© 2004 Science From Israel / LPPLtd., Jerusalem

Israel Journal of Plant Sciences Vol. 52 2004 pp. 155–160

*Author to whom correspondence should be addressed. E-mail:kmtaskin@comu.edu.tr

Apomictic development in Arabis gunnisoniana

KEMAL MELIH TASKIN,a,* KENAN TURGUT,b AND ROD J. SCOTTc

aDepartment of Biology, Çanakkale Onsekiz Mart University, 17100 Çanakkale, TurkeybDepartment of Field Crops, Akdeniz University, 07058 Antalya, Turkey

cDepartment of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK

(Received 11 May 2003 and in revised form 10 September 2003)

ABSTRACT

Apomixis in model systems has been of growing interest for many scientists aroundthe world. A limited number of species belonging to Brassicaceae has been describedas apomictic and considered as a model system to study molecular biology of apomixis.In this study, we report the use of an ovule-clearing technique to characterize embryosac development in Arabis gunnisoniana. The embryo sac development was de-tected by Taraxacum-type diplosporous dyad formation. Hence, the reproductivepathways in Arabis gunnisoniana were revealed by using flow cytometric seedscreen (FCSS) analysis. Arabis gunnisoniana appears to display pseudogamousapomixis.

Keywords: Arabis gunnisoniana, apomixis, embryo sac development, diplospory,ovule-clearing technique

INTRODUCTION

Apomixis is an asexual mode of reproduction that re-sults in embryo formation without fertilization of theegg. Since meiosis is usually absent or modified toprovide unreduced female gametes, the progeny ofapomictic plants are genetically identical to the motherplant. Therefore, it may bring various benefits in cropimprovement; it allows vegetative reproduction throughthe seed and thus can have a major impact not only inseed-propagated crops (cereals, vegetables), but also invegetatively-propagated crops (e.g., potato). It couldeliminate the need for yearly purchase of improved seedand lower the costs of breeding. A major goal forapomixis is its introduction into commercial F1 hybridseed production. It simplifies seed production, becauseisolation is not necessary, and the need to maintain ormultiply parental lines is reduced (Koltunow, 1993;Koltunow et al., 1995). In the genus Arabis(Brassicaceae), there are a variety of reproductivemechanisms (Roy, 1995). Some members of the genusare apomictic and some sexual. Although Arabis

gunnisoniana has been described as a pseudogamousapomict (Roy, 1995), embryo sac development has notbeen surveyed in detail. As a relative of Arabidopsisthaliana, Arabis gunnisoniana is attractive as a potentialmodel species to study the molecular genetics of apo-mixis, and has the advantage compared to other Arabisspp. that it can be genetically transformed (Taskin et al.,2003). Therefore, the introduction of marker genes tofollow segregation and recombination and mutagenicsequences such as T-DNA tags and transposons will bepossible. However, before one can proceed with this, theembryo sac development has to be characterized stageby stage. Here we report the use of an ovule-clearingtechnique to characterize embryo sac development inArabis gunnisoniana. Apomeiotic embryo sac develop-ment was detected by Taraxacum-type diplosporousdyad formation, and the flow cytometric seed screen(FCSS) analyses revealed both unreduced embryo sacand pseudogamous apomixis in Arabis gunnisoniana.

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MATERIALS AND METHODS

Plant materials

Arabis gunnisoniana seeds examined in this study wereoriginally collected from North America (Colorado) andobtained from Dr. Bitty Roy (University of Oregon,USA). The plants were grown in a greenhouse underlong-day conditions at a regime of 16 h light (20 °C): 8 hdark (18 °C) on a peat moss: sand mix (1:3). The plantsstarted to flower after 4 to 6 months.

