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.     

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.     

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.     

In vitro induction of polyploidy in Centella asiatica (L.) Urban

Mutational breeding was conducted using in vitro-grown shoot-tips of Centella asiatica (L.) Urban treated with colchicine (0.025–0.400% for 12–36 h) to induce polyploidy. Treated shoot-tips were grown on Murashige and Skoog (MS) medium supplemented with 4.54 μM thidiazuron (TDZ), and regenerated plantlets were acclimatized and transferred to soil. Two months following transfer to ex vitro conditions, ploidy levels of regenerated plants were determined by flow cytometry and by determining chromosome counts. Treating shoot-tips with colchicine concentrations ranging from 0.050–0.200% for 12–24 h promoted induction of tetraploids. Morphological and growth characteristics and the triterpenoid contents of the polyploids were also measured. Tetraploid plants demonstrated significantly longer stomata and a higher stomatal index compared to those of the diploid control plants. Furthermore, a positive trend in both biomass and triterpenoid production was obtained with the tetraploid and mixoploid plants of C. asiatica.     

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.     

In vitro induction, regeneration and analysis of autotetraploids derived from protoplasts and callus treated with colchicine in Citrus

In the present paper attempts were made to induce chromosome doubling of ‘Meiwa’ kumquat (Fortunella crassifolia) protoplasts and ‘Frost’ navel orange (Citrus sinensis Osbeck) embryogenic callus via colchicine treatment. Colchicine decreased protoplast viability, delayed protoplast division and inhibited callus growth, indicating presence of toxicity to cells. Cell lines established from ‘Meiwa’ protoplasts treated with 0.01 and 0.1% colchicine for 8, 16 and 24 h at each concentration showed different responses when they were cultured on embryoid-induction medium. Flow cytometry (FCM) demonstrated that tetraploids were detected in cell lines and embryoids from all of the treatments, with the highest frequency being 19.23%. As for ‘Frost’, tetraploid cells were only detected when the callus was treated with 0.1% colchicine for either 4 or 8 days, from which plantlets were regenerated. FCM and chromosome counting confirmed them as true tetraploids. The diploid cells were more active in mitotic division during a 12-day culture and smaller in size than their tetraploid counterpart. Potential applications of the novel tetraploid germplasms obtained through in vitro chromosome doubling to citrus cultivar improvement are discussed.     

In vitro induction of tetraploid in pomegranate (Punica granatum)

Tetraploid plants were obtained in pomegranate (Punica granatum L. var. `Nana') by colchicine treatment of shoots propagated in vitro. Shoots cultured on MS medium supplemented with 10 mg l^–1 colchicine, 1.0 mg l^–1 BA and 0.1 mg l^–1 NAA for 30 days produced tetraploids at a high frequency of 20%. No tetraploids were detected by treating the shoots in 5000 mg l^–1 colchicine for 114 h. Shoots treated by 5000 mg l^–1 colchicine for 96 h produced three morphological mutants with narrow leaves, which were later confirmed as mixoploids that separated into diploids and tetraploids after further subculture. In vitro tetraploid plants had shorter roots, wider and shorter leaves than the diploid ones. Tetraploid pomegranate plants grew and flowered normally in pots, but possessed flowers with increased diameter and decreased length compared to diploids. The number of pollen grains per anther was higher in tetraploids, but the viability of pollen decreased significantly.     

Impact of colchicine application during callus induction and shoot regeneration on micropropagation and polyploidisation rates in two <Emphasis Type="Italic">Miscanthus</Emphasis> species

Grasses from the genus Miscanthus have several characteristics that make them very favourable crops for efficient, low input, multifunctional and environmentally friendly biomass production. This study is aimed to improve a polyploidisation method to effectively induce polyploids in Miscanthus sinensis and Miscanthus x giganteus. Colchicine was applied for 2, 4 or 7 d in micropropagation systems using inflorescence segments at two different points: during callus induction (313 and 626 μM colchicine) and during shoot regeneration from callus (313 μM colchicine). Among the tested combinations, the most effective (up to 40%) was the 4-d colchicine treatment of a shoot-forming callus cultured 4 d before the experiment on regeneration medium under light conditions. In vitro colchicine treatment during callus induction and during shoot regeneration from callus resulted in no chimeric polyploids as well as a very low number of albinos (2.5%). Additionally, some combinations using colchicine did not significantly reduce the rates of micropropagation effectiveness. The obtained material is promising for the creation of new high-biomass-yielding forms in the Miscanthus genus. In all genotypes tested, chromosome doubling significantly increased pollen stainability. According to preliminary results, induced tetraploids are fertile and useful in hybrid production. Leaves of polyploid forms of two genotypes demonstrated significantly greater width in comparison to the controls.     

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.     

