Induction of <Emphasis Type="Italic">Anthurium andraeanum</Emphasis> “Arizona” tetraploid by colchicine in vitro

Tetraploids plants of Anthurium andraeanum “Arizona” were successfully induced after treating diploid tissue masses with colchicine. Masses originating from diploid aerial roots were treated with colchicine at three different concentrations (i.e., 0.1, 0.2, 0.3%) for about 3, 5 and 7 h, and then were transferred into Murashige and Skoog medium containing 3 mg/l BAP + 0.2 mg/l 2,4-D. After 60 days, the survival rate and numbers of regenerative shoots were scored. The high concentration and longer duration sharply reduced survival rate. In contrast, the regeneration of plantlets was not noticeably affected by colchicine. Tetraploid plants were obtained in all treatments, but the percentage of induced tetraploids ranged from 0.2 to 7.6%. The best induction was obtained with a 5-h, treatment with 0.3% colchicine. The stomatal size of tetraploid plants was larger than in diploid plants; however, the stomatal density was lower than in diploid plants. Tetraploid plants possessed stronger petioles, thicker and deeper green leaves, and thicker and longer lived spathes in comparison with diploid plants. Abnormal spathes, such as double spathes or those lacking pedicels, were observed in tetraploid plants. Tetraploid plantlets could be regenerated via aerial roots; this technique could be applied to tetraploid plant propagation.     

Identification of tetraploid regenerants from cotyledons of diploid watermelon cultured in vitro

Summary Adventitious shoots were obtained from the diploid watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] cultivars Dixielee, Jubilee II, Mickylee, Minilee, and Royal Sweet by culturing excised cotyledons on shoot regeneration medium for six weeks. Tetraploid and diploid regenerants were identified by counting the number of chloroplasts per guard cell pair from leaves of regenerated plants. Cross fertilization of putative tetraploids with diploid pollinators and the production of triploid seed confirmed the efficacy of this approach. The mean number of chloroplasts for tetraploid regenerants was 19.1 whereas diploids averaged 11.2. These values were similar to tetraploid and diploid plants from seed. Ovary diameter, petal, and anther diameter of male flowers, and leaf length by width ratio were also good indicators of plant ploidy. Progeny obtained from self-fertile tetraploids of Mickylee were crossed with various diploid pollinators to produce triploid hybrid seed. All triploid plants from tissue culture-derived tetraploids produced fruit comparable in quality to fruit produced by currently-available triploid hybrids, demonstrating that in vitro tetraploid induction can be used to produce high quality tetraploid plants for use in triploid hybrid seed production.     

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.     

Comparative study on vegetative,reproductive and qualitative traits of seven diploid and tetraploid watermelon lines

Seven tetraploid watermelon lines developed by colchicine treatments were compared with their diploid counterpart for plant, flower, fruit, seed and qualitative characteristics. Tetraploid genotypes attained statistically higher vine thickness (8.04 mm), leaf area (298.9 cm²) and chlorophyll content (55.6) while internode length and chlorophyll fluorescence was similar to their corresponding diploid. Both pistillate and staminate flower organs (pedicel, anther, ovary, stigma, petals) were larger in tetraploid plants; however, the percent increase in flower components varied across the tetraploid lines. Fruit weight and total sugar content (^∘Brix) in both ploidy fruits was similar. Rind thickness in fruits varied significantly and averaged 12.7 and 17.2 mm in diploid and tetraploid fruits, respectively. Tetraploid genotypes showed sterility, yielded lower number of seed per fruit (37.9), and tetraploid seed was larger and thicker than diploid seed. Overall β-carotene (0.89), lycopene (1.16), fructose (5.43%) and glucose (2.38%) contents were higher in tetraploid than diploid fruits.     

In vitro induction of tetraploid plants from callus cultures of diploid bananas (<Emphasis Type="Italic">Musa acuminata</Emphasis>, AA group) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’

Tetraploid plants were induced successfully from diploid bananas Musa acuminata (AA genome) ‘Kluai Leb Mu Nang’ and ‘Kluai Sa’ (2n = 2x = 22) with in vitro oryzalin treatment. Calluses from in vitro-grown shoot tips of both cultivars were treated with oryzalin at concentrations of 1.5 or 3 mg l⁻¹ for 24, 48 and 72 h, respectively. The oryzalin treatments produced tetraploids at a frequency of 15.6% in ‘Kluai Leb Mu Nang’ and 16.7% in ‘Kluai Sa’ as detected by flow cytometry. Chromosome counting showed that the tetraploid plant chromosome number was (2n = 4x = 44). The selected tetraploid plants were transplanted in the field and variations in the morphological characteristic of leaf shape and fruit bunch compared to normal diploid plants were found under the same growing condition even after 3 years of cultivation.     

