In vitro fertilization from co-cultured pollen tubes and female gametophytes of Douglas fir (Pseudotsuga menziesii)

 Our previous attempt on in vitro fertilization (IVF) in conifers resulted in pollen tube penetration of female gametophytes, but because of the rapid decline in egg viability, no further interaction occurred. In this report, we describe for the first time that IVF has been achieved in conifers. Using Douglas fir (Pseudotsuga menziesii), we describe a two-step process which involved induction of pollen tubes in culture followed by introduction of isolated female gametophytes at the tips of growing pollen tubes. Pollen tubes penetrated the introduced isolated female gametophytes at various places, but a number of tubes entered the egg cell through the neck cells similar to the in vivo condition. Under our current culture conditions, longevity of pollen tubes and eggs has been improved resulting in the release of sperms, fusion of gametes, and initial formation of the proembryo. Continued plasmolysis of the egg limited the number of successful gametic interactions. IVF has been accomplished in flowering plants in several ways, but the gametophyte-gametophyte IVF system described in this paper is unique. IVF offers a novel breeding technology that takes advantage of the sexual reproductive route. When coupled with hybridization and genetic transformation, IVF could result in the development of stable novel genotypes of economically superior trees. Received: 28 October 1997 / Accepted: 9 December 1997     

Genetic variation for in vitro sesame pollen germination and tube growth

  In vitro pollen germination and tube length studies are valuable in elucidating mechanisms (germination capacity and rate, tube growth rate) possibly associated with genetic differences in male transmission. On each of two collection dates, the percentage germination and tube length of the binucleate pollen grains from five diverse sesame (Sesamum indicum L.) genotypes were determined at eight times (30, 60, 90, 120, 150, 180, 240, 300 min) after inoculation on a semisolid medium containing 10% (100 g l^-1) sucrose (C₁₂H₂₂O₁₁), 0.4% (4 g l^-1) purified agar (Fisher Lot 914409), 0.1% (1 g l^-1) calcium nitrate [Ca(NO₃)₂ ⋅ 4H₂O] and 0.01% (100 mg l^-1) boric acid (H₃BO₃). Before heating, the pH of the medium was adjusted to 7.0 with a 0.1 N potassium hydroxide (KOH) solution. Over the five genotypes, 5% germination was found 30 min after inoculation and a maximum of 37% germination 120 min after inoculation with no significant changes thereafter. As indicated by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which germination was initiated and maximum germination attained. Over all five genotypes, the tube length was 91 μm 30 min after inoculation, reaching a maximum of 1000 μm 300 min after inoculation. As shown by the highly significant genotype×time after inoculation interaction, the genotypes differed in the time at which tube length was observed and the maximum tube length was attained. Little or no relationship between percent germination and tube length was observed among the genotypes. For both percent germination and tube length, the statistical significance of collection date and its interactions with genotype and time after inoculation indicated that environment in the form of collection date was also an influencing factor. These results indicated that genetic differences among genotypes were present for in vitro germination capacity, germination rate and tube growth rate and that these factors singly or in combination could alter male transmission of genetic elements. Received: 5 February 1997 / Accepted: 23 June 1997     

Ovary signals for directional pollen tube growth

In angiosperms, the female gametophyte has a secluded life; it is protected by several concentric layers that envelop each other. The embryo sac is surrounded by the nucellus, which in turn is wrapped by the integuments forming the ovule, which is nested in the ovary. These wrappings are not hermetic, but contain little ”gates” the pollen tube must traverse on its way towards the embryo sac. Information is emerging that shows that the ovary and ovule provide signals orienting and directimng the pollen tube on the right course. There are three main bodies of evidence supporting this hypothesis. One relates to developmental changes in the female tissues and how they affect pollen tube growth. The second refers to defective ovule mutants, which induce defective pollen tube guidance. And the third relates to the possible molecules involved in this signalling. Here, information gathered along these three main lines of evidence is reviewed. All converge to the conclusion that different checkpoints exist all along the pollen tube pathway. These checkpoints provide active signalling that guides the pollen tube to its destination, the embryo sac. Received: 15 December 2000 / Accepted: 13 June 2001     