Ovule-clearing procedures

To study ovule development, inflorescences and sili-ques were fixed in FAA (formaldehyde 40%; ethanol70%; acetic acid 98% in the ratio 3:7:1) solution for 24 hand stored in 70% ethanol at 4 °C. Ovaries/siliques weredehydrated in an ethanol series with increasing concen-trations (70%, 80%, 90%, and 100%) for 20 minutes perconcentration, and then cleared in mixtures of ethanol:methyl salicylate in the ratios 1:2, 1:1, 2:1 for 1 h permixture and pure methyl salicylate for 12 h (Naumova etal., 1999). Pistil lengths (from the top of the immaturestigma to the base of the ovary) were measured with aruler, then pistils were grouped according to their sizes(0.5-1, 1–1.5, 1.5–2, and 2–5 mm). Pistils from differentinflorescences of the same plant were analyzed indi-vidually according to their sizes. Cleared ovules from 3plants were investigated using Nomarski optics, andmore than 300 ovules were analyzed per plant. Theembryo sac components (total number of nuclei withinembryo sac, presence of a tetrad or a dyad with a devel-oping chalazal megaspore, whether each embryo saccontained an embryo and endosperm) were recorded.

Flow cytometry

Seeds selected from 10 progenies derived from theArabis gunnisoniana population growing in the green-house were used to analyze ploidy level of the embryoand endosperm by flow cytometry. Seeds were collectedfrom mature siliques of open pollinated individuals.One to 20 bulked samples of 50 seeds were choppedwith a razor blade in DAPI staining buffer (Matzk et al.,2000), filtered, and stored on ice. A Ploidy Analyser(Partec) was used for the measurements (Matzk et al.,2000, 2001).

RESULTS AND DISCUSSION

Embryo sac development was investigated at variousstages of A. gunnisoniana pistil development using anovule-clearing technique (Young et al., 1979; Naumova,1997). The ovule differentiated from placenta when thepistil length reached about 0.5 to 1 mm (Table 1). At this

very early stage, the megaspore mother cell (MMC) waseasy to recognize with its large and prominent nucleusand integuments (Fig. 1). When the pistil length reachedabout 1 to 1.5 mm, MMC divided once and gave adiplosporous dyad (Taraxacum type) consisting of twounreduced megaspore cells, while the inner and outerinteguments enclosed the nucellus (Fig. 2). After that,the megaspore closest to the micropylar degenerated

Table 1Embryo sac development of A. gunnisoniana ovules

Pistil lengths Ovule stages(mm) (number of ovules analyzed)

0.5–1 MMC differentiation (340)1–1.5 Apomeiosis (300)1.5–2 Dyads with a degenerating megaspore (600)

Three plants were analyzed.

Fig. 1. Early phase of ovule development containing megasporemother cell (MMC), outer integuments (OI), and inner integu-ments (II). Scale bar = 10 µm.

Fig. 2. Ovule after meiosis. Scale bar = 10 µm.

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while the remaining chalazal megaspore continued todevelop (Fig. 3, Table 1). The chalazal megaspore un-derwent vacuolization (Figs. 4, 5) and mitosis to form an8-nucleate Polygonum-like embryo sac (Fig. 6) (Craneand Carman, 1987; Leblanc et al., 1995; Naumova et al.,

2001). Embryo sac development was apparently com-pleted when pistil length reached about 5 mm (Fig. 7),since the presence of an embryo and endosperm couldbe observed at this stage in pollinated flowers.

In order to determine the reproductive pathways,

Fig. 3. Ovule with a developing chalazal megaspore (CM) anda degenerating megaspore (DM) of Taraxacum-type dyad(pistil length: 1.5–2 mm). Scale bar = 10 µm.

Fig. 4. Binuclear embryo sac with a large vacoule (V) betweentwo nuclei. Scale bar = 10 µm.

Fig. 5. The four nuclear embryo sac (pistil length: 2–2.5 mm).Scale bar = 10 µm.

Fig. 7. Embryo (Emb) and endosperm (En) development (pistil length: 5 mm). Scale bar = 100 µm.

