Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

Stress-induced microspore embryogenesis in tobacco: an optimized system for molecular studies

Summary Specific stress treatments applied to isolated tobacco (Nicotiana tabacum L.) microspores efficiently induced haploid embryo formation in vitro. A heat shock at 33 or 37°C in the presence of sugar, as well as sucrose-starvation at 25°C, resulted in the formation of embryogenic microspores. A combination of both treatments had an additive effect. Under optimal induction conditions all viable microspores in the culture were embryogenic and developed subsequently into pollen embryos by culture at 25°C in a sugar-containing medium, with induction frequencies of more than 70% with respect to the initial microspore population. A high fraction of the early pollen embryos continued their development in vitro, giving rise to haploid plants. In contrast to other available systems for microspore/pollen embryogenesis, the new protocol allows the production of homogeneous populations of embryogenic microspores and early globular embryos in large-scale cultures, without any purification step, and is therefore well suited for biochemical and molecular work.Abbreviations EDTA ethylenediaminetetraacetate - DAPI 4,6-diamidino-2-phenylindole     

Effects of culture density,conditioned medium and feeder cultures on microspore embryogenesis in Brassica napus L. cv. Topas

Summary In microspore cultures of Brassica napus L. cv. Topas, embryo yield increases with culture density up to about 40,000 microspores per ml. A much higher density (100,000 per ml) appears inhibitory to embryogenesis. A relatively high culture density (30,000 or 40,000 per ml) for the first 2–4 days of culture is crucial for embryogenesis, after which cultures may be diluted to allow better embryo growth.Medium conditioned by culturing microspores at 30,000 or 40,000 per ml for 1 day improved microspore-embryo yield in low density cultures (3,000 or 4,000 per ml) more than 3-fold. In contrast, media conditioned with microspores from 1–4 days or 0–4 days of culture were inhibitory.Use of feeder cultures resulted in up to 10-fold increase of embryo yield in low density microspore cultures, depending on the method used. Filter papers and other membranes placed on top of feeders greatly inhibited embryogenesis in the feeder layer as well as microspores cultured on the feeder, possibly due to poorer gaseous exchange.     

Expression of the BnmNAP subfamily of napin genes coincides with the induction of Brassica microspore embryogenesis

Brassica napus cv. Topas microspores can be diverted from pollen development toward haploid embryo formation in culture by subjecting them to a heat stress treatment. We show that this switch in developmental pathways is accompanied by the induction of high levels of napin seed storage protein gene expression. Changes in the plant growth or microspore culture conditions were not by themselves sufficient to induce napin gene expression. Specific members of the napin multigene family were cloned from a cDNA library prepared from microspores that had been induced to undergo embryogenesis. The majority of napin clones represented three members (BnmNAP2, BnmNAP3 and BnmNAP4) that, along with a previously isolated napin genomic clone (BngNAP1), constitute the highly conserved BnmNAP subfamily of napin genes. Both RNA gel blot analysis, using a subfamily-specific probe, and histochemical analysis of transgenic plants expressing a BngNAP1 promoter--glucuronidase gene fusion demonstrated that the BnmNAP subfamily is expressed in embryogenic microspores as well as during subsequent stages of microsporic embryo development.     

Microspore culture of radish (Raphanus sativus L.): influence of genotype and culture conditions on embryogenesis

A number of factors influencing embryogenesis from isolated microspores of radish (Raphanus sativus) were examined. Of 11 genotypes evaluated, six produced embryos ranging from 8.3 embryos per 10⁵ microspores for Chugoku-ao to 0.2 for Tenshun, but five genotypes were not responsive. An initial culture period at elevated temperature before incubation at 25°C was essential for induction of microspore embryogenesis. However, the optimum period of the treatment varied among genotypes and/or experiments. Bud size also influenced microspore embryogenesis. Though optimum bud size was different between genotypes, the microspore populations represented in these buds contained uninucleate and binucleate microspores. Selection of embryogenic microspores using percoll density gradient resulted in up to 1.3-fold increase of embryo yield. Though almost all embryos failed to develop directly into plantlets, plants were obtained by multiple subcultures. The regenerated plants had hyperploid chromosome numbers.     