Ion dynamics and hydrodynamics in the regulation of pollen tube growth

A coherent picture of pollen tube growth is beginning to emerge that couples ion dynamics with biochemical, biophysical and cytological processes in ordered and controlled feedback circuits that define the nature of polarized apical growth. It is a paradox, however, that complete understanding of the mechanical forces that drive cell elongation in this system still remains to be fully achieved. The results of our recent studies to characterize Cl^– ion dynamics during apical growth in tobacco pollen tubes led us to re-examine this question in the light of a possible force-generating role provided by hydrodynamic flow. Previously we found that oscillatory Cl^– efflux from the apex is closely coupled to oscillatory growth and the cell volume of the apical domain. Cl^– influx occurs in a region of the tube that is distal to the clear zone; hence, a vectorial flow of anion traverses the apical domain and fluxes out of the tip with oscillatory dynamics. Because of the effects that this could induce on charge and osmotic potentials, water could potentially flow through the apical domain, linked to the flux of Cl^–. This conjecture is consistent with studies in other plant cells that demonstrate a pivotal role for flux through anion channels in the control or normalization of osmotic status. In the current report, the relationship between Cl^– efflux oscillations and the physical characteristics of the apical dome during oscillatory growth is examined in closer detail. Evidence is presented that shows a cyclic deformation of the extreme apex occurs during the growth pulse and is correlated with cyclic Cl^– efflux. In addition, there is a dramatic increase in the number and density of clear thread-like zones traversing the apical plasma membrane during the process of tip elongation. Possible functional roles of Cl^– flux and hydrodynamics are discussed in the context of what drives tip elongation during cycles of pollen tube growth. Received: 23 November 2000 / Revision accepted: 19 June 2001     

Morphotype community structure of ectomycorrhizas on Douglas fir (Pseudotsuga menziesii Mirb. Franco) seedlings grown under elevated atmospheric CO2 and temperature

Mycorrhizas alter the acquisition of carbon and nutrients, thereby affecting numerous plant and ecosystem processes. It is important, therefore, to determine how mycorrhizal populations will change under possible future climate conditions. Individual and interactive effects of elevated atmospheric CO₂ concentration and atmospheric temperature were assessed in a 2×2 factorial design [ambient and elevated (200 ppm above ambient) CO₂ concentrations, and ambient and elevated (4°C above ambient) temperatures]. In June 1993, 2-year-old Douglas fir (Pseudotsuga menziesii Mirb. Franco) seedlings were planted in 12 environment-tracking chambers (n=3) containing reconstructed, low-nitrogen, native forest soil. Climate treatments were imposed shortly thereafter, and the seedlings grew until June 1997. Soil cores were taken twice per year during the exposure period. We present findings on changes in the community structure of ectomycorrhizal (ECM) root tips, categorized into morphotypes using gross morphological traits. A diverse and stable community of morphotypes (a total of 40) was encountered; no more than 30 of which were seen at any sampling time. In the first sample, there were only 15 morphotypes found in the 12 chambers. Morphotype numbers increased during the first half of the experiment, remaining fairly constant thereafter. Near the end of the exposure, elevated-temperature treatments maintained more morphotypes than ambient treatments. However, overall, absolute measures (number of ECM tips) were affected primarily by CO₂ treatment, whereas proportional measures (e.g., Simpson’s index) were affected primarily by temperature. While some morphotypes were negatively affected seasonally by higher temperatures (putative Rhizopogon group), others (Cenococcum) seemed to thrive. Underlying the dominant patterns of change in diversity, driven by the Rhizopogon group, subdominant populations responded slightly differently. Community diversity through time tended to increase at a greater rate for all subdominant populations compared with the rate when dominant populations were included. Received: 16 August 1999 / Accepted: 2 March 2000     

Detection and identification of gibberellins in Douglas fir (Pseudotsuga menziesii) shoots

Extracts of Douglas fir ( Pseudotsuga menziesii [Mirb.] Franco) shoots were purified by reversed and normal phase HPLC; gibberellin (GA)-like compounds detected by radioimmunoassay with antibodies against GA₄ and the Tan-ginbozu dwarf rice micro-drop biossay were analyzed by GC-MS. Three major components were identified as GA₄, GA₇, and GA₉ while smaller amounts of GA¹, GA₃ and putative GA⁹-glucosyl ester were also present.     

