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.     

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.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

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.     

Improvement of isolated microspore culture of pepper (<Emphasis Type="Italic">Capsicum annuum</Emphasis> L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remény’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.     

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.     

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.     

Induced parthenogenesis in mandarin for haploid production: induction procedures and genetic analysis of plantlets

This study focused on haploid induction in mandarin through in situ gynogenesis by pollination with irradiated pollen of ‘Meyer’ lemon. Pollination was carried out for three genotypes of mandarin with four levels of gamma-ray-irradiated pollen (150, 300, 600, and 900 Gy). The resulting seeds were characterised by a small size. Embryos were rescued in vitro and the ploidy level of the plantlets was determined by flow cytometry analysis. Haploid, diploid, triploid plantlets were obtained. The haploid parthenogenetic origin was confirmed using microsatellite marker analysis and chromosome count. Diploid and triploid plants were the result of crosses between mandarin and lemon. The induction of gynogenetic haploids of ‘Fortune’ (Citrus clementina Hort ex Tan. × Citrus tangerina Hort ex Tan.) and ‘Ellendale’ (Citrus reticulata Blanco × Citrus sinensis L. Osb) is reported here for the first time.     

The effect of cold and heat pretreatments on anther culture response of Avena sativa and A. sterilis

The effect of stress pretreatments on embryo induction in anther cultures of selected genotypes of Avena sativa and A. sterilis was tested. A heat pretreatment of isolated anthers at +32°C for 5 days was best for the A. sativa line WW 18019 and for A. sterilis line CAV 2648. Genotype dependency may exist since in ‘Stout’ heat pretreatment did not increase embryo production. For A. sterilis 13 green and three albino regenerants were produced, of which five plants (haploids) survived transfer to the greenhouse. For A. sativa, 30 various differentiation media/treatment combinations were used in an attempt to regenerate plants from embryos, with no success. Seven day cold treatment of cut tillers increased slightly the response level in ‘Stout’ and was routinely used in subsequent experiments. Maltose proved to be better then sucrose as a carbon source for the genotypes tested. Fourteen percent maltose promoted the highest induction in A. sterilis, but the quality of embryos was improved in the presence of 10% maltose for both species. Sub-optimal carbohydrate levels did not enhance embryo induction in oats. This revised version was published online in June 2006 with corrections to the Cover Date.     

Barley anther culture: effects of annual cycle and spike position on microspore embryogenesis and albinism

The effect of donor plants annual cycle and anther/spike position on the production of microspore-derived plants and albinism were studied. We used the winter cv. Igri and the spring cv. Cork, known to respond similarly in anther culture but to produce 78% and 2% of green plants, respectively. In both cvs. the number of microspore-derived plants was significantly higher when the anthers were collected from January to July than from August to December. However, during this period the proportion of albino plants was not altered. Conversely, the anther response decreased from 76.6 to 31.5% in Igri and from 58.8 to 32.0% in Cork when the donor spike originates from the main shoot or the fourth tiller. Significantly, anthers collected from spike of the second tiller enabled us to drastically increase the proportion of regenerated green plantlets, by 16% in Igri and 1800% in Cork.     

Effect of amino acids,growth regulators and genotype on androgenesis in barley

The effects of an amino acid mixture and of plant growth regulators added to the FHG barley anther culture medium were examined using three barley cultivars (Cadette, Léger, and Igri) grown in two environments (growth cabinet and glasshouse). ‘Léger’ and ‘Igri’ were known as responsive, and ‘Cadette’ as recalcitrant to androgenesis. Our first experiment showed that the amino acid-supplemented medium was best for embryogenesis and regeneration of ‘Cadette’ and ‘Igri’ in both environments, and if ‘Léger’ in the growth cabinet. The addition of ABA and TDZ did not improve embryogenesis and plant regeneration, and PAA decreased them in the growth cabinet. The addition of the amino acid mixture in the FHG medium also reduced the percentage of albino plants in the growth cabinet, but growth regulators did not improve the percentage of albino plants, and in some cases increased it. In the growth cabinet, disregarding media, ‘Léger’ produced more embryos than ‘Cadette’ and ‘Igri’, and Léger' and ‘Igri’ produced more green plants than ‘Cadette‘. Percentages of albino plants were higher or ‘Cadette’ than for ‘Igri’ or ‘Léger’. In a second experiment, we compared seven hybrids with their parents for androgenic responsiveness. Hybrids had a higher ability to generate green plants than expected based upon the weighted average reflecting the contribution of each parent. This revised version was published online in June 2006 with corrections to the Cover Date.     

Anther culture responsiveness of Hordeum spontaneum derived spring barley lines and a genetic analysis of plant regeneration

Anther culture responsiveness of three H. spontaneum derived spring barley lines, RS170-47(A), RS20-1(B) and 1B-152B(C) was investigated using only one type of culture medium and treatment. The line 1B-152B was identified as highly responsive producing 22.4 total and 12.4 green regenerants per 100 anthers plated. 74% of these green regenerants were spontaneous double haploids. A genetic analysis involving F1 and F2 plants derived from crosses A × B and B × C revealed that the factor(s) determining high anther culture responsiveness in line 1B-152B was heritable and behaved as dominant in the F1. There was an indication that genotypic responsiveness in anther culture for green plant regeneration was different from total or albino plant regeneration.     

