Sex Plant Reprod (1991) 4:12-16 Sexual Plant Reproduction �9 Springer-Verlag 1991

In vitro fertilization of single, isolated gametes of maize mediated by electrofusion E. Kranz*, J. Bautor, and H. L~rz*

Max-Planck-Institut ffir Zfichtungsforschung, Carl-von-Linn6-Weg 10, W-5000 K61n 30, Federal Republic of Germany

Summary. Electrofusion-mediated in vitro fertilization of maize using single sperm and egg cells was performed. Sperm cells were released f rom pollen grains after rup- ture of the latter by osmotic shock in the fusion medium (0.55 M mannitol). Egg cells were isolated by enzyme treatment (pectinase, pectolyase, hemicellulase, and cel- lulase) followed by mechanical isolation. The conditions generally used for the electrical fusion of protoplasts of somatic cells were also applied to the protoplasts of gametic cells of maize. Electrofusion was performed with single pairs o f gametes under microscopic observation. The mean fusion frequency was 79%. Isolated egg cells of maize showed protoplasmic streaming during 22 days of culture, but they did not divide. However, after fusion of the sperm with the egg cells, these fused cells did develop, with a mean division frequency of 83%, and grew to multicellular structures. Egg cells and fusion products were cultivated with a maize feeder-cell system.

Key words: In vitro fertilization - Egg cell - Sperm cell - Electrofusion - Z e a mays

Introduction

Although comprehensive information is available on the female gametophyte and the female and male gametes, little is known about the fertilization processes at the cellular level, as for such studies methods involving the manipulat ion of single cells have to be employed (Kranz et al. 1990). To per form in vitro fertilization at the single cell level, the isolation of viable gametes is a prerequisite. To date, sperm cells of various species have been iso- lated, and viable female gametes have been reported iso-

* Present address: Institut fiir Allgemeine Botanik, UniversitS.t Hamburg, Ohnhorststrasse 18, W-2000 Hamburg 52, Federal Re- public of Germany

Offprint requests to: E. Kranz

lated f rom the embryo sacs of Nicotiana (Hu et al. 1985), Torenia (Mol 1986), Z e a (Wagner et al. /988, 1989) and Plumbago (Huang and Russel 1989). There exists only one report of a micromanipulat ion method for in vitro fertilization in Torenia (at tempt at artificial fertilization), in which sperm cells were injected into the embryo sac (Keijzer et al. 1988).

We now report on the isolation of viable sperm and egg cells o f maize, the culture of single, unfertilized egg cells, the electrical fusion of single egg cells with single sperm cells, and the development of the fusion products in culture. This is to our knowledge the first time that the in vitro fertilization of higher plants with isolated, single gametes has been reported.

Materials and methods

Plant mater ial

For sperm and egg cell isolation, pollen and ears from maize (Zea rnays L.) inbred lines A 188 from A. Pryor, C.S.I.R.O., Canberra, Australia, pollen from inbred lines Ac 718, 1262, 1605, 1635 from P.A. Peterson, Department of Agronomy, Iowa State University, USA, pollen from inbred Ac line Zm651, and ears from inbred 2360 (Origin: Maize Genetics Cooperation) from U. Wienand, Max-Planck-Institut f/Jr Zfichtungsforschung, K61n, FRG, were used. The inbred line 1206 (Dr. W. Schmidt, KWS, Einbeck, FRG) was used for the establishment of a feeder cell suspension. All plants were grown in the greenhouse.

Pollen and ear collection

Pollen was collected between 9 and 12 a.m. from freshly dehisced anthers and used immediately or stored for some hours at about 22~ in 6-cm-diameter plastic dishes sealed with parafilm. The air was moistened by a piece of wet filter paper fixed to the lid of the dish (Kranz and L6rz 1990).

Ears were bagged before silk emergence, and the outer leaves of the ears at various developmental stages (indicated by silk length of emerging stage, 3.5-17.0 cm) were surface sterilized with ethanol (70%). Ovules were selected from the middle part of the ears under a dissecting microscope.

Isolation and selection o f sperm cells and egg cells

Sperm cells were released from the pollen grains after rupture by osmotic shock (Russel 1986; Matthys-Rochon etal. 1987; Cass and Fabi 1988), but without grinding, into the fusion medium (0.55 M mannitol). These were individually selected under micro- scopic observation (inverted microscope IM 35, Carl Zeiss, Ober- kochen, FRG, equipped with Normarski differential interference contrast accessories, objectives Dic 16 x, Dic 40 x ) by taking up with 15 nl of fusion medium into the capillary (diameter of the tip opening about 20 gm), and then transferred into the fusion droplet (Figs. 1, 2d). The capillaries were prepared from micropi- pettes (50 gl, Color code green, Brand, FRG) using a Leitz puller.

