Two pathways of plant regeneration in wheat anther ... - Springer Link

Plant Cell, Tissue and Organ Culture 73: 177–187, 2003.  2003 Kluwer Academic Publishers. Printed in the Netherlands.

177

Two pathways of plant regeneration in wheat anther culture 1 2 1 1, R. Konieczny , A.Z. Czaplicki , H. Golczyk & L. Przywara * 1

´ , Department of Plant Cytology and Embryology, Jagiellonian University, ul. Grodzka 52, 31 -044 Krakow 2 Poland; Plant Breeding and Acclimatization Institute, 05 -870 Blonie, Poland ( * requests for offprints; Fax: 148 -12 -422 -8107; E-mail: przywara@ grodzki.phils.uj.edu.pl) Received 17 December 2001; accepted in revised form 25 November 2002

Key words: Androgenesis, Anther culture, Gramineae, Organogenesis, Plant regeneration, Triticum aestivum

Abstract The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars were used for the induction of androgenesis and plant regeneration. The highest rate of callus induction (9.1%) and green plant production (0.8%) was obtained with the cultivar Apollo that was chosen for histological analysis. The first androgenic division was symmetrical and occurred after 3 weeks of culture. Further divisions of newly formed cells gave rise to multicellular structures which followed two developmental pathways: callus production or direct embryo formation. Plant regeneration was observed in both pathways. Chromosome counting of plantlets regenerated showed that haploid metaphases 2n53x521 were the most frequent. Abbreviations: BA – 6-benzyladenine; IAA – indoleacetic acid; NAA – a -naphthaleneacetic acid

Introduction Anther culture is one of the method commonly used for induction of haploid plants. Its application in breeding programs of crops is of great agronomic importance since it allows fast achievement of genetic homozygosity which is of primary importance in production of new breeding lines and varietes. In wheat anther-derived cultivars: ‘Florin’ (De Buyser et al., 1987), ‘GK Delibab’ (Pauk et al., 1995), ‘Jinghua No.1’ (Hu et al., 1983) and ‘No. 764’ (Hu et al., 1988) have been obtained. As it was shown in numerous papers, the induction of andrognenesis from wheat anther is strongly controlled by genetic and environmental factors with significant interactions between them (Fadel and Wenzel, 1990; Lazar et al., 1990; Moieni and Sarrafi, 1996; Moieni et al., 1997; Stober and Hess, 1997). Thus the screening a large number of genotypes seems to be of crucial importance in final success in androgenic plant regeneration in wheat. Although anther culture is the most common method used for induction of haploid wheat only a limited number of reports describe anatomical

changes associated with regeneration. Additionally, studies published have focused mainly on the early stages of androgenic plant regeneration and the role of the first microspore division plane in further develop´ and ment (Wang et al., 1973; Pan et al., 1983; Szakacs ´ 1988, 1995; Hassawi et al., 1990). To our Barnabas, ´ knowledge, only Ouang et al. (1973) and Rybczynski et al. (1991) described in detail the development of the androgenic embryos from wheat anthers into plants. In contrast, histological events of plant regeneration via somatic embryogenesis in culture of various wheat explants are well documented (Ozias-Akins and Vasil, 1982, 1983a, b; Magnusson and Bornman, 1985; He et al., 1990). Since in cereals induced embryos are often macroscopically similar to shoots or leaves proper identification of regenerative pathways is difficult (OziasAkins and Vasil, 1982, 1983a; He et al., 1990). One way of overcoming these difficulties is detailed histological examination of regeneration; since embryos do not have vascular connection to the original explant (Haccius, 1978). Preliminary experiments performed in Institute of

178 ´ Plant Breeding and Acclimatization in Radzikow revealed that plant regeneration in wheat anther culture of several Polish cultivars cannot be univocally defined as organogenesis or androgenic embryogenesis. Therefore, we decided to study plant differentiation using histological techniques. Moreover, the need for anatomical studies in anther culture is of special importance with respect of the possibility of sporophytic cells involvement in plant regeneration.

