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.     

Plastid differentiation during androgenesis in albino and non-albino producing cultivars of barley (Hordeum vulgare L.)

In order to better understand androgenic albinism in barley, we compared plastid differentiation during anther culture in two cultivars, an albino (spring cultivar Cork) and a non-albino (winter cultivar Igri) producing cultivar. The ultrastructure of plastids and the relative amount of DNA containing plastids were followed in both cultivars during the androgenic process and correlated with the proportion of regenerated chlorophyllous plantlets. For androgenesis, anthers were collected at the uninucleate stage, during mid- or late-microspore vacuolation. At this stage DNA was detected in 15.3 ± 2. 7% of microspore plastid sections in the winter cultivar Igri, compared to 1.7 ± 0.5% in the spring cultivar Cork. In the winter cultivar Igri, starch was broken down after anther pretreatment but plastids divided rapidly during anther culture and thylakoids developed in the stroma. Prior to regeneration, plastids contained 2.0 ± 0.2 thylakoids per plastid and starch represented 26.1 ± 3.3% of the plastid volume. In the spring cultivar Cork, plastids followed a different developmental pathway. After anther pretreatment, microspore plastids differentiated exclusively into amyloplasts, accumulating starch and losing their thylakoids as well as their capacity to divide. This developmental pattern became progressively more marked, so that by the end of anther culture plastids contained 0.5 ± 0.4 thylakoids per plastid and starch represented up to 90.3 ± 4.3% of plastid volume. Following androgenesis, the response was similar in both cultivars except that the winter cultivar Igri provided 87.8% of chlorophyllous plantlets compared to 99.7% albino plantlets in the cultivar Cork. The results presented here suggest that the exclusive regeneration of albino plantlets in the spring cultivar Cork may be due to degradation of microspore plastid DNA during early pollen development, preventing the plastids from differentiating into chloroplasts under culture conditions. Received: 13 March 2000 / Revision accepted: 6 June 2000     

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     

Enhancement of embryogenesis and plant regeneration from barley anther culture by low doses of gamma irradiation

Summary The effects of 0,5 and 10 Gy doses of gamma irradiation on the enhancement of embryogenesis and plant regeneration efficiency of three barley (Hordeum vulgare L.) genotypes, Igri, Arabi Abiad and AECS 76, were evaluated. Embryo yields at 5 and 10 Gy doses were significantly higher than those of the control (OGy). This effect was genotype-dependent. The most responsive genotype was Igri, with 592.8 embryos 32 anthers exposed to 10 Gy. However, despite a high embryo induction rate, the green plant regeneration rate was low. Arbi Abiad had a higher ability to generate green plants produced from, with 28. 13 plantlets obtained from 32 anthers at 10 Gy; irradiation had no significant effect on regeneration of Igri and AECS 76 genotypes. In general, the 10 Gy dose produced a much higher embryo yield than the 5 Gy dose. The root-tip chromosome number and the fertility of 298 regenerating green plants of cv. Igri revealed that 64% of the tested plants were spontaneously doubled haploids (DHs) and fertile.     

Regeneration capacity of microspore-derived structures produced in anther cultures of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.)

The regeneration capacity of microspore-derived structures, with various morphological characteristics produced in anther cultures of maize (Zea mays L.) were studied in order to identify the morphotype resulting in the highest yield of spontaneous doubled haploid regenerants. Parallel to the morphological studies the ploidy level of microspore-derived structures and regenerants was analysed by flow cytometry. Neither the growth conditions of the anther donor plants nor the media used in the experiment had any effect on the frequency distribution of different morphotypes. The highest number of spontaneous doubled haploid plants was regenerated from white compact structures 2–3 mm in size, derived from the anthers of phytotron-grown donor plants.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.     