Induction and verification of autotetraploids in diploid banana (Musa acuminata) by in vitro techniques

Summary Flow cytometry and stomata characteristics were used for screening ploidy levels in a large population of in vitro induced autopolyploids of the Musa acuminata breeding clone SH-3362. Culturing shoot tips in liquid medium stipplemented both with 5.0 mM colchicine for 48 hours or 30 M oryzalin (3,5-dinitro-N4,N-dipropylsulphate) for seven days, both in combination with 2% (v/v) DMSO, resulted in a high (23.1% and 29.1%) frequency of non-chimeric tetraploids in the fourth vegetative generation. Although mixoploidy persisted in subsequent cycles of vegetative propagation, tetraploids as identified by flow cytometry remained solid non-chimeric during two more cycles. These autotetraploids were propagated for field testing. A rough pre-selection of regenerated V4 plants based on their stomata characteristics resulted in a population in which only 56.2% of the plants were solid tetraploids. The somatic polyploidization system reported here can be utilised for banana breeding programmes.Abbreviations FCM Flow Cytometry     

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.     

Artificially induced polyploidization in Humulus lupulus L. and its effect on morphological and chemical traits

Chemically induced polyploids were obtained by the colchicine treatment of shoot tips of Humulus lupulus L. ‘Sybilla’. Flow cytometry revealed that most of the treatments resulted in the production of tetraploids. The highest number of tetraploids was obtained when explants were immersed in 0.05% colchicine for 48 h. A field experiment was conducted to compare diploid and tetraploid plants and assess the effect of genome polyploidization on the morphological and chemical characteristics. Tetraploids showed significant differences in relation to diploids. They had thinner and shorter shoots. The influence of chromosome doubling was also reflected in the length, width and area of leaves. The length of female flowers in the tetraploids was significantly shorter than that observed in diploids. Tetraploids produced a diverse number of lupuline glands that were almost twice as large as those observed in diploids. The most distinct effect of genome polyploidization was a significant increase in the weight of cones and spindles. Contents of major chemical constituents of hop cones was little affected by ploidy level. Total essential oils were significantly lower than those in diploids. However there was a significant increase in the proportion of humulene, caryophyllene and farnesene, oils desired by the brewing industry.     

Flow cytometric identification of sexually derived polyploids in hop (Humulus lupulus L.) and their use in hop breeding

The New Zealand hop breeding programme is based solely on the development of seedless triploid cultivars. This relies on the use of tetraploid parents. While a sexually derived tetraploid parents have been used successfully, sexually derived tetraploids offer a useful alternative. They may have a higher level of heterozygosity and are easier to obtain. Methods for the identification of tetraploids from seedling populations by flow cytometry are described. Two studies were conducted; one on field-grown plants, the other on plants grown in a glasshouse. Approximately 15%of seedlings in the two studies were identified as tetraploids (2n = 4x = 40). Between 70 and 80% were identified as either diploid (2n =2x = 20) or triploid (2n = 3x = 30). Remaining plants were haploid, pentaploid or probably aneuploid. Within triploid and tetraploid populations from 40to 50% of seedlings were female, 30 to40% were monoecious, with the remainder being males and non-flowering plants. Sexually derived tetraploid parents have been used successfully in the breeding programme, from which several promising triploid selections have subsequently been made. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of non-chimeral colchiploids in Rubus species by tissue culture

Summary Diploid Rubus allegheniensis Porter and R. rusticanus Merc. shoot tips were proliferated in tissue culture and treated with 0.02 to 0.08% colchicine to induce non-chimeral polyploidy. About 20 shoot tips of each treatment were transferred to test tubes containing modified Anderson's medium for shoot proliferation. Shoots at the 3- or 4-leaf stage were transplanted into Jiffy-7 peat pellets and placed under intermittent mist for rooting. Root tip squashes were made for chromosome counts. Thirteen R. allegheniensis and 7 R. rusticanus plants proved to be autotetraploids (28 chromosomes). Stomata of the autotetraploids were significantly larger than those of the diploids but stomatal conductance of CO₂ and photosynthesis rate were reduced significantly as compared to diploids. Pollen grains in tetraploid were larger than those in diploid R. allegheniensis plants. These results indicate that tissue culture can facilitate the production of non-chimeral colchiploids in Rubus spp.     

