Effects of relative humidity and temperature conditions on pollen fluorochromatic reaction of Rosmarinus officinalis L. (Lamiaceae)

Summary. Mediterranean ecosystems are characterized by seasonal and annual fluctuations in humidity and temperature which are considered limiting factors for plant growth and might have played a key role in the selection of species that compose the present vegetation. After anther release, pollen is generally exposed to various changes of temperature and humidity conditions, therefore its viability and consequently successful fruit set are strongly affected by these environmental parameters. The aim of this research was to study the effect of different combinations of humidity and temperature on pollen membrane integrity of Rosmarinus officinalis L. in order to investigate possible relations between pollen features and climatic conditions during flowering. This species is an evergreen perennial shrub, occurring among the sclerophyllous vegetation of Mediterranean maquis. In many areas of Southern Italy, it shows a flowering period starting from the beginning of winter and spreading throughout spring months. The analysis of data showed that duration of pollen viability of R. officinalis is enhanced by the co-occurrence of low temperature and high humidity. Moreover, these conditions are able to newly raise the viability of pollen kept at higher temperatures and lower humidity. These observations indicate that reactivation of the pollen membrane depends on low temperature apart from high humidity. Therefore “vernalization” of rosemary pollen enhances its viability, supporting that pollen behavior is adapted to winter condition and allows flowering in winter and early spring. Correspondence and reprints: Laboratorio di Botanica ed Ecologia Riproduttiva, Dipartimento di Arboricoltura, Botanica e Patologia Vegetale, Università degli Studi di Napoli Federico II, Via Università 100, 80055 Portici, Naples, Italy.     

Growth,yield and reproduction of dwarf tomato grown under simulated microgravity conditions

Abstract

A closed hydroponic system combined with a horizontal uniaxial clinostat has been used to grow tomato plants (Solanum lycopersicum L.) under simulated microgravity conditions. The study was carried out to evaluate the quanti-qualitative traits (growth, yield and quality) of the dwarf tomato variety ‘Micro-Tom’ grown under simulated microgravity conditions and to determine if tomato plants would complete their life cycle (‘seed-to-seed’). Morphological and growth characteristics of ‘Micro-Tom’ were modified during clinorotation treatment. The ‘Micro-Tom’ plants grown under simulated microgravity exhibited a spreading growth and an increasing of the internode length. Total fruit yield, small fruit yield, leaf area, leaf dry weight, fruit dry weight, total dry weight and shoot – root ratio were lower in the clinorotated tomato plants than those grown in the control treatment. Foliar amount of carotenoids, and chlorophyll a and b were also substantially reduced under simulated microgravity conditions. Quality parameters (total soluble solids and fruit dry matter) of tomato plants were also negatively affected by clinorotation. The number of flowers per plant was increased by 32% in clinorotated plants versus controls. Fruit setting was reduced by 46% under clinorotation, while no significant difference was recorded for the pollen fertility and the seed number in small and large fruits. Clinorotation-exposed and control seeds were used in a germination trial in order to evaluate whether the seeds so formed were viable and if subsequent generations might be obtained in microgravity. Seeds formed under simulated microgravity proved to be biologically and functionally complete (germination = 78.6%) showing that ‘Micro-Tom’ plants could realize complete ontogenesis, from seed to seed in microgravity.     

Growth and Development in Arabidopsis thaliana through an Entire Life Cycle under Simulated Microgravity Conditions on a Clinostat

Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin-formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes. Received 18 June 1999/ Accepted in revised form 27 August 1999     

Changes in plant growth processes under microgravity conditions simulated by a three-dimensional clinostat

