Somaclonal variation in Tricyrtis hirta plants regenerated from 1-year-old embryogenic callus cultures

The Liliaceous perennial Tricyrtis hirta, sometimes called ‘Japanese toad lily’, has recently become popular as an ornamental for pot and garden uses. Highly embryogenic callus cultures of this plant predominately consisted of diploid cells but also contained tetraploid cells after 1 year of establishment. In the present study, plans regenerated from the 1-year-old embryogenic callus cultures were subjected to ploidy level analysis and morphological characterization following 3 years of cultivation. Among 37 plants examined, 28 kept the diploid level (2n = 2x = 26) but nine were tetraploid (2n = 4x = 52) as indicated by FCM analysis and chromosome observation. Although no morphological alterations were detected in 26 out of 28 diploid regenerants, the remaining two showed noticeable variations: both were severely dwarf and had crimped leaves and many malformed flowers. The tetraploid regenerants had several horticulturally attractive characteristics compared with the diploid controls, such as longer shoots, thicker stems, and larger flowers. Thus regeneration of tetraploid plants from 1-year-old embryogenic callus cultures offers a possibility to improve the horticultural value of T. hirta, although regeneration of trueness-to-type plants is essential for utilizing the cultures for micropropagation and genetic transformation.     

Modeling individual leaf area of ginger (Zingiber officinale Roscoe) using leaf length and width

Leaf area estimation is an important biometrical observation one has to do for comparing plant growth in field and pot experiments. In this study, a leaf area estimation model was developed for ginger (Zingiber officinale Roscoe), using linear measurements of leaf length (L) and maximum width (W). Leaves from five ginger varieties (Varada, Rejatha, Mahima, Maran and Himachal) were used to develop the model in 2006–2007. The actual leaf area (LA) was measured with a leaf area meter (LI-3100, LI-COR, Lincoln, NE, USA) and taken as reference LA. The linear measurements were used to build linear (LA = a + b × L × W) and power models (LA = α × (L × W)^β) for each variety, as the modeling among variety were not different from each other, data for all five varieties have been pooled and compared with earlier models by graphical procedures and statistical criteria such as Mean Square Error (MSE), Root Mean Square Error (RMSE) and Chi-square (χ²). The selected model was validated during 2007–2008. The validation data set was used to produce a validation model for each variety by re-estimating the model parameters to develop the estimation model and the models were compared for consistency. The predicted LA (PLA) was compared with observed LA (OLA) by graphical procedures and lack of agreement was evaluated by calculating the relative bias, estimated by the mean of differences (d) and the standard deviation (SD) of the differences. Normality test was carried out by Spearman's rank correlation coefficient (r_s) and residuals were normally distributed. Finally, the proposed model for leaf area estimation of ginger is LA = −0.0146 + 0.6621 × L × W, R² = 0.997. This model can be reliably used for estimating leaf area of ginger non-destructively. The same equation can be extrapolated to all varieties and land races of ginger as it is vegetatively propagated crop with narrow genetic variability.     

KClO3 applications affect Phalaenopsis orchid flowering

Potassium chlorate (KClO₃) treatments are known to promote flowering in longan plants. Potential effects of KClO₃ on Phalaenopsis orchid flowering were investigated in the present study. However, increasing application concentrations of 2, 4, 8 and 16 mmol/L KClO₃ delayed spike emergence by 5, 6, 18 and 26 days, respectively. Moreover, they reduced final spike length by 2.1%, 4.0%, 16.2% and 46.1%, respectively. Nonetheless, application of KClO₃ at 4 and 8 mmol/L advanced the time to appearance of the first open flower by 13 and 24 days, respectively. Use of 8 mmol/L KClO₃ also increased the number of floral buds by 16%. Treatments with KClO₃ tended to reduce flower size. Overall, the data suggest that application of KClO₃ at an appropriate concentration (e.g. 8 mmol/L) can increase the number of floral buds and advance the time to Phalaenopsis orchid flowering, but may reduce flower size.     

A practical and reliable procedure for in vitro induction of tetraploid tomato

A relatively rapid, practical and reliable procedure for in vitro production of tetraploid tomato using colchicine is described. Using this procedure 11.11% generation of tetraploids was obtained after exposure of seedling shoot meristem explants with 8 mM colchicine for 96 h. Confirmation of tetraploid production (2n = 48), was determined by flow cytometry and verified by cytogenetic analysis of root tip preparations. The results indicate that the procedure is adequate for induction and screening of tetraploid tomato plantlets.     

