Regeneration of the barley zygote in ovule culture

Sex Plant Reprod (1995) 8:49-59 �9 Springer-Verlag 1995

P.B. Holm �9 S. Knudsen �9 P. Mouritzen �9 D. Negri F.L. Olsen. C. Rou6

Regeneration of the barley zygote in ovule culture

Received: 2 May 1994 / Revision accepted: 3 May 1994

Abstract An ovule culture technique has been established for barley that allows the regeneration of plants from zygotes. An average of 1.3 plantlets per ovule could be regenerated from more than 60% of the cultured ovules and about 75 % of the regenerated plantlets developed into normal, fertile plants. The same regeneration frequencies were obtained in intact ovules and in ovules where the two integuments had been removed from the micropylar region. Unfertilized ovules and ovules where the fertilized eggs had been destroyed by a microinjection needle did not give rise to embryo-like structures. Plants could be regenerated from the zygote at the same frequency at developmental stages from immediately after fertilization until the formation of bicellular embryos. This tissue culture system appeared to be largely independent of genotype since similar regeneration frequencies were obtained in two different barley cultivars, Igri and Alexis, that in anther and microspore culture behave differently. The same technique has also been applied successfully in the wheat cultivar Walter.

Key words Hordeum vulgare �9 Egg cell In vitro culture �9 Embryogenesis �9 Plant regeneration

introduction

Regeneration of fertilized egg cells and young zygotic embryos by means of in situ culture of intact ovules is

P. B. Holm ( 9 ) - S. Knudsen - P. Mouritzen �9 F. L. Olsen Carlsberg Research Laboratory, 10, Gamle Carlsberg Vej, DK-2500 Copenhagen, Denmark, FAX no: +45-33274741

D. Negri Secobra Recherches S.A., Centre de Bois Henry, F-78580 Maule, France

C. Rou~ Tepral, Brasseries Kronenbourg, 68, route d'Oberhausbergen, F-67200 Strasbourg, France

well established for many plant species (for review, see Rangan 1982). The initial interests in this tissue culture system were primarily to rescue hybrid embryos and to identify media and conditions supporting embryo mat- uration. In a few cases, this technique has also been used to generate haploids by cultivation of unfertilized ovules (for review, see Yang and Zhou 1982).

In the last few years there has been an increasing interest in developing techniques allowing the manipulation of egg cells and for the regeneration of in vivo or in vitro fertilized egg cells into plants. These techniques are required for the establishment of a more controlled experimental approach to essential proces- ses such as fertilization, zygote development, and early embryogenesis. The egg cell is also considered a pro- mising target for many approaches of relevance for biotechnology and plant breeding such as in vitro fertilization and transformation. Substantial efforts have thus been devoted to the isolation of viable embryo sacs and protoplasts of the egg by combined enzymatic di- gestion and microdissection approaches (for review, see Huang and Russell 1992). In general, however, the vi- ability of isolated cells is reported to be poor and only in two cases have regenerable protoplasts of egg cells been isolated. In maize, Kranz et al. (1991) isolated protoplasts of the unfertilized egg by dissection of enzymat- ically digested ovules. The protoplasts were in vitro fertilized by electrofusion with sperm cells, isolated from mature pollen by osmotic shock. The fusion products were regenerated into plants by co-cultivation with a nonmorphogenic maize suspension (Kranz and L6rz 1993). In barley, protoplasts of the fertilized egg could be isolated by a simple dissection of the ovule and regenerated into fully fertile plants by a co-cultivation with microspores undergoing embryogenesis. The same technique could also be applied to wheat (Holm et al. 1994).

Cultivation and regeneration of the zygote by means of ovule culture represent an alternative to the more complex and tedious isolation and cultivation of isolated egg protoplasts. Many studies have been devoted to

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developing ovule culture systems that also allow the eggs or small embryos to be accessible to manipulation. Keijzer et al. (1988) outlined a strategy for in vitro fertilization of Torenia, where sperm cells were microinjected into the embryo sac. T6pfer and Steinbiss (1985) showed that barley zygotes cultured in ovules could be regenerated into plants and Steinbiss et al. (1985) suggested that a transformation system for barley might be established by microinjection into the fertilized egg. A similar strategy was formulated for wheat (Mathias 1987), involving injection of DNA into the embryo sac on ovaries still attached to the spike. In maize, recent studies have been directed towards mapping the three-dimensional position of the egg cell in ovules as a prerequisite for microinjection of DNA into the egg (Wagner et al. 1990). Culture techniques have also been established for mi- crodissected ovules consisting primarily of the embryo sac (Campenot et al. 1992) or embryo sacs surrounded by a few layers of nucellus cells (M61 et al. 1993). In wheat, Comeau et al. (1992) have described ovule culture techniques allowing the rescue of proembryos 1-6 days after pollination.

