading to the production of hybrid progeny (F₁). F₁ plants completed flowering within only 1 year after sowing. We demonstrate that BpAP1 can be inherited through sexual reproduction. Overexpression of BpAP1 caused early flowering and dwarfism; these lines had an obviously shortened juvenile phase. These results greatly increase our understanding of the mechanisms underlying the flowering transition and enhance genetic studies of birch traits, and they open up new possibilities for the breeding of birch and other woody plants.     

Abscisic acid-regulated Glb1 transient expression in cultured maize P3377 cells

In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression. Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997     

Analysis and optimisation of DNA delivery into wheat scutellum and Tritordeum inflorescence explants by tissue electroporation

Wheat scutella and tritordeum inflorescences were transformed by tissue electroporation with plasmid DNA containing a β-glucuronidase (GUS) gene (gus A) under the control of the rice actin1 promoter. Factors affecting electroporation efficiency were analysed. Important factors were electroporation voltage and pulse length, the volume of electroporation buffer, the osmoticum of electroporation buffer and medium, the osmoticum of pre-electroporation culture medium, and pre-electroporation incubation time and temperature. Maximum transient gene expression was obtained with a single pulse of 550 V/cm from a 960-μF capacitor, using 200 μl of electroporation buffer, after 2–3 h culture on media with 357 mOsm for wheat scutella or 1 day on media with 222 mOsm for tritordeum inflorescences, and 0.5–1 h pre-electroporation incubation with DNA at 24 °C. Under these conditions, up to 90% of the explants showed GUS expression, and up to 149 expression signals were recorded per replicate. Electroporated explants showed high rates of survival and retained the ability to regenerate plants via somatic embryogenesis. Received: 9 August 1996 / Revision received: 11 September 1997 / Accepted: 2 July 1998     

Enhancement of embryogenic culture initiation from tissues of mature sweetgum trees

 Male inflorescences, female inflorescences, and leaves collected from dormant buds of three sweetgum (Liquidambar styraciflua) trees were tested for induction of somatic embryogenesis following treatment with thidiazuron, naphthaleneacetic acid (NAA) or different combinations of the two. Explants were placed into culture either within a few days after collection or following 2 months of storage at –15  °C. Although embryogenic cultures were obtained from all three trees, embryogenesis induction was strongly affected by genotype (source tree), with 100% of the staminate inflorescence explants from one tree producing embryogenic cultures in one experiment. Embryogenesis induction was also influenced by explant type, with staminate inflorescences up to five times more likely to produce an embryogenic culture than female inflorescences. No embryogenic cultures were obtained from leaf explants. While treatment with plant growth regulators was not required for embryogenesis induction from inflorescence explants, culture on medium with NAA alone resulted in the highest production of repetitively embryogenic cultures and cultures producing proembryogenic masses. Dormant buds stored for 2 months at –15  °C were still able to produce embryogenic cultures, although frozen storage decreased this ability by over one-half for staminate inflorescences. Received: 20 January 1999 / Revision received: 18 April 1999 / Accepted: 29 April 1999     

Assessment of expression and inheritance patterns of three transgenes with the aid of techniques for promoting rapid flowering of transgenic apple trees

Rapid flowering of transgenic Royal Gala apple (Malus×domestica) trees was achieved by growing trees under controlled greenhouse conditions. The expression and inheritance of three transgenes were confirmed in the seedling progeny. Grown as single stems on their own roots, the transgenic apple trees produced 80–110 nodes and were 2 m high on average at the end of the 1st year's growth. In the 2nd year, approximately 20% of these trees flowered around node 80. However, when scions collected from the top of 1-year-old trees were grafted onto the dwarfing rootstock Malling 9, 85% produced flowers and fruit within the next year. The grafted trees continued to produce fruit in the following years. Expression of the transgene uidA was monitored by assaying β-glucuronidase (GUS) activity in leaves, flowers and fruit. Inheritance of three transgenes, uidA, neomycin phototransferase II and acetolactate synthase, were demonstrated through the recovery of GUS-positive, kanamycin-resistant and chlorsulfuron-resistant progeny. Segregation patterns fitted a 1:1 ratio in most lines. However, a detailed analysis in one progeny line revealed a complex T-DNA integration pattern. Received: 8 June 1998 / Revision received: 19 November 1998 / Accepted: 26 November 1998     

