In vitro androgenesis of triticale in isolated microspore culture

Culture conditions for triticale (X Triticosecale Wittmack) androgenesis were studied using microspore culture. Sporophytic development of isolated triticale microspores in culture is described in five winter hexaploid triticale genotypes. Microspores were isolated using a microblendor, and embryogenesis was induced in modified 190-2 medium both in the presence and absence of growth regulators. The highest induction of microspore embryogenesis was obtained in a growth regulator-free medium. Adventitious embryogenesis was observed during in vitro development of triticale microspores. Albino and green plantlets were regenerated from embryo-like structures. More than 50% of regenerants were albino. In total, 126 green plantlets were produced, transplanted and established in soil. Cytological evidence revealed that 90% of the transplanted regenerants were haploid. This revised version was published online in June 2006 with corrections to the Cover Date.     

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

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

Colchicine-induced microspore embryogenesis in coffee

A protocol for the induction of androgenesis and plant regeneration from C. arabica cv. Caturra isolated microspores in vitro using colchicine pretreatment has been developed. Microspores were mechanically isolated and then carefully purified. Before colchicine pretreatment, microspores were cultured in a semi-solid medium for further develop and regeneration. Different times of colchicine exposure as well as different concentrations were tested. The best androgenic response was found when microspores were precultured in 100 mg l^–1 colchicine for 48 h. The microspore developmental stages responsive to colchicine were late-uninucleated and early binucleated pollen. Flow cytometry and morphological analyses revealed that 95% of regenerated plants were dihaploids (2n=2x=22). However, some doubled dihaploid plants (2n=4x=44) were also obtained, suggesting that not only androgenic induction but also chromosome duplication could be expected as result of colchicine exposure of coffee microspores. This report represents a new approach in the coffee pollen culture, as well as a major step forward to the utilization of haploid technology in coffee breeding.     

Isolated wheat microspore culture

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

Culture conditions for efficient induction of green plants from isolated microspores of barley

Subculture regime and carbohydrate concentration of the medium had a marked effect on the regeneration of green plantlets from mechanically isolated microspores of Hordeum vulgare L. cv. Kymppi. A sevenfold increase in the yield of green plants was obtained by shortening the suspension culture time of the developing proembryo mass from 4 to 3 weeks. A further twofold increase was obtained by increasing the maltose concentration of the microspore isolation medium and of the culture medium. Under optimal conditions, a mean of 169±97 green plants per spike were regenerated.     

Anther culture of <Emphasis Type="Italic">Lupinus angustifolius</Emphasis>: callus formation and the development of multicellular and embryo-like structures

Androgenesis is an important technique to generate double haploid plants. Anther and microspore cultures are the methods to induce haploid embryogenesis. For culture initiation, it is necessary to select anthers with the appropriate developmental stage of microspores. For lupins, limited reports about the establishment of initial cultures for androgenesis are available. In this study, different parameters of anther culture of three genotypes of Lupinus angustifolius were investigated. For all genotypes, a considerable correlation was observed between the buds and the anthers, depending on their location in the inflorescences. Buds from the central segment of inflorescences had yellowish green anthers that contained the maximum number of microspores at uninucleate stage. Cytological investigation shows that the anthers containing these microspores were the most responsive to induction. Two types of developmental pathways were observed for microspores. In case of cold pre-treated and untreated inflorescences, microspores developed into multicellular and embryo-like structures, respectively. Effects of different factors showed significant differences among: genotypes, pre-treatment, growth regulators (GRs) and genotypes × GRs interaction. Among three genotypes, Emir showed the highest number of multicellular and embryo-like structures on MS medium + 2.0 mg/l 2,4 D + 0.5 mg/l Kinetin (Kin). For all genotypes, anthers produced calli on MS medium containing 2.0 mg/l 2,4 D + 0.5 mg/l Kin. These calli continued their growth on regeneration medium (MS + 2.0 mg/l BA + 0.5 mg/l NAA) and produced roots. Taken together, these results provide a good basis for further research towards the development of haploid plants for L. angustifolius.     

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.     

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.     

