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.     

Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa)

A simple mechanical method has been developed which allows the routine isolation of unfertilized and fertilized egg cells from ovules of Japonica and Indica rice varieties. In the experiments described, the majority of the egg cells and zygotes survived the isolation procedure when the donor plants were in a vigorous state. About 40% of the surviving zygotes underwent sustained development when cultured in Millicell inserts with a non-morphogenic rice feeder-cell culture. Nearly all zygote-derived callus cultures regenerated multiple shoots, which could be subsequently rooted with high efficiency. Zygote-derived plantlets matured to fertile plants when transplanted to soil. So far, about 80 independent plants each from the Japonica variety 'Taipei309' and the Indica variety 'IR58' have been regenerated. The potential of this single-cell regeneration system for marker gene-free transformation is discussed. Received: 26 November 1998 / Revision received: 15 March 1999 / Accepted: 21 March 1999     

Inducing homozygosity in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.     

Efficient regeneration of fertile plants from protoplasts isolated from microspore cultures of barley (Hordeum vulgare L.)

Summary Cultures of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar of malting barley) were used for isolation of protoplasts. The protoplasts were cultured embedded in agarose. The plating efficiency varied from 0.002% to 0.015%. Several hundred green plants were regenerated from the cultures. Plantlets regenerated from protoplasts were potted in soil within 4–5 months of collecting the spikes for microspore culture and the first plants are now setting seed.     

Time-lapse tracking of barley androgenesis reveals position-determined cell death within pro-embryos

Following abiotic stress to induce barley (Hordeum vulgare L.) androgenesis, the development of 794 enlarged microspores in culture was monitored by time-lapse tracking. In total, 11% of the microspores tracked developed into embryo-like structures (type-I pathway), 36% formed multicellular structures (type-II pathway) and 53% of the microspores followed gametophytic divisions, accumulated starch and died in the first days of tracking (type-III pathway). Despite the microspore fate, enlarged microspores showed similar morphologies directly after stress treatment. Ultrastructural analysis, however, revealed two morphologically distinct cell types. Cells with a thin intine layer and an undifferentiated cytoplasm after stress treatment were associated with type-I and type-II pathways, whereas the presence of differentiated amyloplasts and a thick intine layer were associated with the type-III pathway. Tracking revealed that the first morphological change associated with embryogenic potential was a star-like morphology, which was a transitory stage between uninucleate vacuolated microspores after stress and the initiation of cell division. The difference between type-I and type-II pathways was observed during the time they displayed the star-like morphology. During the transition phase, embryo-like structures in the type-I pathway were always released out of the exine wall at the opposite side of the pollen germ pore, whereas in the type-II pathway multicellular structures were unable to break the exine and to release embryo-like structures. Moreover, by combining viability studies with cell tracking, we show that release of embryo-like structures was preceded by a decrease in viability of the cells positioned at the site of exine wall rupture. These cells were also positively stained by Sytox orange, a cell death indicator. Thereby, we demonstrate, for the first time, that a position-determined cell death process marks the transition from a multicellular structure into an embryo-like structure during barley androgenesis.     

Differentiation of isolated wheat zygotes into embryos and normal plants

Efficient and reproducible embryo development has been obtained from fertilized wheat (Triticum aestivum L.) egg cells isolated 3–6 h after hand-pollination of emasculated spikes. It is possible to routinely isolate viable zygotes from about 75% of the excised ovaries from cultivars of both winter and spring types. Co-culture with barley microspores which had been stimulated to sporophytic development resulted in embryonic development of the cultivated wheat zygotes. Within 23 h of pollination; the zygotes underwent their first cell division. They proceeded to develop into club-shaped embryos, most of which turned subsequently to dorsiventral differentiation. The morphological patterns of in-vitro-grown embryos were in accordance with those of normal zygotic embryos growing in planta. The formation of twin or multiple embryos originating from a single zygote was dependent on genotype and exogeneously supplied auxin. Upon transfer onto a suitable solidified medium, zygote-derived embryos usually germinated and developed into plants. After optimizing the feeder system, the nutrient medium and the concentration of 2,4-dichloro phenoxyacetic acid (2,4-D), more than 80 and 90% of the zygotes eventually developed into plants in genotypes Florida and Veery #5, respectively. All regenerated plants were morphologically normal and fertile. The in-vitro development from isolated zygotes of a higher-plant species into typically patterned zygotic embryos is shown here for the first time. Since the entire process, including early zygotic development, is now freely accessible to observation and micromanipulation, the method presented opens up new approaches in fundamental as well as applied fields of reproductive biology. Received: 4 September 1997 / Accepted: 28 November 1997     