Fig. 6. Ovule with a fully developed Taracaxum-type embryo saccontaining synergids (Syn), polar nuclei (PN), and an egg cell (E).Scale bar = 10 µm.

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samples of Arabis gunnisoniana seeds were analyzed byflow cytometric seed screen (FCSS) to measure theDNA content of the different cells (Matzk et al., 2000,2001). A. gunnisoniana seed samples yielded 2C and 6Cpeaks, most likely representing an asexual 2C embryodeveloped from an unreduced (meiosis did not occur)egg cell (2m:0p = 2 maternal:0 paternal) and a hybridendosperm originated by fertilization of the unreducedpolar nuclei by an unreduced sperm (4m:2p) (Fig. 8).Hence, one sample of A. gunnisoniana yielded 2C and5C peaks, most likely representing an asexual 2C em-bryo developed from an unreduced egg cell (2m:0p),

Fig. 9. FCSS histogram obtained from a single seed of A. gunnisonaiana shows 2C and 5C peaks, most likely representingparthenogenetic embryo development from the unreduced egg cell (2C) and a hybrid endosperm development from thefertilization of the unreduced polar nuclei by a reduced sperm (5C).

Fig. 8. FCSS histogram obtained from 20 bulked seeds of A. gunnisonaiana shows 2C, 4C, and 6C peaks, most likelyrepresenting parthenogenetic embryo development from the unreduced egg cell (2C), endopolyploidization in embryo cells(4C), and a hybrid endosperm development from the fertilization of the unreduced polar nuclei by an unreduced sperm (6C).

and a hybrid 5C endosperm developed from 2 unre-duced polar nuclei fertilized by a reduced (meiosis oc-curred) sperm (4m:1p) (Fig. 9). The flow cytometryanalyses of Arabis gunnisoniana revealed that A.gunnisoniana seeds reproduce with pseudogamy (whileembryo formation took place autonomously, endospermdeveloped after fertilization with reduced or unreducedmale gametes) (Table 2).

The breeding systems of Arabis species were investi-gated previously (Böcher, 1951; Roy, 1995; Naumovaet al., 2001). Among these, Taraxacum-type diplosporyin A. holboellii was explained in great detail (Böcher,

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Table 2Reproductive pathways of Arabis gunnisoniana seed samples analyzed by flow cytometry

C values Seed development(embryo:endosperm)

2C:6C (20)1 Pseudogamous, unreduced sperma

2C:5C (1)1 Pseudogamous, reduced spermb

1Number of seed samples used for analyses.aUnreduced embryo sac, egg cell parthenogenesis, and pseudogamous endosperm (fertilization ofcentral cell with unreduced pollen).bUnreduced embryo sac, egg cell parthenogenesis, and pseudogamous endosperm (fertilization ofcentral cell with reduced pollen).

1951; Naumova et al., 2001). In addition, flowcytometry analyses of A. holboellii seeds producedevidence for both pseudogamous and autonomousapomixis (Matzk et al., 2000; Naumova et al., 2001).Although A. gunnisoniana was reported as a pseudo-gamous apomict (Roy, 1995), embryo sac developmenthas not been described to date. Roy (1995) reported thatA. gunnisoniana was triploid (3x:21) and pollensamples showed signs of irregular meiosis, such asimproper pairing and the production of dyads instead oftetrads, indicating apomixis. It was also shown thatA. gunnisoniana could set seed with intraspecific pollenfrom A. holboellii (Roy, 1995). These reports are ingeneral compatible with our results. The flow cytometryresults provided evidence for unreduced sperm and em-bryo sac. Endosperm develops mostly after fertilizationof central cells with unreduced sperm, since only onesample of seeds produced 5C (4m:1p) peaks (Table 2).