We developed a three-dimensional (3-D) clinostat to simulate a microgravity environment and studied the changes in plant growth processes under this condition. The rate of germination of cress (Lepidium sativum), maize (Zea mays), rice (Oryza sativa), pea (Pisum sativum), or azuki bean (Vigna angularis) was not affected on the clinostat. The clinostat rotation did not influence the growth rate of their roots or shoots, except for a slight promotion of growth in azuki roots and epicotyls. On the contrary, the direction of growth of plant organs clearly changed on the 3-D clinostat. On the surface of the earth, roots grow downward while shoots upward in parallel to the gravity vector. On the 3-D clinostat, roots of cress elongated along the direction of the tip of root primordia after having changed the direction continuously. Rice roots also grew parallel to the direction of the tip of root primordia. On the other hand, roots of maize, pea, and azuki bean grew in a random fashion. The direction of growth of shoots was more controlled even on the 3-D clinostat. In a front view of embryos, shoots grew mostly along the direction of the tip of primordia. In a side view, rice coleoptiles showed an adaxial (toward the caryopsis) while coleoptiles of maize and epicotyls of pea and azuki bean an abaxial curvature. The curvature of shoots became larger with their growth. Such an autotropism may have an important role in regulation of life cycle of higher plants under a microgravity environment.     

REGULATION OF THE TIMING OF POLLEN GERMINATION BY THE PISTIL IN TALINUM MENGESII (PORTULACACEAE)

In angiosperms selection among male gametophytes may take place between the time a flower is pollinated and the time its ovules are fertilized. With insect pollination, a surplus of male gametophytes in the form of pollen grains may be deposited on the stigma of a pistil and this provides the potential for gametophytic competition and selection. In all plants examined from two Talinum mengesii populations, pollen germination was delayed for up to two hours after pollination. In plants of two other populations pollen germination was not delayed or the time of germination varied. Reciprocal pollinations between plants with and without the delay trait and pollinations at different times during anthesis revealed that the timing of pollen germination was regulated by the maternal parent. Theoretically, the effect of the delay trait would be to promote the accumulation of pollen on the stigma and induce simultaneous germination. This would intensify intermale competition and provide a mechanism for the maternal parent to influence the paternal parentage of its offspring.     

Growth and development, and auxin polar transport of transgenic Arabidopsis under simulated microgravity conditions on a three-dimensional clinostat.

Growth and development, and auxin polar transport in Arabidopsis thaliana transformed with iaaH gene were studied under simulated microgravity conditions on a three-dimensional (3-D) clinostat. Simulated microgravity conditions on a 3-D clinostat did not affect the number of rosette leaves but promoted the growth and development (fresh weight of plant and the elongation of flower stalk) of transformants. Final growth of transformants under simulated microgravity conditions on a 3-D clinostat was almost equivalent to that grown on 1 g conditions in the presence of 1 micromoles IAM (indole-3-acetamide). The activities of auxin polar transport in the segments of flower stalk (inflorescence axis) of transformants grown on 1 g conditions were significantly promoted by the addition of IAM. Interestingly, simulated microgravity conditions on a 3-D clinostat also promoted the activities of auxin polar transport of transformants grown on the medium with or without IAM. Based on the results in this study, transgenic plants may not have an efficient homeostatic mechanism for the control of growth and development, and auxin polar transport activity in microgravity conditions in space.     

胀果甘草花粉生活力测定及甘草的杂交育种

杂交育种是最为传统的选育新品种的方式之一,花粉生活力能够影响其中遗传物质的传递。胀果甘草是3种药典收录的甘草之一,具有许多特殊活性成分。通过TTC法和离体萌发法对2个胀果甘草(Glycyrrhiza inflata)种质资源GJJ-7和GJJ-9花朵开放5个时期(a~e)的花粉生活力进行测定。结果表明,GJJ-7花粉生活力趋势在5个时期呈现波浪型,在a、d时期达到最高;GJJ-9花粉生活力会随着花瓣展开度的变大而升高,在e时期达到最高。离体萌发结果表明GJJ-7和GJJ-9花粉生活力随花朵开放程度增大呈先升高后降低的趋势,萌发力均在c时期最高。杂交结荚率则与离体萌发结果一致,说明离体萌发法测定花粉生活力比TTC法更可靠。以光果甘草(G.glabra) S-7和S-12为母本的杂交组合结荚率低,但能够获得发芽率高的种子;以乌拉尔甘草(G.uralensis) GDN-16为母本的杂交组合结荚率高,但种子发芽率低,说明在甘草杂交中父母本的选择对结荚率有重要影响。     

Growth and development in Arabidopsis thaliana through an entire life cycle under simulated microgravity conditions on a clinostat.

