The reversible inhibition of pollen germination of Nicotiana tabacum L.: an entry into a transformation system

Summary The hydrodynamics of mature pollen rehydration in Nicotiana tabacum was used to study reversible inhibition of pollen germination in vitro. Tobacco pollen was incubated for various times in media containing calcium, potassium and magnesium salts, boric acid, and exhibiting different osmotic pressures as a function of sucrose concentration. Total inhibition of germination with complete viability preservation was achieved for 56 h by keeping the grains in medium with 80% sucrose, since typical percentages of germination and pollen tube lengths were recovered after this treatment. These effects were considered as consequences of natural osmoregulation of rehydration/germination in mature pollen. The possibility of applying these findings to the incubation of pollen with Agrobacterium tumefaciens to develop a pollen transformation method is discussed.     

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.     

The division of the generative nucleus and the formation of callose plugs in pollen tubes of Aechmea fasciata (Bromeliaceae) cultured in vitro

The effect of different external factors on pollen germination and pollen tube growth is well documented for several species. On the other hand the consequences of these factors on the division of the generative nucleus and the formation of callose plugs are less known. In this study we report the effect of medium pH, 2-[N-morpholino]ethanesulfonic acid (MES) buffer, sucrose concentration, partial substitution of sucrose by polyethyleneglycol (PEG) 6000, arginine (Arg), and pollen density on the following parameters: pollen germination, pollen tube length, division of the generative nucleus, and the formation of callose plugs. We also studied the different developmental processes in relation to time. The optimal pH for all parameters tested was 6.7. In particular, the division of the generative nucleus and callose plug deposition were inhibited at lower pH values. MES buffer had a toxic effect; both pollen germination and pollen tube length were lowered. MES buffer also influenced migration of the male germ unit (MGU), the second mitotic division, and the formation of callose plugs. A sucrose concentration of 10% was optimal for pollen germination, pollen tube growth rate and final pollen tube length, as well as for division of the generative nucleus and the production of callose plugs. Partial substitution of sucrose by PEG 6000 had no influence on pollen germination and pollen tube length. However, in these pollen tubes the MGU often did not migrate and no callose plugs were observed. Pollen tube growth was independent of the migration of the MGU and the deposition of callose plugs. In previous experiments Arg proved to be positive for the division of the generative nucleus in pollen tubes cultured in vitro. Here, we found that more pollen tubes had callose plugs and more callose plugs per pollen tube were produced on medium with Arg. After the MGU migrated into the pollen tube (1 h after cultivation), callose plugs were deposited (3 h). After 8 h the first sperm cells were produced. The MGU moved away from the active pollen tube tip until the second pollen mitosis occurred, thereafter the distance from the MGU to the pollen tube tip diminished. Callose plug deposition never started prior to MGU migration into the pollen tube. Pollen tubes without a MGU also lack callose plugs (±30% of the total number of pollen tubes). Furthermore, we found a correlation between the occurrence of sperm cells in pollen tubes and the synthesis of callose plugs.     

培养基组分及培养条件对蜡梅花粉萌发及花粉管生长的影响

采用液体培养法研究不同培养基组分和培养条件对蜡梅花粉萌发和花粉管生长的影响。结果表明:(1)PEG-4000是蜡梅花粉离体培养所必需的培养基成分,当培养基中无PEG-4000时,花粉不能正常萌发。(2)培养基内低浓度蔗糖对花粉萌发和花粉管的生长无显著影响,但随着蔗糖浓度的升高,则对花粉萌发和花粉管生长表现出强烈的抑制作用,且浓度越高,抑制效应越强。(3)培养基内其它组分分别在一定浓度范围(0～250g/L PEG-4000、0～50mg/L硼酸、0～30mg/L硝酸钙)内对花粉萌发及花粉管生长有促进作用,但超过上述高限值时则起抑制作用。(4)培养基内镁和钾的浓度对花粉萌发及花粉管生长影响不显著。研究表明,蜡梅最适花粉液体培养基组分为250g/L PEG-4000+50mg/L H3BO3+30mg/L Ca(NO3)2.4H2O,且在pH 5.5、温度15℃和600lx的光照培养条件下蜡梅花粉萌发和花粉管生长最佳。     

