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.     

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.     

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.     

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.     

An efficient androgenic embryogenesis and plant regeneration method through isolated microspore culture in timothy (Phleum pratense L.)

A method employing isolated microspore culture was established for the androgenic embryogenesis of timothy (Phleum pratense L). Embryos/calli were obtained and green plants regenerated. The induction medium was PG-96 (1.0 mg l⁻¹ 2,4-D, 0.1 mg l⁻¹ Kinetin) supplemented with 6% maltose monohydrate. Timothy microspore culture was genotype-dependent, among 12 genotypes, 6 produced embryos/calli and 4 produced green plants. Macerating the spikes with a blender and purifying the microspores at a mannitol/maltose monohydrate interface gave a relatively high percentage of cell vitality. The optimum microspore developmental stage was from the very late uninucleate stage to the binucleate stage. Heat shock promoted the initiation of microspore culture. Over 150 regenerated green plants were obtained; in a random sample of 32 of these 65.6% were doubled haploids (6n=42). Albinism was a problem in plant regeneration (9.3–22%). This paper is the first to describe timothy androgenic embryogenesis by isolated microspore culture. Received: 9 September 1999 / Revision received: 6 December 1999 / Accepted: 13 December 1999     

Messenger-RNA and protein changes associated with induction ofBrassica microspore embryogenesis

Brassica napus L. microspores at the late uninucleate to early binucleate stage of development can be induced in vitro to alter their development from pollen to embryo formation. High temperatures or other stress treatments are required to initiate this redirection process. The critical period for induction of microspore embryogenesis is within the first 8 h of temperature-stress imposition. During this period, which precedes the first embryogenic nuclear division, the process regulating the induction and sustainment of microspore embryogenesis is activated. A number of mRNAs and proteins, some of them possibly heat-shock proteins, appear in microspores during the commitment phase of the induction process.Abbreviations SDS sodium dodecyl sulfate - PAGE polyacrylamide gel electrophoresis     

Microsporogenesis and pollen formation in cassava

This article describes the complete microsporogenesis and pollen formation in cassava (Manihot esculenta Crantz) at the various developmental stages (pollen mother cell, meiosis, tetrads, early free spore, mid uninucleate, late uninucleate, binucleate and mature pollen grain). Light microscopy, transmission electron microscopy and confocal laser scanning microscopy were used for the study. Floral bud size and other inflorescence characteristics were correlated with specific stages of the microspore development. This association allows a rapid selection of floral buds with similar microspore developmental stages, useful when a large number of homogeneous cells are needed for analysis and for in vitro induction of androgenesis. This article also compares methods for digestion the exine wall in microspores.     

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.     

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     

Variability and Bimodal Distribution of Size in Microspores of Aesculus hippocastanum

Size variability of uninucleate microspores was studied in horse chestnut (Aesculus hippocastanum L.). Microspores were isolated from buds of different size (3, 4, and 5 mm) taken from lower, middle and upper segments of inflorescences. All analyzed buds showed bimodal distribution of microspore size which confirmed the presence of pollen dimorphism. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Increased frequency of dividing microspores and improved maintenance of multicellular microspores of Coffea arabica in medium with coconut milk

The number of dividing microspores of Coffea arabica L. cv. Catuai and Catimor could be drastically increased in microspore media containing 16% (w/v) coconut milk, allowing cell divisions to continue in the microspore and multicellular microspores to survive until day 60. After a cold treatment, the microspores were mechanically isolated prior to cultivation in Murashige and Skoog medium supplemented with sucrose and maltose and (mg l^-1) 2,4-d: 2, BAP: 1 or a combination of kinetin: .5, 2,4-d: .5 and NAA: .5 as stationary suspension at a density of 1,200/ml. The crucial stage during microsporogenesis suitable for in vitro androgenesis proved to be mid uninucleate till early binucleate in flowerbuds with the size of 13–15 mm two to three days before anthesis. The initial steps of androgenesis were determined.Abbreviations BAP 6-benzylaminopurine - 2,4-d 2,4-dichlorophenoxy-acetic acid - NAA 1-naphthaleneacetic acid     