Chlorophyllian durum wheat plants obtained by isolated microspores culture: importance of the pre-treatments

In Triticum turgidum subsp. durum (Desf.) Husn., the utilization of in vitro anther culture is hampered by the very high frequency of albinism of the regenerated plants reaching in most cases 100%. Only in vitro ovary culture or intergeneric crosses with maize produce gynogenetic green haploid and doubled haploid plants. This paper is concerned with another very interesting method of androgenetic doubled haploid plant production, the in vitro isolated microspore culture. It is shown that this method, associated with cold alone or cold plus mannitol pre-treatments, of the spikes kept within their sheath leaves, during different times, have significant positive effects, not only on embryo production, but also on chlorophyllian plant regeneration. All pre-treatments and control taken together, a total of 16 490 embryos was obtained from 17.4 x 10(6) microspores of two T. durum varieties, among which 9320 embryos were transferred to regeneration medium and developed 150 chlorophyllian plants. Thus a long-term (five weeks) 4 degrees C cold pre-treatment of the microspores could be promising for green regeneration in durum wheat.     

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.     

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     

Biosystematic studies on the genusIsoetes (Isoetaceae) in Japan. III. Variability within qualitative and quantitative morphology of spores

Qualitative and quantitative characteristics of mega- and microspores from all the cytotypes of JapaneseIsoetes are described based on the voucher specimens whose chromosome numbers were known. InI. japonica, the hexaploid possessed reticulate megaspores and levigate microspores, while the octaploid and the heptaploid had echinate microspores. Mega- and microspores of the hexaploid and the octaploid were of normal appearance, while those of the heptaploid displayed polymorphism. The tetraploid and the hexaploid ofI. sinensis resembled each other, since they both possessed cristate megaspores and echinate microspores. Echinate megaspores and levigate microspores characterized the diploidI. asiatica. The spore size was largely variable within each cytotype, while the size of the megaspores varied more than that of the microspores. The microspore length was closely correlated with polyploid level. InI. sinensis, the mean microspore length of the tetraploid was 27.6 μm while that of the hexaploid was 31.9 μm, hence these two cytotypes were easily distinguishable. In the hexaploidI. japonica, variations in mega-and microspore size displayed geoclinal variation showing a positive correlation (r=0.43–0.55) with the longitude and the latitude of the populations. A palynological key for cytotypes is presented.     

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     

l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala)

Isolated microspore culture (IMC) represents a potential alternative technique in the plant breeding process, as it allows the effective production of doubled haploid (DH) homozygous lines. However, the implementation of this technique is limited by a low rate of embryogenesis, high level of embryo death, and low frequency of chromosome doubling. Thus, we investigated the effects of using different concentrations of L-ascorbic acid sodium salt (VcNa), which has never been applied for kale, to enhance the embryogenesis and regeneration by IMC. Specifically, 1 to 5 μM VcNa was added to the NLN-13 medium of four kale genotypes, while control was grown on VcNa-free medium. Overall, 1–4 μM VcNa at pH 5.84 increased embryogenesis, with 4 μM VcNa being the optimum concentration (12.92-fold increase). The proportion of embryo deaths declined when using appropriate VcNa concentrations. To increase the frequency of chromosome doubling, an artificial chromosome doubling protocol was developed for kale microspore-derived haploids. This protocol involved dipping roots of haploid plantlets in colchicine solution and adding colchicine treatment to solid Murashige and Skoog (MS) medium. Optimum chromosome doubling of haploids was achieved by dipping their roots in 750 mg/L colchicine solution for 4–6 h and 1000 mg/L colchicine solution for 2 h (doubling for nearly 50% of haploids). In conclusion, this study delineated an effective tissue culture process in promoting chromosomal ploidy of microspore-derived regenerated plants, allowing more microspores to be maintained that have excellent ornamental characteristics through crossbreeding.