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     

Relationship between the induction of swelling and the inhibition of elongation caused by oryzalin and colchicine in corn roots

In corn seedling roots, colchicine and the dinitroaniline herbicideoryzalin cause swelling and inhibition of elongation which seemto be interconnected responses, yet they can be separated. First,both colchicine (10^–3 M) and oryzalin (10^–6 M) affectelongation (2–4 hr) well before swelling (8–10 hr).Second, a short (4 hr) oryzalin treatment produces maximal inhibitionof elongation but not maximal swelling. Third, a combinationof colchicine and oryzalin (at concentrations which give maximalor saturating effects on swelling and elongation when appliedsingly) produces a greater effect on swelling (but not elongation)than either alone. Thus swelling and inhibition of elongationare not related in a simple reciprocal relationship and maybe, at least in part, separate responses, and colchicine andoryzalin may not act in completely identical ways. (Received June 14, 1977; )     

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 tetraploidy in mulberry (Morus alba L.)

A high frequency of tetraploidy was induced in mulberry (Morus alba L.) through apical bud treatment under in vitro conditions. Apical buds from in vitro-grown plants were treated with three different concentrations (0.05, 0.1 and 0.2%) of colchicine in MS medium for 24 h. Tetraploidy at a frequency of 39.4±4.8% was obtained using 0.1% colchicine, whereas the frequency of tetraploidy was significantly reduced to 16.7±2.3% when 0.2% colchicine was used. Morphological, histological and cytological evidence indicated a phenotypic and genomic similarity of in vitro- with ex vitro-induced tetraploids. Rooting of tetraploids was on basal medium containing 2.6 μm NAA. The recovery of tetraploids was 80.8% more efficient using the in vitro method instead of the ex vitro method. The use of the same colchicine medium for up to 4 weeks with additional explants was found to be equally effective for the induction of tetraploidy. Received: 6 January 1997 / Revision received: 6 October 1997 / Accepted: 12 December 1997     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of protocorm-like bodies in <Emphasis Type="Italic">Cattleya</Emphasis>

A protocol for in vitro induction of crape myrtle tetraploids using nodes from in vitro-grown shoots (2n = 48) was established. Nodal buds were excised from in vitro-grown shoots, maintained on proliferation medium containing Murashige and Skoog medium supplemented with 4.44 μM 6-benzyladenine , 0.54 μM α-naphthaleneacetic acid, and treated with a range of concentrations of colchicine under three different conditions. Nodal bud explants treated in liquid proliferation medium supplemented with either 15 or 20 mM colchicine for 24 h turned necrotic and died; whereas, those cultured on solid proliferation medium supplemented with either 125 or 250 μM colchicine for 30 days survived, but no tetraploid plants were obtained. However, when explants were cultured in liquid proliferation medium containing 250, 500 or 750 μM colchicine for 10 days, tetraploid plants (2n = 96) were obtained. Incubation of explants in medium containing 750 μM colchicine promoted the highest frequency of survival (40%) of explants and of recovered tetraploids (60%). Morphological and anatomical characteristics of leaves, including leaf index, stomata size and number, stomata index (length/width), and number of chloroplasts in guard cells correlated with ploidy of crape myrtle plants. The number of chloroplasts in guard cells of stomata was a stable and reliable marker in discriminating plants of different ploidy levels. Chromosome counts and flow cytometry confirmed these findings.     

Induction of tetraploidy in Buddleia globosa

The aim was to produce a tetraploid form of Buddleia globosa to facilitate introgression of yellow flower colour into B. davidii, which is naturally tetraploid. Protocols were established for the micropropagation of B. globosa and tetraploid plants were obtained by application in vitro of colchicine to pre-cultured excised nodal sections. Three concentrations of colchicine were applied (0.01%, 0.05% and 0.1% w/v) for 1, 2 or 3 days. At 0.01% tetraploids were produced only after 2 days of application. All other treatments produced at least one tetraploid. The colchicine technique was extremely effective: of 29 lines tested, 19 were tetraploid and 5 were mixoploid. The vegetative characteristics of these tetraploids are described and the flowering characteristics of the three that flowered. This revised version was published online in August 2006 with corrections to the Cover Date.     

Magnesium deficiency of kiwifruit (Actinidia deliciosa)

Magnesium deficiency was associated with large yield reductions in a five-year-old commercial kiwifruit (Actinidia deliciosa) orchard. The effect on yield resulted primarily from a reduction in fruit numbers, there being no difference in mean fruit weight between fruit harvested from affected and unaffected vines. Magnesium deficiency had no deleterious effect on postharvest storage characteristics of fruit stored at 0.5–1°C for 18 weeks; fruit from deficient vines were firmer but had slightly lower soluble solids than fruit from control vines. Although deficiency symptoms were first observed on the basal leaves of the non-fruiting shoots mid season, indications of the impending deficiency could be established very early in the season using foliar analysis. Magnesium concentrations in youngest fully expanded leaves (YFEL) on the affected vines were less than 2.0 g kg⁻¹ DM four weeks after budbreak and remained below this value for the rest of the season; concentrations in YFEL on unaffected vines did not decrease below this value and gradually increased after fruitset to 4.5 g kg⁻¹ DM at harvest. To avert potential production losses, it is suggested that soluble magnesium fertilizers (containing at least 200 kg ha⁻¹ Mg) should be broadcast early in the season if foliar magnesium concentrations less than 2.0 gkg⁻¹ DM are measured four–six weeks after budbreak.     

