In vitro propagation using adventitious buds technique as a source of new variability in chrysanthemum

Eleven cultivars of Chrysanthemum × grandiflorum (Ramat.) Kitam.: ‘Richmond’ and its 10 radiomutants, representing the Lady group, were propagated in vitro with shoot tips and leaves as explants. The aim of this study was to investigate if the explant type used for micropropagation affects the genotype and phenotype of chrysanthemums. Plants grown from shoot tips and adventitious buds formed on leaves were rooted in vitro, acclimatized and cultivated in glasshouse up to full-flowering. The colour and shape of inflorescences of plants obtained from two different explant types were compared within the cultivars. All plants derived from shoot-tip explants showed the inflorescence colour and shape typical for the cultivars. Inflorescence colour of plants derived from adventitious buds were true-to-type in four cultivars: ‘Richmond’, ‘Lady Amber’, ‘Lady White’ and ‘Lady Yellow’. All plants of ‘Lady Apricot’ (originally: golden beet) and ‘Lady Salmon’ (salmon) propagated from adventitious buds technique showed altered inflorescence colour (respectively: purple gold; pink and white). ‘Lady Bronze’ (originally: reddish brown), ‘Lady Orange’ (orange brown) and ‘Lady Rosy’ (purple gold) propagated with adventitious buds had both typical and changed inflorescence colours (respectively: yellow; yellow and red; reddish pink). ‘Lady Vitroflora’ showed altered number of ligulate florets grown into tubes in inflorescence when propagated with shoot tips and leaves as explants. Those changes might be an effect of either chimeral structure or somaclonal variation of the plants investigated. The variation appears only if non-meristematical explants were used. The adventitious buds technique might be useful in chrysanthemum breeding as a source of a new variability.     

Adventitious root formation in Anacardium occidentale L. in response to phytohormones and removal of roots

Despite advances in tissue culture techniques, propagation by leafy, softwood cuttings is the preferred, practical system for vegetative reproduction of many tree and shrub species. Species are frequently defined as ‘difficult’- or ‘easy-to-root’ when propagated by conventional cuttings. Speed of rooting is often linked with ease of propagation, and slow-to-root species may be ‘difficult’ precisely because tissues deteriorate prior to the formation of adventitious roots. Even when roots form, limited development of these may impair the establishment of a cutting.     

Growth and flower quality of four Rosa hybrida L. cultivars in response to propagation by stenting or cutting in soilless culture

Performance of four Rosa hybrida L. cultivars (‘African Dawn’, ‘Ilios’, ‘Maroussia’ and ‘Soprano’) was evaluated. They were grown either on their own roots or grafted (stenting) onto Rosa canina L. ‘Inermis’ rootstock in a polyethylene greenhouse with hydroponics system. Parameters of plant growth and flower quality were investigated for two successive harvesting years (2005 and 2006). Results indicated that, all the cultivars were superior for most of the parameters studied when grafted onto rootstock compared to being on their own roots. Flowering stem fresh weight and diameters, flower fresh and dry weight, flower diameter and length, petal number, leaf chlorophyll content and quality index were higher in grafted plants compared to those propagated by cuttings. However, highest flowering stem length and number were observed in plants propagated by cutting, although not significant as compared with stenting method.     

A pollen extender medium technology for seedless watermelon production by pollination with soft X-ray irradiated pollen

The inactivation of pollen by soft X-ray irradiation and subsequent artificial pollination are time-intensive practices used in the production of seedless watermelons (Citrullus lanatus L.). Watermelon generally has a lot of staminate flowers; however, they only have a small amount of pollen. Watermelon pollen cannot be used in pollination under the present situation. Therefore, the objective of this study was to determine which is the most effective pollen extender medium for cultivation of watermelon with soft X-ray irradiated pollen. In this experiment, ‘Agar,’ ‘Marriage powder,’ and ‘Sekishoshi’ (Lycopodium dyed red with safflower pigment) were used as extender media with soft X-ray irradiated pollen at equal or twice the weight of the pollen. When ‘Sekishoshi’ was used as an extender medium, fruit set was very low. A lot of deformed fruit was produced when agar was used with the pollen. On the other hand, when ‘Marriage powder’ was used in equal proportions with the pollen, fruit set was about 70% and, moreover, Brix was high. Thus, the mixture of ‘Marriage powder’ with an equal amount of pollen was the best for use in actual cultivation. We conclude, then, that soft X-ray irradiated pollen in an extender medium can be effectively adapted for producing seedless watermelons.     