Materials and methods Plant material The anthers of 10 Polish winter wheat (Triticum aestivum L.) cultivars, Roma, Kaspar, Java, Rosa, Apollo, PAB 1331 / 85, Wanda, Ramiro, Elena, and Zentos, were used in the experiment. Seeds of donor plants were germinated on petri dishes in the laboratory under natural daylight conditions at 20 8C for 5 days and then vernalized for 6 weeks in a refrigerator at 4 8C. After this time the seedlings were transplanted into 18-cm diameter pots and they were left in the growth chamber with a photoperiod of 16 h day length, light intensity 250 m mol m 22 s 21 provided by cool, white fluorescent light, and temperature 15–12 8C (day–night). The developmental stage of microspores was checked under light microscopy using acetocarmine staining. Immature spikes containing anthers with the microspores at the mid to late uninucleate stage were excised and pretreated in darkness at 4–5 8C for 6–12 days. Next the spikes were surface sterilized with 20% NaOCl for 20 min, washed five times with sterile distilled water and the anthers were excised aseptically and plated on 100310 mm glass petri dishes filled with 10 ml of induction medium.

effect of these supplements on the efficiency of callus induction was tested in previous experiments (Czaplicki, 1993). The medium was solidified with 0.4% (w / v) agarose and adjusted to pH 5.8 with 1 N sodium hydroxide and / or 1 N hydrochloric acid before autoclave sterilization. Approximately 80 anthers originating from single spike were cultured on each petri dish. The dishes were sealed with parafilm and incubated in dark at 28 8C for 6–8 weeks until the explanted anthers produced callus ca. 2–3 mm in diameter. Then the explants with calluses were transferred on 50310-mm glass petri dishes containing 7 ml of 190-2 regeneration medium (Zhuang and Jia, 1983) slightly modified by adding: 0.5 mg l 21 kinetin and 0.5 mg l 21 NAA (R1 medium). Calluses which did not produce shoots, leaves or embryos on R1 medium within 1 week were further subcultured for 7 days on 190-2 medium supplemented with 0.5 mg l 21 BA (R2 medium) and 0.5 mg l 21 IAA (R3 medium). All regeneration media were solidified with 0.6% (w / v) agar (BBL) and adjusted to pH 5.8 before autoclaving. Regeneration cultures were kept at 26 8C in a photoperiod of 16 h day light provided by cool white fluorescent tubes at light intensity of 250 m mol m 22 s 21 . Plantlets obtained on regeneration media were transferred from petri dishes to 150 ml Erlenmeyer flasks containing 50 ml of 190-2 medium without growth regulators (R4 medium) and vernalized for 6 weeks at 6 8C. Then plantlets were transplanted to pots with a soil–sand mixture (3:1) and grown under 16 h light and 8 h dark at day / night and temperature regime 18–15 8C for 4–6 week. The light intensity in a growth chamber was 250 m mol m 22 s 21 . After acclimatization, plants were grown to maturity in the greenhouse at temperature of 25 8C.

Statistical analysis In vitro culture For pollen callus induction we used C-17 medium (Wang and Chen, 1983) with 90 g l 21 sucrose as a carbon source, 1.5 mg l 21 2,4-D and 0.5 mg l 21 kinetin as growth regulators. Medium was additionally modified by addition of some substances (1.0 mg l 21 vitamin C, 0.1 mg l 21 vitamin E, 500 mg l 21 glutamine, 500 mg l 21 casein hydrolysate, 35.6 mg l 21 alanine, 84.3 mg l 21 arginine, 73.1 mg l 21 lysine, 46 mg l 21 proline, 42 mg l 21 serine, 53.2 mg l 21 aspartate acid and 200 mg l 21 mannitol). Positive

The experimental design was randomized block with four replications. Each replication consisted of one pot containing eight plants. In order to normalize the distribution, all the data were transformed by the arcsin Œ]x function before statistical analysis. To compare the different cultivars, the effect of genotype for the following traits was determined: calluses produced per 100 anthers (CA / 100 A) and green plant regeneration per 100 anthers (GR / 100 A). Data were processed by analysis of variance. Genotype differences were tested by the Duncan’s multiple range test.

179 Karyological studies Cytological studies were performed on the Apollo cultivar. Root tips were taken from anther-donor plants (control) as well as from in vitro regenerated plantlets, after 6 weeks of transplantation to pots. Root tips were treated with a saturated solution of a -bromonaphthalene at 4 8C for 24 h, fixed in AA (mixture of glacial acetic acid and absolute ethanol, 1:3, v / v) for 24 h, stained in aceto-orceine (1% solution of orceine dissolved in 45% acetic acid) at room temperature and squashed in a drop of 45% acetic acid. Preparations were frozen by the use of dry-ice, cover glasses removed and dried. Entellan (Merck) was used for making preparations permanent. Two to three root tips from the same plant were squashed on a single slide. Metaphase plates with no apparent signs of fragmentation were selected to minimize the possibility of artifacts (squashing method). A brief cytogenetic analysis of regenerants was based on the ploidy ranges arbitrarily expanded with three chromosomes, e.g., 2x level51463 chromosomes, 3x level52163 chromosomes, 6x level5 4263 chromosomes, etc. (x57 chromosomes). Metaphase plates were analysed in detail under an immersion objective (1003). Images were photographed or transferred to the computer via CCD camera and then processed in Photo Styler.

