Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

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.     

Androgenesis,gynogenesis, and parthenogenesis haploids in cucurbit species

Haploids and doubled haploids are critical components of plant breeding. This review is focused on studies on haploids and double haploids inducted in cucurbits through in vitro pollination with irradiated pollen, unfertilized ovule/ovary culture, and anther/microspore culture during the last 30 years, as well as comprehensive analysis of the main factors of each process and comparison between chromosome doubling and ploidy identification methods, with special focus on the application of double haploids in plant breeding and genetics. This review identifies existing problems affecting the efficiency of androgenesis, gynogenesis, and parthenogenesis in cucurbit species. Donor plant genotypes and surrounding environments, developmental stages of explants, culture media, stress factors, and chromosome doubling and ploidy identification are compared at length and discussed as methodologies and protocols for androgenesis, gynogenesis, and parthenogenesis in haploid and double haploid production technologies.     

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.     

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.     

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.     

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.     

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.     

Production of doubled haploids through anther culture of M<Subscript>1</Subscript> rice plants derived from mutagenized fertilized egg cells

To produce stable mutants from Mankeumbyeo, a japonica rice (Oryza sativa L.) variety, we estimated the mutation efficiency of ethyl methane sulfonate (EMS) and N-methyl-N-nitrosourea (MNU) on fertilized egg cells using doubled haploids (DHs) derived from anther culture of M₁ plants. M₁ seed production and germination were higher in 1 mM MNU than in 94.2 mM EMS. A total of 68 DHs (35.4%) were regenerated by anther culture of M₁ plants. Twenty-one DHs (30.9%) were stable mutants, 14 DHs (20.6%) were unstable mutants, and the remainder (48.5%) were normal. The frequencies of stable mutants following EMS and MNU treatments were 20.7% (three semidwarfs, one early maturation and one glabrous line) and 38.5% (three semidwarfs, two early maturation, four glabrous and one long grain line), respectively. In a field trial of seven stable mutants for yield potential, five mutants did not show a significant difference in yield as compared with the original variety. Among these five, three glabrous mutants (MK-MAC 1, MK-MAC 4 and MK-MAC 26) with a smooth leaf and hull may be considered to be improved mutant lines because of the health benefits (reduced skin damage and generation of less dust compared to the original variety) to farmers handling the plant materials. MK-MAC 26, a glabrous mutant, had also less shattering resistance than that of the original variety. These stable mutants could be used as new breeding materials.Communicated by P.P. Kumar     

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.     

Anther culture for haploid and doubled haploid production

Haploids are plants with a gametophytic chromosome number and doubled haploids are haploids that have undergone chromosome duplication. The production of haploids and doubled haploids (DHs) through gametic embryogenesis allows a single-step development of complete homozygous lines from heterozygous parents, shortening the time required to produce homozygous plants in comparison with the conventional breeding methods that employ several generations of selfing. The production of haploids and DHs provides a particularly attractive biotechnological tool, and the development of haploidy technology and protocols to produce homozygous plants has had a significant impact on agricultural systems. Nowadays, these biotechnologies represent an integral part of the breeding programmes of many agronomically important crops. There are several available methods to obtain haploids and DHs, of which in vitro anther or isolated microspore culture are the most effective and widely used. This review article deals with the current status of knowledge on the production of haploids and DHs through pollen embryogenesis and, in particular, anther culture.     

Detection of androclonal variation in anther-cultured rice lines using rapds

Summary Random amplified polymorphic DNA analysis was used to determine the occurrence and extent of variation in rice (Oryza sativa L.) plants regenerated from anther culture. Androclonal variation in morphologically uniform progenies was detected using 40 10-mer oligonueleotide arbitrary primers. Among 27 plants from nine anther culture-derived lines, variation was detected in three plants from two lines by two primers, namely UBC 160 and UBC 209. Primer UBC 160 amplified a polymorphic band on one of the three progenies from DH-34, while UBC 209 detected polymorphisms on two out of three progenies from line DH-58. Apart from these, the amplification produets were monomorphic across all the regenerants from anther culture-derived plants. Out of 40 tested primers, no difference in the banding pattern was observed in three seed-derived plants. The significance of possible androclonal variation at the DNA level in rice doubled haploid breeding and genetic mapping is discussed.     

Evaluation of haploidization efficiency in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.) through anther culture

The production of large numbers of haploids is the crucial point of the dihaploidisation process. Although in vitro haploid plants were successfully produced by irradiated pollen technique in winter squash (Cucurbita maxima Duch.) and pumpkin (Cucurbita moschata Duch.), the frequency is still insufficient for using in a large-scale breeding programme. Thus, the present study was conducted to determine the efficacy of the anther culture technique on the production of in vitro haploids in the aforementioned species for which there have been no successful reports concerning by androgenesis. The anthers at uninucleate microspore stage were collected at different florescence times and then cultured on a solid MS medium supplemented by different combinations of 2,4-D (2,4-dichlorophenoxyacetic acid), BAP (6-benzylaminopurine), KN (kinetin) with the constant addition of NAA (naphthalene acetic acid) to induce callogenesis, embryogenesis and plantlet initiation. The combination of PGR, genotype and anther collection time played an important role in the androgenic response. The highest response was obtained from 57S?21 and G9 lines with the combination of 2.0 or 4.0 mg/l BAP?+?0.05 mg/l NAA (E6 medium) at the first anther collection time. Plantlets were regenerated and rooted on MS medium supplemented by 0.01 mg/l IAA. In total, 74 plants were recovered and propagated with micro-cuttings. The ploidy analyses revealed that 35 plants (47.3?%) were haploid (n?=?20), and the others (52.7?%) were diploid (2n?=?40).     

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.     

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     

Meiotic metaphase I to telophase II as the most responsive stage during microspore development for callus induction in tomato (<Emphasis Type="Italic">Solanum lycopersicum</Emphasis>) anther cultures

The generation of homozygous doubled haploid lines through induction of androgenesis is a promising alternative to the classical inbreeding and selection programs. However, this technology is poorly developed in tomato, where doubled haploid tomato plants have only been obtained through anther culture. Despite the fact that anther culture is routinely used in a number of economically interesting crops, there are still many drawbacks that prevent tomato breeders from adopting this technique, and improvements in methodology are required. One key issue is the correct identification of the optimal stage for anther excision and culture. In this paper we characterise in vivo microsporogenesis in tomato, defining the different microspore stages and relating them to the length of the donor flower bud. In parallel, we cultured anthers of these stages to obtain embryogenic callus, and followed the microscopic development of the callus contained within the anther. Our data suggest that the stage with the highest response, in terms of callus generation, is meiosis. In particular, we propose the window from metaphase I to telophase II, including tetrad cellularisation, as the timeframe where induction can be accomplished in tomato anther cultures.     

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     

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.     

Anther culture and Hordeum bulbosum-derived barley doubled haploids mutations and methylation

Anther culture and Hordeum bulbosum-derived doubled haploid (DH) lines of barley (Hordeum vulgare L.) were analyzed for RFLP and RAPD polymorphisms. Polymorphisms were not detected in the anther culture-or H. bulbosum-derived DH lines among 273 RFLP and 89 polymerase chain reaction (PCR)-amplified DNA fragments assayed. It was calculated that base substitution or small deletion/insertion mutations had not been induced among 401 640 by screened. Large deletion/insertion mutations were not observed among 33 Mb screened. Polymorphisms were observed when DNA was digested with the methylation-sensitive restriction enzymes HpaII and MspI: these RFLPs originated primarily from the anther culture-derived doubled haploids. The data indicate that heritable DNA methylation changes had occurred during DH production, particularly with the anther culture method.