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.

     

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.     

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.     

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.     

Colchicine-mediated chromosome doubling during anther culture of maize (Zea mays L.)

Efficient methods of chromosome doubling are critical for the production of microspore-derived, doubled-haploid (=DH) plants, especially if, as in maize anther culture, spontaneous chromosome doubling occurs infrequently. In the present study, colchicine (5–1000 mg/l) was added to the induction medium and maize anthers were incubated in the colchicine-containing medium for different durations (1–7 days). In order to improve overall anther culture response, the culture temperature was adjusted to 14°C during the first 7 days. Colchicine applied at low concentration, i.e. 5 mg/l (7 days), or for short duration, i.e. 1–3 days (250 mg/l), showed beneficial effects on the formation of embryolike structures (=ES) and thus led to increased plant production, but was comparatively ineffective regarding chromosome doubling. Optimal doubling effects were observed when anthers had been exposed to culture medium containing 250 and 1000 mg/l of colchicine (7 days); in these treatments the doubling index (=DI), defined as the quotient of the number of DH plants and the number of totally regenerated plants in a specific treatment, rose to 0.56 and 0.53, respectively, compared to 0.20 in the untreated control. However, colchicine administered at concentrations higher than 250 mg/l seemed to be detrimental to general plant production; thus, in spite of a high DI, the overall DH plant production was even lower than in the control treatment. Maximum DH plant production for three different genotypes was accomplished with culture medium containing 250 mg/l of colchicine (7 days). With the best-responding genotype (ETH-M 36) a DH plant production of 9.9 DH plants/100 anthers was accomplished, i.e. a 7-fold increase compared to the non-treated anthers. This is the first report on efficient chromosome doubling in anther culture by subjecting anthers to colchicinecontaining induction medium during a post-plating cold treatment. Chromosome doubling as described here becomes an integral part of the maize anther culture protocol and thus represents a rapid and economical way to produce DH plants.     

植物单倍体的产生、鉴定、形成机理及应用

单倍体(Haploid)是指含有配子染色体数目的个体,对其进行基因组加倍可以快速获得纯合双单倍体(Doubled haploid, DH)。单倍体和双单倍体在植物品种选育、突变体筛选、基因功能鉴定、细胞学研究、遗传群体构建等方面具有重要作用,是近年来植物领域的一大研究热点。本文从单倍体和双单倍体的产生途径、鉴定、形成机理以及应用等方面较全面地综述了单倍体的最新研究进展,为单倍体的研究利用作一定的参考。     

Ploidy of small individual embryo-like structures from maize anther cultures treated with chromosome doubling agents and calli derived from them

Summary In order to determine the ploidy of individual embryo-like structures (ELSs) following chromosome doubling treatments, a method was developed to determine the DNA content (ploidy level) of nuclei from single ELSs weighing as little as 1 mg using flow cytometry. About half (53%) of the ELSs which formed during anther culture of the maize inbred line used in control medium were haploid, 27% mixoploid and 20% diploid. Gibberellic acid (GA₃) increased the diploid percentage to 52% without affecting the mixoploid frequency (26%). A four day treatment with the chromosome doubling agent colchicine (50M) increased chromosome doubling while oryzalin eliminated the diploidy and mixoploidy. When regenerable callus cultures were initiated from the ELSs none were found to be mixoploid but the haploid and diploid proportions were similar to that of the ELSs analyzed. Regenerable cultures could not be initiated from the colchicine treated ELSs, however. These studies show that with the genotype used here, GA₃ and colchicine increased the amount of chromosome doubling of the ELSs while oryzalin and pronamide did not. The mixoploidy which existed in about 25% of the ELSs was never observed in calli apparently because these structures do not initiate callus or cells of only one ploidy level grew.Abbreviations ELS embryo-like structure - GA₃ gibberellin A₃     

同源四倍体水稻原种制备及影响因素分析

本研究已将从全国各地征集的50份水稻优良种质资源(2n=2x=24)诱变为同源四倍体水稻原种。本文讨论不同处理日期、不同秋水仙碱浓度和处理时间、以及不同品种的抗药性等因素对加倍成功率的影响,结果表明:①不同日期处理水稻幼芽,其加倍成功率不同,4月6日～15日加倍效果最好,加倍成功率达19.4%。②不同浓度秋水仙碱和处理时间其加倍成功率存在一定的差别。0.1%秋水仙碱处理48h 和0.2%处理24h 为佳,其芽恢复率超过58%,加倍成功率超过16%。③不同水稻品种抗药性不同。在所加倍的50个水稻品种中,中作9025抗药性最强,芽恢复率达92.4%,加倍成功率38.7%。盐州14抗药性最差,芽恢复率仅为10.6%。     

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.     

