In vitro chromosome doubling of Miscanthus sinensis

The aim was to develop an efficient chromosome doubling method for Miscanthus sinensis to enable the production of triploids and so avoid seed dispersal to the environment. Antimitotic treatments with colchicine or oryzalin were tested in M. sinensis cl. MS‐88‐110 on: (1) in vitro shoots and plants established in soil; (2) during propagation of embryogenic callus; and (c) during the initial stages of callus induction. All systems produced chromosome‐doubled plants. A higher percentage of tetraploids was found after antimitotic treatment at the explant or callus level compared with treatment of in vitro shoots or plants established in soil. In general, oryzalin was more toxic to plant material than colchicine. A higher frequency of chimeras was found among plants with altered ploidy level when the target was formed shoot buds compared with adventitious shoot formation from callus. Antimitotic treatment of embryogenic callus from shoot apices also resulted in a high degree of albinism.     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Doubling the chromosome number of bahiagrass via tissue culture

Crop improvement in bahiagrass (Paspalum notatum Flüggé) is limited by apomixis in most natural tetraploids, however, diploid sexual types occur. Production of sexual tetraploids by chromosome doubling will allow hybridization with apomictic tetraploids. Diploid bahiagrass (Paspalum notatum Flüggé) embryogenic callus tissue was exposed to three concentrations of three antimitotic chemical agents, colchicine, oryzalin and trifluralin. Callus was generated to plants and ploidy was evaluated by stomata size, mitotic chromosome counts, and flow cytometry. A total of 310 plants were verified as tetraploid of 1,432 plants that reached transplanting size. All treatments yielded 4x plants. The mean percentage success over all treatments was 22%, with means of 31% for oryzalin, 24% for colchicine and 16% for trifluralin. The high rates of success indicate that all agents can be successfully used to double chromosome numbers in bahiagrass. The percentage of 4x plants ranged from 9% (20 μM trifluralin) to 43% (20 μM oryzalin). Several treatments adversely affected regeneration. Mitotic chromosome counts are difficult and labor intensive in bahiagrass. Therefore, leaf stomata measurements were used as a preliminary screen. Data gave a bimodal distribution with overlapping tails and based on chromosome counts would have given an error rate of 12%. Flow cytometry analysis of regenerated plants resulted in mean nucleus fluorescence distributions consistent with control diploid or tetraploid values. These values agreed with chromosome counts, and this method is recommended for determining bahiagrass ploidy level. Research goals and available resources should be taken into consideration when selecting a treatment for chromosome doubling in bahiagrass.     

Efficient Production of Doubled Haploid Plants through Chromosome Doubling of Isolated Microspores in Brassica napus

Three methods of chromosome doubling to produce doubled haploid plants from microspore cultures of Brassica napus were compared: colchicine treatment of microspore-derived plants, microspore-derived embryos, and isolated microspores. In the whole plant treatment, 53% of the treated plants set seed, but the treatment delayed plant growth and reduced seed set. When microspore-derived embryos were treated with colchicine, the doubling frequency was 32% (compared to 15% for spontaneous doubling). Direct colchicine treatment of isolated microspores resulted in a doubling efficiency of 70 % of the whole plants. This treatment also stimulated embryogenesis in microspore culture, leading to increased plant regeneration. Thus, direct chromosome doubling of isolated microspores is efficient and more than 10 000 doubled haploid plants have been produced in this manner in the past three years in order to accelerate the plant-breeding process.     

Artificial polyploidy in medicinal plants: Advancement in the last two decades and impending prospects

Medicinal plants are in huge demand since the consumption is widespread and ever-increasing globally. The conventional breeding programs are generally environmental dependent; prone to different biotic and abiotic stresses as well as the secondary metabolite content is too low to harvest. In this context, developing polyploid individuals artificially would be a remarkable approach to increase vigor and attain this objective. Polyploids often exhibit some morphological features that are different or greater in forms than their diploid progenies. Polyploidization can be induced by quite a few antimitotic agents. The most frequently used antimitotic chemicals are colchicine, trifluralin, and oryzalin. The whole method of induced chromosome doubling consists of a series of steps, including an induction phase, regrowth phase, and a confirmation technique to evaluate the rate of achievement. The induction phase depends on different factors, such as explant types, antimitotic agents, its different concentrations, and exposure durations. To evaluate the accomplishment of polyploidization, morphological or anatomical observations are recorded as a rapid method. However, chromosome count and flow cytometry are the most eminent method for absolute confirmation. Despite significant prospects of polyploidization, there has been very little research on medicinal plants. The current review gives an overview of the different parameters of in vitro chromosome doubling, the history of the technique, and progress made over the last two decades.     