Effects of cadmium and copper on pollen germination and fruit set in pea (Pisum sativum L.)

In this work, pea (Pisum sativum) plants exposed to increasing cadmium and copper concentrations were tested for heavy metals accumulation in flowers and for ‘in vivo’ pollen germination. Based on the Cd and Cu accumulation amounts in the flowers, an evaluation of the same metals effects on ‘in vitro’ pollen germination was achieved. Moreover, the effects of both metals on fruits number and weight and on seed set and yield at individual plant level were examined. While cadmium concentrations did not affect ‘in vivo’ pollen germination, only higher copper concentrations rendered a significant reduction. This is in contrast with the clear negative effect on pollen germination in vitro and might be explained by the different dynamic and bioavailability of both metals. A clear effect of Cd and Cu was observed on two important yield components ie, fruit weight and seed set. Although results obtained herein cannot give a clear cut relationship between the effect of Ca and Cu on reproductive development and its consequences on yields, they represent emerging results on the potential consequences of metals contamination on reproductive development in plants.     

Adventitious shoot regeneration in a bioreactor system and EST-PCR based clonal fidelity in lowbush blueberry (Vaccinium angustifolium Ait.)

Adventitious shoots were regenerated from leaf explants of three lowbush blueberry (Vaccinium angustifolium Ait.) clones: ‘QB1’, ‘QB2’ and ‘PB1’ by culturing on a gelled basal medium (BM) with 2.3–4.5 μM thidiazuron (TDZ) for four weeks followed by in a bioreactor system containing the same liquid medium but with 1.2–2.3 μM TDZ for another four weeks. Young expanding basal leaf segments with the adaxial side touching the culture medium and maintained for two weeks in darkness, produced the best results. Callus development and shoot regeneration were genotype dependent. Adventitious shoots were elongated in the liquid BM with 1 μM zeatin and rooted on a three peat: two perlite (v/v) medium. Acclimatized plantlets were grown actively in the greenhouse with an apparent normal leaf and shoot morphology. Ten random ‘QB1’ regenerated plants were screened using 14 expressed sequence tag-polymerase chain reaction (EST-PCR) markers and showed similar monomorphic amplification profiles confirming clonal fidelity of in vitro-derived ‘QB1’ plants. Results obtained suggested the possibility of adventitious shoot regeneration and true-to-type lowbush blueberry micropropagation using a bioreactor system combined with gelled medium.     

Correlation of phytoplasma concentration in Hydrangea macrophylla with green-flowering stability

The interaction between phytoplasma concentration and green-flowering stability was studied in hydrangea cultivars. Three green and 18 nongreen cultivars were subjected to polymerase chain reaction (PCR) analysis to determine Japanese hydrangea phyllody (JHP) phytoplasma infection. The results showed that JHP-phytoplasma was detected only in ‘Midori’ plants, which have green sepals. ‘Midori’ plants were propagated, and from 29 rooted cutting plants, they were grouped into three types on the basis of sepal color, that is, green (75.9%), blue-green (13.8%) and blue (10.3%) sepals. To clarify the variability in the sepal color of ‘Midori’ plants, JHP-phytoplasma concentration in the sepals and leaves of green-, blue-green- and blue-flowering plants was determined by PCR analysis. The semiquantitative PCR comparisons of 370 bp DNA fragments showed that the JHP-phytoplasma concentrations in green sepals were 16 times higher than that in blue-green sepals. JHP-phytoplasma could not be identified by PCR analysis in blue sepals and leaves. These results showed that JHP-phytoplasma concentration correlated with green sepal stability in ‘Midori’ plants. A histological observation of sepals showed that epidermal cells of blue and blue-green sepals had a dome shape. Otherwise, green sepals were leaflike with flat epidermal cells, and palisade parenchyma cells with numerous chloroplasts.     

Effects of genotype and stigma development stage on seed set following intra- and inter-specific hybridization of Dianthus spp.

