Single cell origin of adventitious buds

Summary Objections are raised against the view of Norris et al. (Science 220, 1983), that adventitious buds from African violet leaf explants are of multicellular origin.The experimental results of these authors can be interpreted differently and may therefore support as well the opposite standpoint that the adventitious buds are (ultimately) of single-cell origin.     

Chromosome doubling of mature haploid Brussels sprout plants by colchicine treatment

Summary The effect of colchicine concentration, and the mode and time of colchicine application in doubling the chromosomes of haploid Brussels sprout plants was assessed by scoring treated plants for the presence of diploid flowers and seed set after self-pollination.When colchicine treatments were applied after the plants had been vernalised, using 2 dose rates and 3 methods of application, only 38.1% became doubled and only 13.8% produced seed. Treatment with 0.1% colchicine with or without the addition of 2% dimethyl sulphoxide gave doubling rates similar to those reached with 0.05% colchicine, but resulted in more damage to the apices.When 0.05% colchicine solution was injected into the plants' apices at varying times during vernalisation, the rate of doubling was 71.2% on average and 50.7% of plants gave seed on selfing.Overall doubling rates of plants where the apex was easily accessible were 79.0%, while for plants with dense terminal heads they were 35.0% and for plants with visible buds 31.4%.     

Short-duration colchicine treatment for in vitro chromosome doubling during ovule culture of Beta vulgaris L.

Colchicine uptake into ovules of sugar beet after 7 days of culture and its chromosome-doubling effect on ovule-derived plants were studied with high colchicine concentrations (0.4–6.0%) and short treatment duration (0–5 h). The best result of 4.2 diploid plants per 100 ovules was produced by treatment with 0.4% colchicine for 2.5 h. Both colchicine concentration and treatment time of ovules showed toxic effects on embryo formation, but it was stabilized at a low level with short exposure. The chromosome-doubling effect, by contrast, was unchanged with the colchicine concentrations used, but highly affected by the duration of exposure studied. A maximum percentage of 60% diploid plants was obtained after 3–5 h of uptake, which corresponds to only 31–39% of the total capacity for colchicine uptake in the ovules. Further uptake of the drug produced mainly toxic effects. Flow-cytometric measurements of the ploidy level in plantlets in vitro and of the same plants before flowering in soil were similar in about 80% of cases. Thus, flow-cytometric selection of diploid plants in vitro may be an efficient tool.     

Effect of gamma and ultraviolet irradiation on adventitious regeneration from in vitro cultured pear leaves

Summary Irradiation of in vitro explants and subsequent adventitious regeneration has been tested for 4 commercially important varieties of pear (Pyrus communis) with the aim to create mutants with a reduced susceptibility to fire-blight (Erwinia amylovora). The effect of gamma and ultraviolet irradiation of leaves on adventitious regeneration ability has been studied. The LD50 (50% decrease of regeneration) after gamma irradiation was genotype-dependent and was between 20 and 50 grays. The curves of regeneration showed a threshold dose underneath which none or a very slight decrease was registered. The decrease might result from cumulative events. After an ultraviolet irradiation as low as 62.5 J/m², the leaves became crumbly and rolled up, and their metabolism seemed to be altered. The LD50 was about 125 J/m² for all varieties and the decrease of regeneration was linear. Histological investigations showed leaves with flattened epidermal cells after ultraviolet irradiation and slack spongy parenchyma after gamma irradiation.Abbreviations Gy grays - J joules - LD lethal dose     

Oryzalin as an Efficient Agent for Chromosome Doubling of Haploid Apple Shoots in vitro

In an outbreeding species such as apple, haploid plants may be especially useful in breeding programmes for the production of homozygous material. However, methods must be available to induce chromosome doubling in the haploid plants. Two antimitotic agents, colchicine and oryzalin, were compared as regards their efficiency in inducing chromosome doubling of in vitro haploid apple shoots. Three colchicine levels (0.025, 0.25 and 1.25 mM) and three oryzalin levels (5, 15 and 30 μM) were evaluated. Three techniques were also used and compared. Survival rate and chromosome counts were determined. Differences were observed between the two antimitotic agents and between the three techniques. This study demonstrates that oryzalin could be a better choice than colchicine for chromosome doubling on haploid apple shoots in vitro.     