Manipulation of the fertilized egg in situ in isolated ovules by microinjection requires that the egg is accessible and can be viewed with high resolution. For labor- intensive manipulations such as microinjection, it is also essential that plants can be regenerated from the egg cell with a high frequency. The present study has been ad- dressed to developing an ovule culture technique for barley that can meet these two requirements. It has been an additional objective of this study to investigate if the regeneration capacity of the egg cell in situ relates to its developmental stage from immediately after fertilization until the first division of the zygote. The analyses were performed in two barley cultivars, the winter barley Igri and the spring malting barley Alexis that show very different performances in anther and microspore culture.

Materials and methods

Plant material

Barley plants of the winter barley cultivar Igri and the spring malting barley cultivar Alexis were grown in growth cabinets at 15~176 day/night temperatures using a 16-h light period at a light intensity of ca 350 laE x m -2 x s 1. The florets were emasculat- ed 3-4 days before anthesis and pollinated by hand using a fine- tipped brush.

mixture (no. K3129, Sigma Chemical Company, St. Louis, Mo., USA). Vitamin-free casamino acids (no. 2238, Merck, Darmstadt, Germany) were added at a concentration of 250 mg/l together with 20 ml/1 coconut water (no. 570-5180AJ, Gibco Laboratories, Life Technologies, Grand Island, N.Y., USA). The medium was made up 2 x concentrated, the pH adjusted to pH 5.8 with 1 N NaOH and the solution sterilized by filtration (0.22 i, tm pore size). Solid Kao 90 medium was made by mixing the double concentrated Kao 90 medium with 0.8% autoclaved SeaPlaque agarose (FMC BioProducts, Rockland, Me., USA) in a 1 : i ratio. For regeneration, the MS medium described above was used but containing only 0.4 mg/1 BAP and 30 g/1 maltose. The medium was also solidified with SeaPlaque agarose as described above. Unless otherwise stated, all chemicals were from Merck or Sigma.

Isolation of ovules

Spikes were harvested 1-24 h after pollination and the ovaries removed from the florets and placed on Kao 90 medium. Ovaries from one spike were sterilized for 10 rain in 5 ml 10% sodium hypochlorite (no. 23039, BDH, Poole, UK) with 50 gl 10% Tween 20 (no. 82218, Merck, Schuchardt, Germany). After two washes in sterile water, the ovaries were transferred to Kao 90 and dissected under a dissection microscope (40x magnification, transmitted light). Ovaries were placed with the placental side facing downwards and the pedicel and the two lodicules were removed by using a pair of fine-tipped forceps. A cross section though the ovarian tissue was made with the tip of the forceps at a median position and along one side down to the micropylar end of the ovary. The tip of the ovary was removed, exposing the micropylar end of the ovule. In some preparations, the inner and outer integuments were dissected away from the micropylar part of the ovule. A few experiments were performed where the tip of the ovule containing the egg was cut off and cultured on liquid Kao 90 with 20% Ficoll 400 (no. 17-0400-02, Pharmacia LKB Biotechnology AB, Uppsala, Sweden). The Ficoll was used as a buoyancy increasing agent, which allowed the ovules to float on top of the liquid medium. In the majority of cases, the dissected ovaries were transferred to 2.5-cm Whatman filter paper discs (no. 1004025, Whatman, Maidstone, UK) on agarose-solidified medium. The ovaries were opened by cleaving them slightly to the one side of their longitudinal axis and the ovules lifted out and placed on filter paper with their placental side facing downwards. Steritin culture dishes (5 cm, Bibby Sterilin, Stone, Staffs, UK) were sealed with Nesco film (Nippon Shoji Kaisha, Ltd., Osaka, Japan), wrapped in aluminum foil and incubated at 23~ After 3 weeks, the dishes were opened and the developing embryo-like structures were dissected out of the ovule. After 3-4 weeks, when the embryos started to germinate, they were transferred to regeneration medium in Sterilin dishes and cultured under low light conditions at 23~ When additional growth had occurred, the plantlets were moved to fresh regeneration medium in 10-cm-high cylindrical containers (Greiner, Bibby Sterilin). For the winter barley Igri, the plantlets were transferred to 4~ for 8 weeks for vernalization when large plantlets had formed. Thereafter, the plantlets were transferred to soil and cultured in a greenhouse equipped with artificial light and heating/cooling facilities (15/10~ 16 h light period).

