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Microspore embryogenesis is the most commonly used method to produce doubled haploids. It is based on the ability of a single haploid cell, the microspore, to de‐differentiate and regenerate into a whole plant after being exposed to stresses, such as low or high temperatures, carbon starvation and colchicine. Some stresses such as temperature treatments and carbon starvation have been used with success in many plant species, whereas others such as colchicine had limited application in a few species. Reports on the application of whole plant treatments with feminizing agents on inflorescences and buds are scarce. Furthermore, the technical means to apply some stresses such as γ‐irradiation are not readily available. Recently, novel stresses such as pH, inducer chemicals, carrageenan oligosaccharides and heavy metals were reported to induce microspore embryogenesis. It remains to be seen, however, whether these stresses are effective in a wider range of species. Finally, pretreatment of cultured cells with high concentrations of 2,4‐D efficiently induces somatic embryogenesis in several species (carrot, alfalfa). However, reports on the use of this particular chemical stress are not available in microspore embryogenesis. The paper presented here gives an overview of various stresses and mechanisms of action of these stresses in inducing microspore embryogenesis.

SUMMARYThe plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole-3-acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin-related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate-limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester-conjugated IAA. Activation of the auxin-regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1-naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum-localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxindependent transcriptional activity.

The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type- microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo.

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

Reverse breeding (RB) is a novel plant breeding technique designed to directly produce parental lines for any heterozygous plant, one of the most sought after goals in plant breeding. RB generates perfectly complementing homozygous parental lines through engineered meiosis. The method is based on reducing genetic recombination in the selected heterozygote by eliminating meiotic crossing over. Male or female spores obtained from such plants contain combinations of non-recombinant parental chromosomes which can be cultured in vitro to generate homozygous doubled haploid plants (DHs). From these DHs, complementary parents can be selected and used to reconstitute the heterozygote in perpetuity. Since the fixation of unknown heterozygous genotypes is impossible in traditional plant breeding, RB could fundamentally change future plant breeding. In this review, we discuss various other applications of RB, including breeding per chromosome.

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