The effects of simulated microgravity conditions produced by a horizontal clinostat on the entire life cycle of Arabidopsis thaliana ecotype Columbia and Landsberg erecta were studied. Horizontal clinorotation affected little germination of seeds, growth and development of rosette leaves and roots during early vegetative growth stage, and the onset of the bolting of inflorescence axis and flower formation in reproductive growth stage, although it suppressed elongation of inflorescence axes. The clinorotation substantially reduced the numbers of siliques and seeds in Landsberg erecta, and completely inhibited seed production in Columbia. Seeds produced in Landsberg erecta on the clinostat were capable of germinating and developing rosette leaves normally on the ground. On the other hand, growth of pin formed mutant (pin/pin) of Arabidopsis ecotype Enkheim, which has a unique structure of inflorescence axis with no flower and extremely low levels of auxin polar transport activity, was inhibited and the seedlings frequently died during vegetative stage on the clinostat. Seed formation and inflorescence growth of the seedlings with normal shape (pin/+ or +/+) were also suppressed on the clinostat. These results suggest that the growth and development of Arabidopsis, especially in reproductive growth stage, is suppressed under simulated microgravity conditions on a clinostat. To complete the life cycle probably seems to be quite difficult, although it is possible in some ecotypes.     

Effect of enhanced UV-B radiation on pollen quantity,quality, and seed yield in Brassica rapa (Brassicaceae)

We conducted three experiments to examine the influence of ultraviolet-B radiation (UV-B; 280–320 nm) exposure on reproduction in Brassica rapa (Brassicaceae). Plants were grown in a greenhouse under three biologically effective UV-B levels that simulated either an ambient stratospheric ozone level (control), 16% (“low enhanced”), or 32% (“high enhanced”) ozone depletion levels at Morgantown, WV, USA in mid-March. In the first experiment, we examined whether UV-B level during plant growth influenced in vivo pollen production and viability, and flower production. Pollen production and viability per flower were reduced by ≈50% under both enhanced UV-B levels relative to ambient controls. While plants under high-enhanced UV-B produced over 40% more flowers than plants under the two lower UV-B treatments, whole-plant production of viable pollen was reduced under high-enhanced UV-B to 17% of that of ambient controls. Whole-plant production of viable pollen was reduced under low-enhanced UV-B to 34% of ambient controls. In the second experiment, we collected pollen from plants under the three UV-B levels and examined whether source-plant UV-B exposure influenced in vitro pollen germination and viability. Pollen from plants under both enhanced-UV-B treatments had initially lower germination and viability than pollen from the ambient level. After in vitro exposure to the high-enhanced UV-B levels for 6 h, viability of the pollen from plants grown under ambient UV-B was reduced from 65 to 18%. In contrast, viability of the pollen from plants grown under both enhanced UV-B treatments was reduced to a much lesser extent: only from ≈43 to 22%. Thus, ambient source-plant pollen was more sensitive to enhanced UV-B exposure. In the third experiment, we used pollen collected from source plants under the three UV-B levels to fertilize plants growing under ambient-UV-B levels, and assessed subsequent seed production and germination. Seed abortion rates were higher in plants pollinated with pollen from the enhanced UV-B treatments, than from ambient UV-B. Despite this, seed yield (number and mass) per plant was similar, regardless of the UV-B exposure of their pollen source. Our findings demonstrate that enhanced UV-B levels associated with springtime ozone depletion events have the capacity to substantially reduce viable pollen production, and could ultimately reduce reproductive success of B. rapa.     