Media

In all ovule cultures, a medium referred to as Kao 90 was used. This contains the macro- and micro-elements of the modified Mu- rashige and Skoog (MS) medium (Murashige and Skoog 1962) described by Olsen (1987). In this medium, the NH4NO 3 concentration is reduced to 165 mg/l while 750 mg/1 of glutamine is added. The medium also contained 1 mg/1 6-benzylaminopurine (BAP), maltose (90 g/l) as the only carbohydrate source, and the vitamins and organic acids from the Kao medium (Kao and Michayluk 1975). The vitamins were purchased as a ready-to-use

Optical analysis of the egg in vivo

Ovaries where one or both integuments had been removed from the micropylar part of the ovule were mounted for optical analysis in the following way: the outermost 2-3 mm of a 24 x 40 mm cover slip was covered with a rim of silicone glue (Fugendichtung Silicon, Practicus Chemie,Grevenbroich, Germany) and the cover slips sterilized by placing them under UV illumination for a minimum of 30 min. The ovaries were mounted in the glue with the exposed ovule tip towards the center of the cover slip and oriented to ensure that the longitudinal axis of the egg cell was parallel to

the surface of the coverslip. Kao 90 medium was added in suffi- cient amounts to cover the ovule tip. The dissected ovules were analyzed under differential interference contrast on a Zeiss Ax- iovert microscope using a 40X objective with a variable focal distance. The microscope was equipped with a VE-1000 video enhancement system (DAGE-MTI, Inc., Ind., USA). Needles to be used for disrupting the egg were pulled from borosilicate micro- capillaries (microelectrode capillaries no. 1403512, 1.5 mm in outer diameter, Hilgenberg, Malsfeld, Germany) using a vertical puller (Bachofer, Reutlingen, Germany) and the tips bevelled with a Narishige EG-4 microgrinder (Narishige, Setagaya-Ku, Tokyo, Japan).

Regeneration in relation to the stage of the zygote

Ovules were sampled at 1 h intervals from 2 h after pollination until 28 h after pollination and the integuments removed from the micropylar part of half the ovules of each spike. The analysis was performed for both Igri and Alexis. To ensure optimal synchrony between the individual zygotes, florets from the tip and the base of the spike were not used, since they are delayed in development compared to the florets of the median part of the spike. Fifteen to 20 ovules were cultured for each interval of each of the four com- binations, i.e., Igri and Alexis with and without integuments in the micropylar part. The embryo-like structures were dissected out after 21-28 days of culture and the number of ovules with embryo- like structures recorded as responding ovules. The number of embryo-like structures in each ovule was counted and if loose aggregates of embryo-like structures were present, the individual structures were separated. Individual embryo-like structures from each ovule were cultured separately and the number of plants regenerated from each ovule was counted.

Light microscopy

Freshly isolated as well as cultured ovules were fixed in 4% glu- taraldehyde in 50 mM phosphate buffer, pH 7.0 for 90 min. After three washes in the same buffer, the material was stored in buffer at 4~ overnight and the next day postfixed for 2 h in 1% OsO4 in phosphate buffer. After a wash for 15 min in buffer, the cells were dehydrated in a graded ethanol series (30 min in each concentration), washed twice in absolute ethanol, once in propytenoxide, and thereafter infiltrated with Spurr's low viscosity epoxy resin (Spurr 1969). The resin was polymerized at 70~ overnight in flat embedding molds. The material was sectioned at 2-5 txm thick- ness with a glass knife, using a Reichert OmU3 ultramicrotome. The sections were placed on water droplets on slides which were then baked on a hot plate. Permanent slides were made by mount- ing in Eukitt (acrylic resin in xylene, O. Kindler, Freiburg, Ger- many). The sections were analyzed under phase contrast optics in a Zeiss Axioplan microscope.