Amphidiploids between Cyclamen persicum Mill. and C. purpurascens Mill. induced by treating ovules with colchicine in vitro and sesquidiploids between the amphidiploid and the parental species induced by conventional crosses

Summary We cultured colchicine-treated hybrid ovules in vitro to produce fertile amphidiploids of C. persicum (2n=2x=48. referred to as AA) × C. purpurascens (2n=2x=34, referred to as BB). Seedlings and mature plants were obtained from the ovules without colchicine and those exposed to 50 mg/l colchicine for 5, 10 and 15 days, whereas they were not obtained from the ovules exposed to 50 mg/l colchicine for 20 days and 500 mg/l for 5, 10, 15 and 20 days. Although 8 mature hybrids derived from the ovules without colchicine produced a few fertile pollen grains, they failed to produce viable seeds by self-fertilization. The hybrids had 41 somatic chromosomes. Four and 3 mature plants were derived from ovules exposed to 50 mg/l colchicine for 10 and 15 days, respectively. One each among 4 and 3 mature plants showed a high frequency of pollen grain fertility, produced several seeds by self-fertilization, and had 82 somatic chromosomes which is twice the number of hybrid chromosomes (2n=41, AB). These findings indicated that these plants are amphidiploids (2n=82, AABB) between C. persicum and C. purpurascens. Three and 2 viable seeds were derived by the conventional crosses of diploid C. persicum × the amphidiploid and the amphidiploid × C. purpurascens, respectively. Flowering plants that developed from the seeds of diploid C. persicum × the amphidiploid were barely fertile and had 65 somatic chromosomes (2n=65, AAB), whereas those that developed from the seeds of the amphidiploid × C. purpurascens were barely fertile and had 58 somatic chromosomes (2n=58, ABB). The somatic chromosomes indicated that these plants are probably sesquidiploids between the amphidiploid and either C. persicum or C. purpurascens. The interspecific cross-breeding of cyclamen using the amphidiploids and the sesquidiploids is discussed.     

Chromosome doubling through adventitious shoot formation on in vitro cultivated leaf explants from diploid interspecific potato hybrids

Summary Nearly 450 plantlets were produced from 51 diploid Solanum etuberosum x S. pinnatisectum F₁ hybrids through adventitious shoot formation on in vitro cultivated rachis and petiole explants.On the basis of phenotypical assessment of the ploidy level of 425 plants, 84.7% of the plants were scored as doubled or doubled twice. A cytological analysis of ploidy in the three layers L₁.L₂ and L₃ of 112 plants revealed 83.9% complete doubling: periclinal ploidy chimeras were not found and only two sectorial ploidy chimeras were detected. Doubled plants were obtained from all 51 clones.Various flower colours and epinastic leaves (in 1 clone) may be indications of mutagenesis through the treatment.     

Chromosome numbers of Tulipa species and the occurrence of hexaploidy

Summary Chromosome numbers of Tulipa species were determined. For the first time hexaploidy was found in the genus. It became clear that in many species or subspecies several ploidy levels may occur.     

Improved techniques for the induction and isolation of polyploids in the genus Fragaria

Summary The sensitivity of Fragaria seedlings to colchicine is dependent on the plant organ that is treated. Complete immersion of seedlings in a 1.5% colchicine solution results in total lethality, whereas the survival rates were more than 75% even at concentrations of 3.0% when only shoot apices were treated. High proportions of polyploids were isolated by treating shoot apices of seedlings with a 2.0% colchicine solution for 24–28 h. The dropper method is preferred to the tube method as it involves a minimum of manipulation and requires simple equipment. A differential response to colchicine was observed within and between different diploid species, diploid and tetraploid hybrids.     