Anther culture as an effective tool in winter wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) breeding

The aim of this study was to determine the effect of genotype and induction medium in anther culture of wheat (Triticum aestivum L.). Ten F1 winter wheat genotypes were tested in anther culture (AC) to compare the two most frequently applied induction media (W14mf and P4mf). Androgenesis was induced during the treatment of each tested genotypes and green plants were produced from them using both media. Based on statistical analysis, the genotypes significantly influenced (at the 0.001 probability level) the efficiency of AC (embryo-like structures (ELS), albinos, green plantlets and transplanted plantlets) and the media also had a significant effect on the number of ELS and albino plantlets. Both media can be used for AC in wheat doubled haploid (DH) plant production. The production of ELS and green plantlets was higher in P4mf medium (48.84 ELS/100 anthers, 4.82 green plantlets/100 anthers) than in W14mf medium (28.14 ELS/100 anthers, 4.59 green plantlets/100 anthers). However, the green plant regeneration efficiency of the microspore-derived structures was 16.9% when using W14mf medium, while this value was 9.6% in the case of ELS induced with P4mf medium. The application of W14mf medium thus proved to be time- and labour-saving medium in the large-scale production of DH wheat plants. In our experiments, 267 DH plants were produced for our winter wheat breeding program. The spontaneous rediploidization rate was 32.72%.     

Microspore embryogenesis and programmed cell death in barley: effects of copper on albinism in recalcitrant cultivars

Albinism remains a major problem in cereal improvement programs that rely on doubled haploid (DH) technology, and the factors controlling the phenomenon are not well understood. Here we report on the positive influence of copper on the production of DH plants obtained through microspore embryogenesis (ME) in recalcitrant cultivars of barley (Hordeum vulgare L.). The presence of copper sulphate in the anther pre-treatment medium improved green DH plant regeneration from cultivars known to produce exclusively albino plants using classical procedures. In plastids, the effect of copper was characterized by a decrease in starch and a parallel increase in internal membranes. The addition of copper sulphate in the ME pre-treatment medium should enable breeders to exploit the genetic diversity of recalcitrant cultivars through DH technology. We examined programmed cell death (PCD) during microspore development to determine whether PCD may interfere with the induction of ME and/or the occurrence of albinism. By examining the fate of nuclei in various anther cell layers, we demonstrated that the kinetics of PCD in anthers differed between the barley cultivars Igri and Cork that show a low and a high rate of albinism, respectively. However, no direct correlation between PCD in the anther cell layers and the rate of albinism was observed and copper had no influence on the PCD kinetic in these cultivars. It was concluded that albinism following ME was not due to PCD in anthers, but rather to another unknown phenomenon that appears to specifically affect plastids during microspore/pollen development.     

Stimulation of embryogenesis and haploid production in Brassica campestris anther cultures by elevated temperature treatments

Summary Culture of Brassica campestris anthers at 35°C for one or three days prior to culture at 25°C significantly stimulated the yield of microspore-derived embryos. More than 100 plants were regenerated from cultured embryos and haploids were identified amongst them. The haploid frequency was greater than 70% if all small-flowered sterile plants were considered to be haploid. The yield of microspore-derived plants in B. campestris is approaching the level where anther culture may be utilized as a practical breeding tool.     

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.     

Differential development of plastids during microspore embryogenesis in barley

Summary In order to understand and limit albino plantlet formation during pollen embryogenesis in barley (Hordeum vulgare L. cv. Igri), plastid feature was followed during pollen embryogenesis under two anther culture conditions and compared to plastid development in the zygotic embryo. The first condition was characterized by cold pretreatment and maltose in the induction medium. Both embryos and calli were then obtained. During pollen embryo development, up to 30% of plastids had abnormal features. Disruptions mainly affected the plastid size, the feature of plastid envelopes, thylakoid and granum organization, as well as starch accumulation. In pollen calli, superficial cells had meristematic features. Up to 50% of plastids exhibited the above mentioned abnormalities. Internal cells were highly vacuolated with amyloplast-like plastids; envelopes had normal features but no internal membrane was detected. Pollen embryo-derived plantlets had a green-to-albino ratio (G/A) being equal to 1.0, whereas calli-derived embryos only formed albino plantlets. The second condition was characterized by mannitol pretreatment and the presence of both maltose and mannitol in the induction medium. No callus was formed but most of microspore-derived structures developed haploid embryos and then the green plantlets (200 plantlets per 100 responding anthers, G/A=9.4). In this case, plastid development in zygotic and pollen embryos were similar and almost no albino plantlets were formed.     

The influence of genotype and temperature pre-treatment on anther culture response in barley (Hordeum vulgare L.)