Results

The in vitro development of the barley zygote

The barley embryo sac is of the Polygonum type (Ma- heshwari 1950). The central cell constitutes mainly the embryo sac and contains a giant vacuole surrounded by a thin band of cytoplasm. The two central or polar nuclei are located next to the synergids and the egg cell. In the barley cultivar Bomi grown at 18/13~ day/night temperature with a 16 h light period, the egg is fertilized 45 rain after pollination. At 55-60 rain after pollination, the two polar nuclei and the associated sperm nucleus

51

move away from the egg cell and into the central cell (Engell 1989). The absence of polar nuclei associated with the egg apparatus thus provides an excellent control indicating that fertilization has taken place. This can easily be seen in dissected ovaries using a dissection microscope equipped with transmitted light (Holm et al. 1994).

Initial experiments were directed towards excising and culturing the micropylar tip of the ovule. By this approach, the egg would also be accessible for manipulation. The ovule tips were cultured floating on liquid Kao 90 medium with 20% Ficoll. The addition of Ficoll proved to be important , since the increased density of the medium prevented the ovule tip from sinking into the medium, in which case the development of embryo- like structures was generally arrested. Several series of experiments revealed that about 20% of the egg cells of Igri as well as Alexis developed into embryo-like structures which produced plants. Ovule tips with embryos ranging in stage from bicellular to 0.2-0.3 mm in length were also cultured, and there was an almost linear, pos- itive correlation between the time after pollination and the frequency of embryo formation.

Cultivation of ovule tips on solid agarose-Kao 90 medium resulted in a slight increase in the frequency of embryo-like structures, but the structures which developed appeared more embryo-like than when grown on liquid medium. It was also apparent that the egg was often damaged when cutting off the micropylar tip of the ovule. To improve embryo quality and to minimize dissection-induced damage, all subsequent experiments were performed on whole ovules. This meant that the egg cell would only be accessible by penetrating the integuments and the nucellus layers.

Removal of one integument drastically improved the optical resolution of the egg apparatus, in particular if interference contrast microscopy was performed. When both integuments were dissected away from the micropylar tip, it was possible to see details such as the nucleus of the egg at a high resolution (Fig. 1A) in most of the mounted ovaries. After a few hours of culture, the nucellus tissue became more opaque and the further development of the egg and the embryo-like structures could only be moni tored in dissected ovules or in fixed and sectioned material. The series of photographs in Fig. 2 illustrates the development of the egg into embryogenic cell masses. Polyembryony was often encoun- tered and separation of cells or cell clusters giving rise to two or more embryo-like structures may occur early in development (Fig. 2B). Endosperm formation was not observed but the antipodal cells appeared to stay alive for a few days. At later stages of culture, the developing structures often consisted of aggregates of cells that were either completely or partly separated from each other (Fig. 2C,D). The smaller aggregates and the outer layers of the larger aggregates consisted of cells with dense cytoplasm, large nuclei, and few vacuoles, while the central part of the large aggregates was parenchy- ma-like with large vacuoles.

52

Fig. 1A-F Ovule culture in barley. A The morphology of the egg cell in situ as seen in interference contrast. Both integuments have been removed and only the nucellus cell layers remain. Bar 30 gin. B A club-shaped proembryo-like structure developed within the ovule. Bar 0.5 ram. C An aggregate of embryo-like structures. Bar 0.5 ram. D Different types of embryo-like structures emerging from the ovular remains. Bar 1.0 mm. E A club-shaped proembryo dissected out of the ovule. Bar 0.5 ram. F A well-defined embryo-like structure emerging from the remains of the ovule. Bar 0.5 mm

After 3 4 weeks of culture, the emerging embryo-l ike structures were dissected out of the ovules, which had acquired a dark color, p robab ly due to the accumula- tion of polyphenols (Fig. 1B,D,F). At an early stage of development , the embryo-l ike structures were club- shaped (Fig. 1E) with apical meris tems and a r im of tissue later to become the scutellum. These structures developed further into well defined embryo-l ike structures with a scutellum and a coleopti le-coleorhiza (Fig. 1F). In several cases, however, the embryo-l ike structures possessed scutella with a quite irregular outline and

