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.     

Isolated microspore culture of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): induction of androgenesis and cytological analysis of early haploid divisions

Routine production of haploid plants has not been reported for any legume, despite the major role these species play in sustainable farming systems and human nutrition. It is within this context that we report a protocol for the induction of haploid development in chickpea (Cicer arietinum L.) using isolated microspore culture. The cultivars “Rupali”, “Narayen”, and “Kimberley Large” were identified as responsive to isolated microspore culture. Flower bud length and microspore developmental stage were correlated for these cultivars. Depending on the cultivar, buds 2.85–3.5 mm in length contained uninucleate microspores. Microspores from donor plants grown in winter and spring were more responsive than those grown in summer. A cold treatment (4°C) of between 24 and 48 h enhanced microspore response in winter- and spring-grown material but was not effective in summer-grown material. A medium developed by the authors was effective for microspore induction and early-stage embryo development. The addition of hormones to this medium was promotive of microspore induction in winter- and spring-grown material, but not in summer-grown material. The initial haploid division predominantly occurred via symmetrical division of the vegetative nucleus. Further research is under way to convert pro-embryos into plants.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).     

Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus

Microspores of Brassica napus L. cv. Topas, undergo embryogenesis when cultured at 32.5 °C for the first 18–24 h and then at 25 °C. The first division in heat-treated microspores is a symmetric division in contrast to the asymmetric division found after the first pollen mitosis in-planta or in microspores cultured continuously at 25 °C. This asymmetric division is unique in higher plants as it results in daughter cells separated by a non-consolidated wall. The cytoskeleton has an important role in such morphological changes. We examined microtubule (MT) organization during the first 24 h of heat induction in the embryogenic B. napus cv. Topas and the non-embryogenic B. napus breeding line 0025. Preprophase bands (PPBs) of MTs appeared in cv. Topas microspores in late uninucleate microspores and in prophase figures after 4–8 h of heat treatment. However, more than 60% of the PPBs were not continuous bands. In contrast, PPBs were never observed in pollen mitosis; MT strands radiated from the surface of the nuclear envelope throughout microspore maturation to the end of prophase of pollen mitosis I, during in-planta development and in microspores cultured at 25 °C. Following 24 h of heat treatment, over 95% of the microspores appeared to have divided symmetrically as indicated by the similar size of the daughter nuclei, but only 7–16% of the microspores eventually formed embryos. Discontinuous walls were observed in more than 50% of the divisions and it is probable that the discontinuous PPBs gave rise to such wall abnormalities which may then obstruct embryo development. Preprophase bands were not formed in heat-treated microspores of the non-embryogenic line 0025 and the ensuing divisions showed discontinuous walls. It is concluded that the appearance of PPBs in heat-induced microspores marks sporophytic development and that continuous PPBs are required for cell wall consolidation and embryogenesis. It follows that induced structures with two equally condensed nuclei, do not necessarily denote symmetric divisions. Received: 22 October 1998 / Accepted: 28 November 1998     

Effects of aluminum on DNA synthesis, cellular polyamines, polyamine biosynthetic enzymes and inorganic ions in cell suspension cultures of a woody plant, Catharanthus roseus

Individual buds of Brassica napus cv. Topas, near the first pollen mitosis, were used for microspore culture. Bud and petal lengths were recorded. Microspores isolated from the individual buds were plated and small samples were fixed for cytology. Following embryo induction and three weeks of culturing, numbers of embryos were scored. Bud and petal lengths did not accurately indicate which buds would supply microspores that would form embryos at high frequencies. Fluorescence microscopy was used to examine nuclei stained with Hoechst 33258 and vacuolar morphology of microspores was revealed by the weaker fluorescence due to glutaraldehyde fixation. Following isolation, nuclear and vacuolar characteristics were used to stage the microspores as miduninucleate, late uninucleate vacuolate, late uninucleate, mitotic, or binucleate. The relationship of developmental stage to the frequency of microspore-derived embryos was evaluated. A classification scheme was developed which uses the relative proportions of microspores at each of the stages to identify microspore isolations that would form embryos at high frequencies. It was found that when 1 to 87% of the isolated microspores were binucleate, 21.4 ± 3.0% of the viable microspores developed into embryos. This was a significant ( P < 0.001) increase over the other 3 classes. The ability to select highly embryogenic microspore isolations is of great advantage for developmental cell biology studies.     