The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

In vitro induction of tetraploids in ornamental <Emphasis Type="Italic">Ranunculus</Emphasis>

This study investigates the capacity of the antimitotic agents colchicine, oryzalin and trifluralin for inducing polyploidisation of Ranunculus asiaticus ‘Alfa’ in vitro shoots. Flow cytometry was used to evaluate the optimal concentration of each antimitotic agent for polyploidisation. Trifluralin at a concentration of 2 μM resulted in the highest percentage of polyploidisation (27.5%), followed by a colchicine treatment of 200 μM, which induced 23.3% of polyploids. For oryzalin the highest percentage was achieved using a concentration of 1 μM. Different exposure periods were tested and turned out to be an important factor. The maximal exposure period tested (10 weeks) resulted in a significant increase in polyploidisation by oryzalin and trifluralin. In contrast, for colchicine (100 μM) exposure times of either 16 or 24 h did not significantly influence polyploidisation. Additionally the effect of the antimitotic agents on the viability was analysed. For colchicine no significant effect on the survival rate was observed, for trifluralin only a concentration of 10 μM affected viability whereas for oryzalin, concentration as well as exposure period were significant parameters. Flow cytometric data were confirmed by counting chromosomes in root tip cells.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.     

Polyploidization and early screening of Rhododendron hybrids

Polyploid induction represents a useful tool for breeders of floral crops as larger flowers, longer flowering period and deeper colors can be achieved through chromosome doubling. This study aimed at testing the efficiency of colchicine and oryzalin in inducing polyploidy in three Rhododendroncultivars grown in vitro. The chemicals were used in two concentrations with 24 h and 48 h treatment durations. The survival of the plants was better in colchicine than in oryzalin solutions. The higher concentration of both chemical skilled more plantlets. The treatment duration in oryzalin did not affect the survival, but 48 h in colchicine was more destructive than 24 h. The low survival rate may not be a disadvantage, if the treatment induces desired ploidy. The ploidy levels were screened with flow cytometry. Oryzalin was more efficient than cochicine in inducing polyploidy, the treatment duration and the concentration did not have significant effects as main factors. The biggest proportion of solid tetraploids (18.2% of the survived plants) was obtained from the 24 h treatment in 0.005% oryzalin. Immediately after the treatment the polyploids grew very slowly, whereas most of the unaffected diploids were vigorous from the very beginning. More mixoploids than solid tetraploids were obtained in all treatments. Most of the mixoploids retained their chimerism, one third shifted todiploidy and one single plant to tetraploidy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Antimicrotubule herbicides for in vitro chromosome doubling in Beta vulgaris L. ovule culture

In vitro chromosome doubling during ovule culture of sugar and fodder beets (Beta vulgaris L.) was studied with four anti-microtubule herbicides: amiprophos-methyl (APM), oryzalin, pronamide, and trifluralin at concentrations of 0–300 μM. Best chromosome doubling results were obtained by treatment of the ovules with 100 μM APM which produced 4.7 diploid plants per 100 ovules. Highest chromosome doubling was found with oryzalin using 1 μM, with trifluralin at 10 μM, and with pronamide at 10 μM producing 2.8, 2.0, and 2.0 diploid plants per 100 ovules, respectively. The APM treatments showed relatively low toxicity on embryo formation which in combination with a high chromosome doubling effect, resulted in up to 89 diploids per 100 plants regenerated. Oryzalin and trifluralin had more severe toxic effects, which reduced embryo formation, thereby lower percentages of chromosome doubled plants were obtained from these treatments. Pronamide had no significant toxic effect but it induced chromosome doubling at lower frequencies. Compared to colchicine, APM seems to be as efficient for chromosome doubling during beet ovule culture, but at molar concentrations 100 times lower than those used for chromosome doubling with colchicine. This revised version was published online in July 2006 with corrections to the Cover Date.     