结球甘蓝游离小孢子胚胎发生

以结球甘蓝品种“强夏”为材料进行游离小孢子培养,对与胚胎发生关系密切的因子进行探讨。研究结果表明,在盛花前期取材最适宜;单核晚期至双核期的小孢子才能发育成胚状体;含17%蔗糖的培养液在培养初期有利于小孢子存活;培养3d后胚胎诱导则以14％蔗糖浓度为最好;高浓度（17％）蔗糖培养3d后添加低浓度（11％）蔗糖培养液能大大提高胚胎发生能力,比一直在14％蔗糖培养液培养的提高282．4％,比更新培养液培养的提高126．1％。     

花药培养过程栽培稻花粉不育杂种F1与亲本台中65的细胞学研究

利用活体压片、半薄及超薄切片技术,对栽培稻(Oryza sativa L.)花粉不育杂种F1及育性正常的亲本台中65离体培养前后的小孢子及花药壁进行细胞学研究.结果表明:与台中65相比,杂种F1的花粉小孢子在发育至单核中-晚期,出现比例较高的胞质凝聚小孢子和少量星型小孢子,正常小孢子和淀粉化小孢子比例降低.在离体条件下,胞质凝聚小孢子、星型小孢子、正常小孢子、液泡化小孢子、淀粉化小孢子的发育主要沿着胞质凝聚败育过程、孢子体发育过程、配子体发育过程、液泡化过程和淀粉化过程进行;药壁组织在离体条件下,杂种F1比台中65的绒毡层降解速度快,中层膨大程度高.杂种F1与台中65在离体培养下小孢子发育及药壁细胞学的差异主要是受到S-a座位内等位基因互作及离体培养环境的影响.     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

Efficient male germ line transformation for transgenic tobacco production without selection

Microspores at late uninucleate/early binucleate stages were isolated from flower buds of tobacco (Nicotiana tabacum L.) and in vitro culture methods optimised for their maturation to fully functional viable pollen which, after application to the stigma of emasculated plants in situ, led to the generation of large numbers of seed. Efficient protocols were established for the biolistic introduction of a construct containing a reporter gene and selectable marker into these microspores and hence, after in vitro maturation and in situ fertilisation, for the generation of transgenic plants. Stable transformants of low copy number were generated by this procedure. The efficiency of transformation achieved allows the production of large numbers of transgenic plants without selection, dispensing with the requirement for a selectable marker in plant transgenesis.     

Morphogenesis in Isolated Microspore Cultures of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Androgenesis is a phenomenon in which microspores are made to bypass the sexual pathway and follow the sporophytic mode of development to generate new plants without the intervention of fertilization under specialized in vitro conditions. Microspore culture provides an ideal system, with a large, relatively uniform population of haploid cells, for use in mutant selection, genetic transformation and in studies on the molecular mechanism of induction of androgenesis and embryogenesis. This paper involves a study on establishing a reproducible and efficient protocol for microspore embryogenesis in various varieties of Brassica juncea. The genotype had a pronounced effect on androgenic response in microspore cultures. The cultivar Rajat exhibited the most response, producing around 3500 embryos/100 buds. The microspores of B. juncea cv. PR-45 from ed plants maintained at a day/night temperature of 10 °C/5 °C form embryos with suspensors with varied morphology. The microspore embryos germinated to produce plants with frequencies. These plants exhibited 52% survival and 74% fertility.     

Genetic manipulation of microspores and microspore-derived embryos

Summary Recent advances in plant cell and molecular biology have furthered the genetic manipulation of many plant species and advanced the options for crop improvement. Among the many targets for genetic manipulation, microspores offer several unique advantages: they are haploid, single-celled, and highly synchronized. In many plant species microspores develop into haploid embryos, and eventually haploid and doubled haploid plants, after in vitro anther or microspore culture. This induced in vitro developmental pathway of microspores, termed microspore embryogenesis, can be used to recover individual homozygous plants from microspores and microspore-derived embryos after genetic manipulation such as mutagenesis and gene transfer. The highly efficient microspore embryogenesis system inBrassica napus has been used successfully to obtain various mutants after microspore mutagenesis, and to achieve gene transfer mediated byAgrobacterium tumefaciens. Presented in the Session-in-Depth In Vitro Gametophyte Biology at the 1991 World Congress on Cell and Tissue Culture held in Anaheim, California, June 16–20, 1991.     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.