Improvement in efficiency of microspore culture to produce doubled haploid lines of Ethiopian mustard

Factors affecting microspore embryogenesis of Ethiopian mustard (Brassica carinata A. Braun) were evaluated, including flower bud length, pollen developmental stage, and microspore density. An embryogenic frequency of 300 embryos per Petri plate was observed with NLN (Nitsch-Lichter-Nitsch) medium supplemented with 13% sucrose, 3.0–3.4-mm-long buds, and a plating density of 65,000 microspores/ml. About 65% of the microspores from buds 3.0–3.4-mm long were at the late uninucleate stage. Microspore-derived embryos were successfully transferred to solid medium for germination. After 4 wk, the resulting plantlets were transplanted to a soilless potting mixture and grew well under greenhouse conditions.     

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.     

Plasticity in Cell Division Patterns and Auxin Transport Dependency during in Vitro Embryogenesis in Brassica napus

In Arabidopsis thaliana, zygotic embryo divisions are highly regular, but it is not clear how embryo patterning is established in species or culture systems with irregular cell divisions. We investigated this using the Brassica napus microspore embryogenesis system, where the male gametophyte is reprogrammed in vitro to form haploid embryos in the absence of exogenous growth regulators. Microspore embryos are formed via two pathways: a zygotic-like pathway, characterized by initial suspensor formation followed by embryo proper formation from the distal cell of the suspensor, and a pathway characterized by initially unorganized embryos lacking a suspensor. Using embryo fate and auxin markers, we show that the zygotic-like pathway requires polar auxin transport for embryo proper specification from the suspensor, while the suspensorless pathway is polar auxin transport independent and marked by an initial auxin maximum, suggesting early embryo proper establishment in the absence of a basal suspensor. Polarity establishment in this suspensorless pathway was triggered and guided by rupture of the pollen exine. Irregular division patterns did not affect cell fate establishment in either pathway. These results confirm the importance of the suspensor and suspensor-driven auxin transport in patterning, but also uncover a mechanism where cell patterning is less regular and independent of auxin transport.     

Characterization of polarity development through 2- and 3-D imaging during the initial phase of microspore embryogenesis in Brassica napus L

Isolated microspores of B. napus in culture change their developmental pathway from gametophytic to sporophytic and form embryo-like structures (ELS) upon prolonged heat shock treatment (5 days at 32 °C). ELS express polarity during the initial days of endosporic development. In this study, we focussed on the analysis of polarity development of ELS without suspensor. Fluorescence microscopy and 3-D confocal laser scanning microscopy (CLSM) without tissue interfering enabled us to get a good insight in the distribution of nuclei, mitochondria and endoplasmic reticulum (ER), the architecture of microtubular (MT) cytoskeleton and the places of 5-bromo-2′-deoxy-uridine (BrdU) incorporation in successive stages of microspore embryogenesis. Scanning electron microscopy (SEM) analysis revealed, for the first time, the appearance of a fibrillar extracellular matrix-like structure (ECM-like structure) in androgenic embryos without suspensor. Two types of endosporic development were distinguished based upon the initial location of the microspore nucleus. The polarity of dividing and growing cells was recognized by the differential distributions of organelles, by the organization of the MT cytoskeleton and by the visualization of DNA synthesis in the cell cycle. The directional location of nuclei, ER, mitochondria and starch grains in relation to the MTs configurations were early polarity indicators. Both exine rupture and ECM-like structure on the outer surfaces of ELS are supposed to stabilize ELS's morphological polarity. As the role of cell polarity during early endosporic microspore embryogenesis in apical–basal cell fate determination remains unclear, microspore culture system provides a powerful in vitro tool for studying the developmental processes that take place during the earliest stages of plant embryogenesis.     

Isolated microspore culture of oat (Avena sativa L.) for the production of doubled haploids: effect of pre-culture and post-culture conditions

The production of doubled haploid (DH) plants from microspores is an important technique used in plant breeding programs and basic research. Although doubled haploidy efficiencies in wheat and barley are sufficient for breeding purposes, oat (Avena sativa L.) is considered recalcitrant. The objective of this project was to develop a protocol for the production of microspore-derived embryos of oat and further develop these embryos into fertile DH plants. A number of experiments were conducted evaluating the factors influencing microspore embryogenesis, i.e. donor plant conditions, pretreatments, media composition, and culture conditions. The initial studies yielded little response, and it was not until high microspore densities (10⁶ microspores/mL and greater) were used that embryogenesis was achieved. Depending on the treatment, yields of over 5,000 embryos/10⁶ microspores were obtained for breeding line 2000QiON43. The doubled haploidy protocol includes: a 0.3 M mannitol pretreatment of the tillers for 7 days, culture in W14 basal medium with a pH of 6.5–7.5, a microspore density of 10⁶ microspores/mL, and continuous incubation at 28 °C incubation. The resulting embryos observed after 28 days were plated onto solidified W14 medium with 0.8 or 1.0 g/L activated charcoal. A colchicine treatment of 0.2 % colchicine for 4 h resulted in conversion of 80 % of the plants from haploid to DH. This protocol was successful for the production of oat microspore-derived embryos and DH green plants with minimal albinism. DH seed was produced and planted for evaluation in a field nursery.     

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.     

