Induction of adventitious shoots and tetraploids in Antirrhinum majus L. by treatment of antimitotic agents in vitro without plant growth regulators

We examined the effects of five antimitotic agents using Antirrhinum majus L. ‘Maryland True Pink’ on the induction of adventitious shoots resulted in increase of frequencies of chromosome doubling without plant growth regulators. Seeds were treated in vitro with 0, 16.5, 32.9, 65.8, 131.6, or 263.2 µM oryzalin (ORY), amiprofos-methyl (APM), butamifos (BUT), or propham (IPC) or 800, 1,600, 3,200, 6,400, or 12,800 µM colchicine (COL) for 7 day. ORY, COL and APM promoted induction of adventitious shoots on the hypocotyls at maximum frequencies of 57.6% with 16.5 µM ORY, 5.6% with 800 µM COL and 88.8% with 131.6 µM APM. ORY and COL also induced adventitious shoots on the epicotyls adjacent to the cotyledons, particularly at high concentrations, with a maximum frequency of 26.0% at 12,800 µM COL. APM treatment increased frequencies of tetraploids from 0.0 to 93.1%, with a positive correlation between the frequency and concentration. By contrast, ORY and COL induced tetraploids at frequencies of 16.0 to 54.6% and 4.0 to 59.4%, respectively, with peaks at both low and high concentrations of each. Correlation analysis revealed that frequencies of adventitious shoot formation could be useful as an index for the induction of tetraploids. These results showed that three of the antimitotic agents tested induced both adventitious shoot and tetraploid without plant growth regulators, indicating that antimitotic action may play a common role in the induction of adventitious shoot.     

Colchicine and oryzalin mediated chromosome doubling in different genotypes of Miscanthus sinensis

Different explant materials were treated with antimitotic agents to induce chromosome doubling in several Miscanthus sinensis clones. In vitro propagated plants established in soil, in vitro shoots, embryogenic callus, shoot apices and leaf explants were treated with different concentrations of colchicine or oryzalin. No tetraploids were obtained after antimitotic treatment of plants established in soil. The percentage of chromosome doubled plants after antimitotic treatment of single in vitro shoots was genotype dependent. Rooted in vitro plantlets were not a suitable target for antimitotic treatment, due to a high frequency of ploidy chimeras. Many tetraploid plants were regenerated after antimitotic treatment at the callus and explant level, but the efficiency was genotype dependent, primarily due to differences in the ability to form regenerable callus and to regenerate plants from embryogenic callus. Treatment of shoot apices with colchicine was the most efficient and reproducible system in the four genotypes tested. It was possible to repeatedly use the same colchicine-containing medium without any reduction in the induction of regenerable callus or in the percentage of tetraploids, thereby minimising the handling of this very toxic compound.     

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.     

The relationship between induction of embryogenesis and chromosome doubling in microspore cultures

Summary. The objective of this paper is to review the relationship between induction of microspore embryogenesis and chromosome doubling. It has been augmented with relative data on chromosome doubling by nuclear fusion. Some of the treatments used for induction of embryogenesis may also lead to doubling of the chromosome number, either through nuclear fusion or endomitosis. High frequencies of spontaneous chromosome doubling in cereal species appear to be induced by treatments that block cell wall formation during the first cell divisions, resulting in coenocytic cells in which the nuclei are able to fuse. The use of mannitol as a pretreatment for induction of embryogenesis in barley, wheat, and maize microspore cultures provides examples of nuclear fusion. The use of antimicrotubule agents for embryo induction via treatments during the first few hours of microspore culture has also resulted in high frequencies of chromosome doubling. Factors such as the doubling agent concentration, temperature during treatment, and duration of treatment may be critical for individual species. Actin filament as well as microtubule assembly studies related to new cell wall formation provide further evidence at the molecular level for the relationship between microspore embryogenesis and chromosome doubling. Correspondence and reprints: Department of Plant Agriculture, University of Guelph, Crop Science Building, Guelph, ON N1G 2W1, Canada.     

