Normal Pollen Tube Research Articles

Heat Shock Proteins and Survival of Germinating Pollen of Lilium longiflorum and Nicotiana tabacum

Summary Protein synthesis as well as pollen tube development of pollen of Lilium longiflorum and Nicotiana tabacum , incubated in vitro , are affected by a heat shock applied after germination for 30 min. Pollen tube elongation ceases and a protrusion is formed at the tip. The protein pattern in germinating pollen of lily is slightly changed by heat shock; cordycepin inhibited heat shock-induced de novo synthesis of two proteins, indicating that heat shock-induced regulation occurs at the level of transcription. In tobacco, however, the change in the pattern of protein synthesis is more pronounced, and the production of more heat shock proteins (HSPs) could be established. Some of those HSPs in tobacco are already present before heat shock: their synthesis at the control temperature of 27 °C can be inhibited by cordycepin, indicating that transcriptionally controlled synthesis of HSP-mRNAs occurs during normal pollen tube growth. Furthermore, some of the heat shock induced changes in the overall pattern of protein synthesis in tobacco seem to be similar to those in lily. The alterations in protein synthesis due to heat shock gradually disappear during the subsequent period of 1.5 h following heat shock. This period is characterized both by the recovery of the synthesis of the normal proteins and the restart of pollen tube growth. The recovery of the overall pattern of protein synthesis can be retarted by cordycepin as is the restart of the pollen tube growth. In lily and tobacco, a pre-heat shock of 3 min immediately after imbibition has a similar effect on the protein pattern as cordycepin: no synthesis of the HSPs when the actual heat shock of 30 min is applied.

Ultrastructural research on cell wall regeneration by cultured pollen protoplasts of Lilium longiflorum

Protoplasts from pollen grains of Lilium longiflorum regenerate amorphous cellulosic cell walls in culture, during which some precursors of cellulose are polymerized, thus producing progressively harder cellulosic cell walls as the period of culture continues. It is presumed that the components of the cell wall regenerated during 1 week in culture differ from those of the intine of the pollen grain wall. The regenerated cell wall is formed by means of large smooth vesicles; in addition, numerous coated vesicles and pits aid in wall regeneration. The pollen tube that germinates from the 8-day-old cultured protoplast has numerous Golgi bodies and many vesicles which build the pollen tube wall. The tube wall has two layers just like a normal pollen tube wall.

The effect of ε-aminocaproic acid on self-incompatibility and incongruity inPhaseolus

SummaryThe effects of e-aminocaproic acid (EACA) on self-incompatibility was tested in a Phaseolus coccineus line which failed to form pods in controlled pollinations. Scanning electron microscope (SEM) and fluorescence miscroscopy confirmed problems in pollen-germination and pollen-tube growth but EACA-treated self- pollinations resulted in fairly normal pollen germination and pollen tube growth. In a few cases, pollen tubes were seen entering the embryo sac and occasionally pods were formed. Incongruity barriers also exist in crosses of P. coccineus × P. vulgaris, P. acutifolius × P. coccineus and their reciprocals. Effects of EACA on flower abscission, pod development and pod abscission were studied. Increased pod formation was observed in EACA treated materials, except in P. acutifolius × P. coccineus and P. coccineus × P. vulgaris cv. Jacobs Cattle. EACA seems to act at the stigmatic and stylar levels, thereby enhancing pollen germination and pollen tube growth and delaying flower abscission. The net...

Intersubgeneric Crossability in the Genus Glycine Willd.

AbstractIn intersubgeneric crosses between wild perennial Glycine species of submenus Glycine and soybeans, Glycine max (L.) Merr., of subgenus Soja, all the putative hybrid pods aborted from 9 to 30 days after pollination (DAP). Spraying of 70 ppm gibberellic acid on hybridized gynoecia facilitated pod retention. Fluorescence microscopy revealed normal pollen germination and pollen tube growth in all the intersubgeneric crosses except in the G. cyrtoloba × G. max cross.

