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     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

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     

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.     

Impact of abscisic acid in overcoming the problem of albinism in horse chestnut androgenic embryos

Horse chestnut (Aesculus hyppocastanum L., Hyppocastanacea) is a relict species with a slow and complex reproductive cycle considered to have horticultural and medical importance. The cycle maybe circumvented via in vitro androgenesis. Androgenesis of horse chestnut was induced in microspores and anther culture on MS media. Some of the horse chestnut androgenic embryos were albinos. Addition of abscisic acid in media (in concentrations of 0.01, 0.1, 0.5, 1, 2, 5, 10, and 20 mg l^?1) with horse chestnut androgenic embryos has circumvented the reproduction cycle barriers. The best results were achieved on medium with the lowest abscisic acid concentration (0.01 mg l^?1) in microspore culture. The microspore culture proved to be a better model system for embryo production and albino embryo reduction than anther culture. Flow cytometry analysis after maturation treatments induced by ABA showed that 88 % of green embryos originating from microspore culture were haploid. However, 50 % of green embryos from anther culture were haploid. The remaining analyzed androgenic embryos, from both types of cultures were diploid.     

Improvement of green plant regeneration by manipulation of anther culture induction medium of hexaploid wheat

Anthers of three hexaploid wheat (Triticum aestivum L.) genotypes with high frequencies of albino regenerants in anther culture were compared to DH after inoculation on medium supplemented with ficoll, colchicine or maltose separately, pair-wise or combined, in an attempt to increase green plant regeneration. Maltose treatment produced more green regenerated plants than sucrose for all of the genotypes. The three chemicals combined in anther medium either reduced green plant regeneration or did not yield significantly different numbers of green regenerated plants compared to the maltose treatment. With DH fewer embryo-like structures (ELS) were obtained per 100 cultured anthers on all medium containing colchicine but greater frequencies of green plants per 100 ELS were obtained. It appeared that the increase in green regenerated plants per 100 ELS was due to a better quality of embryos that were capable of regenerating into green rather than albino plantlets. Smaller increases in green plants per 100 ELS were observed in ICR 4 and V-15 on colchicine containing medium compared to DH. Genotypic differences in anther culture response were observed for ELS per 100 cultured anthers (increased for V-37, decreased for DH and approx. the same for ICR 4 and V-15 in medium with all three chemicals compared to the sucrose control).     

High frequency androgenesis in indica × Basmati rice hybrids using liquid culture media

An improved procedure has been developed for high frequency androgenesis in indica × Basmati rice hybrids using a liquid culture medium. Anthers from fourteen genotypes comprising of indica × Basmati rice F₁ hybrids, F₂ plants and the parental rice cultivars, were floated in liquid RZM, N6M, and Heh5M media. Anther culture frequencies (percentage of anthers forming calluses) in most of the genotypes were significantly higher in RZM medium (16–75%) compared to those obtained in N6M (7–29%) and Heh5M (7–41%) media. Agarose (1.0% w/v)-solidified MSR1 medium containing 3.0% (w/v) maltose, 1 mg l⁻¹ kinetin, 1 mg l⁻¹ 6-benzyladenine (BA) and 0.5 mg l⁻¹α-naphthalene acetic acid (NAA) induced green shoot regeneration at high frequencies compared to the medium (MSR2) lacking BA. In all the genotypes, microspore calluses initiated in RZM medium regenerated green shoots with over tenfold higher frequencies compared to the calluses initiated in other two media. High plant regeneration frequencies (up to 270 green plants/1000 anthers) were obtained from microspore-derived calluses of some of the F₁ hybrids (Gobind × Basmati 370, Gobind × Taraori Basmati) and F₂ plants (Gobind × Basmati 370, Gobind × Taraori Basmati, HKR86-3 × Taraori Basmati) as compared to their actual parents. Cytological analysis of the root tips of the progeny seedlings of the microspore-derived plants revealed haploids at a frequency of about 50%; 22% of the microspore- derived plants had > 5% spikelet fertility and were diploid. Use of RZM liquid and MSR1 media, respectively for anther culture and plant regeneration resulted in several fold increase in the recovery of green plants from recalcitrant indica × Basmati rice F₁ hybrids/F₂ plants which were comparable to those reported for japonica rice varieties/hybrids leading to the improved feasibility of using doubled haploids in genetic, breeding and mapping research with indica rice. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Enhanced induction of microspore embryogenesis after <Emphasis Type="Italic">n</Emphasis>-butanol treatment in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) anther culture

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.     

