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     

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.     

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.     

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.     

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.     

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.     

In vitro induction of polyploidy in yacon (<Emphasis Type="Italic">Smallanthus sonchifolius</Emphasis>)

In vitro chromosome doubling was induced in octoploid (2n = 58) yacon using oryzalin and colchicine as mitotic spindle inhibitors. Nodal segments of in vitro cultured plants, 5–15 mm long, were exposed to 20, 25, or 30 μM oryzalin and 1, 3, or 5 mM colchicine for 24 or 48 h. The resulting ploidy level was determined by chromosome counting and flow cytometry. Out of 240 nodal segments, 3.33% hexadecaploid (2n = 116) plants were regenerated after the application of oryzalin. The greatest proportions of hexadecaploid plants (1.6%) were obtained after 48 h of 25 μM oryzalin treatment. With the colchicine treatment, only 0.42% hexadecaploid plants were detected and their survival rate was significantly lower in comparison with the oryzalin treatment. In hexadecaploid yacon, significantly higher levels of saccharides were detected (FOS 13.9 g/100 g FM, fructose 4.6 g/100 g FM and glucose 2.1 g/100 g FM) compared to the octoploid control (FOS 5.3 g/100 g FM, fructose 2.9 g/100 g FM and glucose 1.0 g/100 g FM). These results indicate that in vitro treatment of nodal segments with oryzalin solution could be an effective procedure for chromosome doubling and the polyploidy breeding can help to increase the FOS content in the tuberous roots.     

Doubled haploid plants following colchicine treatment of microspore-derived embryos of oilseed rape (<Emphasis Type="Italic">Brassica napus L.</Emphasis>)

An efficient method for producing doubled haploid plants of oilseed rape (Brassica napus L.) was established using in vitro colchicine treatment of haploid embryos. Haploid embryos in the cotyledonary stage were treated with one of four colchicine concentrations (125, 250, 500 and 1,000 mg/L); for one of three treatment durations (12, 24 and 36 h) at one of the two temperatures (8 and 25°C) and were compared to control embryos (without colchicine treatment). The number of chromosomes, seed recovery, size and density of leaf stomata, and pollen grain size from regenerated plants were determined. No doubled haploid plants were regenerated from control embryos; however, the doubled haploid plants were regenerated from colchicine-treated embryos. A high doubling efficiency, 64.29 and 66.66% of regenerated plants, was obtained from 250 mg/L colchicine treatment for 24 h and 500 mg/L colchicine treatment for 36 h, respectively, at 8°C. Following 500 mg/L colchicine treatment for 36 h, a few plants regenerated (9 plants). At the higher colchicine concentration (1,000 mg/L), no plant regenerated. These results indicate that the colchicine treatment of embryos derived from microspores can induce efficient chromosome doubling for the production of doubled haploid lines of oilseed rape.     

Antimitotic and hormone effects on green double haploid plant production through anther culture of Mediterranean japonica rice

Rice double haploid (DH) plants are produced mainly through anther culture. In order to improve the anther culture protocol, microspores of two japonica rice genotypes (NRVC980385 and H28) were subjected to three growth regulator combinations and four colchicine treatments on induction medium. In addition, a post anther culture procedure using colchicine or oryzalin was tested to induce double haploid plantlets from haploid plantlets. A cold pre-treatment of microspores for 9 days at 10 °C increased callus induction 50-fold in the NRCV980385 genotype. For both genotypes, 2 mg L^?1 2,4-D and 1 mg L^?1 kinetin on colchicine-free induction medium gave the best culture responses. The culturability of both genotypes changed on colchicine-supplemented induction media. A high genotype dependency was recorded for callus induction, callus regenerating green plantlets and regeneration of green double haploid plantlets. Colchicine at 300 mg L^?1 for 48 h enhanced callus induction 100-fold in H28. Colchicine-supplemented media clearly improved green double haploid plantlet regeneration. We showed that the post-anther culture treatment of haploid plantlets at 500 mg L^?1 of colchicine permitted fertile double haploid plantlets to be generated. Finally, an enhanced medium-throughput flow cytometry protocol for rice was tested to analyse all the plantlets from anther and post anther culture.     