Further attempts to induce ?egg transformation? using irradiated pollen

Numerous pollination treatments involving heavily irradiated (40-100 krad) pollen of diverse plant species failed to produce any clear cut "egg transformants" of the type reported by Pandey in Nicotiana. Genetic stocks of pea, rapeseed, and apple, bearing multiple Mendelian markers, were employed to detect any possible transformation events. For each plant species, an optimal level of irradiation was determined which would allow normal pollen tube growth leading to fertilization, but which would prevent the formation of normal hybrids due to the "pulverized" condition of the chromosomes contributed by the irradiated pollen. Pollination treatments included selfing, pollination with donor pollen mixed with self pollen, pollination with irradiated donor pollen mixed with self pollen, pollination with irradiated pollen followed by a delayed self pollination, and pollination with irradiated pollen by itself. None of these treatments produced clearly transformed seedlings. The total number of potential transformation events screened was in excess of 6,046 including 2,268 for pea, 3,309 for rapeseed, and 469 for apple. It is concluded that if egg transformation occurs outside of Nicotiana it is a rare event, and its frequent occurrence in Nicotiana must be, at best, an isolated phenomenon.

Membrane-associated calcium during pollen grain germination: a microfluorometric analysis

Pear (Pyrus communis L.) pollen was germinated and grown in hanging drop cultures containing phenothiazine drugs, trifluoperazine and chlorpromazine, potent inhibitors of the Ca2+-calmodulin complex. Responses for the two drugs were similar: at 1.0–2 10.0 ΜM pollen germination and tube growth were inhibited. Inhibition of tube growth was not uniform; at 10.0 ΜM growth of about half of those tubes which had germinated was inhibited while the remaining half of the population grew normally. This bimodal distribution of tube growth was noted, but to a lesser extent, at lower concentrations of the drugs as well. Microfluorometric analysis of membrane calcium using chlorotetracycline, a fluorescent chelate probe for membrane calcium, revealed that upon pollen grain hydration there was a sharp decrease in the concentration of calcium associated with membranes and that membrane calcium became localized primarily at the periphery of the newly hydrated pollen grain. Prior to germination there was a reloading of calcium onto membranes in the region of the germination aperture through which the pollen tube would emerge. The release of Ca2+ from membrane sites upon hydration was partially inhibited by treatment with 10 ΜM trifluoperazine. Distribution of membrane calcium in populations of the inhibitor-treated pollen grains was not bimodal however; approximately half of the population had CTC-fluorescence emissions exceeding the maximum value found in the control population. These results suggest that there is a maximum concentration of membrane-associated calcium consistent with normal pollen germination and tube growth and that phenothiazines interfere with the unloading of Ca2+ from membrane sites.

Stigma-surface Esterase Activity and Stigma Receptivity in Some Taxa Characterized by Wet Stigmas

Stigma-surface esterase activity and stigma receptivity through a sequence of developmental stages of the pistil have been studied in four taxa characterized by having wet stigmas-Petunia hybrida, Nicotiana tabacum, Crinum defixum and Amaryllis vittata. The style is solid in the first two and hollow in the latter two taxa. In all the taxa, stigma-surface esterase could be detected in a thin surface layer (pellicle) from a very early stage of pistil development, irrespective of the presence or absence of the exudate. However, the taxa showed variation in stigma receptivity. In Petunia and Nicotiana, stigmas from pistils of all the stages supported pollen germination and tube growth. In Amaryllis and Crinum, stigmas of only the mature pistils, when the exudate is present on the stigma, supported normal pollen germination and tube growth. It is inferred that in taxa characterized by a wet stigma and solid style, the factors required for pollen germination are present from an early stage of pistil development and the exudate per se is not involved in pollen germination. In taxa characterized by a wet stigma and hollow style, however, the pellicle does not carry the factors required for pollen germination and tube growth; they appear to be present in the exudate.

Interspecific hybridization in Cucumis L. II. The crossability of species, an investigation of in vivo pollen tube growth and seed set

The crossability of 12 Cucumis species of African and Asiatic origin was studied in a diallel cross, in order to find ways to realise the cross between the common cucumber (C. sativus L.) and its wild relatives which carry resistances against diseases and pests. Self-pollinations and cross-pollinations within species gave normal pollen tube growth and seet set. The different accessions within a species, as a rule reacted alike in interspecific crosses. In crosses between African species different crossing patterns were found, viz. bilateral congruity, bilateral incongruity and unilateral incongruity. Within C. sativus all accessions intercrossed freely, except one, which displayed unilateral incongruity. Good seed was harvested from several crosses and in some cases embryo culture was needed for further development of seeds. No good seeds were obtained from any cross between a species of the African group and C. sativus L.