Pollen biology in four Mediterranean Quercus species

Potential pollen production, viability and germination were studied in the most important species of Quercus in the mountains of Córdoba to determine the contribution of each species to the total amount of airborne pollen. The results were compared over two consecutive years with different rainfall patterns. The viability of pollen grains was determined at anther opening, and during the pollination period, in order to determine potential pollination capacity. Results indicated that there were differences in the number of pollen grains produced by stamen in the four species. Equally, there were differences in the number of flowers among the species, being Q. suber the species with higher number of catkins groups and flowers per individual tree. Total pollen production per tree can be summarized in the following proportion: 1: 3: 3: 6 (Q. coccifera, Q. ilex ssp. ballota, Q. faginea and Q. suber). Potential pollen viability was estimated using the Fluorochromatic Reaction (FCR) and a germination assay. The results have shown that Quercus potential pollen viability is high and declines slowly with time. Q. coccifera was the species with the highest percentage of germination, with Q. suber being the lowest.     

EXPERIMENTAL NON-STIGMATIC POLLINATIONS IN CLARKIA UNGUICULATA LINDL. (ONAGRACEAE)

Within angiosperms, the stigma-style apparatus provides a barrier to direct contact between ovules and the external environment. In traversing this barrier, male gametophytes presumably are subject to intense competition and natural selection. Successful fertilizations and resultant seed set can be accomplished following surgical decapitation of the stigma and style, provided that these normally receptive sites are replaced with an optimal environment suitable for pollen germination and growth. Unlike other methodologies, these in situ-in vitro pollinations are comparatively easily accomplished, and provide a means for circumventing natural fertility barriers; additionally, they may provide a simple means of artificially selecting pollen genomes. In Clarkia unguiculata 23.0% of attempted non-stigmatic pollinations produced outcrossed F₁ progeny.     

Molecular cloning,functional expression and characterization of two serine/threonine-specific protein kinases from<Emphasis Type="Italic"> Nicotiana tabacum</Emphasis> pollen

The existence of different kinds of kinases in pollen and pollen tubes suggests that kinase-mediated signaling pathways are likely involved in regulating pollen germination and pollen tube growth during the life cycle of higher plants. We have used RT-PCR and RACE to isolate full-length cDNAs for two pollen-expressed kinases, named NtPK1 and NtPK2, of Nicotiana tabacum. NtPK1 and NtPK2 encode proteins of 365 and 369 amino acids with calculated molecular masses of 39.2 kDa and 39.5 kDa, respectively, and both proteins possess the 12 sub-domains that are conserved among protein kinases. The nucleotide and deduced amino acid sequences of NtPK1 and NtPK2 share 88% and 91% identity, respectively, with the C-terminal region being the most conserved. RT-PCR analysis revealed that NtPK1 was specifically expressed in pollen and pollen tubes, and that NtPK2 was also expressed in pistil and petal. Immunoblot analysis using anti-NtPK1 and anti-NtPK2 antibodies confirmed that both NtPK1 and NtPK2 were produced in pollen and pollen tubes, and that NtPK2 was also produced in developing male gametophytes and other floral tissues. Biochemical fractionation experiments showed that, in all the tissues examined, NtPK1 and NtPK2 were present in the cytosolic fraction and not in the microsomal fraction. NtPK1 and NtPK2 were found to autophosphorylate on threonine and, for NtPK2, on serine as well. All the results taken together suggest that NtPK1 and NtPK2 are novel receptor-like cytosolic serine/threonine kinases, and could mediate signaling pathways required for pollen germination and/or pollen tube growth.The nucleotide sequence data of NtPK1 and NtPK2 reported in this paper will appear in the EMBL/GenBank/DDBJ nucleotide sequence databases under the accession numbers AJ608156 and AJ608157, respectively     

Control of morphogenesis of woody plant by gravity on earth]

Using the weeping branch of Japanese flowering cherry tree and its woody stem of the seedling grown under simulated microgravity condition by three dimensional clinostat, it was elucidated that the morphogenesis of its secondary xylem supporting the plant itself to grow upward is seriously controlled by gravity on earth with a sedimentable amyloplast as its sensor. Space experiment of woody plant is expected to elucidate such problem.     

