Sex Plant Reprod (2000) 13:95–104
© Springer-Verlag 2000
O R I G I N A L PA P E R
S. Caredda · C. Doncoeur · P. Devaux · R.S. Sangwan C. Clément
Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)
Received: 13 March 2000 / Revision accepted: 6 June 2000
Abstract In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri proS. Caredda · C. Doncoeur · C. Clément (✉) Université de Reims Champagne Ardenne, UFR Sciences, Biologie et Physiologie Végétales, BP 1039; 51687 Reims Cédex 2, France E-mail:
[email protected] P. Devaux Florimond Desprez Ind., Section Biotechnologies, BP 41; 59242 Cappelle en Pévèle, France R.S. Sangwan Université de Picardie Jules Verne, Androgénèse et Biotechnologies, 33, rue Saint-Leu; 80039 Amiens, France
vided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Key words Androgenesis · Albinism · Hordeum vulgare L. · Plastid differentiation · Plastid DNA · Pollen development
Introduction One goal of recent plant biotechnology is to obtain doubled haploid plants. Doubled haploids can be used by plant breeders to produce homozygous plants and to develop agronomically improved varieties. Hordeum vulgare L. is one of the most commonly used species for doubled haploid production, with haploids mostly derived from androgenesis following microspore or anther culture (Luckett and Darvey 1992; Pickering and Devaux 1992; Jähne and Lörz 1995). Androgenesis consists of in-vitro regeneration of haploid plantlets from microspores initially programmed to develop into pollen grains. Androgenesis has been widely used and welldeveloped in barley and now is succesfully combined with genetic transformation (Kasha et al. 1995; Yao and Kasha 1997; Yao et al. 1997; Hönicka et al. 1999). However, the androgenic process induces formation of albino plantlets in varying proportions, depending on the cultivar. This phenomenon occurs most commonly in Poaceae (Caredda and Clément 1999) and greatly limits the potential yield of androgenesis in the most sensitive species such as barley (Jähne and Lörz 1995). Actually, several cultivars used in breeding programmes because of their agronomic importance can not be used following androgenesis because they produce exclusively albino plantlets (Knudsen et al. 1989; Jähne et al. 1991).
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The plastid is the main structure involved in the occurrence of albinism since chlorophyll synthesis and photosynthesis-related metabolism occur in the stroma (von Wettstein et al. 1995; Reinbothe and Reinbothe 1996). Plastids of haploid androgenic plant cells are derived from proplastids of the microspore as they develop into the androgenic embryo (Sunderland and Huang 1985). Therefore, the state of microspore plastids at sampling is a key to obtaining chlorophyllous androgenic plantlets. During the androgenic process, the microspore undergoes redifferentiation induced by pretreatment and is reoriented towards the alternative sporophytic programme (Touraev et al. 1997). At the time that the microspore is redifferentiating, micropore plastids are following the same pathway, leading finally to their differentiation into chloroplasts within the androgenic green plantlet (Sunderland and Huang 1985). Albino plantlets are formed when plastids fail to follow the chloroplast pathway development pathway, instead remaining undifferentiated and, containing a prolamellar body, unorganized thylakoids and numerous plastoglobules (Caredda et al. 1999). Most studies aimed at improving the androgenesis protocol or elucidating the androgenic mechanisms in barley are performed using the winter cultivar Igri as a control (Kumlehn and Lörz 1999; Wang et al. 1999a). In this cultivar optimizing the different steps of androgenesis makes it possible to greatly reduce the proportion of albino regenerated plantlets under both anther and microspore culture (Ziauddin et al. 1990; Jähne et al. 1991; Hoekstra et al. 1993; Caredda et al. 1999). However, numerous varieties remain recalcitrant in producing chlorophyllous plantlets following androgenesis (Knudsen et al. 1989; Jähne et al. 1991) and little information is available on why only albino plantlets are produced in these cultivars. When albinism in microspore-derived plantlets was investigated at the molecular level, it was reported that albino androgenic plants have deletions in their both nuclear and plastid genomes (Dunford and Walden 1991). Data dealing with the earlier steps of androgenesis remain scarce and fragmentary, however, especially in terms of the embryonic development of the microspore. In order to better understand the occurrence of albino plantlets following pollen embryogenesis in barley, plastid development and the presence of plastid DNA were followed and compared during the androgenic process in two barley cultivars. One is the winter cultivar Igri, which produces 87.8% chlorophyllous androgenic plantlets (Caredda et al. 1999) and the other is the spring cultivar Cork which produces only albino plantlets.
