Production and conversion of haploid embryos in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.) anther cultures using high 2,4-D and silver nitrate containing media

Due to recalcitrant nature of chickpea (Cicer arietinum L.) to androgenesis, the production of double haploid plants has been only reported by Grewal et al. (Plant Cell Rep 28:1289–1299, 2009) using some physical stresses such as anther centrifugation and electrical shock. In the present study, we successfully obtained haploid plants from cultured anthers of two chickpea cultivars, Bivanij and Arman, using high 2,4-D and silver nitrate containing media without applying of these time and labor consuming stresses. For induction of androgenesis, different concentrations of 2, 4-D (0, 2, 5 and 10 mg/l) and silver nitrate (0, 5, 10, 15, 25 and 50 mg/l) were used in embryo development medium. In Bivanij cultivar, anther induction medium containing 10 mg/l 2,4-D and 15 mg/l silver nitrate produced the highest number of embryos (0.188) and regenerated plants (0.1) per each cultured anther, while the highest frequencies of embryos (0.1) and regenerated plants (0.075 and 0.063) were obtained from Arman cultivar when 10 mg/l 2,4-D was combined with 15 and 50 mg/l silver nitrate in anther culture medium, respectively. In second part of this study, different cold (4 °C for 4 and 7 days) and heat (30 °C for 10 days, 32 °C for 2 days and 35 °C for 8 h) pretreatments were applied on cultured anthers of Bivanij cultivar. Incubation of cultured anthers at 32 °C for 2 days significantly enhanced the rate of embryo formation up to 0.222 embryos per each anther, while the highest number of regenerated plants/anther (0.0332) was obtained when cold treated anthers at 4 °C for 7 days incubated at 30 °C for 10 days. Taken together, these results provide a good basis for large-scale generation of DH plants in this important legume species.     

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.     

Plant regeneration from carrot (<Emphasis Type="Italic">Daucus carota</Emphasis> L.) anther culture derived embryos

The research concerned of the regeneration of plants from embryos obtained from anther cultures of seven carrot (Daucus carota L.) cultivars. The aim was to determine the influence of the regeneration medium on the efficiency of the regeneration process. The optimization of the adaptation of the obtained plants was also carried out. Embryogenesis occurred on four of the tested media: B5 and MS without hormones, MS with charcoal, and MS with 1 mg dm⁻³ BA and 0.001 mg dm⁻³ NAA. Embryos obtained from the anther cultures produced secondary embryos, from which the regenerations of plants was observed. Secondary embryos were formed most extensively on the B₅ medium without hormones. The efficiency of the regeneration process depended on the cultivar. Most of the secondary embryos were formed by androgenetic embryos of the cultivar ‘Feria F₁’. The highest number of plants (102) regenerated from one embryo during 12 weeks of culture was also obtained in case of the cultivar ‘Feria F₁’. Secondary embryogenesis and plant regeneration from embryos allow to omit the difficult stage of root induction applied when plants are regenerated form shoots' explants. This makes the plant regeneration process quicker and easier. The plants regenerated by the conversion of embryos are better adapted to the ex vitro conditions than those obtained in the two-stage organogenesis involving the regeneration of shoots and in second stage roots induction.     

Androgenic potential in coconut (Cocos nucifera L.)

Conditions for induction of androgenesis in coconut cv. Sri Lanka Tall were studied. Anthers collected from inflorescences at four maturity stages were given heat (38°C) or cold (4°C) pretreatments for 1, 3, 6 and 14 days, either prior to or post inoculation. Three different basal media and different anther densities were also tested. Androgenesis was observed only in anthers collected from inflorescences 3 weeks before splitting (WBS) and after a heat pretreatment at 38°C for 6 days. Modified Eeuwens Y₃ liquid medium supplemented with 100 μM 2,4-dichlorophenoxyacetic acid (2,4-d), 0.1% activated charcoal and 9% sucrose was effective in inducing an androgenic response. The lowest anther density tested, 10 per petri plate, was found to be the optimal density. When androgenic calli or embryos were subcultured to Y₃ medium containing 66 μM 2,4-d, followed by transfer to Y₃ medium without plant growth regulators and finally to Y₃ medium containing 5 μM 6-benzyladenine (BA) and 0.35 μM gibberellic acid (GA₃), plantlets regenerated at a frequency of 7%. Histological study indicated that the calli and embryos originated from the inner tissues of the anthers. Ploidy analysis of calli and embryos showed that they were haploid. This is the first report of successful androgenesis yielding haploid plants from coconut anthers.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35?°C for a few days and by then incubating them in the dark at 25?°C. Pre-culturing anthers at 35?°C for 4?days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15?°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Improved callus formation and plant regeneration for shed microspore culture in asparagus (Asparagus officinalis L.)