The caryophyllaceae is a family of flowering plants commonly known as the pink or carnation family. Members of this family are widely grown as ornamental plants. The species Dianthus chinensis, Dianthus barbatus, and Dianthus superbus include notable examples of such ornamental varieties. Intra- and inter-specific hybridization is important for the breeding of improved varieties and, therefore, it is desirable to optimize the efficiency of cross-pollination procedures within this family. Here, we conducted intra-specific pollination using four lines of D. chinensis, and inter-specific pollination between D. chinensis, D. barbatus and D. superbus genotypes. For each of these crosses we recorded information of pollen viability, stigma development stage at time of pollination, pollen germination on the stigma surface and pollen tube elongation within the stigma, and final seed set. Pollen viability levels were determined at the start of anther dehiscence, which occurred on day 1 of flower opening. The genotypes D. chinensis var. ‘Bz2’, D. barbatus var. ‘Xb’, and D. superbus wild species ‘Qm’ all displayed good levels of pollen viability (i.e. between 68% and 75%); D. chinensis vars. ‘H58III’, ‘H68I’ and ‘H6II’ showed lower levels of pollen viability (i.e. between 32% and 51%). In each Dianthus line, the stigma organs were observed to undergo morphological changes during the 1–7 d following flower opening. We have characterized these changes as five distinct development stages, namely, ‘column’ (day 1), ‘Y-like’ (day 2), ‘Y-shaped with slight curve’ (days 3–4), ‘Y-shaped horn-like’ (days 5–6), and ‘wilting’ (day 7). Pollen was artificially applied to stigma organs at each of these development stages and aniline blue staining was used to follow pollen tube germination on the stigma surface and pollen tube elongation down the length of the stigma. This showed that the ‘Y-shaped slightly curved’ stigma stage (3–4 d) was optimal for pollination. The ‘Y-shaped horn-like’ stage (5–6 d) was also receptive to pollination. By contrast, the earlier ‘Y-shaped’ (2 d) and ‘column’ (1 d) stages, and also the later ‘wilting’ (7 d) stage, were far less receptive. Parallel field experiments were conducted in which seed set was measured following the artificial pollination of flowers at different development stages. These data also indicated that the optimal time for pollination was during 3–4 d of flower opening. This conclusion held true for both intra- and inter-specific crosses of the various Dianthus lines. Thus, stigma receptivity in Dianthus does not become optimal until 3–4 d after flower opening. Since anther dehiscence occurs at 1 d (i.e. when stigma are at the unreceptive ‘column’ stage), this suggests that Dianthus employs a delayed self-fertilization strategy. We also found a significant effect of genotype on pollen tube growth and seed-set values. The highest numbers of seed per flower were achieved when the D. superbus wild species ‘Qm’ was the female parent in inter-specific crosses. Thus, genotype and stigma development are identified as key factors that determine the success of cross-pollination in Dianthus lines.     

The effect of forcing temperature on peony shoot and flower development

Pre-chilled potted plants of Paeonia ‘Coral Sunset’, ‘Monsieur Jules Elie’, ‘Sarah Bernhardt’, and ‘Karl Rosenfeld’ were placed in a range of controlled temperature regimes to ascertain the effect of temperature on the timing of shoot emergence and floral development. For all cultivars, warmer temperatures up to 25 °C lead to more rapid shoot emergence and flower development. Linear temperature responses adequately described the rate of development from shoot emergence to flower bud appearance, and from bud appearance to flower opening, but a curvilinear response was required to describe the time taken for shoots to emerge. There were significant differences between cultivars in the number of heat units required for shoot emergence, with the shoots of the slowest-developing cultivar, ‘Monsieur Jules Elie’, taking 50% longer to emerge than those of the most rapid, ‘Coral Sunset’. No significant differences were found among cultivars in the time taken from shoot emergence to flower opening, although the ‘split’ stage (when the bud opens sufficiently for petal colour to be observed) was slightly earlier in ‘Karl Rosenfeld’.     