Sexual specialization in two tropical dioecious figs

Ficus species (figs) and their species-specific pollinator wasps are involved in an intimate mutualism in which wasps lay eggs in some ovaries of the closed inflorescences (syconia), and mature, inseminated offspring carry pollen from mature syconia to fertilize receptive inflorescences. In monoecious species, each syconium produces seeds and wasps. In functionally dioecious fig species, making up approximately half the figs worldwide, male and female functions are separated; hermaphrodite (functionally male) trees produce wasps and pollen only, while female trees produce seeds only. This sexual separation allows selection to act independently on the reproductive biology of each sex. Examining sexual specialization in a tight mutualism allows us to determine aspects of the mutualism that are flexible and those that are canalized. In this study, we quantified the phenology of two species of dioecious figs, F. exasperata and F. hispida, for 2 years by following the fates of several thousand syconia over time. In studying each of these species in a dry and a wet site in south India, we tested specific predictions of how dioecious figs might optimize sexual function. On female trees of both species, more inflorescences matured during the wet (monsoon) season than in any other season; this fruiting period enabled seeds to be produced during the season most suitable for germination. In F. exasperata, functionally male trees released most wasps from mature syconia in the dry season, during peak production of receptive female syconia, and thus maximized successful pollination. In F. hispida, “male” trees produced more syconia in the dry and monsoon seasons than in the post-monsoon season. In both species, male and female trees abscised more unpollinated, young inflorescences than pollinated inflorescences, but abscission appeared to be more likely due to resource- rather than pollinator- limitation. The phenology of F. exasperata requires that male inflorescences wait in receptive phase for scarce pollinators to arrive. As expected, male inflorescences of this species had a longer receptive phase than female inflorescences. In F. hispida, where pollinators are rarely scarce, duration of receptive phase was the same for both sexes. Duration of developing phase was longer in female syconia of both species than in male syconia, most likely because they need a longer period of investment in a fleshy fruit. Variation in developing phase of female syconia in one species (F. exasperata) was also greater than that in male syconia, and enabled female trees to sample a variety of germination environments in time. The strong sexual differences in both fig species support the hypothesis that selection for sexual specialization has strongly influenced the reproductive biology of these species. Received: 28 May 1997 / Accepted: 2 February 1998     

Integration of flowering dates in phenology and pollen counts in aerobiology: analysis of their spatial and temporal coherence in Germany (1992–1999)

We studied the possibility of integrating flowering dates in phenology and pollen counts in aerobiology in Germany. Data were analyzed for three pollen types (Betula, Poaceae, Artemisia) at 51 stations with pollen traps, and corresponding phenological flowering dates for 400 adjacent stations (< 25 km) for the years 1992–1993 and 1997–1999. The spatial and temporal coherence of these data sets was investigated by comparing start and peak of the pollen season with local minima and means of plant flowering. Our study revealed that start of birch pollen season occurred on average 5.7 days earlier than local birch flowering. For mugwort and grass, the pollen season started on average after local flowering was observed; mugwort pollen was found 4.8 days later and grass pollen season started almost on the same day (0.6 days later) as local flowering. Whereas the peak of the birch pollen season coincided with the mean flowering dates (0.4 days later), the pollen peaks of the other two species took place much later. On average, the peak of mugwort pollen occurred 15.4 days later than mean local flowering, the peak of grass pollen catches followed 22.6 days after local flowering. The study revealed a great temporal divergence between pollen and flowering dates with an irregular spatial pattern across Germany. Not all pollen catches could be explained by local vegetation flowering. Possible reasons include long-distance transport, pollen contributions of other than phenologically observed species and methodological constraints. The results suggest that further research is needed before using flowering dates in phenology to extrapolate pollen counts.     