 To establish an efficient asparagus microspore culture system, experiments were conducted to investigate the effects of medium components, period of cold pretreatment for flower buds, and period of anther co-culture on culture response. All factors affected the frequency of asparagus microspore division and the yields of microspore-derived calli. The best results were obtained by pretreating genotype G459 flower buds at 4  °C for 7–9 days, co-culturing anthers with shed microspores for 14 days, and including 6% sucrose, 2 mg l^–1α-naphthaleneacetic acid and 1 mg l^–1 N⁶-benzylaminopurine in the culture medium. After 4 days of culture, most shed microspores contained starch-like bodies and died. The 2% of shed microspores lacking these structures divided to produce microcalli. For the best treatments in the different experiments, about 140 calli per 100 anthers were recovered. Cultured on four different regeneration media, 19.6–21% and 3.9–8.0% of microspore-derived calli produced shoots and embryos, respectively, and ultimately plantlets, among which 49% were haploid, 34% diploid, 4% triploid and 11% tetraploid. Received: 3 September 1998 / Revision received: 25 November 1998 / Accepted: 5 December 1998     

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     

Improved embryogenesis from anther culture and plant regeneration in timothy

Timothy (Phleum pratense L.) is an important forage grass grown in northern temperate areas. Development of haploid cell culture techniques for timothy has been limited due to the recalcitrance of timothy in tissue culture. In this study, timothy anther culture techniques were established. Liquid PG-96 (Pulli and Guo, 1996) induction medium significantly promoted embryo yield; the best result was 800–1000 embryos (calli) per 100 anthers. Genotype was an important factor in androgenetic embryogenesis of timothy. Embryos were obtained from 16 genotypes out of the 28 genotypes tested. The optimum stage for microspore development was between the very late uninucleate stage and the binucleate stage. Cold pretreatment applied to the donor plants (spikes) increased embryo yield. Despite a high embryo induction rate, green plant regeneration rate was relatively low. The frequency of albinos was reduced by use of low light intensity conditions during regeneration. Over 300 green plants were recovered. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microspore embryogenesis induced through in vitro anther culture of almond (<Emphasis Type="Italic">Prunus dulcis</Emphasis> Mill.)

Anther culture is one of the most widely used methods to induce gametic embryogenesis. The aim of this investigation was to induce microspore embryogenesis in almond (Prunus dulcis Mill.), through this technique. Anthers were cultured at the vacuolated developmental stage, and seven cultivars, two culture media and two temperature treatments were assessed. Although evidence of the microspore induction was observed in all the genotypes and treatments tested (symmetrical nucleus division and multinucleated structures), calli were produced merely by anthers cultured in the medium P and the regeneration of embryos was detected only in anthers of the cultivars Filippo Ceo, Lauranne and Genco, placed on medium P and subjected to the Control treatment (direct culture at 25?±?1?°C, without the hot thermal shock at 35?±?1?°C for 7 days). Characterization by SSR marker analysis of the embryo genotypes revealed that the regenerants had a single allele for each locus whereas the parent cultivar was heterozygous, indicating their development from haploid microspores. This study reports the evidence of gametic embryogenesis and, particularly, of microspore embryogenesis through in vitro anther culture, in almond, and, for the first time to our knowledge, the production of homozygous embryos.     