Effect of distance from early flowering pollinizers ‘Maypole’ and ‘Dolgo’ on ‘Fiji’ fruit set

An apple orchard consisting of a single cultivar under the condition of natural pollination must have suitable pollinizers such as Crab apples to ensure stable fruit production. We selected ‘Maypole’ and ‘Dolgo’ as pollinizers for the cultivar ‘Fuji’, and investigated the rate of fruit and seeds in ‘Fuji’ fruits produced by pollen of the pollinizers. We developed a method for tracing pollen flow based on the leaf color of progeny and S-RNase allele of ‘Maypole’, and on Simple Sequence Repeat (SSR) analyses of ‘Maypole’ and ‘Dolgo’. These were powerful tools for determining the distance insects (mainly Osmia cornifrons) carry pollen from the pollinizers to ‘Fuji’. Although the fruit set of ‘Fuji’ apples was not reduced with increasing distance between ‘Fuji’ apple trees and pollinizers (probably due to pollen flow from other commercial cultivars planted outside the area), the rate of ‘Fuji’ apple fruit produced by the pollen of the pollinizers decreased with increasing distance. The rate of fruit produced by the pollinizers was 84% and 77% when ‘Fuji’ was 2.5 m from ‘Maypole’ and ‘Dolgo’, respectively, and 71% and 64% when ‘Fuji’ was 5 m from ‘Maypole’ and ‘Dolgo’, respectively, but was reduced to 47% and 39% when ‘Fuji’ was 10 m from ‘Maypole’ and ‘Dolgo’, respectively. However, the spacing could cause reduced fruit size, and require extra fruit thinning for producing large fruits. It could also cause reduction of the yield for increasing the planting area of pollinizers. We recommend that pollinizers should be planted not more than 10 m from ‘Fuji’.     

Ray florets color and shape mutants induced by C ion beam irradiation in chrysanthemum

Ion beam irradiation is attracting attention in floriculture as a means of inducing mutations. We investigated the effect of ion beam irradiation on induction of ray florets color/shape mutants from two strains of chrysanthemum to create new flower cultivars. The ray florets and leaf explants of chrysanthemum cultivars, ‘Shiroyamate’ and ‘H13’, respectively, cultured on MS medium were irradiated with ¹²C⁵⁺ ion beam at doses of 1, 2, 4 and 8Gy. The frequency of shoot primordia formation on ray florets explants of ‘Shiroyamate’ and shoot bud formation on leaf explants of ‘H13’ was decreased by 8Gy and 4Gy irradiation, respectively. The effective dose of ion beam was less than 4Gy in ‘Shiroyamate’ and less than 2Gy in ‘H13’. After exposure to an effective dose of ion beam, regenerated plants were grown in a green-house and the field. Yellow ray florets mutants from ‘Shiroyamate’, and various ray florets color mutants (dark-red, light red, pink, pink spray) and a flower shape mutant (double-ray florets) from ‘H13’ were induced by ion beam irradiation. Furthermore, a white mutant was obtained from a chimeric mutant. These results suggest that the combination of ion beam irradiation and tissue culture would be an effective means of generation mutants at a high efficiency.     

Anther wall development,microsporogenesis and microgametogenesis in male fertile and sterile chrysanthemum (Chrysanthemum morifolium Ramat., Asteraceae)

Anther wall development, microsporogenesis and microgametogenesis were compared between a normal male fertile chrysanthemum cultivar ‘NJAU04-29-2’ and the two male sterile selections ‘rm20-11’ (anther indehiscent) and ‘NJAU05-52-2’ (anther aborted). In both of the two male sterile types, the tapetum enlarged abnormally and showed signs of disorganization of walls at the onset of meiosis, the pollen was aborted, the anthers appeared shrunken, and the anther vascular bundle and connective tissue were degenerated by anthesis. In ‘rm20-11’, the two smaller pollen sacs began to degenerate at the microsporogenesis stage, so that only one or two microsporangia developed, while in ‘NJAU05-52-2’, only one or two microsporangia were formed following the archesporial cell stage, and most of the microspore mother cells degenerated during the course of meiosis.     

Field performance characterization of strawberry (Fragaria × ananassa Duch.) plants derived from cryopreserved apices

This study compares agronomic traits of two cultivars of strawberry in plants derived from conventional propagation, micropropagation and cryopreserved apices. The agronomic traits evaluated were fruit production and fruit quality. Differences were found in some of the fruit production traits studied in the plants post-micropropagation and post-cryopreservation when compared to conventionally propagated plants of the cultivar ‘Andana’; showing the first two groups higher second class yield and smaller fruits. Plants of the cultivar ‘Camarosa’ post-cryopreservation showed differences in traits related to fruit quality when compared to the other two groups of plants (internal colour, firmness and soluble solids). No adverse effect on agronomic traits could be associated with the use of cryopreservation. Plants from in vitro and cryopreservation origin were phenotypically similar to the conventional propagated plants in production field.     

