Spontaneous chromosome doubling results from nuclear fusion during in vitro maize induced microspore embryogenesis

A multidisciplinary study was carried out to analyse the chromosome doubling process during the early stages of in vitro maize microspore embryogenesis. The main stages (microspore derivatives) that were formed in the course of the culture were analysed. Chromosome number was determined from squashed cells, and DNA content was measured by cytometry. In parallel, an ultrastructural analysis of the microspore derivatives demonstrated the occurrence of a nuclear fusion process. It seems likely that nuclear fusion ensures chromosome doubling at early stages of induced microspore embryogenesis. It occurs precisely at the 5/7 day stage in the embryonic domain and probably leads to polyploidy in the endosperm domain of the microspore derivatives. As a conclusion a scheme summarises the results and proposes an interpretation of the sequence of chromosome doubling events during early maize microspore embryogenesis. Understanding of this process will be important for future efforts to increase the percentage of homozygous plants for crop improvement.Communicated by G. Almouzni     

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     

Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production

An isolated microspore culture provides an excellent system for the study of microspore induction and embryogenesis, provides a platform for an ever-increasing array of molecular studies, and can produce doubled haploid (DH) plants, which are used to accelerate plant-breeding programs. Moreover, isolated microspore cultures have several advantages over anther culture, wherein presence of the anther walls can lead to the development of diploid, somatic calli and plants. Although protocols for isolated microspore culture vary from laboratory to laboratory, the basic steps of growing donor plants, harvesting floral organs, isolating microspores, culturing and inducing microspores, regenerating embryos, and doubling the chromosomes, remain the same. Over the past few years, a large proportion of the research reports on isolated microspore culture have focused on cereal and Brassica species. For some of these species, isolated microspore culture protocols are well established and routinely used in laboratories around the world for developing new varieties, as well as for basic research in areas such as genomics, gene expression, and genetic mapping. Although these species are considered highly responsive to microspore culture, improvements in efficiency are still being made. However, with many species, isolated microspore culture is simply not yet efficient enough at producing DH plants to be cost-effective for breeding programs. There has been a recent resurgence of haploidy research with response being reported in some species once considered recalcitrant. Future research programs aimed at elucidating pathways involved in microspore induction and embryogenesis will be of benefit, as will novel approaches to improve the efficiency of microspore culture for DH production. With many species, anther culture has proven to be more effective than isolated microspore culture, necessitating more research to clarify the contribution of the anther wall to embryogenesis. The development of molecular markers for use in determining the gametic origin of regenerated plants, irrespective of their ploidy, would also be beneficial. In this review, we aim to provide an overview of the basic isolated microspore culture protocol with an emphasis on recent progress in several crop species.     

甘蓝型油菜双单倍体高效诱导技术体系的优化研究

本研究通过对甘蓝型油菜小孢子培养过程影响成胚成苗效果效率最关键的因素,包括取材时期、选蕾大小、培养密度、启始诱导培养基和加倍处理等技术环节及其流程的优化研究,建立了不受大田、温室和人工气候室生长条件影响和基因型的限制,规模化大批量获得甘蓝型油菜双单胚体的诱导技术体系,为甘蓝型油菜基础研究和生物技术育种奠定基础。     

Effects of brassinosteroids on microspore embryogenesis in <Emphasis Type="Italic">Brassica</Emphasis> species

Summary Experiments were conducted to determine the effects of brassinosteroids on microspore embryogenesis in Brassica species. Two compounds, 24-epibrassinolide (EBR) and brassinolide (BL), were evaluated. An increase in embryogenesis was observed in all Brassica napus lines evaluated, including Topas 4079 and several recalcitrant cultivars: Garrison, Westar, and Allons. Microspore embryogenesis, calculated as the number of embryos at 21 d of culture, was increased in the recalcitrant cultivars up to 12 times that of the control. An increase in microspore embryogenesis was also observed for B. juncea when EBR or BL was added to the culture medium. In constrast, no significant increases in embryogenesis was observed for several other Brassica species evaluated (i.e. B. carinata, B. nigra, and B. rapa). The addition of brassinosteroids to the induction media did not affect the subsequent conversion of the embryos to plantlets, but did appear to influence chromosome doubling.     

Stress-related variation in antioxidative enzymes activity and cell metabolism efficiency associated with embryogenesis induction in isolated microspore culture of triticale (<Emphasis Type="Italic">x Triticosecale</Emphasis> Wittm.)