Somatic embryogenesis and plant regeneration in date palm <Emphasis Type="Italic">Phœnix dactylifera</Emphasis> L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus

Plant regeneration through somatic embryogenesis from young leaf explants (5–10 mm long) adjacent to the apex of 5–6 year old offshoots of Tunisian date palm (Phœnix dactylifera L.), cultivar Boufeggous was successfully achieved. Factors affecting embryogenic callus initiation, including plant growth regulators and explant size, were investigated. The highest induction frequencies of embryogenic calli occurred after 6–7 months on MS medium supplemented with 10 mg l⁻¹ 2,4-D and 0.3 mg l⁻¹ activated charcoal. The subculture of these calli onto maintenance medium resulted in the formation of proembryos. Fine chopping and partial desiccation (6 and 12 h) of embryogenic calli with proembryos prior to transfer to MS medium supplemented with 1 mg l⁻¹ ABA stimulated the rapid maturation of somatic embryos. Maturated somatic embryo yield per 0.5 g FW of embryogenic callus was 51 embryos with an average maturation time of 55 days. This was increased to 422 with finely chopped callus, and 124 and 306 embryos following 6 and 12 h desiccation treatments, respectively. The average time to maturation for these 3 treatments was 35, 43 and 38 days, respectively. Subsequent substitution of ABA in MS medium with 1 mg l⁻¹ NAA resulted in the germination and conversion of 81% of the somatic embryos into plantlets with normal roots and shoots. The growth of regenerated somatic plants was also monitored in the field.     

Induction of somatic embryogenesis from female flower buds of elite <Emphasis Type="Italic">Schisandra chinensis</Emphasis>

Somatic embryogenesis (SE) was induced in female flower buds from mature Schisandra chinensis cultivar ‘Hongzhenzhu’. Somatic embryo structures were induced at a low frequency from unopened female flower buds and excised unopened on Murashige and Skoog (MS) agar medium containing 4.0 mg l⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D). Friable embryogenic calli were induced from somatic embryo structures after three to four subcultures on initiation medium. The frequencies of mature somatic embryo germination and plantlet conversion were low, but increased in the presence of gibberellic acid (GA₃). Some germinated somatic embryos could form friable embryogenic calli on medium without plant growth regulators (PGRs). The germination and conversion frequencies of somatic embryos from embryogenic calli induced using PGR-free medium were higher than for somatic embryos from embryogenic calli induced on medium containing 2,4-D. Most somatic embryos from 2,4-D-induced embryogenic calli had trumpet-shaped embryos, and most somatic embryos from PGR-free medium–induced embryogenic calli had two or three cotyledons. Histological observation indicated that two- and three-cotyledon embryos had defined shoot primordia, but most of the trumpet-shaped embryos yielded plantlets that lacked or had poorly developed meristem tissue. Cytological and random amplification of polymorphic DNA (RAPD) analyses indicated no evidence of genetic variation in the plantlets of somatic embryo origin.     

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.     

Haploid and doubled haploid plants from developing male and female gametes of Gentiana triflora

Protocols were developed for the generation of haploid or doubled haploid plants from developing microspores and ovules of Gentiana triflora. Plant regeneration was achieved using flower buds harvested at the mid to late uninucleate stages of microspore development and then treated at 4°C for 48 h prior to culture. Anthers and ovaries were cultured on modified Nitsch and Nitsch medium supplemented with a combination of naphthoxyacetic acid and benzylaminopurine. The explants either regenerated new plantlets directly or produced callus that regenerated into plantlets upon transfer to basal media supplemented with benzylaminopurine. Among seven genotypes of different ploidy levels used, 0–32.6% of cultured ovary pieces and 0–18.4% of cultured anthers regenerated plants, with all the genotypes responding either through ovary or anther culture. Flow cytometry confirmed that 98% of regenerated plants were either diploid or haploid. Diploid regenerants were shown to be gamete-derived by observing parental band loss using RAPD markers. Haploid plants were propagated on a proliferation medium and then treated with oryzalin for 4 weeks before transfer back to proliferation medium. Most of the resulting plants were diploids. Over 150 independently derived diploidised haploid plants have been deflasked. The protocol has been successfully used to regenerate plants from developing gametes of seven different diploid, triploid and tetraploid G. triflora genotypes.     

