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.     

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.     

A brief temperature pulse enhances the competency of microspores for androgenesis in Datura metel

Androgenesis may be induced in plants by a stress application on microspores or anthers. Temperature stress treatments have generally been confined to a single temperature regime (above or below ambient) lasting from a few hours to days. We introduced a gradient with two temperature pulses (30 s each) in the stress application on anthers of Datura metel L. by stepping the temperature up and down for a total period of 60 s. Anthers were immersed in sterile water preheated and cooled to the desired temperature and cultured on Nitsch medium. The temperature pulse gradient significantly improved androgenesis compared to single temperature treatments, resulting in increased mean embryogenesis of 128% over control for 45°/15°C, 110% for 45°/10°C, 113% for 40°/10°C and 96% for 45°/5°C. The 45°/10°C gradient also significantly increased the number of dividing microspores observed, after 14 days of anther culture. Besides the differential of the gradient, the temperature limit was important, with anthers not tolerating temperatures beyond 45°C. The temperature pulse gradient applied at an early stage of culture may increase the window of competency of microspores for androgenesis.     

Effect of various exogenous and endogenous factors on microspore embryogenesis in Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern and Coss)

Summary The influence of donor plant growth environment, microspore development stage, culture media and incubation conditions on microspore embryogenesis was studied in three Indian B. juncea varieties. The donor plants were grown under varying environments: field conditions, controlled conditions, or a combination of the two. The correlation analysis between the bud size and microspore development stage revealed that the bud size is an accurate marker for donor plants grown under controlled conditions, however, the same does not hold true for the field-grown plants. The buds containing late uninucleate microspores collected from plants grown under normal field conditions up to bolting stage and then transferred to controlled environment were observed to be most responsive with genotypic variability ranging from 10 to 35 embryos per Petri dish, irrespective of the other factors. NLN medium containing 13% sucrose was found to be most suitable for induction of embryogenesis The fortification of this medium with activated charcoal, polyvinylpyrrolidone, colchicine, or growth regulators (6-benzylaminopurine and 1-naphthaleneacetic acid) was observed to be antagonistic for microspore embryogenesis, while silver nitrate (10 μM) had a significant synergistic effect. A post-culture high-temperature incubation of microspores at 32.5±1°C for 10–15 d was found most suitable for high-frequency production of microspore embryos. The highest frequency of microspore embryogenesis (78 embryos per Petri dish) was observed from the late uninucleate microspores (contained in bud sizes 3.1–3.5 nm irrespective of genotype) cultured on NLN medium containing 13% sucrose and silver nitrate (10 μM), and incubated at 32.5°C for 10–15 d.     

Isolated microspore culture of Chinese flowering cabbage (Brassica campestris ssp. parachinensis)

Microspores of several genotypes of Brassica campestris ssp. parachinensis have been cultured in vitro and induced to undergo embryogenesis and plant formation. Conditions favourable for embryogenesis in this species include a bud size of 2–2.9 mm, NLN-13 culture medium (Nitsch and Nitsch 1967; Lichter 1981, 1982; Swanson 1990), and an induction through exposure to 32°C for a period of 48 h. Longer periods of an elevated temperature for induction of embryogenesis resulted in embryo abortion at early developmental stages. With the protocol developed here, microspores of 60–80% of donor plants could be induced to produce embryos, although embryo yields were low, i.e. 2–5 embryos per 10 buds. Some genotypes responded to culture conditions with high numbers of embryo formation (100–150 embryos per 10 buds) but most of these subsequently failed to mature. The pattern of cell division and morphological changes of the microspores in culture were studied using various microscopic techniques.     

Effect of thermal shock in the development of androgenic plants of <Emphasis Type="Italic">Anemone coronaria</Emphasis> L.: influence of genotype and flower parameters