禾谷类花器官机械游离小孢子培养研究

以大田及温室生长的植株为材料,成功地建立了直接从禾谷类花器官(大麦穗切段、水稻颖花、小麦小穗)机械游离小孢子的程序及培养系统。从供试的二个大麦材料上重复获得大量游离小孢子再生植株,从一个水稻广亲和品种上得到游离小孢子再生植株,以及从三个小麦品种(系)上获得小孢子形成的多细胞结构(MCS)和早期胚状体(ELS)。相对较长时间的低温预处理有利于提高ELS(大麦)及MCS(小麦)的得率,改善培养物的通气状况,以及提早再分化有利绿色植株再生。     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

In vitro culture of isolated living sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) embryo sacs

Ovules of sunflower tubular florets were observed histologically by serial sectioning and clearing to study the correlation between flower morphology and developmental stage. On the basis of these data, florets with visible stigma and receptive "curling" surfaces were chosen for embryo sac (ES) isolation. In such florets, ~20% of the ovules were unfertilized; fertilized zygote-stage ES (~75% of ovules) or ES containing two-celled proembryo occurred in the remaining ovules. ES were dissected manually using needles under a stereomicroscope or were treated with enzymes for 4–5 h after manual isolation. The viability of ES isolated without enzymes was more than 90% as assessed by fluorescein diacetate staining, and decreased to ~3% after 72 h of culture. The use of enzymes during isolation resulted in diminished ES viability, suggesting that removal of the protective layers of ovular cells may be part of the problem. Another factor affecting ES viability is medium osmolality. Living ES were then cultured in vitro. On liquid medium containing 9% sucrose, globular embryos developed in ~10% of zygote-stage ES, but further growth of the embryos was abnormal and callus was produced. Transfer of embryo-derived callus to solid Murashige-Skoog medium supplemented with 1-naphtaleneacetic acid and kinetin resulted in organogenesis. When ES were co-cultured with androgenetic microspores and microspore-derived embryos of Brassica napus, ~11% of the ES showed growth and development. Embryological study of the cultured ES revealed outgrowth of endothelium.     

Fertile Indica rice plants regenerated from protoplasts isolated from microspore derived cell suspensions

Rice plants (Oryza sativa L., Chinsurah Boro II var. Indica) were regenerated from protoplasts isolated from microspore derived cell suspensions. A simple procedure for the establishment of such cell suspension cultures from embryogenic microcallus derived from cultured isolated microspores of Indica-type rice is described. Regenerating protoplasts could readily be isolated from 5–12 months old cell suspensions showing visible colony formation in the range of 180–1050 colonies/10⁶ protoplasts after about one month in culture. More than 100 independent green plantlets were regenerated via secondary embryogenesis from ca 20×10⁶ protoplasts. Out of 32 plants grown to maturity under greenhouse conditions 24 were fertile.Abbreviations CH casein hydrolysate - 2,4-D 2,4-dichlorophenoxyacetic acid - ECS embryogenic cell suspension - NAA naphthaleneacetic acid     

Differentiation ex ovulo of Embryos and Plantlets in Stem Tissue Cultures of Tylophora indica

Tylophora indica callus tissue repeatedly subcultured every month on a series of different nutrient media in sequence regenerated roots, shoots and typical, bipolar embryos in vitro from unorganized callus parenchyma. The embryo-like structures developed from somatic cells grew into normal plantlets when isolated and cultured on appropriate milieu of nutrients and hormones bypassing the normal sexual method of reproduction. Likewise, free cells in suspension also passed through embryonic stages reminiscent of development from fertilized egg.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