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Fig. 2A-D Sections of ovules at the different stages in the formation of embryo-like structures. A Ovule isolated 3 h after pollination and cultured for 6 h (E egg cell, DS degenerating synergid, N nucellus tissue, I I inner integument, OI outer integument). Bar 50 gin. B Ovule isolated 24 h after pollination and cultured for 4 days. Bar 50 gin. C Ovule isolated 7 days after pollination. Bar 100 gin. D Ovule isolated 14 days after pollination. Bar 100 gm

with more apical meris tems (Fig. 1B). Compac t callus- like structures with little structural differentiation were frequently observed. Aggregates of embryo- l ike structures (Fig. IC) were generally easy to dissociate, while more callus-like structures could be separated only to a limited extent. Plantlets were obta ined mainly f rom the more defined embryo- l ike structures but sometimes also f rom morphogen ic calli. In a few cases, the c lub-shaped

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Table 1 Cultivation of Igri ovules on seven different media

Medium Ovules Ovules with formation of embryo-like structures

Number %

Plantlets Plantlets after 33-35 days after 41-45 days

Number % Number %

Full Kao 90 29 27 93 Kao 90 16 11 69 without casein hydrolysate Kao 90 16 14 88 without coconut water Kao 90 30 17 57 without BAP Kao 90 without BAP 29 25 86 + 1 rag/1 2,4-D Kao 90 without BAP 28 21 75 + 2 mg/1 2,4-D Kao 90+ 1 mg/l 2,4-D 19 9 47

27 93 28 97 7 44 l l 69

10 63 12 75

3 10 7 23

2 7 22 76

8 29 20 71

6 32 7 37

Table 2 Cultivation of Alexis ovules on four different media

Medium Ovules Ovules with formation of embryo-like structures

Number %

Plantlets Plantlets after 32-34 days after 49-54 days

Number % Number %

Kao 90 20 13 65 Kao 90 without BAP 10 9 90 Kao 90 without BAP 10 6 60 + 2 rag/1 2,4-D Kao 90 + 1 rag/1 2,4-D 20 8 40

6 30 16 80 1 10 4 40 0 0 3 30

2 10 10 50

structures germinated precociously. The shoots and roots sometimes developed simultaneously but usually rooting was delayed until the plantlets had been transferred to regeneration medium.

Embryo-l ike structures were not obtained from unfertilized ovules or ovules where the egg cell had been disrupted. Ovules were isolated from ten spikes of the cultivar Igri, ranging in development from 2 weeks before flowering until immediately before anthesis and cultured on Kao 90-agarose. In none of the cultured ovules was there any indication of embryo formation after several weeks of culture. Forty ovaries from two spikes of Igri were isolated 8 h after pollination, the integuments removed from the tip of the ovules and the dissected ovaries mounted for microinjection. A bevelled microinjection needle was inserted through the nucellus tissue into the embryo sac, and the egg cell punc- tured while leaving the rest of the embryo sac, including the persistent synergid, intact. After several weeks of culture, there were no indications of embryo formation in the ovules. Ovules isolated and cultured immediately after poll ination were also sterile.

Effect of different media

Ovules of Igri, isolated 8 h after pollination were grown on seven different media to identify media components

and hormones essential for optimal regeneration. The analyses were performed as split spike experiments where ovules from a given spike were placed at random on at least four different media. The results presented in Table 1 represent the means of three separate experiments. The fastest growth and the best embryo formation were obtained on Kao 90 medium. Kao 90 without BAP resulted in a very slow embryo formation, while nearly all ovules on Kao 90 medium responded with the formation of embryo-like structures that germinated in- to plantlets. Only seven plantlets were regenerated from 30 ovules in the absence of BAP. The most prominent effect of the omission of casein hydrolysate and coconut water was slower development. Cultivation on Kao 90 with 1 or 2 rag/1 2,4-D but omitting BAP resulted in the formation of very compact callus-like structures that primarily formed roots. Germinat ion was delayed by about 10 days compared to that observed on Kao 90. If BAP was included together with 2,4-D, only half the ovules responded and gave rise to compact calli that tended to darken and die. However, a few plants were recovered from ovules grown on this medium.