Direct regeneration of shoots from immature inflorescence cultures of turmeric

Microspore-derived embryo (MDE) induction was evaluated following the culture of wheat (Triticum aestivum L.) anthers in a basal medium with a range of supplements. Several supplements were evaluated, including a commercial bovine haemoglobin solution (Erythrogen™), Ficoll and the co-polymer surfactant Pluronic F-68, but these did not enhance MDE induction. However, phenylacetic acid, replacing both 2,4-dichlorophenoxyacetic acid and kinetin, increased MDE induction by a factor of 1.6 (p<0.05), producing 257±29 (mean±s.e., n=3) MDEs per 100 anthers cultured. Twenty-four plants regenerated from MDEs induced via anther culture were used for ploidy determinations; 33% were sterile haploids, while the remainder were fertile. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.     

Factors affecting somatic embryogenesis in anther cultures of Chinese pink <Emphasis Type="Italic">(Dianthus chinensis</Emphasis> L<Emphasis Type="Italic">.)</Emphasis>

In this study, we aimed to maximize the rates of somatic embryogenesis achievable in anther cultures of Chinese pink (Dianthus chinensis L.) (2n = 2x = 30). The genotype of the donor plant was found to be a major factor in determining the success rate. Conditions imposed during anther culture (notably medium composition and light conditions) and pretreatments (namely, cold, heat, and mannitol incubations) were also found to influence somatic embryo induction. For example, the highest levels of embryogenic callus induction were achieved when the donor buds had been cold pretreated and the subsequent anther culture was maintained in darkness. Furthermore, there appeared to be an interaction of genotype with culture conditions. Thus, in cultures of the cultivar (cv.) ‘Carpet’, the highest rates of embryogenesis were obtained when the anthers had received a 5-d heat-shock, but such a thermal treatment did not generally produce a significant effect. Likewise, a 3-d mannitol pretreatment was optimal only for the cross-hybrid line ‘HC’. Assessment of the ploidy of the plants regenerated from the anther cultures revealed both diploid and tetraploid plants. Histological and cytological observations showed that all of these (both from n-pollen and 2n-pollen lines) derived from anther wall cells. Spontaneous chromosome doubling was inferred to have occurred during the embryogenic callus culture period.     

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.     

Conservation and storage of a haploid cucumber (Cucumis sativus L.) collection under in vitro conditions

 Over 3 consecutive years (1992–1994), a collection of cucumber haploids was obtained from three different lines and one hybrid. Attempts were made to maintain and store a subcollection of these haploids for 3 years. Cucumber haploids appeared to be stable when cultured in vitro. There were no instances of spontaneous doubling and only one morphologically changed plant. During the first year of storage, between 30% and 80% the clones were lost, due to disturbances in plant development, increased levels of endogenous bacteria, and physiological changes resulting in continuous flowering. After 2 years of storage haploids showed reduced vigour. Therefore, plants were regenerated directly from primordial leaf microexplants. Haploid plants were obtained from nearly all of the previous haploid plants. The rejuvenated haploids possessed the same ploidy level and morphological traits as the old collection. The only new characteristic was faster vegetative growth. Received: 17 March 1998 / Revision received: 14 April 1999 / Accepted: 10 May 1999     

Activated charcoal induced high frequency microspore embryogenesis and efficient doubled haploid production in Brassica juncea

Three Indian Brassica juncea cultivars were studied for embryogenic response of microspores, microspore embryo regeneration, ploidy assessment of microspore-derived plants and their diploidization. Genotype dependence for microspore totipotency was observed and a significant effect of genotype by bud size selection was established. The addition of activated charcoal in NLN medium containing 13% (w/v) sucrose and 10 μM silver nitrate resulted in a fourfold increase in microspore embryogenesis, ranging from 100 to 405 embryos per Petri dish corresponding to 2,700–10,935 embryos per 100 buds. Conversion/germination of embryos produced in presence or absence of activated charcoal was similar but air-drying of microspore embryos was essential. Incubation of microspore embryos at 4 ± 1°C for 10 days in dark resulted in 82.3% conversion. The majority of plants produced from these embryos was haploid. Treating microspore-derived plants at the 3–4 leaf growth stage with 0.34% colchicine for 2–3 h resulted in greatest survival (70%) and chromosome doubling (75%) frequencies. Doubled haploid plants were self-pollinated and grown to maturity under field conditions.     

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.     

l-Ascorbic acid sodium salt promotes microspore embryogenesis and chromosome doubling by colchicine in ornamental kale (Brassica oleracea var. acephala)

Isolated microspore culture (IMC) represents a potential alternative technique in the plant breeding process, as it allows the effective production of doubled haploid (DH) homozygous lines. However, the implementation of this technique is limited by a low rate of embryogenesis, high level of embryo death, and low frequency of chromosome doubling. Thus, we investigated the effects of using different concentrations of L-ascorbic acid sodium salt (VcNa), which has never been applied for kale, to enhance the embryogenesis and regeneration by IMC. Specifically, 1 to 5 μM VcNa was added to the NLN-13 medium of four kale genotypes, while control was grown on VcNa-free medium. Overall, 1–4 μM VcNa at pH 5.84 increased embryogenesis, with 4 μM VcNa being the optimum concentration (12.92-fold increase). The proportion of embryo deaths declined when using appropriate VcNa concentrations. To increase the frequency of chromosome doubling, an artificial chromosome doubling protocol was developed for kale microspore-derived haploids. This protocol involved dipping roots of haploid plantlets in colchicine solution and adding colchicine treatment to solid Murashige and Skoog (MS) medium. Optimum chromosome doubling of haploids was achieved by dipping their roots in 750 mg/L colchicine solution for 4–6 h and 1000 mg/L colchicine solution for 2 h (doubling for nearly 50% of haploids). In conclusion, this study delineated an effective tissue culture process in promoting chromosomal ploidy of microspore-derived regenerated plants, allowing more microspores to be maintained that have excellent ornamental characteristics through crossbreeding.