About 20 pieces of ovular tissue (containing the embryo sac) were collected in a mannitol solution (570 mosmol/kg HzO) follow- ing initial digestion in 3.5-cm-diameter plastic dishes with 1.5 ml enzyme solution (0.75% pectinase (Serva, Heidelberg, FRG); 0.25% pectolyase Y23 (Seishin, Tokyo, Japan); 0.5% hemicellulase (Sigma, Deisenhofen, FRG); and 0.5% cellulase "Onozuka" RS (Yakult Honsha, Tokyo, Japan), pH 5.0, adjusted to 570 mosmol/ kg H20 with mannitol). The ovular tissue-enzyme mixture was agitated on a rotary shaker at 140 rpm and 240_+0.5 ~ C for 50_+ 10 min, followed by mechanical isolation of the embryo sacs, the egg cells and the non-gametic cells (Fig. 2a-c) with a microcapill- ary. Embryo sacs, egg cells, synergids, and central cells were indi- vidually selected and transferred to the fusion droplet by microca- pillaries as described below, but with hand-drawn tip openings of about 200 gm.

Sperm and egg cells were selected by use of a hydraulic system: the microcapillaries were connected by means of Teflon tubing filled with mineral oil to a computer-controlled dispenser/dilutor (Microlab M. Hamilton, Darmstadt, FRG) (Koop and Schweiger 1985a).

In vitro fertilization Isotation

@ ~ egg cell sperm cell

| Etectrofusion

| fertilized egg ce[[

Cu[ture

1

L ~ l f e e d e r IVelrm"~ =~1 cells

mil[icell Fig. 1. Electrofusion-mediated in vitro fertilization method. Single, isolated sperm and egg cells were transferred into the fusion drop- lets, and pairs of gametes were fused electrically after dielectrophor- etical alignment on one of the electrodes. For culture, the fusion products were transferred individually into "Millicell" inserts sur- rounded by feeder cells as described

13

Fig. 2a-~l. Isolation of embryo sac, egg and sperm cells of Zea mays L. E Egg cell, S synergid, C central cell. Bars= 50 gin. a Remnant cell wall and adherant cells have to be removed mechani- cally, b Unit of central cell (C), synergids (S) and egg cell (E) after mechanical isolation. Note the rounding-off in the fusion medium, e Isolated egg cell. d Isolation of sperm cells by a microca- pillary. Arrows indicate sperm cells

Electro fusion

The controlled electrical fusion of selected single gametes was per- formed using the method described by Koop et al. (1983), Koop and Schweiger (1985b), and Schweiger et al. (1987). Ten fusion droplets of 1000 nl each, each consisting of 0.55 M mannitol, were overlayered by mineral oil on a coverslip, and selected single ga- metes were transferred to these droplets for fusion (Fig. l). Con- trolled electrofusion was performed with a pair of electrodes (plati- num wire, diameter 50 gm) that were fixed to an electrode support mounted under the condensor of the microscope. The distance between the electrodes was adjusted by lowering them along the z-axis onto the coverslip. This was accomplished by a step motor controlled by a computer and a positioning system (MCC 13 JS, Lang, H/ittenberg, FRG). Fusion was induced by single or multiple (2-3) negative DC-pulses (50 gs; 0.9-1.0 kV-cm-1) after dielectro- phoretic alignment (1 MHz, 71 V-cm-1) on one of the electrodes for a few seconds with an electrofusion apparatus CFA 400, Kriiss, Hamburg, FRG).

