The effect of anther-derived substances on pollen function was studied using pollen produced by in vitro culture of immature pollen of tobacco (Nicotiana tabacum L.) and petunia (Petunia hybrida). Addition of conditioned medium consisting of diffusates from in situ matured pollen strongly increased pollen germination frequency and pollen tube growth, as well as seed set after in situ pollination. Thin-layer chromatography and depletion of phenolic substances by Dowex treatment indicated that flavonols are present in the diffusate and may be the active compounds. When added to the germination medium, flavonols (quercetin, kaempferol, myricetin) but not other flavonoids strongly promoted pollen germination frequency and pollen tube growth in vitro. The best results were obtained at very low concentrations of the flavonols (0.15-1.5 Mm), indicating a signaling function. The same compounds were also effective when added during pollen development in vitro.Male gametophyte and gamete formation in plants occurs by a close interaction with the surrounding sporophytic tissues, particularly the tapetum (1,14,28). A variety of factors have been suggested to play a role in this interaction. However, their function is still largely unknown.Flavonoids are secondary plant products that include pigments (chalcones, anthocyanins) and colorless compounds (flavanones, flavones, and flavonols) that are involved in pollination, seed dispersal, UV light protection, and plant/ pathogen interaction (9,11,23). Flavonoids are present in pollen of many species of angiosperms and gymnosperms, as well as in spores of ferns and mosses (29). Flavonoid biosynthesis is initiated by chalcone synthase, followed by the synthesis of flavanones by chalcone isomerase. Recently, it was shown that the chs and chi genes are coordinately In vitro culture of isolated microspores has shown that an important function of the anther for the development of the microspores and pollen grains is the stepwise provision of low mol wt nutrients and anabolic precursors (2, 4,18,20). Germination frequency and seed set of this pollen are, however, lower compared with mature pollen taken directly from the plant (2, 20). Obviously, in vitro pollen lacks factors that are provided in vivo by the sporophyte. The in vitro pollen can thus be considered as 'minimal pollen' that fulfills the minimal requirements for pollination and fertilization but lacks factors for optimal reproductive success (28). It should, therefore, react very sensitively to added compounds that in vivo are provided by the anther wall. Here, we show that flavonols but not other flavonoids produced by the anther are present in diffusates of mature tobacco (Nicotiana tabacum L.) pollen and have a strong stimulatory effect on in vitro pollen development, pollen germination, and pollen tube growth. MATERIALS AND METHODS
We have detected a plant beta-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric beta-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45 degrees C. It is relatively resistant to proteases and insensitive to -SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by the Escherichia coli beta-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay the E. coli beta-glucuronidase in transgenic plants without interference from the endogenous plant activity.
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.
Immature pollen of two varieties of Triticum aestivum, at the stage right after the first pollen mitosis, was isolated from individual anthers and cultured in microcultures of microliter droplets. In a specifically designed medium, some of the pollen grains developed to maturity. These were applied to excised stigmas on agar, where they produced pollen tubes. Application to flowers in vivo led to seed set. Pollen was matured in vitro from a variety that produced a different protein banding pattern on SDS-PAGE as compared to the variety that was pollinated. The protein banding in the produced seeds showed the hybrid pattern, demonstrating that the seeds were not produced by self-pollination in this in-breeding species but by pollination with the in-vitro-matured pollen.
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