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.     

Doubled haploid production in Flax (Linum usitatissimum L.)

There is a requirement of haploid and double haploid material and homozygous lines for cell culture studies and breeding in flax. Anther culture is currently the most successful method producing doubled haploid lines in flax. Recently, ovary culture was also described as a good source of doubled haploids. In this review we focus on tissue and plants regeneration using anther culture, and cultivation of ovaries containing unfertilized ovules. The effect of genotype, physiological status of donor plants, donor material pre-treatment and cultivation conditions for flax anthers and ovaries is discussed here. The process of plant regeneration from anther and ovary derived calli is also in the focus of this review. Attention is paid to the ploidy level of regenerated tissue and to the use of molecular markers for determining of gametic origin of flax plants derived from anther and ovary cultures. Finally, some future prospects on the use of doubled haploids in flax biotechnology are outlined here.     

Effect of the 1B/1R translocation on anther culture ability in wheat (Triticum aestivum L.)

Using two varieties, their reciprocal hybrids, F₈ lines and doubled haploids, results confirmed that three genetic components are involved in wheat anther culture ability, viz embryo induction frequency, regeneration ability and the frequency of albinism. In these experiments, no significant maternal effects were noticed. For embryo yields, transgressive lines were obtained from hybrids between distant genotypes. Regeneration of green plants depended upon two independent traits: regeneration ability and the frequency of albinos. F₈ lines and two doubled haploids equaled the 50% regeneration rate of the hybrids, but they only regenerated green plants. Based upon cytological examination and gliadin patterns, it is suggested that genes favoring regeneration ability could be linked to the 1BL-1RS translocated chromosome from Aurora.Abbreviations DH doubled haploids - MS Murashige and Skoog - MPG multicellular pollen grains     

Should maize doubled haploids be induced among F<Subscript>1</Subscript> or F<Subscript>2</Subscript> plants?

Maize (Zea mays L.) doubled haploid lines are typically produced from F₁ plants. Studies have suggested that the low frequency of recombinants in doubled haploids may reduce the response to selection. My objective was to determine if, for sustaining long-term response, doubled haploids should be induced in F₁ or F₂ plants during maize inbred development. In simulation experiments, I examined the response to multiple cycles of testcross selection among doubled haploid lines derived from F₁ plants (denoted by DH), doubled haploid lines derived from F₂ plants (DH_F2), and recombinant inbred (RI) lines derived by single-seed descent. For a trait controlled by 100 or more quantitative trait loci (QTL), the cumulative responses to selection were up to 4–6% larger among DH_F2 lines than among DH lines. The cumulative responses were up to 5–8% larger among RI lines than among DH lines. The QTL become unlinked as the number of QTL in a finite genome decreases, and the responses among RI, DH, and DH_F2 lines were equal or nearly equal when only 20 QTL controlled the trait. Metabolic-flux epistasis reduced the differences in the response among RI, DH, and DH_F2 lines. Overall, the results indicated that doubled haploids should be induced from F₂ plants rather than from F₁ plants. If year-round nurseries are used and new F₁ crosses for inbred development are initially created on a speculative basis, the development of doubled haploids from F₂ rather than F₁ plants should not cause a delay in inbred development.     

The agronomic performance of anther culture derived plants of barley produced via pollen embryogenesis

Anther culture was used to generate microspore-derived doubled haploid (DH) plants from four spring barley crosses. The culture medium used contained maltose as the sole carbohydrate source and the mode of plantlet regeneration was mainly via pollen embryogenesis. Both haploid and spontaneously doubled regenerants were produced and the doubled haploids were compared to recom-binant inbred lines generated by several rounds of selfing (single seed descent). Parental, DH and single seed descent (SSD) lines were grown in randomised, replicated field trials and the samples were scored for a range of agronomic traits. The mean performance and phenotypic distribution of the DH and SSD samples were similar and there was little evidence to support the conclusion that anther culture derived lines exhibit a reduction in vigour. Where significant differences were detected between groups these were mainly confined to crosses which were segregating for the denso dwarfing gene. The differential transmission of particular regions of the barley genome may therefore influence and confound the expression of agronomic traits in DH populations. This is the first report of the agronomic performance of anther culture lines produced via pollen embryogenesis and the results are discussed in relation to the exploitation of anther culture technology in barley breeding.     

Gynogenetic haploid plants analysis for agronomic and enzymatic markers in maize (Zea mays L.)