POLLEN TUBE‐SYNERGID INTERACTIONS IN PROBOSCIDEA LOUISIANICA (MARTINEACEAE)

The ultrastructure of the synergids of Proboscidea louisianica was investigated from just before fertilization until 48 hr after pollination. It was found that the cytoplasm of one synergid consistently begins to degenerate before arrival of the pollen tube at the embryo sac, and that it is always this synergid which receives the pollen tube tip and its discharge. The other synergid (persistent synergid) remained unchanged throughout the study period. Polysaccharide vesicles of pollen tube origin were observed fusing with the pollen tube wall as well as contributing to cell wall formation of the degenerate synergid. In one ovule (48 hr after pollination) two pollen tubes had entered and grown the length of the micropyle, but only the first tube penetrated the degenerate synergid and discharged normally. The second pollen tube was abutting against the persistent synergid, but had not entered or discharged. In another exceptional case (18 hr after pollination), a pollen tube had grown the length of the micropyle, but did not discharge, or enter either synergid. Both synergids of this ovule were observed to be completely intact. It is concluded that synergid and pollen tube cytoplasmic degeneration is the result of a very specific interaction between these two cells and that this degeneration is probably a prerequisite for normal pollen tube entrance and discharge into the embryo sac, and for male gamete transfer to the egg and central cell.

FUNCTION OF THE POLLEN COAT IN DIFFERENT STAGES OF THE FERTILIZATION PROCESS

SUMMARY The weight of released proteins after four subsequent washings of pollen of Petunia hybrida L. in germination medium (containing 10% saccharose and 0.01 % boric acid) is calculated to be c. 18% of the original dry pollen weight. Although washed-and-dried pollen did germinate very badly after replacement in the germination medium, it produced normal pollen tube growth in the style and normal seed setting in the ovary. It is concluded that released pollen coat substances are not functional in the in vivo fertilization process. Another conclusion is that these eluted pollen coat substances have no role as recognition factors in the gametophytically-determined incompatibility system of Petunia.

Dimethyl Sulfoxide: Reversible Inhibitor of Pollen Tube Growth

Five percent dimethyl sulfoxide (DMSO) completely inhibited tube initiation, stopped tube growth and suppressed the high respiration associated with tube growth of lily pollen. The effect of DMSO on respiration was indirect because uncoupling concentrations of 2,4-dinitrophenol abolished the inhibition of respiration. Five percent DMSO did not inhibit rapid starch synthesis during the first 30 minutes of incubation, nor did DMSO inhibit the period of high respiration associated with rapid starch synthesis. DMSO did not cause permanent damage to the cells since normal pollen tube growth occurred after its removal. Dimethyl sulfoxide is not a general inhibitor of pollen metabolism, but it may be a specific inhibitor of a process required for tube growth.

Effect of ionizing radiation on microsporogenesis in Pinus Rigida mill

A Cesium 137 (9500 c) source was placed in a natural pitch pine—oak forest at the Brookhaven National Laboratory. Beginning on November 22, 1961, the forest was exposed to gamma rays during 20 hr each day. Pinus rigida trees were exposed to radiation levels ranging from a high of 960 r/day to a level of normal background radiation. Microsporangiate strobili were collected and evaluated at regular intervals to follow the effects of gamma radiation on the cytology of microsporogenesis. For comparison, mature pollens of Pinus rigida and Pinus strobus were exposed to acute γ radiation for a period of 16 hr from a Cobalt 60 source of approximately 3200 c at levels ranging from 1 to 300 kr. Trees receiving 82 r/day and higher from Cs 137 were unable to resume cell division and differentiation during the spring of 1962, while microspore mother cells were formed on trees receiving 75 r/day. At 56 r/day meiosis with susbequent formation of tetrads took place, and the highest level at which viable microspores were collected was 11 r/day. A slight reduction in the length of the strobili was observed at 0·4 r/day, and at 3·7 r/day they were significantly shorter than the controls. The percentage of cells with visible chromosome aberrations increased with an increase in exposure, e.g. 0·4 r/day=1 per cent; 7 r/day=2 per cent; 11 r/day=12 per cent; and 17 r/day=76 per cent. Pollen abortion increased with an increase in radiation, and at 7 r/day, approximately 50 per cent of the pollen grains were aborted. At 3 to 5 r/day, germination in vitro was reduced to 50 per cent of the controls. Pollen tube length was not related to exposure. Mature pollen of Pinus rigida and Pinus strobus produced normal pollen tubes in vitro after 16 hr exposure to 300 kr Co 60 radiation. Illustrations of the various observations are given, and the results are discussed in relation to physiological and genetic implications.