Embryogenesis and plant regeneration from isolated microspores of Brassica rapa L. ssp. Oleifera

Summary Conditions favourable to embryogenesis from isolated microspores of Brassica rapa L. ssp. oleifera (canola quality) were identified. A population with enhanced responsiveness for microspore embryogenesis (C200) was synthesized by crossing individual plants showing microspore embryogenic potential. For optimal microspore embryogenesis, buds (2–3mm in length, containing mid-late uninucieate microspores) were collected from older plants (2 months old) and microspores isolated and washed in iron-free B5 medium. NLN medium with its iron content reduced to half was beneficial for initial microspore culture. An elevated temperature(33–35°C) during the first day of culture, followed by maintenance at 25°C resulted in dozens of embryos from each isolation (about 100 buds). Seeds were obtained from plants regenerated from microsporederived embryos after colchicine treatment.     

A novel stress-related gene in developing pepper anthers

To study gene expression patterns and to find genes related with microspore embryogenesis during pepper (Capsicum annuum L.) anther development, mRNA expression patterns were investigated at four developmental stages distinguished according to the size of flower bud, the color of anthers, and the cytological feature of microspores. Through GeneFishing using 120 random primers, 81 genes were found to be differentially expressed as anthers develop. We directly sequenced seven of them, which were either up- or down-regulated at stage 2, since microspores at stage 2 are known to be responsive to the induction signals for microspore embryogenesis. Nucleotide sequence analysis of the isolated differentially expressed genes (DEGs) and the comparison of these sequences with the GenBank data indicate that DEG13 is a novel gene, which is highly homologous to a stress-related gene of potato, POACT88 (≈91%) and to alcohol dehydrogenase gene of Arabidopsis (≈70%), whose expression is also tightly related to stresses. In vitro data also showed that DEG13 was more abundantly expressed in heat-treated microspores than in untreated microspores. Here, we report developmental stage-specific gene expression patterns during anther development and a novel stress-related gene, DEG13, which may be involved in microspore embryogenesis in response to heat treatment.     

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 pH, MES, arabinogalactan-proteins on microspore cultures in white cabbage

The objective of this study was to improve induction of embryogenesis in white cabbage (Brassica oleracea var. capitata) microspore cultures. The effect of NLN-13 liquid medium pH on isolated microspore embryogenesis was investigated in five white cabbage genotypes. Relatively high pH (6.2 or 6.4) was more effective on microspore embryogenesis in most of the white cabbage genotypes than the pH of 5.8, especially for inducing microspore-derived embryos in recalcitrant genotype ??Zhonggan No. 8??. Based on this, 2??(N-Morpholino) ethanesulfonic acid (MES) and the arabinogalactan-protein from gum arabic were tested on four out of five genotypes to see if they could increase embryo yield in microspore cultures. Adding MES or gum arabic alone was effective for these four genotypes, but the frequency of embryos derived from microspores was still low. However, the combination of 10?mg?l^?1 gum arabic and 3?mM MES in NLN-13 at pH 6.4 significantly enhanced microspore embryogenesis efficiency (with embryo production of 4.57?C222.97 embryos per bud), especially with recalcitrant genotype ??Zhonggan No. 8?? for which it was increased by about 35-fold.     

A high throughput <Emphasis Type="Italic">Brassica napus</Emphasis> microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.     

Scanning electron microscopy of microspore embryogenesis inBrassica spp.

Scanning electron microscopy was employed to study and compare microspore embryogenesis in vitro with pollen development in planta inBrassica napus andB. oleracea. An exine with its specific pattern had already been formed, when microspores were released from tetrads. During subsequent pollen development, microspores increased in size and continued to strengthen the exine. Upon in vitro culture, all microspores, i.e., embryogenic and nonembryogenic, initially showed the same morphological features. After 24 h in culture, the microspores had increased in size. Thereafter, embryogenesis was indicated in some microspores by two different morphological changes. One featured an expansion in volume of the cell cluster around the germination aperture (type I), the other showed cell cluster volume expansion over the entire microspore surface (type II). Two-thirds of embryogenic microspores in bothB. napus andB. oleracea demonstrated type I development. When followed by fluorescence microscopy, in vitro culture of microspores revealed cultures with a high embryo frequency were those with a high frequency of symmetrical division.Abbreviations SEM Scanning electron microscopy - TEM Transmission electron microscopy     