Culture procedures

Fusion products were cultured on the transparent, semipermeable membrane of "Millicell-CM" dishes (diameter 12 ram, Millipore). These dishes were inserted in the middle of 3.5-cm plastic dishes previously filled with 1.5 ml of a maize feeder suspension, or feeder cells were separately cultivated in additional Millicell inserts sur- rounded by medium (Fig. 5 a). The fusion products as well as the feeder cells were cultivated in a modified MS medium (Murashige and Skoog 1962) containing 1.0 mg/t 2,4-D and 0.02 rag/1 kinetin, adjusted to 600 mosmol/kg H20 with glucose, pH 5.7 (medium I) or in a different modified MS medium containing 0.4 mg/1 thia- mine, 183 mg/1 IMysine, 147 rag/1 L-glutamic acid, 105 mg/1 L-serine (Gengenbach and Green 1975), 0.5 g/1 caseinhydrolysate, 1.0 rag/1 2,4-D, 0.02 mg/1 kinetin, adjusted to 530 mosmol/kg H20 with glu- cose, pH 5.8 (medium II). The culture conditions were: 24~

14

Fig. 3a-e. Electrofusion sequence of a selected pair of egg and sperm cell of Zea mays L. Arrows indicate fusion site of sperm cell with the egg cell. After application of the DC pulse the time interval from a to e was 4 s, but in most experiments this interval was less than i s. E Egg cell, S sperm cell. Bar = 50 pm

0.5 ~ C, a light/dark cycle of 16/8 h, and a light intensity of approxi- mately 50 gEm-z s- i .

The non-morphogenic maize cell suspension used as a feeder was established using immature embryos about 1.0 mm in size. These were cultured on solid CC medium (Potrykus et al. 1979) for about 10 days. Embryos with developing callus were individual- ly cultivated in macroplates for up to 7 months in 2 ml liquid Kao medium (Kao 1977) following the modifications of Nakamura and Keller (1982) and using the micronutrient composition of Schenk and Hildebrandt (1972) supplemented with 8 mg/1 2,4-D, pH 5.6. The suspension was then transferred onto MS medium containing 1.0 mg/1 2,4-D, 0.02 mg/1 kinetin, and 30 g/1 sucrose (190 mosmol/ kg HzO), pH 5.7. After a further 8 months the suspension was transferred onto the same medium, but instead of sucrose, glucose was used, and the medium was adjusted to 600 mosmol/kg H20 and cultured on a rotary shaker at 140 rpm and 24_+0.5~ C in the dark.

Fig. 4. DAPI-staining of the fusion product of sperm cell and egg cell of Zea mays L. Arrow indicates the sperm cell inside the egg cell. Bar = 50 gm

Staining procedures

4',6-diamidino-2-phenylindole (DAPI) staining was performed us- ing 10 gl stock solution (1 mg DAPI/ml distilled water) added to 0.5 ml citrate-phosphate buffer, pH 4.0, (Coleman and Goff 1985), adjusted to 600 mosmol/kg H20 with mannitol. Egg cells were transferred with 500 nl medium into 500 nl droplets of staining solution.

Results

Egg cell isolation

A yield of up to 50% isolated egg cells could be obta ined. Rout ine ly , 5 egg cells per 20 ovule pieces could be iso- lated (mean isola t ion f requency 25%). This could be achieved with ovular tissue f rom var ious deve lopmenta l stages ( indicated by silk emergence lengths of 3.5, 5 . 5 , 8.0, 10.0, 12.0, and ~7.0 cm, line A 188).

Electrofusion o f sperm and egg cells

Condi t ions general ly used for the electrofusion of p ro to- plasts of somat ic cells were also applied to the gametic pro toplas ts o f maize (Fig. 4). Sperm cells fused very quickly with egg cells. The d u r a t i o n of the r o u n d i n g - o f f process did no t exceed 1 s in mos t exper iments (Fig. 3). The m e a n fus ion f requency was 79%, bu t 100% could also be ob ta ined (total n u m b e r of fusions per formed was 186). The a l ignmen t of the two gametes in the se- quence so that the egg cell was in contac t with the elec-

Fig. 5a-e. Culture method and development of fusion products of defined pairs of egg and sperm cells of Zea mays L. a Culture of the fusion products in "Millicell" dishes as indicated by arrow. Feeder cells can be inoculated in additional "Millicell" dishes as shown, or the feeder suspension can be inoculated into the outer dish. Bar= 1.5 cm. b Unfertilized egg cells 6 days after isolation on the semipermeable membrane of a "Millicell" dish. Culture with feeder cells. Medium I. Bar= 50 gm. c Fertilized egg cells developed to multicellular structures 6 days after fusion with sperm cells. Same cultures conditions as in h. Bar = 50 gm. d Cell division 2.5 days after fusion. Culture conditions as in h. Bar=lO0 gin. e Multicellular structures 18 days after fusion. Culture conditions as in b. Bar = 500 gm

15

Table 1. Electrofusion and development of the fusion products of sperm and egg cells of different maize lines. Medium I

Lines Multicellular structure Sperm cell + Egg cell formation (total 122)

Mean frequency (%) n

A 188 + A 188 83 22 Ac 1262 +A 188 80 23 Ac 718 +A 188 77 16 Ac 1635 § 188 82 20 Ac Zm 651 +A 188 83 24 Ac 1605 +2360 77 17

trode (Fig. 3) proved to be necessary in order to obtain high fusion frequencies. In most cases fusion was in- duced by a single DC pulse. No differences in fusion frequency could be found between egg cells derived f rom ovules of different developmental stages (line A 188).