In Alexis, similar effects of these two hormones were observed but relatively high regeneration frequencies were observed on Kao 90 + 1 mg/l 2,4-D (Table 2). On Kao medium with 60 g/l and 120 g/1 maltose, formation of embryo-like structures and plantlet regeneration

Alexis

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Fig. 3 Histograms showing the frequency of ovules with embryo- like structures, the frequency of ovules giving rise to plantlets, the number of embryo-like structures per responding ovule and the

55

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number of plantlets per embryo-like structure in Alexis and Igri without integuments (white columns) and with integuments (hatched columns)

56

Table 3 Summary table: frequency of formation of embryo-like structures and plantlet regeneration in Alexis and Igri with and without integuments in micropylar region

Cultivar Ovules Ovules giving rise to Embryo-like structures Plantlets

Embryo-like Plantlets Per Per responding Per embryo- Per responding structures % % ovule ovule like structure ovule

Igri 232 87 without integuments

Igri 205 92 with integuments

Alexis 246 72 without integuments

Alexis 261 77 with integuments

72 1.05 1.21 0.80 1.16

55 0.94 1.02 0.62 1.05

63 1.18 1.65 0.81 1.53

61 1.12 t.46 0.75 1.39

were also frequent. It remains to be determined whether regeneration frequencies are inferior or superior to those found on Kao 90.

Relationship between the age of the egg and regeneration frequency

The first division of the barley zygote takes place 24- 28 h after fertilization. A large-scale experiment was carried out to evaluate if the frequency of formation of embryo-like structures and plantlet regeneration de- pended on the developmental stage of the zygote.

The histograms in Fig. 3 illustrate the frequencies of ovules giving rise to embryo-like structures and plants, the number of embryo-like structures per responding ovule, and the number of plants per embryo-like structure from 2 to 30 h after pollination in dissected and intact ovules of Igri and Alexis. The observations were grouped for 4 h intervals for the period from 2 to 18 h after pollination while the period from 18 h to 30 h was divided into 6 h intervals. This was performed to facili- tate the statistical analysis of the data and to minimize variation originating from small sample sizes. The means of the data for each of the four experimental conditions are given in Table 3.

Statistical analysis using a Tukey's Studentized Range (HSD) test for variable showed the following: in Igri the frequency of ovules with embryo-like structures was significantly higher than in Alexis (significant at the 1% level) but there was no effect of removing the integuments. In contrast, the number of embryo-like structures per ovule was significantly higher in Alexis than in Igri. In Alexis 68, 19, 7, and 4% of the ovules contained 1, 2, 3, and 4 embryo-like structures respectively, while for Igri the corresponding frequencies were 87, 12, 1, and 0%. Individual embryo-like structures of the aggregates were as viable as those developed as single structures in an ovule. In a few cases, up to seven embryos and plantlets were obtained from single ovules and all germinated into plantlets. There was in both cultivars a significant increase in the number of embryo-like structures per ovule when integuments were removed. Re-

moval of the integuments also resulted in an improved quality of the embryos as measured by their ability to form plants.

Minor differences among the four experimental conditions, (better response in Igri, a greater tendencey to polyembryony in Alexis, and the increase in polyembryony as well as embryo quality following removal of the integuments) tended to balance each other. There was thus no significant difference between the two varieties with respect to the frequency of ovules giving rise to plants and the effect of removing the integuments was only at the limit of the 5% significance level. Finally, statistical analysis did not reveal significant differences between the six intervals from 2 to 30 h after pollination for any of the measured parameters with the exception of the parameter "Ovules giving rise to plantlets" (significant at the 5% level) where there was a reduction in frequency in the interval from 24 to 30 h after pollination. It can be concluded from the combined data on the culture of 944 ovules of Igri and Alexis that 81% of the ovules gave rise to embryo-like structures and 63% to plants. On average, 1.3 embryo-like structure formed per responding ovule and 70% of these germinated into plantlets.

Fertility of the regenerated plants

Several hundred plantlets were regenerated from each of the two genotypes but albino plants were never observed among the primary regenerants. In a few cases only did an albino shoot develop from callus present at the base of the primary regenerant.