Tracking individual wheat microspores in vitro: identification of embryogenic microspores and body axis formation in the embryo

 The development of isolated, defined wheat microspores undergoing in vitro embryogenesis has been followed by cell tracking. Isolated wheat (Triticum aestivum L.). microspores were immobilized in Sea Plaque agarose supported by a polypropylene mesh at a low cell density and cultured in a hormone-free, maltose-containing medium in the presence of ovaries serving as a conditioning factor. Embryogenesis was followed in microspores isolated from immature anthers of freshly cut tillers or from heat- and starvation-treated, excised anthers. Three types of microspore were identified on the basis of their cytological features at the start of culture. Type-1 microspores had a big central vacuole and a nucleus close to the microspore wall, usually opposite to the germ pore. This type was identical to the late microspore stage in anthers developing in vivo. Microspores with a fragmented vacuole and a peripheral cytoplasmic pocket containing the nucleus were defined as type 2. In type-3 microspores the nucleus was positioned in a cytoplasmic pocket in the centre of the microspore. Tracking revealed that, irrespective of origin, type-1 microspores first developed into type 2 and then into type-3 microspores. After a few more days, type-3 microspores absorbed their vacuoles and differentiated into cytoplasm-rich and starch-accumulating cells, which then divided to form multicellular structures. Apparently the three types of microspore represent stages in a continuous process and not, as previously assumed, distinct classes of responding and non-responding microspores. The first cell division of the embryogenic microspores was always symmetric. Cell tracking also revealed that the original microspore wall opened opposite to a region in the multicellular microspore which consisted of cells containing starch grains while the remaining cells were starch grain-free. The starch-containing cells were located close to the germ pore of the microspore. In more advanced embryos the broken microspore wall was detected at the root pole of the embryo. Received: 27 December 1999 / Accepted: 11 May 2000     

大麦HvLEC1基因的克隆及其表达特征分析

植物LEC蛋白是NF-Y转录因子的一类B亚基,在植物胚状体形成过程中起重要作用。为了研究大麦小孢子体外培养形成胚状体的机理,本研究利用RACE技术在大麦中克隆了一个新的LEC基因,该基因cDNA全长为1004 bp,开放阅读框全长为597 bp,编码198个氨基酸,其蛋白1~59位氨基酸含有LEC结构域,命名为HvLEC1。HvLEC1在大麦的根、茎、叶和小孢子培养过程中均能表达,其中小孢子培养7 d时表达量最高,且HvLEC1在大麦品系BI04中的表达量比基19高,BI04愈伤产量也比基19高,表明HvLEC1表达量和愈伤产量有相关性,受盐胁迫后HvLEC1在大麦的根中快速上调表达,提示HvLEC1可能不仅参与小孢子胚状体发生,而且参与盐胁迫响应。     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

Induction of microspore embryogenesis inBrassica napus L. by gamma irradiation and ethanol stress

Summary Gamma irradiation and ethanol stress treatments redirected pollen development to an embryo formation pathway inBrassica napus. Less than 0.01% of microspores developed into embryos at 25°C compared to approximately 2% at 32°C. However, subsequent to gamma irradiation and ethanol treatments up to 1% and 0.7% of microspores formed embryos at 25°C, respectively. Gamma irradiation also enhanced embryogenesis at 32°C. The possible importance of these findings is discussed in relation to microspore embryogenesis.     

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.     

Cytological analysis of early microspore divisions leading to gametic embryo formation in <Emphasis Type="Italic">Quercus suber</Emphasis> L. anther cultures

The correlation between the phenologic stage of the inflorescence and the microspore development stage was studied. Cytological examinations of the development of microspores during in vitro anther culture of cork oak (Quercus suber L.), were carried out during the first four weeks of culture. To observe the division occurring in the microspores, anthers were taken randomly from the cultures after heat shock treatment and were stained with DAPI. Most of the anthers responding to a heat stress treatment contained 91 % vacuolated microspores, indicating that this developmental stage is responsive to embryogenesis induction in cork-oak microspores. After the heat shock treatment some cork-oak microspores were induced and initiated the embryogenic pathway with the occurrence of numerous symmetric mitosis, producing structures with two to ten or more nuclei. These lead to the formation of high numbers of multicellular cork-oak microspores (pro-embryos). Twenty-forty days after induction, small white globular and cotyledonal embryos were observed, which further developed root and shoot, regenerating plantlets.     