Histological observations Since the best morphogenic response was obtained in culture of Apollo cv. only this material was used for histological study. Tissues for sectioning were taken after 2, 3, 4, 5, 6, 8, and 10 weeks of culture and embedded in paraffin as well as in Technovit 7100 (2-hydroxyethylmetacrylate) (Heraeus Kulzer). Paraffin sections at 8–10 m m were cut from samples fixed in FAA (formalin, acetic acid, 50% ethanol, 5:5:9, v / v / v) for 72 h and dehydrated in a graded ethanol series. The sections were stained with Heindenhain’s hematoxylin. For Technovit embedding, the material was fixed in 5% glutaraldehyde in 0.1 M phosphate buffer at pH 7.2 for 2 h. After dehydration in ethanol series the tissues were embedded in methacrylate with addition of 5% PEG 400 to soften the Technovit and increase the ease of sectioning. The sections (3 m m thick) were stained with 1% aquatic solution of toluidine blue solution or Periodic acid–Schiff’s re-

agent (PAS). The anthers before explantation were used as a control.

Results Anther culture The general results of in vitro culture are presented in Table 1 and Figure 1. The analyses of variance showed significant differences between the genotypes used for two characters: CA / 100 A and GR / 100 A studied ( p,0.0001). A total of 23899 anthers were plated and 1145 calluses (4.8%) were formed from them. The highest frequency (9.1%) of callus induction occurred in Apollo cv. while the lowest (0.2%) was with the Wanda genotype. The means of the two traits compared by the Duncan’s multiple range test are shown in Table 1. In all cultivars studied callus formation was not observed earlier than 6 weeks of culture on induction medium. Initially, calluses were homogenous in structure with no visible signs of differentiation. Most of them were yellowish and hard, only occasionally spongy and greenish tissue was produced. No relation between cultivars used and nature of the callus produced was found. After 6 weeks of culture single translucent outgrowths emerged from some yellowish calluses. These expanded and most of them acquired milky white color by the end of culture on induction medium During the subculture on R1 medium, the observed outgrowths gradually took on the appearance of embryos or developed into shoots and leafy structures. The embryogenic and organogenic structures emerged also de novo from callus during the culture on R2 and R3 medium. From cultured anthers 47 green plants were obtained. The frequency of plant regeneration was highest in Apollo and Kaspar cultivar. However the callus production was significantly lower in Kaspar genotype, so for histological studies Apollo cultivar was chosen. Karyological observations In total 370 metaphase plates from nine regenerants (18–76 metaphases per plant) were analyzed (Table 2). Chromosome complement in root tip cells of four control plants was typically of bread wheat and comprised 2n542 chromosomes (Figure 2A). Our results indicate that within regenerants numerical chromo-

180 Table 1. Results of anther culture of Polish wheat cultivars Cultivar

Roma Kaspar Java Rosa Apollo PAB 1331 / 85 Wanda Ramiro Elena Zentos Total

No. of anthers inoculated

No. of calluses produced

No. of plants regenerated

Trait CA / 100 A

Trait GR / 100 A 0.00 0.65 0.05 0.00 0.79 0.12 0.00 0.07 0.18 0.12

3806 1223 2182 1385 3550 2472 3732 1361 1683 2505

284 65 174 104 322 10 7 52 12 115

0 8 1 0 28 3 0 1 3 3

7.46 5.31 7.97 7.51 9.07 0.40 0.19 3.82 0.71 4.59

23899

1145

47

4.79

b c b b a g h e f d

c a b c a b c b b b

0.20

(CA / 100 A) – calluses produced per 100 anthers. (GR / 100 A) – green plant regeneration per 100 anthers. Means followed by different letters are significantly different at p50.05 as analysed by the Duncan’s multiple range test. Data were transformed by the arcsin vx function before statistical analysis.