Summary Experiments were conducted to investigate whether selection occurs during the processes involved in the production of doubled haploids. Haploid plants produced from two hybrids, each heterozygous for isozyme markers, were subjected to genetic analysis. The distributions of doubled haploid lines and pedigree lines derived from the hybrid C123 x Oh7 were compared with regard to agronomic character. The results suggest that the populations of haploid plants obtained by in vivo gynogenesis represent a random gametic array. Thus, in order to introduce haploid plants into breeding programmes in maize, maternal haploidy seems to be a very attractive method.     

Induction conditions for somatic and microspore-derived structures and detection of haploid status by isozyme analysis in anther culture of caraway (Carum carvi L.)

Plant regeneration was obtained from cultured anthers and hypocotyl segments of caraway (Carum carvi L.). Microspore- and somatic tissue-derived embryos were compared by observation of the regeneration process under identical induction conditions. Fluorescent microscopy with DAPI staining showed initiation of cell divisions and formation of embryogenic callus and somatic embryos from anther sacs, with production of embryos of both microspore and somatic origin. Induction of somatic embryos from hypocotyl-derived callus was also demonstrated. Isozyme native polyacrylamide gel electrophoresis was used to identify haploids and doubled haploids, and to determine the frequency of spontaneous diploidization of regenerated plants of microspore origin. Donor plants (2n = 20) and their anther-derived derivative plants (n = 10, 2n = 20, 4n = 40) in callus stage or leafy rosette stage were compared. The esterase (EST) band patterns of regenerated plants differed from the heterozygous parental material, suggesting that the regenerated plants were microspore-derived haploid/doubled haploid plants. The similar profile of EST bands between the diploid anther-derived plants and a sample of the donor plants corresponded to a somatic regeneration pathway. Although the selected induction conditions revealed no preference for induction of microspore embryogenesis, the anther culture protocol established for caraway utilizing isozyme segregating EST loci markers is suitable for DH production.     

Rapid attainment of a doubled haploid line from transgenic maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) plants by means of anther culture

Summary We present a strategy for establishing a transgenic doubled haploid maize line from heterozygous transgenic material by means of anther culture. Compared to conventional inbreeding, the in vitro androgenesis technique enables a faster generation of virtually fully homozygous lines. Since the androgenic response is highly genotype-dependent, we crossed transgenic, non-androgenic plants carrying a herbicide resistance marker gene (pat, encoding for phosphinothricin acetyl transferase) with a highly androgenic genotype. The transgenic progenies were used as donor plants for anther culture. One transgenic and three non-transgenic doubled haploid lines have been established within approximately 1 yr. The homozygosity of all four doubled haploid lines was tested by analysis of simple sequence repeat (SSR) markers at 19 different loci. Polymorphisms were found between the lines but not within the lines indicating the homozygous nature of the entire plant genome gained by anther culture. Southern blot analysis revealed that the transgenic donor plants and their doubled haploid progeny exhibited the same integration pattern of the pat gene. No segregation of the herbicide resistance trait has been observed among the progeny of the transgenic doubled haploid line.     

A milestone in the doubled haploid pathway of cassava

This study was aimed at inducing androgenesis in cultured anthers of cassava (Manihot esculenta Crantz) to develop a protocol for the production of doubled haploids. Microspore reprogramming was induced in cassava by cold or heat stress of anthers. Since the anthers contain both haploid microspores and diploid somatic cells, it was essential to verify the origin of anther-derived calli. The origin of anther-derived calli was assessed by morphological screening followed by histological analysis and flow cytometry (FCM). Additionally, simple sequence repeat (SSR) and amplified fragmented length polymorphism (AFLP) assays were used for the molecular identification of the microspore-derived calli. The study clearly demonstrated the feasibility of producing microspore-derived calli using heat- or cold-pretreated anthers. Histological studies revealed reprogramming of the developmental pathway of microspores by symmetrical division of the nucleus. Flow cytometry analysis revealed different ploidy level cell types including haploids, which confirmed their origin from the microspores. The SSR and AFLP marker assays independently confirmed the histological and FCM results of a haploid origin of the calli at the DNA level. The presence of multicellular microspores in the in vitro system indicated a switch of developmental program, which constitutes a crucial step in the design of protocols for the regeneration of microspore-derived embryos and plants. This is the first detailed report of calli, embryos, and abnormal shoots originated from the haploid cells in cassava, leading to the development of a protocol for the production of doubled haploid plants in cassava.     

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F₁ crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C₁₇ induction culture medium with ovary co-culture. The optimum regeneration medium J25–8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.     