On average, 75% of the regenerated plantlets survived and developed further. In nearly all cases these plantlets also survived the vernalization (Igri) and the transfer to soil. Most regenerated plantlets were dis- carded, but 43 Igri and 25 Alexis plants were grown to maturity in the greenhouse. The fertility of the individual spike was measured by counting the number of florets with and without grains. In Alexis, 19, 65, 15, 1, and 0% of the spikes showed a fertility of 100, 75-100, 50-75, 25 50, and 0-25% while in Igri the corresponding fre-

quencies were 8, 41, 21, 14, and 17%. The slightly lower fertility in Igri resulted because all spikes of each plant were evaluated for their fertility, while in Alexis only the first 15 spikes formed on each plant were included. Igri regenerants formed about three times as many spikes per plant as seed-grown control material and spikes ap- pearing late generally had low fertility. The high number of spikes resulted in part from a cutting back of the Igri regenerants prior to vernalization which facilitates the production of more tillers. It may also be attributed to the presence of multiple apical meristems in the embryo-like structures. The fertility of the Alexis and Igri regenerants was similar to that observed in seed-grown control material and there was only a slight variation in fertility among the regenerants. No completely sterile plants were found.

Regeneration of plants from ovule cultures of the wheat zygote

A few experiments were carried out to ascertain if the same ovule culture technique could be applied to wheat. The wheat cultivar Walter was grown and the ovules processed and cultured as described for barley. In three experiments spikes were hand-pollinated and the ovaries removed (26, 8, and 8 h after pollination). Thir- teen, 18, and 14 ovules were cultured and in each series an embryo-like structure formed. The structures germinated readily and the three plants were grown to maturity. All were morphologically normal and fertile.

Discussion

The present study has devised an ovule culture technique that allowed an efficient regeneration of the barley zygote into fertile plants. Similar regeneration frequencies were obtained from ovules cultured immediately after fertilization until the formation of bicellular embryos, while unpollinated ovules did not give rise to embryos and plants. Removal of the integuments around the micropylar region of the ovule induced a slightly higher frequency of polyembryony, and the embryo-like structures from the dissected ovules tended to give rise to more plants than embryo-like structures from undissected ovules. The culture technique may also be largely genotype independent, since similar regeneration frequencies were observed in the two cultivars Igri and Alexis. These two cultivars show in contrast very difference performances in microspore culture where a mean of 11.7 green plants and 0.1 albino plants could be regenerated per anther of Igri while 0.06 green plants and 0.61 albino plants were regenerated per anther of Alexis (P. Mouritzen and P.B. Holm, manuscript submitted). A similar difference was observed when whole anthers were cultured (P. Mouritzen and P.B. Holm, unpublished observation). While albinism in the regenerants was a major problem in the microspore-

57

derived plants of Alexis, albino plants were never observed among the primary regenerants from the egg cell.

These data confirm and extend the results and con- clusions presented by T6pfer and Steinbiss (1985). In their study, small samples of ovules were cultured from 11 different spring and winter cultivars using liquid Bar- ley Medium II (Norstog 1973). Poorly differentiated embryos were found in 5-28% of the ovules and about 20% of these developed into plants. Embryos could be obtained from ovules isolated 90 rain after pollination while unpollinated ovules were sterile. Cultivation of ovules of cross-pollinated material and genetic analysis of the regenerants proved that the regenerants arose from the zygote and not from maternal tissue.

The significant increase in the formation of embryo- like structures and plantlets obtained in this study compared to that obtained by T6pfer and Steinbiss (1985) may to a large extent can be attributed to cultivation on solid medium. In our experience, submerged ovule cultures seldom produced embryogenic structures of a good quality, while acceptable results were obtained if the ovules were floating on the surface of Kao 90 + 20% Ficoll. Cultivation on Whatman filter paper discs was also believed to have an effect since it reduced the accu- mulation of liquid around the ovules. The different media used in the two studies may also be important. T6pfer and Steinbiss (1985) found no effect from adding casamino acids, coconut water, or BAP, while these proved important in the present study. Casamino acids, coconut water and cytokinins are often used for ovule culture (for review, see Rangan 1982) and particular concentration ranges of coconut water, casamino acids, glutamine, ammonium ions, and abscisic acid have also been found important for sustaining the growth and development of isolated barley embryos (Ziebur et al. 1950; Norstog 1961, 1970, 1973; Norstog and Klein 1972; Cameron-Mills and Duffus 1977; Umbeck and Norstog 1979).

