In vitro culture of Arabidopsis embryos within their ovules - Sauer - 2004 - The Plant Journal - Wiley Online Library.pdf

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  TECHNICAL ADVANCE In vitro   culture of  Arabidopsis   embryos within their ovules Michael Sauer and Jir ˇ ı´  Friml * Zentrum fu ¨ r Molekularbiologie der Pflanzen, Auf der Morgenstelle 3, 72076 Tu ¨ bingen, Germany  Received 28 April 2004; revised 19 August 2004; accepted 6 September 2004. * For correspondence (fax þ 49 7071 295 797; e-mail jiri.friml@zmbp.uni-tuebingen.de). SummaryEmbryogenesis of flowering plants establishes a basic body plan with apical–basal, radial and bilateralpatterns from the single-celled zygote.  Arabidopsis   embryogenesis exhibits a nearly invariant cell divisionpattern and therefore is an ideal system for studies of early plant development. However, plant embryos aredifficult to access for experimental manipulation, as they develop deeply inside maternal tissues. Here wepresent a method for the culture of zygotic  Arabidopsis   embryos  in vitro  . The technique omits excision of theembryo by culturing the entire ovule, thus greatly facilitating the time and effort involved. It enables externalmanipulation of embryo development and culture from the earliest developmental stages up to maturity.Administration of various chemical treatments as well as the use of different molecular markers isdemonstrated together with standard techniques for visualizing gene expression and protein localization in invitro  cultivatedembryos.Thepresentedsetoftechniquesallowsforsofarunavailablemolecularphysiologyapproaches in the study of early plant development.Keywords:  in vitro   culture, embryogenesis, immunolocalization, physiological studies.Introduction Flowering plants vary remarkably in their overall appear-ance. However, mature embryos look much the same,because only basic body parts are formed in the course of embryogenesis. A mature embryo, which develops from thesingle-celled zygote, is composed of a shoot and a rootmeristem placed at opposite poles of the apical–basal axis.The shoot meristem is located at the top, flanked by thecotyledons (embryonic leaves). It is connected with the rootmeristem at the bottom by the hypocotyl (embryonic stem)and the root itself. The apical–basal axis, radial and lateralsymmetry, as well as patterns of different cell fates alongthese symmetries, are established during embryogenesis,laying the foundations for all post-embryonic development(Ju ¨rgens, 2001).In the model plant  Arabidopsis thaliana  , the embryodevelops through a highly uniform series of cell divisionsand cell fate changes (reflected also by molecular markers),leadingto morphologically distinct intermediatestages (e.g.Friml  et al. , 2003; Vroemen  et al. , 1996). The initial zygoticdivision is asymmetric, giving rise to a smaller apical and alarger basal cell. The basal cell and its lineage divideanticlinally and form the suspensor, an extraembryonic fileof cells which connects the embryo to the maternal tissue.After the 32-cell stage, the uppermost suspensor cellbecomes incorporated into the embryo as the hypophysiscell, from which most of the future root meristem is derived.All other embryonic parts are derived from the apical cell,which divides peri- and anticlinally to first form an eight-cellstage proembryo, then establishes the protoderm (at the16-cell stage) and finally gives rise to the round globularembryo. At this stage, bilateral symmetry is established,with the positions of the cotyledon primordia becomingapparent at the triangular stage. At the heart stage, thepatterning is complete and during later development thispattern is merely elaborated upon (Ju ¨rgens and Mayer,1994).In both plant and animal systems, initial patterningevents, and therefore embryogenesis, are topics of greatinterest to developmental biology. However, morphologicalcharacterization alone is not sufficient to elucidate theunderlying mechanisms. In order to scrutinize embryogen-esis thoroughly, it is necessary to employ additional ª  2004 Blackwell Publishing Ltd 835 The Plant Journal   (2004)  40 , 835–843 doi: 10.1111/j.1365-313X.2004.02248.x  techniques such as the analysis of mutants and transgeniclines, microsurgery or external manipulation of