Efficient induction of microspore embryogenesis using abscisic acid,jasmonic acid and salicylic acid in Brassica napus L

The stress hormones abscisic acid (ABA), jasmonic acid (JA) and salicylic acid (SA) play an important role in the regulation of physiological processes and are often used in tissue culture to promote somatic embryogenesis and to enhance the quality of somatic embryos. Despite many studies on Brassica napus microspore culture, the effects of stress hormones (ABA, JA and SA) on microspore embryogenesis are not well explored. In this study, the effects of three incubation periods (6, 12 and 24 h) at different levels of ABA, JA and SA (0, 0.2, 0.5, 1.0, 2.0 and 5.0 mg l^?1) on microspore embryogenesis of rapeseed (B. napus L.) cv. ‘Regent’ were investigated. ABA (0.5 mg l^?1 for 12 h) enhanced microspore embryogenesis by about threefold compared with untreated cultures and increased normal plantlet regeneration by 68 %. ABA treatment also effectively reduced secondary embryo formation at all concentrations tested but enhanced callusing at high levels, for example 67 % at 1.0 mg l^?1 for 24 h. Highest embryo yield (286.0 embryos Petri dish^?1) was achieved using 1.0 mg l^?1 JA for 24 h and highest normal plantlet regeneration (54 %) was observed in cultures exposed to 0.5 mg l^?1 JA for 12 h. JA (5.0 mg l^?1 for 24 h) also reduced the germination of microspore-derived embryos on regeneration medium by 21 %. SA at 0.2 and 0.5 mg l^?1 for 6 h increased microspore embryogenesis (184.0 and 193.4 embryos Petri dish^?1) relative to the control (136.2 embryos Petri dish^?1). However, SA did not improve normal regeneration, secondary embryo formation or callusing. Microspore embryogenesis and plant regeneration could be improved by ABA, JA as well as SA when the appropriate level and duration of incubation were selected.     

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.     

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     

Microspore embryogenesis induced through in vitro anther culture of almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis> Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25?±?1?°C, without the hot thermal shock at 35?±?1?°C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.     

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     

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     

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.     

Plant growth environment effects on rapeseed microspore development and culture : a flow cytometric study

The influence of donor plant growth conditions on microspore embryogenesis in rapeseed (Brassica napus) was studied for plants grown at 23/18°C (16/8 hours) under continuous light, 23/18°C (16/8 hours) with a light/dark (16/8 hours) cycle, 15/12°C (16/8 hours) under continuous light and 15/12°C (16/8 hours) with a light/dark (16/8 hours) cycle. Significantly higher embryo yields were obtained from microspore cultures initiated from donor plants grown at 15/12°C instead of 23/18°C. Flow cytometric measurements of the microspores isolated from 2.5- to 5.0-millimeter buds showed that the microspores isolated from low-temperature-grown plants had significantly lower log 90-degree light scatter to forward angle light scatter and log 90-degree light scatter to time of flight ratios than those isolated from high-temperature-grown plants, suggesting that the former are more translucent than the latter. Thus, the effect of donor plant growth temperature on microspore embryogenesis may be mediated by a change in the physiology of the microspore cell, which results in the reduction of its cytoplasmic granularity and/or exine density.     

Effects of genotypes and culture conditions on microspore embryogenesis and plant regeneration in several subspecies of Brassica rapa L.

A number of factors influencing microspore embryogenesis and plant regeneration were examined in five subspecies (rapa, oleifera, niposinica, perviridis, broccoletto) of B. rapa. Addition of 6-benzylaminopurine (BA) in 1/2 NLN-10 medium improved the embryo yield by 2?C12 fold. Addition of activated charcoal (AC) in the medium was not effective for microspore embryogenesis. Moreover, AC canceled the positive effect of BA, when the medium containing both BA and AC was used. Of 24 genotypes examined for microspore embryogenesis, 22 genotypes of all five subspecies produced embryos ranging from 0.02 to 15.0 per 2?×?10⁵ microspores, but two genotypes were not responsive. Low temperature pretreatment of flower buds significantly improved the microspore embryogenesis. When cotyledonary embryos were subcultured on a filter paper placed on top of 0.8?% agar-solidified B5-2 medium and 1.6?% agar B5-2 medium, plant regenerations were increased 4?C8 fold compared to 0.8?% agar medium. The ploidy levels of regenerated plants in three genotypes were determined by flow cytometry, revealing that 66?C100?% of them were diploid. The results enable the advancement of breeding programs and genetic studies in B. rapa.     

Identification of potentially embryogenic microspores in Brassica napus

Studies were undertaken with Brassica napus L. cv. Topas to identify buds containing microspores predisposed to embryogenesis in vitro and to investigate bud and microspore development in relation to this process. No significant correlation was found between the final embryo number and bud components. There appears to be a developmental window of less than 8 h duration during which microspores are very likely to form embryos: over 70% of the microspores can undergo division and up to 70% of these can form embryos. Embryos were mainly obtained from late uninuucleate to early binucleate microspores: the former contained mainly a G2 or M phase nucleus located at the microspore periphery and the latter a generative nucleus (associated with the intine) and a vegetative nucleus. Observations indicated that only the vegetative nucleus contributed to embryo formation. The first embryogenic division occurred between 8 and 16 h for uninucleate- and between 8 and 48 h for binucleate-derived embryos.     

Effect of boron on microspore embryogenesis and direct embryo to plant conversion in <Emphasis Type="Italic">Brassica napus</Emphasis> (L.)

The NLN-medium has been successfully used, since 1982, for microspore culture in Brassica napus and other Brassica species. Changes to the media composition were restricted to carbohydrate and nitrogen sources and growth regulators while micro-nutrients have not been optimized. The NLN-medium contains boron at a concentration of 162 µM. Boron is required for diverse physiological and metabolic processes in the cell. This study investigated the effect of seven- and 13-fold increased boron concentration on the induction of embryos in microspore cultures of four genotypes of B. napus. A significant improvement of microspore embryogenesis was achieved by both enhanced boron concentrations in the NLN medium. No effect on the regeneration of embryo to plant conversion was observed.     

Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern and Coss)

Summary The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5±1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 nm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.     

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.