Insights into a Key Developmental Switch and Its Importance for Efficient Plant Breeding Mei Wang*, Sandra van Bergen, and Bert Van Duijn - Perspective asupra unui comutator de dezvoltare cheie i importana acesteia pentru eficient Ameliorarea plantelor Mei Wang * , Sandra van Bergen , iar Bert Van Duijn

Sexual reproduction of plants starts with the formation of gametes and a 2-fold reduction in the number of chromosomes (ploidy) of these cells. Fertilization, the union of sperm and egg cell restores the ploidy level to its normal value again. During the formation of pollen grains, the structures that contain the male gametes, the reduction in the number of chromosomes happens when the vegetative cells undergo meiosis and give rise to microspores. In a diploid plant with 2n chromosomes the microspores are haploid with n chromosomes. Microspores normally develop into pollen grains, but as a result of an unusual event, microspores can also undergo a developmental process that leads to the formation of a haploid embryo. This androgenesis pathway is also called microspore embryogenesis or regeneration. During androgenesis the haploid microspore divides and develops into an embryo and subsequently into a new haploid plant. Haploid plants are not fertile because they cannot make gametes again since this would require another halving of the chromosome number. If chromosome doubling occurs at some stage during androgenesis, the regenerated plants from these microspores are completely homozygous (doubled haploid) fertile individuals. Such doubled haploid plants from haploid microspores provide excellent material for research, plant breeding, and plant transformation. However, androgenesis is not a naturally occurring event in angiosperms and only seems to be induced as a result of certain chemical and physical stimuli. - Reproducerea sexual a plantelor ncepe cu formarea de gamei i o reducere de 2 ori a numrului de cromozomi (ploidiei) ale acestor celule. Fertilizare, unirea de spermatozoizi i ovule restabilete nivelul ploidie la valoarea normal din nou. Pe parcursul formrii granulelor de polen, structurile care conin grneilor masculini, reducerea numrului de cromozomi se ntmpl cnd celulele vegetative supuse meiozei i dau natere la microspori. ntr-o instalaie diploid cu cromozomi 2n microsporilor sunt haploid cu n cromozomi. Microspori dezvolte normal n granule de polen, ci ca urmare a unui eveniment neobinuit, microspori poate suferi, de asemenea, un proces de dezvoltare care duce la formarea unui embrion haploid. Aceasta cale androgenesis se mai numete i embriogeneza microsporului sau regenerare. n timpul androgenesis se divide microspor haploizi i dezvolt ntr-un embrion i, ulterior, ntr-o nou fabric haploid. Plantele haploide nu sunt fertile, deoarece acestea nu pot face gamei din nou, deoarece acest lucru ar necesita un alt njumtire a numrului de cromozomi. Dac dublarea cromozomilor are loc la un moment dat n timpul androgenesis, plantele regenerate din aceste microsporii sunt complet homozigote (dublat haploid) persoanele fertile. Astfel de plante haploide dublat de microspori haploide ofer excelent material pentru cercetare, creterea plantelor, i de transformare a plantelor. Totui, androgenesis nu este un eveniment care are loc in mod natural in angiosperme i numai pare a fi indus ca urmare a unor stimuli chimici i fizici. Since androgenesis involves the control and reprogramming of developmental switches, it provides opportunities to investigate key elements in developmental control. Moreover, via androgenesis, fertile homozygous progeny from a heterozygous parent can be obtained in a single generation, thus significantly reducing time required in breeding programs and providing a major advantage in preparing F1 hybrid seeds as well. Both the possibilities for studying basic processes in plant development and the economic importance have motivated numerous research groups to investigate androgenesis in different crops. Here, the basic steps in androgenesis induction protocols, different processes and the signals involved in reprogramming of the pollen development pathway, the possible molecular markers, and the evolutionary perspective are considered. - Deoarece androgenesis implic controlul i reprogramarea de switch-uri de dezvoltare , ofer oportuniti de a investiga elemente-cheie n controlul de dezvoltare . Mai mult dect att , prin androgenesis , descendeni homozigot fertil dintr -un printe heterozigot poate fi obinut ntr-o singur generaie , reducnd astfel semnificativ timpul necesar n programele de ameliorare i oferind un avantaj major n pregtirea semine hibride F1 , de asemenea. Att posibilitile de a studia procesele de baz n dezvoltarea plantelor i importana economic au motivat multe grupuri de cercetare pentru a investiga androgenesis n diferite culturi . Aici , paii de baz n androgenesis protocoale de inducie , diferite procese i semnalele implicate n reprogramarea calea dezvoltrii polenului , posibile markeri moleculari , iar perspectiva de evolutie sunt luate n considerare. LABORATORY METHODS FOR INDUCING ANDROGENESIS The induction of androgenesis has been tested in a significant number of economically important plants. In monocotyledonous plant species such as wheat, rice, maize, barley, rye, and sorghum, different protocols for anther/microspore regeneration have been investigated. For the dicots, many reports concern Brassica sp., and some other plant species such as Datura sp., Nicotiana sp., potato, apple, and sunflower. Most protocols for androgenesis induction/ microspore regeneration for breeding purposes have been developed by trial and error. In general, such protocols consist of different phases. As a typical example, the barley microspore regeneration protocol can be divided into the following major steps as shown in Figure 1: (a) pretreatment of the plant material: the right developmental stages of anthers are used for a stress pretreatment for approximately 4 d (Fig. 1, AD); (b) microspore culture: microspore isolation from pretreated anthers and subsequent microspore culture (Fig. 1D). After 4 d, cell division can be observed (Fig. 1E), and after 14 d, formation of multicellular structures takes place (Fig. 1F); and (c) development of multicellular structures into embryos or embryo-like structures (Fig. 1G) and further development into young plants (Fig. 1H), which requires an additional 21 d. - METODE DE LABORATOR pentru inducerea ANDROGENESIS Inducerea androgenesis a fost testat ntr-un numr semnificativ de plante importante punct de vedere economic. La speciile de plante monocotiledonate, cum ar fi grul, orezul, porumbul, orzul, secara i sorg, diferite protocoale de antere / regenerare microsporului au fost investigate. Pentru dicotiledonate, multe rapoarte se refer Brassica sp., i alte specii de plante, cum ar fi Datura sp., Nicotiana sp., Cartofi, mere, i floarea soarelui. Cele mai multe protocoale pentru androgenesis regenerare inducie / microspor pentru reproducere au fost dezvoltate prin ncercare i eroare. In general, aceste protocoale constau din diferite faze. Ca un exemplu tipic, protocolul orz microsporului