Production of tetraploid plants of non apomictic citrus genotypes

Ploidy manipulation in Citrus is a major issue of current breeding programs aiming to develop triploid seedless mandarins to address consumer demands for seedless fruits. The most effective method to obtain triploid hybrids is to pollinate tetraploid non apomictic cultivars with pollen of diploid varieties. Such non apomictic tetraploid lines are not found in the citrus germplasm and need to be created. In this work we describe a new methodology based on in vitro shoot-tip grafting combined with treatment of the micro-grafted shoot-tip with colchicine and oryzalin to achieve chromosome doubling and a dechimerization procedure assisted by flow cytometry. Stable tetraploid plants of Clemenules, Fina and Marisol clementines and Moncada mandarin have been obtained directly from shoot tip grafting combined with colchicine and oryzalin treatments or after dechimerization of mixoploids plants (2x–4x). These stable tetraploid plants have been used in 4x × 2x hybridizations, to recover over 3,250 triploid hybrids in 3 years.     

In vitro induction of autotetraploids from diploid yellow passion fruit mediated by colchicine and oryzalin

In vitro chromosome doubling from hypocotyl segments of yellow passion fruit (Passiflora edulis Sims.) was carried out in the presence of either colchicine (0, 25, 250 and 1,250 μM) or oryzalin (0, 5, 15 and 30 μM). Murashige and Skoog (in Physiol Plant 15:473–497, 1962)(MS)-based regeneration medium containing 250 or 1,250 μM colchicine markedly affected explant development leading to browning and death of the hypocotyl segments. Oryzalin has similar effect to colchicine in inducing polyploidy. In vitro regenerated autotetraploid plants induced by 25 μM colchicine or 15 μM oryzalin were further acclimatized and cultivated in hydroponics system in greenhouse. Autotetraploids plants were more vigorous than the control diploids. The chromosome number of diploid plants was 2n = 2x = 18, whereas that found on autotetraploid plants were 2n = 4x = 36. The stomata sizes of the autotetraploids were significantly larger than those on the diploid counterparts, while the frequency of stomata was significantly reduced. Similarly, the chloroplast number of guard cells of autotetraploid plants increased significantly. Two albino plants (4%) were generated in medium with 25 μM colchicine, indicating phytotoxic effects. These plants are being grown to full maturity in order to test their potential to use in a breeding program.     

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     

In vitro induction of polyploidy in japanese quince (Chaenomeles japonica)

In vitro conditions for Japanese quince polyploidisation were investigated. Microshoots and isolated cotyledons were treated with colchicine and oryzalin. Morphogenesis was more dependent on the concentration of colchicine or oryzalin than on the duration of exposure, genotype differences were observed. Low oryzalin concentrations had no impact on morphogenesis. Plants with changed chromosome numbers were obtained at 0.25–38 mM colchicine and 10–50 μM oryzalin concentrations. It was determined that stomata length is a suitable parameter for identifying putative Japanese quince tetraploids. Stomata of tetraploid shoots of the same clone were approximately 1/3 longer than in the diploids. It was shown that through polyploidisation gigas effect was not obtained in fruit size but tetraploids have reduced seed set and an increased proportion of fruit flesh.     

In vitro induction of tetraploids in an interspecific hybrid of Calanthe (Calanthe discolor × Calanthe sieboldii) through colchicine and oryzalin treatments

Tetraploids were successfully produced from diploid seeds obtained through interspecific crossing between Calanthe discolor and Calanthe sieboldii by treating with colchicine or oryzalin. Colchicine was tested at concentrations of 0.05 and 0.1 % for 0, 3, or 7 days and oryzalin was tested at a concentration of 0.003 % for 1, 2, 4, and 7 days, and the ploidy of the seedlings was determined by flow cytometry. Tetraploids (4×) were obtained from the interspecific hybrid seeds treated with all colchicine and oryzalin concentrations. The most efficient condition for inducing tetraploids seemed to be treated with 0.003 % oryzalin for 1 or 2 days. Cytological and morphological evidence confirmed the results of flow cytometric analysis. The stomatal density and sizes of the tetraploid plants were significantly higher and larger than those of the diploid plants. Differences in leaf shape were found between the tetraploid and diploid plants under the same growing conditions: the leaves of the diploids were elongated and those of the tetraploids were round.     