Antimitotic agents increase the production of doubled-haploid embryos from cork oak anther culture

The objective of this study is to induce the nuclear DNA duplication of anther-derived embryos of cork oak (Quercus suber L.) to obtain doubled-haploid plants. Anther culture of this species produces a low percentage (7.78%) of spontaneous diploids, as assessed by flow cytometry. Therefore, three antimitotic agents, colchicine, oryzalin and amiprophos-methyl (APM), were applied in vitro to anther-derived cork oak haploid embryos from six genotypes at different concentrations and for different treatment durations. Antimitotic toxicity was determined by embryo survival. Efficiency in inducing chromosome doubling of haploid embryos was evaluated by flow cytometry measurements and differences were observed between treatments. Nuclear DNA duplication and embryo survival of cork oak haploid embryos was most efficiently induced with oryzalin 0.01 mM for 48 h. Around 50% diploid embryos were obtained. The rate of chromosome duplication induced by APM 0.01 mM was also acceptable but lower than that induced by oryzalin, regardless of the duration of the treatment. Colchicine 1.3 or 8.8 mM was the least efficient, with the induction of necrosis and only a small rate of nuclear DNA duplication.     

Oryzalin combined with adventitious regeneration for an efficient chromosome doubling of trihaploid kiwifruit

Summary Chromosome doubling of one parthenogenetic trihaploid from cultivar Hayward ofActinidia deliciosa was investigated. Two antimitotic agents, colchicine and oryzalin, appliedin vitro on shoots and leaves at different concentrations were compared with regard to their efficiency. Survival and regeneration rates were determined and ploidy level of regenerated plantlets was evaluated by flow cytometry. Differences were observed between the two antimitotic agents depending on whether shoots or leaves were treated. Hexaploid plantlets were obtained with highest efficiency by adventitious regeneration from leaves treated by oryzalin at 5 M, constituting an original and promising result which was corroborated for another trihaploid clone. Dodecaploid plantlets were also induced but only from oryzalin treated leaves. On the other hand, colchicine applied to leaves was very phytotoxic. This study demonstrates that oryzalin combined with adventitious regeneration is particularly efficient to induce chromosome doubling of trihaploid kiwifruit.Abbreviations MS Murashige and Skoog - IBA indole-3-butyric acid - DMSO dimethylsulfoxide - PBS phosphate buffer salin - DTT dithiothreitol     

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.     

Patterns of DNA and chromosome variation during in vitro growth in various genotypes of potato

Patterns of variation in nuclear DNA content and chromosome number were analysed in a temporal sequence, during in vitro growth of calli and cell suspensions in two monohaploids, a dihaploid and a tetraploid of potato (Solanum tuberosum). The results showed that both polyploidization and aneuploidy occurred during the initial stages of callus induction in all the genotypes. With further growth of callus, the frequency and extent of polyploidy and aneuploidy increased. In addition, the patterns of DNA and chromosome variation in cell suspension cultures revealed continued mitotic activity and transmission of cells with higher ploidy levels and aneuploidy. The results suggest that endoreduplication as well as endomitosis are important mechanisms of polyploidization, and that chromosome lagging and non-disjunction contribute to the production of aneuploidy.The various genotypes cultured under the same in vitro growth conditions differed in genetic instability, as assessed from the rate and degree of polyploidization and aneuploidy. Monohaploids showed more rapid rate of polyploidization than the dihaploid and tetraploid potatoes. It was concluded that the differences in genetic stability were due to different ploidy levels and genetic make-up of the genotypes.     

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.     

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.     

In vitro induction of tetraploid garlic with trifluralin

Garlic (Allium sativum) is propagated asexually. Since sexual cross breeding is almost impossible, means for effective breeding are not currently available and the available production cultivars are seriously aged and degenerated. A possible alternative for breeding is chemical induction. Trifluralin, a type of herbicide, has been reported to provoke chromosome doubling. However, this chemical had not been tested on garlic. We tested various trifluralin concentrations and treatment durations for efficiency in the induction of tetraploid garlic. A clove base of garlic with a stem cv. Gailiang was used as the ex-plant to induce calluses on Murashige and Skoog (MS) medium; the calluses were then inoculated onto MS medium containing different levels of trifluralin and cultured to induce chromosome number variation in vitro. Garlic calluses were effectively induced via the ex-plant and both shoots and roots differentiated well on MS medium containing 6-benzylaminopurine at 3.0 mg/L and indole-3-acetic acid at 0.1 mg/L. However, increases in trifluralin concentration and treatment duration reduced the survival rate and differentiation rate of calluses. Garlic callus cultured for 15 days on medium containing 100 μM trifluralin gave the highest rate of chromosome doubling. Through observation of chromosome number in the root apical cells and the morphology of guard cells on the leaf epidermis of the regenerated plantlets, it was clear that chromosome number variation was induced and tetraploids were produced in vitro by trifluralin treatment.     