Elimination of a new ampelovirus (GLRaV-Pr) and Grapevine rupestris stem pitting associated virus (GRSPaV) from two Vitis vinifera cultivars combining in vitro thermotherapy with shoot tip culture

A new virus species designated as Grapevine leafroll associated virus-Pr (GLRaV-Pr), which is classified in a distinct phylogenetic group of the genus Ampelovirus (Closteroviridae), was recently characterized from Greek grapevine cultivars. Elimination studies of GLRaV-Pr were carried out in two grapevine cultivars, ‘Mantilaria’ and ‘Prevezaniko’, co-infected with Grapevine rupestris stem pitting associated virus (GRSPaV, Flexiviridae). Both viruses were detected by nested RT-PCR assays. Virus elimination was achieved by combining in vitro thermotherapy with meristem (≤0.2 mm) or shoot tip culture (≤0.5 cm). The survival and regeneration rate of meristems was very low. On the other hand, high survival rates were observed in the cultured shoot tips accompanied with high elimination rates for both viruses. Data obtained in this study indicate that virus elimination depends on the genotype of grapevine. The results confirmed that sanitation is easier for species of the Closteroviridae family than for GRSPaV, whereas it seems that eradication of GLRaV-Pr and GRSPaV is feasible even with larger plant tissue parts if combined with an appropriate thermotherapy profile in vitro.     

Diploid female gametes induced by colchicine in Oriental lilies

The young flower buds of diploid Oriental cultivars: ‘Con. Amore’ and ‘Acapulco’ (Lilium) were treated with 0.02, 0.05, 0.1, and 0.2% colchicine to induce diploid egg (female gamete). The treated young buds between the two cultivars were crossed reciprocally as female parents with normal pollen respectively. The polyploid plants were identified by measuring stoma size and chromosome number. The results showed: that triploid progenies could be obtained through this way, and the fact that the treated young flower buds were successfully used as female parents indicated the formation of 2n or 2x egg cells. The above results implied that polyploidization by artificially induced diploid female gametes could be a powerful method to create novel variations in the breeding of Oriental lilies.     

两个柱花草品种根系构型差异及其对间作柑橘砧木实生苗磷营养的竞争

 根系构型与植物的养分吸收密切相关,可能影响植物不同个体间的养分竞争。比较了‘格 拉姆’和‘184 号’柱花草（Stylosanthes Sw.）的根系构型差异,并探讨了两者在间作条件下对柑橘砧木 红藜檬（Citrus limonia）实生苗的生长和养分吸收的影响。结果表明,‘格拉姆’和‘184 号’在单作条 件下生物量、总根长、主根长和根尖数相似,但是‘184 号’的基根角度、根系表面积、平均根直径均小 于‘格拉姆’;在与柑橘实生苗间作条件下,‘184 号’有41.4%的根系进入柑橘根区,而‘格拉姆’仅有 0.8%的根系进入柑橘根区,因此与‘184 号’间作的柑橘砧木实生苗的生物量和对磷养分的吸收均显著低 于与‘格拉姆’间作的柑橘砧木实生苗。本研究表明,与根系垂直生长型的‘格拉姆’相比,根系水平 生长型的柱花草‘184 号’不宜用于果园生草栽培,根系构型应该成为草种筛选的重要指标。     

Production and characterization of new triploid seedless progenies for mandarin improvement

Eighty-six triploid Citrus plants were recovered from interploid crosses between a natural tetraploid selection of the tangerine ‘Dancy’ (Citrus reticulata Blanco, cultigroup ‘Tangerine’), used as the pollen parent, and two seedy selections of diploid mandarins (C. reticulata Blanco) as well as one clementine (Citrus clementina Hort. ex Tan.): ‘Fortune’ mandarin, ‘Wilking’ mandarin and ‘Monreal’ clementine. Flow cytometric analysis was used for screening the triploid plantlets and the ISSR-PCR technique was used to characterize the obtained triploids through a double approach, confirming the hybrid nature of the offspring and allowing the analysis of the genetic pool obtained. Selection among triploid genotypes generated from elite seedy parents will give us a better chance to obtain superior mandarin cultivars characterized by true seedlessness.     