Effects of pollen competitive environment on pollen performance in Mirabilis jalapa (Nyctaginaceae)

 We examined the influence of pollen competitive environment on pollen performance in Mirabilis jalapa. We used the number of pollen grains and the number of pollen tubes per pistil as measures of pollen competition. Pollen germination, pollen tube penetration into the style, and pollen tube growth rates were used as measures of pollen performance. All three measures of pollen performance were affected by the competitive environment. Pollen germination was greatest at intermediate pollen load sizes. The percentage of germinated pollen grains that penetrated the stigma and grew into the style decreased with pollen load size. Pollen tube growth rate in the style was greater and more variable with larger numbers of pollen tubes in the style. Controlling for the degree of selection at the stigma indicated that pollen-pollen or pollen-style interactions were the likely causes of increased growth rates. Received: 28 October 1996 / Revision accepted: 24 January 1997     

Observations of pollen tube growth in Nicotiana alata and their implications for the mechanism of self-incompatibility

 Style squashes and stylar grafts were used to examine the growth of Nicotiana alata pollen tubes in self-compatible and self-incompatible styles. Compatible tubes typically showed a uniform layer of callose deposition in the walls and in small plugs spaced at regular intervals within the tube. Incompatible tubes were characterised by the variability of callose deposition in the walls and by larger, closer and more irregularly spaced plugs. There was no difference in the growth rate of compatible and incompatible tubes during growth through the stigma, but within the style most compatible tubes grew 20–25 mm day^-1 (maximum 30 mm day^–1), whereas incompatible tubes grew 1.0–1.5 mm day^-1 (maximum 5 mm day^–1). Many incompatible tubes continued to grow until flowers senesced, and only a small proportion died as a consequence of tip bursting. Grafting compatibly pollinated styles onto incompatible styles showed that the incompatible reaction could occur in pollen tubes between 2 and 50 mm long, and that inhibition of pollen tube growth occurred in both the upper and lower parts of the transmitting tract. Grafting incompatibly pollinated styles onto compatible styles showed that the incompatible reaction was fully reversible in at least a proportion of the pollen tubes. The findings are not consistent with the cytotoxic model of inhibition of self-pollen tubes in solanaceous plants, which assumes that the incompatible response results from the degradation of a finite amount of rRNA present in the pollen tube. However, if pollen tubes do in fact synthesise rRNA, the findings become consistent with this model. Received: 23 May 1996 / Revision accepted: 22 August 1996     

In vitro double fertilization in Nicotiana tabacum (L.): fusion behavior and gamete interaction traced by video-enhanced microscopy

In vitro double fertilization in tobacco was carried out with attention to fusion behavior and gamete interaction. Structural and cytological events indicating possible reaction to the fusion of sperm-egg and especially sperm-central cell were recorded by video-enhanced microscopy. Generative cells were fused with the egg cell or central cell as a control system to better understand gamete interaction. As early as adherence of the male cell, the female cell showed response by means of cytoplasm strand formation. After gamete fusion, cytoplasm activation in the egg cell was observed as long distance movement of organelles. In fertilized central cells, however, fusion did not result in notable cytological change within 30 min. Male nuclear movement recorded in the female cell illustrated two different patterns of movement which showed similarity to organelle movement. The dynamics of male and female nuclear fusion after in vitro fertilization was also recorded in the central cell. It revealed that the fusion process requires only a few seconds and is similar to that of gamete fusion in vitro. This may offer a new clue for understanding how female and male nuclei attract, adhere and finally fuse each other. Received: 13 October 1999 / Revision accepted: 6 December 1999     

Germination and early tube development in vitro of Lycopersicum peruvianum pollen: Ultrastructural features

Morphologic changes occurring during pollen grain activation and ultrastructural features of Lycopersicum peruvianum Mill. pollen tube during the first stages of growth in vitro have been studied. The more evident morphologic changes during activation, in comparison to those already described for mature inactive pollen, concern dictyosomes, rough endoplasmic reticulum (RER), and ribosomes. The dictyosomes are very abundant and produce large and small vesicles. Near the germinative pores both types of vesicles are present, while all along the remaining cell wall only the large type is observed. These latter react weakly to Thiéry's test and probably contain a callose precursor necessary for the deposition of a callosic layer lining at first only the inner side of the functioning pore and occasionally the other two pores, and subsequently the entire inner surface of the cell wall. The small vesicles, highly positive to Thiéry's test, are present only near the pores and could be involved in the formation of the pectocellulosic layer of the tube wall. The setting free of RER cisterns, which in the mature inactive pollen were aggregated in stacks, coinciding with polysome formation and resumption of protein synthesis, is in accord with the hypothesized role of RER cistern stacks as a reserve of synthesizing machinery. The pollen tube reaches a definitive spatial arrangement soon after the generative cell and vegetative nucleus have moved into it. At this stage four different zones that reflect a functional specialization are present. In the apical and subapical zone two types of dictysosome-originated vesicles, similar to those found in the activated pollen grain, are present. Their role in the formation of the callosic and pectocellulosic wall layers seems to be the same as in the activated pollen grain.Abbreviations ER endoplasmic reticulum - RER rough endoplasmic reticulum Research performed under CNR program Biology of Reproduction     