some variability exists in root meristems. Among nine plantlets studied chromosome numbers ranged from 11 to 73 (2x–10x). Most of metaphase plates represented 3x and 2x level (45.4 and 36.5%, respectively) (Figure 2B,C). Only 7% of metaphases counted contained chromosome number 6x or higher. Intermediate ploidy level (4x and 5x) was found in 11% of metaphase plates. Histological observations Observations of the anthers before culture revealed that microspores of the control material were of similar size and morphology with no detectable starch (Figure 3A–C). In transverse sections most of the microspores were closely attached to well-developed tapetum already at the ameboid stage (Figure 3B). At the beginning of the second week of culture the tapetum was not present and the microspores were in contact with the endothecium, clearly distinguishable by its cell wall thickenings. Staining with PAS showed abundant starch accumulation in occasional microspores located close to the anther wall (Figure 3D). The first androgenic division occurred after 3 weeks of culture giving rise to two cells of identical size and morphology (Figure 3E). With continued culture further divisions within the microspore led to the formation of multicellular structures surrounded by continuous, thick microspore wall (Figure 3F,G). The number of dividing cells varied between different anthers and decreased with time of culture. Until the

fourth week the multicellular structures consisted of no more than 10 cells and were still surrounded by continuous microspore wall (Figure 3H,I). Non-responding microspores enlarged compared to the control and became irregular in shape. Their cytoplasm was less stainable or completely non-stainable indicating degeneration (Figure 3J). In degenerating microspores starch grains were not found (Figure 3K). Between the fourth and sixth week of culture most of the multicellular structures increased in cell number that led to exine disruption and formation of fast growing endogenous callus (Figure 3L). One week later the anther wall was ruptured and the explants were covered with a yellowish callus. The peripheric regions of the callus consisted of small, densely staining cells, whereas the inner regions were occupied by more vacuolated cells of parenchymatous nature (Figure 4A). All cells of the callus contained numerous, large starch granules (Figure 4B). When maintained on the induction medium (C-17) the callus formed small meristematic centres, scattered throughout the tissue. From centres located deep in the callus adventitive roots arose. At the same time some of the superficial cells of the callus underwent regular division giving rise to numerous protuberances with no visible vascular connection to the whole callus. The morphology of some of these outgrowths, especially those narrowly based, resembled embryo (Figure 4C– E). The embryo-like structures could be easily removed and induced to form plantlets when subcul-

181

Figure 1. Triticum aestivum (L.) cv. Apollo. In vitro culture of anthers. (A) anthers plated on C-17 induction medium; (B) callus observed on anthers after 6 weeks of culture; (C, D) embryo-like structures differentiated on R1 medium, 8-week-old culture; (E, F) leafy structures differentiated from callus on R1 medium, 8-week-old culture; (G) young regenerant on R4 medium, 10-week-old culture; (H) 12-week old plantlet on R4 medium; (I) young plant in soil. Arrows in (C, D) point androgenic embryoids. Bar in A55 mm, B–G51 mm, H515 mm, I520 mm.

182 Table 2. Ploidy status of root meristem cells found in 370 metaphase plates from nine anther-derived wheat plantlets regenerated of Apollo variety Plant

No. of

Ploidy level

number

plates studied

2x 3x No of chromosomes

4x

5x

6x

7x

8x

9x

10x

11–17

25–31

32–38

39–45

46–52

53–59

60–66

67–73

9 – – 2 – – – – 3

19 – – – – 1 – 1 6

1 – 1 – – – 6 6 3

– – – – – – – – –

6 – – – – – – – –

– – – – – – – – 2

– – – – – – 1 – –

14 (3.8%)

27 (7.3%)

17 (4.6%)

–

6 (1.6%)

2 (0.5%)

1 (0.3%)

1 2 3 4 5 6 7 8 9 Total

72 18 19 21 18 45 70 76 31 370

23 10 6 6 8 21 19 32 10 135 (36.5%)

18–24 14 8 12 13 10 23 44 37 7 168 (45.4%)

tured on regeneration media (R1, R2 or R3). The basic mode of regeneration was organogenesis, the de novo shoot and / or leaf induction from the subepidermal regions of transferred explants. Sporadically, they developed as bipolar structures with shoot apex opposite the root pole. The non-regenerative calluses differentiated into shoots and leaves when transferred from induction to regeneration media. After 1 week of subculture, as a result of the uneven distribution of meristematic activity the peripheric regions of the callus became compact and nodular, whereas the inner parts remained parenchymatous. From the meristematic regions at the callus surface, numerous shoots and / or leaves formed (Figure 4F,G). Leaf formation often preceded shoot regeneration so that shoot apices differentiated from a broad meristematic zone at the base of the leaves. Very sporadically, distinct shoot buds were also found. The leaves obtained on regeneration media usually had an obvious vascular connection to the callus.

