Plastid ultrastructure and DNA related to albinism in androgenetic embryos of various barley (Hordeum vulgare L.) cultivars

In order to understand the occurrence of albinism during androgenesis in barley, a number of plastid parameters were analyzed in microspore-derived embryos and androgenetic plantlets, and the results were compared in albino and non-albino producing cultivars. In the winter cv. Igri, plastids in microspore-derived embryos are characterized by numerous divisions, differentiated thylakoids, low amount of starch and a high DNA content examined by immunoelectron microscopy. After regeneration, the androgenetic plantlets were mostly chlorophyllous. In contrast, in the spring lines tested, the plastids of microspore-derived embryos were rarely dividing amyloplasts in which thylakoids and DNA were scarce and albino plantlets were mainly regenerated. After 2 weeks on the regeneration medium, plastids of Igri chlorophyllous androgenetic plantlets were typical chloroplasts, whereas in spring lines plastids of albino androgenetic plantlets were proplastids with the same characteristics as those in the corresponding microspore-derived embryos. These results strongly suggest that the origin of androgenetic albinism differs in winter and spring cvs.: in the winter cv. Igri plastid alteration may take place during the regeneration step of androgenesis whereas in the tested spring lines plastids are already affected in the microspore-derived embryos meaning that albinism is not initiated during regeneration but originates earlier during the androgenetic process likely as early as the sampling stage.     

Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism

The effect of donor plants annual cycle and anther/spike position on the production of microspore-derived plants and albinism were studied. We used the winter cv. Igri and the spring cv. Cork, known to respond similarly in anther culture but to produce 78% and 2% of green plants, respectively. In both cvs. the number of microspore-derived plants was significantly higher when the anthers were collected from January to July than from August to December. However, during this period the proportion of albino plants was not altered. Conversely, the anther response decreased from 76.6 to 31.5% in Igri and from 58.8 to 32.0% in Cork when the donor spike originates from the main shoot or the fourth tiller. Significantly, anthers collected from spike of the second tiller enabled us to drastically increase the proportion of regenerated green plantlets, by 16% in Igri and 1800% in Cork.     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

Microspore embryogenesis and programmed cell death in barley: effects of copper on albinism in recalcitrant cultivars

Albinism remains a major problem in cereal improvement programs that rely on doubled haploid (DH) technology, and the factors controlling the phenomenon are not well understood. Here we report on the positive influence of copper on the production of DH plants obtained through microspore embryogenesis (ME) in recalcitrant cultivars of barley (Hordeum vulgare L.). The presence of copper sulphate in the anther pre-treatment medium improved green DH plant regeneration from cultivars known to produce exclusively albino plants using classical procedures. In plastids, the effect of copper was characterized by a decrease in starch and a parallel increase in internal membranes. The addition of copper sulphate in the ME pre-treatment medium should enable breeders to exploit the genetic diversity of recalcitrant cultivars through DH technology. We examined programmed cell death (PCD) during microspore development to determine whether PCD may interfere with the induction of ME and/or the occurrence of albinism. By examining the fate of nuclei in various anther cell layers, we demonstrated that the kinetics of PCD in anthers differed between the barley cultivars Igri and Cork that show a low and a high rate of albinism, respectively. However, no direct correlation between PCD in the anther cell layers and the rate of albinism was observed and copper had no influence on the PCD kinetic in these cultivars. It was concluded that albinism following ME was not due to PCD in anthers, but rather to another unknown phenomenon that appears to specifically affect plastids during microspore/pollen development.     

Anther plastids in angiosperms

In the anther of angiosperms, all types of plastids are found in the course of pollen development. They are located in the different cell layers of the microsporangium and have various functions that contribute to the formation of the functional male gametophyte. This includes photosynthesis, stomata opening, sugar storage and/or mobilization, lipid synthesis and secretion for pollenkitt formation, as well as serving as a physiological buffer under stress conditions. They are also involved in plastid inheritance, but to different extents, according to the species. The plastid is a semi-autonomous organelle. Plastid division in the anther is synchronous with cell division, except in the vegetative cell during pollen maturation. Furthermore, recent data seem to show that plastids are affected by programmed cell death and DNA degradation, which occur in the whole anther throughout pollen development. However, the timing of plastid disappearance fluctuates in the different cell layers and also depending on species. In vitro, following androgenesis, plastids that originate in the microspore are responsible for the occurrence of albino plantlets in Poaceae. This trait reflects the relative independence of the plastid genome when compared with that of the nucleus. In this family, microspore plastids may become so involved in programmed cell death that they are unable to follow the alternative sporopohytic program. The different pathways of plastid differentiation in neighboring anther cell layers require an accurate regulation of cell development that remains widely unknown in the anther.     