2,4-Dichlorophenoxyacetic Acid as an aid to Seed Production when widely separated Solanum Species are crossed

AFTER repeated unsuccessful attempts to duplicate Swaminathan's1 technique for overcoming the cross incompatibility between Solanum species, a study was undertaken to determine the causes of seed failure when widely separated Solanum species are crossed. This work showed that some pollen germinated in all attempted crosses between several tuber-bearing series of the genus. Apparently normal pollen tubes were even observed at the bases of S. melongena L. styles when that species was pollinated by S. phureja Juz. and Buk.

Studies on the fertility of artificial poliploid plants II. Germination tests of stored pollen grains of Capsicum annuum L..

Germination tests of stored pollen grains of Capsicum annuum L. on the artificial media (1.5% agar +0.3Mol sucrose, pH. 5.2) were carried out using diploids and autotetraploids induced by colchicine treatment. The results were as follows. (a). Pollen fertility in 2x (70-95%) is about 10-20% higher than that in corresponding 4x. (b). Germintion percentage of stored pollen grains in 2x is always higher than that in 4cc. The germination power is observed to be lost in about 72 hours in 2x and in 48 hours in 4x. (c). The pollen-tubes in 4x often show deformity as forked or swollen, so that the germination percentage of pollen grains with normal pollen-tubes is below 20% in 4x, while it is above 40% in 2x. (d). The growth of pollen-tubes in 2x is faster than in 4x.

OVERCOMING BARRIERS TO CROSSABILITY DUE TO STYLE LENGTH

FOR SOME YEARS investigators working with lJatura have been trying to obtain crosses between all possible combinations of the ten species. Often in these attempted crosses some part of the process which ordinarily would lead to the formation of seed fails, and the resulting elimination of hybrid embryos leads us to refer to this cross as incompatible. It has been found, while trying to obtain hybrid embryos of Datura species, that several barriers to crossability cause the elimination of either the gametes or young hybrid embryos. In some cases hybrid embryos can be removed from their maternal environment when this surrounding tissue proves unfavorable and can be cultured in vitro and grown to maturity (Blakeslee, 1945). This technique has added a considerable number of species hybrids to those obtained by ordinary crossing methods. There is also another group of barriers which is considered to be independent of the former. In this group the incompatibility is due to the presence of the male gametophyte in a foreign style. Incompatibilities of pollen tube and grain include such factors as non-germination of pollen, slower growth rates and bursting of pollen tubes. Such conditions are found not only in interspecific crosses but also in gametophytic elimination of both balanced and unbalanced extra-chromosomal types (Buchholz and Blakeslee, 1929, 1932) and of some single gene mutations (Tricarpel, Buchholz and Blakeslee, 1927). Attempted intergeneric crosses using Datura as the female parent showed similar pollen-tube abnormalities (Sanz, 1945). Buchholz et al. (1935) investigated pollen-tube growth in both selfs and crosses of the ten species of Datura, charting the relative numbers of normal and burst pollen tubes and calculating the growth rates. By comparing these data with the results of interspecific crosses of Datura which have yielded fertile hybrid seed or dissectible embryos, we find that the greatest number of crosses yet to be obtained lies in the group which exhibits slow growth rate and high percentage of burst pollen tubes. Another factor contributing to incompatibility of these crosses is style length. In Datura species the styles range from 35-190 mm. in length. Most of the crosses which are yet to be' obtained involved the growth of the pollen of a short-styled flower in a much longer style of a different species.