In the second developmental pathway, the callus formation at the early stages of culture was accompanied by the formation of globular units similar to young embryos (Figure 5A). These globular structures, clearly visible after 5–6 weeks of culture could be found both in non-callusing anthers and among prolifering callus. However, further development of these globular units became disturbed, so after 6–7 weeks of culture a complicated branched structures with various meristematic and parenchymatous nodules were formed (Figure 5B,C). Such structures, enclosed by continuous epidermis were regularly observed inside the non-callusing and callusing anthers maintained on induction medium. When harvested from the explant and subcultured on the regeneration medium they showed organogenic proliferation. During the whole period of culture including induction phase on C-17 medium and regeneration on R1, R2 and R3 media no cell division in the sporophytic tissue of the explants was observed.

Figure 2. Triticum aestivum (L.) cv. Apollo. Metaphase plates. (A) root tip squash of control plant with 42 chromosomes; (B) regenerant with 21 chromosomes; (C) regenerant with 14 chromosomes. Arrows in (A) indicate two chromosome arms separated by squashing. Bar512 m m.

183

Figure 3. Triticum aestivum (L.) cv. Apollo. Sections of anthers. (A–C) anthers prior to culture; (A) longitudinal section; (B, C) transverse sections, arrow on Figure B indicates ameboid tapetum; (D–L) in vitro cultured material; (D) starch accumulation in a single microspore at the second week of culture; (E) first microspore division after 3 weeks of culture, arrow points cell wall formation; (F–I) successive stages of multicellular structure formation, 3–4 weeks in culture, (J, K) degeneration of non-dividing microspores at the fourth week of culture; (L) development of endogenous callus, 5-week-old culture. Sections in (A) and (H) were stained with Heidenhain’s hematoxylin, (B, E–G, J and L) with toluidine, and (C, D, I, K) with PAS reaction. Bar580 m m.

Discussion The response of anthers used in this experiment depends mainly on the genotype used. Cultivars with high callus induction and low or no green plant regeneration (e.g., Java, Roma, Rosa), and cultivars with low callus induction but relatively high green plant regeneration (e.g., Elena) were observed. Relatively high callus induction and green plant regeneration was characterized by cultivar Kaspar. These

observations are in agreement with the postulation of Lazar et al. (1984) and Henry and de Buyser (1985) confirmed by Stober and Hess (1997) that the ability of wheat anthers for callus production and subsequent plant regeneration could not be correlated. The qualitative data of androgenesis in this experiment were comparable to that found by ForoughiWehr and Zeller (1990) for some German wheat varietes and by Holme et al. (1999) for north-western European lines. Callus induction in our experiment

184

Figure 4. Triticum aestivum (L.) cv. Apollo. Callus and androgenic structure formation in anther culture. (A, B) disruption of anther wall and outgrowth of the callus after 6 weeks of culture, arrow indicates starch grains; (C–E) embryo-like structures occurred on callus after 6 (C, E) and 8 (E) weeks of culture, (F, G) shoot buds and leafy structure formation, 10-week-old culture. Sections in (A) and (D) were stained with Heidenhain’s hematoxylin, (C, E–G) with toluidine, and (B) with PAS reaction. Abbreviations: SA – shoot apex; SC – scutellum; LS – leafy structure; RP – root pole. Bar580 m m.

was comparable to that obtained by Stober and Hess (1997), however the frequency of plant regeneration was significantly lower than obtained by these authors. Also Armstrong et al. (1987) in American wheat varietes and Holme et al. (1999) for eastern European lines of wheat reported a higher frequency of androgenic plant regeneration than obtained in our study. Karyological analysis of nine regenerated plantlets showed different chromosome numbers in root tip meristems ranging from |14 to |70 chromosomes

(2x–10x). The low frequency of 6x cells and cells of higher ploidy levels, and high frequency of 3x metaphase cells suggest that chromosome doubling could happen only incidentally in the material studied. Moreover, relatively large fraction of 2x cells indicates the existence of some chromosome reductional events, possibly contributing to numerical chromosome variability observed in regenerated plants. Chromosome number variability within root meristems is not unusual for wheat plants derived in vitro and in general such plants are rather frequently obtained via

185

Figure 5. Triticum aestivum (L.) cv. Apollo. Direct androgenesis in anther culture. (A) androgenic structure resembling globular embryo, 5-week-old culture; (B, C) mature androgenic structures with high organogenic potential, after 8 weeks of culture, arrow indicates leaf formation. All sections stained with toluidine blue. Bar580 m m.