Anti-microtubular herbicides and fungicides affect Ca2+ transport in plant mitochondria

The herbicides amiprophosmethyl (APM) trifluralin, and oryzalin as well as the fungicides methylbenzimidazolyl carbamate (MBC), O-isopropyl N-phenyl carbamate (IPC), and chlorisopropyl N-phenyl carbamate (CIPC), which are known to cause the destruction of microtubules in vivo but do not interfere with tubulin polymerization in vitro, have been examined with respect to their ability to affect Ca²⁺ transport in isolated cell organelles. In contrast to colchicine which has no effect on Ca²⁺ transport in isolated mitochondrial and microsomal fractions, all of the substances investigated caused considerable reduction of ca²⁺ net uptake into mitochondrial but not into microsomal fractions. This reduction has been shown to be due to an increase in passive Ca²⁺ efflux. These results have been extrapolated to in vivo situations where they are postulated to act by raising cytoplasmic Ca²⁺ levels.Abbreviations APM amiprophosmethyl - CIPC chlorisopropyl N-phenyl carbamate - IPC O-isopropyl N-phenyl carbamate - MBC methylbenzimidazolyl carbamate - Mops 3-(N-Morpholino) propanesulfonic acid - DMSO dimethylsulfoxide     

A comparison of the effects of various spindle toxins on metaphase arrest and formation of micronuclei in cell-suspension cultures ofNicotiana plumbaginifolia

The effects of the spindle toxins colchicine, oryzalin and amiprophos-methyl (APM) on metaphase arrest, chromosome scattering, and on the induction and yield of micronuclei were compared in suspension cells ofNicotiana plumbaginifolia (kanamycin-resistant “Doba” line). The inhibition of spindle formation is stronger with oryzalin and APM than with colchicine, which resulted in a more efficient accumulation of meta-phases with well-scattered chromosomes, allowing the isolation of single chromosomes. Further, APM and oryzalin treatments resulted in a higher frequency of micro-nucleated cells and greater yield of micronuclei than after colchicine treatment. The different actions of the chemicals on the functioning of the spindle, development of nuclear membranes around the chromosomes, formation of micronuclei and fusion of micronuclei, resulting in restitution nuclei, are discussed.     

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.     

Colchicine, an efficient genome-doubling agent for maize (Zea mays L.) microspores cultured in anthero

The construction of maize genotypes with high haploid induction capacity made it possible to study the effect of colchicine on maize androgenesis in vitro. Anther cultures of three hybrids were treated with 0.02% and 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine added to the induction medium had no negative influence on the androgenic responses (anther induction, induction of structures of microspore origin and their regeneration ability) of the genotypes examined. However, significantly higher fertility was observed in plants originating from colchicine-treated microspores, especially at 0.03%. Cytological examinations showed that colchicine treatment before the first microspore division efficiently arrested mitosis and resulted in homozygous doubled-haploid microspores. Under the experimental conditions, the antimitotic drug had no later effect on the division symmetry of the microspore nucleus, and unequal divisions remained dominant. Callus formation from the induced microspores seemed to be more typical (ranging between 60–70%), but embryo frequency was increased by approximately 10%, especially at the higher colchicine concentration. These results suggest that the mechanism of colchicine action in premitotic maize microspores may differ from that previously observed in wheat. Received: 15 June 1998 / Revision received: 17 September 1998 / Accepted: 3 December 1998     

Use of doubled haploid technology for development of stable drought tolerant bread wheat (Triticum aestivum L.) transgenics

Anther culture–derived haploid embryos were used as explants for Agrobacterium‐mediated genetic transformation of bread wheat (Triticum aestivum L. cv CPAN1676) using barley HVA1 gene for drought tolerance. Regenerated plantlets were checked for transgene integration in T₀ generation, and positive transgenic haploid plants were doubled by colchicine treatment. Stable transgenic doubled haploid plants were obtained, and transgene expression was monitored till T₄ generation, and no transgene silencing was observed over the generations. Doubled haploid transgenic plants have faster seed germination and seedling establishment and show better drought tolerance in comparison with nontransgenic, doubled haploid plants, as measured by per cent germination, seedling growth and biomass accumulation. Physiological evaluation for abiotic stress by assessing nitrate reductase enzyme activity and plant yield under post‐anthesis water limitation revealed a better tolerance of the transgenics over the wild type. This is the first report on the production of double haploid transgenic wheat through anther culture technique in a commercial cultivar for a desirable trait. This method would also be useful in functional genomics of wheat and other allopolyploids of agronomic importance.     

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.