Haploid plant regeneration via embryogenesis from anther cultures of Hepatica nobilis

An anther culture technique for the production of haploid plants was developed in Hepatica nobilis. Embryos with bipolar meristem regions were induced from microspores within the cultured anthers. Embryo formation was promoted by first culturing anthers on NN medium (Nitsch and Nitsch, 1969) supplemented with 1% activated charcoal (AC) at 5 or 35°C for a few days and by then incubating them in the dark at 25°C. Pre-culturing anthers at 35°C for 4days (thermal-shock treatment) led to the best embryo formation (45 embryos/Petri dish with 30 anthers). Plant regeneration was achieved by culturing the anther-derived embryos on NN medium without AC at 15°C. Flow cytometric analysis of anther-derived embryos and chromosome counts in regenerated plants showed that they were haploid plants.     

Isolated microspore culture and plant regeneration in rye (Secale cereale L.)

 An isolated microspore culture and green plant regeneration method for rye (Secale cereale L.) was established. Rye isolated microspore androgenesis was genotype-dependent. PG-96M medium supplemented with 6% maltose gave the highest microspore survival rate after 48 h of culture and the highest embryo/callus yield (930 embryos/calli per 100 anthers from cv. Florida 401). Osmotic pressure in the induction medium played an important role. Pretreatment of the anthers with mannitol was beneficial for the microspore culture. Embryos/calli of a relatively younger age and smaller size had a higher regeneration ability, with the best green plant regeneration rate being 6%. Over 150 microspore-derived green plants have been obtained so far. About 90% of the regenerated plants were spontaneous doubled haploids. This is the first report of isolated microspore culture in true rye resulting in androgenic embryogenesis and plant regeneration. Received: 26 April 1999 / Accepted: 23 November 1999     

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.     

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.     

Histological study of somatic embryogenesis induction on zygotic embryos of macaw palm (<Emphasis Type="Italic">Acrocomia aculeata</Emphasis> (Jacq.) Lodd. ex Martius)

The aim of this paper was to describe the histological events that led to somatic embryogenesis in macaw palm (Acrocomia aculeata (Jacq.) Lodd. ex Martius). Zygotic embryos were inoculated on Y₃ medium containing 9 μM 4-amino-3,5,6-trichloropicolonic acid (picloram). Somatic embryos regenerated from nodular callus on induction medium with activated charcoal under photoperiod or without activated charcoal under dark. Many proembryos originated from the fundamental meristem after 10–20 days of culture. When transferred to medium containing activated charcoal, under photoperiod, calli regenerated into somatic embryos of unicellular origin. These embryos had protoderm, plumule and procambial strands and some of them could germinate. After 30–40 days of culture, meristematic masses grew from procambial cells. The masses generated nodular callus, and after transfer to medium without activated charcoal, under dark, they generated somatic embryos of multicellular origin. Those embryos did not regenerate into plants.     

Plantlet regeneration from protoplast-derived haploid embryogenic calli of wheat

To search for an alternative method for protoplast culture, regenerable embryogenic calli were obtained from anther culture of three wheat cultivars, Karl 92, Jinghua #1, and Pavon 76. Protoplasts were isolated directly from the haploid embryogenic calli and cultured in modified PMI and LM8P media without going through cell suspension culture. After 8–11 days of subculture, the embryogenic calli produced the maximum yield of protoplasts and cell division was at the highest frequency when plated at a density of 3–4 × 10⁵ protoplasts ml⁻¹. Frequency of colony formation varied from 0.2% to 0.5% for Jinghua #1 and from 0.1% to 2% for Pavon 76, while Karl 92 failed to produce colonies, even though its embryogenic calli were friable and fast-growing on the maintenance medium. Green haploid plantlets of Jinghua #1 and Pavon 76 have been regenerated from protoplasts, which were cultured on a differentiation medium first and then on a rooting medium. This revised version was published online in June 2006 with corrections to the Cover Date.     

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.