Isolated microspore cultures of two spring triticale (x Triticosecale Wittm.) cultivars were used to examine the effect of various stress treatments (either high—32°C or low—5°C temperature with or without nitrogen/carbohydrate starvation) applied to excised anthers on the effectiveness of microspore embryogenesis induction. To quantify the effects of pretreatment conditions, the activity of antioxidative enzymes (catalase, peroxidase and superoxide dismutase) together with respiration rate and heat emission were measured. It was observed that heat shock treatment applied as the only one stress factor increased the activity of antioxidative enzymes which suggests intensive generation of reactive oxygen species. Such pretreatment effectively triggered microspore reprogramming but drastically decreased microspore viability. After low temperature treatment, the activity of antioxidative enzymes was similar to the control subjected only with the stress originated from the transfer to in vitro culture conditions. This pretreatment decreased the number of microspores entering embryogenesis but sustained cell viability and this effect prevailed in the final estimation of microspore embryogenesis effectiveness. For both, low- and high-temperature treatments, interaction with starvation stress was beneficial increasing microspore viability (at 5°C) or efficiency of embryogenesis induction (at 32°C). The latter treatment significantly reduced cell metabolic activity. Physiological background of these effects seems to be different and some hypothetical explanations have been discussed. Received data indicate that in triticale, anther preculture conditions could generate oxidative stress and change the cell metabolic activity which could next be reflected in the cell viability and the efficiency of microspore embryogenesis.     

Maintenance of gametophytic development after symmetrical division in tobacco microspore culture

Isolated tobacco (Nicotiana tabacum L.) microspores maturing in vitro can be induced to undergo symmetrical divisions, instead of the normal asymmetrical first pollen mitosis, by addition of anther extracts to the culture medium. The two daughter cells in symmetrically divided pollen resemble vegetative pollen cells in cytological characteristics, nuclear size and chromatin condensation, are separated by a cell wall and remain viable during in vitro maturation. After transfer to a germination medium, only one of the two vegetativelike cells forms a pollen tube in vitro. Therefore, apparently normal gametophytic development can be maintained after symmetrical microspore division. These results are discussed in relation to current models for induction of microspore embryogenesis.     

Nuclear fusion in cultured microspores of barley

Fusion of the generative and vegetative nuclei physically separated by a wall has been observed in cultured microspores of barley. The generative cell appears to play an active role in fusion as it elongates toward the vegetative nucleus, becomes detached from the microspore wall, and finally completely encloses the vegetative nucleus. The generative cell wall disappears before nuclear fusion takes place. Since these events have been known to occur during pollen development in vivo, it is hypothesized that the occurrence of nuclear fusion in cultured microspores is the result of continued expression of the genes for gametophytic development.     

Changes in pectins and MAPKs related to cell development during early microspore embryogenesis in Quercus suber L

The occurrence and significance of changes in cell wall components and signalling molecules has been investigated during early microspore embryogenesis in cork oak (Quercus suber L.) in relation to cell proliferation and cell differentiation. Microspore embryogenesis has been induced in in vitro anther cultures of Q. suber by the application of a stress treatment of 33 degrees C. After the treatment, microspores at the responsive developmental stage of vacuolate microspore switched towards proliferation and the embryogenesis pathway to further produce haploid plantlets. Ultrastructural and immunocytochemical analysis revealed changes in cell organisation after induction at different developmental stages, the cellular features displayed being in relation to the activation of proliferative activity and the beginning of differentiation in young and late proembryos. Immunogold labelling with JIM5 and JIM7 antibodies showed a different presence of pectin and level of its esterification in cell walls at different developmental stages. Non-esterified pectins were found in higher proportions in cells of late proembryos, suggesting that pectin de-esterification could be related to the beginning of differentiation. The presence and subcellular distribution of Erk 1/2 MAPK homologues have been investigated by immunoblotting, immunofluorescence and immunogold labelling. The results showed an increase in the expression of these proteins with a high presence in the nucleus, during early microspore proembryos development. The reported changes during early microspore embryogenesis are modulated in relation to proliferation and differentiation events. These findings provided new evidences for a role of MAPK signalling pathways in early microspore embryogenesis, specifically in proliferation, and would confer information for the cell fate and the direction of the cell development.     

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

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

Pathways to doubled haploidy: chromosome doubling during androgenesis

Production of doubled haploid (DH) plants through androgenesis induction is a promising and convenient alternative to conventional selfing techniques for the generation of pure lines for breeding programs. This process comprises two main steps: induction of androgenesis and duplication of the haploid genome. Such duplication is sometimes indirectly induced by the treatments used to promote androgenic development. But usually, an additional step of direct chromosome doubling must be included in the protocol. Duplication of the haploid genome of androgenic individuals has been thought to occur through three mechanisms: endoreduplication, nuclear fusion and c-mitosis. In this review we will revise and analyze the evidences supporting each of the proposed mechanisms and their relevance during androgenesis induction, embryo/callus development and plant regeneration. Special attention will be devoted to nuclear fusion, whose evidences are accumulating in the last years.     