embryodevelopment. The latter, in particular, requires the possibil-ity of   in vitro   embryo cultivation.Insomeanimalsystemssuchas Drosophila  ormicethisispossible (for a review see New, 1990). In contrast, suchexperimental approaches are not very well established inplant systems, and extremely limited in  Arabidopsis  .One possible way to culture plant embryos is associatedwith somatic embryogenesis. Under certain culture condi-tions plants will form somatic embryos that undergo anembryogenesis-like development  in vitro   and eventuallybecome mature plants (e.g. De Vries  et al. , 1988; Huang andYeoman, 1984; Luo and Koop, 1997; Mordhorst  et al. , 1998).There are two main limitations to this approach for studyingembryogenesis. The first is that the culture media usuallycontain high levels of phytohormones, or that specific Arabidopsis   mutants, such as the  primordia timing (pt)   areused, both of which affect normal zygotic embryogenesis(Friml  et al. , 2003; Liu  et al. , 1993a). The second limitationlies in the question of whether somatic embryogenesisfollows the same developmental program as zygoticembryogenesis. Although somatic embryogenesis requiresgenes also known to be required for zygotic embryogenesis(Mordhorst  et al. , 2002), all experiments based on somaticembryogenesissufferfromtheabsenceofwelldefinedearlystages. Somatic  Arabidopsis   embryos (derived from zygoticembryos) up to the four-cell stage have been described (Luoand Koop, 1997), however, the subsequent cell divisionpatternbecomesveryirregular.Moreover,somaticembryosderived from leaf protoplasts exhibit some resemblance tonormal zygotic embryos, but they are less regular andnaturally lack a suspensor. In summary, somatic embryoculture appears to be a limited substitute for the study of thenatural course of early zygotic embryogenesis.A better approach is the culture of zygotic embryos. In Brassica juncea  , successful methods for  in vitro   embryoculture and treatment with substances have been shown(Hadfi  et al. , 1998; Liu  et al. , 1993a,b). A similar method hasalso been described in  Arabidopsis   (Kost  et al. , 1992).However, these works incorporated removal of the embryosfrom their ovules, which especially in the case of   Arabidop- sis  , makes it almost impossible to analyze stages youngerthan the globular stage. Nevertheless, the use of   Arabidop- sis   is highly favorable, due to the obvious advantages of afullysequencedgenome,easy transformationtechniquesaswellasreadilyavailablemutant,T-DNAinsertionandmarkerlines.To overcome the manual problems of embryo excision,welookedforalternativeapproaches.Methodsbasedontheculture of whole ovules are widespread in the plant field.However, most published procedures do not focus onembryogenesis itself, but were developed in order to rescuespecific hybrids or to investigate fiber development (e.g. forgrapes:Cain et al. ,1983;forcotton:Beasley,1971;forwheat:Kumlehn  et al. , 1997). Their greatest advantage is therelative ease of ovule preparation in contrast to embryoexcision, thus allowing access to early stages and the use of large sample sizes. Additionally, these protocols do notrequire phytohormones in the medium, nor are they limitedto specific mutants.Here we present a method whereby  Arabidopsis   embryoscan be cultivated in their ovules,  in vitro  , for prolongedperiods of time from the very first developmental stagesonward. We provide examples of its application for embry-ological studies incorporating exogenous treatments withsubstances, reporter gene-based markers as well as immun-ocytochemical methods. Results and discussion Procedure of   in vitro  culture  To establish the procedure, we had to optimize ovule isola-tion under semi-sterile conditions, culture conditions suchas light and temperature, and especially, medium compo-sition. For the culture, we first selected siliques of theappropriate stages and sterilized them by a short dip in 70%EtOH. The siliques were allowed to dry and placed ontodouble adhesive tape that was likewise sterilized. Under adissecting scope, the siliques were cut open along thereplum with needles and the ovules were carefully trans-ferred onto plates containing  in vitro   culture medium (ICM).Simple shielding of the dissecting scope from air draughtsdecreased fungal contamination