RAPD analysis of sporting and chimerism in chrysanthemum

Summary The potential of colchicine and the microtubule depolymerizing herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling during B. napus microspore culture was studied. Colchicine was administered during the first 6, 12 or 24 h of culture with 8 different concentrations up to 3 mM, and herbicides at 6 different concentrations up to 30 M for 12 h.Treatments with moderate concentrations of colchicine (3–100 M) produced a small increase in embryo production, while concentrations above 300 M were toxic. Colchicine treatment for 12 h resulted in higher embryo production than treatment for 6 and 24 h. Duration of treatment and concentration of colchicine both had a significant effect on the chromosome doubling. The highest diploidization rates (94% diploid regenerants) were seen after 24 h treatment with 1 mM colchicine.All three herbicides were similar to colchicine in terms of their effect on embryo formation and chromosome doubling comparable to the one of colchicine, but at concentrations approximately 100 times lower. APM was less toxic than trifluralin and oryzalin, but no significant difference in chromosome doubling efficiency was detected between the compounds. The 12 h treatment resulted in a maximum of approximately 65% diploid regenerants with all three herbicides, but APM may have an advantage because of its less toxic effects. Prolonged treatment with APM (20–24 h) may produce 95–100% diploid regenerants.Abbreviations APM amiprophos methyl - DMSO dimethyl sulfoxide     

Chromosome doubling of pear haploid plants and homozygosity assessment using isozyme and microsatellite markers

The improvement of perennial fruit trees through traditional breedingmethods is a long-term effort because of their long generation time. Theproduction of haploid and doubled haploid plants should offer newpossibilities for genetic studies and breeding work. In this study, haploidclones of pear were treated in vitro by oryzalin for chromosomedoubling; the level of ploidy was assessed by flow cytometry. Oryzalinappeared to be an efficient agent for chromosome doubling, the optimalconcentrations range from 200 to 300 M. For homozygosityassessment, analyses of isozyme markers were carried out, together withmicrosatellite markers PCR-amplified with primers initially developed forapple. The use of isozyme markers confirmed homozygosity of all thedoubled haploid clones except for one. The microsatellite markers can beused earlier than isozymes for checking homozygosity during theprogramme of haploid and doubled haploid clones production. Truedoubled haploid clones of pear were obtained in less than one year andtheir acclimatisation in greenhouse has already started.     

Improving in vitro induction of autopolyploidy in grapevine seedless cultivars

The efficiency of in vitro polyploidization depends on several variables associated to the plant, the antimicrotubule agent and the interactions between them. In the present work, we have used response-surface methodology to determine the best operating conditions for plant recovery in polyploidization assays for shoot apices and somatic embryos of two seedless grape cultivars, employing colchicine and oryzalin. Explant type, tubulin-interfering compound and concentration were the critical factors determining plant recovery. Linear reduction in viable plant regeneration via organogenesis and somatic embryogenesis was obtained by increasing oryzalin concentrations and treatment time, whereas the effects of colchicine were better described by a quadratic design for both explants types. The conditions promoting higher rates of plant recovery were used in chromosome doubling experiments with oryzalin and colchicine for shoot apices and somatic embryos of ‘Crimson seedless’ and ‘BRS Clara’. The established protocols allowed the recovery of non-chimerical autotetraploid plants at rates higher than 30 % for both cultivars. Stomata size parameters statistically correlate to the ploidy level of the regenerants and were effective for preliminary polyploidy screening. Autotetraploid lines of seedless grapes were incorporated into the Vitis germplasm bank for further agronomical evaluations. To our knowledge, this is the first report of in vitro oryzalin induced polyploidization of grapevine and of the use of mathematical modeling to optimize chromosome doubling in plants.     

