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%.     

Organelle antioxidants improve microspore embryogenesis in wheat and triticale

Low frequency of green plant production and albinism limits the use of isolated microspore culture (IMC) in cereal breeding programs. The present study was conducted in triticale and bread wheat IMC to increase the production of green plants and minimize albinism. NPB-99?+?10% Ficoll induction medium was supplemented with mitochondrial or plastid antioxidants, in a completely random design, to evaluate their contribution to successful microspore embryogenesis and green plant production. Each group of antioxidants was tested independently: first in triticale and then validated in various spring wheat genotypes. While the response differed by wheat genotype, induction medium supplemented with proline (10 mM) yielded a greater number of embryos/embryo-like structures and green plants in both triticale and wheat. No differences were found with respect to albinism in triticale or wheat except for the cv. Sadash. Among plastid antioxidants tested, glutathione (2 μM) proved to be the best antioxidant to increase embryo and green plant production. Salicylic acid also helped to reduce the number of albino plants in triticale and the wheat genotype SWS366. Overall, induction medium supplemented with proline or glutathione enhanced microspore embryogenesis in both triticale and wheat and increased the number of green plants in the recalcitrant genotypes.     

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.     

Screening South African wheat germplasm for androgenic competence

Microspore and anther cultures provide an opportunity to create haploid and doubled haploid plants within a single season, thereby reducing the time and cost of cultivar development. Microspore and anther culture has been widely used and incorporated into wheat breeding programs in many countries, but little is known about the effectiveness of these techniques on South African germplasm. By using two responsive genotypes, isolated microspore culture was shown as more effective at revealing androgenic competence, and was used to evaluate the response of four South African inbred lines and two hybrids. Inbred lines A and B were highly responsive (336 and 207 embryo-like structures [ELS] per 100 anthers, respectively), line D was slightly responsive (5.1 ELS per 100 anthers) while line C was recalcitrant. The hybrid A × C was highly responsive (274 ELS per 100 anthers), and B × D did not respond at all. Green plant regeneration in a local genotype was very low (1% for line B) compared to that of foreign genotype (17% for Pavon 76). Similarly to other wheat genotypes grown around the world, the responsiveness of the South African varieties is also very variable. Thus, more efforts are needed so that isolated microspore culture can become a general tool in breeding programs.     

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.     

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

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

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).     

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     

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.     

Effects of colchicine on anther and microspore culture of bread wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

The aim of this work was to study the effects of colchicine application on chromosome doubling and androgenic response in anther and microspore culture of different bread wheat genotypes. Colchicine was applied during a mannitol stress pretreatment or during the first 48 h of culture at concentrations of 0, 150 and 300 mg l⁻¹. When colchicine was applied during stress pretreatment, the percentage of doubling depended on genotype and concentration. A significant increase in doubling was observed with 300 mg l⁻¹ in the low androgenic responding cv. Caramba. Colchicine incorporation during the first hours of culture improved percentage of doubling in all genotypes, in both anther and microspore culture. Application of 300 mg l⁻¹ colchicine improved the percentage of doubling in the two low responding genotypes, to 118% of control in DH24033, and 75% in Caramba in microspore and anther culture, respectively. Concerning the androgenic response, the effect of colchicine on embryo formation and percentage of green plants depended on the genotype and on the culture method. In cv. Pavon, a 2- and a 3-fold increase in percentage of embryogenesis and green plants, respectively, were obtained with 300 mg l⁻¹ colchicine in microspore culture. However, no significant differences in these two variables were observed in anther culture. The number of green doubled haploid (DH) plants reflects the index of success of the procedure. Regardless of the culture method, when colchicine was incorporated during the first hours of culture, the number of green DH plants increased significantly in three of four genotypes. These results confirm the usefulness of colchicine application during the first hours of culture in wheat breeding programs.     

Microspore development in cultured maize anthers

The present study follows in vivo and in vitro microspore development utilizing an anther culture-responsive maize genotype (Pa91×FR16) and a DNA-specific fluorescent dye (mithramycin). Cultured anthers were sampled at various times and scored for abnormal microspore divisions, multicellular masses, and embryo-like structures. The frequency of abnormal microspore divisions reached a peak during the first 7 days in culture and then declined. The vegetative nucleus was mitotically active in culture with over 50% of the induced microspores exhibiting this type of division. Multicellular masses and embryo-like structures first appeared in the 14 and 25 day samples, respectively. Most of the microspores did not reach the multicellular stage and an even greater mortality occurred during the formation of embryo-like structures.     

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.     