Direct effect of colchicine on the microspore embryogenesis to produce dihaploid plants in wheat (Triticum aestivum L.)

Summary In the present experiment, different chromosome reduplication techniques were applied to microspore-originated Triticum aestivum L. (cv Ciano) haploids. In addition to the conventional treatment (whole plant exposure to colchicine solution), spontaneously redoubled haploids were also examined. As an experimental treatment, different concentrations (0.01, 0.02, 0.04%) of colchicine were added directly to the induction media. Colchicine did not affect the anther response or the plant regeneration capacity. The success and stability of genome redoubling was estimated on the basis of the fertility of the regenerated (R₀) plants and their progeny (R₁). Chromosome doubling produced by colchicine before the first microspore mitosis was significantly more efficient than the conventionally used techniques.     

Induction of tetraploid poplar and black locust plants using colchicine: chloroplast number as an early marker for selecting polyploids in vitro

Tetraploid plants were produced by inducing chromosome doubling using colchicine in in vitro shoot tips of poplar and black locust clones. Many of the plants treated with colchicine showed modified morphological characteristics like stunted growth, thicker leaves and modified leaf morphology. The counting of chloroplast number in the epidermal guard cells of stomata was used for the rapid screening of tetraploids. The differences in mean chloroplast numbers between diploid and tetraploid plants were highly significant. For all plants tested, the tetraploid genotype had almost double the number of chloroplasts per guard cell compared to the diploid origin. Some plants were further analysed by flow cytometry to verify their ploidy status that was determined by chloroplast numbers. The results of this study demonstrated for the first time that chloroplast counting in poplar and black locust could be an effective and reliable method for pre-screening large numbers of plants for their ploidy level. The protocol might be applicable in a wide scope of breeding programs.     

Chromosome doubling of the bioenergy crop,Miscanthus×giganteus

The perennial grass, Miscanthus×giganteus is a sterile triploid, which due to its growth rate and biomass accumulation has significant economic potential as a new bioenergy crop. The sterility associated with the triploid genome of this accession requires labor‐intensive vegetative, instead of seed propagation for potential commercial production. Chromosome doubling was used to produce hexaploid plants in an effort to restore fertility to M×giganteus. Tissue culture derived calli from immature inflorescences were treated with the antimitotic agents, colchicine and oryzalin in liquid and solid media. Calli survival rate decreased with increasing concentrations and durations of colchicine or oryzalin treatments and ranged from 0% to 100%. Nuclear DNA content, as determined by flow cytometry, indicated that the frequency of chromosome‐doubled calli varied between compounds and concentrations with the greatest proportion of callus doubling observed using 2‐day treatments of 15 μm oryzalin (78%) or 939 μm colchicine (67%). Liquid media treatments were more effective than solid gels for chromosome doubling. Although oryzalin was effective at chromosome doubling, it inhibited callus growth and plant regeneration frequency. Seven hexaploid plants with doubled DNA content were generated, which displayed increased stomata size (30.0±0.2 μm) compared with regenerated triploid M. ×giganteus plants (24.3±1.0 μm). Following clonal replication these plants will be evaluated for growth rate, biomass accumulation, and pollen viability. Successful chromosome doubling and plant regeneration of M.×giganteus suggests that ploidy manipulation of this plant and its parental species (Miscanthus sinensis and Miscanthus sacchariflorus) could be a means to access genetic variability for the improvement of Miscanthus as a biofuel/bioenergy crop.     

大花蕙兰多倍体的高效诱导

以大花蕙兰‘红瀑布’无菌苗丛芽为材料、秋水仙素为诱变剂,采用不同的处理浓度、时间诱导大花蕙兰体细胞加倍。通过形态学和细胞学观察、统计等方法对其进行倍性鉴定。结果表明：秋水仙素浓度0.05%,处理时间24 h的条件下,诱导率高达28.2%;多倍体苗外部形态、叶绿体数目、气孔数目和大小与二倍体差异大,加倍后的细胞核明显变大,染色体倍数增加。     

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.     