According to cytoembryological data, when the pistillength reached 1–1.5 mm, most of the embryo sacs(about 95%) contained one nuclei that enteredapomeiosis and gave dyads instead of tetrads. Later,dyads were confirmed with the degenerating megasporeclose to the micropylar end of the ovule when the pistillength reached about 2 mm (Table 1). At this stage,while about 84% of embryo sacs contained dyads withdegenerating megaspore, the rest contained 1 or morenuclei. The additional reproductive pathways such as mei-otic embryo sac and autonomous endosperm develop-ments, recorded in A. holboellii (Naumova et. al., 2001;Matzk et al., 2000), were not seen in our work. However,our results may not represent other A. gunnisoniana popu-lations since only one population was analyzed in ourstudy. A variety of reproductive mechanisms were al-ready reported in different Arabis species (Roy, 1995).

According to our results, A. gunnisoniana showsTaraxacum-type diplospory and appears to display

pseudogamous apomixis, so it could be used as a modelplant to study molecular biology of apomixis.

ACKNOWLEDGMENTS

The authors thank TUBITAK (The Scientific and Techni-cal Research Council of Turkey), Akdeniz University Sci-entific Research Projects Unit, and The British Council forfinancial support. The authors also thank Dr. F. Matzk(Institut für Pflanzengenetik and Kulturpflanzenforschung(IPK), Gatersleben, Germany) for the flow cytometryanalyses.

REFERENCES

Böcher, T.W. 1951. Cytological and embryological studies inthe Amphi-apomictic Arabis holboellii complex. K. Dan.Vidensk. Selsk. Biol. Skr. VI. 7:1–57.

Crane, C.F., Carman, J.G. 1987. Mechanisms of apomixis inElymus rectisetus from eastern Australia and New Zealand.Am. J. Bot. 74: 477–496.

Koltunow, A.M. 1993. Apomixis: embyo sacs and embryosformed without meiosis or fertilization in ovules. PlantCell 5: 1425–1437.

Koltunow, A.M., Bicknell, R.A., Chaudhury, A.M. 1995.Apomixis: molecular strategeis for the generation ofgenetically identical seeds without fertilization. PlantPhysiol. 108: 1345–1352.

Leblanc, O., Peel, M.D., Carman, J.G., Savidan, Y. 1995.Megasporogenesis and Megagametogenesis in severalTripsacum species (Poaceae). Am. J. Bot. 82: 57–63.

Matzk, F., Meister, A., Schubert, I. 2000. An efficient screenfor reproductive pathways using mature seeds of monocotsand dicots. Plant J. 21: 97–108.

Matzk, F., Miester, A., Brutovska, R., Schubert, I. 2001. Re-construction of reproductive diversity in Hypericumperforatum L. opens novel strategies to manage apomixis.Plant J. 26: 275–282.

Naumova, T.N. 1997. Apomixis in tropical fodder crops: cyto-

Israel Journal of Plant Sciences 52 2004

160

logical and functional aspects. Euphytica 96: 93–99.Naumova, T.N., Hayward, M.D., Wagenvoort, K. 1999. Apo-

mixis and sexualty in diploid and tetraploid accessions ofBrachiaria decumbens. Sex. Plant Reprod. 12: 43–52.

Naumova, T.N., van der Laak, J., Osadtchiy, J., Matzk, F.,Kravtchenko, A., Bergervoet, J., Ramulu, K.S., Boutilier,K. 2001. Reproductive development in apomict popula-tions of Arabis holboellii. (Brassicaceae). Sex. PlantReprod. 14: 195–200.

Roy, B.A. 1995. The breeding systems of six species of Arabis(Brassicaceae). Am. J. Bot. 82: 869–877

Taskin, K.M., Turgut, K., Ercan, A.G., Scott, R.J. 2003.Agrobacterium-mediated transformation of Arabisgunnisoniana. Plant Cell, Tiss. Org. Cult. 72: 173–180

Young, B.A., Sherwood, R.T., Bashaw, E.C. 1979. Cleared-pistil and thick-sectioning techniques for detectingaposporous apomixis in grasses. Can. J. Bot. 57: 1668–1672.