Role of mitotic inhibitors and genotype on chromosome doubling of <Emphasis Type="Italic">Rosa</Emphasis>

Dinitroanilines represent a class of compounds that are widely used in herbicide formulations as they depolymerise plant microtubles, causing chromosome doubling. The potential of microtubule depolymerising herbicides trifluralin, oryzalin, and amiprophosmethyl (APM) for in vitro chromosome doubling of Rosa was studied. Five concentrations (0, 3, 6, 12 and 24 μM) and three exposure periods (12, 24 and 48 h) for each of the compounds were compared. Oryzalin, trifluralin and APM were not significantly different in their ability to induce chromosome doubling of R. hybrida cv Iceberg. At concentration of 6 μM and exposure period of 24 h, chromosome doubling of R. hybrida cv Iceberg was not significantly different with each of the polyplodising agents. At higher concentration (24 μM) and longer exposure period (48 h), 66.7% and 62.5% chromosome doubling was achieved with APM and trifluralin, respectively. However, the application of 6 μM oryzalin to R. persica (2n = 2x), R. hybrida cv Iceberg (2n = 3x) and R. hybrida cv Akito (2n = 4x), resulted in 60.0%, 6.3% and 0% chromosome doubling, respectively, which suggest that chromosome doubling is genotype dependent and plants with lower ploidy level have a higher propensity for chromosome doubling. Flow cytometry results at 18 and 24 weeks after herbicide treatment, indicated that the best time to test the treated plants was after 24 weeks.     

Effect of chromosome-doubling agents on somaclonal variation in the progeny of doubled haploids of maize

This study was carried out to determine whether the treatment of anther-culture-derived haploid callus of maize (Zea mays) with chromosome-doubling agents, such as colchicine or the herbicides pronamide and amiprophos-methyl (APM), induces higher than normal levels of somaclonal variation. A total of 79 R₁ families produced by diploid regenerated plants resulting from chromosome-doubling treatments were evaluated in the field in comparison with the three parental inbreds. Four qualitative variant phenotypes — male sterility, chlorophyll deficiency, earless plants, and short plants with narrow leaves and thin stalks —– were observed. The last phenotype (narrow leaves and thin stalks) was also found in the inbreds FR16 and H99 grown from seed, so it may not be directly related to the tissue-culture conditions or the anti-microtubule-agent treatments. The frequency of R₁ families segregating for the other three mutations was 3.8%, which is no higher than the somaclonal variation frequencies observed previously in tissue-culture-derived maize plants. Observations of three quantitative traits—–days to anthesis, days to silk emergence, and plant height—– also failed to detect any extra variation that could be related to the treatments with anti-microtubule agents. These studies indicate that the anti-microtubule agents APM, pronamide and colchicine can be used to induce chromosome doubling of anther-culture-derived callus to produce a high proportion of doubled haploid plants without causing increased rates of mutation (somaclonal variation).     

In vitro polyploidisation of <Emphasis Type="Italic">Helleborus</Emphasis> species

Helleborus species are members of the family of the Ranunculaceae. These popular perennials are all diploids (2n = 2x = 32). This study investigates polyploidy induction by different antimitotic agents. Colchicine, oryzalin and trifluralin were tested in vitro on shoots of Helleborus niger, H. orientalis and H. × nigercors. Furthermore the effect of the antimitotic agents on the viability and the multiplication rate of cultured plantlets were analyzed. Flow cytometry demonstrated that polyploidisation was genotype dependent: using H. niger, tetraploids were obtained using either oryzalin (3 μM) or trifluralin (3 or 10 μM), whereas for H. × nigercors only trifluralin (3 or 10 μM) induced polyploidisation. For H. orientalis neither treatment was effective to produce tetraploids or mixoploids. For these three species, colchicine (100 μM) was ineffective. The polyploidisation events in H. niger and H. × nigercors were confirmed by chromosome counts of mounted nuclei derived from root tips (2n = 4x = 64).     

Development of novel chromosome doubling strategies for wheat × maize system of wheat haploid production

Two chromosome doubling strategies were evaluated for producing wheat doubled haploids from wheat x maize crosses: (i) in vitro colchicine application to haploid embryos and (ii) colchicine treatment through postpollination tiller injections. In the in vitro approach the haploid embryos were rescued on medium containing colchicine (at concentrations of 0.2, 0.3, 0.4 and 0.5%) and moved to a colchicine‐free regeneration medium 48 h later. Embryos exposed to 0.5% colchicine had 91.67% of their regenerated plants showing chromosome doubling. In the tiller injection approach, different concentrations (0.5, 0.75 and 1.0%) of colchicine solution, which also contained 2,4‐D (100 ppm), were injected into the uppermost inter‐node of crossed tillers 48 and 72 h after pollination. The chromosome doubling efficiency varied from 33 to 100%, with 1% treatment being the most effective. No chimeras of doubled/haploid sectors were observed in the case of the tiller injection treatment and all the florets showed seed set in the doubled plants. Stomatal guard cell length provided rapid, early‐stage and unambiguous analysis of ploidy level on the basis of 10 guard cell observations per plant.     