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.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

In vitro androgenesis of triticale in isolated microspore culture

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

Cefotaxime prevents microbial contamination and improves microspore embryogenesis in wheat and triticale

Key message

Cefotaxime (100 mg/l) mitigate occasional gram negative bacterial contamination in wheat and triticale microspore culture and most importantly it increases cell growth and green plant production.

Abstract

Isolated microspore culture is a promising option to rapidly fix the product of meiotic recombination of F₁ hybrids, in the process of varietal development. Clean culture and high embryogenesis rate are essential to commercial triticale and wheat microspore cultures. So, this study investigated (1) contaminants from isolated microspores cultures, (2) two antibiotics to control bacterial growth, and (3) the contribution of antibiotics to increased microspore-derived embryo-like structures (ELS), green and albino plants. Five species of bacteria were identified in contaminated cultures (Erwinia aphidicola, Pantoea agglomerans, Pseudomonas sp., Staphylococcus epidermis and Staphylococcus warneri) using fatty acid analysis and 16S ribosomal RNA sequences analysis, and yeast. Antibacterial susceptibility test using Cefotaxime and Vancomycin resulted in strong inhibition of 24 bacterial isolates, using Cefotaxime at 100 mg/l, but not Pseudomonas sp. Other antibiotic treatments inhibited bacterial growth at least partially. Microspore induction medium supplemented with the same antibiotics treatments resulted in successful microspore embryogenesis and green plant production. Antibiotic treatments were first tested in triticale and then validated in wheat cultivars AC Carberry and AC Andrew. Induction medium supplemented with Cefotaxime at 50 and 100 mg/l substantially increased the formation of ELS and green plants in triticale and wheat, respectively. Incidentally, it also affected the occurrence of albinism in all genotypes. Our results demonstrated dual purpose of Cefotaxime for isolated microspore culture, most importantly it increases cell growth and success of microspore cultures in triticale and wheat genotypes, but would also prevent accidental loss of cultures with most common bacterial contaminants.     

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.     

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.     

Isolated microspore culture overperforms anther culture for green plant regeneration in barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.)

Barley isolated microspore culture (IMC) was compared to anther culture (AC) for its efficiency in green plant (GP) regeneration. With six cultivars investigated, IMC resulted in significantly more GPs (3.6–287 per 100 anthers) than AC (0–29.6), which was on average 9.3-fold more efficient (113.7 vs 12.2). GPs were produced via IMC from all the genotypes tested, whereas no green shoot was generated by AC in two of the cultivars. In spite of genotype dependency for regeneration rates, the average GP percentage of IMC was just slightly higher than that of AC. Effects of microspore developmental stages and medium-sterilization methods on IMC were examined with the aim of optimizing culture conditions. We found that the optimum stage for cold pretreatment of spikes was different from that for mannitol starvation of anthers. Significant variations in microspore embryogenesis and regeneration were observed among five stages tested. Optimal stages for the two pretreatments were accordingly determined. Percentages of viable microspores were strongly influenced by protocols of medium-aseptisation. Although filter-sterilized media yielded two-time higher frequencies of living microspores and significantly more GPs than autoclaved ones, the filtration protocol was rather labor-intensive and time-consuming. Therefore a new procedure by combining filtering with autoclaving was subsequently developed as it was more effective than autoclaving and more convenient than filter-sterilization. The method described here could be useful for large-scale preparation of culture media.     

Microspore culture of small grain cereals

Androgenesis of wheat, rice and triticale was studied in isolated microspore culture. It is the first publication which studies microspore culture reaction of Hungarian rice varieties. The effect of different basic media, lack and absence of growth regulators in culture media were tested on important parameters of microspore culture. Direct embryogenesis was observed in microspore culture of wheat and triticale genotypes. In the case of rice, calli were induced in isolated rice microspore culture and haploid rice plantlets were regenerated via organogenesis.In wheat, the effect of basic media (W₁₄, A2, CHB₃, P4-m) was compared and among them the W₁₄, and A2 had a superior effect on embryo production and albino and green plantlet regeneration. In rice the C, CHB₃ and MS_m media were tested in microspore culture and the significantly highest numbers of calli were achieved by using C and CHB₃ media depending on the genotypes. The lack of exogenous growth regulators was observed in isolated microspore culture of triticale and rice. Growth regulator-free medium had a positive effect on embryo production and plant regeneration of triticale genotypes, whereas in rice microspore culture multicellular structures did not continue their division without growth regulators from the third week of microspore culture. Developing of microspore-origin calli was maintained by supplement of 2,4-D and Kinetin combination in the microspore culture medium.