Recent progress on sperm characterization in flowering plants

The organization, isolation and physiology of the angiosperm male gamete has recently emerged as an area of special interest. Detailed in vivo studies have revealed: (1) that the functional unit of male reproduction is a'male germ unit,'composed of two sperm cells and a physically associated vegetative nucleus; (2) that the two sperm cells are often cytoplasmically dimorphic, and (3) that some sperm cells appear to undergo preferential fertilization, preferentially fusing with either the egg or the proendospermaric central cell. Male gamete isolation has provided enrichments of up to 10⁷ cells ml^–1, with sperm integrity and viability demonstrated by the flunrochromatic reaction and ATP levels. Although results from biochemical and hybridoma antibody characterization are still preliminary, they indicate that sperm cells possess polypeptide expression patterns that are different from those of surrounding cells. These findings suggest the existence of an independent developmental program in the angiosperm male gamete.     

Specific phosphoproteins in the initial period of tobacco pollen embryogenesis

Several phosphoproteins specifically correlated with the induction of embryogenic cells were detected in immature pollen grains of Nicotiana tabacum L. By regulating the concentration of glutamine in the medium the developmental pathways of immature pollen grains isolated at the mid-bicellular stage could be controlled, resulting in the formation of either mature pollen grains or embryogenic cells. Different phosphoproteins, designated as a-d and as e-i, respectively, were detected when the pollen grains either became embryogenic cells in glutamine-free medium, or when they were allowed to mature in glutamine-containing medium. The formation of embryogenic cells was suppressed by adding glutamine or cytokinin to the glutamine-free medium, nor did it occur with pollen grains at younger or older stages, and in these cases the phosphoproteins a-d were detectable only partially or faintly. The phosphoproteins a-d and e-i thus may be one of the factors necessary to direct the developmental pathway of immature tobacco pollen grains to embryogenic cells and to mature pollen grains, respectively.The authors thank Dr. V.S. Jaiswal (Botany Department, Banaras Hindu University, Varanasi, India) for his valuable suggestion in the preparation of the paper. This work was supported by a Grantin-Aid for special project research from the Ministry of Education, Science and Culture of Japan.     

Microspore ofSecale cereale as a transfer cell type

Summary In the mature microspore ofSecale cereale, a set of wall ingrowths deposited as the first (outer) intine layer between exine and the microspore plasma membrane, are revealed by electron microscopy. The wall ingrowths form a girdle in the vicinity of the apertural region at the external pole of microspore which is in contact with the tapetum, so the microspore can be considered as a transfer cell which is polarized. After microspore division the second (inner) intine layer is deposited by the vegetative cell and forms a labyrinth of branched wall ingrowths. As a result, the periphery of a vegetative cell is also irregular and appears as very thin plasmatubules or evaginations delimited by plasma membrane and penetrating the pollen wall.The possible functions of the microspore as a transfer cell and the wall-membrane system of the vegetative cell are discussed.     

Characterization of the translocator associated with pollen tube organelles

Summary In pollen tubes, the motive force of cytoplasmic streaming is assumed to be generated by the sliding of the translocator associated with cell organelles along actin filaments. In the present study, the characteristics of the translocator were studied by reconstituting the movement of pollen tube organelles along characean actin bundles. Movement of pollen tube organelles proceeded from the pointed end to the barbed end of the actin filaments of the characean cells. The reconstituted movement was not inhibited by vanadate. KCL at higher concentrations inhibited the movement. Furthermore, heavy meromyosin (HMM) prepared from rabbit skeletal muscle myosin partially inhibited the reconstituted movement and pCMB-modified HMM inhibited it completely. The present results strongly support our previous conclusion that the translocator which generates the motive force of cytoplasmic streaming in pollen tube is myosin.Abbreviations AMP-PNP adenylyl-imidodiphosphate - ATP adenosine-5-triphosphate - ATP--S adenosine-5-0-(3-thiotriphosphate) - BSA bovine serum albumin - CCCP carbonylcyanide m-chlorophenylhydrazone - DTT dithiothreitol - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - HB homogenization buffer - HMM heavy meromyosin - NEM N-ethylmaleimide - pCMB p-chloromercuribenzoic acid - PIPES piperazine-N,N-bis-(2-ethanesulfonic acid) - PPi pyrophosphate     

Generative cell composition and its relation to male plastid inheritance patterns inMedicago sativa

Summary Genetic studies have recently shown that plastids are inherited biparentally in alfalfa; yet most crosses produce high frequencies of progenies containing only paternal plastids, and certain genotypes have been characterized as relatively strong or weak transmitters of male plastids. The objective of the present study was to determine whether the structure of generative cells differs among genotypes known to differ in male plastid transmission pattern. Using the techniques of serial ultrathin sectioning and three-dimensional reconstruction, we found that mature generative cells of the genotypes investigated have basically similar morphology, and contain numerous plastids in each end of the spindle shaped cell. Since the morphological variation that does occur is as great within a genotype as it is between genotypes, it does not appear that generative cell structure can be used to predict male plastid transmission behavior in a particular genotype. The number of mitochondria in generative cells, which is much less than that of plastids, varies considerably among genotypes. However, comparable genetic studies between genotypes are not yet available on male mitochondrial inheritance in alfalfa.     