Chromosome doubling in sterile Syringa vulgaris × S. pinnatifolia hybrids by in vitro culture of nodal explants

The aim was to produce tetraploid forms of hybrids of Syringa vulgaris × S. pinnatifolia to restore fertility and enable further breeding to be undertaken. Excised nodal sections of three selections were pre-cultured for 5 days then treated with a range of colchicine concentrations for 1, 2 or 3 days, after which they were washed three times in sterile distilled water and cultured on shoot proliferation medium. Frequent movement to fresh medium was beneficial to survival. Three successive experiments established the range of concentrations of colchicine, 0.05 mM to 0.25 mM, at which tetraploids were likely to be produced. Thus a protocol for the production of tetraploids was established. Cytometric analysis showed that tetraploid forms of two selections were produced. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effects of colchicine on carrot callus — growth and energy status

Colchicine effects on growth, oxygen uptake, and adenosine phosphates level of carrot (Daucus carota L.) callus tissue were determined over a period of 18 days after subculture. Colchicine at 10⁻³ and 10⁻⁴ M inhibited the increase in oxygen consumption which was observed with the initiation of callus tissue. Initially the same levels of colchicine stimulated AMP and ADP levels above those of the control, but later the levels returned to those of the control. Colchicine at 10⁻⁴ and 10⁻⁶ M reduced ATP levels; however, at 10⁻³ M there was no effect. Growth was not severely affected by the same concentrations during the same treatment period. Oxygen consumption and nucleotide metabolism were more sensitive to colchicine than was growth. Energy charge was calculated to be 0.7–0.8 in both treated and control tissue during 3–15 days in culture. South Carolina Agricultural Experiment Station Journal No. 1840.     

Effect of colchicine induced tetraploidy on morphology,cytology, essential oil composition,gene expression and antioxidant activity of Citrus limon (L.) Osbeck

Citrus limon (L.) Osbeck cultivated all over the world is a valuable source of aromatic essential oil. To develop tetraploids of C. limon, four different concentration of colchicine (0.025, 0.05, 0.1 and 0.2%; w/v) and three varied exposure time (12, 24 and 48 h) were employed. The ploidy level of diploids (2n = 2x = 18) and tetraploids (2n = 4x = 36) were determined by direct chromosome count and confirmed by flow cytometric analyses. Successful result with maximum tetraploidy frequency was observed in plantlets developed from seeds treated with 0.025% colchicine for 24 h. Morphological and stomatal characteristics indicated that tetraploids were taller with increased leaf and root length. On the other side, the leaves of tetraploids had fewer and larger stomata with a greater number of chloroplasts in guard cells in contrast with diploids. GC–GC/MS analyses showed cyclic monoterpene, limonene had increased significantly in tetraploids and was further confirmed by HPLC quantification. RT-PCR analyses revealed unaltered expression of monoterpene synthase, sesquiterpene synthase and flavone synthase and remarkable upregulation of genes such as limonene synthase, chalcone synthase and phenylalanine ammonia lyase in tetraploids. Antioxidant activity of essential oil of tetraploids was higher than diploids in all the five test systems studied. Overall, the findings of the present study prove that colchicine induced tetraploidy in C. limon could be a greater source of essential oil with improved composition and of economic significance.     

In vitro induction and identification of tetraploid plants of <Emphasis Type="Italic">Paulownia tomentosa</Emphasis>

Polyploidization is a major trend in plant evolution that has many advantages over diploid. In particular, the enlargement and lower fertility of polyploids are very attractive traits in forest tree breeding programs. We report here a system for the in vitro induction and identification of tetraploid plants of Paulownia tomentosa induced by colchicine treatment. Embryonic calluses derived from placentas were transferred to liquid Murashige and Skoog (MS) medium containing different concentrations of colchicine (0.01, 0.05, or 0.1%) and incubated for 24, 48, or 72 h on an orbital shaker at 110 rpm. The best result in terms of the production of tetraploid plantlets was obtained in the 48 h + 0.05% colchicine treatment, with more than 100 tetraploid plantlets being produced. The ploidy level of plantlets was verified by chromosome counts, flow cytometry, and morphology. The chromosome number of tetraploids was 2n = 4x = 80 and that of diploid plantlets was 2n = 2x = 40. The relative fluorescence intensity of tetraploids was twofold higher than that of diploids. The tetraploid and diploid plantlets differed significantly in leaf shape, with those of the former being round and those of the latter pentagonal. The mean length of the stomata was longer in tetraploid plants than diploid plants, and stomatal frequency was reduced with the increased ploidy level. The tetraploids had large floral organs that were easily distinguishable from those of diploid plants.