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     

Androgenic response to preculture stress in microspore cultures of barley

Summary. Various stresses such as starvation and cold or heat shocks have been identified as triggers in the induction of the microspore embryogenesis. This study attempts to quantify the effects of different pretreatment conditions for successful microspore culture of malting barley (cv. Scarlett). While the sporophytic microspore development could be induced from treated and nontreated microspores, abiotic stress was essential for embryo formation and plant regeneration. The type of stress treatment applied affected the numbers and the ratios of albino and green plants regenerated, as well as their fertility. The highest number of green plants was obtained after the treatment of anthers in 0.3 M mannitol at 32 °C for 24 h before microspore culture. Correspondence and reprints: Department of Plant Biotechnology and Cytogenetics, Institute of Plant Breeding and Acclimatization, Radzików, 05-870 Blonie, Poland.     

Enhancement of microspore culture efficiency of recalcitrant barley genotypes

The culture response of isolated microspores of seven recalcitrant cultivars of barley has been largely improved by identifying an appropriate pretreatment and utilizing ovary co-cultivation. After comparison of three pretreatment media, medium B was shown to be most efficient for inducing microspore embryogenesis, while 0.3 M mannitol frequently used for the responsive cv. Igri was found to be ineffective for recalcitrant genotypes. A further significant improvement of embryogenesis was achieved by using ovary co-culture, which resulted in an overall 2.1-fold increase in embryo formation and 2.4-fold increase in green plant regeneration from all cultivars compared with the control. Optimal co-culture conditions were identified as 5 ovaries/ml medium kept over 20 days in induction culture. Microspore plating densities in cultures with and without co-culture were found to be optimal at 4᎒⁴/ml and 8-12᎒⁴/ml, respectively. The most effective and reproducible method for culturing microspores of recalcitrant genotypes appeared to be the combination of medium B pretreatment with ovary co-culture. By using this procedure, the genotypic difference in microspore embryogenesis could be reduced. It was found that medium B mainly enhanced percent live embryogenic microspores, and ovary co-culture subsequently improved cell division and embryogenic development. The method described here is important for the application of the microspore culture technique to barley breeding and biotechnology.     

Effects of n-butanol on barley microspore embryogenesis

Doubled haploid (DH) production is an efficient tool in barley breeding, but efficiency of DH methods is not consistent. Hence, the aim of this study was to study the effect of n-butanol application on DH barley plant production efficiency. Five elite cultivars of barley and thirteen breeding crosses with different microspore embryogenesis capacities were selected for n-butanol application in anther and isolated microspore cultures. Application of 0.1 % n-butanol after a mannitol stress treatment in anther culture significantly increased the number of embryos (up to almost twice) and green plants (from 1.7 to 3 times) in three low-responding cultivars: Albacete, Astoria and Majestic. No significant differences on microspore embryogenesis efficiency were observed in medium and high responding cultivars. The application of n-butanol treatment to isolated microspores from cold treated spikes in thirteen spring breeding crosses with a low or very low androgenetic response did not have a significant effect on the overall number of green plants. Nevertheless, an increase in the number of green plants was observed when 0.2 % n-butanol was applied in four out of seven low-responding crosses. Therefore, application of n-butanol could be routinely applied to anther cultures using mannitol treatment, in low-responding material. However, further studies are needed to determine optimal conditions in protocols using cold treatment and isolated microspore cultures.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

Inducing homozygosity in transgenic barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) by microspore culture

Homozygosity was induced in transgenic barley by microspore culture. Spikes of transgenic barley plants carrying microspores in the late uni-nucleate stage were cold pretreated. Teflon rod maceration and a density of 100 000 viable micropores per plate were used. The developed calli were regenerated and plantlets were treated with colchicine. The microspore culture of 16 mother plants (three transgenic lines) resulted in 927 green regenerants. Of these plants, 476 were transferred to soil, 380 were transgenic, 358 reached maturity and 350 were fertile with a normal seed-set carrying a yield of 6.9 kg. A production efficiency of 0.8 fertile transgenic doubled haploid barley plants per spike used for microspore isolation was recorded. The produced transgenic seeds were used in malting experiments.     

Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green-fluorescent protein

The green-fluorescent protein (GFP) gene from the Pacific Northwest jellyfish, Aequorea victoria, was used as a screenable marker in the production of transgenic barley plants. Isolated barley microspore culture was biolistically transformed with two synthetic forms of GFP, sgfp and pgfp. Thirty-seven fluorescing multicellular structures were isolated using epifluorescent microscopy. Sixteen structures developed shoots, but only five regenerated into green plants. Three events had been co-bombarded with #-glucuronidase (gus) and assayed positive for gus expression in the leaves, and all five events were positive for gfp expression. The expected transgene band size was PCR-amplified from all five plants, and Southern blots performed on three plants revealed unique patterns of gfp transgene integration. Fluorescent in situ hybridization also revealed the transgenic status and hemizygous nature of all the events. GFP-based visual screening provides a viable alternative method to chemical selection of transgenic plants from barley microspore culture.     

