Plant Physiol. (1989) 90, 9-120032-0889/89/90/0009/04/$01 .00/0

Received for publication September 13, 1988and in revised form November 30, 1988

Communication

Isolation of Fixed and Viable Eggs, Central Cells, andEmbryo Sacs from Ovules of Plumbago zeylanica'

Bing-Quan Huang and Scott D. Russell*Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019

ABSTRACT

Three altemative protocols for light microscopy, electron mi-croscopy, and biochemical characterization of isolated megaga-metophytic tissues are described employing enzymic macerationand microdissection of living and fixed ovular tissue of Plumbagozeylanica. Morphologically well preserved megagametophytesare obtained using fixed ovules in two different regimes (nearly40 and 60% yield, respectively). Fluorescein diacetate-positivemegagametophytic cells are recovered in nearly 20% of unfixedovules using the third regime.

phytes. These protocols reliably provide fixed isolated eggs,central cells, and female gametophytes ofPlumbago zeylanicasuitable for LM, FM, and EM, and viable tissues suitable,with modification, for tissue culture.

MATERIALS AND METHODS

Plant Material

Plants of Plumbago zeylanica L. were maintained in 16 hdaylength at 18 to 24°C in growth chambers and greenhousesat the University of Oklahoma.

The gametophyte of angiosperms represents the reductionofindependent haploid generations of plants that produce thegametes. The semiautonomous nature of male gametophyticcells is evident in the occurrence of unique mRNAs (13) andmale germ unit polypeptides in two-dimensional PAGE stud-ies of pollen (3). Since male gametophytic cells are releasedfrom the plant as pollen, these cells have been readily availablefor biochemical and physiological characterization. The fe-male gametophyte, however, is typically produced singly inthe ovule and is retained within numerous surrounding spo-rophytic cells throughout maturation, sexual function, andgermination. This has prevented all but the most rudimentaryphysiological characterization of the gametophytic cells thatproduce the functional female germ unit (2).

Ultimately, fertilization and embryogenetic developmentare likely to be controlled by a shared responsibility of bothmale and female gametophytic cells (8, 9). Gene productsresponsible for gametic function and recognition are probablyrestricted both in activity and expression to gametes andgametophytic cells (8). Therefore, methods to isolate bothtypes of gametic cells en masse will be required to elucidatethe physiological basis of sexual reproduction in floweringplants (2).The present study evaluates three protocols specifically for

producing fixed and viable isolated ES2 cells and gameto-

'Supported by grants from the United States Department of Ag-riculture (86-CRCR-1-1978) and the National Science Foundation(DCB-840915 1).

2Abbrevations: ES, embryo sac/megagametophyte; LM, light mi-croscopy; FM, fluorescence microscopy; FPA, formalin-propionicacid-alcohol; GA, glutaraldehyde; FA, paraformaldehyde; IPCM, in-verted phase contrast microscopy; IVEM, intermediate voltage elec-tron microscopy; CPD, critical point drying; DIC, differential inter-ference contrast microscopy; FDA, fluorescein diacetate.

Isolation of Fixed Material

Individual ovules were dissected from flowers of P. zeylan-ica (Fig. 1) and fixed using either: (a) FPA (formalin:propionicacid:50% ethanol [v/v]) mixed at 1:1:18 ratio 24 to 26 h or(b) GA-FA (2.5% GA-2% FA) mixture in 0.1 M Pipes buffer(pH 6.8) 12 h (for evaluation of fixation quality of other EMbuffers, see ref. 1 1). Tissue fixed in FPA was transferred to70% ethanol, then stored at 4°C or rehydrated to water usinga graded ethanol series, and rinsed in water overnight. Tissue

Isolate ovules

by Light/Electron microscopy

E26hr Fix 12 hr in 2.5%GA-2.5% FA in 0.1 MPipes buffer pH 6.8

Mount and observe I Mount on foldinggrid and observe

Figure 1. Protocol for isolation of fixed ES cells using FPA and GA-FA.