problems considerably,with typically about 5–8% of the plates contaminated. Thislow contamination rate, given the relative ease and speed of preparation, does not justify more elaborate sterilization orovule preparation protocols. Microscopic examination of afew ovules is sufficient to determine the developmentalstage of the embryos, as embryos within one silique do notdiffer much in their developmental stage (Bowman, 1994).Theplateswerethensealedwithparafilmandturnedupsidedown,topreventcondensatingwaterfromdrippingontotheovules. The plates were kept in the dark. It did not matter if the ovules were placed on the medium singly or in smallclusters.Furthermore,itdidnotsignificantlyaffectsurvivalif thefuniculusorevenmorematernaltissuewasstillattachedto the ovule (not shown). However, submerging the ovulesin the medium seemed to have adverse effects on survival.Of particular importance was the composition of the culturemedium. In previous experiments, we used a lower sucrosecontent of only 2%, but the survival rates were much lower,probably due to osmotic problems as the ovules lookedbloated (not shown). Using a higher (10%) sucrose concen-tration improved survival rates. However, the high sucrosecontent negatively affects the later steps of embryogenesisand plant development (see below). Thus for longer culture 836  Michael Sauer and Jir ˇ ı ´  Friml  ª  Blackwell Publishing Ltd,  The Plant Journal  , (2004),  40 , 835–843  durations (typically exceeding 5 days), we found it bestto transfer the ovules from ICM and darkness to normal Arabidopsis   medium (AM) and dim light under sterileconditions. Survival rate  To assess the efficiency of our culture system, we culturedovules for 5 days on ICM. The initial embryonic stage wasdetermined as mentioned above. The ‘young’ group con-tained mostly embryos between four- and 16-cell stage,the ‘old’ group 32 cell and globular stage embryos at thestart of culture. Using a stereoscope, the vitality of theovules was easily evaluated. The viable ones were bigger,translucent or pale white and round compared with nec-rotic ovules of brownish color or flat appearance(Figure 2b). The survival rate was influenced by the start-ing stage, older ovules being less sensitive than youngerones, probably due to their greater mechanical stability(Figure 1a). Overall, the vitality of the embryo, determinedby microscopic analysis, correlated with the vitality of theovule (Figure 1a). We confirmed this correlation by stain-ing with propidium iodide (PI), which stains dead cellsand fluorescein diacetate (FDA), which stains living cells(Rotman and Papermaster, 1966). As expected, embryosfrom necrotic ovules were strongly stained by PI (Figure 2c),whereas embryos from viable ovules were not stained byPI but by FDA (Figure 2d). Thus the fast analysis with astereomicroscope is a reasonable way to non-invasivelydetermine the vitality of the cultured embryos. Keeping theplants on ICM medium for periods longer than 5 daysaffected the survival rate negatively (Figure 1b). For evenlonger culture durations (>10 days), ICM abolished thedevelopment of real leaves and a root system, theplants rather formed many leaf primordia and producedincreased amounts of anthocyans (Figure 2e). The transferto AM after 5 days improved the general survival rate(Figure 1b). When ovules were cultured on AM mediumfor the rest of embryogenesis, the embryos were able togerminate, developed rosette leaves and a root system,finally bolted and flowered (Figure 2a). The duration untilthey started to bolt varied in the range of 40–50 days afterstart of culture. Based on these analyses, approximately20% of the plants will reach maturity using the methodwith the transfer from ICM to AM after 5 days of culture.Fortheveryearlystagesupto24 hafterpollination,whenthe zygotes or embryos are still not detectable, our methodis lessefficient.About50%oftheovulesweredead(smallorflat) already after 1 day ( n   > 50), which did not change afterlonger culture duration.In summary, this analysis shows that complete embryo-genesis starting from a zygote and finally yielding a matureplant can be readily reproduced in our culture system. Developmental speed  Next we addressed the speed of development in our culturesystem. We cultivated ovules from young siliques of  (a)(c)(b) Figure 1.  