regenerare poate fi mprit n urmtoarele etape principale dup cum se arat n Figura 1: (a) pre-tratament al materialului vegetal: drepte etapele de dezvoltare ale anterele sunt folosite pentru o pretratare stres timp de aproximativ 4 d ( Figura 1, A-D).; (B) cultura microspor: izolare microspor de antere pretratate si cultura microspor ulterioare (Fig 1D.). Dup 4 d, diviziunea celular poate fi observat (Figura 1E.), Iar dup 14 d, formarea unor structuri multicelulare are loc (figura 1F.); i (c) dezvoltarea unor structuri multicelulare n embrioni sau structuri-embrion ca (Fig. 1G) i dezvoltarea n continuare n plante tinere (Fig. 1 H), care necesit o suplimentare de 21 d. Depending on the plant species, and even the plant variety, many variations of the basic protocol exist. The variations can be found in the type of plant material used for the pretreatment (e.g. whole flowers, isolated anthers, or isolated microspores), the type of stress pretreatment (e.g. temperature, starvation, or osmotic stress), and the type of cultures (e.g. microspore culture or anther culture). However, these protocols always follow the same basic scheme as summarized in Figure 1. The main bottlenecks for the practical approach are the quality of the donor material, the type of pretreatment, recognition of the occurrence of the developmental switch, the right culture conditions for the induction of embryogene- - n funcie de speciile de plante , i chiar varietatea de plante , multe variante ale protocolului de baz exist . Variaiile pot fi gsite ntipul de material vegetal folosit pentrupretratarea ( ex flori ntregi , antere izolate, fie microspori izolate ) ,tipul de stres pretratare ( de exemplu temperatur , nfometare , sau stres osmotic ) , itipul de culturi ( de exemplu, cultura microspor sau antere cultur ) . Cu toate acestea , aceste protocoale urmeaz ntotdeauna aceeai schem de baz ca rezumate n figura 1. Principalele blocaje pentru abordarea practic sunt calitatea materialului donator , tipul de pretratare , recunoaterea apariia comutatorului de dezvoltare , condiiile de cultur adecvate pentru inducerea embryogene-sis, and subsequent embryo development, germination, and outgrowth to a new plant. Donor Material The results of microspore regeneration experiments are highly dependent on the variety used, the growth condition of the plants, and the quality of the donor material. The conditioning of the plant growth is important, as exemplified by the significant effects of season on regeneration efficiency (Foroughi-Wehr and Mix, 1979). The natural flowering conditions (light intensity, day-length, temperature regime, humidity, etc.) are normally the best environment for donor plants to produce anthers to be used in successful regeneration experiments. Any infection or stress to the donor plants will lead to less success or complete failure for induction of androgenesis and further regeneration. The age of the plant material can also highly influence the regeneration efficiency. For example, in barley, the first five spikes show 15% to 20% higher regeneration efficiency than the later spikes. However, in the tree Aesculus carnea the older trees (60 and 100 years) give a higher success rate of androgenic anthers (approximately 20%) than the younger trees (2040 years; approximately 9%) (Marinkovic and Radojevic, 1992). Besides plant age, the microspore developmental stage that is for the best regeneration varies between different species. For barley, the microspores should be at the late uninucleate stage (Hoekstra et al., 1997, and refs. therein), whereas in rye the best regeneration efficiency is obtained from microspores at the starch granule stage (Immonen and Anttila, 1998). - sis, si dezvoltarea embrionului ulterioare, germinare, i rod a unei noi fabrici. Donator Material Rezultatele experimentelor regenerare microsporului sunt foarte dependente de soiul folosit, starea de cretere a plantelor, precum i calitatea materialului donator. Condiionarea creterea plantelor este important, aa cum este exemplificat de efectele semnificative ale sezonului privind eficiena de regenerare (Foroughi-Wehr i Mix, 1979). Condiiile nflorire naturale (intensitate lumina, zi-lungime, regimul de temperatur, umiditate, etc.) sunt n mod normal cel mai bun mediu pentru plante donator pentru a produce antere pentru a fi utilizate n experimente de regenerare de succes. Orice infecie sau stres plantele donatoare va conduce la mai puin succes sau eec total pentru inducerea androgenesis i regenerare n continuare. Vrsta de material vegetal pot, de asemenea, extrem de influenta eficienta de regenerare. De exemplu, la orz, primele cinci piroane arat eficiena regenerare 15% pn la 20% mai mare dect piroane ulterioare. Cu toate acestea, n arborele Aesculus carnea copacilor mai mari (60 si 100 de ani) ofera o rata de succes mai mare anterelor androgenice (aproximativ 20%) dect copacii tineri (20-40 de ani; aproximativ 9%) (Marinkovic i Radojevi 1992) . n afar de vrst a plantelor, stadiul de dezvoltare microspor care este pentru cel mai bun regenerarea variaz ntre diferite specii. Pentru orz, microsporilor trebuie s fie cel etapa uninucleat trzie (Hoekstra i colab., 1997, i referinele. Aici), n timp ce n secar se obine cea mai bun eficien de regenerare de microspori la etapa de granule de amidon (Immonen i Anttila, 1998). Variety-dependent success rates are well known. Different and sometimes closely related cultivars show different responsiveness to the same regeneration protocol. For example, the barley cv Igri has a regeneration efficiency 8.5%, whereas with the same regeneration protocol in the cv Digger only 0.85% regeneration efficiency was obtained (Van Bergen et al., 1999). Pretreatments/Induction of the Developmental Switch Once the donor material containing the microspores has been selected, microspore development - Ratele de succes depind de soi sunt bine cunoscute . Diferite i uneori strns nrudite soiuri arat reacie diferit laacelai protocol de regenerare . De exemplu ,cv orzul IGRI are o eficien de regenerare 8,5 % , n timp ce cuacelai protocol de regenerare ncv fost obinut excavator doar 0,85 % randament regenerare ( Van Bergen colab . , 1999 ) . Pretratamente / Inducereaswitch Developmental Dupmaterialul coninnd donor microsporilor a fost selectat , dezvoltarea microsporuluiFigure 1. Scheme of microspore regeneration in barley. A, Developing spike within ensheathed leaves, original size is approximately 10 cm; B, isolated spike, original size approximately 9 to 10 cm; C, dissected flower with ovary (o) and anthers (a), original flower size is approximately 1 cm, original anther size is approximately 0.4 cm; D, isolated microspores after 4 d of anther pretreatment with mannitol, the enlarged embryogenic microspore (g) with red interference (diameter approximately 60 mm) and nonembryogenic microspore (b) with blue/black interference (diameter approximately 2030 mm); E, dividing microspore, diameter approximately 70 mm; F, multicellular structure derived from microspore, diameter approximately 0.2 cm; G, formation of a young plant from an embryo-like structure derived from a