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Plant Physiol. Vol. 90, 1989

fixed in GA-FA was rinsed in Pipes buffer and enzymicdigestion conducted in Pipes buffer at pH 6.8. The enzymeincubation medium was mixed using (w/v): 3% cellulase, 3%hemicellulase, 3% pectinase, and 1% ,B-glucuronidase (SigmaChemical Co.) in either 0.2 M Tris-HCl (pH 4.7 for FPAmaterial) or Pipes buffer (pH 6.8 for GA-FA material) for l/2to 1 h at 37°C (200 ovules per 200 uL) and subsequentlyrinsed in buffer three times. GA-FA-fixed material typicallyrequired microdissection to remove integuments and adher-ent nucellar cells. Ovules were transferred to microfuge tubesand mixed for 2 to 5 min on a Vortex mixer. Eggs, centralcells, and intact ES liberated by the enzyme treatment werelocated using IPCM and collected using a flame-drawn micro-pipette (Fig. 1). Recovery rates were calculated by taking thenumber ofES recovered using IPCM, dividing by the numberof ovules used, and multiplying by 100.For EM, material was postfixed in 2% buffered OS04 for 2

h, rinsed in buffer, dehydrated in a graded ethanol series, andrinsed repeatedly in absolute ethanol over molecular sieves.Whole-mounted material for IVEM was subjected to CPDusing liquid CO2 and then mounted on formvar-coated fold-ing grids.

Isolation of Living Material

Ovules were removed from fresh flowers of P. zeylanica(Fig. 2), bisected transversely, and immersed in a stabilizationsolution consisting of 0.6 M sorbitol, 4% D-glucose (w/v), and0.05 M CaCl2 (pH 5.8). Ovules were then transferred to anenzymic maceration medium consisting of stabilization so-lution with (w/v): 2% cellulase, 2% hemicellulase, 2% pecti-nase, 1% i3-glucuronidase, and 0.5% pectolyase (Sigma Chem-ical Co.) at room temperature for approximately 2 h (100-150 ovules per 200 uL of solution). Material was rinsed inthree changes of stabilization solution, isolated ES cells wereselected using IPCM, and rinsed twice more in the samesolution. Isolated ES cells were identified using IPCM andthen recovered using a micropipette (Fig. 2). Recovery rateswere determined by ES cell type, as calculated above.

LIsolate ovules in stabilization solution(0.6 M sorbitol + 4% D-glucose + 0.05 M

CaC12)

L

Enzyme maceration: Incubate 2 hrin 2% cellulase, 2% hemicellulase,

2% pectinase, 0.5% pectolyaseand 1% B-glucuronidase

Rinse and dissect using invertedphase contrast microscope

Rinse and test viability by addingfluorescein diacetate (5 pg/ml)

Observe using light andfluorescence microscopy

Figure 2. Protocol for isolation of unfixed ES cells.

Microscopy

Light microscopy was conducted using a Leitz Dialux 20equipped for DIC and epifluorescence microscopy. Nucleiwere localized in fixed material using 4 h RNase extraction(0.1% [w/v] RNase, type II-A [Sigma Chemical Co.] in 0.1 Mcitric acid, 0.2 M Na2HPO4, and 200 mM KCI [pH 5.5]),stained 10 h in 1 Ag/mL Hoechst 33258 in the same bufferand mounted for observation in glycerol. Viability of unfixedmaterial was tested using FDA (0.1% [w/v] FDA in acetone,diluted 1:50 in stabilization solution) diluted 1:4 with stabili-zation solution on the microscope slide for a final concentra-tion of 5 ,g/mL. IVEM observations were made using a JEOL2000-FX at 200 kV.

RESULTS

Fixed Material

The isolated ES of P. zeylanica contains an egg, a centralcell, and a variable number of accessory cells (7, up to twolateral cells and one antipodal cell), which are often lost duringprocessing (Figs. 3 and 4). FPA-fixed material (Fig. 3) is wellsuited for LM, but GA-FA fixed material is better preservedin both LM (Fig. 4) and EM (Fig. 6), if cytoplasmic detail iscarefully examined.The prominent central cell contains numerous transvacu-

olar strands (Figs. 3 and 4, arrowheads) and a centrally locatednucleus, as is evident in LM. In IVEM, these structures areevident (Fig. 6) in addition to the native electron density ofthe ES. Cytoplasmic strands emanating from the perinuclearcytoplasm to the periphery of the ES (Fig. 6) vary in shapefrom cylindrical structures to flattened sheets (11). The eggcontains a chalazally placed nucleus and a micropylar cellwall structure, known as a filiform apparatus (Figs. 3, 4, and6). Nuclear position during early embryogenesis is illustratedusing FM with the DNA fluorochrome Hoechst 33258 (Fig.5).Up to 200 ovules were used per isolation, with recovery of

nearly 60% in FPA-fixed material and 40% for GA-FA fixedfiaterial using IPCM.

Unfixed Material

Megagametophytic cells isolated from fresh, unfixed mate-rial are spherical and appear similar to protoplasts (Figs. 7-10). These apparently lack cell walls after enzymic digestionas evaluated using DIC and 0.01% calcofluor white M2Rstaining observed in FM (not shown). Up to 100 ovules wereused per isolation, with recovery of approximately 10% eggs,4% lateral cells, and 6% central cells. FDA-fluorescence,indicative of viability, could be achieved in isolated eggs (Fig.8) for 33 h, central cells (Fig. 10) for 29 h, and intactmegagametophytes for 54 h.

