Pollen Protoplasts Research Articles

Exploding pollen in Montrichardia arborescens (Araceae)

Pollen grains of Montrichardia are inaperturate with psilate ornamentation. The pollen wall is formed by a thin ectexine and an extraordinarily thick intine. In living as well as in dead pollen grains contact with water leads to a rapid swelling of the intine followed by an explosive opening of the exine. Within a few seconds a thick tube is formed, which is not the pollen tube. The pollen protoplast is situated at the tip of the tube. These intine tubes are interpreted as pollen connecting tools to keep pollen grains together and adhere them to the cuticle of the hairless pollinators.

Quantum Dot-Mediated Detection of γ-Aminobutyric Acid Binding Sites on the Surface of Living Pollen Protoplasts in Tobacco

gamma-Aminobutyric acid (GABA) is an inhibitory transmitter in the central nervous system of mammals. Recent investigations showed that it also plays an important role in regulating pollen tube growth and orientation in plants. To determine whether GABA receptors are also present on the membrane of pollen protoplasts, a fluorescence probe of quantum dots (QDs) was constructed and applied. The water-soluble CdSe-ZnS (core-shell) QDs were first synthesized and verified to possess good optical properties. GABA was then bioconjugated to the QDs in the presence of 1-ethyl-3-(3)-dimethylaminopropyl carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to make the fluorescence probe. Using the probe, GABA binding sites were detected on the protoplast membrane of both pollen and somatic cells. Both the fluorescent signals on the surface of the protoplasts and the Ca(2+) oscillation assayed via the Ca(2+) probe Fluo-3/AM inside the protoplasts provided evidence that the potential GABA(B) receptors are present on the plant protoplast membrane.

Differentiation of generative and vegetative cells by unequal cell division

Summary: Cellular differentiation of a generative and a vegetative cell during pollen development is requisite for sexual plant reproduction and is initiated by an unequal(asymmetric)cell division. The smaller, generative cell produces two sperm cells, or male gametes, whereas the larger, vegetative cell produces an elongated pollen tube, agametophytic cell, to deliver the male gametes to the embryo sac. The distinct differences between two kinds of cell are found in organelle composition and cytoskeletal organization in their cytoplasm. Recent transcriptome analysis also revealed the difference in gene expression profiles between both cells. We pay attention to the difference of theirchromatin configuration, that is assumed to be associated with the difference in gene activity between the two types of nucleus. Namely, the generative nucleus has highly condensed chromatin, as do the nuclei of animal sperm, whereas the vegetative nucleus contains diffuse chromatin. For this reason, by developing an efficient method to isolate the generative and vegetative nuclei from lily pollen protoplasts, their histone composition was investigated biochemically and immunocytochemically. Expression analysis of isolated histone genes was also done. As a result, we detected, in addition to 1)replication-dependent, somatic types of histones common to both nuclei, 2)replication-independent, generative and sperm nuclei-specific histone variants(gH 2 A, gH 2 B and gH 3)and 3)replication-independent, vegetative nucleus-specific histone variant(H 3. 3). The significance of this discovery will be discussed with respect to the function and developmental fates of both cells in the process of sexual plant reproduction.

Characterization of Phosphatidylinositol-Specific Phospholipase C (PI-PLC) from Liliumdaviddi Pollen

The phosphatidylinositol-specific phospholipase C (PI-PLC) activity is detected in purified Lilium pollen protoplasts. Two PI-PLC full length cDNAs, LdPLC1 and LdPLC2, were isolated from pollen of Lilium daviddi. The amino acid sequences for the two PI-PLCs deduced from the two cDNA sequences contain X, Y catalytic motifs and C2 domains. Blast analysis shows that LdPLCs have 60-65% identities to the PI-PLCs from other plant species. Both recombinant PI-PLCs proteins expressed in E. coli cells show the PIP(2)-hydrolyzing activity. The RT-PCR analysis shows that both of them are expressed in pollen grains, whereas expression level of LdPLC2 is induced in germinating pollen. The exogenous purified calmodulin (CaM) is able to stimulate the activity of the PI-PLC when it is added into the pollen protoplast medium, while anti-CaM antibody suppresses the stimulation effect caused by exogenous CaM. PI-PLC activity is enhanced by G protein agonist cholera toxin and decreased by G protein antagonist pertussis toxin. Increasing in PI-PLC activity caused by exogenous purified CaM is also inhibited by pertussis toxin. A PI-PLC inhibitor, U-73122, inhibited the stimulation of PI-PLC activity caused by cholera toxin and it also leads to the decrease of [Ca(2+)](cyt) in pollen grains. Those results suggest that the PPI-PLC signaling pathway is present in Lilium daviddi pollen, and PI-PLC activity might be regulated by a heterotrimeric G protein and extracellular CaM.