multicellular structure, diameter approximately 0.4 cm; H, young regenerated barley plant in culture medium. - Figura 1. Schema de regenerare microsporului n orz . A, Spike dezvoltarea n frunze ensheathed , dimensiunea original este de aproximativ 10 cm ; B , Spike izolat , dimensiunea original aproximativ 9-10 cm ; C , floare disecat cu ovar ( o) i anterele ( a) , dimensiune floare iniial este de aproximativ 1 cm , originalul anterei dimensiune este de aproximativ 0,4 cm ; D , microspori izolai dup 4 d din antere pretratare cu manitol ,microspor embriogenic extins ( g ) cu interferen rou ( diametru aproximativ 60 mm ) i microspor nonembryogenic ( b ) cu albastru interferen / negru ( cu diametrul de aproximativ 20-30 mm ) ; E , mprind microspor , diametru de aproximativ 70 mm ; F , structura multicelulare derivat din microspor , diametru de aproximativ 0,2 cm ; G , formarea unei plante tinere de la o structur - embrion ca derivat dintr -o structur multicelular , diametru de aproximativ 0,4 cm ; H , plante tinere de orz regenerat n mediu de cultur . has to be switched from the gametophytic pathway to a sporophytic development. Induction of this switch requires specific pretreatment conditions, which usually consist of the application of different stresses to the plant material. The pretreatment can be applied at different levels of explants, such as intact flowers (e.g. for barley complete spikes), isolated anthers, or isolated microspores. With regard to different explants, the type, levels, and duration of pretreatments are different, and the regeneration efficiencies vary as well. The pretreatment of barley can be a cold treatment of spikes or a pretreatment in a mannitol solution of anthers. These different treatments result in different regeneration efficiencies (cold pretreatment of spikes results in a 1% regeneration efficiency, whereas pretreatment of anthers with a combination of starvation and osmotic stress results in a 8.5% regeneration efficiency). When the pretreatment was applied to isolated barley microspores, no plants could be obtained by regeneration. However, mannitol pretreatment of isolated tobacco microspores (Touraev et al., 1997) or heat shock pretreatment of freshly isolated Brassica napus microspores (Custers et al., 1996) have proven to be successful methods for induction of microspore regeneration. - trebuie s fie trecut de la calea gametofitic unei dezvoltri sporofitice. Inducerea acest parametru sunt necesare condiii de pretratament specifice, care constau de obicei din aplicarea diferitelor solicitrile la materialul vegetal. Pretratare poate fi aplicat la diferite niveluri de explante, cum ar fi flori intacte (de exemplu, pentru orz crampoane complet), antere izolate, fie microspori izolate. n ceea ce privete diferite explante, tipul, nivelul i durata de pretratamente sunt diferite, iar eficiena de regenerare variaza, de asemenea. Pretratarea orz poate fi un tratament la rece de crampoane sau o pretratare ntr-o soluie de manitol anterelor. Aceste tratamente diferite ca rezultat randamente diferite regenerare (pretratare rece de vrfuri are ca rezultat o eficien de regenerare 1%, n timp ce tratamentul prealabil anterelor cu o combinaie de nfometare i rezultatele stres osmotic ntr-o eficien regenerare 8,5%). Cnd pretratamentul a fost aplicat la microspori orz izolate, fr plante ar putea fi obinut prin regenerare. Cu toate acestea, pretratare manitol de microspori tutun izolate (Touraev colab., 1997) sau pretratare oc termic al proaspt izolate microspori Brassica napus (Custers et al., 1996) s-au dovedit a fi metode de succes pentru inducerea regenerrii microsporului. Cell Culture and Embryogenesis After the pretreatment, the microspores are cultured in a specific culture medium where cell division and differentiation occur. It has been suggested that in this phase the doubling of chromosomes (forming a doubled haploid) is taking place due to aberrations in the mitosis in callus formation or due to nuclear fusion in early division. The type of culture medium, the cell density, the application of plant growth regulators, and conditioned media all affect the regeneration efficiency (e.g. Hoekstra et al., 1997, and refs. therein; Manninen, 1997). At this stage of androgenesis, the induction of cell division is a bottleneck with a variety of possible solutions for different species and cultivars. Once a multicellular structure is formed, two different developments can occur. In the first, the structure develops into an embryo-like structure (Fig. 1, F and G) and via an in vitro zygotic-like embryo development pathway, a plant is formed. In the second, the structure will give rise to a callus from which secondary embryogenesis can be induced to form plants. We found that the number of structures showing cell division is much higher than the number of embryos formed (S. Van Bergen and M. Wang, unpublished data). So, the pathway to undifferentiated callus formation is more likely to occur than the route to embryo formation. Culture condition variations, especially hormones and sugars, have important effects on these two separate pathways (e.g. Hoekstra et al., 1997, and refs. therein; Manninen, 1997). - Cell Cultura si embriogeneza Dup pretratarea, microsporilor sunt cultivate ntr-un mediu de cultur specific n care apar diviziunea i diferenierea celular. Sa sugerat c n aceast faz dublarea cromozomilor (formarea unei dublat haploid) are loc din cauza aberaiilor n mitozei n formarea de calus sau ca urmare a fuziunii nucleare n diviziunea devreme. Tipul de mediu de cultur, densitatea celular, aplicarea regulatorilor de cretere a plantelor, precum i mediu condiionat toate afecteaz eficiena de regenerare (de exemplu Hoekstra i colab, 1997, i refs acesta;.. Manninen, 1997). n acest stadiu al androgenesis, inducerea diviziunii celulare este o strangulare cu o varietate de soluii posibile pentru diferite specii i soiuri. Odat ce o structura multicelulare este format, pot aprea dou evoluii diferite. n primul rnd, structura se dezvolt ntr-o structur-embrion ca (Fig 1, F i G.) i printr-o dezvoltare a embrionilor cale-zygotic ca in vitro, o plant se formeaz. n dou, structura va da natere unui calus de unde embriogeneza secundar poate fi indus pentru a forma plante. Am constatat c numrul de structuri care prezint diviziunii celulare este mult mai mare dect numrul de embrioni de format (S. Van Bergen i M. Wang, date nepublicate). Astfel, calea spre formarea de calus nedifereniat este mai probabil s apar dect drumul spre formarea embrionului. Variaii stare cultur, n special hormoni i zaharuri, au efecte importante asupra acestor dou ci separate (de exemplu, Hoekstra i colab, 1997, i ref acesta;.. Manninen 1997). Once an embryo is formed from the microsporederived multicellular structure, this embryo will usually be transferred to a solid culture medium for induction of embryo growth (Fig. 1, G and H). MORPHOLOGICAL CHANGES DURING REPROGRAMMING OF POLLEN DEVELOPMENT In the three major steps discussed so far (pretreatment processes, microspore culture into multicellular structures, development of embryos) for microspore regeneration, the