The influence of temperature stress pre-treatment on anther culture response has been examined in eight commercially desirable barley cultivars. Spikes were pre-treated in darkness at 4°C for periods of 0, 7, 14, 21 and 28 days. Overall, the optimum pre-treatment period was 21 days, although there were large genotype by pre-treatment interactions. The most responsive cultivar was Igri, with a mean of 38% anthers responding, and relatively little effect of pre-treatment. The greatest effect of pre-treatment was in cv. Heriot, which had 3% response with no pre-treatment and 52% response from 14 days pre-treatment.     

BA对大麦花药培养中药壁的衰退和植株再生频率的影响

用含20ppm 6-BA的0.1％吐温-80溶液喷施花粉为单核前期的大麦上部叶片和穗部,明显影响大麦花药培养效率。实验结果表明:1)BA处理可明显延缓培养花药的药壁衰退进程。2)BA处理后的花药,在培养期间,其死亡的花粉数比对照大大减少,相反其双核或多核的花粉数比对照明显增加。3)BA处理虽然没有促进大麦花粉愈伤组织的诱导率,但显著地促进愈伤组织的生长。提高愈伤组织成长率,增加可转入分化培养的愈伤组织块数。4)BA处理促进愈伤组织的再分化,尤其是绿苗的分化。     

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     

Increased induction and chromosome doubling of androgenetic haploid rye

Summary Further progress of studies aimed at increasing production of androgenetic Secale cereale plants via the culture of anthers is described. Two culture media initially developed for rice and wheat anther culture have been shown to have pronounced influence on rye. It has been possible to increase the average percentages of responsive anthers (i.e. those producing embryoids or calluses) from 0.26% to 10% with a maximum in certain experiments of over 40 %. Of nearly 400 plants produced in 1976, 1/4 are green and can be grown further by transfer to potting compost; 3/4 are albino. Stable green haploid lines were present amongst the plants, and after vegetative propagation of the lines representative samples have been treated with colchicine resulting in diploid, triploid and tetraploid plants. The influence of the genetic background of the donor plants on the success rate of anther culture and on the percentage of albino formation is discussed.     

The influence of several factors on the efficiency of androgenesis in carrot

The influence of cultivar, donor plant and culture procedure on the efficiency of androgenesis was studied in carrot anther culture. Experiments were carried out on five carrot cultivars: CxC 9900 F1, Lucky B F1, HCM, Beta III and Perfekcja, which were chosen because of their high carotene contents. Two procedures of anther culture were compared: (1) incubation in darkness for two weeks, followed by exposure to continuous light and transfer onto a fresh medium of the same composition; and (2) incubation in darkness until embryos appeared, without transfer onto a fresh medium. Temperature was +27 degrees C all the time. Genotype played an important role in the process of androgenesis in carrot anther culture.The efficiency was the highest in cv. HCM - 5.6 embryos per 100 anthers. Considerable differences in the capacity for androgenesis were observed between individual donor plants. The ratio of embryos obtained per 100 anthers for cv. HCM varied from 0.0 to 48.9. The second procedure of anther culture proved to be more efficient, cheaper and less complicated.     

The effect of osmotic potential on anther culture in spring wheat (Triticum aestivum)

This study was conducted to determine the effect of osmotic potential in a modified 85D12 medium on both callus induction and plant regeneration in the anther culture of two wheat genotypes, cv. Chris and cv. Pavon. Altering the medium osmotic potential by changing the carbohydrate source and concentration or by adding a non-metabolized osmoticum appeared to have the greatest potential for improving anther-derived green plant production. The medium osmotic potentials were varied (-0.67 to –2.30 MPa) by altering sucrose and PEG concentration. Both osmotica affected callus production, with –0.9 to –1.4 MPa media producing the most calluses. Callus production depended on genotype and osmoticum. Only PEG concentration affected green plant regeneration. The greatest number of green plants (11.5 plants per 100 anthers in cv. Chris) was obtained with 0.0125 M of PEG. This experiment suggested that a low level of PEG in the medium was beneficial for producing green plants from wheat anthers.     