Microspore development in cultured maize anthers

The present study follows in vivo and in vitro microspore development utilizing an anther culture-responsive maize genotype (Pa91×FR16) and a DNA-specific fluorescent dye (mithramycin). Cultured anthers were sampled at various times and scored for abnormal microspore divisions, multicellular masses, and embryo-like structures. The frequency of abnormal microspore divisions reached a peak during the first 7 days in culture and then declined. The vegetative nucleus was mitotically active in culture with over 50% of the induced microspores exhibiting this type of division. Multicellular masses and embryo-like structures first appeared in the 14 and 25 day samples, respectively. Most of the microspores did not reach the multicellular stage and an even greater mortality occurred during the formation of embryo-like structures.     

Induction medium osmolality improves microspore embryogenesis in wheat and triticale

The objective of this study was to determine the effect of induction medium osmolality on embryogenesis and green plant production in wheat and triticale. Isolated microspores of wheat and triticale were subjected to a range of osmolality (300–500 mOsm kg^?1) using mannitol. In both species, the maximum number of embryo-like structures (ELS) and green plants were obtained at 350 mOsm kg^?1 when the induction medium was supplemented with 9.1 g L^?1 of mannitol. A sharp decline in microspore response was observed at higher osmolality. These results demonstrate the effect of osmolality on induction of ELS and production of green plants indicating that the process of microspore embryogenesis can be improved in wheat and triticale by increasing osmolality of the induction medium to 350 mOsm kg^?1.     

Regeneration of fertile plants from protoplasts derived from embryogenic cell suspensions of barley (Hordeum vulgare L.)

We report regeneration of fertile plants from barley (Hordeum vulgare L. cv. Igri) protoplasts isolated from regenerable suspension cultures initiated from anther-derived embryogenic callus. Plants were routinely regenerated from these suspension cultures, which maintained their regenerative capacity for several months. It was first possible to isolate protoplasts from suspensions after three months of culture and after four months protoplasts capable of division could be isolated. Protoplasts maintained the regenerative capacity of the donor cells and formed embryogenic callus. Green plants were regenerated from protoplast-derived calli, although the proportion of albino plantlets was high. Viable regenerants were transferred to soil and fertile plants were recovered.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 6-BAP 6-benzylaminopurine - PP Protoplasts     

Cell division and differentiation in protoplasts from cell cultures of Glycine species and leaf tissue of soybean

Protoplasts were isolated from cell cultures of G. soja and G. tabacina, respectively. The isolation procedure employed Percoll for the separation and concentration of protoplasts. The cultured protoplasts formed cells which developed into embryo-like structures. Protoplasts also were isolated from leaf tissue of soybean cv. Williams 82. Upon culture, the protoplasts regenerated cell walls and divided to form cell cultures.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid BA|Benzyladenine - BA Benzyladenine     

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.     

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.     

Isolated wheat microspore culture

The use of doubled haploid plants in a wheat breeding program requires an efficient haploid production system. While the techniques for producing doubled haploids from anther culture are well established, those for isolated microspores are complicated and inefficient. Four methods of isolating microspores from anthers (blending, stirring, macerating, and floating) were compared. Isolated microspores were washed and cultured in liquid medium. The effects of pre-isolation mannitol conditioning, cell density, culture dilution, and sucrose centrifugation on microspore viability were evaluated. Isolation by blending gave the highest initial microspore viability (75%). Mannitol conditioning and purification by sucrose centrifugation had a detrimental effect on initial viability. An initial microspore density of 2 × 10⁵ microspores per ml was necessary for continued microspore viability. One hundred and nine haploid or spontancously doubled haploid plants were regenerated from microspores isolated without mannitol conditioning using the blending method. Based on this research, blender isolation with an initial density of 2 × 10⁵ microspores per ml is recommended for isolated microspore culture.Abbreviations LSmean least square mean - MES 2-N-morpholinoethane sulfonic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphtaleneacetic acid