Evaluation of survival rate and devel-opmental speed of   in vitro   cultured embryos.(a) Percentage of surviving ovules and embryosafter 5 days of   in vitro   culture. Ovule survivalcorrelates with embryo survival. Older embryosdisplay slightly increased survival rates.(b) Percentage of surviving embryos after10 days of   in vitro   culture with and withouttransfer to  Arabidopsis   medium (AM) after5 days. Culturing on AM medium increases thesurvival rate.(c) Distribution chart of embryo stages at start of embryo culture and after 5 days. The ovuleswereisolatedfromanumberof‘young’and‘old’siliques, respectively. The graphs indicate theprogress of embryo development after 5 days of  in vitro   culture. Y axis: percentage of observedstage. In vitro  embryogenesis in   Arabidopsis 837 ª  Blackwell Publishing Ltd,  The Plant Journal  , (2004),  40 , 835–843  approximatelythesameage.Atthestart,andafter12,24,48,72 and 96 h of culture, we took a random sample anddetermined the youngest embryo stages in each sample. Atthe start the youngest stage was one cell, after 12 h,embryos had reached two or four-cell stage, after 24 h eightto 16-cell stage, after 48 h early to mid globular stage, after72 htriangularstageandafter96 hyoungtomidheartstage(Figure 2a).As a complementary approach, we cultured ovules fromsiliques roughly divided into a ‘young’ and an ‘old’ group.We determined the developmental stages of randomlyselected embryos at the start and after 5 days of culture,and calculated the distribution of developmental stages(Figure 1c). The later stages (torpedo and beyond) are lesswell defined and include embryos with different cell num-bers but similar shape, consequently the graph is com-pressed toward the later stages. At the start, the mostrepresented stages were eight- and 16-cell stages in the‘young’ group. After 5 days of culture, most embryos wereat late heart stage. For the ‘old’ group, the most abundantstarting stage was the globular stage, after 5 days mostembryos had reached walking stick to bent cotyledon stage.Compared with uncultured embryos, this seems to beslightly slower. It may be that already after 5 days adverseeffects of the high sucrose concentration in ICM becomeapparent, as keeping the ovules on ICM for prolongedperiods of time leads to aberrant and delayed development(see above). The presence or absence of dim light (cultureplates covered with a sheet of plain paper or aluminum foil,respectively) did not influence the speed of development,but the embryos cultured in light developed chlorophyll atlater stages and the ovules became green (not shown).To verify the somewhat reduced speed of development,we compared the timing of development  in planta   with ourculture system. To synchronize embryo development, weused ovules from emasculated and manually pollinatedsiliques. Twenty-four hours after pollination, we startedculturing the ovules. We then compared the culturedembryos with embryos from a control group whose siliquesremained on the plant. After 4 days, 46% ( n   ¼  28) of thecontrol embryos reached the late globular stage and 50%reached the triangular stage. Of the surviving cultured Figure 2.  Different stages of embryo development during  in vitro   culture.(a) Progress of embryo development from one-cell stage to adult plant. Hours (h) and days (d) of culturing indicated in the upper right corner.(b) Stereoscopic assessment of ovule vitality: upper two vital, lower three examples of dead or dying ovules.(c) Propidium iodide stains dead cells of an embryo from a necrotic ovule, inset shows unstained embryo extracted from a viable ovule.(d) Fluorescein diacetate stains living cells of an embryo from a viable ovule, inset shows unstained and abnormally shaped embryo from a necrotic ovule.(e) Aberrations of germinated seedling cultured on high sucrose medium.(f–i) Example of aberrations induced by  in vitro   culture. Basal aberration (f, arrowhead indicates abnormal division of hypophysis), wide heart stage (g). Severeaberrations are often associated with brownish necrotic ovule tissue (h). ‘Folded’ hypocotyl after long time culture (i). 838  Michael Sauer and Jir ˇ ı ´  Friml  ª  Blackwell Publishing Ltd,  The Plant Journal  , (2004),  40 , 835–843
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