Anemone is an allogamous species marketed as a cut flower. Pure lines formation in this species is expensive and inefficient because progeny suffers from inbreeding depression. Pure lines obtained from plant anthers are a widely used material to produce new hybrids in short time. The aim of this study is the optimization of production of A. coronaria androgenic plants. Flower buds of eight cultivars were measured, disinfected for 20′ in 1% sodium hypochlorite and rinsed with distilled sterile water. Development stage of microspores and their size were evaluated. Anthers were distributed in Petri plates containing a double layer constituted of Nitsch and Nitsch agarized substrate enriched with activated charcoal (1%) and with or without sucrose (3%) and the same substrate in liquid phase without activated charcoal. Anthers were shocked with a preculture at 5 or 33 °C for 5 days and then incubated in the dark at 23 °C. After 21 weeks, the number of regenerants was counted. Plants were transferred into the flask with modified MS substrate and placed at 18 °C. After 3 months of in vitro culture, the survival rate of plants was evaluated. Among the varieties, significant differences in anther number, microspore size, and presence of abnormal microspores were observed. Heat shock pretreatment at 33 °C increases the number and survival of embryos and plants, but the varieties respond to the thermal shock in different ways. The influence of genotype and the maturation phase of flower buds, anthers and microspores are discussed related to the androgenic plant development.     

Plant regeneration from isolated microspore cultures of Chinese cabbage (Brassica campestris spp. pekinensis)

Isolated microspores of Chinese cabbage (Brassica campestris ssp. pekinensis) were incubated in modified NN medium containing 10% sucrose in darkness at 33°C for one day followed by culture at 25°C. After 14 days of culture, microspores developed into embryos ranging from globular to cotyledonary stage. Plants were regenerated after transfer of embryos to medium containing 3% sucrose and no plant growth regulators.Abbreviations NN Nitsch and Nitsch - MS Murashige and Skoog - NAA naphthaleneacetic acid - BA 6-benzylaminopurine     

Successful development of a shed-microspore culture protocol for doubled haploid production in Indonesian hot pepper (Capsicum annuum L.)

Various systems of anther and microspore cultures were studied to establish an efficient doubled haploid production method for Indonesian hot pepper (Capsicum annuum L.). A shed-microspore culture protocol was developed which outperformed all the previously reported methods of haploid production in pepper. The critical factors of the protocol are: selection of flower buds with more than 50% late unicellular microspores, a 1 day 4°C pretreatment of the buds, followed by culture of the anthers in double-layer medium system for 1 week at 9°C and thereafter at 28°C in continuous darkness. The medium contained Nitsch components and 2% maltose, with 1% activated charcoal in the solid under layer and 2.5 μM zeatin and 5 μM indole-3-acetic acid in the liquid upper layer. All the ten genotypes of hot pepper tested, responded to this protocol. The best genotypes produced four to seven plants per original flower bud. This protocol can be used as a potential tool for producing doubled haploid plants for hot pepper breeding.     

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.     

Production of microspore-derived plants by anther culture of an interspecific F1 hybrid between Cyclamen persicum and C. purpurascens

Anthers of a F1 hybrid (2n=41) between Cyclamen persicum (2n=2x=48) and C. purpurascens (2n=2x=34) were cultured to produce microspore-derived plants. Embryoids were produced when anthers, containing microspores at the early uninucleate stage of pollen development, were cultured in B5 medium containing sucrose (90 g l-1) and NAA (0.1, 1 mg l-1) or 2,4-D (0.1 mg l-1) in the dark at 5 °C for 4 days, then at 25 °C for 60 days. The embryoids usually developed into plantlets when cultured in B5 medium containing sucrose (30 g l-1) in the dark at 25 °C. At meiosis, the F1 hybrid, used as source for anther culture, formed some cells with restitution nuclei at telophase and dyads at the tetrad stage, which resulted in the production of viable pollen grains as unreduced gametes. Plants produced by anther culture were grouped into sterile plants with 2n=41 chromosomes and fertile plants with 2n=82 chromosomes. The present findings suggested that the sterile plants were polyhaploids originating from unreduced microspores (n=41) of the F1 hybrid and that the fertile plants were amphidiploids induced by a spontaneous doubling during culture of chromosomes of such unreduced microspores. This revised version was published online in June 2006 with corrections to the Cover Date.     