Development of egg cells and fusion products

Our at tempts to induce cell division of unfertilized egg cells have so far failed. The egg cells of maize Showed protoplasmic streaming during 22 days of culture, but no cell division was observed (Fig. 5 b). Under compara- ble culture conditions, however, the fusion products of a sperm cell with an egg cell started to divide within 2.5-3 days (Fig. 5d). Microcalli developed with a mean frequency of 83% (Fig. 5c, e). A difference in the divi- sion rate, when cultured in medium I and II, respectively, could not be observed. High frequencies of format ion of multicellular structures could be found using sperm cells and egg cells f rom different lines (Table 1).

Discuss ion

In vitro fertilization of plants using isolated gametes of maize was performed for the first time. We accomplished the initial steps of the in vitro development of the artifi- cial, putative zygote. Without any cell-wall degrading enzyme t reatment prior to fusion, sperm cells could effi- ciently be fused with the egg cell, which is a further indication that the sperm cells in a mature pollen grain of maize represent true protoplasts (Dupuis et al. 1987; Mat thys-Rochon et al. 1988; Cass and Fabi 1988).

We could not observe any difference in the yield of isolated embryo sacs and egg cells using ovules of a wide range of different developmental stages. This is in contrast to the results of Wagner et al. (1989), who worked with a different line. Higher concentrations of cell-wall degrading enzymes than described above gave suboptimal yields of isolated embryo sacs and egg cells. Additionally, as found by Wagner et al. (1989), the exact timing of incubation of ovule tissue of maize in the en- zyme mixture was important . The yield was ultimately determined by the subsequent manual isolation step.

To date the development of egg cells of higher plants in culture has not been described in the literature. Our

observat ion that unfertilized egg cells do not divide under conditions allowing the fusion products of sperm and egg to develop at a high frequency may facilitate studies on cell division factors. Tha t the fusion products will grow in culture will also facilitate studies on the differentiation of artificially produced zygotes in vitro. These cells, with the i r " na tu ra l " competence for division and regeneration, might be useful for t ransformat ion studies involving the microinjection of DNA. In prelimi- nary studies we have observed high survival and division rates (83 % and 67 % respectively) in artificially produced putative zygotes injected with plasmid D N A (E. Kranz, unpublished data). Additionally, it could be shown that electrofusion-mediated in vitro fertilization, using iso- lated, single gametes, is a suitable system for transmit- ting chloroplasts and mitochondria via cytoplasts through the fertilization process (Kranz et al. 1991), which is of basic interest for the study of cytoplasmic inheritance.

Acknowledgements. The authors thank Dr. U. Wienand (MPI, K61n, FRG) for providing material of the Ac lines, Dr. A. Pryor (C.S.I.R.O., Canberra, Australia) for seeds of line A188, Dr. W. Schmidt (KWS, Einbeck, FRG) for seeds of line 1206, Prof. H.U. Koop, Dr. G. Spangenberg, Mr. D. Wolff, and Mr. J. Dirk for help on the single cell culture and electrofusion techniques. The critical reading of the manuscript by Dr. P.A. Lazzeri, the prepara- tion of the photographic material by Mrs. C. Adami, and the finan- cial support by the BMFT Bonn (grant BCT 0390-2) for this re- search project are gratefully acknowledged.