DISCUSSION

Isolated ES and gametophytic cells provide a source forpure female cell collections that can be used for structural orphysiological experimentation. Structurally, isolated fixed ESprovide detailed information on the three-dimensional orga-

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EGG AND EMBRYO SAC ISOLATION IN PLUMBAGO

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Figure 3. FPA-fixed isolated ES of P. zeylanica. Arrowheads indicate transvacuolar strands in central cell (CC). E, egg; FA, filiform apparatus(DIC, x240).Figure 4. GA-FA fixed isolated ES. Arrowheads indicate transvacuolar strands in central cell (CC). E, egg. DIC. (x240).Figure 5. Hoechst 33258 staining of a FPA-fixed ES, showing the nuclei of the 4-celled embryo (Em) and 4-nucleate endosperm (En) (2 Ennuclei are not in plane of focus) (FM, x240).Figure 6. IVEM of GA-FA, OS04 fixed ES. Note transvacuolar strands (arrowheads) and filiform apparatus (FA). CC, central cell; E, egg (IVEM,x400).Figure 7. Unfixed isolated egg (E) and two lateral cell (LC) protoplasts (DIC, x21 0).Figure 8. FDA reaction reveals localized esterase activity in egg and one lateral cell cytoplasm suggesting retention of viability (same cells asshown in Fig. 7). The larger lateral cell was apparently damaged during preparation. FM; 6 h after isolation (x21 0).Figure 9. Central cell (CC) protoplast following isolation (DIC, x21 0).Figure 10. FDA reaction reveals localized esterase activity in isolated central cell (same cell as shown in Fig. 9). FM; 9 h after isolation (x21 0).

nization of the egg and central cells that may simplify thetechniques needed to study the cellular organization of em-bryogenic cells in angiosperms. The detailed information thatthese techniques provide cannot be equalled by ovule clearing(4), morphometry, serial sectioning, or three-dimensional re-construction (9). Isolated ES have also been embedded inplastic resin, thin-sectioned, and examined by conventionalEM ( 11) to evaluate the effect that preparation may have oncellular structure in the gametophyte. Except for some chem-ical extraction and the normal shrinkage expected of CPDmaterial, the native ultrastructure of the ES of P. zeylanicaappears intact when compared to electronmicrographs avail-able elsewhere (see 9, for references). This attests to an excel-lent quality of preservation and the immediate applicabilityof this technique to basic embryological research. Physiolog-ically, this technique provides a concentrated fraction of EScells that may be used for in vitro study or biochemicalcharacterization.Although it is likely that different enzymes will be required

for different species, the basic protocol presented here shouldbe applicable to many if not all flowering plant ovules. In the

present study, cellulose, hemicellulase, pectinase, and ,B-glu-curonidase were required to break down the major cross-linking elements of the cell wall in P. zeylanica, and digestiontimes were kept to a minimum by removing the integumentsby hand. Driselase (5), Macerozyme R10 (6), and snailase (5,14-16) have also been used in other formulae for ES isolation.Successful isolation of unfixed ES cells appears to require

that turgor is retained within the cells. Viability in P. zeylanicawas optimal using glucose as a metabolic sugar and sorbitolas the major osmoticum. Previous researchers have used 100mM glycine, 1% hexylene glycol, 1% propan-2-ol, 0.1% Tri-ton, Ca(OH)2 to pH 8.4 (1); 0.65 M mannitol (5); 2 to 14%sucrose (depending on species) and 0.2% potassium dextransulfate (15, 16); or 20% sea water and 0.7 or 0.57 M mannitolas major osmoticums (6) to isolate unfixed material; the latterfour reports described FDA-fluorescent cells. Only M61 (6)noted extended viability of ES cells, reporting FDA-fluores-cence for up to 2 weeks in cultures supplemented with 10%coconut water, 2 mg/L 2,4-dichlorophenoxyacetic acid andmannitol to 720 or 840 mOs/kg; however, cell wall reestab-lishment was not observed (6). In the present study, intact