A rapid, efficient method for the mass production of pollen protoplasts from Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast.

An optimized protocol was established to isolate large numbers of mature living pollen protoplasts of Pinus bungeana Zucc. ex Endl. and Picea wilsonii Mast. Intact pollen grains of P. bungeana or pollen with short tubes were incubated with gentle agitation in a solution of 2% cellulase R-10, 1.5% macerozyme R-10, 15% sucrose, 0.01% H 3BO 3, and 0.01% CaCl 2. Intact pollen protoplasts with diameters of 40 μm were liberated, with an isolation rate of up to 70% after 6 h of enzymatic incubation. The optimal pH and temperature for the reaction were 5.8 and 24 °C, respectively, and the optimal enzymatic digestion conditions were 6 h of incubation in the above solution. The method for isolating pollen protoplasts from P. wilsonii was similar to that for P. bungeana, except that the incubation medium contained 12% rather than 15% sucrose and the optimal enzyme concentrations were 3% cellulase and 2% macerozyme. The isolated pollen protoplasts were demonstrated to be living by microscopy in a fluorochromatic reaction with fluorescein diacetate (FDA).

Ca2+ Influx into Lily Pollen Grains Through a Hyperpolarization-activated Ca2+-permeable Channel Which Can be Regulated by Extracellular CaM

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.

Ca2+-permeable channels in the plasma membrane of Arabidopsis pollen are regulated by actin microfilaments.

Cytosolic free Ca2+ and actin microfilaments play crucial roles in regulation of pollen germination and tube growth. The focus of this study is to test the hypothesis that Ca2+ channels, as well as channel-mediated Ca2+ influxes across the plasma membrane (PM) of pollen and pollen tubes, are regulated by actin microfilaments and that cytoplasmic Ca2+ in pollen and pollen tubes is consequently regulated. In vitro Arabidopsis (Arabidopsis thaliana) pollen germination and tube growth were significantly inhibited by Ca2+ channel blockers La3+ or Gd3+ and F-actin depolymerization regents. The inhibitory effect of cytochalasin D (CD) or cytochalasin B (CB) on pollen germination and tube growth was enhanced by increasing external Ca2+. Ca2+ fluorescence imaging showed that addition of actin depolymerization reagents significantly increased cytoplasmic Ca2+ levels in pollen protoplasts and pollen tubes, and that cytoplasmic Ca2+ increase induced by CD or CB was abolished by addition of Ca2+ channel blockers. By using patch-clamp techniques, we identified the hyperpolarization-activated inward Ca2+ currents across the PM of Arabidopsis pollen protoplasts. The activity of Ca2+-permeable channels was stimulated by CB or CD, but not by phalloidin. However, preincubation of the pollen protoplasts with phalloidin abolished the effects of CD or CB on the channel activity. The presented results demonstrate that the Ca2+-permeable channels exist in Arabidopsis pollen and pollen tube PMs, and that dynamic actin microfilaments regulate Ca2+ channel activity and may consequently regulate cytoplasmic Ca2+.

Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

The formation of the polarity of pollen protoplast and the dynamics of actin cytoskeleton were observed by non-fixation, Alexa-Phalloidin probing and confocal laser scanning microscopy. Our results showed that the protoplast obtained from stored pollen contained numerous crystalline fusiform bodies to constitute a storage form of actin. When dormant pollen was hydrated, the actin cytoskeleton forms a fine network spreading uniformly in the protoplast. In the process of polarity formation and germination of pollen protoplast, actin filaments marshaled slowly to the brim, and then formed multilayer continuous actin filament bundles surrounding the cortical of the protoplast. When the protoplast was exposed to actin filament-disrupting drugs, such as Latrunculin A and Cytochalasin D, continuously arranged actin bundles were disturbed and in this condition, the protoplast could not germinate. But when exposed to actin filament stabiling drug-phalliodin, the dynamics of actin filaments in the protoplasts behaved normally and the protoplasts could germinate normally. These results were also confirmed by the pharmacology experiments on pollen grains. And when Latrunculin A or Cytochalasin D was washed off, the ratio of pollen germination was resumed partly. All the results above show that the dynamic organization of the actin cytoskeleton are critical in the cell polarity formation and germination of pollen protoplast, and that the reorganization of actin cytoskeleton is mainly due to the rearrangement of actin filament arrays.

Identification and characterization of stretch-activated ion channels in pollen protoplasts.

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.

Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

Dynamic organization of actin cytoskeleton during the polarity formation and germination of pollen protoplasts

Lily ( Lilium longiflorum L.) pollen protoplast adhesion is increased in the presence of the peptide SCA

The style of lily produces a specialized extracellular matrix (ECM) in the transmitting tract epidermis that functions to guide pollen tubes to the ovary. This adhesive ECM contains low esterified pectins and a peptide, SCA (stigma/stylar cysteine-rich adhesin). Together they form a matrix to which pollen tubes adhere as they grow through the style. Pollen tubes also adhere to each other but only when grown in vivo, not in vitro. Pollen does not produce detectable SCA, but when SCA is added to an in vitro growth medium, it binds to pollen tubes that have esterified and low-esterified pectins in their walls. Since adhesion of the pollen tube to the stylar matrix requires tip growth, we hypothesized that the pectin wall at the pollen tube tip interacted with the SCA protein to initiate adhesion with the stylar pectin [Lord (2000) Trends Plant Sci 5:368–373]. Here, we use a pollen protoplast system to examine the effect of SCA on protoplast adhesion when it is added to the growth medium in the absence of the stylar pectin. We found that SCA induces a 2-fold increase in protoplast adhesion when it is added at the start of protoplast culture. This effect is less when SCA is added to the medium after the cell wall on the protoplast has begun to regenerate. We show that among the first components deposited in the new wall are arabinogalactan proteins (AGPs) and highly esterified pectins. We see no labeling for low esterified pectins even after 3 days of culture. In the pollen protoplast culture, adhesion occurs in the absence of the low esterified pectin. The newly formed wall on the protoplast mirrors that of the pollen tube tip in lily, which is rich in AGPs and highly esterified pectins. Thus, the protoplast system may be useful for isolating the pollen partner for SCA in this adhesion event.

Outward K+ channels in Brassica chinensis pollen protoplasts are regulated by external and internal pH.

Patch-clamp whole-cell and single-channel recording techniques were used to investigate the regulation of outward K(+) channels by external and internal protons in Brassica chinensispollen protoplasts. Outward K(+) currents and conductance were insensitive to external pH (pH(o)) except at pH 4.5. Maximal conductance (G(max)) for the outward K(+) currents was inhibited at acidic external pH. Half-activation voltage ( E(1/2)) for the outward K(+) currents shifted to more positive voltages along with the decrease in pH(o). E(1/2) can be described by a modified Henderson-Hasselbalch equation expected from a single titratable binding site. The activation kinetics of the outward K(+) channels was largely insensitive to pH(o). An internal pH (pH(i)) of 4.5 significantly increased outward K(+) currents and conductance. G(max) for the outward K(+) currents decreased with elevations in pH(i). In contrast to the effect of pH(o), E(1/2) was shifted to more positive voltages with elevations in pH(i). The outward K(+) currents, G(max) and E(1/2) can be described by the modified Henderson-Hasselbalch equation. Furthermore, acidifying pH(i) accelerated the activation of the outward K(+) currents significantly. The differences in electro-physiological properties among previously reported and currently described plant outward K(+) channels may reflect differences in the structure of these channels.