Efficient production of doubled haploid wheat plants by in vitro treatment of microspores with trifluralin or APM

Isolated microspore cultures from two doubled haploid (DH) lines of wheat, Triticum aestivum L., were used to develop an in vitro chromosome-doubling protocol. During the initial 24 h or 48 h of culture the microspores were treated with either of the two antimicrotubule herbicides trifluralin or amiprophos-methyl (APM) in concentrations ranging from 0.1 μM to 10μM. Untreated control cultures yielded 209 embryos per 100000 microspores, which is the equivalent of one spike. Among the regenerated plantlets 67% were green, and 15% of the flowering plants were spontaneously chromosome doubled. Treatments with both the herbicides had a significant effect on chromosome doubling, measured as the percentage of fertile regenerants. With the best combination of treatment duration (48 h) and herbicide concentration (10/μM) the percentage of fertile plants among regenerants could be increased up to 74% with APM and up to 65% with trifluralin. The largest numbers of DH plants per spike could be obtained with herbicide concentrations at 1–3 μM. Treatments with either herbicide at these concentrations resulted in an estimated average between the two genotypes of 27 DH plants per 100 000 microspores. These results demonstrate the high potential of APM and trifluralin as chromosome-doubling agents in isolated microspore cultures. The in vitro treatment integrated into tissue culture procedures will constitute an efficient method for chromosome doubling in future wheat breeding     

Efficient production of doubled haploid Brassica napus plants by colchicine treatment of microspores

Summary The effect of colchicine on isolated microspore cultures of Brassica napus was evaluated in order to combine a positive effect of colchicine on the induction of embryogenesis with the possibility to induce chromosome doubling at an early developmental stage, thus avoiding the production of haploid or chimeric plants. Colchicine was added to the culture medium immediately after isolation of B. napus microspores. The cultures were incubated from 6 to 72 h with various concentrations of colchicine. Samples were taken from the regenerating embryoids after 6 weeks for ploidy determination by flow-cytometry.The highest diploidization rate was obtained after a 24 h treatment of microspores with 50 mg/l colchicine, leading to 80–90% diploid embroids. A concentration of 100 mg/l colchicine applied for the same duration resulted in a lower diploidization rate (76–80%). Treatment durations of 6 h were not long enough to induce a high rate of diploidization, whereas the application of 10 mg/l for 72 h was also very effective.A sample of the plants regenerated from the colchicine treated microspores was transferred to the greenhouse. The plants looked similar to normal diploid rapeseed plants and showed reasonable pod and seed set. Thus, an additional generation for seed increase in the greenhouse is rendered unnecessary. The advantage of applying a minimum volume of colchicine under controlled in vitro conditions means a considerable saving of time and labour in DH-breeding programs.     

Nuclear DNA content and in vitro induced somatic polyploidization cassava (Manihot esculenta Crantz) breeding

Summary The diploid (2C) amount of DNA in cassava (Manihot esculenta Crantz) is 1.67 picograms (pg) per cell nucleus. This value corresponds to 772 mega-base pairs in the haploid genome. The size of the nuclear genome in cassava is very small in comparison with other Angiosperms. Flow cytometry techniques were used to screen ploidy levels in a large population of in vitro plantlets treated with colchicine and oryzalin (3,5-dinitro-N⁴,N-dipropylsulphate). Culture of axillary node cuttings for 48 hours in liquid medium supplemented with 2.5 to 5.0 mM colchicine in combination with 2% dimethyl sulfoxide (DMSO) resulted in a high frequency (23 to 42%) of non-chimeric tetraploids in the V₃ generation. Although mixoploidy may persist in as many as four cycles of vegetative propagation of node cuttings, solid (non-chimeric) tetraploids can be identified by flow cytometry among in vitro plantlets and then rapidly propagated for field testing. A somatic polyploidization system is proposed for implementation in cassava breeding programmes.Dedicated to the memory of the late Dr. Novak. Correspondence to M. van Duren