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.     

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm³ 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm³ kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

     

A refined method for ovule culture in sugar beet (Beta vulgaris L.)

Induction of gynogenesis through ovule culture is a valuable tool to produce haploid and doubled haploid plants in sugar beet (Beta vulgaris L.). However, there is still large room for refining the method. In this study we investigated the gynogenic response of cultured ovules of three sugar beet genotypes, the effect of the application to inflorescences of different pretreatments with mannitol at 4ºC and with 5-azacytidine and 2,4-D, and the effect of the use of different basal culture media and sucrose concentrations. The response was evaluated in terms of percentages of induction of gynogenesis, embryogenesis and callogenesis, as well as of regenerated plants. We showed that a pretreatment with 0.5 M mannitol at 4 °C for 4 days, and with 50 µM 5-AzaC for 1 h, notably improved the percentage of embryogenesis and plant regeneration. Besides, the use of MS basal medium and 60 g/L sucrose was also found beneficial. This study provides new ways to improve the efficiency of haploid induction and plant regeneration through ovule culture in sugar beet, and is potentially applicable to ovule culture in other crops.

     

The effect of gamma irradiation dose on cucumber (Cucumis sativus L.) haploid embryo production

Pollination with irradiated pollen was the only effective way for the induction of haploid embryo in cucurbits. The possibility of using lower doses of γ rays (Co⁶⁰ source) was studied. The effect of 0.2 and 0.3 kGy of rays was tested on five cucumber lines and three hybrids in the first experiment. It was found that there was hardly any difference between the total number of embryos produced by all studied forms. The highest number of isolated embryos was obtained from hybrids Gy-3 M and BxOg (111 and 188 respectively). The plant regeneration was estimated at 3.3 %. The best two lines and one hybrid were used in the second experiment to find the lowest possible dose of irradiation. The dose 0.05 kGy produced only diploid embryos and was rejected as too low. Other three doses (0.1; 0.2; and 0.3 kGy) effected embryo development in relation to the irradiation applied with the highest number obtained at the lowest dose. However the number of plants regenerated from each combination was similar. The plant regeneration in this experiment was 7.7 %. The effect of 0.1 and 0.3 kGy was tested during the next two years on one highly vigorous variety. It was confirmed that 0.1 kGy stimulated the development of higher number of haploid embryos.     

In vitro production of haploid and doubled haploid plants from pollinated ovaries of maize (Zea mays)

Haploid induction has potential application for maize breeding. This paper reports that maize haploid plants have been induced by in vitro culture of pollinated ovaries. From a total of 26,400 cultured ovaries, 24 haploid plants were obtained and two of them were doubled after colchicine treatment. The maximum frequency of gynogenesis was 0.17% at 19.5 h post-pollination (HPP). The results showed that HPP was an important factor affecting plant induction from ovaries. Regenerated diploid R₀ plants were then subjected to genetic analysis using SSR molecular markers. One R₀ plant, whose progeny revealed a high level of homogeneity for several agro-morphological traits, was homozygous at 20 loci tested, with 11 showing paternal and 9 maternal banding pattern. This demonstrates that it is feasible to induce maize haploid plants by in vitro culture of pollinated ovaries.     

Improved chromosome doubling of parthenogenetic haploid plants of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) using colchicine,trifluralin, and oryzalin

An effective chromosome doubling protocol was established in essential garden crop of cucumber (Cucumis sativus L.) Cv. Hi Power. The different concentrations of colchicine (0, 250, 500, 750, and 1500 mg/L), oryzalin (0, 5, 15, 25, 50, 75, and 150 mg/L) and trifluralin (0, 5, 15, 25, 50, 75, and 150 mg/L) were applied on parthenogenesis-induced haploid nodal and shoot tip explants of cucumber for 18 and 38 h in three independent factorial experiments. Increasing concentrations of applied antimitotic agents led to the significant reduction in the survival rate of both shoot tip and nodal explants, especially in longer exposure duration. Three ploidy levels including haploid, mixoploid, and doubled haploid were regenerated form both explant types treated with colchicine, oryzalin, and trifluralin. Flow cytometry analysis proved successful chromosome doubling of haploid plants. Based on the results obtained, the highest number of regenerated doubled haploid plants (92.31%) and fruit set (86.21%) were related to immersion of nodal explants in 50 mg/L oryzalin for 18 h. The highest doubled haploid regeneration for colchicine and trifluralin antimitotic agents were 58.33 and 83.33%, respectively. The leaf size of doubled haploid plants was larger than their correspond haploids. The optimized chromosome doubling protocol would be applicable for doubled haploid production in garden crops of Cucurbitaceae family, which is recalcitrant to the spontaneous doubling, and also for in vitro polyploidy induction studies.     

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.

     

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.     