Mapping of quantitative trait loci for microspore embryogenesis-related traits in the oilseed rape doubled haploid population DH4069 × Express 617

Oilseed rape (Brassica napus L.) is among dicotyledonous plants, a model species for microspore embryogenesis. Tremendous differences exist among oilseed rape genotypes in their embryogenic response and direct embryo to plant conversion. Despite some attempts to identify relevant genes, the genetic basis of these traits remains largely unknown. The objective of this work was to develop and to provide to the scientific community a doubled haploid (DH) population derived from a cross of the reported highly embryogenic genotype DH4079 and the low embryogenic inbred line Express 617. A population of 198 DH-lines was generated and genotyped with the Brassica 60 K Illumina Infinium? SNP array. The parental and the F1 genotypes as well as between 81 and 107 DH-lines were characterized for their number of microspores, number of microspore-derived embryos, embryo survival rate, direct embryo to shoot conversion, and related traits. The results obtained for the F1 genotype were mostly in between the two parents. SNP markers in the DH population showed to 49% distorted segregation and of those 63% were in favor of DH4079. Significant genotypic differences were found for all traits and heritabilities ranged from 66 to 88%. Together, 13 quantitative trait loci (QTL) for the different traits were identified on linkage groups A01, A02, A05, A10, C04, and C06, and candidate genes were identified within their QTL confidence intervals.     

Development of asparagus microspores in vivo and in vitro is influenced by gametogenic stage and cold treatment

Summary Development of asparagus microspores in cold-treated buds of varying sizes and shed microspores from these buds in in vitro culture were observed cytologically for the G459 genotype. Before cold pretreatment, more than 75% of the microspores in flower buds of the 1.4–1.6, 1.7–1.9, 2.0–2.2, 2.3–2.5, and 2.6–2.8 mm size classes were at the early-, mid-, late-uninucleate, early-, and late-binucleate stages, respectively. After 7 d in cold treatment, percentages of microspores at different stages changed in all flower buds. Most notable was the appearance of binucleate microspores resulting from symmetric rather than asymmetric division. For flower buds of 1.7–1.9, 2.0–2.2, and 2.3–2.5 mm size classes, 4.9%, 27.2%, and 11.4% of the microspores had divided symmetrically, respectively. When microspores from buds of each size category were cultured in androgenesis induction medium, only microspores completing symmetric pollen mitosis I during cold treatment were observed to divide further, and calluses were only obtained from microspores of flower bud size classes where symmetric divisions were observed after several days of cold treatment. Significant correlations existed among microspore callus yield, the percentage of late-uninucleate microspores in vivo before cold treatment, and the frequency of symmetric pollen mitosis I after 7 d of cold treatment. Consequently, asparagus microspore androgenesis may occur through one developmental pathway, where a symmetric first mitotic division is a prerequisite for continued development.     

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.     

Selection of Brassica napus L. embryogenic microspores by flow sorting

Flow cytometry can be used to select and sort microspore subpopulations of Brassica napus cv. Topas. Data obtained from embryogenic microspore populations were used to identify potentially embryogenic microspores from developmentally heterogeneous microspore populations based on differences in forward light scatter and green autofluorescence. Culture enrichment for embryogenic microspores is possible. Frequencies of 8 and 14% microspore embryogenesis were obtained when selected 16 h and 72 h after culture initiation. This represents 5- and 13-fold increase in microspore embryogenesis compared to non-sorted controls.     

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.     

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.     

Development of microsporesin vivo andin vitro inBrassica napus L.

Summary Populations of highly homogeneous uninucleate and binucleate microspores ofBrassica napus cv. Topas were obtained by bud selection and percoll fractionation. The development of the uninucleate and the binucleate microspores in culture was compared to thosein vivo using the fluorochrome DAPI to stain DNA. The major developmental pathway of the uninucleate microsporesin vitro resulted in embryo formation. The characteristic of this pathway was that the first division produced two diffusely stained nuclei and subsequent divisions gave rise to a multinucleate embryoid. The second pathway which occurred in a small number of the uninucleate microspores led to callus formation. The majority of the binucleate microsporesin vitro followed the developmental pattern of their counterpartsin vivo and were not embryogenic. The embryogenic binucleate microspores produced embryos through the divisions of the vegetative nucleus.Plant Research Centre Contribution # 1147     

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     

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.     

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.     

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.     

Efficient microspore embryogenesis in wheat (Triticum aestivum L.) induced by starvation at high temperature

We have established an efficient method to induce embryo formation from isolated wheat (Triticum aestivum L.) microspores. Culture of excised anthers under starvation and heat shock conditions induced the formation of embryogenic microspores at high frequency in nine Austrian winter wheat genotypes, including cultivars that had been considered as recalcitrant in anther culture. Percoll gradient centrifugation of the mechanically isolated microspores allowed us to obtain homogeneous populations of embryogenic microspores in all genotypes which, after transfer to a rich medium containing immature ovaries for conditioning, divided and produced globular embryos. Thousands of embryos were produced in one petri dish. Many of these embryos developed into plantlets after transfer to a solid medium without ovaries.