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.     

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.     

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.     

Improving the efficiency of isolated microspore culture in six-row spring barley: I-optimization of key physical factors

Key message

An improved isolated microspore culture protocol alleviating the recalcitrance typically observed in six-row spring barley was developed by optimizing four key physical factors to increase embryogenesis and reduce albinism.

Abstract

Doubled haploid (DH) plants are completely homozygous individuals that can be generated in just a few months via androgenesis in vitro. DHs are useful tools in genetic research and in plant breeding. Isolated microspore culture (IMC) is the most efficient way to produce DHs, but a strong genotype dependency imposes limitations to its wide application. Six-row, spring barley genotypes are considered as particularly recalcitrant due to a low frequency of embryogenesis and a high rate of albinism. Seeking to develop an efficient IMC protocol for this type of barley, we explored four important factors: (1) the harvest stage of immature spikes, (2) the type of pretreatment applied, (3) the osmotic potential in the induction medium, and (4) the plating density of microspores. This work was first performed using four barley genotypes: two typical six-row spring cultivars (ACCA and Léger), a two-row spring (Gobernadora) and a two-row winter (Igri) cultivar. First, by optimizing the harvest stage for each genotype we obtained a twofold to fourfold increase in the yield of embryogenic microspores. Second, two pretreatments (0.3 M mannitol for 2 days, or a combination of cold and heat over 15 days) both performed significantly better than the commonly used cold pretreatment (28 days at 4 °C). Third, an induction medium-containing mannitol (32 g/l) doubled green plant regeneration. Fourth, a plating density of 10⁶ microspores/ml yielded the highest number of green regenerated plants. Our most important findings were then confirmed using sets of F1s from a six-row, spring-type breeding program.     

Regeneration of Fertile Barley Plants from Mechanically Isolated Protoplasts of the Fertilized Egg Cell

A simple procedure is described for the mechanical isolation of protoplasts of unfertilized and fertilized barley egg cells from dissected ovules. Viable protoplasts were isolated from ~75% of the dissected ovules. Unfertilized protoplasts did not divide, whereas almost all fertilized protoplasts developed into microcalli. These degenerated when grown in medium only. When cocultivated with barley microspores undergoing microspore embryogenesis, the protoplasts of the fertilized egg cells developed into embryo-like structures that gave rise to fully fertile plants. On average, 75% of cocultivated protoplasts of fertilized egg cells developed into embryo-like structures. Fully fertile plants were regenerated from ~50% of the embryo-like structures. The isolation-regeneration techniques may be largely genotype independent, because similar frequencies were obtained in two different barley varieties with very different performance in anther and microspore culture. Protoplasts of unfertilized and fertilized eggs of wheat were isolated by the same procedure, and a fully fertile wheat plant was regenerated by cocultivation with barley microspores.     

Improved plant regeneration from wheat anther and barley microspore culture using phenylacetic acid (PAA)

Summary The effect of the auxin phenylacetic acid (PAA) on wheat anther and on barley anther/microspore culture was investigated. With PAA the induction response was not usually significantly different from controls but a significantly higher number of green plants were produced in wheat anther and barley microspore culture. For wheat anther culture 100 mg/L PAA was beneficial. For barley microspore culture the optimum levels were from 1 to 100 mg/L, depending on genotype. In barley anther culture there were no improvements using PAA. In wheat anther culture, 145 green plants/100 anthers were obtained with cultivar VeeryS, while the average response from twelve F₁ hybrids in the breeding program was 332 green plants/100 anthers. At least 1000 green plants were obtained using isolated microspores from 100 anthers in barley cv. Igri. With cv. Bruce, regeneration occurred only when 100 mg/L PAA was used. The influence of PAA appears at the embryogenic phase of the culture system. The possible mechanisms by which PAA may improve regeneration are discussed.     

大麦不同基因型游离小孢子的直接培养再生及培养体系的优化

以微搅拌法建立了小孢子直接游离的预处理和培养程序。在大田生长的４个对培养反应不同的大麦基因型上,以新鲜幼穗游离小孢子进行直接培养,均成功地诱导了胚状体并获得再生绿色植株。小孢子的发育进程说明,直接游离的小泡子在预处理过程中的发育要慢于在花药中预处理的小孢子,而且其培养效率也较低。直接游离小孢子的培养密度以０．８～１．０×１０５／ｍｌ较理想,至少应不低于６×１０４／ｍｌ．８％－１０％的糖浓度可明显提高小孢子分裂频率和胚状体诱导频率。实验结果也表明两种培养基ＦＨＧ和ＭＮ６无明显差异,均适宜于直接游离的小孢子培养,并对游离小孢子直接培养在理论和应用上的意义进行了讨论     

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.     

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     

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     

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 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.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999