Dynein-related polypeptides in pollen and pollen tubes

 Microtubules in pollen tubes are evident within the vegetative and generative cell cytoplasm. This observation led to the formulation of several hypotheses regarding the role of microtubules in cytoplasmic movement and the migration of the vegetative nucleus/generative cell along the pollen tube. The study of microtubular motor proteins in pollen tubes followed the discovery and characterization of an immunoreactive homolog of mammalian kinesin in tobacco pollen tubes. Recent identification of dynein-related polypeptides in pollen tubes of Nicotiana tabacum and pollen of Ginkgo biloba is a significant step in the definition of the role of microtubule function within pollen and pollen tubes. Received: 31 May 1996 / Revision accepted: 26 July 1996     

Structural aspects of in vitro pollen tube growth and micropylar penetration inGasteria verrucosa (Mill.) H. Duval andLilium longiflorum Thunb.

Summary In vitro penetration of the micropyle of freshly isolatedGasteria verrucosa ovules by pollen tube was monitored on agar medium. 40–60% of the micropyles were penetrated, comparable with in vivo penetration percentages. When germinated on agar,Gasteria pollen tube elongation lasts for up to 8 h while plasma streaming continues for about 20–24 h. The generative cell divides between 7 and 20 h after germination, and after 20 h the pollen tube arrives at one of the synergids. The sperm cells arrive after 22 h. The whole process takes more time in vitro than in vivo. In fast growing pollen tubes, a pulsed telescope-like growth pattern of tube elongation is observed. The formation of pollen tube wall material precedes tube elongation and probably prevents regular enlargement of the pollen tube tip-zone. Rapid stretching of the new pollen tube wall material follows, probably due to gradually increased osmotic pressure and the use of lateral wall material below the tip. The stretching ceases when the supplies of plasma membrane and excretable wall material are exhausted. Multiple pollen tube penetration of the micropyle occurs in vitro as it does in vivo. Most pollen tube growth ceases within the micropyle but, if it continues, the pollen tubes curl. Inside the micropyle the pollen tube shows haustorial growth. At the ultrastructural level, the wall thickening of in vitro pollen tubes is quite similar to that in vivo. Before transfer of pollen tube cytoplasm a small tube penetrates one of the synergids. Sperm nuclei with condensed chromatin are observed in the pollen tube and the synergid. In vivo prometaphase nuclei are found in the most chalazal part of a synergid, against the egg cell nucleus and nucleus of the central cell at a later stage. Using media forLilium ovule culture,Gasteria ovules were kept alive for at least 6 weeks. Swelling of the ovule depends on pollen tube penetration. The conditions for fertilization to occur after in vitro ovular pollination seem to be present.     

Micromanipulation and in vitro fertilization with single pollen grains of maize

Summary The manipulation of single pollen grains of maize was studied. The effects of delivering substances both locally to the grain wall, tube or tip by a microcapillary and directly into the pollen grain by microinjection, and single grain pollination were investigated. Germination was induced by adding small amounts of water locally to the grains with either a microcapillary or with a waterdelivering emulsion without any other ingredients in the medium. The grains were overlayered by mineral or silicone oil so that tube growth proceeded without the grains bursting. There was no apparent penetration of high-molecular-weight substances (FITC-dextran, ethidium bromide labelled DNA) into the living grain either before or after pollination. Neither could the penetration of these substances be detected in both dry, viable and hydrated grains, tubes and tube tips, with or without treatment with Triton X-100 and dimethyl sulfoxide. By microinjection, however, the delivery of high-molecular-weight substances into grains was possible. Such injected grains successfully pollinated stigmas of cultured ear segments. Pollination with pore-injected grains was most efficient (mean 26%). No difference in fertilization rates between mass pollination (mean 41%) and single grain pollination (mean 39%) could be found. A mean fertilization rate of 29% could be obtained after microinjection. Seedlings developed 3 weeks after being pollinated by means of the in vitro pollination and fertilization method.     