Correction to: Shoot regeneration process and optimization of <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation in <Emphasis Type="Italic">Sinningia speciosa</Emphasis>

The objective of this research was to induce mitotic chromosome doubling in Anemone sylvestris L. The mitosis inhibitor oryzalin was directly added to the induction medium at 1, 2, 5, 10 and 15 μM for 8, 10 or 12 weeks of cultivation. Three tetraploid plants (2n?=?4x?=?32), 0.8% (polyploidization efficiency), were obtained from diploid plants (2n?=?2x?=?16) in three treatments (1 μM for 10 weeks, 5 μM for 8 weeks and 8 μM for 10 weeks). Ploidy level was confirmed by flow cytometry. Morphological characteristics (e.g. flower diameter, total plant height, leaf area) and chlorophyll content differences between diploid and tetraploid A. sylvestris were observed together with polyphenol content and antioxidant activity. The inter primer binding sites markers were used for evaluation of polymorphism. New genotypes with different morphological and biological characteristics were obtained through somatic polyploidization. The tetraploid plants were stronger, more vigorous and had an early flowering, which is essential for its use as an ornamental plant. The iPBS analysis showed unique amplicons that can be used for the purposes of molecular identification of tetraploid plants of A. sylvestris in the future. The results demonstrate the first report of in vitro induction of tetraploids of A. sylvestris.     

Pathways to doubled haploidy: chromosome doubling during androgenesis

Production of doubled haploid (DH) plants through androgenesis induction is a promising and convenient alternative to conventional selfing techniques for the generation of pure lines for breeding programs. This process comprises two main steps: induction of androgenesis and duplication of the haploid genome. Such duplication is sometimes indirectly induced by the treatments used to promote androgenic development. But usually, an additional step of direct chromosome doubling must be included in the protocol. Duplication of the haploid genome of androgenic individuals has been thought to occur through three mechanisms: endoreduplication, nuclear fusion and c-mitosis. In this review we will revise and analyze the evidences supporting each of the proposed mechanisms and their relevance during androgenesis induction, embryo/callus development and plant regeneration. Special attention will be devoted to nuclear fusion, whose evidences are accumulating in the last years.     

植物离体组织染色体加倍诱导同源四倍体

随着生物技术的迅速发展,通过植物离体组织人工诱导多倍体已经成为获得多倍体植株的有效途径。本文就植物离体组织染色体加倍诱导同源四倍体的研究进展做一介绍,详细评述了植物离体组织细胞加倍的途径、影响植物离体组织加倍的因素、利用不同诱导剂进行处理效果比较及离体组织材料的早期倍性鉴定技术等,并展望了植物离体诱导同源四倍体的前景。     

广藿香毛状根多倍体诱导及其植株再生

为了提高药用植物广藿香的次生物质广藿香醇含量,采用秋水仙素人工诱导染色体加倍技术,进行了广藿香毛状根多倍体诱导及其植株再生、倍性鉴定和挥发油组分广藿香醇含量的测定。结果表明,广藿香毛状根多倍体诱导的最佳条件为0.05%秋水仙素处理36 h,其多倍体诱导率可达40%以上;经秋水仙素加倍的广藿香毛状根在MS+6-BA 0.2 mg/L+NAA 0.1 mg/L培养基中培养60 d后可获得毛状根多倍体再生植株。与对照(二倍体植株)相比,广藿香毛状根多倍体再生植株根系更发达、茎更粗、节间变短、叶片的长度、宽度和厚度均较二倍体明显增大。根尖细胞染色体压片观察证实,所获得的广藿香毛状根多倍体再生植株为四倍体,其根尖细胞染色体数约为128;同时,其叶片的气孔保卫细胞体积及其叶绿体数目均约为对照的两倍;但其气孔密度则随着倍性增加而下降,二倍体植株叶片的气孔密度约为四倍体植株叶片的1.67倍。GC-MS测定结果表明,广藿香毛状根多倍体再生植株的广藿香挥发油组分广藿香醇的含量为4.25 mg/g干重,约为二倍体植株的2.30倍。该结果证实毛状根多倍体化可提高药用植物广藿香的广藿香醇含量。     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

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.     