Unfertilized barley eggs in ovule culture were not regenerable. The same conclusion was reached by T6pfer and Steinbiss (1985). Isolated, unfertilized protoplasts of maize (Kranz et al. 1991) and barley (Holm et al. 1994) were never observed to divide. In contrast, in vitro fertilized egg protoplasts of maize (Kranz et al. 1991) and in vivo fertilized egg protoplasts of barley (Holm et al. 1994), grown in medium only, divided a few times and formed microcalli that thereafter degenerated. This indi- cates that division of the egg cell requires the stimulus of fertilization. However, haploids can in some species be regenerated from ovules and ovaries, isolated and cultured at different developmental stages (see Yang and Zhou 1982 for review). In barley, haploid plants have been regenerated from ovaries of florets where the pollen was either binucleate or young trinucleate (San Noeum 1979; Castillo and Cistu6 1993). tn the former study the best response was obtained from a doubled haploid, where 0.11% of the ovaries produced one or more plants. The embryogenic structures appeared to originate primarily from the egg but regeneration of

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structures from antipodals and synergids was also reported. Castillo and Cistu6 (1993) obtained callus formation from about 8% of cultured barley ovaries when using N6 medium supplemented with 4-chloro-2- methylphenoxyacetic acid, indole-3-acetic acid, 6-ben- zyladenine, 90 g/1 sucrose, and 100 g/1 coconut water. Plants could be regenerated from half the calli and were confirmed to be haploid by chromosome counts. The best response was obtained when using ovaries from flowers at the trinucleate stage of pollen.

Protoplasts of in vitro and in vivo fertilized egg of maize and barley respectively could only undergo an embryogenic development if they were co-cultured with a maize suspension or barley microspores undergoing embryogenesis (Kranz and L6rz 1993; Holm et al. 1994). In barley, co-cultivation was required for up to 30 days, at which stage 1-2 mm large embryo-like structures had developed. The development of the egg in ovule culture, which is very similar to in vivo development, must thus depend upon a similar support from the ovule tissues. The fact that an embryo-like development of the fertilized egg also can be achieved by culturing tips of ovules further suggests than only a minimum of integument and nucellus tissue is required. In addition, the embryogenic development of the egg does not appear to depend on the egg cell being left undisturbed in its original loca- tion. The mechanical isolation of the egg protoplasts involved a puncturing of the giant vacuole of the central cell followed by a gentle tapping on the micropyle with a pair of forceps. This resulted in the liberation of the egg as a spherical protoplast into the central cell and the egg protoplast could be squeezed out of the ovule following removal of the nucellus tissues in the micropylar region (Holm et al. 1994). Ovules treated in a similar way and thereafter cultured gave rise to embryo-like structures at the same frequency as ovules where the egg had been left in situ (unpublished observation).

The present results show that it is possible to affect the embryogenic development of the egg by changing the media and adding hormones such as BAP and 2,4-D. This suggests that ovule cultures may be used as sensitive bioassays for analyzing the effect of particular compounds expected to affect embryogenesis. The fact that the cells of the egg apparatus and the polar nuclei can be seen with high optical resolution in the dissected ovules may also allow a manipulation of these cells by, e.g., microinjection where DNA, sperm cells or other materials are introduced into the egg, the synergids, and the central cell. Microinjection into the egg is further facilitated by the fact that the barley egg in vivo is largely a protoplast that is covered only by wall material in the micropylar region (Holm et al. 1994). Since the regeneration from the zygote appeared to be independent of its developmental stage from fertilization to the first division of the zygote, this technique also allows the manipulation of the egg at particular stages of its cell cycle.

Acknowledgements We are indebted to Bente Jensen and Judita Gartner for expert technical assistance and to Marie Dam for taking excellent care of the plants. Ann Soft Steinholz is thanked for producing the color prints and Jan Ruud Hansen for perform- ing the statistical analysis. Seeds of the wheat variety Waiter were generously provided by Dr. Sven Bode Andersen, Department of Crop Science, The Royal Veterinary and Agriculture University, Denmark. The work was supported by Eureka grant EU 270 and is ABIN (Adaptation of Barley for Industrial Needs) publication no. 139.

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Regeneration of the barley zygote in ovule culture

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