Root architecture,early development and basal sweep in containerized and bare-rooted Douglas fir (Pseudotsuga menziesii)

Seedlings grown in containers often have deformed, spiralling, kinking root systems (reported especially in pine growing in colder climates like Fenno-Scandinavia). The current study examines the effect of containers on the root systems of Douglas fir planted in Ireland. A sample of sixteen 10-year-old Douglas fir trees, planted as either bare-rooted transplants (2/1) or containerized seedlings (Paperpot 610 (2/0)) on an acid brown earth site in Ireland, were excavated for root achitectural studies. In addition to thorough above-ground measurements, an assessment of basal sweep was also carried out. Root systems were systematically described and horizontal angles, cross-sectional areas (CSA), and maximum depths of all roots>5 mm in diameter were recorded. Various biomass ratios and estimates for dominant roots were also calculated. Symmetry of the root systems, max. and min. numbers, and CSA of roots for sectors of various sizes were compared for the two plant types. The initially (when planted) bigger bare-rooted transplants were still larger than the container-planted trees after ten years. Of the total above-ground fresh weight, the container stock had allocated more to the crown and less to the stem compared to bare-roots. The difference in root weights between stock types was small. Basal sweep occurred on average in 50% of the bare-roots and 35% of the container trees. The direction of the basal sweep leaning was concentrated to the NE, which coincided with the direction of the slope and the prevailing wind direction. No effect of planting position in relation to the direction of site preparation was found for basal sweep. The size of the root system, fresh weight and total cross-sectional area was on average for all trees correlated to both DBH and shoot fresh weights. For containers separately, however, only root area was correlated to DBH and stem fresh weight. In spite of the difference in the above-ground size, there were no significant differences in root numbers and root area (CSA) between the two plant types. No difference in rooting depth between plant types was found. When splitting the root system into 120° horizontal sectors (1/3 of the root system) the highest numbers of roots were concentrated in the NW direction. The highest amount of root area tended to be concentrated along a NW-SE diagonal, with a dominance for the latter (SE). Sectors without dominant roots (expected to be the future main structural, stabilizing roots), varied in size (94–178°) but were on average wider in bare-roots and on average concentrated in the NE direction and the downhill side of the slope. This revised version was published online in June 2006 with corrections to the Cover Date.     

Dose effect of UV-B irradiation on pollen tube growth and seed-specific promoter activities in irradiated pollen grains of Nicotiana plumbaginifolia

Low doses of UV-B irradiation applied to mature Nicotiana plumbaginifolia pollen grains stimulated pollen tube growth. The most pronounced effect was achieved after 1.5 min of irradiation. Using transgenic N. plumbaginifolia plants expressing the GFP reporter gene under the control of the seed-specific promoters USP (unknown seed protein) or LegB4 (legumin B4) genes, it was shown that these promoters are also inducible by UV-B irradiation of the pollen grains. The improvement of pollen viability and germination by UV-light is discussed with respect to effects on plant flowering and reproduction. Received: 10 November 1999 / Revision accepted: 14 February 2000     

In vitro double fertilization in Nicotiana tabacum (L.): the role of cell volume in cell fusion

The role of cell volume in regulating cell fusion was investigated by in vitro fertilization to elucidate mechanisms involved in double fertilization. The results revealed that in our model gamete fusion was more efficient than the fusion of somatic cells under the same conditions. The fusion of selected mesophyll protoplasts of different sizes and their fusion with chloroplasts demonstrated that cell volume ratio played a role in this process: as the ratio increased, fusion was more efficient and faster. When one of the cells was as small as a sperm, the formation of a round fusion product was faster. This might explain why gamete fusion was highly efficient in all in vitro germ cell fusion systems. This finding may also explain why sperm evolved as small cells. The results reported here will be useful for interpreting and evaluating data of in vitro fertilization experiments and for distinguishing gamete-specific characters. Received: 12 September 2000 / Revision accepted: 6 December 2000     

Effects of water stress on male gametophyte development in plants

 Male reproductive development in plants is highly sensitive to water deficit during meiosis in the microspore mother cells. Water deficit during this stage inhibits further development of microspores or pollen grains, causing male sterility. Female fertility, in contrast, is quite immune to stress. The injury is apparently not caused by desiccation of the reproductive tissue, but is an indirect consequence of water deficit in the vegetative organs, such as leaves. The mechanism underlying this stress response probably involves a long-distance signaling molecule, originating in the organs that undergo water loss, and affecting fertility in the reproductive tissue, which conserves its water status. Much research has been focused on the involvement of abscisic acid in this regard, but the most recent evidence tends to reject a role for this hormone in the induction of male sterility. Stress-induced arrest of male gametophyte development is preceded by disturbances in carbohydrate metabolism and distribution within anthers, and an inhibition of the key sugar-cleaving enzyme, acid invertase. Since invertase gene expression can be modulated by sugar concentration, it is possible that decreased sugar delivery to reproductive tissue upon inhibition of photosynthesis by stress is the signal that triggers metabolic lesions leading to failure of male gametophyte development. Received: 31 October 1996 / Revision accepted: 18 February 1997