Studies on the Germination of Pine Pollen (Pinus mugo) in vitro. II. Effects of Different Ions

Studies on the initial germination of pollen of Pinus mugo showed no significant influence of ions on O₂ uptake and uptake of ³²P-labelled phosphate. At the onset of tube growth O₂ uptake decreased in the absence of calcium. In inorganic media tube growth and ³²P uptake were reduced in the absence of calcium or boric acid. In the absence of calcium a requirement for magnesium was observed. When the medium was deprived of polyvalent ions with EDTA, growth and ³²P uptake ceased. The presence of calcium in the medium was found to be essential for the maintenance of the structural and functional integrity of the cell membranne. — The ion requirement was more pronounced when tube growth was stimulated with sucrose. Calcium, magnesium, boric acid, and nitrate (as nitrogen source) were essential constitutents of the medium. The stimulation due to calcium required either magnesium or boric acid. — A density effect was observed which can be related to diffusible substances from the pollen into the medium. This was not observed when calcium and magnesium were present in the medium. The phenomenon is explained as an enrichment of the medium with diffusible substances from non-germinated dead pollen. — Germination and the tube growth were found to be greatly dependent on a short period of equilibration of pollen at room temperature before sowing.     

Effects of Calcium Ion and the Protective Action on Survival and Growth Inhihition of Pollen

Studies were conducted on the protective action of Ca against growth inhibition and death of pollen from Crinum asiaticum and Catharanthus roseus. Ca ions not only promoted pollen germination and pollen tube growth, but also were antagonistic to pollen injuries induced by various chemical and physical treatments. Most of the chemicals used seemed to act as osmotic inhibitors. Among the chemical agents tested, DNP inhibited pollen germination more strikingly with Ca than without it, whereas pollen tube growth was enhanced. Another metabolic inhibitor, low temperature, showed a similar effect on pollen germination. Insignificant or no protective action of Ca was observed when IAA, TIBA and coumarin, were applied. Large populations of pollen when grown in cultural media witbout Ca showed a partial protective action. The protective action of Ca in pollen growth against various inhibitors required other cations such as Mg and K ions. The promoting of pollen growth by Ca also required tbese otber cations. The protective action of Ca is considered to be based on its binding in tbe pollen cell walls, particularly in the pectic regions. This gives rise to a decreased permeability and increased structural rigidity against tbe chemical and physical inhibitors.     

Effect of smoke derivatives on in vitro pollen germination and pollen tube elongation of species from different plant families

Plant‐derived smoke stimulates seed germination in numerous plant species. Smoke also has a positive stimulatory effect on pollen germination and pollen tube growth. The range of plant families affected my smoke still needs to be established since the initial study was restricted to only three species from the Amaryllidaceae. The effects of smoke‐water (SW) and the smoke‐derived compounds, karrikinolide (KAR₁) and trimethylbutenolide (TMB) on pollen growth characteristics were evaluated in seven different plant families. Smoke‐water (1:1000 and 1:2000 v:v) combined with either Brewbaker and Kwack's (BWK) medium or sucrose and boric acid (SB) medium significantly improved pollen germination and pollen tube growth in Aloe maculata All., Kniphofia uvaria Oken, Lachenalia aloides (L.f.) Engl. var. aloides and Tulbaghia simmleri P. Beauv. Karrikinolide (10^?6 and 10^?7 m ) treatment significantly improved pollen tube growth in A. maculata, K. uvaria, L. aloides and Nematanthus crassifolius (Schott) Wiehle compared to the controls. BWK or SB medium containing TMB (10^?3 m ) produced significantly longer pollen tubes in A. maculata, K. uvaria and N. crassifolius. These results indicate that plant‐derived smoke and the smoke‐isolated compounds may stimulate pollen growth in a wide range of plant species.     