first step is key for the determination of the efficiency of regeneration (observed as cell division and differentiation). Early markers for induction of androgenesis, being morphological, biochemical, or molecular, and more information about the molecular basis of stress-induced cell division are essential for both understanding the mechanism of androgenesis and breeding applications. Only understanding of the androgenesis process and unequivocal markers will enable us to control microspore regeneration in a species-independent manner to significantly reduce the time span of a wide variety of breeding programs. From the protocols it can be concluded that for the production of doubled haploid plants from microspores via androgenesis, a specific developmental stage of the microspores in combination with a stress treatment of anthers and microspores cultured at a specific density is essential (e.g. Heberle-Bors, 1989; Hoekstra et al., 1997, and refs. therein). These conditions induce a reprogramming of the pollen development that is barely understood. To gain a better understanding of the processes and working mechanisms involved in the control of this developmental switch, studies on the morphology and cell biological aspects of anthers and microspores during androgenesis were carried out. These studies not only resulted in useful parameters for protocol design but also show the complexity of the process and, moreover, the involvement of different cell types present in anther tissue. - Odat ce un embrion este format din structura multicelulare microsporederived, acest embrion, de obicei, va fi transferat la un mediu de cultur solid pentru inducerea creterii embrion (Fig. 1, G i H). MORFOLOGICE modificri n timpul REPROGRAMAREA polen dezvoltrii n trei pai importani discutat pn acum (procese de pretratare, cultura microspore n structurile multicelulare, dezvoltarea de embrioni) pentru regenerarea microspore, primul pas este cheie de determinare a eficienei de regenerare (observ diviziunea celular i difereniere). Markeri devreme pentru a androgenesis, fiind morfologic, biochimic sau molecular i mai multe informaii despre baza molecular a diviziunii celulare induse de stres sunt eseniale pentru nelegerea mecanismului de androgenesis i aplicaii de reproducere. Doar intelegerea procesului de androgenesis i fr echivoc markeri ne va permite s controlai microspore regenerare n mod independent de specii pentru a reduce semnificativ durata de timp de o mare varietate de programe de reproducere. La protocoalele se poate concluziona c pentru producia de plante haploizi dublat de microspores prin intermediul androgenesis, un stadiu de dezvoltare specifice de microspores n combinaie cu un tratament de stres anterele i microspores de cultur la o densitate specifica este esenial (ex. Heberle-Bor, 1989; Hoekstra et al., 1997, i refs. acolo). Aceste condiii determina o reprogramare polen de dezvoltare, care este abia neles. Pentru a obine o mai bun nelegere de procese i mecanisme de lucru implicate n controlul de acest comutator dezvoltare, studii de morfologie i celule aspecte biologice ale anterele i microspores n timpul androgenesis au fost efectuate. Aceste studii nu numai a dus la parametri de utile pentru Protocolul de proiectare dar, de asemenea, Arat complexitatea procesului i, mai mult dect att, implicarea diferite celule tipurile prezente n antere esutului.Microspore Morphology During pretreatment, part of the population of microspores changes into cells that are called embryogenic microspores. These microspores have the potential to complete sporophytic development. The embryogenic microspores can be recognized microscopically by their increased size (swollen to 5060 mm in diameter for barley; Fig. 1D), whereas nonembryogenic microspores (the microspores that develop either into pollen or enter a cell death pathway) have a much smaller size (30 mm in diameter for barley; Fig. 1D). Other parameters vary for different varieties. For instance barley embryogenic microspores have a red interference color of the exine wall (Fig. 1D), whereas the nonembryogenic microspores can - Microspor Morfologia timpul pretratare , o parte a populaiei de microsporilor modificari in celule , care sunt numite microspori embriogenice . Aceste microsporii au potenialul de a finaliza dezvoltarea sporofitice . Microsporilor embrionare pot fi recunoscute la microscop prin mrimea lor a crescut ( umflate la 50-60 mm n diametru pentru orz , . Figura 1D ) , n timp ce microsporii nonembryogenic ( microsporilor care se dezvolta , fie n polen sau introducei o cale de celule moarte ) au o mult mai mic dimensiune ( 30 mm diametru pentru orz ; . figura 1D ) . Ali parametri variaz pentru diferite soiuri . De exemplu orz microspori embriogenice au o culoare roie interferen aperetelui numit exin ( fig. 1D ) , n timp ce microsporilor nonembryogenic potbe recognized microscopically by their blue/black interference color of the exine wall (Fig. 1D). After successful pretreatment, the swollen microspores form an extremely large vacuole that pushes the nucleus to the periphery of the cell close to the cell wall (Fig. 2). - fie recunoscute microscopic de albastru / negru culoarea interferen a peretelui numit exin (Fig . 1D ) . Dup pretratare succes , microsporilor umflate fac vacuole extrem de mare care mpingenucleul laperiferiacelulei aproapeperetelui celular (Fig . 2) . Pretreatment-Induced Changes in Anther Tissue During successful pretreatment of whole anthers, changes in cell wall properties of anther tissue are observed (Sunderland et al., 1984). The developing barley microspores (or mature pollen grains) are localized within the anther loculus (Fig. 2, L), the wall of which consists of three layers of cells (Fig. 3A). The loculus is lined by a layer of nutritive cells, the tapetum (Fig. 3A, T). The cells in the middle layer of the loculus wall contain chlorophyll. A rapid loss of chlorophyll and degeneration of tapetum and anther wall cells were observed in stress pretreated anthers (Hoekstra, 1996; Fig. 3B). After 4 d of pretreatment, the tapetal cells have disappeared, and the innermost layer of loculus cells is crushed (Fig. 3B). The death of anther tapetal cells is much more rapid during pretreatment conditions than during non-pretreatment conditions (Wang et al., 1999). The tapetum is a specialized cell layer between the sporogenous tissue and the anther wall, which functions as a source of nutrients for developing pollen grains (e.g. Pacini et al., 1985). The ultimate fate of these cells is death when pollen grains mature. During stress pretreatment, these type of cells are apparently most sensitive to stress and enter a cell death pathway. The death of these cells may release signals that are able to trigger susceptible microspores to enter a sporophytic development pathway (dedifferentiation and division). The degradation products of cell wall components from the tapetal cells - Indus de pretratare Schimbri n esutul anterei timpul pretratare succes anterelor ntregi, se observ modificri ale proprietilor ale peretelui celular al esutul anterei (Sunderland .a., 1984.). Cele