Rapid plant regeneration through organogenesis and somatic embryogenesis from cultured protoplasts of Brassica juncea

Protoplasts derived from hypocotyls of seedlings grown on half-strength MS medium containing 1% sucrose were cultured at a density of 5×10⁴ ml^-1 in Kao's medium supplemented with 1.0 mgl^-12,4-D, 0.1 mgl^-1 NAA and 0.5 mgl^-1 zeatin riboside. After three days of culture in darkness at 25±1°C, cultures were transferred to light (70 Em^-2s^-1) in a 16/8 h ligø ht/dark cycle. Cultures were diluted on the 7th, 10th and 13th day with Kao's medium containing 3.4% sucrose, 0.1 mgl^-1 2,4-dichlorophenoxyacetic acid and 1.0 mgl^-1 benzyladenine. On the fifteenth day, microcalli were plated on K₃ medium gelled with 0.5% agarose (Type 1, low EEO, Sigma). After a further period of two weeks, transfers were made to specific media to achieve either organogenesis or somatic embryogenesis. Time taken from plating protoplasts to obtaining plantlets is 8–10 weeks. Using this procedure, several hundred regenerated plants have been hardened in a growth chamber and transferred to soil.     

The effects of ruthenium red,lanthanum, fluorescein isothiocyanate and trifluoperazine on vesicle transport,vesicle fusion and tip extension in pollen tubes

The effects of ruthenium red, lanthanum, fluorescein isothiocyanate and trifluoperazine, all antagonists of Ca²⁺ function in cells, have been studied in growing pollen tubes of Tradescantia virginiana. All four drugs inhibit pollen-tube growth but bring about different ultrastructural changes at the growing tips and within the cytoplasm. The results strongly support the hypothesis that Ca²⁺ plays a vital role in the mechanism of pollen-tube tip growth. The effect of ruthenium red provides evidence that sequestration of Ca²⁺ by mitochondria critically adjusts the concentration of these ions at tube tips. Fluorescein isothiocyanate appears to be a potent inhibitor of vesicle fusion at the plasma membrane, with vesicles accumulating in the tip at rates equivalent to those determined previously for their production. Both vesicle fusion and tip extension are regulated by Ca²⁺ but appear to be independently controlled processes.     

Plant regeneration through somatic embryogenesis in protoplast cultures of sandalwood (Santalum album L.)

Summary Protoplasts were isolated from embryogenic cell suspension cultures derived from proliferating shoot segments of a 20-year-old sandalwood tree (Santalum album Linn.). Under appropriate conditions, isolated protoplasts divided in liquid culture medium and produced embryogenic cell aggregates and globular embryos. Plating of cell aggregates on a fresh medium facilitated the differentiation of somatic emryos which further developed into plantlets.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - GA₃ Gibberellic acid - IAA indoleacetic acid - IBA indolebutrytic acid - MES 2-(N-morpholino)ethane sulfonic acid - MS Murashige and Skoog's medium as modified in the text     

“Egg transformation” induced by irradiated pollen in Nicotiana: a re-examination

Summary We have attempted to confirm previous reports of egg transformation in seven Nicotiana species, including genetic stocks obtained from the original experimenter. The methods employed in the original experiments were duplicated as closely as possible. In total, 1,622 pollinations were made involving irradiated pollen and mixtures of irradiated and self pollen. Consequently, 995 seedlings from 9,052 seeds were screened for 1,594 potentially detectable transformation events. A very low frequency of unexpected progeny resulted, but these results were not repeatable and appear to have arisen by mechanisms other than transformation. These results are strongly at odds with previous claims, when 50% of offspring were found to be transformed. We conclude that the previous observations of high frequency egg transformation are not reproducible. However, due to the ambiguous nature of the markers employed, it is very difficult to prove that such transformation events do not occur as very rare events.     

In-situ seed production after pollination with in-vitro-matured,isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.Abbreviations cms cytoplasmic male sterility; AMGLU, MS, M2S, MR26 - GK culture media, see Material and methods