PCIB an Antiauxin Enhances Microspore Embryogenesis in Microspore Culture of Brassica juncea

An efficient protocol to improve microspore embryogenesis is established in an important oleiferous crop, Brassica juncea (Indian mustard). Colchicine was used for enhancing microspore embryogenesis and also to obtain doubled haploid embryos. Colchicine at high concentrations (>10 mg l⁻¹), for 24 h, proved convenient for direct recovery of diploid embryos. Higher temperature treatment and an antiauxin PCIB (p-chlorophenoxyisobutyric acid) enhanced microspore embryogenesis significantly as compared to colchicine. An increase in temperature from 32°C to 35°C proved very efficient in increasing embryogenesis by 10-fold. The highest embryogenesis rate was obtained when PCIB was added at 35°C in the culture after 1 day of culture initiation. 20 μM PCIB could enhance microspore embryogenesis by 5-fold. Different abnormal shapes of embryos like lemon, banana, flask and fused cotyledons were observed. Both normal and fused cotyledonous embryos showed normal germination when transferred on the B₅ basal medium.     

Production of doubled-haploid plants from tritordeum anther culture

The effects of different media and cold pretreatment of spikes on the androgenic response and regeneration capacity from anther culture of tritordeum was studied. L5 medium gave the highest frequency of anther response. The frequency of cultures regenerating green or albino plantlets was not affected by the composition of the medium tested. Cold pretreatment of the spikes significantly increased the frequency of anther response and also the percentage of cultures giving albino plantlets. A mean of four green plants was obtained per 100 subcultured calli/embryos. The percentage of spontaneous chromosome doubling was only 1%. The addition of colchicine at 0.02% to the induction medium significantly increased the frequency of doubled haploids regenerated without any effect on regeneration capacity. This technique proved more efficient than a conventional chromosome-doubling method.     

Haploid plantlets derived by anther culture of Cucurbita pepo

This work was conducted to study the effect of sucrose and 2,4-D combinations on induction of haploid plants of a summer squash cultivar through anther culture; therefore, sucrose was used at 30, 60, 90, 120 and 150 g l⁻¹and 2, 4-D was used at 0.1, 1.0, 2.5 and 5.0 mg l⁻¹on solid MS anther culture medium. Anthers at the mid or late uninucleate microspore stage without filament were excised from sterilized buds and plated on 20 different induction media. The most plantlets resulted from the induction medium supplemented with 150 g l⁻¹sucrose and 5 mg l⁻¹2, 4-D. Root tips from 20 plantlets were cytologically examined under a light microscope. The results revealed ten diploid (2n>= 2x= 40) and ten haploid (2n= x= 20) plants. This revised version was published online in June 2006 with corrections to the Cover Date.     

The improvement in regenerated doubled haploids from anther culture of wheat by anther transfer

This study was conducted to determine the most suitable method of regeneration by comparing two approaches: transfer of anthers (with and without embryo-like structures) to regeneration conditions after a period of two to four weeks on induction medium (= anther-transfer treatment) and transfer of embryo-like structures to regeneration conditions after five to eight weeks on induction medium. The early transfer of anthers brought about a significant reduction in the number of embryos formed, but nevertheless significantly improved the frequency of plant regeneration. Combining an optimal date of anther transfer with the early addition of colchicine to the induction medium (100 mg l⁻¹ for 1 and 3 days) led to an increase in the number of doubled haploid regenerants. The results indicate that transferring the anthers after 28 days and adding 100 mg l⁻¹ colchicine to the induction medium on one day only caused a significant improvement in the ability of green plants to regenerate (7.0 compared to 0.50) as well as in chromosome doubling (success index: 4.0 compared to 0.33). This revised version was published online in August 2006 with corrections to the Cover Date.     