Chromosome numbers and microsporogenesis in Brachiaria brizantha (Gramineae)

The genus Brachiaria, native to the African tropical savannas, has achieved significance as a pasture grass in many tropical and subtropical countries, including Brazil. Many species and accessions are polyploid and apomictic, which complicates the improvement of breeding stocks through hybridization. In support of breeding programs, cytogenetic characterization, including chromosome counts and evaluation of the meiotic behavior in the accessions of the Brachiaria has been undertaken at the Embrapa Beef Cattle Center. In this study, 22 accessions of B. brizantha were analyzed of which one was found to be diploid (2n = 2x = 18), 18 were tetraploid (2n = 4x = 36) and three were hexaploid (2n = 6x = 54). The meiotic chromosome behavior was slightly irregular in the diploid and in some tetraploid accessions, and highly irregular in most tetra- and hexaploid accessions. Meiotic abnormalities were those common to polyploidy, i.e., multivalent chromosome association at diakinesis and irregular chromosome segregation leading to micronuclei formation in the tetrad stage. Low frequencies of multivalent chromosome associations among polyploids suggest that they may be segmental allopolyploids. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inheritance studies of caffeine and theobromine content of Mate (Ilex paraguariensis) in Misiones, Argentina

In order to start a genetic improvement program for quality in the Mate crop (Ilex paraguariensis), it was attempted to estimate the actual genetic parameters of caffeine, theobromine and related quality traits, using available progeny tests in Misiones, Argentina. Using cluster analysis, eight groups of similar characteristics could be identified, and individual within full-sib family selection for quality was performed. Additive effects were strong for caffeine but weak for theobromine. Co-heritability between caffeine and theobromine was positive, suggesting that additive genetic correlations and dominance correlations are concurring. The strong influence of paternal progenitors regarding caffeine needs more research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Genetic variability in plants regenerated from in vitro culture of sunflower (Helianthus annum L.)

In vitro regenerated sunflower (Helianthus annuus L.) plants (R₁) were self-fertilized and the R₂ generation was evaluated for qualitative traits. A broad range of phenotypic variation was observed and mutation frequencies were calculated. Some in vitro induced variant phenotypes were similar to known spontaneous or induced mutations in sunflower, while others were new. Chlorophyll and carotenoid deficiencies, chimaerical variegation, fasciated stem and capitulum, abnormal shoot development, and other morphological variations, were noted. Substitution of anthers with petaloid structures in a disk-floret mutant indicates a possible homeotic mutation induced by in vitro tissue culture.     

肝素浓度和作用时间对绵羊精子体外获能和体外受精的影响

摘 要：通过研究添加不同的肝素浓度(0、5、10、20、50μg/ml)及不同作用时间（0、30、45、60、90、120min）对绵羊精子体外获能、存活时间和相应体外受精的影响,为改善绵羊精子体外获能效果及研究获能的机理提供一定的参考。在获能液中添加10μg/ml肝素作用45min时,其获能率最高,顶体反应率最低（40.92％和16.59％）（P<0.05）,存活时间18h,能够提高受精卵的囊胚发育;单独使用咖啡因或与10μg/ml肝素联合使用,其获能效果均不如单独使用10μg/ml肝素。说明绵羊精子获能时肝素的适宜浓度是10μg/ml,作用时间是45min,咖啡因无协同肝素促进获能的作用。     

Increasing embryogenesis and doubling efficiency by immediate colchicine treatment of isolated microspores in spring Brassica napus

The effect of colchicine on induction of embryogenesis andchromosome doubling during microspore culture was evaluated in twoF₁ hybrids of spring oilseed rape (Brassica napus L.). Immediatecolchicine treatment of isolated microspores with the concentrations 50 and500 mg/L for 15 h stimulated embryogenesis and produced largeamounts of healthy-looking embryos. These normal embryos germinatedwell at 24 ^°C after being transferred to solid regeneration mediumand an initial period of low temperature (2 ^°C) for 10 days, andcould directly and rapidly regenerate vigorous plants. A high doublingefficiency of 83–91% was obtained from 500 mg/L colchicinetreatment for 15 h with low frequency of polyploid and chimeric plants.The present experiment showed that a treatment duration of 30 h revealedless positive effects on embryogenesis and doubling efficiency, especially athigher colchicine concentration (1000 mg/L). Poor embryogenesis andembryo germination were observed from ordinary microspore culturewithout change of induction medium and colchicine treatment, and severalsubcultures were required for induction of secondary embryogenesis andplant regeneration.     