Comparison of media for their aptitude in wheat anther culture

Different media were evaluated with anthers of five spring wheat (Triticum aestivum L.) genotypes for their ability to produce embryos and green plants in anther culture. Our first experiment showed that the addition of a combination of 19 amino acids significantly increased the number of embryos and green plants obtained. The mean number of green plants per 100 anthers for the two genotypes in this experiment, HY320 and B723, went from 28.2 without amino acids in the medium, to 46.7 with addition of amino acids. Our second experiment with the genotypes HY320, Wim and Laval-19 showed that liquid medium with Ficoll is more efficient for anther culture (9.9 green plants/100 anthers) than solid (0 green plants), gelationous media (2.5 green plants/100 anthers) or liquid medium with Membrane Rafts (0 green plants; Hoechst Celanese Corp.). Our third experiment revealed that the effect of replacement of sucrose by maltose varied with the genotype of the donor plant. Maltose partially inhibited the androgenesis of three responsive genotypes, HY320, Wim and Reliance (40.3 green plants/100 anthers instead of 43.9 with sucrose), while maltose significantly increased the androgenesis of the recalcitrant genotype Laval-19 (10.8 green plants/100 anthers instead of 5.4 with sucrose). An amino acid x maltose interaction was also observed. Amino acids without maltose increased androgenesis, but the addition of maltose to the amino acid-enriched medium eliminated this positive effect of the amino acids.     

In vitro induction of androgenic haploids in Safflower (Carthamus tinctorius L.)

The influence of culture medium on induction of androgenic calli was examined with five different basal media. MS medium was the most responsive in inducing callus. Differences in induction of calli among ten genotypes revealed that the most responsive genotype was a local cultivar, Mangira, with 48.6% anthers initiating callus formation. The influence of temperature pre-treatment (5°±1°C) for varying periods (0 to 15 days) on immature capitula prior to inoculation of anthers on the medium revealed that the percentage of anthers inducing callus increased till 3–5 days of pre-treatment. The effect of physiological conditions of anther donor plants grown in the field and in green houses on induction and re-differentiation have shown that the field grown anther donor plants exhibited optimum response. Shoot regeneration was observed on MS supplemented with BAP (2.0 mg/l) and NAA (0.5 mg/l) and rhizogenesis on MS (half-strength) medium, supplemented with NAA (0.1 mg/l) and 1% sucrose. Cytological studies of anther derived plants showed two ploidy levels, where the haploids were predominant (64%).     

The Role of Abscisic Acid in Induction of Androgenesis: A Comparative Study Between Hordeum vulgare L. Cvs. Igri and Digger

Under the same mannitol pretreatment and culture conditions, regeneration efficiency in the barley cultivar (cv.) Igri was about 10 times higher than in the cv. Digger, a difference only partially reflected by a difference in viable microspores after anther pretreatment. Therefore, a comparative study between cvs. Igri and Digger was carried out under various pretreatment conditions. For both cultivars, under water, CPW buffer and mannitol pretreatment conditions, there was a positive correlation between microspore viability and regeneration efficiency in that mannitol > CPW buffer >> water. Mannitol pretreatment of cv. Igri produced a much higher endogenous abscisic acid (ABA) level than as to Digger. Addition of ABA stimulated both percentages of viability and regeneration efficiency except in the case of mannitol pretreatment. Under CPW buffer pretreatment conditions, addition of ABA significantly stimulated regeneration efficiency and was ABA concentration dependent. However, cv. Digger was less responsive to ABA than cv. Igri. In both cultivars, under less optimal pretreatment conditions (e.g., water and CPW buffer), the effect of ABA was to stimulate increased percentages of viability and/or to reduce the number of binucleate microspores. Moreover, in cv. Igri, direct culture of anthers for 4 days without pretreatment caused an increased number of binucleate microspores compared with microspores with pretreatment for 4 days. These binucleate microspores showed DNA degradation in the nuclei. However, with mannitol pretreatment binucleate microspores and DNA fragmentation in the nuclei of microspores was rarely observed. On the basis of our observations, we suggest that the difference in regeneration efficiency in cv. Igri and cv. Digger is related to the differences in endogenous ABA production levels under mannitol pretreatment and responsiveness to ABA. One of the effects of ABA is likely due to an inhibition of cell death. Received May 21, 1999; accepted October 5, 1999