Extension of anther culture to several genotypes of cultivated oats

 To improve plant regeneration from oat anther culture, the basic medium, hormonal supplements and genotype effect were studied. Six of the 14 genotypes tested regenerated plants. Cultivars Kolbu, Katri, Stout and naked oat Lisbeth produced green plants, cultivars Virma and line OT 257 only albinos. The total number of green plantlets regenerated was 22, of which 13 (11 haploid, 2 doubled haploid) survived into the greenhouse, and 37 albinos. Regenerable-type embryos were induced from heat-pretreated anthers on media containing 2, 3 or 5 mg l^–1 2,4-dichlorophenoxyacetic acid and 0.2 or 0.5 mg l^–1 kinetin as hormonal supplements. 6-Benzylaminopurine promoted albino plant regeneration especially in W₁₄ medium. Colchicine treatment was applied successfully to haploid regenerants. Received: 12 April 1999 / Revision received: 19 August 1999 / Accepted: 8 September 1999     

Positive effects of cold pretreatment, iron source, and silver nitrate on anther culture of strawberry (Fragaria × ananassa Duch.)

Androgenesis by anther culture or isolated microspore culture is the most efficient method for haploid production. In this study, the effects of cold pretreatment (4 °C), silver nitrate, and iron source in the medium were investigated on anther culture of five strawberry cultivars (Camarosa, Selva, Pajaro, Paros, and Gaviota) in three independent experiments, and three traits including percentage of androgenic anthers, embryogenesis, and callogenesis were determined. In the first experiment, cold pretreatment of 4 °C for 2 and 3 days produced the highest percentage of androgenic anthers in three cultivars (Camarosa, Selva, and Pajaro) and cold pretreatment of 4 °C for 2 days produced the highest percentage of androgenic anthers in cultivar Paros. In the second experiment, the effect of Fe-EDDHA was more effective than Fe-EDTA, and increased the percentage of androgenic anthers and embryogenesis. The use of 57.5 mg l^?1 Fe-EDDHA produced the highest percentage of androgenic anthers in cultivar Camarosa and the best embryogenesis in cultivars Camarosa and Gaviota. In the third experiment, the use of 15 mg l^?1 silver nitrate in medium significantly increased the percentage of androgenic anthers and embryogenesis in cultivar Camarosa.     

Improvements in the production of doubled haploids in durum wheat (<Emphasis Type="Italic">Triticum turgidum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C₁₇ induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars ‘Ciccio’ and ‘Claudio’ an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F₁ crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named ‘Lanuza’, has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.     

Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.)

Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F₁ hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microspore-derived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5 mg l⁻¹) and kinetin (0, 0.2 and 0.5 mg l⁻¹) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microspore-derived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microspore-derived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.     

Comparison of Anther and Microspore Culture in the Embryogenesis and Regeneration of Rye (Secale cereale)

Anther culture in solid and liquid medium and isolated microspore culture were compared in rye genotypes with potential agronomic characteristics. Some important factors influencing androgenic capacity were optimised. Three weeks cold pre-treatment of spikes and two days mannitol pre-treatment of anthers maximized callus and green plant yield in both culture methods. Intensity order of the culture methods in callus and green plant production was: isolated microspore culture, anther culture in liquid medium and anther culture in solid medium. Genotype ability of embryogenesis followed the same pattern in both cultivation methods. Kinetin (BA) with genotype dependent concentrations created the most effective regeneration conditions.     

Arabinogalactan proteins improve plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) anther culture

Androgenesis-based methods of doubled haploid (DH) production show considerable variation in efficiency in different barley genotypes. Arabinogalactan proteins (AGPs) have been shown to play a key role in several developmental processes, including embryogenesis, in different plant species. In this study we investigated the effect of exogenous AGPs from gum arabic on androgenesis and the regeneration efficiency in barley anther culture. Supplementation of the induction medium with 10 mg l^?1 gum arabic increased the total plant regeneration rate up to 2.8 times; when exposure to GA was extended to also include the pretreatment step, the regeneration rate was up to 6.6-times higher than in control. The effect of gum arabic was reversed by the Yariv reagent, an AGPs antagonist. This suggests a direct involvement of AGPs in androgenic development from barely microspores. Addition of gum arabic reduced cell mortality, increased the frequency of mitotic divisions of microspores and the number of multicellular structures (MCSs) when compared to control. The positive effect of gum arabic also included reduction in time required for the androgenic induction and substantially improved the quality of formed embryos. Observations made in this study imply a complex role of AGPs during androgenic development and confirmed the usefulness of gum arabic in production of barley androgenic plants.