In vitro pollen tube growth and penetration of female gametophyte in Douglas fir (Pseudotsuga menziesii)

 Pollen tube and female gametophyte interactions in Douglas fir (Pseudotsuga menziesii) were examined in vitro. Formation of pollen tubes in Douglas fir occurred on a modified Murashige and Skoog medium in which concentrations of H₃BO₃ and Ca(NO₃)₂ were altered and supplemented with sucrose and polyethylene glycol. Addition of 100 μg/ml H₃BO₃ and 300 μg/ml Ca(NO₃)₂ resulted in optimum pollen viability. Lack of H₃BO₃ inhibited pollen tube formation. Addition of H₃BO₃ and Ca(NO₃)₂ significantly increased pollen tube formation within one week in culture. Using a medium supplemented with mannitol, viability of Douglas fir pollen can be sustained for 7 weeks in culture, about the same length of time as in vivo. However, pollen tubes are not formed. This suggests that the factors responsible for tube formation reside in the external environment of the pollen. Culture of female gametophytes to examine egg viability and longevity had not been done previously. We found that egg viability in culture is short-lived, and therefore the window to study and manipulate events of fertilization in Douglas fir is very limited. In spite of this, about 7% of the female gametophytes that were co-cultured became penetrated by pollen tubes. In vitro archegonial penetration has been repeatedly achieved, but pollen tubes also penetrated other parts of the female gametophytes. Pollen tubes also penetrated non-viable eggs. Most female gametophytes were not penetrated because of pollen tube branching and swelling, failure of tubes to orient towards the female gametophytes, or premature pollen tube death due to plasmolysis. This report outlines the first attempt towards in vitro fertilization in conifers. Received: 13 March 1997 / Revision accepted: 6 June 1997     

EFFECT OF SALINITY ON POLLEN I. POLLEN VIABILITY AS ALTERED BY INCREASING OSMOTIC PRESSURE WITH NaCl,MgCl2, and CaCl2

Petunia (Petunia hybrida Vilm. cv. ‘Snowstorm') plants were grown in saline solution (NaCl, MgCl₂, and/or CaCl₂) of 0, 1, 2, and 3 bars osmotic pressures. Pollen viability was tested by tetrazolium chloride staining and by germination (by the hanging drop method, using 15 % sucrose and 0.01 % boric acid as the nutrient medium, at 27 ± 1 C). Pollen viability decreased with increased salinity. Pollen from plants grown in single salt solutions of NaCl, MgCl₂, and CaCl₂ (each at 0, 1, 2, or 3 bars osmotic pressure) was germinated in base culture medium. Pollen viability decreased more with NaCl than with MgCl₂ or CaCl₂. In vitro studies of the effects of three salts, viz., NaCl, MgCl₂, and CaCl₂, on pollen germination and tube growth showed that NaCl inhibited germination and pollen tube growth more than did MgCl₂ or CaCl₂. MgCl₂ was least injurious, and even promoted tube growth at 0.5 and 0.75 bars osmotic pressure. Adding low concentrations of MgCl₂ reduced the toxic effect of NaCl and increased the percentage of germination. CaCl₂ reduced the effect of NaCl less than did MgCl₂. We conclude that specific ion effects were more important than osmotic pressure.     

Ionophore A 23187 stops tip growth,but not cytoplasmic streaming,in pollen tubes ofLilium longiflorum

Summary The effects of the cationophore A 23187 on growing pollen tubes ofLilium longiflorum and on pollen germination were testedin vitro, and measured light microscopically. The ionophore is a very potent inhibitor of pollen tube growth: ionophore contentrations down to 10^–7 M stop tip growth. Cytoplasmic streaming is less sensitive: Only with added external Ca²⁺ and higher concentrations of the ionophore the cytoplasmic streaming is stopped. Pollen germination is less sensitive to ionophore than pollen tube growth at later stages. The ionophore inhibition is partially reversible in a medium containing no added external Ca²⁺, but is not reversible in a Ca²⁺-enriched medium. EDTA addition to the medium prevents pollen germination and growth totally. It is hypothesized that the pollen ofLilium longiflorum needs Ca²⁺ to sustain oriented exocytosis at the pollen tube tip. The ionophore A 23187 seems to interfere with the electrical pulse/Ca²⁺-orientation mechanism of exocytosis by equilibration of the Ca²⁺-gradient.     