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Plant Physiol. Vol. 90,1989

ES retained FDA-fluorescence for 54 h without additionalsupplements.Optimal recovery ofunfixed materials seems to depend also

on minimizing exposure to macerating enzymes and main-taining pH within physiological limits. In P. zeylanica, 2 h indigestive enzymes at room temperature was optimal in recov-ery of FDA-fluorescent protoplasts at a pH of 5.8. Previousresearchers have obtained FDA-fluorescent cells using enzymeexposures of 3 to 3.5 h at 28 to 30TC for ES, 8 h for ES-cellprotoplasts (pH not given; 5); 2 to 6 h agitated at 28 to 30C(pH 5-5.5) (15); and 5 h at room temperature (pH 5.6) (6).Enzyme activity optima differ for each specific enzyme, butranged from 4.7 to 6.0 in the present work. The naturallyoccurring pH of the fluid within the ES, from 5.5 to 6.0 indicot species and 4.8 to 5.2 in monocot species (12), suggeststhat the isolation solution is within the physiologically ac-ceptable range. As evidenced by FDA-fluorescence ofES cells,the membranes of fixed and unfixed ES cells remain essen-tially intact.The major limitation of this technique is that ES isolations

require manual dissection steps that ultimately determineyield. These techniques are less time-consuming than methodspreviously used to study ES organization (9) and provideunique data unavailable by other methods (1 1). Physiologicalexamination of ES cells and the basis of gametic recognitionand specificity (8) have been essentially impossible before thedevelopment of ES isolation. This technique complementsprevious studies that have provided protocols for isolating themale gamete (10).

ACKNOWLEDGMENTS

We thank Greg Strout for assistance with EM preparations. Use offacilities of the Samuel Roberts Noble Electron Microscopy Labora-tory is gratefully acknowledged.

LITERATURE CITED

1. Allington PM (1985) Micromanipulation of the unfixed cerealembryo sac. In GP Chapman, SH Mantell, RW Daniels, eds,Experimental Manipulation of Ovule Tissues. Longman, Es-sex, pp 39-51

2. Duinas C, Knox RB, McConchie CA, Russell SD (1984) Emerg-ing physiological concepts on sexual reproduction in angio-sperms. What's New Plant Physiol 15: 17-20

3. Geltz NR, Russell SD (1988) Two-dimensional electrophoreticstudies ofthe proteins and polypeptides in mature pollen grainsand the male germ unit of Plumbago zeylanica. Plant Physiol88: 764-769

4. Herr JM Jr (1972) A new clearing-squash technique for thestudy of ovule development in angiosperms. Am J Bot 58:785-790

5. Hu SY, Li LG, Zhou C (1985) Isolation of viable embryos sacsand their protoplasts in Nicotiana tabacum. Acta Bot Sin 27:337-344

6. M61 R (1986) Isolation of protoplasts from female gametophytesof Toreniafournieri. Plant Cell Rep 3: 202-206

7. Russell SD (1983) Fertilization in Plumbago zeylanica: gameticfusion and fate of the male cytoplasm. Am J Bot 70: 416-434

8. Russell SD (1985) Preferential fertilization in Plumbago: ultra-structural evidence for gamete-level recognition in an angio-sperm. Proc Natl Acad Sci USA 82: 6129-6132

9. Russell SD (1986) Dimorphic sperm cells, cytoplasmic transmis-sion and preferential fertilization in Plumbago zeylanica. InSH Mantell, GP Chapman, PFS Street, eds, The Chon-driome-Chloroplast and Mitochondrial Genomes. Longman,Essex, pp 69-116

10. Russell SD (1986) Isolation of sperm cells from the pollen ofPlumbago zeylanica. Plant Physiol 81: 317-319

11. Russell SD, Huang BQ, Strout GW (1989) Preliminary inter-mediate voltage electron microscopic observations of the iso-lated embryo sacs and eggs of Plumbago zeylanica L. (Plum-baginaceae). In J Pare, M Bugnicourt, J Mortier, eds, Festscriftfor Prof Dr A Lebegue (in press)

12. RyczkowskiM (1965) The pH ofthe sap surrounding the embryoin the developing ovules. Bull Acad Polon Ser Sci Biol 13:479-483

13. Stinson JR, Eisenberg AJ, Willing RP, Pe ME, Hanson DD,Mascarenhas JP (1987) Genes expressed in the male gameto-phyte of flowering plants and their isolation. Plant Physiol 83:442-447

14. Zhou C (1987) A study of fertilization events in living embryosacs isolated from sunflower ovules. Plant Sci 52: 147-151

15. Zhou C, YangHY (1985) Observations on enzymatically isolated,living and fixed embryo sacs in several angiosperm species.Planta 165: 225-231

16. Zhou C, Yang HY (1986) Isolation ofembryo sacs by enzymaticmaceration and its potential in haploid study. In H Hu, HYYang, eds, Haploids ofHigher Plants in Vitro. Springer-Verlag,Berlin, pp 192-203

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