PeanutDB: an integrated bioinformatics web portal for Arachis hypogaea transcriptomics

Background

Doubled haploid production is a key technology in triticale research and breeding. A critical component of this method depends on chromosome doubling, which is traditionally achieved by in vivo treatment of seedlings with colchicine.

Results

In this study we investigated the applicability of an in vitro approach for chromosome doubling based on microspore culture. Our results show a pronounced increase in the proportion of doubled haploid triticale plants compared to the spontaneous doubling rate, but also compared to the doubling obtained by the standard in vivo approach. In addition, the frequency of plants surviving from culture medium to maturity is also much higher for the in vitro approach. Colchicine concentrations of 1?mM for 24?h or 0.3?mM applied for 48 or 72?h during the first hours of microspore culture performed best.

Conclusions

Our results suggest that for triticale, in vitro chromosome doubling is a promising alternative to the in vivo approach.     

Cytogenetics of Lolium perenne

Summary A number of diploid inbred lines of Lolium perenne were treated with colchicine at the early seedling stage to induce chromosome doubling. In each inbred line the colchicine-treated undoubled diploids were kept as controls, as well as the normal untreated diploids. Comparisons of vegetative growth and development, involving the three treatments within each line, revealed that colchicine treatment of seedlings has long-lasting effects upon plant growth and development independent of chromosome doubling, and that for certain characteristics the effects of chromosome doubling are confounded with other effects of the treatment used to produce tetraploids. This colchicine induced variation in the diploids is transmitted through the seed generations in at least one of the inbred lines. The variation appears to be non-random and also shows a strong genotypic component. In so far as the effects of chromosome doubling could be determined, they showed the usual gigas response but were again strongly influenced by genotype.     

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.     

Novel technologies in doubled haploid line development

haploid inducer line can be transferred (DH) technology can not only shorten the breeding process but also increase genetic gain. Haploid induction and subsequent genome doubling are the two main steps required for DH technology. Haploids have been generated through the culture of immature male and female gametophytes, and through inter‐ and intraspecific via chromosome elimination. Here, we focus on haploidization via chromosome elimination, especially the recent advances in centromere‐mediated haploidization. Once haploids have been induced, genome doubling is needed to produce DH lines. This study has proposed a new strategy to improve haploid genome doubling by combing haploids and minichromosome technology. With the progress in haploid induction and genome doubling methods, DH technology can facilitate reverse breeding, cytoplasmic male sterile (CMS) line production, gene stacking and a variety of other genetic analysis.     

Somatic chromosome number doubling of selected potato genotypes using callus culture or the colchicine treatment of shoot nodes in vitro

Experiments were carried out to double the somatic cell chromosome numbers of a monoploid and dihaploid of Solanum tuberosum and a genotype of S. circaeifolium subsp. quimense. Colchicine was used in vitro on shoot nodes from which the axillary meristems had been removed. Plants with doubled chromosome numbers were obtained from shoots grown from the tertiary, sub-axillary meristems of all three genotypes. The callus culture of stem and leaf explants was found to produce more shoots with doubled chromosome numbers than the colchicine treatment in the case of the dihaploid and quimense genotypes but no shoots were obtained from callus culture of the monoploid. Fifty-two % of the shoots from the dihaploid and 63% from the quimense clone were ploidy doubled in the case of the best callus culture system. Using a sub-lethal dose of colchicine, the dihaploid yielded 37% ploidy-doubled shoots whereas all the shoots produced from the monoploid were doubled and the quimense clone produced 27% doubled plants. Callus culture was highly dependent upon the type of growth medium and other, unknown, factors. The colchicine treatment, although yielding fewer products, was more reliable for achieving ploidy doubling in selected clones if the number of plants produced is not important.     

In vivo haploid induction in maize

Two haploid-inducing lines, MHI and M741H, were used for the production of maternal haploids. Haploids were obtained from all maternal genotypes involved in the experiment, including dent, flint and flint×dent maize. The maternal genotype had a significant influence on the frequency of haploids obtained. The frequency ranged from 2.7% to 8.0%. For chromosome-doubling seedlings were treated with colchicine solution, and 49.4% of the haploid plants produced fertile pollen, 39.0% could be selfed and 27.3% produced seeds after selfing. Synthetic populations, improved by haploid sib recurrent selection, were tested in a field trial. The results show that the utilization of maternal haploid plants has great potential for maize breeding and maize genetics. Received: 14 May 2001 / Accepted: 3 August 2001