dezvoltare microsporilor orz (sau granulele de polen mature) sunt localizate n interiorul loculus anterei (fig. 2, L), a crei perete este format din trei straturi de celule (Fig. 3A). Loculus este cptuit cu un strat de celule nutritive, tapet (fig. 3A, T). Celulele din stratul de mijloc al peretelui loculus conin clorofil. O pierdere rapid de clorofil i degenerare a tapetum i antere celule de perete au fost observate n stres pretratate antere (Hoekstra, 1996, fig. 3B). Dup 4 d din pretratare, celulele tapetale au disprut, iar stratul intern al celulelor loculus este zdrobit (fig. 3B). Moartea anterei celulelor tapetale este mult mai rapid dect n condiii de pretratament n condiii non-pretratare (Wang et al., 1999). Tapet este un strat de specialitate celul ntre esutul sporogene i peretele anterei, care funcioneaz ca o surs de nutrieni pentru dezvoltarea granule de polen (ex Pacini et al., 1985). Soarta final a acestor celule este moartea, atunci cnd granulele de polen matur. Timpul stresului pretratare, acest tip de celule sunt aparent cel mai sensibil la stres i introducei o cale de celule moarte. Moartea acestor celule pot elibera semnale care sunt capabile s declaneze microsporii susceptibile de a introduce o cale de dezvoltare sporofitice (dedifereniere i diviziune). Produsele de degradare ale componentelor peretelui celular din celulele tapetalecould be considered as signal molecules in this process. SIGNALS AND SIGNAL TRANSDUCTION DURING ANDROGENESIS Complete control over androgenesis in a speciesindependent protocol obviously requires full understanding of the signals and signal transduction involved in the reprogramming of pollen development. From the above it may be concluded that essential signal molecules may be produced by different tissues in the pretreated anther as well as by the nonsporophytic microspores in a cell density-dependent way. As pretreatment involves the application of stresses, the stress hormone abscisic acid (ABA) and its signal transduction pathways are mainly considered as signaling factors of importance. Although no other signaling molecules strongly involved in the regulation of androgenesis have been reported, new factors still may be found in e.g. conditioned medium of microspore cultures. - ar putea fi considerate ca molecule semnal n acest proces . Semnale i de transmitere a semnalului TIMPUL ANDROGENESIS controlul complet asupra androgenesis ntr -un protocol speciesindependent necesit n mod evident nelegere deplin a semnalelor i traducerea semnalului implicate n reprogramarea dezvoltrii polenului . Dinsus , se poate concluziona c moleculele eseniale semnalului pot fi obinute prin diferite esuturi n antereitratat n prealabil , precum i de microsporilor nonsporophytic ntr-un mod dependent de densitate celular . Ca pretratare implic aplicarea subliniaz ,hormonul stresului acidul abscisic ( ABA ) i cile sale de transmitere a semnalului sunt considerate in principal ca factori de importan semnalizare . Dei nici alte molecule de semnalizare puternic implicate in reglementarea androgenesis au fost raportate , noi factori nc pot fi gsite de exemplu n mediu condiionat de culturi microsporului . Figure 2. Transmission electron microscope photograph of ultrastructure of a pretreated microspore within an anther loculus (L). Part of the microspore with large vacuole (V) pushing the nucleus (N) to the periphery of the cell can be seen. For the orientation of the tissue, the orbicules (O) are indicated. Asterisk indicates an anther wall cell. Bar 5 0.7 mm. - Figura 2. Transmisie microscop electronic fotografie a ultrastructura unui microsporului pretratate ntr-o loculus anter (L ) . O parte dinmicrosporului cu vacuolar mare ( V ) mpingndnucleul ( N) pentru aperiferiacelulei poate fi vzut . Pentruorientareaesutului , sunt indicate orbicules ( O) . Asterisc indic o celul perete anter . Bar 5 0,7 mm . Role of ABA As the pretreatments applied to anthers and microspores are stress treatments generally associated with ABA signaling, a role for this stress hormone in the reprogramming of pollen development has been postulated. As early as 1980, Imamura and Harada proposed that a specific level of ABA was required for induction of androgenesis because they found a peak in endogenous ABA level after 24 h of mannitol pretreatment in tobacco anthers. Similarly, in barley anthers, the starvation and mannitol stress pretreatment induces a peak in ABA levels within 24 h (Van Bergen et al., 1999). Results showing that ABA addition in combination with suboptimal pretreatment - Rolul ABA Deoarece pretratare aplicate anterele i microspori sunt tratamente de stres n general asociate cu ABA semnalizare , un rol pentru acest hormon de stres nreprogramarea dezvoltrii polenului a fost postulat . nc din anul 1980, Imamura i Harada a propus , care a fost nevoie de un nivel specific de ABA pentru inducerea androgenesis deoarece au gsit un vrf n nivelul ABA endogen dup 24 de ore de manitol pretratare din antere tutun . In mod similar , n antere orz ,pretratarea nfometare i manitol stresul induce un vrf n nivelurile ABA decurs de 24 ore ( Van Bergen et al., 1999 ) . Rezultatele arat c adugarea ABA , n combinaie cu pretratare suboptimalrestores regeneration efficiency (Van Bergen et al., 1999) and that fluridone inhibition of de novo ABA synthesis reduces regeneration efficiency (Hoekstra et al., 1997) strongly indicate a role for ABA in androgenesis. In addition, positive effects of ABA addition during pretreatment were shown for wheat androgenesis (Hu et al., 1995) and tobacco androgenesis (Kyo and Harada, 1986). Furthermore, endogenous ABA levels are important for morphogenic competence, and ABA application enhances somatic embryogenesis, whereas the inhibition of ABA synthesis in donor plants causes the loss of capacity for embryogenesis in Pennisetum sp. (Rajasekaran et al., 1987). The difference in regeneration efficiency of the barley cv Igri and cv Digger (high and low efficiency, respectively) is also reflected in different pretreatmentinduced ABA peak levels in these cultivars (high followed by slow decrease and low followed by sharp decrease for cv Igri and cv Digger, respectively) (Van Bergen et al., 1999). It was recently demonstrated in barley that there is a strong correlation between stress-induced ABA production and microspore viability and that addition of ABA to suboptimal pretreatment levels enhanced microspore viability (Van Bergen et al., 1999; Wang et al., 1999) and repressed pollen tube formation (S. Van Bergen and M. Wang, unpublished data). It is likely that by preventing cell death, the stressinduced increase in ABA affects the regeneration efficiency - reface eficienta de regenerare (Van Bergen et al., 1999) i c fluridone inhibarea sintezei de novo ABA de reduce eficiena de regenerare (Hoekstra et al., 1997) puternic indica un rol pentru ABA n androgenesis. n plus, efectele pozitive ale ABA plus timpul pretratarea erau indicat pentru gru androgenesis (Hu i colab., 1995) i tutun androgenesis (Kyo i Harada, 1986). n plus, endogene ABA niveluri sunt importante pentru morphogenic competen, i ABA cererea mbuntete embriogeneza