Low responsiveness of six-rowed genotypes to androgenesis in barley does not have a pleiotropic basis.

A heterozygous mutant for the two- and six-rowed character was isolated in the barley cultivar Igri through application of sodium azide to isolated microspore cultures and posterior regeneration. Six-rowed and two-rowed homozygotic plants were subsequently identified in the self-pollinated M2 progenies of the original heterozygous M1. Detailed molecular markers confirmed the isogenic nature of this recovered mutant and the original cultivar Igri. A comparative study of the anther culture response of this six-rowed induced mutant vs. diploid 'Igri' was performed to assess whether the two- or six-rowed gene influences anther culture response in barley through a pleiotropic effect or via linkage disequilibrium. No significant differences for any of the recorded variables throughout the in vitro regeneration process were detected between the 'Igri' six-rowed mutant and any of their two-rowed isogenic lines. This suggests that row-type association with anther culture response in barley cultivars is due to the effect of a tight linkage with other genes directly responsible for androgenic response.     

Induction of in vitro androgenesis in anther and isolated microspore culture of different spelt wheat (<Emphasis Type="Italic">Triticum spelta</Emphasis> L.) genotypes

This is the first report on isolated microspore culture—derived spelt wheat. The efficiency of anther- and isolated microspore was compared using four genotypes (‘Franckenkorn’, ‘GK Fehér’, ‘Mv Martongold’, ‘Oberkulmer Rotkorn’). In anther culture, genotype dependency was observed, and cold pre-treatment enhanced the efficiency of the method. In isolated microspore culture, the ovary co-culture supported the development of embryo-like structures. The presence of growth regulators (0.5 mg/l 2,4-D and 0.5 mg/l kinetin) were not essential for the induction of androgenesis, but these increased the production of embryo-like structures, green and albino plantlets. The low plant regeneration rate and high number of albinos hinder the practical application of isolated microspore culture while anther culture was efficient for in vitro green plantlets production in spelt wheat. The mean of green plantlets production was 41.45/100 anthers (from 20.93 to 83.07 depending on genotype). The phenomenon of albinism was mitigated in anther culture (3.48 albinos/100 anthers). Altogether, 1720 anther culture—derived green plantlets were produced from the four genotypes.     

Impact of abscisic acid in overcoming the problem of albinism in horse chestnut androgenic embryos

Horse chestnut (Aesculus hyppocastanum L., Hyppocastanacea) is a relict species with a slow and complex reproductive cycle considered to have horticultural and medical importance. The cycle maybe circumvented via in vitro androgenesis. Androgenesis of horse chestnut was induced in microspores and anther culture on MS media. Some of the horse chestnut androgenic embryos were albinos. Addition of abscisic acid in media (in concentrations of 0.01, 0.1, 0.5, 1, 2, 5, 10, and 20 mg l^?1) with horse chestnut androgenic embryos has circumvented the reproduction cycle barriers. The best results were achieved on medium with the lowest abscisic acid concentration (0.01 mg l^?1) in microspore culture. The microspore culture proved to be a better model system for embryo production and albino embryo reduction than anther culture. Flow cytometry analysis after maturation treatments induced by ABA showed that 88 % of green embryos originating from microspore culture were haploid. However, 50 % of green embryos from anther culture were haploid. The remaining analyzed androgenic embryos, from both types of cultures were diploid.     

Effect of the 1BL.1RS wheat-rye translocation on the androgenic response in spring bread wheat

The study was conducted to investigate the effect of the 1BL.1RS wheat-rye-translocation on the androgenic response in spring bread wheat. Therefore, four bread wheat cultivars carrying the translocation, four Greek and three Canadian bread wheat cultivars without the translocation were used. An equal number of anthers from each cultivar, containing microspores in the mid (MU) to late uninucleate (LU) microspore developmental stage, were cultured after cold pre-treatment for seven days at 4°C. W14, 190-2 and the basic MS were used as induction, regeneration, and rooting media respectively. The best androgenic response was recorded in two cultivars carrying the translocation. Only two cultivars lacking the translocation responded to anther culture. It is concluded that the positive effect of the 1BL.1RS translocation on anther culture response of bread wheat cultivars cannot be attributed entirely to its presence because the genetic background of the cultivars carrying the translocation could be also important.     