Materials and methods Plant material The barley (Hordeum vulgare L.) winter cultivar Igri and the spring cultivar Cork were grown in the greenhouse at 12°C in Florimond Desprez Ind. For anther culture, spikes were collected at the uninucleate stage during microspore vacuolation.
Anther culture Anthers were harvested and surface sterilized in 70% ethanol. They were then pretreated at 4°C in the dark for 3 days in mannitol (30 g/l). After pretreatment, anthers were cultured on the Olsen (1987) medium composed of macroelements including KH4NO3 (0.166 g/l), KNO3 (1.9 g/l), MgSO4 · 7H2O (0.374 g/l), KH2PO4 (0.17g/l), CaCl2 (0.022 g/l); microelements including H3BO3 (6.2 mg/l), ZnSO4 · 4H2O (8.6 mg/l), KI (0.83 mg/l), Na2MoO4 · 2H2O (0.25 mg/l), CuSO4 · 5H2O (0.025 mg/l), MnSO4 · 4H2O (22.3 mg/l), Fe-Na-EDTA (40 mg/l); and aminoacids including glutamine (752 mg/l). This medium was supplemented with 32 g/l mannitol as an osmoticum and 60 g/l of maltose as a carbohydrate source. The pH was adjusted to 5.6 before the addition of agarose (6 g/l). After autoclaving, filter-sterilized vitamins, including myo-inositol (0.1 mg/l), thiamine-HCl (0.4 mg/l), and growth substances, including AIA (2 mg/l) and BAP (1 mg/l) were added to the medium. Twenty five anthers were placed in each 5-cm petri dish. Dishes were sealed with a double layer of parafilm and maintained at 26°C, with 85% relative humidity, in the dark for 21 days. Embryo-like structures (ELS) larger than 1 mm were then transferred to a regeneration medium, according to Olsen (1987), containing 30 g/l sucrose as a unique source of carbohydrate and AIA/BAP (0.4/0.4 mg/l) as phytohormones. The petri dishes were maintained at 26°C, 85% relative humidity, and 60 µE/m2/s for 14 days with a 16/8 photoperiod. Albino and green androgenic plantlets were counted after 2 weeks on the regeneration medium. Chlorophyllous plantlets were then transferred in tubes onto the same medium without BAP. Electron microscopy For transmission electron microscopy, anthers, ELS and plantlets were fixed for 24 h at room temperature in a solution of 2% (w/v) glutaraldehyde in 0.1 M phosphate buffer (pH 7.24) with 2% (w/v) sucrose and 1‰ (v/v) Tween 20. Each sample was rinsed three times for 5 min each time in the 0.1 M phosphate buffer (pH 7.24) with 2% (w/v) sucrose. The samples were post-fixed for 4 h in 1% (w/v) osmium tetroxide in 0.1 M phosphate buffer (pH 7.24) with 2% (w/v) sucrose, dehydrated in an alcohol series and embedded in araldite. Ultra-thin sections were harvested on 150-mesh copper grids with a diamond knife on a Reichert U2 ultratome, stained with uranyl acetate/lead citrate, and examined on a JEOL JSM 201 electron microscope at 60 or 80 kV. Quantitative cytology Several androgenesis steps were investigated. Plastid density, division frequency, the number of thylakoids per plastid as well as the plastid maximal length were determined from two-dimensional sections of microspores or microspore-derived embryo cells. Since plastids are unequally distributed in the cell and since the threedimensional structure of plastids has to be taken into account, the number of cells or plastids investigated was selected to obtain the most valuable data. In microspores before and after pretreatment, ultrastructural parameters of plastids were estimated from observation of at least ten microspores in three different anthers collected from three different spikes; this means that at least 30 microspores were examined in detail. Similarly, 30 microspore-derived embryos were studied after 7 days of culture, which corresponds to a mean of 120 cells. In older embryos and in microsporederived plantlets, data were collected from observations of at least 100 embryo cells obtained from at least three different embryos, representing 300 cells. Plastid density and the division frequency were expressed per 100 µm2 of cytoplasm; the vacuole surface was not taken into account. For plastid length, only the highest measures were registered because of random plastid sections. Starch proportion in the plastid was measured from image analysis of serial section profiles, using the analysis software from Olympus, assisted by a CCD camera. At each stage of the androgenic process, at least 100 plastids were examined.