Regeneration of adventitious shoots from mature stored cotyledons of Japanese plum (Prunus salicina Lind1)

Adventitious shoot regeneration from mature stored cotyledons of Japanese plum (Prunus salicina Lind1) was achieved in vitro. The influences of the presence and absence of the light, different concentrations of thidiazuron (TDZ) and benzyladenine (BA) in the culture media, TDZ pretreatments and different basal salts on shoot regeneration were evaluated. TDZ was more effective in inducing shoot regeneration from mature stored cotyledons than BA. Dark incubation significantly increased the regeneration frequencies. Quoirin/Lepoivre (QL) basal salts stimulated shoot regeneration more than woody plant (WPM) or B5 salts did. The frequency of adventitious shoot formation varied among the varieties and the regeneration ability appeared to be genotype depended. The frequency of regeneration under the optimum tested conditions for ‘Bruce’, ‘Shiro’, ‘Redheart’, ‘Gladstone’ and ‘Early Golden’ cotyledons were 66.7%, 46.7%, 43.3%, 26.7% and 6.7%, respectively.     

Investigation of the fertilization biology of some sweet cherry cultivars grown in the Central Northern Anatolian Region of Turkey

The experiments were carried out between 2003 and 2005 in the Province of Amasya. The first objective of the study was to determine the best pollinizer for the cultivar ‘0900 Ziraat’ which is the most popular sweet cherry cultivar (Prunus avium L.) in Turkey. The ‘0900 Ziraat’ was used as a female parent and ‘Türko?lu’, ‘Köro?lu’, ‘Kargayüre?i’, ‘Hac? Ali’, ‘Starks Gold’ and ‘Geçkiraz’ were used as pollinizer cultivars. In 2003, for the hand cross pollination combinations, fruit set was between 31.25% and 42.80%. In 2004, fruit set was reduced by spring frost and was between 9.69% and 15.24%. Additionally, the blooming periods of the experimental cultivars, fruit set of open pollination and hand self pollination were examined. The fruit set ratios of all tested cultivars under open pollination conditions varied between 26.73% and 53.49% in 2003 and 4.38% and 38.35% in 2004. ‘Geçkiraz’ had the highest fruit set owing to its late flowering date. For open pollination, fruit set values of ‘0900 Ziraat’ were lower than hand cross pollination with other cultivars in 2003 when the results of both treatments are compared. On the contrary, fruit set was higher than hand cross pollination in 2004. In the present study, pollen germination values varied greatly among year, cultivar and sucrose concentration. Viability percentages were generally high, not less than 66%. According to phenological observations, partial or complete overlapping of the flowering period of ‘0900 Ziraat’ and its possible pollinizer cultivars were observed in 2003 and 2005. But in 2004, the flowering period overlapped for only ‘0900 Ziraat’ and ‘Geçkiraz’ cultivars. When blooming times are taken into consideration, ‘Geçkiraz’ was found to be the best pollinizer cultivar.     

Characterization of sequence-related amplified polymorphism markers analysis of tree peony bud sports

Bud sports have been widely exploited for creating novel cultivars in vegetatively propagated plants. However, bud sports of tree peony have rarely occurred nor been applied to breeding. We used sequence related amplified polymorphism (SRAP) markers to characterize two tree peony groups with bud sports. ‘Er Qiao’ and ‘Luo Yang Hong’ formed group I and are derived from Chinese cultivars, while ‘Shima-nishiki’ and ‘Taiyō’ formed group II and are related to Japanese cultivars. The DNA samples were evaluated with 25 different primer pairs. This resulted in a total of 149 different amplicons in Group I, of which 80.5% were polymorphic. Group II generated a total of 57 amplicons, of which 63.2% were polymorphic. The two groups shared 43 amplicons. The genetic similarity among the cultivars ranged from 0.33 to 0.60. The two bud sports groups were apparently separated on the unweighted pair-group method with arithmetic averages cluster analysis tree. The results demonstrated that this marker system appeared effective in detecting molecular differences between original parents and their bud sports in tree peony. This study provides a technical base for characterizing plants with bud sports, which are valuable genetic resources for further breeding.