Regeneration of the barley zygote in ovule culture

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.     

Positive effects of cold pretreatment, iron source, and silver nitrate on anther culture of strawberry (Fragaria × ananassa Duch.)

Androgenesis by anther culture or isolated microspore culture is the most efficient method for haploid production. In this study, the effects of cold pretreatment (4 °C), silver nitrate, and iron source in the medium were investigated on anther culture of five strawberry cultivars (Camarosa, Selva, Pajaro, Paros, and Gaviota) in three independent experiments, and three traits including percentage of androgenic anthers, embryogenesis, and callogenesis were determined. In the first experiment, cold pretreatment of 4 °C for 2 and 3 days produced the highest percentage of androgenic anthers in three cultivars (Camarosa, Selva, and Pajaro) and cold pretreatment of 4 °C for 2 days produced the highest percentage of androgenic anthers in cultivar Paros. In the second experiment, the effect of Fe-EDDHA was more effective than Fe-EDTA, and increased the percentage of androgenic anthers and embryogenesis. The use of 57.5 mg l^?1 Fe-EDDHA produced the highest percentage of androgenic anthers in cultivar Camarosa and the best embryogenesis in cultivars Camarosa and Gaviota. In the third experiment, the use of 15 mg l^?1 silver nitrate in medium significantly increased the percentage of androgenic anthers and embryogenesis in cultivar Camarosa.     

Plant regeneration from embryogenic cell suspensions derived from anther cultures of barley (Hordeum vulgare L.)

Summary We have established embryogenic cell suspension cultures of barley (Hordeum vulgare L. cultivars Igri, Gimpel, Princesse, and Baronesse) from anther-derived embryogenic callus. Suspension cultures of cultivars Igri and Gimpel were regenerable. The most successful cultivar was Igri, from which a number of independent cell lines producing plantlets were established. Plants could be transferred to soil; up to now, 50% of more than 200 regenerated plants were morphologically normal and fertile. The relative frequency of sterile plants increased as suspensions aged. Suspensions older than 1 year produced embryogenic callus but only albino plantlets could be regenerated.     

Two pathways of plant regeneration in wheat anther culture

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 2n=3x=21 were the most frequent.     

Enhanced post-germinative growth of encapsulated somatic embryos of Siberian ginseng by carbohydrate addition to the encapsulation matrix

Based on a protocol for microspore culture in apple (Malus domestica Borkh.), the embryo induction phase has been improved with regard to pretreatment of microspores for initiation of microspore embryogenesis, the concentration of carbon source in the induction medium and the microspore density in the suspension. Furthermore, the effect of the genotype was studied. To determine the efficiency of in vitro androgenesis, both methods, via anther and microspore culture, were investigated using the same bud material. A comparison of the efficiency of embryo induction in anther and microspore cultures showed that microspore culture resulted in an increase up to 10 times, depending on the genotype. The regeneration route in microspore culture is similar to that of androgenic embryos via anther culture and showed adventitious shoot formation in most cases after a long period of secondary embryogenesis.Communicated by H. Lörz     

Effects of free and chelated iron on in vitro androgenesis in barley and wheat

A suitable form of iron supplement in the induction medium was found to be important for further development of induced pollen embryos in barley and wheat cultivars (genotypes), especially those providing few green plants viain vitro androgenesis. Genotypes able to regenerate many green plants were less susceptible to the lack of iron in induction medium. Although Fe-EDTA was found to be a suitable form of iron in the induction medium, androgenesis was also induced on media containing non-chelated iron (Fe²⁺ and Fe³⁺ ions). EDTA alone without iron inhibited the androgenic response even in the wheat cv. Florida, a model cultivar for androgenesis in wheat. In all barley cultivars under study including cv. Igri, a model cultivar for androgenesis in barley, EDTA alone caused an almost total suppression of androgenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

Barley anther culture: assessment of carbohydrate effects on embryo yield, green plant production and differential plastid development in relation with albinism