97 Immunocytochemistry
In the pretreated microspore
Sections were fixed, dehydrated in alcohol series, and embedded in araldite, as for electron microscopy, except that post-fixation was omitted. Ultrathin sections were harvested on nickel grids (300 mesh) coated with collodion (1% v/v). Each grid was then floated on glycine (0.1 M) in distilled water for 10 min and then in BSA (0.5% w/v) in 0.1 M phosphate buffered saline (PBS) pH 7.2 for 45 min. Sections were immunolabelled using a monoclonal primary antibody (10 µg/ml in 0.5% BSA/PBS: 1/3) raised against DNA (Boehringer, Mannheim) at 4°C overnight. Grids were then rinsed in PBS four times for 5 min each time. Next, grids were immersed in 0.5% BSA in PBS for 15 min and the primary antibody was marked using goat anti-mouse IgG bearing 10-nm gold particles (British Bio Cell International) in PBS (1/50) for 60 min. Grids were then rinsed in PBS four times for 5 min each time. The primary-secondary antibody complex was fixed on immersed grids in 2.5% glutaraldehyde in PBS for 5 min and grids were rinsed in PBS four times for 5 min each time. Control was performed by omitting the primary antibody. After washing in distilled water, the grids were stained in 5% (w/v in double-distilled water) uranyl acetate for 30 min and observed on a JEOL JSM 201 electron microscope at 60 or 80 kV. The presence of DNA labelling was examined from twodimensional plastid sections. Quantitative data were obtained by determining the percentage of plastid sections showing labelled DNA. At least 300 plastid sections from three different samples were observed, so that 900 plastids were examined at each stage of the androgenic process.
The pretreatment induced drastic modifications of microspore plastids with some differential effects between cultivars. In both cultivars, plastid density decreased sharply, from 72.3 ± 2.4 to 2.7 ± 0.6 plastids per 100 µm2 cytoplasm in winter cultivar Igri and from 28.7 ± 5.8 to 1.6 ± 0.4 plastids per 100 µm2 cytoplasm in spring cultivar Cork (Fig. 2A). In winter cultivar Igri, frequency of plastid divisions increased (Figs. 1H, 2A) but the amount of starch per plastid decreased from 71.5 ± 2.6% to 26.0 ± 1.4% of plastid volume (Figs. 1I, 2E). DNA was still present in the stroma (Fig. 1J). In contrast, in spring cultivar Cork, plastid divisions were reduced (Fig. 2B) and most plastids accumulated starch, from 44.3 ± 5.8% to 59.8 ± 3.7% of plastid volume (Figs. 2E, 3A, B). Plastids with double starch grains appeared in the pretreated microspore, which was not observed during in-vivo development. In both cultivars, the number of thylakoids per plastid was not significantly modified but plastid size slightly increased in the microspore (Figs. 2C, D).
Results Plastid features in the microspore at sampling Anthers were collected during microspore vacuolation. At this stage, in the winter cultivar Igri, most plastids were elongated and included starch and thylakoids (Fig. 1A). Some of them were organized in a circle (Fig. 1B) indicating that they were ready to divide. In the spring cultivar Cork, plastids were scarce (Fig. 1C) but had similar features to those observed in the microspore of the winter cultivar Igri: most plastids included a small single starch grain and a single thylakoid (Fig. 1D). Plastids organized in a circle (Fig. 1E) and plastid divisions (Fig. 1F) were also detected. Up to 15.3 ± 2.7% of plastid sections exhibited DNA in the winter cultivar Igri (Fig. 1G), compared with 1.7 ± 0.5% in the spring cultivar Cork. When structural data of plastids was further quantified (Figs. 2A-E), additional differences appeared between the two cultivars. Plastids density was twice as high in winter cultivar Igri (72.3 ± 2.4 plastids per 100 µm2) as in spring cultivar Cork (28.7 ± 3.8 plastids per 100 µm2) and plastid divisions were ten times more frequent. An average of 0.97 ± 0.4 thylakoids per plastid were detected in the microspore of winter cultivar Igri, but only 0.22 ± 0.1 thylakoids per plastid were formed in spring cultivar Cork. The length of plastids was 0.76 ± 0.15 µm in the winter cultivar Igri against 0.61 ± 0.05 µm in the spring cultivar Cork. Starch represented up to 71.5 ± 2.6% of plastid volume in the microspore of the winter cultivar Igri and 44.3 ± 5.8% in the spring cultivar Cork.
After 7 days of culture Plastid density in the microspore and thylakoid density in the plastid remained unchanged in microspore-derived structures of both cultivars. In winter cultivar Igri, division affected most plastids (Fig. 3C), occurring at a rate of 4.3 ± 0.5 plastid divisions per 100 µm2 cytoplasm (Fig. 2B), whereas in the spring cultivar Cork the frequency of plastid division decreased (Fig. 3D) to 0.06 ± 0.02 per 100 µm2 cytoplasm (Fig. 2B). Plastid length progressively increased in both cultivars. The relative proportion of starch in the plastid stroma was stable in winter cultivar Igri but it increased in spring cultivar Cork (Fig. 3E), reaching 68.1 ± 3.3% of plastid volume (Fig. 2E). After 12 days of culture At this stage, pollen-derived embryos were 0.5 mm in length in both cultivars. Plastid structural differences between the two tested cultivars were more pronounced. In winter cultivar Igri, the intensity of plastid divisions was 5.5 ± 0.2 per 100 µm2 cytoplasm (Fig. 2B), an average of 1.5 ± 0.15 thylakoids per plastid were found (Figs. 2C, 3F), plastid length reached 2.2 ± 0.43 µm (Fig. 2D) and starch represented 25.0 ± 2.6% of plastid volume (Fig. 2E). In contrast, in spring cultivar Cork, the most important changes were a slight increase in plastid length (Fig. 2D) and the growth of starch in the plastid, up to 80.5 ± 5.5% of plastid volume (Figs. 2E, 3G). After 21 days of culture In winter cultivar Igri, microspore-derived embryos exhibited elongated plastids, including reduced starch re-
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Fig. 2 A–E Quantification of plastid ultrastructure during androgenesis. ● Winter cv. Igri; ■ Spring cv. Cork A Plastid density. B Frequency of plastid divisions. C Number of thylakoids per plastid. D Plastid length. E Starch volume per plastid. VM Vacuolated microspore; T0 beginning of anther culture after pretreatment; T7 7 days of culture; T 12 12 days of culture; T 21 21 days of culture. Bars represent standard errors
Fig. 1 A–J Microspore plastid features during pollen embryogenesis in winter cultivar Igri and spring cultivar Cork. A Winter cultivar Igri at sampling. High plastid (P) density in microspore cytoplasm. Most plastids elongated with single starch grains (asterisk) and single thylakoid (arrowheads). I Intine ×23 000. B Winter cultivar Igri. Higher magnification shows circular plastids (P). M Mitochondria. ×36 000. C Spring cultivar Cork at sampling. Low plastid (arrowheads) density in microspore. N Nucleus; n nucleolus; V vacuole. ×3 100. D Spring cultivar Cork at sampling. Plastid with unique starch grain (asterisk) and single thylakoid (arrowhead). ×43 000. E Spring cultivar Cork at sampling. Circular plastid with starch (asterisk). ×48 000. F Spring cultivar Cork at sampling. Plastid division (opposite arrowheads). Asterisk starch. ×40 000. G Winter cultivar Igri at sampling. Immunolabelling of DNA (arrowhead) in plastid stroma. Asterisk starch. ×52 500. H Winter cultivar Igri after pretreatment. Numerous plastid divisions (arrowheads) in microspore. N Nucleus; n nucleolus; V vacuole. ×4 000. I Winter cultivar Igri after pretreatment. Higher magnification shows plastid division (opposite arrowheads). Asterisk starch; M Mitochondria. ×37 000. J Winter cultivar Igri after pretreatment. Immunolabelling (arrowhead) of DNA in the plastid stroma of the dividing plastid (P). Asterisk starch. ×35 000
serves and well-developed thylakoids. The intensity of plastid division was still increasing (Figs. 2B, 4A), as was plastid length (Fig. 2E), but the relative proportion of starch was not modified (Fig. 2D). DNA was immunodetected in 21.3 ± 2.1% of plastid sections (Fig. 4B). In spring cultivar Cork, there were few plastids in the cells of microspore-derived embryos (Fig. 4C) and most of them were amyloplasts (Fig. 4D). The tested parameters were not significantly modified (Figs. 2A-E), except that starch continued to accumulate in the stroma, representing up to 90.3 ± 4.3% of plastid volume. Plastid DNA could be detected in 2.6 ± 1.3% of plastid sections (Fig. 4E), which was approximately one-tenth that in winter cultivar Igri. At this stage, microspore-derived embryos of both cultivars were transferred onto the regeneration medium.
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101 Table 1 Structural parameters of plastids in androgenic plantlets
Chlorophyllous Albino
Plastid density (plastids/100 µm2)
Thylakoid density (thylakoids/plastid)
Plastid length (µm)
Starch (Percent plastid volume)
4.8 ± 0.7 1.5 ± 0.6
12.6 ± 1.3 1.0 ± 0.3
4.5 ± 0.2 1.9 ± 0.2
0.5 ± 0.1 0
Table 2 Anther response in winter cultivar Igri and spring cultivar Cork. RP/100 RA: regenerated plantlets per 100 responding anthers; G/A: green to albino
Winter cultivar Igri Spring cultivar Cork
Anthers plated
Percent responding anthers
RP/100 RA
G/A ratio
500 500
49.3 45.8
108.7 109.2
7.2 0.03
In haploid plantlets In androgenic plantlets, chloroplasts and albino plastids had similar features in winter cultivar Igri and spring cultivar Cork. After 2 weeks of regeneration, chlorophyllous plantlets derived from microspores included chloroplasts characterized by developed thylakoids and huge grana containing up to 40 saccules (Fig. 4F). Their density was 4.8 ± 0.4 per 100 µm2 cytoplasm, their length was 4.5 ± 0.2 µm, and starch represented only 0.5 ± 0.1% of plastid volume (Table 1). Plastid DNA was immunodetected in a about 30% of plastid sections (Fig. 4G). In the albino plantlets derived from microspores of spring cultivar Cork, plastids contained mainly a prolamellar body and numerous plastoglobules (Fig. 4H). Their density was reduced to 1.5 ± 0.6 plastids per 100 µm2 cytoplasm, their length was 1.0 ± 0.2 µm, and no starch was found. Anther response
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Percentage of responding anthers and yield of regenerated haploid plantlets were similar in both cultivars (Table 2). But in winter cultivar Igri, the ratio of green to albino anthers (G/A ratio) was 7.2, showing 87.8% chlorophyllous Fig. 3 A-G Plastid features in microspores and microspore-derived embryos during pollen embryogenesis in winter cultivar Igri and spring cultivar Cork. A Spring cultivar Cork after pretreatment. Low magnification of pretreated microspore showing that plastids (arrowheads) were non-dividing amyloplasts. I Intine. ×6 000. B Spring cultivar Cork after pretreatment. Higher magnification showing accumulation of starch (asterisks) and absence of thylakoids. M Mitochondria. ×38 000. C Winter cultivar Igri, microspore-derived embryo after 7 days of culture. High density of plastids bearing numerous divisions (arrowheads). M Mitochondria. ×20 000. D Spring cultivar Cork, microspore derived embryo after 7 days of culture. All plastids are amyloplasts (a). ×7 500. E Spring cultivar Cork, microspore derived embryo after 7 days of culture. Higher magnification of amyloplasts filled mainly with starch (asterisks) and scarce thylakoids (arrowhead). ×40 000. F Winter cultivar Igri, microspore-derived embryo after 12 days of culture. Plastid division, starch (asterisk) reduction and thylakoid (arrowheads) development in the stroma. ×22 000. G Spring cultivar Cork, microspore-derived embryo after 12 days of culture. Plastids were amyloplasts with starch (asterisks). ×40 000
regenerated plantlets, whereas in spring cultivar Cork, the G/A ratio was 0.03, showing 99.7% albino plantlets.
Discussion During the entire androgenic process, plastids followed opposite pathways in the two tested cultivars. This is detectable as early as the sampling stage, especially in terms of plastid division and differentiation. Later on, the pretreatment further enhanced the difference between the two cultivars. In spring cultivar Cork, proplastids differentiate into amyloplasts during anther culture: they progressively accumulate starch and lose their thylakoids as well as their division potential. Such differentiation corresponds to the development pathway observed in the pollen grain when it develops in vivo (data not shown). The main function of pollen plastids in barley is restricted to the accumulation of starch during pollen maturation, and pollen plastids have a limited lifespan. After pollination, starch is mobilized, providing monomers of glucose during pollen tube growth; plastids disappear soon after fertilization (Mogensen 1996). In winter cultivar Igri, plastids follow another pathway: they hydrolyse starch during pretreatment, maintain their division activity and progressively develop thylakoids through anther culture. During regeneration, they differentiate into chloroplasts as described in the zygotic embryo (von Wettstein et al. 1995; Caredda et al. 1999). These data could mean that the fate of microspore plastids is pre-determined as early as the microspore stage. In spring cultivar Cork, the amyloplast programme seems to be irreversible and only albino plantlets are formed since plastids are unable to differentiate into chloroplasts. In contrast, in winter cultivar Igri, microspore plastids conserve the ability to switch towards the alternative chloroplast programme, enabling microspore-derived embryos to produce androgenic chlorophyllous plantlets. The state of plastid DNA in the microspore may have a role in determining whether albinism occurs following androgenesis. In winter cultivar Igri, it was recently shown that DNA is degraded in the anther during pollen development, indicating apoptosis in this organ (Wang et al. 1999a). Barley is a species with maternal cytoplasmic
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Fig. 4 A–H Plastid features in microspore-derived embryos and plantlets during androgenesis in winter cultivar Igri and spring cultivar Cork. A Winter cultivar Igri, microspore derived embryo after 21 days of culture. Elongation of plastids correlated with reduction of starch (asterisk) and differentiation of thylakoids (arrowheads). M Mitochondria. ×20 000. B Winter cultivar Igri, microspore-derived embryo after 21 days of culture. Immunolocalization of DNA (arrowhead) in plastid (P) and nucleus (N). Asterisk starch. ×51 000. C Spring cultivar Cork, microspore-derived embryo after 21 days of culture. Low density of plastids (asterisks) in embryo cells. Most plastids were amyloplasts. N Nuclei. ×4 600. D Spring cultivar Cork, microspore-derived embryo after 21 days of culture, higher magnification. Plastids were amylop-