In vitro germination and growth of lily pollen tubes is affected by calcium inhibitor with reference to calcium distribution

The calcium inhibitors A23187, EGTA and La³⁺ inhibit pollen grain germination and growth of pollen tubes of Lilium davidii var. unicolor at different concentrations. Treatment with 10⁻⁴ or 10⁻⁵ M ionophores A23187 reduced germination rate and resulted in distortion of pollen tube. Addition of 2 or 10 mM of the chelator EGTA disturbed the direction of pollen tube growth and extended the diameter of pollen tube as observed by light and confocal microscopy. The Ca²⁺-channel blocker lanthanum chloride (La³⁺) restrained germination or markedly caused transformation of pollen tube. Furthermore, all treatments led to disappearance of any calcium gradient. Calcium distribution in pollen grain and pollen tube was altered as shown by confocal microscopy for each treatment. This indicates that the inhibitors influence pollen development by affecting the calcium gradient which may play a critical role in germination and tube growth. Fourier transform infrared (FTIR) spectra indicated slight increases in contents of amide I and a substantial decrease in the content of aliphatic esters and saturated esters in treated pollen tubes compared with normal pollen tubes. The FTIR analysis confirmed that EGTA and La³⁺ weakened the accumulation of ester in pollen tubes, which may be associated with an increased content of amide I.     

A cytochemical study on the role of ATPases during pollen germination in Agapanthus umbelatus L'Her.

Summary Cytochemical detection of ATPase activity in the pollen grain (PG) and pollen tube (PT) of Agapanthus umbelatus showed that the enzymes concerned presented specific patterns of membrane distribution according to their ionic dependencies and to the timecourse of germination and tube growth. In the pollen tubes Ca²⁺-ATPases were mainly localized in mitochondria and ER membranes, while Mg²⁺-ATPases were found especially in the tonoplast and in the membrane of the P-particles. K⁺-ATPases showed a high activity at the plasma membrane. In the pollen grain similar patterns of ATPase activity were observed. The highest activity of all three types was observed at the plasma membrane of the grain and at the intine and inner exine layers of the cell wall. The activity observed in the pollen grain cell wall decreased with germination time. In vivo germination studies in the presence of specific inhibitors of the ATPases showed patterns of inhibition that could be correlated with the corresponding ATPase putative role.The results are discussed in terms of the ultrastructural organization of the PG and PT, especially those correlated with (1) formation and maintenance of ionic gradients throughout the PT, (2) polarized growth and (3) hydrodynamics of PT elongation.Abbreviations PT Pollen tube - PG pollen grain - PTW pollentube wall - PGW pollen-grain wall - ER endoplasmic reticulum - NEM N-ethylmaleimide     

桃花粉离体萌发和花粉管生长特性研究

采用花粉离体萌发法研究不同培养基组分和培养条件对桃花粉萌发和花粉管生长的影响,同时对不同贮藏温度下的桃花粉寿命进行研究.结果表明:固体培养基与液体培养基对桃的花粉萌发率和花粉管长度影响差异不显著;10%蔗糖是大多数桃品种花粉的最适萌发条件;硼能提高桃花粉的萌发率,但对花粉管的生长没有促进作用;桃花粉在20℃～25℃的培养温度下萌发率最高,花粉管最长;桃花粉萌发率和花粉管长度在培养前3 h内上升最快,3～5 h上升趋势减弱,5 h后基本停止;随着贮藏温度的升高和贮藏时间的延长,花粉生活力呈降低的趋势.     

Inhibition of in Vitro Pollen Tube Growth by Isolated S-Glycoproteins of Nicotiana alata

Pollen from three S-genotypes of Nicotiana alata was grown in vitro in the presence of S-glycoproteins isolated from styles of the same three genotypes. Pollen germination was not affected by the presence of the S-glycoproteins, but pollen tube growth of all genotypes was inhibited. S2 pollen was preferentially inhibited by the S2-glycoprotein and S3 pollen by the S3-glycoprotein. The S6-glycoprotein preferentially inhibited growth of both S2 and S6 pollen over S3 pollen. Heat treatment dramatically increased the inhibitory activity of the S-glycoproteins as inhibitors both of pollen germination and tube growth; after heat treatment, S-allele specificity of pollen tube inhibition was not detected.     