染色体数据的挖掘及其在植物多样性进化研究中的利用

多倍化（或全基因组加倍）是植物物种形成的重要途径,现存的被子植物可能都发生过一次甚至多次多倍化事件。多倍化传统的定义是染色体数目相对于祖先类群呈整倍性增加。其中最常用的研究方法是核型分析,核型能够提供物种的基本细胞学参数,包括染色体数目、倍性水平、核型不对称性、核型变异系数等。目前核型研究的趋势表现出从物种基本核型参数分析逐渐演化到多类群、多学科交叉融合的特点：一方面植物核型分析从种群、物种、科属的类群到生命之树,探讨染色体核型在各支系的进化特征、趋势以及驱动植物系统进化的细胞学机制;另一方面探讨和分析区域或生态系统植物区系的染色体谱或倍性等细胞学特征,可以探究区域地质环境变化或生态环境对染色体倍性等的影响,或通过区域染色体谱的构建,分析区域植物区系的形成和进化历史。因而,植物核型研究为系统发育、分子系统进化、生命之树以及植物区系地理的起源和演化研究提供了新思路。越来越多的新方法、新手段在植物核型分析与多倍化研究中得到运用,从而揭示了植物类群或植物区系的染色体进化以及细胞地理特征。今后植物细胞学研究趋势会向多学科交叉融合,整合各研究领域证据,从不同水平角度综合分析植物核型多样性形成的原因及意义,从而更加全面地认识和理解植物物种多样化与物种形成原因。     

染色体数据的挖掘及其在植物多样性进化研究中的利用

多倍化(或全基因组加倍)是植物物种形成的重要途径,现存的被子植物可能都发生过一次甚至多次多倍化事件。多倍化传统的定义是染色体数目相对于祖先类群呈整倍性增加。其中最常用的研究方法是核型分析,核型能够提供物种的基本细胞学参数,包括染色体数目、倍性水平、核型不对称性、核型变异系数等。目前核型研究的趋势表现出从物种基本核型参数分析逐渐演化到多类群、多学科交叉融合的特点:一方面植物核型分析从种群、物种、科属的类群到生命之树,探讨染色体核型在各支系的进化特征、趋势以及驱动植物系统进化的细胞学机制;另一方面探讨和分析区域或生态系统植物区系的染色体谱或倍性等细胞学特征,可以探究区域地质环境变化或生态环境对染色体倍性等的影响,或通过区域染色体谱的构建,分析区域植物区系的形成和进化历史。因而,植物核型研究为系统发育、分子系统进化、生命之树以及植物区系地理的起源和演化研究提供了新思路。越来越多的新方法、新手段在植物核型分析与多倍化研究中得到运用,从而揭示了植物类群或植物区系的染色体进化以及细胞地理特征。今后植物细胞学研究趋势会向多学科交叉融合,整合各研究领域证据,从不同水平角度综合分析植物核型多样性形成的原因及意义,从而更加全面地认识和理解植物物种多样化与物种形成原因。     

Regeneration of amphidiploid plants from tissue cultures of Allium wakegi

Callus was induced from the bulb of Allium wakegi Araki on MS semisolid medium supplemented with several growth regulating substances. The calli were subcultured every 40 days. At the time of every subculture the callus was subdivided to be used for chromosome studies, plant regeneration, or continuous callus multiplication. The chromosome constitution of cells in callus and regenerated plants varied over the culture period, and at the 3rd subculture amphidiploid plants were obtained. They appeared even more frequently than amphihaploid plants in the 4th subculture. Hypoamphihaploid regenerants appeared as stumpy shoots but none of these shoots proceeded further to form a normal plant. By Giemsa C-banded karyotype, the chromosome constitution of amphidiploid plants was found to result from exact doubling of the chromosome sets of amphihaploid common species. Amphidiploid plants show better viability and growth than common plants. The possibility and the expectation of new crop plants to be developed from amphidiploid plants will be discussed.