Production of doubled haploid rice plants (Oryza sativa L.) by anther culture

The results of anther culture of F₂ pollen issued from 23 single crosses are presented. A relation between the morphology of the panicle and the microspore stage was established. After cold-pretreatment (8 days at 4°C), the anthers were cultured on the callus-induction medium N6 supplemented with 1 mg l^–1 naphthaleneacetic acid. The calli were transferred to MS plant regeneration medium supplemented with 3 mg l^–1 kinetin + 0.5 mg l^–1 naphthaleneacetic acid. The induction frequency varied from 0.22% to 29% and the regeneration frequency from 0% to 144.4%, dependent upon the crosses used. On average, 27% of the plants obtained were albinos and 59% of the green plants underwent spontaneous chromosome doubling. Thirtynine doubled haploid lines were evaluated and multiplied in the field. Lines with an excellent behaviour in upland culture conditions were selected from two crosses.     

Improvement of anther culture methods for doubled haploid production in barley breeding

There is potential to accelerate cultivar development with a doubled haploid system for breeding line production. Anther culture methodology was evaluated for U.S.A. spring barley (Hordeum vulgare L.) breeding applications. Gelrite was found to be an acceptable replacement for ficoll in the induction medium to reduce costs while maintaining embryoid and plant production levels. Beneficial effects of 28 d cold pretreatment of donor spikes for anther culture were confirmed with Pacific Northwest USA barley genotypes. A 3 d mannitol solution pretreatment of fresh anthers was shown to be less effective for green plant production compared to 28 d cold pretreatment of donor spikes. Extended donor spike cold pretreatment from 28 to 42 d did not reduce anther culture productivity. Based on this research, anther culture techniques show promise for economical and convenient application in spring barley breeding.Abbreviations DH doubled haploid - LS Linsmaier and Skoog basal medium - BAP benzylaminopurine - GLM Generalized Linear Model - SAS Statistical Analysis System     

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.     

Efficient tissue culture and <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of haploid poplar derived from anthers

Calli were induced from anthers of Populus simonii × P. nigra. Haploid plants were then regenerated from the callus and multiplied efficiently by culturing leaf explants. The presence of both haploid and diploid cells in the same plant revealed spontaneous chromosome doubling in haploid cells. The haploid plants were transformed with the nptII gene by Agrobacterium-mediated method using leaf explants, and five independent kanamycin-resistant lines were obtained, with a transformation frequency more than 6%. Further PCR test indicated that the exogenous betA gene was transferred into these kanamycin-resistant lines, which were still haploid. Thus, the efficient tissue culture system and transformation of haploid poplar plants were achieved. Our study will contribute to forest improvement via the haploid culture and transgenic technology. Published in Russian in Fiziologiya Rastenii, 2007, Vol. 54, No. 4, pp. 629–633. The text was submitted by the authors in English.     

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α-trifluoro-2,6-dinitro-N,N-dipropyl- p -toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18-h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μ M trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.     

Enhanced recovery of doubled haploid lines from parthenogenetic plants of melon (<Emphasis Type="Italic">Cucumis melo</Emphasis> L.)

Recovery of doubled haploid (DH) progeny from haploid melon plants for use in breeding programs requires efficient chromosome doubling procedures. We describe improved procedures for recovery of fruits and viable seeds from parthenogenetic melon plants. Plant regeneration from nodal explants treated with 500 mg/L colchicine for 12 h was increased from 40 to 88% by transferring the treated explants to medium supplemented with a combination of growth regulators [5 μM IAA; 5 μM BA; 1 μM ABA; 30 μM AgNO₃). Prolonged exposure (2–7 days) to colchicine inhibited regeneration from nodal explants but had less effect on shoot tip explants. Many colchicine-treated plantlets flowered in vitro, allowing early assessment of their male fertility. Production of stained pollen in plants from nodal explants was highest after 0.5–2 days of colchicine treatment and on plants from shoot tips after 1–2 days. In vitro pollen counts correlated well with counts from greenhouse grown plants and with fruit set. The fruit set rate for colchicine-treated plants with a high pollen number was 47%. Appropriate colchicine treatment and culture of nodal explants as well as tip explants can substantially increase the number of fertile plants and DH lines recovered from parthenogenetic melons.     

Spontaneous versus colchicine-induced chromosome doubling in maize anther culture

The range of genetic variation of spontaneous chromosome doubling frequency of maize haploid plantlets derived from in vitro anther culture was evaluated. When regeneration is obtained by direct embryo-genesis, bypassing the callus phase, it appears that the frequency of spontaneous doubling may exceed 40 of the regenerated plantlets. This high frequency may be one consequence of the use of doubled haploid lines derived from anther culture and spontaneous chromosome doubling. We also report an increase, by more than 50, of the productivity of diploid fertile regenerated plantlets produced by colchicine supplemented medium during the cold shock pretreatment of the microspores inside the anthers. Optimization of the treatments and the anther culture procedure are discussed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Regeneration of fertile green plants from oat isolated microspore culture

Regeneration of fertile green plants from isolated oat microspores is reported for the first time. Factors critical for microspore growth and regeneration include cold pre-treatment, pH of culture medium and the use of conditioned culture medium. It was found that cold pre-treatment at 4°C in the dark for a minimum of 6 weeks was necessary to consistently achieve microspore growth into multicellular structures (MCS). Longer pre-treatments of up to 9 weeks were tested and found to be positively correlated with the number of MCS produced. Microspore culture medium with pH 8.0 produced significantly more MCS larger than eight cells in size than media with pH 5.8. The use of medium conditioned by actively growing barley microspores significantly increased the numbers of MCS larger than eight cells in size compared to non-conditioned media. Plants were regenerated only from cultures using conditioned medium. A total of 2 green plants and 15 albinos were regenerated. Of the green plants, one had the haploid chromosome complement (n = 3x = 21) and the other had the parental hexaploid chromosome complement (2n = 6x = 42) which may be due to spontaneous chromosome doubling. The hexaploid plant set seed naturally and the haploid plant set seed after its chromosome complement was doubled with colchicine.     