anther culture in cereals (Ahmed and Sagi, 1993). In ´ natural populations this phenomenon is rare (Cieslak et al., 2000; Joachimiak et al., 2001). Histological observations showed that microspores were the only responsive cells within the explanted anthers. Only those lying in the vicinity of the anther wall started to divide and gave rise to plants. Similar relationship between position of the microspore within the anther locule and its ability to follow sporophytic development has been reported in Hyoscamus niger (Raghavan, 1978) and barley (Idzikowska et al., 1982). In maize, the effect of the anther wall on induction of androgenesis was confirmed at the biochemical level by the identification of a specific protein in somatic tissues of the anther that triggered androgenic differentiation of the microspores (Vergne et al., 1993). It seems that in our material the competence for androgenesis could be also affected by the anther wall. It is very difficult to separate androgenic and noninducible pollen inside the anther under the light microscope. In wheat (Zhou, 1980; Heberle-Bors and Odenbach, 1985) and rice (Cho and Zapata, 1988,

1990) androgenic microspores could be distinguished from the non-inducible ones by their size. In our material, however, all the microspores from the control anthers were of similar size and staining properties. Nevertheless, after 2 weeks of culture some of the microspores started to accumulate starch. In some species, e.g., tobacco (Touraev et al., 1996) starch accumulation in plastids is regularly observed only in non-responsive microspores, whereas in other species, e.g., Datura innoxia (Sangwan and Sangwan-Norreel, 1987a, b) or rape (Zaki and Dickinson, 1990) starch could be found both during typical pollen development and in the androgenic competent cells. In wheat Indrianto et al. (2001) found that all embryogenic microspores distinguished by their ultrastructural features passed through the starch rich stage followed by the induction of the first sporophytic mitosis. Furthermore in wheat (Indrianto et al., 2001) as well as in rape (Hause et al., 1994) starch accumulation was found to be involved in the determination of the polarity within the multicellular structures and further orientation of the body axis of the embryoids. It cannot be excluded that in our material the starch

186 bearing microspores were the only competent cells for androgenesis. Because of the low number of microspores that entered androgenic development it was difficult to trace in detail the earliest stages of embryo and callus formation. However, from our data it seems that the first androgenic division gave rise to two cells of equal size and staining properties. The newly formed cells divided repeatedly forming multicellular structures. The role of the first microspore division is widely discussed in literature and is regarded by many authors as the crucial event influencing the pathway of development. However in wheat, as in numerous other species (for review see Raghavan, 1997) both symmetrical and asymmetrical mitosis were found to lead to androgenic differentiation. Therefore no general rule could be defined. For example Hassawi et al. ´ (1990) and Rybczynski et al. (1991) showed that embryos in wheat anthers were formed after asymmetric divisions of microspore nucleus, whereas ´ and Barnabas ´ Wang et al. (1973) as well as Szakacs (1988, 1995) revealed the origin of the pollen embryos from symmetrically dividing ones. Additionally, in some anther culture experiments (Pan et al., 1983; Reynolds, 1993) embryos could be obtained by multiple pathways: after symmetric and asymmetric divisions of microspore nucleus. Recent experimental studies (Eady et al., 1995; Touraev et al., 1995) showed that the symmetry of first pollen mitosis is not essential for determining the developmental fate of the pollen and may be under strong influence of the culture conditions. Also from our data it seems that entering the pathway of regeneration (callus or globular units resembling proembryo production) by multicellular structures was not dependent upon the orientation of the first androgenic division. The presence of globular structures consisting of small meristematic cells can be regarded as evidence of direct pollen embryogenesis. However, in our material further differentiation of these globular units into typical embryos was not found. Instead, they formed a compact tissue of high organogenic potential that was surrounded by an apparent distinct outer cell layer. The reasons for this aberration observed in embryo development could be due to exposure to 2,4-D which was found to be responsive for various abnormalities both in somatic (Ozias-Akins and Vasil, 1982, 1983a, b) and androgenic (Armstrong et al., 1987) embryos morphology and development. The data obtained in the present study indicate that our system for haploid wheat plant regeneration could

be improved. It could be expected that by more precise definition of culture conditions (growth regulator concentration and time of subculture) the quality of the embryos will be improved and the process of plant regeneration enhanced toward embryogenesis.

Acknowledgements Financial support from the State Committee of Scientific Research (KBN project no. PB 0302 / P04 / 2001) is acknowledged. The authors are grateful to Prof. J. Zimny for discussion and valuable suggestions.

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