Prevention of flower formation in dicotyledons

Prevention of flower formation is important, for example for preventing the spread of transgenes from genetically modified plants or the spread of non-native species, for increasing vegetative growth or preventing the formation of allergenic pollen. The aim of this study was to determine whether flowering of dicotyledonous plants can be prevented by genetic manipulation without harmful effects on vegetative growth. Here we describe isolation of the BpMADS1 gene (similar to SEP3, formerly AGL9) from birch and show that it is expressed only in the inflorescences. In tobacco and Arabidopsis, the expression of BpMADS1::GUS was also virtually inflorescence-specific. Transgenic tobacco and Arabidopsis containing a BpMADS1::BARNASE construct grew well. In one tobacco line the formation of the inflorescence was completely prevented; in several other lines the flowers lacked stamens and carpels and therefore were sterile. The final dry weights of the shoots of the sterile tobacco lines were 140–200% of those of controls. In Arabidopsis, some of the transgenic lines containing the BpMADS1::BARNASE construct formed inflorescences. Some of these lines formed never flowers and some others formed occasionally single fertile flowers. Some other lines did not form inflorescences, but formed up to about one hundred leaves, even in long-day conditions. These results suggest that formation of flowers or inflorescences in widely different dicotyledonous plants could be prevented using the BpMADS1::BARNASE construct and that prevention of flowering may lead to increased vegetative mass.     

Agrobacterium tumefaciens– mediated transformation of embryogenic avocado cultures and regeneration of somatic embryos

Embryogenic avocado cultures were genetically transformed with the uidA (GUS) and nptII genes, and transformed somatic embryos were recovered from these cultures. Embryogenic avocado cultures derived from zygotic embryos of `Thomas' and consisting of proembryonic masses were gently separated and co-cultivated with disarmed, acetosyringone-activated Agrobacterium tumefaciens strain A208, which contained the cointegrative vector pTiT37-ASE::pMON9749 (9749 ASE). Kanamycin-resistant embryogenic suspension cultures were selected in two steps: (1) initial selection in maintenance medium, consisting of MS basal medium, supplemented with 0.1 mg l^–1 picloram and 50 mg l^–1 kanamycin sulfate for 2–4 months and (2) subsequent selection in maintenance medium with 100 mg/ml kanamycin sulfate for 2 months in order to eliminate chimeras. Somatic embryo maturation was initiated by subculture onto semisolid maturation medium (without picloram) followed by transfer to maturation medium with 100 mg l^–1 kanamycin sulfate. Genetic transformation of embryogenic cultures and somatic embryos was confirmed by the X-gluc reaction, and integration of nptII and uidA into the avocado genome was confirmed by PCR and Southern hybridization, respectively. Received: 2 June 1997 / Revision received: 26 September 1997 / Accepted: 11 October 1997     

Comparisons of the efficiency of some promoters in silver birch (Betula pendula)

The efficiency of several promoters (pin2 from potato, ubiquitin from sunflower, rolC from Agrobacterium rhizogenes, act1 from rice and CaMV 35S from cauliflower mosaic virus) fused to the uidA reporter gene was measured after biolistic bombardment of birch leaves (Betula pendula L.). The highest level of β-glucuronidase (GUS) activity was achieved with the pin2 promoter and the lowest activity with the CaMV 35S promoter. The activity of the potato wound-inducible promoter (pin2) was also tested in stably transformed birch. The promoter showed induced activity after mechanical wounding and feeding by leaf weevils. The systemic effect was confirmed by enhanced GUS activity in non-wounded leaves. The results of this study indicated that the potato wound-inducible promoter maintains its function in birch and would be a suitable promoter in studies of insect-birch interaction at the molecular level. Received: 17 October 1996 / Revision received: 7 February 1997 / Accepted: 1 March 1997     

Regeneration of transgenic plants by Agrobacterium-mediated transformation of somatic embryos of juvenile and mature Quercus robur