Stresses applied for the re-programming of plant microspores towards in vitro embryogenesis

Microspore embryogenesis is the most commonly used method to produce doubled haploids. It is based on the ability of a single haploid cell, the microspore, to de-differentiate and regenerate into a whole plant after being exposed to stresses, such as low or high temperatures, carbon starvation and colchicine. Some stresses such as temperature treatments and carbon starvation have been used with success in many plant species, whereas others such as colchicine had limited application in a few species. Reports on the application of whole plant treatments with feminizing agents on inflorescences and buds are scarce. Furthermore, the technical means to apply some stresses such as γ-irradiation are not readily available. Recently, novel stresses such as pH, inducer chemicals, carrageenan oligosaccharides and heavy metals were reported to induce microspore embryogenesis. It remains to be seen, however, whether these stresses are effective in a wider range of species. Finally, pretreatment of cultured cells with high concentrations of 2,4-D efficiently induces somatic embryogenesis in several species (carrot, alfalfa). However, reports on the use of this particular chemical stress are not available in microspore embryogenesis. The paper presented here gives an overview of various stresses and mechanisms of action of these stresses in inducing microspore embryogenesis.     

Efficient production of doubled haploid plants by immediate colchicine treatment of isolated microspores in winter Brassica napus

The effect of colchicine on embryogenesis induction and chromosomedoubling during microspore culture was evaluated in two F₁ hybridsofwinter oilseed rape (Brassica napus L.). Colchicinetreatment (50 and 500 mg/L) of isolated microspores during thefirst 15 h in culture stimulated embryogenesis and produced large amounts ofhealthy-looking embryos. These normal embryos germinated well at 24°C after being transferred to solid regeneration medium and aninitial period of low temperature (2 °C) for 10 days, andcoulddirectly and rapidly regenerate vigorous plants. A high doubling efficiency of84–88% was obtained from 500 mg/L colchicine treatment for15h with low frequency of polyploid and chimeric plants. Acolchicinetreatment duration of 6 h was less effective on embryogenesis anddoubling efficiency. The present experiment also showed that changing of induction medium 15h after microspore isolation produced higher spontaneous doublingefficiency, as compared with medium change 6 h after isolation.     

Effects of light conditions and 2,4-D concentration in soybean anther culture

The morphogenic response of anther walls and connective tissue is the greatest obstacle to androgenesis in soybean anther culture. Whereas induction to microspore embryogenesis occurs in the dark in almost all plant species, soybean anthers have been cultured under light. In an attempt to establish culture conditions that simultaneously stimulate microspore embryogenesis and inhibit epidermal and connective cell proliferation, the effect of light and two 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations (2 and 10 mg l^–1) on the induction process was investigated. Higher 2,4-D concentration speeded up microspore plasmolysis and did not improve androgenesis. Callogenesis and embryogenesis induction from sporophytic cells were significantly lower in the dark, and some microspores showed major alterations in the sporoderm. Auxin 2,4-D and induction under light contributed to the morphogenic response of the anther walls and connective tissue under the conditions previously recommended to trigger microspore embryogenesis.     

Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis

The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids `germinate' to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research.     

Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.     

Early markers of in vitro microspore reprogramming to embryogenesis in olive (Olea europaea L.)

Microspore embryogenesis to form haploid and double-haploid embryos and regenerated plants is an efficient method of producing homozygous lines for crop breeding. In trees, the process is of special interest since classical methods are impractical in many cases, as in Olea europaea L. Recently, a convenient method has been developed for microspore embryogenesis induction by stress in olive isolated microspores in vitro cultures. In the present work, the switch of the microspore developmental pathway and the formation of microspore-derived multicellular proembryos have been achieved and a cytochemical and immunocytochemical analysis was performed in the early stages. The young microspore proembryos displayed defined features different to both, the in vivo gametophytic, and the in vitro non-responsive microspores. Reprogrammed microspores showed an absence of starch, the occurrence of a first symmetrical division and cytokinesis, the presence of an abundant ribosomal population, and changes in cellulosic and pectic cell wall components which constituted early markers of the embryogenic microspore process. They provided new insights on the molecular and cellular events associated with the microspore reprogramming of woody plants, and specifically in olive, providing interesting knowledge which could guide future selection and regeneration strategies in this fruit tree of high economic interest.     

植物游离小孢子胚胎形成机理

植物小孢子胚胎的形成是基于具有单套染色体的小孢子在外界胁迫条件下通过脱分化和再生而形成一个完整植株的过程,是产生双单倍体的常用方式.它体现了植物细胞的全能性,为植物组织培养和胚胎发育的基础研究提供了一个独特的模式.从细胞学水平、代谢水平和分子水平3个方面综述在植物游离小孢子胚胎形成机制方面已取得的研究进展,并提出目前尚待解决的问题,以及对未来的展望,为进一步研究小孢子胚胎发生机制提供理论依据和奠定技术基础.     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores.