Morphological events in cultures of mechanically isolated maize mcirospores

Summary In tis androgenic response, maize is considered to be a recalcitrant plant. We used mechanically isolated microspores of maize genotype A18 to establish a responsive microspore culture of maize. Morphological events occurring during the first days of maize androgenesis in a microspore culture were observed and described, and some morphological markers for distinguishing between embryogenic microspores and nonembryogenic microspores were identified. It was found that the enlargement of microspores during the first days in culture and the ‘star-like’ organization of the cytoplasm inside the microspore are connected with reprogramming of the developmental pathway in maize microspores. Some differences were also found in the surface wall architecture of embryogenic microspores. Fertile plants were successfully recovered from microspore-originated structures.     

Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat. Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998     

Genetic transformation of barley microspores using anther bombardment

Bombardment of intact anthers of commercial barley (Hordeum vulgare) varieties resulted in 0.5–1.0% of transformed microspores of which 20–40% continued in androgenic development (0.2% of all bombarded microspores). Using a system based on bombardment of anthers is therefore likely to be more technically efficient than the use of a microspore isolation, transformation and regeneration system. Bombardment of anthers has a number of technical and scientific advantages over existing systems for gene transfer and can be considered as a alternative method to existing methods for genetic transformation in barley.     

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     

Prospects of androgenetic induction in <Emphasis Type="Italic">Lupinus</Emphasis> spp.

Anthers cultures of six Polish cultivars of pasture lupin (Lupinus L.) were examined for their androgenic response. Anthers with microspores at the uninucleate stage were isolated from flower buds and cultured in liquid media. Better viability of androgenetic structures was obtained when donor plants had grown under field as opposed to greenhouse conditions. A density of five anthers per 0.5 ml medium was more conducive to androgenetic induction than 25 anthers per 0.5 ml medium. Addition of 5% maltose to the induction medium and culture at 25°C without pre-treatment of flowers, buds or anthers promoted microspore release and division. The greatest frequency of androgenic callus, ~70% was developed from cvs. Katon, Wat (white lupin), in contrast to cvs. Legat, Juno (yellow lupin), Polonez and Sonet (narrow-leafed lupin) with callus induction ~30–40%. Despite various combinations of media tested, plant regeneration was not obtained from anther derived callus.     

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.     

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     

Factors affecting the regeneration capacity of isolated barley microspores (Hordeum vulgare L.)

The culture of isolated microspores of barley (Hordeum vulgare L. cv. Kymppi, an elite malting barley cultivar) was studied. A careful choice of culture steps resulted in an average regeneration frequency of 300 green plants per starting material spike. Strong seasonal variation in regeneration capacity was observed. The choice of a cold pretreatment method affected the viability of microspores. A cold pretreatment of the collected starting material at +4°C for 4 weeks was needed for the efficient regeneration of green plants from isolated microspore cultures. Glutamine omission from and copper additions to microspore culture were studied. The omission of glutamine did not affect the number of regenerated green plants but did result in an increase in the number of regenerated albino plants. The addition of copper did not improve the regeneration capacity of isolated barley microspores. Transformation by particle bombardment of isolated microspores did not result in the production of transgenic plants.     

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.     

High frequency androgenesis from isolated microspores of maize

Anthers from a highly androgenic genotype of maize (139/39-02), when cultured in a modified, liquid YP medium, dehisced within 2–7 days resulting in a stationary suspension of microspores. After 12–15 days, the microspore suspension was found to contain multicellular masses which went on to produce macroscopic embryo-like structures within 20–25 days of culture initiation. Embryogenic callus could be obtained by transferring microspore-derived embryos onto a modified N6 medium supplemented with 2.5 mg/l dicamba and 0.1 mg/l 2,4-D. Subculture onto hormone-free medium resulted in plant regeneration. Over 400 embryo-like structures per 100 anthers cultured have been obtained from liquid induction medium as compared to 55 embryos per 100 anthers cultured on an agar-solidified medium. Approximately 5–25% of these embryo-like structures went on to produce callus from which plants could be recovered. Mechanical isolation of microspores from anthers precultured for 0, 3, and 7 days also resulted in embryo production and plant regeneration. This represents the first report of plant recovery from isolated maize microspores. The use of a liquid induction medium applied to a highly androgenic genotype allows for the production of large numbers of microspore-derived plants and provides a single, haploid cell regeneration system for maize.     

Induction of Haploid Morphogenic Calluses from in vitro Cultured Anthers of Prunus Armeniaca cv. ‘Harcot’

Apricot (Prunus armeniaca) ‘Harcot’ anthers, were cultured in vitro for the production of haploid plants. The best androgenic response was achieved with Nitsch and Nitsch (1969) medium, supplemented with 4.52 μM 2,4-D, 4.52 μM zeatin, 2.85 μM IAA and 40 g l⁻¹ sucrose. Cultures were maintained in the dark for 8 days, at 28°C, followed by transfer to a 16-h photoperiod, with 35 μm m⁻² s⁻¹ light intensity and 24/22°C day/night temperature. The androgenic response was correlated with the floral bud size, its phenologic stage and the level of microspore evolution. Anthers containing microspores at the tetrad/uninucleate stage were the most appropriate. The ploidy level of the calluses was evaluated by flow cytometry revealing that they range from haploid to octaploid. Mixoploid calluses have also been identified. Histological studies showed that the haploid calluses have their origin in the microspores. Nodular structures consisting of cells with dense cytoplasm and differentiated xylem elements were observed and were surrounded by an autofluorescent layer, probably due to cutin deposition.     

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.     

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.     

Improved plant regeneration from shed microspore culture in barley (Hordeum vulgare L.) cv. igri

This report describes rapid regeneration of green plants from microspores of the barley cultivar Igri. Use of 0.3 M mannitol during maceration and isolation was essential for response from mechanically isolated microspores of barley cv. Igri grown under our conditions. A shed microspore culture system proved to be simple and gave a fast response; plants were obtained as early as 25 days after the material was taken from the donor plant. A 28-day cold-pretreatment of spikes can also be replaced with a 3–4 day pretreatment of anthers in mannitol. Shed microspores from 100 anthers produced an average of 292 plants with 91% of them green. Approximately 80% of the regenerated plants were spontaneously doubled-haploids.Abbreviations IAA Indole-3-acetic acid - FHG Hunter's media (1988) - MS Murashige and Skoog     

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.     

Isolated microspore culture of maize: effects of isolation technique,reduced temperature,and sucrose level

Improvements in ab initio microspore culture of maize are presented using a modified isolation technique, reduced temperature during early stages of culture, and an elevated sucrose level in the culture medium. Blending-isolation, using excised anthers, was less stressful on microspores than pressing anthers against a stainless steel sieve and resulted in a 3-fold increase in the yield of embryo-like structures (ELS). Exposure to reduced temperature (15°C) during the first 4 days of culture improved microspore viability and increased by 2-fold the number of ELS produced. Higher levels of sucrose (8.0–9.5%) also resulted in improved response. Maximum yield in the present study was 92 ELS per 100 anther equivalents, exceeding previously reported values of 15 ELS per 100 anther equivalents for ab initio microspore culture of maize. The increase in the total number ELS produced had no observable effect on their quality as evidenced by the frequency of formation of callus capable of regenerating plants.     

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.     

Improvement of isolated microspore culture of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.     

Zinc sulphate improved microspore embryogenesis in barley

The effect of ZnSO₄ concentration on barley (Hordeum vulgare L.) microspore embryogenesis was investigated using cultivars of different androgenetic response. Concentrations from 0 (control) to 600 μM in the stress pre-treatment medium alone or in combination with 30 (control) to 600 μM in the embryo induction medium were assayed in anther culture. Incorporation of Zn²⁺ in the pre-treatment medium itself did not affect microspore embryogenesis. The optimum concentration in the stress pre-treatment and induction media was 180 μM for cultivars (cvs.) Igri and Reinette, and 90 μM for cv. Hop. A significant increase of 30 and 300% in cv. Igri and Reinette, respectively, were produced with 180 μM ZnSO₄ in both the number of embryos and green plants. In order to confirm the effect of Zn²⁺ on microspore embryogenesis this micronutrient was incorporated in the induction medium of isolated microspore cultures of cv. Igri. Concentrations of 90–300 μM ZnSO₄ resulted in an increase of 40–53% in the number of embryos and green plants. All these results indicate that the beneficial effect of Zn²⁺ is exerted mainly during the culture phase, increasing the number of embryos, leading to an increased number of green plants, but it had no effect on percentage of regeneration or green plants.