Somatica, ntruct inhibarea ABA sinteza n donator plante provoac pierderea de capacitate pentru embriogeneza n Pennisetum sp. (aurel et al., 1987). Diferena n eficiena de regenerare de orz cv Igri i CV-ul excavator (nalt i redus de eficien, respectiv), de asemenea, se reflect n nivelurile de vrf de diferite pretreatmentinduced ABA in aceste soiuri (nalt urmat de scderea lent i mic urmat de scderea accentuat pentru cv Igri i CV-ul Digger, respectiv) (Van Bergen et al., 1999). Acesta a fost demonstrat recent n orz c exist o corelaie puternic ntre producia de Alessandro induse de stres i microspore viabilitatea i acel plus de ABA la niveluri sub nivelul optim de amplasament microspore mbuntit viabilitatea (Van Bergen et al., 1999; Wang et al., 1999) i reprimate polen tube formarea (S. Van Bergen i M. Wang, nepublicate date). Este probabil c, prin prevenirea moartea celulelor, creterea stressinduced ABA afecteaz eficiena regenerareapositively by increasing the number of viable microspores available for androgenesis. Moreover, the effect of ABA was not only a viability increase, but also a reduction in the number of binucleate microspores. These binucleate microspores show DNA degradation and are likely to be in the process of dying (Van Bergen et al., 1999). It is likely that the effects of ABA are on at least two different processes: (a) preventing the death of microspores, thus increasing the number of viable microspores during pretreatment and (b) repressing further development of microspores into mature pollen. As the experiments involving ABA measurements during pretreatment were carried out in anther tissue, it is not clear which cells produce the stressinduced ABA. Further investigations may reveal the ABA origin and a possible role for specific anther cells in the induction of the developmental switch. - pozitiv prin creterea numrului de microspori viabile disponibile pentru androgenesis . Mai mult dect att ,efectul ABA a fost nu numai o cretere viabilitate , ci i o reducere anumrului de microspori binucleate . Aceste microsporii binucleate arat degradarea ADN-ului si sunt susceptibile de a fi n procesul de moarte ( Van Bergen et al . , 1999) . Este probabil ca efectele ABA sunt pe cel puin dou procese diferite : ( a) care mpiedicmoartea microspori , crescnd astfelnumrul de microspori viabile n timpul pretratare i ( b ) reprim dezvoltarea ulterioar a microspori n polen matur . Ca experimentele care implic msurtori ABA timpul tratamentul prealabil au fost efectuate n esutul anterei , nu este clar care celulele producABA stressinduced . Investigaiile suplimentare pot dezvluioriginea ABA i un posibil rol pentru celulele antere specificeinducereacomutatorului de dezvoltare . Figure 3. Transmission electron microscope photograph of ultrastructure of developing (A) and pretreated (B) anther wall cells (as indicated by asterisks). L indicates the anther loculus in which the microspores are localized. For the orientation of the tissue, the orbicules (O) are indicated. Barley anthers have four loculi with a central vascular region. Each loculus is composed of three layers of sterile anther wall cells (A, asterisks), which surround a single layer of tapetal cells (T). The tapetal cell layer separates the wall layers from the interior-most layer of microspores. The wall cells provide structural support for the anther loculus. Chlorophyll (C) can be seen in the middle layer of developing anther wall cells and the majority of this chlorophyll is lost after 4 d of mannitol pretreatment (B). In this pretreated anther, tapetal cells are lost and the inner-most layer of anther wall cell layer is crushed and only the rest of debris can be seen (see in B). The cell content (asterisks in B) is also lost in pretreated anther wall cells. A, Bar 5 2 mm. B, Bar 5 1 mm. - Figura 3. Transmiterea microscop electronic fotografie a ultrastructura dezvoltrii (A) i pretratate (B) antere celulele peretelui (indicat prin asteriscuri). L indic loculus anterei n care microsporilor sunt localizate. Pentru orientarea esutului, sunt indicate orbicules (O). Anterele orz au patru loculi cu o regiune vascular central. Fiecare loculus este compus din trei straturi de sterile antere celulele peretelui (A, asteriscuri), care nconjoar un singur strat de celule tapetale (T). Stratul celular tapetal separ straturile peretelui din stratul interior cea mai mare parte microspori. Celulele de perete ofer sprijin structural pentru loculus anterei. Clorofila (C) poate fi vzut n stratul de mijloc de a dezvolta antere celulele peretelui i majoritatea acestui clorofilei se pierde dup 4 d din pretratare manitol (B). In acest antere pretratate, celulele tapetale se pierd i stratul interior, de cele mai multe anterei strat de celule de perete este zdrobit i doar restul de resturi poate fi vzut (vezi n B). Coninutul de celule (asteriscuri n B) este, de asemenea, a pierdut n pretratate antere celule de perete. O, Bar 5 2 mm. B, Bar 5 1 mm. ABA Signaling Although the involvement of ABA in microspore regeneration is apparent, the involvement of ABA signal transduction components and ABA-induced genes is scarcely reported. Reynolds and Crawford (1996) demonstrated that there was a direct and positive correlation with an increase of ABA and expres- - ABA Convertoare de semnalizare Deiimplicarea ABA n regenerarea microsporului reiese ,implicarea componentelor transducie semnal ABA i gene induse cu ABA este abia raportat . Reynolds i Crawford ( 1996) au demonstrat c exist o corelaie direct i pozitiv cu o cretere de ABA i exprimaresion of an early metallothionein gene in developing pollen embryoids. As the mitogen-activated protein (MAP) kinase signaling cascade is associated with abiotic stresses, ABA signaling, cell cycle, and growth control in plants (e.g. Hirt, 2000), it is tempting to speculate that the stress-induced ABA signaling may activate a MAP kinase cascade inducing cell division and differentiation in microspores. A first indication for MAP kinase involvement in microspore development was reported by Prestamo et al. (1999) who demonstrated an increased expression and cellular distribution of a MAP kinase in strongly vacuolated pepper microspores. These microspores do not enter androgenesis but are at the verge of a mitotic division just before the developmental switch could be made. Stress-induced MAP kinase activation might induce equal cell division instead of asymmetric cell division at this point. - sion unei gene metalotionein devreme n dezvoltarea embrioizi polen . Ca proteina activat mitogen- ( MAP ) kinaza cascada de semnalizare este asociat cu tensiuni abiotici , ABA semnalizare , ciclului celular , i controlul creterii n instalaii (de exemplu, Hirt , 2000 ) , este tentant sa speculeze caABA semnalizare indus de stres pot activa o diviziune celulara MAP kinaza cascada inducerea i diferenierea n microspori . Un prim indiciu pentru implicarea MAP kinaza in dezvoltarea microsporului a fost raportat de Pre'stamo et al . ( 1999 ) care a demonstrat o exprimare crescut i distribuia celular a unei kinaze MAP n microspori piper puternic vacuolizat . Aceste microsporii nu intr androgenesis , dar sunt la un pas de o diviziune mitotic nainte comutatorul de dezvoltare ar putea fi . MAP activare induse de stres kinaza ar putea induce diviziunea celulara egal n loc de diviziunii celulare asimetrice n acest moment . GENE EXPRESSION AND MARKER GENES FOR ANDROGENESIS To understand the mechanism of the androgenic pathway and to find specific markers, it is important to identify genes that are developmentally specific for androgenesis, i.e. the developmental switch from the gametophytic to the sporophytic pathway. There are many reports on gene expression related to natural microspore development, zygotic, or somatic embryogenesis. These will not be considered here as the major bottleneck in microspore regeneration is the induction of the developmental switch into androgenesis. To date there is only limited information concerning this developmental switch in microspore regeneration. During induction of androgenesis, there is a requirement for altered synthesis and accumulation of mRNAs and proteins in embryogenic microspores, leading to a sporophytic type of cell division. Raghavan (1981) demonstrated that the synthesis of de novo mRNA in Hyoscyamus niger androgenic microspores occurred during the 1st h of culture, and Pechan et al. (1991) identified mRNAs and proteins that appear to be associated with embryogenesis of Brassica sp. microspores. Up to now, only a few genes have been isolated that are specifically expressed during the early stage or switch of microspore embryogenesis (Touraev et al., 1997). Reynolds and Crawford (1996) identified an ABA-responsive gene that is expressed in the early stage of wheat microspore embryogenesis. In Brassica sp. microspores, a high expression of seed storage napin genes, and synthesis of a specific heat shock protein are correlated with embryogenic induction (Boutilier et al., 1994; Custers et al., 1996). It has been demonstrated that a peroxiredoxin anti-oxidant is predominately expressed in anther-derived cells that show embryogenic potential (Stirn et al., 1995). - Expresia genelor si gene marker PENTRU ANDROGENESIS Pentru a nelege mecanismul de calea androgen i de a gsi markeri specifici, este important s se identifice genele care sunt n dezvoltare specifice androgenesis, adic trecerea de dezvoltare la gametofitic la calea sporofitice. Exist multe rapoarte privind expresia genelor legate de dezvoltarea natural microsporului, zygotic sau embriogeneza somatic. Acestea nu vor fi luate n considerare aici ca principala piedic n regenerarea microspor este inducerea comutatorului de dezvoltare n androgenesis. Pn n prezent exist doar informaii limitate cu privire la acest comutator de dezvoltare n regenerarea microsporului. Pe parcursul inducerea androgenesis, exist o cerin pentru sinteza alterat i acumularea de ARNm i proteine in microspori embriogenice, ceea ce duce la un tip sporofitice de diviziune celular. Raghavan (1981) au demonstrat c sinteza de novo ARNm n Hyoscyamus niger microsporii androgen avut loc n timpul 1 h de cultur, i Pechan et al. (1991) a identificat ARNms i proteine care par a fi asociate cu embriogeneza de Brassica sp. microspori. Pn n prezent, numai cteva gene au fost izolate, care sunt exprimate n mod specific n timpul stadiu incipient sau comutatorul de microsporului embriogenezei (Touraev et al., 1997). Reynolds i Crawford (1996) a identificat o gena-ABA receptiv, care este exprimat n stadiul incipient al microsporului gru embriogenezei. In Brassica sp. microspori, o expresie nalt a genelor napin depozit din semine, i sinteza unei proteine specifice oc termic sunt corelate cu inducerea embriogen (Boutilier .a., 1994;.. Custers .a., 1996). S-a demonstrat c un anti-oxidant peroxiredoxin este exprimat predominant n antere-derivate din celule care prezint potenial embriogen (Stirn 1995 colab.). More recently, using differential screening of barley cDNA libraries, three cDNAs have been identified that might be markers for early microspore embryogenesis (Virnten et al., 1999). One of them has homology to lipid transfer proteins. In carrot, a lipid transfer protein cDNA EP2 (embryogenesis protein 2) has been reported as a marker for embryogenic potential in somatic cell suspension (Sterk et al., 1991). It is clear that the identification of genes involved in microspore embryogenesis is still in its infancy. More genes specific for the early processes in the microspore regeneration process need to be identified and their function established. For this, model systems that are well characterized at the molecular level and the regeneration protocol level are needed. Although the Arabidopsis system is powerful for gene identification and studying gene function, the bottleneck of using this system is the lack of an efficient Arabidopsis microspore regeneration protocol. - Mai recent , utiliznd selecia diferenial a coleciilor ADN orz , trei ADNc au fost identificate care ar putea fi markeri pentru timpurie embriogeneza microsporului ( Virnten et al., 1999 ) . Unul dintre ei are omologie cu proteinele de transfer lipidic . ntr morcov , o protein de transfer a lipidelor ADNc EP2 ( protein embriogeneza 2 ) a fost raportat ca un marker pentru potenialul embriogen n suspensie de celule somatice ( Sterk colab . , 1991 ) . Este clar c identificarea genelor implicate in microspor embriogeneza este nc n faz incipient . Mai multe gene specifice pentru primele procese n procesul de regenerare microspor trebuie s fie identificate i funcia lor stabilit . Pentru aceasta, este nevoie de sisteme de modele care sunt bine caracterizate lanivel molecular inivel de protocol de regenerare . Dei sistemul Arabidopsis este puternic pentru identificarea genelor si functia genei studierea , gtuire de folosirea acestui sistem este lipsa unui protocol eficient de regenerare Arabidopsis microsporului . EVOLUTIONARY POINT OF VIEW: COULD MOSSES AND FERNS SHOW THE WAY TO A UNIVERSAL PROTOCOL? In the life cycle of plants, the diploid phase that produces spores is called the sporophyte, whereas the haploid phase that produces the gametes is called the gametophyte. In an evolutionary development perspective from algae to flowering plants, the prominence in the appearance of the plant shifted from the gametophyte to the sporophyte. In mosses, the haploid gametophyte is the dominant structure, whereas in the monocots and dicots, the gametophytes are reduced to extremely small structures, embryo sac and germinated pollen, embedded in the highly dominant sporophyte. In mosses and ferns the gametophyte is formed from the haploid spores. In both mosses and ferns the spores divide to form a multicellular structure called protonema. In mosses the protonema develops further into plants consisting of leave and stem structures, whereas in the ferns the protonema remains small with gameteforming structures. The spore development in mosses and ferns shows clear parallels with the androgenesis process. In both cases the haploid spore produced by the sporophyte divides mitotically and develops into a (haploid) multicellular structure, and both processes start with an increase of the cell volume. So we may hypothesize that during the evolution of plants, the spore development pathway into multicellular structures was greatly shortened in favor of direct gamete formation, but that this pathway is still present and can be activated as is shown in androgenesis. If so, the available knowledge about spore germination and development of mosses and ferns may be very useful to understand and control androgenesis in monocots and dicots plants. In addition, mosses and ferns may - PUNCT de vedere evolutiv: POT muchi i ferigi arata calea pentru o UNIVERSAL PROTOCOL? In ciclul de via al plantelor, faza diploid produce spori care se numete sporofitul, n timp faza haploid care produce gameii se numete gametofitul. ntr-o perspectiv de dezvoltare evolutiv din alge la plante cu flori, proeminena n aspectul plantei mutat din gametofitul la sporofitul. n muchi, gametofitul haploid este structura dominant, n timp ce n monocotiledonate i dicotiledonate, gametofiilor sunt reduse la structuri extrem de mici, de embrioni sac i polenului germinat, ncastrate n sporofite puternic dominante. n muchi i ferigi gametofitul este format din sporii haploizi. In ambele muchi i ferigi sporii divid pentru a forma o structur multicelulare numit protonema. n muchii protonema dezvolt n continuare n plante compuse din structuri de concediu i stem, n timp ce n ferigi protonema rmne mic cu structuri gameteforming. Dezvoltarea spori n muchi i ferigi prezinta paralele clare cu procesul androgenesis. n ambele cazuri, sporul haploid produs de sporofitul mparte mitotic i dezvolt ntr-o (haploid) structura multicelulare, i ambele procese s nceap cu o cretere a volumului de celule. Deci, am putea emite ipoteza c n timpul evoluia plantelor, calea de dezvoltare spori n structurile pluricelulare a fost mult scurtat n favoarea formrii grneilor direct, dar c aceasta cale este nc prezent i poate fi activat dup cum este prezentat n androgenesis. Dac este aa, cunotinele disponibile despre germinarea sporilor si dezvoltarea muchilor i ferigi poate fi foarte util pentru a nelege i de a controla androgenesis la monocotiledonate i plante dicotiledonate. n plus, muchi i ferigi mayprovide valuable model systems for research on developmental switches involved in androgenesis. Several mosses are currently studied as model systems for plant development and reproduction, and mutants with defects in spore development and protonema formation are available (for reviews, see Cove and Knight, 1993; Cove et al., 1997). In addition, external factors affecting spore germination have been studied for a long time. Light, calcium, auxin, and cytokinin all play a role in the initial phases in the formation of the multicellular structure of Physcomitrella sp. (Cove and Knight, 1993). In spore germination of different mosses and ferns a stimulating effect of red light, low pH, gibberellins, nitrate, and low temperature has been reported (e.g. Haas et al., 1992; Whittier and Moyroud, 1993). In the light of the above these factors may also be considered in the development of microspore regeneration protocols. - furniza sisteme valoroase model pentru cercetarea pe switch-uri de dezvoltare implicate n androgenesis . Mai multe muschi sunt n prezent studiate ca sisteme model pentru dezvoltarea plantelor si reproducere , i mutani cu defecte de dezvoltare spor i formarea protonema sunt disponibile ( pentru comentarii, vezi Cove i Knight , 1993; Cove et al ., 1997 ) . In plus , factorii externi care afecteaz germinarea sporilor au fost studiate pentru o lung perioad de timp . Lumina , calciu , auxin , iar cytokinin toate joac un rol n fazele iniiale nformareastructurii multicelulare de Physcomitrella sp . ( Cove i Knight , 1993) . n spori germinarea diferite muchi i ferigi un efect stimulator de lumin roie , cu pH sczut , gibereline , nitrat i temperatur sczut a fost raportat ( de exemplu, Haas et al , 1992; . Whittier i Moyroud , 1993 ) . Avnd n vedere cele de mai sus aceti factori pot fi , de asemenea, luate n considerare n dezvoltarea de protocoale de regenerare microsporului . CONCLUSIONS Although the first observations of androgenesis in barley were made in the 1930s, protocols that can be applied to breeding programs have only recently become available. Progress in understanding and controlling androgenesis has been slow. Most economically relevant crops are still recalcitrant, and a universal species-independent protocol for androgenesis induction is not within sight. In the last few years some progress was made in identifying genes that may be markers for androgenesis, and the roles for ABA and anther tissue have become clear. However, an integration of different essential disciplines (i.e. molecular biology, signal transduction research, cell biology, and practical experience in androgenesis protocol design) in a single suitable model system is lacking. Modern genomics and proteomics technologies are promising in this respect and will be useful if a model system is available that can be fully controlled. Hence, in the coming years our progress in understanding and controlling androgenesis will depend largely on combining the different new technologies with the available practical expertise from the trial and error androgenesis protocol design. In addition, mosses and ferns may provide an alternative model for research into the secrets of androgenesis. ACKNOWLEDGMENTS We thank Gerda Lamers and Wessel de Priester for kindly providing the electron microscopical photographs and Christa Testerink, Martien Caspers, and Sylvia de Pater for critical comments on the manuscript. Received May 10, 2000; accepted June 6, 2000. - CONCLUZII Dei primele observaiile androgenesis din orz s-au fcut n 1930, protocoale care pot fi aplicate la programe de reproducere au devenit disponibile recent. Progresele n nelegerea i androgenesis controlul a fost lent. Cel mai economic culturi relevante sunt nc recalcitrant, i un protocol specii independent universal pentru androgenesis inducie nu este n vedere. n ultimii ani a fost realizat unele progrese n identificarea genelor care pot fi markeri pentru androgenesis, iar rolurile de ABA i esutul anterei au devenit clare. Cu toate acestea, o integrare a diferitelor discipline eseniale (de exemplu, biologie moleculara, de cercetare transmiterea semnalului, biologie celular, i experien practic n androgenesis proiectare protocol) ntr-un singur sistem de model adecvat lipsete. Genomica moderne i tehnologii proteomica sunt promitoare n acest sens i va fi util dac un sistem de model este disponibil care poate fi controlat n totalitate. Prin urmare, n urmtorii ani progresul n nelegerea i androgenesis control vor depinde n mare msur pe combinarea diferitelor noile tehnologii cu experienta practica disponibile la proces i de eroare de proiectare androgenesis protocol. n plus, muchi i ferigi poate oferi un model alternativ pentru cercetare in tainele androgenesis. MULUMIRI Mulumim Gerda Lamers i Wessel de Priester pentru furnizarea cu amabilitate fotografii microscopice de electroni i Christa Testerink, Martien Caspers, i Sylvia de Pater pentru comentarii critice de pe manuscrisul. Primit 10 mai 2000; acceptat 06 iunie 2000.