The creation of tetraploid beets

Summary Tetraploid beets have produced interesting results abroad. This is the reason that in 1950 the Foundation for Agricultural Plant Breeding initiated a program for the development of tetraploid strains. By treating germinating seed with colchicine enough tetraploids can be secured, provided the material be examined during the first and the second year.Cytological studies made in 1951 with the aid of the phase-contrast microscope showed that a 0.1% colchicine solution is preferable to a 0.2% solution. The cytological examination has the object of purifying the material. If diploid cells are found the plants are discarded. In order to increase the number of tetraploid seeds it is necessary to examine the beets again during the second year. In 1951 this was done by studying the young leaves in the rosette stage and by taking the axillary leaflets of young stalks. As the number of tetraploids in the offspring was disappointingly small in 1952 the work was organized somewhat differently. Only one tetraploid stalk of each plant was kept, all other stalks were removed.The investigations are being continued.     

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).     

不结球白菜同源四倍体苏州青的诱导鉴定

为获得高产优质的不结球白菜四倍体苏州青,本研究使用浓度分别为0.1%,0.2%及0.3%的秋水仙素溶液,点滴处理二倍体苏州青幼苗植株的子叶生长点,进行5次处理后,通过形态学和细胞学方法筛选鉴定四倍体苏州青植株,并比较二、四倍体苏州青的农艺性状及品质差异。结果表明：秋水仙素浓度为0.2%时加倍效果最好。形态解剖学鉴定结果表明：四倍体在株型、叶片、花、种荚、种子大小都有较明显的变大趋势;四倍体植株叶片气孔较二倍体变大,气孔密度变小;同时四倍体植株花粉粒比二倍体更膨大,更不规则;细胞学鉴定结果表示,四倍体植株染色体数为40条,二倍体植株为20条;通过流式细胞仪鉴定,四倍体植株荧光强度约为二倍体的2倍。通过品质比较分析,结果显示四倍体苏州青的各项生理指标含量较于二倍体均有不同。通过农艺性状比较,单株质量、叶宽、叶厚都有一定程度的增加。本研究利用秋水仙素操作技术,抑制有丝分裂后期纺锤丝的形成,实现染色体加倍,创制不结球白菜新种质,为选育四倍体不结球白菜提供理论基础和种质资源。     

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.     

Thebaine content and yield in induced tetraploid and triploid plants of Papaver bracteatum Lindl.

Summary The effect of colchicine induced autotetraploidy on the thebaine content and yield of the capsules of Papaver bracteatum was studied. A significant increase in the thebaine content of the capsules was found in tetraploid plants during two consecutive growing seasons, but the thebaine yield per plant was slightly reduced in the autotetraploids. Autotriploid plants showed a very high concentration of thebaine (8.8%) only in the first growing season.Preferential bivalent pairing and a relatively high pollen fertility were observed in the tetraploids, thus making possible the stabilization of tetraploidy by selfing.Tetraploidy seems a most promising approach for developing clones (by vegetative propagation) or lines rich in thebaine.     

Cytology and digoxin production in hybrids between Digitalis lanata and D. grandiflora

Summary Digitalis lanata is the only commercial source of the cardiac glycoside digoxin. It is an outbreeder and displays considerable variation. An attempt to increase uniformity was made by making D. lanata × D. grandiflora diploid hybrids and from these produce allotetraploid hybrids using colchicine treatment. Such tetraploid plants would be homozygous. Tetraploid hybrids were shown to contain digoxin but at lower levels than in D. lanata. Progeny of selfed tetraploid hybrids were significantly more uniform than D. lanata grown from commercial seed, but were considerably less vigorous.     