Effect of Salt Stress on Viability, Germination and Endogenous Levels of Some Metabolites and Ions in Maize (Zea mays L.) Pollen

Maize plants, subjected to 0, 80, 120 and 160 meq l^–1salinity using NaCl, showed adverse effects on viability, germinationand tube growth of pollen, besides enhancing the bursting ofpollen. The endogenous levels of various metabolites in pollenwere also affected. Pollen grains from salinized plants hadmore soluble carbohydrates, free amino acids, especially proline,phenols and DNA and less starch, protein and RNA compared tothe non-saline controls. Salinity also resulted in the accumulationof ions such as Na⁺, K⁺ and Cl^– while it caused a reductionin the boron content of pollen. These metabolic disturbancespossibly lead to decreased viability, germination and tube growthof pollen thereby resulting into a reduction in reproductivecapacity of the plants under salt stress. Zea mays L., maize, pollen, viability, germination, salt stress     

POLLINATION,FERTILIZATION AND OVULE ABORTION IN OXALIS MAGNIFICA

Pollination, fertilization and ovule abortion were studied in Oxalis magnifica (Rose) Knuth, a strongly self-incompatible herb that regularly matures only a fraction of its ovules. Examination of cleared ovules indicated that among 9 individuals the average number of ovules fertilized ranged from 48–92%. The remaining ovules either failed to produce female gametophytes, or more commonly contained unfertilized female gametophytes, despite large numbers of compatible pollen grains that were placed on stigmas. Abortion of fertlized ovules could be detected first by the flattened and enlarged appearance of the endosperm nuclei, followed by visible deterioration of the embryo. Among individuals the rate of embryo abortion varied from 3.4–47.9%. At lower levels of pollination an almost one-to-one relationship existed between the number of pollen grains placed on stigmas and the number of seeds matured in the capsule. No threshold number of pollen grains necessary for successful pollen tube growth and fertilization could be demonstrated. Reduction in seed number through embryo abortion provides an opportunity for selection among developing seeds. The potential for this form of selection varies widely among individuals of Oxalis magnifica, which showed a 14-fold variation in the average percentage of aborted ovules.     

Variation in pollen viability among Picea abies genotypes – potential for unequal paternal success

An in vitro germination method was used to study variation in pollen viability, that is pollen-tube growth rate together with germination percentage, among the Picea abies genotypes in a seed orchard. The method permits easy, rapid screening of large numbers of genotypes. Significant variation in pollen viability among the genotypes was evident, the differences among the pollen-lot means being 7–10–fold in different years. No correlation was found between the average pollen viability and the phenology, growth or growing-site characteristics of the pollen donors. However, there appeared to be pollen lots that either benefit from a higher germination temperature or else germinate faster at lower temperatures. The significant variation in pollen viability among the pollen donors indicates a potential for male gametophyte competition. This, together with the observed genotype-environment interactions in pollen performance, may contribute to the variable genetic composition of seed produced in the seed orchard. Received: 9 December 1999 / Accepted: 22 December 1999     

Low temperature effect on selective fertilization by pollen mixtures of wild and cultivated tomato species

Summary In vitro pollen germination of cultivated tomato, Lycopersicon esculentum Mill., is inhibited by an ambient temperature of 5°C, more so than pollen from a Peruvian ecotype of Lycopersicon hirsutum Humb. & Bonpl. originating from an altitude of 3200 m. The frequency of L. hirsutum gametes contributing to hybrid zygote formation is more than doubled when controlled fertilizations with pollen mixtures of the two species occurs at 12/6°C as compared to crosses with the same mixtures at 24/19°C. The results suggest that differential selection at the gametophytic level occurs in response to low temperature regimes. To our knowledge this is the first time in higher plants that alteration of an environmental factor has been demonstrated to change selection values of male gametophytes in a fashion predicted by the ecology of the parental sporophytes.