Induction and Growth of 'Microspore-Derived' Embryos of Brassica napus ssp. oleifera

Three cultivars of spring rape (Brassica napus ssp. oleifera),Tower, Willi and Duplo, were used for a study of induction andgrowth of ‘microspore-derived’ embryos, Buds, 2.0mm in length, containing uninucleate microspores were harvestedand stored for 14 d at 4 ?C in darkness. Anthers were then removedand cultured on a liquid medium based upon that of Murashigeand Skoog and containing 8% sucrose, 0.5 mg l^–1 naphthylaceticacid and 0.05 mg l^–1 benzylaminopurine. Cultures werepre-incubated at 35 ?C for 0–3 d and then incubated at30 ?C. After a total of 42 d incubation, cultures were scoredfor the presence of macroscopic embryos (1–2 mm in length)and for the presence of anthers containing abortive embryoidswhich had not developed further. The cultivars differed greatly in terms both of the frequencyof anthers showing induced embryoids and of the final yieldof embryos. Tower showed the highest frequency of induction(maximum 38% of cultured anthers with induced embryoids) whereasthe highest yield (equivalent to 1.1 embryo per cultured anther)was obtained from anthers of the cv. Duplo after a 3 d treatmentat 35 ?C. Yields from the other cultivars were much lower andwere relatively unaffected by the 35 ?C treatment. Key words: Brassica napus, Rape, Anther culture, Pollen, Haploid     

A microspore embryogenesis protocol for <Emphasis Type="Italic">Camelina sativa,</Emphasis> a multi-use crop

Camelina [Camelina sativa (L.) Crantz], a member of the Brassicaceae family, has a unique oil profile that has potential both for biofuels and as a food crop. It is essential to have a doubled haploidy protocol in order to enhance breeding of this crop for prairie conditions as well as improve the yield and quality characteristics. Microspore-derived embryos have been produced from Camelina sativa. Buds 1–3 mm in length were selected for culture. The microspores were isolated and purified in full-strength B5 extraction medium and cultured in NLN medium with 12.5% sucrose and 12.5% polyethylene glycol 4000 (PEG) without glutamine, at a density of 10,000 microspores per mL. Glutamine was added to the cultures 72 h after extraction to give a final concentration of 0.8 g/L. The microspore cultures were maintained at 24°C in the dark. After 28 days embryos were observed and these were regenerated to plants and selfed seed was produced. The highest embryogenic frequency achieved was 38 microspore-derived embryos from 100,000 microspores.     

Microspore embryogenesis of high sn-2 erucic acid Brassica oleracea germplasm

Brassica oleracea accessions possess traits that would be useful in commercial Brassica crops. These traits can be studied more effectively through the production of doubled haploid plants. Nineteen B. oleracea accessions from several subspecies possessing significant sn-2 erucic acid were screened for suitability for microspore culture using techniques well established for Brassica. Fifteen of the 19 accessions produced embryos. Genotypic differences were observed with embryogenesis ranging from 0 to 3000 embryos/100 buds. Embryogenesis was improved for two of four accessions by initiating cultures in NLN medium with 17% sucrose, then reducing sucrose to 10% after 48 h. An increase in embryogenesis for the same two accessions was observed when microspores were cultured at a density of 100 000/ml rather than 50 000 microspores/ml. A culture temperature of 32 °C for 48 h was beneficial for three of the four accessions when compared to a longer incubation period (72 h) or a higher temperature (35 °C). One accession line, Bo-1, was found to produce microspore-derived embryos which contained triacylglycerols with significant proportions of erucic acid at the sn-2 position. This revised version was published online in June 2006 with corrections to the Cover Date.     

Embryogenesis and haploid induction using anther culture in lovage (Levisticum officinale W.D.J. Koch)

An efficient and robust protocol to induce embryogenesis in lovage (Levisticum officinale W.D.J. Koch) has been developed. Immature anthers, with most of the microspores at the late uninucleate stage, were used as explants, and embryogenesis was induced in medium with combinations of plant growth regulators including α-naphthalene acetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and 6-benzylaminopurine (BAP). The frequencies of in vitro embryogenesis ranged from 0.42 to 18.25% depending on the combinations of plant growth regulators in the induction medium. Induced globular embryos successfully developed into heart and torpedo-staged embryos. Fresh anther explants produced the highest embryo formation rate (17.75%). Anthers treated at 4?ºC for 3, 5, or 8 d, significantly reduced the embryogenic response (to 3.52–7.85%). More embryos were induced when the sucrose content in the medium was increased from 3 to 6% (w/v), but significantly fewer embryos were produced when sucrose was 8% or more. Nearly 20% of fresh anthers were able to produce embryogenic structures when cultured on Murashige and Skoog medium supplemented with 10.74 μM NAA, 8.80 μM BAP, 9.05 μM 2,4-D, and 6% sucrose. Furthermore, when silver nitrate was added to the embryo induction medium at 90 μM, the frequency of anther browning decreased by 30% and the embryo formation rate increased to 24.75% of anthers cultured. In total, 418 plants were regenerated and cytological analysis confirmed 11 haploid lines from 187 samples randomly selected.     