A protocol was developed for genetic transformation of somatic embryos derived from juvenile and mature Quercus robur trees. Optimal transformation conditions were evaluated on the basis of the results of transient GUS expression assays with five oak embryogenic lines and a strain of Agrobacterium tumefaciens (EHA105) harbouring a p35SGUSINT plasmid containing a nptII and a uidA (GUS) genes. For stable transformation, embryo clumps at globular/torpedo stages (4–10 mg) were inoculated with EHA105:p35SGUSINT bacterial cultures, cocultivated for 4 days and selected in proliferation medium with 75 mg/l of kanamycin. Putatively transformed masses appeared after 20–30 weeks of serial transfers to selective medium. Histochemical and molecular analysis (PCR and Southern blot) confirmed the presence of nptII and uidA genes in the plant genomes. Transformation efficiencies ranged from up to 2% in an embryogenic line derived from a 300-year-old tree, to 6% in a juvenile genotype. Twelve independent transgenic lines were obtained from these oak genotypes, and transgenic plantlets were recovered and acclimatized into the soil. This is the first demonstration of the production of transformed somatic embryos and regenerated plants from juvenile and mature trees of Q. robur and suggests the possibility of introducing other genetic constructions to develop trees that are tolerant/resistant to pathogens and/or biotic stresses.     

The seasonal activity and the effect of mechanical bending and wounding on the PtCOMT promoter in Betula pendula Roth

In this study, 900-bp (signed as p including nucleotides –1 to –886) and partly deleted (signed as dp including nucleotides –1 to –414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th–13th week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin. Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding. The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including nucleotides −415 to −886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees.     

A satellite-based map of onset of birch (<Emphasis Type="Italic">Betula</Emphasis>) flowering in Norway

Birch (Betula pubescens L.) is by far the most common deciduous tree in Norway and birch forests define the forest line both northwards and upwards. Because of its mountainous topography, long fjords, and long length from north to south, Norway is climatically and ecologically very diverse. Therefore, developing pollen forecasts in Norway is a challenging task. In this study we use MODIS-NDVI (normalized difference vegetation index) satellite data with 250 m spatial resolution and 16-days time resolution for the period 2000–2007, and birch pollen counts from ten Burkad traps distributed throughout Norway, to characterize the onset of birch flowering in Norway. Four of the seven trap stations with long-term series show significant values at the 5% level or better between the MODIS-NDVI defined onset and the date when the annual accumulated birch pollen sum reaches 2.5% of the annual total. A map of Norway that shows the eight-year mean (2000–2007) onset of birch flowering was produced. It reveals large differences in the timing of the onset of birch flowering along the north–south and altitude gradients. The map provides useful general information that can be utilized by the Norwegian pollen forecast service. This study shows that remote sensing is a useful tool for not only characterizing the onset of the birch pollen season but also revealing regional differences not easily detected by pollen stations alone.     

Agrobacterium tumefaciens-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants

An efficient system for Agrobacterium-mediated transformation of Eucalyptus camaldulensis and production of transgenic plants was developed. Transformation was accomplished by cocultivation of hypocotyl segments with Agrobacterium tumefaciens containing a binary Ti-plasmid vector harboring chimeric neomycin phosphotransferase and β-glucuronidase (GUS) genes. A modified Gamborg's B5 medium used in this study was effective for both callus induction and regeneration of transgenic shoots. This medium could also effectively maintain the organogenic capability of callus for more than a year. Culturing transgenic shoots in Murashige and Skoog medium supplemented with 0.1 mg ⋅ l^–1 benzylaminopurine prior to root induction in rooting medium markedly increased the rootability of shoots that were recalcitrant to rooting. Histochemical assay revealed the expression of the GUS gene in leaf, stem, and root tissues of transgenic plants. Insertion of the GUS gene in the nuclear genome of transgenic plants was verified by genomic Southern hybridization analysis, further confirming the integration and expression of T-DNA in these plants. Received: 1 August 1997 / Revision received: 11 December 1997 / Accepted: 24 January 1998     