In vitro and in vivo genetic parameters and character associations in potato

Relative behaviour of genetic parameters and character associations for in vitro and in vivo systems was studied by evaluating 22 potato genotypes for seven morpho-agronomic characters under six in vitro and four in vivo conditions. There was considerable similarity between in vitro and in vivo systems with regard to pattern of coefficient of variation (both phenotypic and genotypic), heritability (broad sense), genetic advance (as% of mean) and correlation coefficients (both phenotypic and genotypic) between characters. The magnitude of various genetic parameters was higher under in vitro than in vivo conditions, particularly for tuber yield and its components. Treatment-to-treatment variation in genetic parameters and character associations was higher under in vivo than in vitro conditions. In vitro selection for agronomic characters in potato may be possible. This revised version was published online in July 2006 with corrections to the Cover Date.     

鸡冠花离体快繁及多倍体诱导

以鸡冠花顶芽为外植体,研究其离体快繁程序并在无菌体繁殖条件下探索了秋水仙素溶液不同浓度和时间处理对鸡冠花的诱变效应.快繁研究结果显示:最适初代培养基为MS 6-BA 1.0 mg/L NAA 0.2 mg/L;以芽和茎段为培养对象诱导产生不定芽的最适培养基分别是MS 6-BA 1.0 mg/L NAA 0.1 mg/L,MS 6-BA1.0 mg/L NAA 0.02 mg/L,最佳生根培养基是1/2 MS IBA 0.4 mg/L.采用浸泡法将鸡冠花营养芽在0.05%、0.1%、0.15%和0.2%不同秋水仙溶液浓度下分别处理12 h、24 h、36 h和48 h,并对诱导处理后获得的再生植株进行鉴定,得出以0.15%秋水仙素溶液浸泡36 h为诱导多倍体的最佳处理组合,诱导率达23.3%.     

In vitro induction of tetraploid plants from colchicine-treated diploid daylily callus

Summary Daylily plantlets with tetraploid or octaploid chromosome numbers were induced from colchicine-treated diploid callus (2n=22). Callus tissue of the daylily Hemerocallis flava L. was placed on a morphogenetic induction medium, a modified MS formula supplemented with 1 mg 2,4-D and 1 mg kinetin/liter, plus 0,10, 20, or 40 mg/liter colchicine in the dark at 12°C for 3 days and recuperated for 1 week under the same environmental conditions except that colchicine was left out. The calluses were then returned to the normal growth temperature 25°C. Ploidy was identified by chromosome counts of squashed root-tip cells collected from the newly potted plantlets and later by measurements of stomates and pollen grains. Over 50% of the plants initiated from the colchicine-treated calluses were completely tetraploid. All of the plantlets differentiated from untreated callus had a diploid number of 22. Of the 3 colchicine levels applied, the 20 mg/liter treatment appeared to be the most effective in production of tetraploidy.     

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.     

Improvement of snow mould resistance by conventional and in vitro techniques

Summary Under our climatical conditionsMicrodochium nivale is the main pathogen which causes snow mould in grasses. Like manyFusarium species,M. nivale is able to produce toxins like Deoxynivalenol (DON). In vitro selection with callus cultures from two populations of perennial ryegrass and DON as selective agent was carried out in a one step (subculture) or two step procedure (induction plus subculture). The regenerants from in vitro selection and a seed derived control from the two populations were artificially inoculated withM. nivale. Significant improvement of snow mould tolerance in the high DON variant could be found. Additionally, a toxin test was established which allows to screen large numbers of mature embryos without laborious callus culture. The sexual progenies (R1) of the regenerants were subjected to both the snow mould and the toxin test. Toxin and pathogen resistance were not always in good accordance. The application of both tests in practical plant breeding is discussed.     

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.