Pollen coat proteins after two-dimensional gel electrophoresis and pollen wall ultrastructure of Secale cereale and Festuca pratensis

This paper reports results of two-dimensional gel electrophoresis analysis of pollen coat and pollen protoplast proteins of self-incompatible and self-fertile Secale cereale as well as pollen collected from Festuca pratensis populations and selected self-sterile plants. Washing pollen 10 times in isotonic buffer showed that the first and second fractions contained the majority of the pollen coat proteins. Results of protein analysis are discussed against the background of pollen wall ultrastructure. A fraction of peptides found in the pollen coat were also present in the protein patterns of protoplasts; however, numerous pollen coat peptides were not detected in the protoplast and vice versa. The self-incompatible S. cereale had 23 pollen coat peptides and 46 from protoplasts that differed in molecular weight (MW) and isoelectric point (IP) in comparison to those of pollen coat and protoplasts of self-fertile S. cereale. Similarly, self-sterile F. pratensis had 60 pollen coat peptides and 11 protoplast peptides different from those of the self-sterile/self-fertile F. pratensis. The pollen coat fraction of the self-incompatible S. cereale and the self-sterile plants of F. pratensis had three peptides with very similar MW and IP, whereas in their protoplasts two peptides with similar MW and IP were found. The possible relationship between pollen ultrastructural organisation and rate of protein elution is discussed.

First patch, then catch: measuring the activity and the mRNA transcripts of a proton pump in individual Lilium pollen protoplasts

Combining the patch-clamp method with single-cell reverse transcription polymerase chain reaction (scRT-PCR) a fusicoccin-induced current reflecting the activity of the plasma membrane H + ATPase of lily pollen protoplasts was measured and subsequently, the ATPase-encoding mRNAs were collected and amplified. Southern blot signals were observed in all ‘patch–catch’ experiments and could be detected even in 2560-fold dilutions of the pollen contents. H + ATPase mRNAs were detectable only in the vegetative but not in the generative cell of pollen as confirmed by immunolocalisation. In 15% of the scRT-PCR experiments, a random non-reproducibility of the PCR was observed, probably caused by varying amounts of ATPase mRNAs in the protoplasts.

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.

Identification and characterization of the inward K+ channel in the plasma membrane of Brassica pollen protoplasts.

Patch clamp techniques have been used to identify and characterize the whole-cell currents carried by inward K+ channels in isolated matured pollen protoplasts of Brassica chinensis var. chinensis. The whole-cell inward currents in the isolated pollen protoplasts were activated at hyperpolarized membrane potentials more negative than -100 mV. The magnitudes of the whole-cell inward currents were strongly dependent on the external K+ concentration, and were highly selective for K+ over other monovalent cations. The inward currents were not observed when external K+ was replaced with the same concentration of Cs+ or Na+. The addition of 1 mM or 10 mM Ba2+ in external solutions resulted in 30% or 80% inhibition of the inward currents at -180 mV, respectively. These results demonstrated that the inward K+ currents mainly account for the recorded whole-cell inward currents in Brassica pollen protoplasts. Increase of cytoplasmic Ca2+ concentrations from 10 nM to 30 microM or even 5 mM did not affect the inward K+ currents. Decrease of external Ca2+ concentrations from 10 mM to 1 mM inhibited the inward K+ currents by 25%, while the increase of external Ca2+ from 10 mM to 50 mM almost completely blocked the inward K+ currents. Physiological importance of K+ transport into pollen and its possible regulatory mechanisms are also discussed.

Identification of pollen components regulating pollination-specific responses in the stigmatic papillae of Brassica oleracea.