Evidence for stabilising selection acting on flowering time in Arum maculatum (Araceae): the influence of phylogeny on adaptation

The relationship between flowering time and reproductive success was investigated in the fly-pollinated, monoecious perennial herb Arum maculatum L. (Araceae). This species temporarily traps its principle pollinator, a psychodid midge. Probability of fruit set was analysed in relation to early, peak and late periods of the flowering phenology of four British populations between 1992 and 1997. In three out of five cases, plants which flowered during early and late periods were significantly less likely to set fruit. In addition, one population showed a similar relationship for percentage fruit set of individual inflorescences, and seeds from peak-flowering plants were significantly heavier. There was no variation in number of female flowers per inflorescence over the flowering season. Probability of fruit set appears to be mediated by the likelihood of trapping psychodid midges that have previously been trapped and picked up pollen, an unlikely event during early and late flowering periods when few inflorescences are open. The majority of plants in all populations produce only one inflorescence which means that timing of flowering may be crucial to reproductive success. We interpret our findings as evidence that stabilising selection may be acting on some populations and/or during some years. The ultimate cause, however, can be related to the very short (12–18 h) female phase of each inflorescence, a phylogenetically conservative trait within the Araceae. Received: 19 August 1998 / 15 February 1999     

Peculiarities of sexual reproduction inFagus crenata forests in relation to annual production of reproductive organs

Annual production rates of reproductive organs inFagus crenata forests in the lower area of the species' range were studied using 10 litter traps in 1980–1986. The production rates of dispersed pollen were estimated by multiplying the number of fallen male inflorescences per ha per year by the mean amount of pollen per inflorescence before anthesis. Large annual fluctuations in the production rates of male and female inflorescences were recognized, whereas their annual trends were synchronized with each other. Pollen production rates were within the range 1.0–6900 (mean: 1630)×10⁹ha⁻¹ yr⁻¹, the maximum/minimum ratio attaining 7000.F. crenata was the lowest producer of pollen among seven tree species studied: the number of pollen grains equivalent to a single ovule was in the range 6.0–14×10⁴. Furthermore, the mean dry weight of a single pollen grain (3.77×10⁻⁵mg) was higher than for wind-pollinated species. Three factors seemed to cause the low seed fertility ofF. crenata. The dry-matter production rate in the best seed year reached 3252 kg ha⁻¹ yr⁻¹, of which pollen accounted for 259 kg ha⁻¹ yr⁻¹. Unproductive years with less than 10% of the maximum production occurred four times in a 7-yr period. In such years there were fewer male and female inflorescences, and more fruit dropped as a result of insect damage. Lower nut dissemination would play an important role in suppressing any increase in nut predators, and fewer flowers would be produced to avoid wastage of photosynthates in a cool-temperate climate.     

Agrobacterium-mediated transformation and plant regeneration from hypocotyl segments of Japanese persimmon (Diospyros kaki Thunb)

Hypocotyl segments from the seeds of Japanese persimmon (Diospyros kaki Thunb) were cultured on a modified Murashige and Skoog medium supplemented with N-(2-chloro-4-pyridyl)-N′-phenylurea, zeatin or 6-benzylaminopurine. The highest frequency of shoot regeneration was observed when the segments were cultured on medium containing 2 mg/l of zeatin. This culture system was adapted to Agrobacterium-mediated transformation. The hypocotyl segments were inoculated with Agrobacterium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the β-glucuronidase gene. Regenerated shoots were selected on a medium containing kanamycin. Histochemical GUS assay showed that the shoots regenerated from the segments inoculated with EHA101/pSMAK251 expressed the gus gene. The presence and integration of the gus gene was confirmed by polymerase chain reaction (PCR) and Southern blot analysis. The regeneration frequency of transformed shoot was 11.1%. The transgenic shoots were rooted and developed into whole plants within 4–5 months. Received: 18 August 1997 / Revision received: 8 October 1997 / Accepted: 11 November 1997