Pretreatment of seeds with thidiazuron delimits its negative effects on explants and promotes regeneration in chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.)

Thidiazuron (TDZ) is an important cytokinin-like substance used in plant tissue culture. However, long exposure of plant tissues to TDZ results in abnormal regeneration. The present study reports an efficient regeneration protocol that avoids the TDZ overexposure to chickpea (Cicer arietinum L.) tissues. Two separate experiments were designed for seed pretreatment with TDZ, of which the first one involved short-term pretreatment with various concentrations of TDZ (15, 20 and 25 µM), whereas the second one lacked TDZ pretreatment. Axillary meristem explants prepared from TDZ-pretreated and TDZ-non-pretreated seeds were then analyzed in shoot induction medium (SIM) with or without 4 µM TDZ. Thus, four conditions were chosen to analyze the effect of TDZ: (i) TDZ pretreatment only without TDZ in SIM, (ii) TDZ pretreatment along with TDZ in SIM, (iii) no TDZ pretreatment but there was TDZ in SIM, and (iv) there was neither TDZ pretreatment nor TDZ supplied in SIM (control). The response in terms of percent regeneration (69%), number of shoots per explant (20.66?±?0.5), minimum number of days taken for multiple shoot induction (7.3?±?0.5) and effective rooting was the highest under the condition (i) with 20 μM TDZ pretreatment without subsequent TDZ supplementation into the SIM. In addition, shoot elongation medium (SEM-3) having 5 μM benzyladenine, 2 μM kinetin and 2 μM gibberellic acid showed the highest branching and maximum shoot length. In conclusion, we reported an easy and efficient regeneration method in chickpea using TDZ pretreatment only, which showed qualitative multiple shooting and effective rooting.     

Heterozygous diploid plants regenerated from anther culture of F1 rice plants

Summary Plants derived from anther culture are theoretically haploid, but diploid plants are also known to arise. Anther culture-derived diploid plants are usually homozygous and are believed to be due to spontaneous doubling of chromosomes in either microsporocytes or callus cells during the culture process. However, heterozygous diploid regenerants may also arise from a) regeneration from cultured somatic cells, b) mutation occurring during or after a spontaneous doubling event, c) fusion of unlike haploid cells in chimeric callus, and d) regeneration from diploid microsporocytes resulting from aberrant meioses. This study was designed to elucidate the frequency and origin of diploid regenerants from rice anther culture. Regenerants were obtained from 11 F₁ genotypes. Progeny testing detected heterozygosity in 7 out of 211 regenerants. Each of the heterozygous regenerants were from ‘Calrose 76’/waxy ‘M-101’, Half of the diploid regenerants from this cross were heterozygous. No heterozygous regenerants arose from the other 10 F₁ genotypes. Progeny testing indicated that two of the heterozygous regenerants were as heterozygous as the F₁ plants for three parental characters. The other five regenerants exhibited decreased levels of heterozygosity. One of the heterozygous regenerants exhibited evidence of mutation for a non-parental character. However, mutation is an unlikely cause of the observed high levels of parental-type heterozygosity. No evidence for the occurrence of chimeric callus was detected, making this an unlikely cause as well. The most likely origin of the observed partial heterozygosity is regeneration from diploid microspores, which could also produce plants exhibiting complete parental-type heterozygosity.     

Isolated microspore culture of chickpea (<Emphasis Type="Italic">Cicer arietinum</Emphasis> L.): induction of androgenesis and cytological analysis of early haploid divisions

Routine production of haploid plants has not been reported for any legume, despite the major role these species play in sustainable farming systems and human nutrition. It is within this context that we report a protocol for the induction of haploid development in chickpea (Cicer arietinum L.) using isolated microspore culture. The cultivars “Rupali”, “Narayen”, and “Kimberley Large” were identified as responsive to isolated microspore culture. Flower bud length and microspore developmental stage were correlated for these cultivars. Depending on the cultivar, buds 2.85–3.5 mm in length contained uninucleate microspores. Microspores from donor plants grown in winter and spring were more responsive than those grown in summer. A cold treatment (4°C) of between 24 and 48 h enhanced microspore response in winter- and spring-grown material but was not effective in summer-grown material. A medium developed by the authors was effective for microspore induction and early-stage embryo development. The addition of hormones to this medium was promotive of microspore induction in winter- and spring-grown material, but not in summer-grown material. The initial haploid division predominantly occurred via symmetrical division of the vegetative nucleus. Further research is under way to convert pro-embryos into plants.     

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.