When components of the mature pollen grain of Brassica oleracea are applied to the cuticularised surfaces of the stigmatic papilla, a number of changes can take place in the architecture of the subjacent cell wall. Treatment of the papillar surface with isolated pollen coating evokes a rapid and extensive expansion of the outer of the two stigmatic wall layers. This response occurs within 4 h but is restricted to those regions where no callose is formed and where the coating exhibits a characteristic increase in electron opacity. This study establishes that the coating alone is responsible for initiating the expansion of the outer wall, which is considered to be an essential step preceding penetration of the stigma surface by the pollen tube. Vesicle-like inclusions, some staining intensely, occasionally occur in regions of the wall expanded by isolated coating, sometimes fusing to form a subcuticular 'boundary layer'. These structures are not observed under compatible grains in vivo and their presence is regarded as artefaetual. However, close examination of the plasma membrane at sites beneath areas of expanded cell wall reveals membrane-bound structures resembling the vesicles being generated by the cytoplasm and moving into the apoplast. These data indicate that signals from the coating initiate the loosening of the wall matrix by stimulating an unusual form of localized secretion which appears to be an essential prerequisite for stigmatic penetration. Isolated pollen coating contaminated with fragments of the grain itself engender a very different response, including the formation of densely-staining vesicles accompanied by extensive accumulations of callose; wall expansion never occurs under these circumstances. Self-pollinations in B. oleracea are frequently accompanied by the synthesis of stigmatic callose, the presence of which has, in the past, been interpreted as forming a structural barrier to incompatible pollen tubes. However, callose elicited both by self-pollinations and coating supplemented by killed grains is independent of protein synthesis and occurs in the presence of the phosphatase inhibitor, okadaic acid. Since both these inhibitors also overcome self-incompatibility (SI) in B. oleracea, these data strongly suggest that callose synthesis is unrelated to the operation of SI, and it is proposed that callose, when formed, is elicited by molecules released from the necrotic pollen protoplast. Interestingly, isolated pollen coating of self- and cross-genotypes does not readily elicit callose. The significance of these changes in the stigmatic wall is discussed in the perspective of current views on pollination and self-incompatibility in Brassica.

High level of GUS gene expression driven by pollen-specific promoters in electroporated lily pollen protoplasts

Gene constructs that contained the β-glucuronidase (GUS) gene under the control of a pollen-specific Zm13 promoter from maize and a LAT52 promoter from tomato were introduced by electroporation into pollen protoplasts isolated from bicellular pollen grains of Lilium longiflorum. After 20 h in culture, the pollen protoplasts exhibited the apparent expression of GUS in a fluorometric assay. The GUS activity induced under the control of the Zm13 promoter was over 10 000 times higher than activity in the control (with no DNA or without electroporation). By contrast, the GUS gene was nearly silent in the lily microspore protoplasts and generative cell protoplasts. The GUS activity driven by the Zm13 and LAT52 promoters was also detected by a cytochemical assay. The frequency of blue-staining pollen protoplasts was about 70% in the case of the Zm13 promoter. The efficiency of gene transfer by electroporation was much higher than by particle bombardment. This protoplast-specific electroporation system is suitable for rapid and reliable examination of pollen-specific promoters, being as good as the particle bombardment system.

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg(-1) H2O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca(2+) method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.

Regeneration and characterization of plants produced from mature tobacco pollen protoplasts via gametosomatic hybridization

Mature pollen protoplasts (n) isolated from kanamycin resistant plants of Nicotiana tabacum (2n = 4x = 48) were fused with somatic mesophyll protoplasts (2n) of Nicotiana plumbaginifolia (2n = 20) to produce plants. A total of 3.6·10(6) mature pollen protoplasts were fused with 7·10(6) mesophyll protoplasts using a PEG/Ca(2+) method. Mature pollen protoplasts did not divide in our culture conditions, and N. plumbaginifolia protoplasts stopped dividing when the protoplast-derived colonies were transferred to a selection medium containing paromomycine (20 mg·l(-1)). A total of 133 actively growing colonies were recovered on the selection medium containing kanamycin (100 mg·l(-1)). Plants from twenty resulting cell lines were confirmed as hybrids (17) or cybrids (3) based on leaf and floral morphology and fertility analysis. Isozyme pattern analysis confirmed the nuclear hybrid and cybrid nature, respectively, for 2 and 3 typical gametosomatic selected plants. Root tip squashes of 6 of the gametosomatic hybrid plants revealed chromosome numbers ranging from 44 to 68; the 3 selected cybrid plants had 48 chromosomes. Evidence for organelle transmission from the mesophyll partner in the gametosomatic plants is shown. From the analysis it can be concluded that the gametosomatic fusion involving mature pollen protoplasts (n) carrying a dominant selection marker can be convenient for synthesis of either hybrids or cybrids. Such gametosomatic fusion is therefore considered as a new approach towards the production of androgenetic plants with a choosen cytoplasm.