In-vitro germination and growth of lily pollen tubes is affected by protein phosphatase inhibitors

Inhibitors of type-2A protein phosphatase (PPase-2A), calyculin A (cal A) and okadaic acid (OA), inhibit pollen grain germination and growth of pollen tubes of Lilium longiflorum Thunb. at nanomolar concentrations. Half-maximal inhibition of cytoplasmic PPase-2A activity was below 0.1 nM for cal A and at 0.7 nM for OA. Other protein phosphatase inhibitors (tautomycin, cypermethrin, and dephostatin) were less effective. The OA- and cal A-sensitive as well as dephostatin-sensitive PPase activity in the cytoplasm did not change during germination and growth of pollen tubes. Addition of cal A and OA disturbed the direction of pollen tube growth and the distribution of cytoplasmic organelles and caused cell wall thickenings as observed by light and electron microscopy. Inhibition of PPase-2A caused multiple effects at the cellular level, cytoskeletal elements being a putative target of PPase-2A activity. Received: 30 March 1998 / Accepted: 6 July 1998     

The involvement of hydrogen peroxide in UV-B-inhibited pollen germination and tube growth of Paeonia suffruticosa and Paulownia tomentosa in vitro

Previous studies have shown that UV-B could affect pollen germination and tube growth. However, the mechanism of response of pollen to UV-B has not been clear. The purpose of this study was to investigate the role of hydrogen peroxide (H₂O₂) in the UV-B-induced reduction of in vitro pollen germination and tube growth of Paeonia suffruticosa Andr. and Paulownia tomentosa Steud. Exposure of pollen of the two species to 0.4 and 0.8 W m⁻² UV-B radiation for 3 h resulted in not only the reduction of pollen germination and tube growth, but also the H₂O₂ production in pollen grain and tube. Also, exogenous H₂O₂ inhibited pollen germination and tube growth of the two species in a dose-dependence manner. Two scavengers of H₂O₂, ascorbic acid and catalase, largely prevented not only the H₂O₂ generation, but also the reduction of pollen germination and tube growth induced by UV-B radiation in the two species. These results indicate that H₂O₂ is involved in the UV-B-inhibited pollen germination and tube growth.     

In vitro chemotropism of pearl millet pollen tubes to stigma tissue: a response to glucose produced in the medium by tissue-bound invertase

Summary Water-homogenized stigma pellets of pearl millet and precipitates resulting from dialysis of their salt extracts were observed to: (1) chemotropically attract pearl millet pollen tubes on a sucrose-containing pollen germination and growth medium, (2) have acid invertase activity as assayed by the arsenomolybdate method, (3) hydrolyze sucrose in the pollen germination and growth medium to glucose as assayed by coupled glucose oxidation with Nitro Blue Tetrazolium, and (4) lose chemotropic and invertase activities upon heat treatment. The results indicate that the in vitro chemotropic attraction of pearl millet pollen tubes to water-homogenized stigma pellets is a response to glucose produced by homogenate-pellet-bound invertase hydrolyzing the sucrose present in the pollen germination and growth medium. Yeast and tomato invertases used as controls verified this conclusion. Water extracts of whole stigmas contained water-soluble acid invertase. The results are discussed in relation to the identification of possible in vivo chemotropic factors of pearl millet and other plants by in vitro assays.Abbreviations dH₂O Deionized, house-distilled water - NBT Nitro Blue Tetrazolium, NBT-medium - PGG medium, pollen germination and growth medium (10% sucrose, 1 mM H₃BO₃, and 1% agarose); - WHS pellet, water-homogenized stigma pellet On Specific Cooperative Agreement 58-6612-8-002 with the Department of Biochemistry, University of Georgia, Athens, GA 30602, USA     