     

Doubled haploid plants following colchicine treatment of microspore-derived embryos of oilseed rape (<Emphasis Type="Italic">Brassica napus L.</Emphasis>)

An efficient method for producing doubled haploid plants of oilseed rape (Brassica napus L.) was established using in vitro colchicine treatment of haploid embryos. Haploid embryos in the cotyledonary stage were treated with one of four colchicine concentrations (125, 250, 500 and 1,000 mg/L); for one of three treatment durations (12, 24 and 36 h) at one of the two temperatures (8 and 25°C) and were compared to control embryos (without colchicine treatment). The number of chromosomes, seed recovery, size and density of leaf stomata, and pollen grain size from regenerated plants were determined. No doubled haploid plants were regenerated from control embryos; however, the doubled haploid plants were regenerated from colchicine-treated embryos. A high doubling efficiency, 64.29 and 66.66% of regenerated plants, was obtained from 250 mg/L colchicine treatment for 24 h and 500 mg/L colchicine treatment for 36 h, respectively, at 8°C. Following 500 mg/L colchicine treatment for 36 h, a few plants regenerated (9 plants). At the higher colchicine concentration (1,000 mg/L), no plant regenerated. These results indicate that the colchicine treatment of embryos derived from microspores can induce efficient chromosome doubling for the production of doubled haploid lines of oilseed rape.     

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.     

Improvement of isolated microspore culture of barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.): the effect of floret co-culture

Barley microspores from five field-grown breeding lines were isolated using an ultra-speed blender and the effect of co-culture with young florets was investigated. Floret co-culture in the induction stage increased the formation of MCS, ELS and green plant regeneration. The florets of teraploid plant were more effective than ones of diploid plant. For line S23, co-culture with florets from tetraploid plants gave rise to 2.6 and 7.8 times more MCS and ELS, respectively, than non-co-culture control, whereas co-culture with florets from diploid plants resulted in 1.8 and 6.1 times more MCS and ELS, respectively, than non-co-culture control (Table 2). Florets subjected to cold treatment for 10–20 days induced a greater response than fresh ones, and florets with uninucleate microspores surpassed binucleate microspores. For microspores culture from 15-day cold pre-treated spikes, 93A floret co-culture gave rise to 3.6 and 6.8 times more MCS and ELS, respectively, than the non-co-cultured control, while SD1 floret co-culture resulted in 1.9 and 4.0 times more, respectively. Similarly, for microspore culture from 20-day cold pre-treated spikes, 93A floret co-culture gave rise to 2.6 and 5.1 times more MCS and ELS, respectively, than non-co-cultured control, while SD1 floret co-culture resulted in 1.5 and 3.0 times more, respectively (Table 3). Some microspores formed dense MCS that did not develop further. Compared with the control, floret co-culture resulted in less dense MCS formation, indicating that the isolated florets were beneficial to the normal development of MCS. Floret co-culture was only effective when the spikes were cold pre-treated before microspore isolation. Spike cold pre-treatment before microspore preparation was crucial for dedifferentiation of cultured isolated microspores, and this could not be replaced by floret co-culture. It is postulated that the florets provided essential substances for in vitro cultured isolated microspores to undergo dedifferentiation and embryogenesis. Both the genotype selection and the physiological status (developmental status and cold treatment) adjustment of the florets for co-culture could improve barley microspore culture. Compared with ovary co-culture, floret co-culture is more efficient. The technique is of simple application in breeding programs and can be a solution for coping with recalcitrant genotypes and or plant donor condition.     

A microspore embryogenesis protocol for <Emphasis Type="Italic">Camelina sativa,</Emphasis> a multi-use crop

Camelina [Camelina sativa (L.) Crantz], a member of the Brassicaceae family, has a unique oil profile that has potential both for biofuels and as a food crop. It is essential to have a doubled haploidy protocol in order to enhance breeding of this crop for prairie conditions as well as improve the yield and quality characteristics. Microspore-derived embryos have been produced from Camelina sativa. Buds 1–3 mm in length were selected for culture. The microspores were isolated and purified in full-strength B5 extraction medium and cultured in NLN medium with 12.5% sucrose and 12.5% polyethylene glycol 4000 (PEG) without glutamine, at a density of 10,000 microspores per mL. Glutamine was added to the cultures 72 h after extraction to give a final concentration of 0.8 g/L. The microspore cultures were maintained at 24°C in the dark. After 28 days embryos were observed and these were regenerated to plants and selfed seed was produced. The highest embryogenic frequency achieved was 38 microspore-derived embryos from 100,000 microspores.     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

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

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