lasts with few thylakoids (arrowhead). asterisk starch. ×32 000. E Spring cultivar Cork, microspore-derived embryo after 21 days of culture. Immunolocalization of DNA (arrowheads) in plastid and nucleus (N). Asterisk starch. ×50 000. F Winter cultivar Igri, microspore-derived chlorophyllous plantlet after 14 days regeneration. Plastids have differentiated into chloroplasts. Arrowheads Intergranal thylakoids; asterisk starch; G grana. ×40 000. G Microspore-derived chlorophyllous plantlet after 14 days regeneration. Immunolocalization of DNA (arrowhead) in plastid with unspecific labelling in starch grain (asterisk). ×22 000. H Microspore-derived albino plantlet after 14 days regeneration. Albino plastid including mostly plastoglobules (white star) and a prolamellar body (pb). ×15 000
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inheritance and microspore plastids are particularly affected by DNA degradation during pollen development (Mogensen 1996). In winter cultivar Igri, alteration of anther DNA is stopped when the anthers receive the androgenic pretreatment and DNA integrity is restored during anther pretreatment (Wang et al. 1999b). It is therefore likely that anther apoptosis is not synchronous in various barley cultivars. The results presented here regarding the presence of DNA in microspore plastids at the sampling stage and during anther culture strongly suggest that the process of DNA degradation occurs earlier in spring cultivar Cork than in winter cultivar Igri. The consequence may be that the microspores of spring cultivar Cork remain unable to produce green androgenic plantlets whatever the pretreatment and the culture conditions. In this cultivar, the dramatic reduction of plastid division as early as the microspore stage and later in microspore-derived embryos is amazing, since plastid division is usually correlated with cell division (Pyke 1997). This could represent an additional sign of plastid DNA alteration in the microspore of this cultivar. In barley, in-vitro culture of various tissues usually induces alterations of plastid physiology which result in production of albino plantlets (Jähne-Gärtner and Lörz 1996; Cho et al. 1998). This means that plastids in Poaceae are particularly sensitive to culture conditions, suggesting that there is discrimination between nuclear and plastid behavior under in vitro conditions. When albino plantlets are formed, the nucleus may conserve totipotency, whereas the plastids may lose the ability to dedifferentiate and follow a new sporophytic programme during the culture process. Winter cultivar Igri and spring cultivar Cork represent an attractive set of cultivars for studying the fundamental bases of androgenic albinism in barley. Both cultivars behave identically in response to anther culture conditions except for the G/A ratio. This indicates that the microspores of spring cultivar Cork are competent up to androgenesis, although regenerated plantlets remain unable to synthesize chlorophyll. It has been shown in winter cultivar Igri that both nucleus and plastid genomes are affected by deletions in albino microspore-derived plantlets, especially in respect to genes related to chlorophyll and photosynthesis (Dunford and Walden 1991). In this cultivar, it seems that plastid genome deletions appear during the regeneration phase (Mouritzen and Holm 1994). The results provided in this study, indicate that it is possible plastid genome alterations occur earlier in spring cultivar Cork, and this may also be the case in other albino regenerating cultivars. Providing information on the state of microspore genomes at the sampling stage could help in obtaining molecular markers of anther culturability in terms of albino androgenic plantlet regeneration. This could also be applied to other Poaceae where genotype-dependent androgenic albinism is an important issue, such as with wheat (Day and Ellis 1984; 1985) and rice (Sun et al. 1979). Acknowledgements The authors would like to thank Professor M.M. Peet for corrections to the English manuscript.
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