Sugars and polyols were tested at different steps of anther culture in barley (Hordeum vulgare L.) to elucidate their influence on both the overall yield of androgenesis and the structure of plastids in relation to albinism. During the pretreatment period, the osmotic regulation in the medium was beneficial to microspore embryogenesis regardless of the type and concentration of the tested osmoticum. The use of mannitol (300 mOsm/kg), sorbitol (180 mOsm/kg), PEG (240 mOsm/kg) and sucrose (180 mOsm/kg) gave the best results in terms of green plant production, although the influence of each substance differed according to the studied parameter. Similarly, during anther culture the regulation of the osmotic pressure in the medium had various effects, according to the osmoticum used. The best results were obtained using mannitol (364 mOsm/kg), providing 139.7 green plants per 100 plated anthers. Plastids were examined by electron microscopy following both pretreatment and culture. In the presence of mannitol and PEG, plastids did not accumulate starch at any stage of the protocol but they started to differentiate into chloroplasts in the microspore-derived embryos. Using sorbitol and sucrose, plastids differentiated poorly but accumulated large amounts of starch, suggesting that these sugars are metabolized by micropores and microspore derived structures. However, the accumulation of starch was not correlated with the occurrence of albinism. These results indicated that, in barley, the osmotic regulation was favourable to switch the microspore gametophytic program toward a sporophytic program regardless of the nature of the osmoticum. In addition, during the pretreatment period, mannito was found to be the most suitable osmoticum for subsequent embryo development.     

水稻花粉白苗的若干特性及其成因的探讨

水稻花粉白苗的再分化所生成的再生植株全为白苗,这说明花粉白苗的形成是不可逆的,可能是基因突变的结果。不同品种或杂交组合的水稻花粉白苗叶片丙酮提取液的吸收光谱相互间显示出了相似性,这种相似性就进而提示突变可能发生在基因组的特定位点上。有可能花粉白苗是质体DNA在花粉的脱分化过程中发生缺失形成的。这种缺失主要影响质体rRNA的合成水平,并不导致蛋白质和叶绿素合成能力的全面丧失。花粉植株细胞中的质体可能是不均一的。     

Seasonal rhythmics of the leucoplast structure in bulb scales of Scilla sibirica L

A study was made of seasonal changes in plastids of ground tissue cells of bulb scales in early-spring ephemeroid Scilla sibirica L. In summer, plastids are represented by typical amyloplasts, with their main volume (97.0 +/- 4.3%) being occupied by one large starch grain. The volume fraction of plastid stroma is at its minimum. The stroma contains small plastoglobuli and no thylakoids. The same structure is characteristic of plastids in October. However, no starch is found in December, when some thylakoids are seen at the plastid periphery. In the early spring (March), when leaves still remain below the ground, the volume fraction of starch grains is 53.0 +/- 2.2%. In the stroma some structures superficially similar to those of microtubuli are revealed. The thylakoid system is fairly well developed, some of thylakoids being concentrically arranged. Some electron-opaque material is seen in the thylakoid lumen. Many plastids are sheathed with elements of the smooth endoplasmic reticulum. Based on the analysis of these and literature data, a conclusion is made that plastids of bulb scales not only store starch, but also seemingly participate in phytohormone biosynthesis.     

Cytological Basis of Plastid Inheritance in Phaseolus vulgaris-Investigations of Plastids,Mitochondria and Their DNA Nucleoids During Pollen Development

Electron microscopic and DNA fluorescence microscopic observations of the plastids, mitochondria and their DNA in the developing pollen of Phaseolus vulgaris L. have demonstrated that the male plastids were excluded during microspore mitosis. The formed generative cell was free of plastids because of regional localization of plastids in early developing microspore and the extremely unequal distribution during division. The fluorescence observations of DNA showed that cytoplasmic (plastid and mitochondria) nucleoids degenerated and disappeared during the development of microspore/pollen, and were never presented in the generative cell at different development stages. These results provided precise cytological evidence of maternal plastid inheritance in Phaseolus vulgaris, which was not in accord with the biparental plastid inheritance identified from early genetic analysis. Based on authors' previous observations in a variety of common bean that the organelle DNA of male gamete was completely degenerated, the early genetic finding of the biparental plastid inheritance was unlikely to be effected by genotypic difference. Thus those biparental plastid inheritance might be caused by occational male plastid transmission, and plastid uniparental maternal inheritance was the species character of Phaseolus vulgaris.