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.     

Donor Plant Growth Factors Affecting Anther Culture of Maize and Sweetcorn (Zea mays)

Anthers from seven unselected commercial sweetcorn lines andten experimental maize lines were cultured on a liquid/solidbi-layer culture medium, containing 13 % maltose as the carbonsource. Mean anther efficiencies (number of embryos per 100anthers plated) of 0 to 27.6 % were recorded, with the maximumefficiency of 57.1 % from one plant. The anther efficiency wasfound to be dependent on genotype, microspore developmentalstage and the growth temperatures of donor plants. Immaturemicrospores were found to continue their development duringthe cold pretreatment of the spikes, and this in turn influencedthe level of response to culture. Direct regeneration of embryoidsto plants occurred most frequently when well formed uni- orbi-polar embryos were produced. The quality of embryo producedwas apparently inversely correlated with the number of embryosproduced. Zea mays, haploid culture, embryos, microspores     

Efficient embryogenesis and regeneration in freshly isolated and cultured wheat (Triticum aestivum L.) microspores without stress pretreatment

The major advantage of doubled haploids in plant breeding is the immediate achievement of complete homozygosity. Desired genotypes are thus fixed in one generation, reducing time and cost for cultivar or inbred development. Among the different technologies to produce doubled haploids, microspore embryogenesis is by far the most common. It usually requires reprogramming of microspores by stress such as cold, heat, and starvation, followed by embryo development under stress-free conditions. We report here the development of a simple and efficient isolated microspore culture system for producing doubled haploid wheat plants in a wide spectrum of genotypes, in which embryogenic microspores and embryos are formed without any apparent stress treatment. Microspores were isolated from fresh spikes in a nutrient-free medium by stirring and cultured in medium A2 in the dark at 25°C. Once embryogenic microspores were formed, ovaries and phytohormones were added directly to the cultures without changing the medium. The cultures were incubated in the dark at 25–27°C until the formation of embryos and then the embryos were transferred to regeneration medium. The regeneration frequency and percentage of green plants increased significantly using this protocol compared to the shed microspore culture method.Communicated by W. Harwood     

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.     

Microspore embryogenesis and the development of a double haploidy protocol for cow cockle (Saponaria vaccaria)

Factors affecting microspore embryogenesis of cow cockle (Saponaria vaccaria) were evaluated including donor plant growing conditions, genotype, bud size, density, medium composition, and culture conditions. Of the two donor plant (day/night) temperature regimes evaluated (10/5°C and 20/15°C), plants grown at 20/15°C were the most embryogenic. An embryogenic frequency of greater than 350 embryos/100 buds was observed in the most embryogenic genotype, cv. ‘White Beauty’. Buds from 3–9 mm in length were evaluated for their embryogenic potential; buds that were 4–7.9 mm produced the most embryos/100 buds. Of all the media compositions evaluated, NLN medium with 15% sucrose resulted in the most embryos. Cow cockle microspores required an initial period of 32°C for 3 days for production of microspore-derived embryos (MDEs).     

Embryogenesis from microspores of Ginkgo biloba L., a medicinal woody species

Summary The present work establishes that isolated microspores of Ginkgo biloba L. cultured at densities of 1.5 to 5·10⁴ per milliliter in Bourgin and Nitsch (1967) liquid medium are able to divide, both in the presence and in the absence of exogenous growth regulators, and to germinate by growing a pollen tube. In all experiments the microspores exhibited various modes of division leading to embryo formation in the liquid medium. Four weeks later, the microspores which had been previously submitted to various electrical stresses showed pro-embryo development earlier than those which had not. After ten weeks the number of embryos was found to be 300 to 5300 ml^–1 following the experiments. When the embryos exhibited a slower growth in liquid medium, they were transferred onto various solid media for maturation. Two months later, embryos had proliferated visibly.Abbreviations BN Bourgin and Nitsch (1967) medium - IAA Indole-3-acetic acid - KIN Kinetin - GS Growth substances