1-Aminocyclopropane-1-Carboxylic Acid Enhanced Direct Somatic Embryogenesis from Oncidium Leaf Cultures

Influence of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and two ethylene inhibitors, silver nitrate (AgNO₃) and cobalt chloride (CoCl₂), on direct somatic embryogenesis were tested in vitro using leaf cultures of Oncidium cv. Gower Ramsey. Leaf cells of tips, adaxial sides and cut ends could directly form somatic embryos on a hormone-free 1/2-strength MS medium. The frequency of embryo-producing explants was 55, 52.5 and 30 %, respectively. The embryo numbers per embryo-producing explant was 20.3. ACC at lower concentrations (5 and 10 μM) significantly retarded direct embryo formation from cut ends. However, higher concentrations of ACC (20 and 50 μM) significantly promoted embryogenic response of leaf tips and adaxial sides. All concentrations of AgNO₃ and CoCl₂ significantly retarded direct embryo formation. The best response was found on 20 μM of ACC, and the frequency of embryo-producing explants were 90, 85 and 35 % on leaf tips, adaxial sides and cut ends, respectively. The embryo numbers per embryo-producing explant was 32.2. This revised version was published online in July 2006 with corrections to the Cover Date.     

Identification of loci associated with embryo yield in microspore culture of <Emphasis Type="Italic">Brassica rapa</Emphasis> by segregation distortion analysis

Key message

We identified three physical positions associated with embryo yield in microspore culture of Brassica rapa by segregation distortion analysis. We also confirmed their genetic effects on the embryo yield.

Abstract

Isolated microspore culture is well utilized for the production of haploid or doubled-haploid plants in Brassica crops. Brassica rapa cv. ‘Ho Mei’ is one of the most excellent cultivars in embryo yield of microspore culture. To identify the loci associated with microspore embryogenesis, segregation analysis of 154 DNA markers anchored to B. rapa chromosomes (A01–A10) was performed using a population of microspore-derived embryos obtained from an F₁ hybrid between ‘CR-Seiga’, a low yield cultivar in microspore-derived embryos, and ‘Ho Mei’. Three regions showing significant segregation distortion with increasing ‘Ho Mei’ alleles were detected on A05, A08 and A09, although these regions showed the expected Mendelian segregation ratio in an F₂ population. The additive effect of alleles in these regions on embryo yield was confirmed in a BC₃F₁ population. One region on A08 containing Br071-5c had a higher effect than the other regions. Polymorphism of nucleotide sequences around the Br071-5c locus was investigated to find the gene possibly responsible for efficient embryogenesis from microspores.

     

Microtubule configurations and nuclear DNA synthesis during initiation of suspensor-bearing embryos from Brassica napus cv. Topas microspores

In the new Brassica napus microspore culture system, wherein embryos with suspensors are formed, ab initio mimics zygotic embryogenesis. The system provides a powerful in vitro tool for studying the diverse developmental processes that take place during early stages of plant embryogenesis. Here, we studied in this new culture system both the temporal and spatial distribution of nuclear DNA synthesis places and the organization of the microtubular (MT) cytoskeleton, which were visualized with a refined whole mount immunolocalization technology and 3D confocal laser scanning microscopy. A ‘mild’ heat stress induced microspores to elongate, to rearrange their MT cytoskeleton and to re-enter the cell cycle and perform a predictable sequence of divisions. These events led to the formation of a filamentous suspensor-like structure, of which the distal tip cell gave rise to the embryo proper. Cells of the developing pro-embryo characterized endoplasmic (EMTs) and cortical microtubules (CMTs) in various configurations in the successive stages of the cell cycle. However, the most prominent changes in MT configurations and nuclear DNA replication concerned the first sporophytic division occurring within microspores and the apical cell of the pro-embryo. Microspore embryogenesis was preceded by pre-prophase band formation and DNA synthesis. The apical cell of the pro-embryo exhibited a random organization of CMTs and, in relation to this, isotropic expansion occurred, mimicking the development of the apical cell of the zygotic situation. Moreover, the apical cell entered the S phase shortly before it divided transversally at the stage that the suspensor was 3–8 celled.