Semi in vivo pollen tube growth of Aechmea fasciata

With semi in vivo pollen tube growth assays, stigmas are pollinated in vivo and, after a fixed time interval, the styles are isolated from the ovary and placed on culture medium in vitro. Semi in vitro pollination includes isolation of the stigma and style complex, followed by pollination and placing the stylar end on nutrient medium. After semi in vivo pollination more and longer pollen tubes protruded from the cut end of the styles into medium, in comparison to semi in vitro pollination. Medium with 3 g l^–1 agar was better than that with 6 g l^–1 agar for pollen tube growth after the tubes emerged from the cut style. Semi in vitro pollination of the reversed style indicated that pollen tube growth was not influenced by the direction of the style. Fructose and glucose inhibited pollen tube growth compared to sucrose. Swollen tips characterized tube growth inhibition. After semi in vivo pollination all generative nuclei had divided to give two sperm nuclei. The average distance between the last sperm nucleus and the pollen tube tip as well as the distance between the two sperm nuclei diminished in growing pollen tubes between 24 and 48 h after pollination. The arrangements between the vegetative and the generative nuclei did not differ in semi in vivo and in vitro cultured pollen tubes of Aechmea fasciata. This information is important to explain why fertilization rate is low after placental pollination in comparison to placental grafted style pollination of Aechmea fasciata. The data may also contribute to the improvement of in vitro fertilization methods in Bromeliaceae and other higher plants.     

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.     

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.     

Germination of trinucleated pollen: formulation of a new medium for Capsella bursa-pastoris

Summary In Brewbaker and Kwack's medium (BK) only 16% of the pollen grains germinated, and these produced pollen tubes having a maximum length of 25 m. With a solution based on Monnier's medium 47% germination and 160-mlong pollen tubes were observed. Calcium was shown to be essential for germination; the optimal concentration was 880 mg/l calcium chloride. The optimal concentrations of magnesium sulphate and boric acid were 360 and 50 mg/l, respectively. Germination at pH 4.0 but also pH 8.0 and the presence of vitamins B1 and B6 (1 mg/l each) were stimulatory. Polyethylene glycol (PEG) was superior to sucrose as an osmoticum and germination and tube length were significantly improved using PEG 4000 at a concentration of 120 g/l (0.03 M). Equimolar concentrations of PEG 400 and PEG 600 gave inferior results. Combining PEG with sucrose in the medium did not improve germination or increase tube length.     

Nucleotide Metabolism during Pine Pollen Germination

The metabolism of purine- and pyrimidine nucleotides in pine pollen (Pinus mugo) grown in suspension cultures have been examined. In the ungerminated dehydrated pollen, the presence of ATP has been demonstrated. Incubation of the pollen in a germination medium leads to an exhaustion of the ATP pool, which is restored with the onset of oxygen uptake. By labelling pollen cultures with ³²P-orthophosphate, it has been possible to quantitate the nucleotide components of the pollen, and thereby to measure changes in the nucleotide pattern at various growth stages. The most marked changes occur during the initial phase of tube growth when a large increase in the ribonucleoside triphosphate and the sugar nucleotide pools is observed. The contents of ATP and UDP-glucose are further increased if starch synthesis is initiated by the addition of sucrose to the culture medium. In order to determine whether nucleotides in pine pollen are synthesized from de novo pathways or via reutilization pathways, from breakdown products of nucleic acids, pollen was incubated with ¹⁴C-labelled precursors of both the de novo and the reutilization pathways. Incorporation experiments established de novo synthesis of ATP and GTP from glycine, and de novo synthesis of CTP and UTP from orotic acid. The operation of pathways for the utilization of exogenous nucleosides was also demonstrated. While uridine, cytidine and adenosine are incorporated into nucleoside triphosphate to a great extent, only minor incorporation of inosine and guanosine is observed. These reutilization pathways might be of importance for the synthesis of nucleotides during tube growth in situ. Addition of inhibitors of glycolysis and oxidative phosphorylation drastically reduces the level of ribonucleoside triphosphates, indicating a rapid turnover of the nucleotide pool.