Tests for the presence of gametoclonal variation in barley and wheat doubled haploids produced using the Hordeum bulbosum system

Summary To investigate whether the Hordeum bulbosum system of doubled haploid production generates gametoclonal variation, populations of second generation doubled haploid lines were developed from first generation doubled haploid lines of two barley varieties and three wheat genotypes. In barley, no variation between doubled haploids from doubled haploids was detected for a range of quantitative characters, suggesting the absence of any gametoclonal effects. However, the original selfed-seed stocks were shown to contain cryptic allelic variation for some of the characters investigated. In wheat, gametoclonal variation was detected for ear emergence time, plant height and yield, and its components for two out of the three genotypes investigated. The type and range of variation was similar to that reported from studies of somaclonal variation from immature embryos and gametoclonal variation from anther culture. Generally, the effects appeared to reduce the yield performance of individual lines. The difference in response between the two species and the consequences for the use of the doubled haploid system in breeding programmes are discussed.     

Efficient production of doubled haploid plants through colchicine treatment of anther-derived maize callus

Summary A chromosome doubling technique, involving colchicine treatment of an embryogenic, haploid callus line of maize (Zea mays L., derived through anther culture), was evaluated. Two colchicine levels (0.025% and 0.05%) and three treatment durations (24, 48, and 72 h) were used and compared to untreated controls. Chromosome counts and seed recovery from regenerated plants were determined. No doubled haploid plants were regenerated from calli without colchicine treatment. After treatment with colchicine for 24 h, the callus tissue regenerated about 50% doubled haploid plants. All of the plants regenerated from the calli treated with colchicine for 72 h were doubled haploids, except for a few tetraploid plants. No significant difference in chromosome doubling was observed between the two colchicine levels. Most of the doubled haploid plants produced viable pollen and a total of 107 of 136 doubled haploid plants produced from 1 to 256 seeds. Less extensive studies with two other genotypes gave similar results. These results demonstrate that colchicine treatment of haploid callus tissue can be a very effective and relatively easy method of obtaining a high frequency of doubled haploid plants through anther culture.     

Anther cultures of Nicotiana tabacum L. mutants

Summary The theoretically expected and experimentally observed phenotypic ratios have been compared in populations of haploids derived from chlorophyll mutants of Nicotiana tabacum L. with a known genotypic constitution. The frequencies of mutant genotypes were significantly lower than the expected values, proving the existence of selection in a system of haploid embryoids developing in the anther.The anthers from M₁ plants of a diploidized Nicotiana tabacum haploid cv. Samsun, treated with various concentrations of N-nitroso-N-methylurea and n-butylmethane sulphonate, were cultivated in vitro. The number of anthers which gave rise to haploids (embryogenic anthers) was stimulated by lower concentrations of both the mutagens. The stimulation at the level of M₁ sporophyte is explained by internal genetic heterogeneity induced by adequate mutagen concentration. The average number of haploids per embryogenic anther decreased in all the treatments. The frequency of haploid plants of the mutant phenotypes increased with increasing mutagen concentration.     

Gametic and somatic embryogenesis through in vitro anther culture of different Citrus genotypes

In vitro tissue culture represents a useful technique for advancing Citrus breeding and propagation. Among in vitro regeneration systems, anther culture is commonly used to produce haploids and doubled haploids for a fast-track producing homozygous lines, in comparison with the traditional self-pollination approach, which involves several generations of selfing. In addition, anthers culture can produce somatic embryos that can also be used for clonal propagation. In this study, two thermal shocks were applied to the anthers of six Citrus genotypes (two clementine and four sweet oranges), just after they were put in culture. The response obtained was different depending on the genotype: both clementines, namely Hernandina and Corsica, produced homozygous and triploid regenerants (microspore-derived embryos), whereas all of the analyzed regenerants from sweet oranges, three cultivars of Tarocco and Moro, produced heterozygous and diploid regenerants similar to the parental genotypes (somatic embryos).     

Doubled haploid production via anther culture in annual,winter type of caraway (<Emphasis Type="Italic">Carum carvi</Emphasis> L.)