References

Cass DD, Fabi GC (1988) Structure and properties of sperm cells isolated from the pollen of Zea mays. Can J Bot 66:819-825

Coleman AW, Goff LJ (1985) Applications of fluorochromes to pollen biology. I. Mithramycin and 4',6-diamidino-2-phenyl- indole (DAPI) as vital stains and for quantitation of nuclear DNA. Stain Technol 60:145-154

Dupuis I, Roeckel P, Matthys-Rochon E, Dumas C (1987) Proce- dure to isolate viable sperm cells from corn (Zea mays L.) pollen grains. Plant Physiol 85 : 876-878

Gengenbach BG, Green CE (1975) Selection of T-cytoplasm maize callus cultures resistent to Helminthosporium maydis Race T pathotoxin. Crop Sci 15 : 645-649

Hu SY, Li LG, Zhu C (1985) Isolation of viable embryo sacs and their protoplasts of Nicotiana tabacum. Acta Bot Sin 27: 337-344

Huang BQ, Russel SD (1989) Isolation of fixed and viable eggs, central cells, and embryo sacs from ovules of Plumbago zeylani- ca. Plant Physiol 90:9-12

Kao KN (1977) Chromosomal behaviour in somatic hybrids of soybean - Nicotiana glauca. Mol Gen Genet 150:225-230

Keijzer CJ, Reinders MC, Leferink-ten Klooster HB (1988) A mi- cromanipulation method for artificial fertilization in Torenia. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Berlin Heidelberg New York, pp 119 124

Koop HU, Schweiger HG (1985a) Regeneration of plants from individually cultivated protoplasts using an improved microcul- ture system. J Plant Physiol 121 : 245-257

Koop HU, Schweiger HG (1985b) Regeneration of plants after electrofusion of selected pairs of protoplasts. Eur J Cell Biol 39: 4649

Koop HU, Dirk J, Wolff D, Schweiger HG (1983) Somatic hybrid- ization of two selected single cells. Cell Biol Int Rep 7: 1123- 1128

Kranz E, L6rz H (1990) Micromanipulation and in vitro fertiliza-

16

tion with single pollen grains of maize. Plant Sex Reprod 3:160-169

Kranz E, Bautor J, L6rz H (1990) In vitro fertilization of single, isolated gametes, transmission of cytoplasmic organelles and cell reconstitution of maize (Zea mays L.). In: Nijkamp HJJ, Van der Plas LHW, Van Aartrijk J (eds) Progress in plant cellu- lar and molecular biology. Proceedings of the VIIth Interna- tional Congress on Plant Tissue and Cell Culture, Amsterdam, The Netherlands, 24-29 June 1990. Kluwer Academic Publish- ers Dordrecht, Boston, London, pp 252-257

Kranz E, Bautor J, L6rz H (1991) Electrofusion-mediated transmis- sion of cytoplasmic organdies through the in vitro fertilization process, fusion of sperm cells with synergids and central cells, and cell reconstitution in maize. Sex Plant Reprod 4:17-21

Matthys-Rochon E, Vergne P, Detchepare S, Dumas C (1987) Male germ unit isolation from three tricellular pollen species: Brassi- ca oleraeea, Zea mays, and Triticum aestivum. Plant Physiol 83: 464466

Matthys-Rochon E, Detchepare S, Wagner V, Roeckel P, Dumas C (1988) Isolation and characterization of viable sperm cells from tricellular pollen grains. In: Cresti M, Gori P, Pacini E (eds) Sexual reproduction in higher plants. Springer, Berlin Hei- delberg New York, pp 245-250

Mol R (1986) Isolation of protoplasts from female gametophytes of Toreniafournieri. Plant Cell Rep 3 : 202-206

Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497

Nakamura C, Keller WA (1982) Callus proliferation and plant regeneration from immature embryos of hexaploid Triticale. Z Pflanzenzfichtg 88 : 137-160

Potrykus I, Harms CT, L6rz H (1979) Callus formation from cell culture protoplasts of corn (Zea mays L.). Theor Appl Genet 54:209-214

Russel SD (1986) Isolation of sperm cells from the pollen of Plum- bago zeylanica. Plant Physiol 81:317-319

Schenk RU, Hildebrandt AC (1972) Medium techniques for induc- tion and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199-204

Schweiger HG, Dirk J, Koop HU, Kranz E, Neuhaus G, Spangen- berg G, Wolf D (1987) Individual selection, culture and manipu- lation of higher plant cells. Theor Appl Genet 73:769-783

Wagner VT, Song Y, Matthys-Rochon E, Dumas C (1988) The isolated embryo sac of Zea mays: structural and ultrastructural observations. In: Cresti M, Gori P, Pacini E (eds) Sexual repro- duction in higher plants. Springer, Berlin Heidelberg New York, pp 125-130

Wagner VT, Song YC, Matthys-Rochon E, Dumas C (1989) Obser- vations on the isolated embryo sac of Zea mays L. Plant Sci 59:127-I 32