Chromosome doubling of Allium fistulosum × A. cepa interspecific F1 hybrids through colchicine treatment of regenerating callus

Regenerating calluses of Allium fistulosum × A. cepa interspecific F₁ hybrids were treated in vitro with colchicine. A factorial experiment was designed to test the effects of colchicine concentration and time on the recovery of tetraploid plants from in vitro-colchicine-treated calluses. Shoot production of regenerating calluses following in vitro colchicine treatment decreased with increasing colchicine concentration and treatment time. Cytological analyses of root tip cells from regenerated plantlets showed that chromosomes of control plantlets (not treated with colchicine) were not doubled. Chromosome number of some plantlets regenerated from in vitro-colchicine-treated calluses were doubled, resulting in tetraploids. Calluses treated with 0.1 or 0.2% colchicine in BDS liquid medium for 48 or 72 hours yielded the highest numbers of tetraploid plantlets. Chromosome bridges at anaphase or early telophase were observed in diploid and tetraploid plants; their potential use is discussed. These results demonstrate that in vitro-colchicine treatment of regenerating calluses of interspecific F₁ hybrids is effective in recovering tetraploid plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Amiprophosmethyl-induced efficient in vitro production of polyploids in raphanobrassica with the aid of aminoethoxyvinylglycine (AVG) in the culture medium

Optimum conditions for obtaining tetraploid were investigated in raphanobrassica, the intergeneric hybrid between radish (Raphanus sativus) and kale (Brassica oleracea var. acephala) by treating in vitro plants with an anti-mitotic agent, amiprophosmethyl (APM). Initially, no tetraploids but hexaploids and octaploids were induced by the treatments. Although the leaves of these polyploids of raphanobrassica showed chlorosis during subcultures in in vitro conditions, the chlorosis could be successfully prevented by the ethylene inhibitors, both AVG and AgNO₃. Based on this result, AVG was added into medium used for the culture after the chromosome doubling treatment, which subsequently resulted in increased survival rates of the treated plant materials as well as increased production rates of polyploids including tetraploid. These polyploid plants showed obviously different characters from the original diploid plant. The tetraploid plant had bigger sizes in shoot, flower and leaf, and more number of leaves than the diploid. On the other hand, the hexaploid and octaploid plants had smaller sizes in shoots and leaves, and less number of leaves than the diploid. Concentration of glucosinolates, functional substances of Brassicaceae crops, did not significantly differ between diploid and tetraploid of raphanobrassica, but reduced in hexaploid and octaploid.     

Intra‐ and inter‐specific crosses of Solanum materials after mitotic polyploidization in vitro

Mitotic polyploidization in vitro was used in selected wild Solanum species and Solanum tuberosum dihaploids. The efficiency of polyploidization by colchicine was compared with that of oryzalin. Oryzalin was more effective than colchicine (P = 0.1). The rate of non‐affected to mixoploid to tetraploid regenerants was 22 : 2.5 : 1 (colchicine) and 14 : 2 : 1 (oryzalin). Optimal concentrations and durations were 3.5 mm /24 h for colchicine and 25 or 30 μm for 24 or 48 h for oryzalin (variations in concentration and duration are necessary owing to possible diversity of responses in selected genotypes). Tetraploids were obtained from S. berthaultii, S. bulbocastanum, S. pinnatisectum, S. verrucosum and eleven S. tuberosum dihaploids. The yield of tetraploids derived from tbr dihaploids was lower than that from the wild species (P = 0.01). Tetraploid regenerants were tested in intra‐ and inter‐specific crosses. Three of 43 intra‐specific combinations (298 pollinated flowers) were successful and yielded 440 seeds. Inter‐specific crosses (138 combinations, 1672 pollinated flowers) yielded 48 seedless berries.     

Colchicine-induced chromosome doubling in Platanus acerifolia and its effect on plant morphology

We report the production of tetraploid plants of Platanus acerifolia, with the ultimate aim of improving the ornamental qualities of this important urban landscaping tree. Chromosome doubling was achieved by the application of colchicine to either pre-soaked seed or to the apical meristems of young seedlings. Treatment of the ungerminated seed was the more efficient method in terms of numbers of tetraploid seedlings (up to 40%, as determined by chromosome counting of the root-tip nuclei) but this method produced no mature tetraploid plants due to the deleterious effect of colchicine on subsequent root growth. When colchicine was applied directly to the apical growing tip of cotyledon-stage seedlings, leaf and stem growth was temporarily affected but the plants eventually recovered. We conducted a preliminary screen for putative tetraploids based on the observation in other plant species of a correlation of stomatal size and distribution with ploidy. Plants containing significantly larger stomata and at a lower density across the lower leaf epidermis, were selected for further analysis by flow cytometry and chromosome counting. These techniques confirmed that, of the 12 putative polyploids, four were tetraploid, five were mixoploid and three were, in fact, diploid. Morphological differences of the tetraploids included a more compact growth habit and broader, thicker leaves. These plants are being grown to full maturity in order to test their potential for use in a breeding programme aimed at producing sterile triploid lines.