Caraway (Carum carvi L.) is a traditional medicinal and spice cross-pollinated plant species. Although in vitro techniques are recently extensively applied in plant breeding programmes, these are not commonly utilized in caraway. Therefore, based on the protocol for anther culture in carrot (Daucus carota L., a closely related species of caraway in Daucaceae family), in vitro androgenesis in caraway has been studied with the aim to produce completely homozygous inbred lines. Various induction conditions, such as temperature pretreatments, carbon sources and combination of growth regulators in a culture medium as well as the effect of genotype on in vitro androgenesis were examined. Ten breeding lines of winter caraway representing third generation of forced (artificial) self-pollination were used as donor plant material. Cultured anthers produced embryogenic calli, and subsequently two types of regenerated plants were obtained, namely haploids with evident microspore origin, and diploids which may represent somatic (anther wall) regenerants or spontaneous doubled haploids. The ploidy status of regenerated plants was determined by flow cytometry. This is the first report on androgenic doubled haploid production in caraway.     

Karyological characterization of sugar beet gynogenetic lines cultured in vitro

Flow cytometry was used to screen ploidy levels in 47 cultured in vitro sugar beet gynogenetic lines of various origin and age, obtained after plant regeneration from unfertilized ovules. When donor plants were diploid, the majority of regenerants were found to have cells with 1C, 2C and 4C relative DNA content (mainly haploid and diploid) and there were large differences in the rate of spontaneous in vitro chromosome doubling between individual homozygous lines. Six ovule-derived lines regenerated from fertile and sterile diploid donors of forty-five lines were solid diploids from the very early stages of their in vitro cultivation, and thus could not be classified as doubled haploids. In the case of tetraploid donor plants, the gynogenetic regenerants demonstrated 2x-ploidy level. The results obtained in chimeric plants with both haploid and diploid cells indicated the possibility to overcome mixoploidy by their re-cultivation through generative shoot tip culture. The flow cytometry method confirmed data obtained by conventional microscopic chromosome counting in dividing leaf cells and was found very useful for screening of a large number of regenerants and for characterizing the process of in vitro gynogenetic lines formation in sugar beet.     

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.     

Embryogenesis and doubled haploid production from anther culture in gentian (<Emphasis Type="Italic">Gentiana triflora</Emphasis>)

The overall goal of this study is to develop an anther culture system to produce doubled haploid (DH) lines of gentian (Gentiana triflora), an ornamental flowering plant, for use in an F1 hybrid breeding program. Embryogenesis was induced from anther cultures incubated on half-strength modified Lichter (NLN) medium containing a high concentration of sucrose (130 g/l) and subjected to heat shock treatment. Among the various parameters investigated, anthers collected from buds 9–12 mm in length induced the highest frequency of androgenesis. Moreover, among three genotypes tested, cvs. Ashiro-no-Aki and Ashiro-no-Natsu produced 21.3 and 3.7 embryos per 100 anthers, respectively, whereas, cv. Lovely-Ashiro failed to produce embryos. Among a total of 427 embryos transferred to a regeneration medium consisting of Murashige and Skoog (MS) medium, 138 plants were regenerated. The ploidy levels of regenerants were determined by flow cytometry and chromosome counts, revealing the presence of 5% haploids, 25% diploids, and 70% triploids. Inter simple sequence repeat (ISSR) analysis using the 6PS line obtained following self-pollination of the diploid plant obtained from anther culture confirmed that the diploid plant was indeed a DH.     

Doubled Haploid Production Using an Improved Anther Culture Protocol for Sorghum [<i>Sorghum bicolor</i> (L.) Moench]

Sorghum [Sorghum bicolor (L.) Moench] can benefit from accelerated breeding and release of improved varieties through doubled haploid technology. The technology has been used in speeding up the breeding of other major cereals such as wheat, maize and rice, for which generally widely applied optimised protocols exist. A reproducible protocol for the crop, that can overcome genotype dependency and other species-specific challenges such as phenolic exudation is however lacking. This study aimed at sorghum doubled haploids production thereby contributing to the development of an improved protocol. From the 28 hybrid genotypes, both F₁ registered- and experimental hybrids involved, this study successfully produced haploids from five genotypes and subsequently, four confirmed doubled-haploid lines on W14mf medium or its modification with 1.0 gl⁻¹ L-proline, 1.0 gl⁻¹ L-asparagine and 1.0 gl⁻¹ KH₂PO₄. Medium 190-2Cu was used for regeneration and rooting, which occurred successfully, if the calli were transferred on to it less than 7 days after induction, and temperature was maintained at 25˚C under light condition. Genotype dependency was not wholly overcome; however, sorghum’s high tillering ability and abiotic stress tolerance were observed to contribute to attainment of haploid plantlets. Spontaneous diploids producing seeds at rates of upto 80.5% were obtained, therefore eliminating the need for colchicine duplication.