Seasonal variation in pigmentation and anthocyanidin phenetics in commercial Eustoma flowers

The seasonal change in petal color and pigmentation of 29 commercial Eustoma cultivars was studied. The flowers are basically divided into four groups according to the major anthocyanidin phenotype in association with petal coloration, i.e., delphinidin (Dp)-based (purple flower), cyanidin (Cy)-based (reddish purple flower), pelargonidin (Pg)-based (pink flower), and none (white flower) groups. The constitution of petal anthocyanidins was not changed by forcing treatment in most of the flowers. Lightness (L^*) and chroma (C^*, color saturation) showed a change along with the increase/decrease of hue angle difference (ΔH^*), thus simultaneously the chromatic tonalities tended to move to redder and bluer, respectively. Floral pigment clustering described two flower groups in a dendrogram, based on anthocyanidin constitutions as phenetic markers, which are apparently the Dp- and Pg-based phenotypes of anthocyanidin syntheses. The Cy-based flowers made a subcluster with the Pg-based flowers, indicating a close relationship in the biosynthesis of the two anthocyanidins, and suggesting the Dp- and Pg-syntheses complement one another.     

Establishment of in vitro tissue cultures from Echinacea angustifolia D.C. adult plants for the production of phytochemical compounds

The establishment of in vitro cultures of Echinacea angustifolia D.C. was obtained directly from sections of flower stalks of adult plants. The shoot formation was obtained from this plant material placed on a modified MS basal medium named CH supplemented with 0.5 mg L⁻¹ 6-benzylaminopurine (BA). The in vitro propagation procedure of E. angustifolia consisted of three distinct phases: an initial regeneration phase from stalk sections (IP shoots on basal medium with 0.25 mg L⁻¹ BA), an elongation phase on active charcoal and an axillary proliferation of the shoots (AP shoots on basal medium with 0.5 mg L⁻¹ BA).Regenerating calli were established from leaves of in vitro shoots cultured on CH medium supplemented with 3 mg L⁻¹ BA and 0.5 mg L⁻¹ indole-3-butyric acid (IBA). Developed shoots from the callus cultures were subcultured on the CH medium with 0.5 mg L⁻¹ BA (leaf regenerated shoots: LR shoots). The secondary metabolite content of the in vitro plant material was compared with that of the greenhouse growing plants. The quali-quantitative LC-DAD-ESI-MS analysis on the extracts from axillary proliferation shoots (AP shoots) showed significant production of caffeic acid derivatives while leaf callus and LR shoots, accumulated mainly alkamides. These results showed that the proper choice of the procedures for in vitro multiplication allowed us to obtain plant biomass able to produce the active compounds typical of E. angustifolia plants.     

Tetraploid muskmelon alters morphological characteristics and improves fruit quality

Tetraploid muskmelon plants were induced successfully from diploid Cucumis melo inbred M01-3 (2n = 24) by colchicine. The morphological characteristics and fruit qualities of diploid and tetraploid muskmelons were investigated. The results showed that the leaves and flowers of the tetraploid plants were markedly larger, the plants were obviously higher, and the stems were thicker than those of the diploid plants. Transmission electron microscope observation revealed that the numbers of chloroplast, granule and grana, and the length of chloroplast and granule of the tetraploid plant leaves were significantly more or longer than those of the diploid plants. The soluble solid, soluble sugar and vitamin C contents in the tetraploid fruit were distinctly higher than those in the diploid fruit. The weight of the tetraploid fruit was 30% heavier than that of the diploid fruit. And the seeds from the tetraploid plants were broader and thicker than those from the diploid plants. Together, our results suggest that the tetraploid muskmelon exhibited better agronomical characteristics than the diploid muskmelon and the tetraploid muskmelon could be used as the improved variety and a potential germplasm for the development of triploid fruit.     

In vitro induction of the amphiploid in interspecific hybrid of blueberry (Vaccinium corymbosum × Vaccinium ashei) with colchicine treatment

Amphiploids were produced from pentaploid hybrids between Vaccinium corymbosum (4x) and Vaccinium ashei (6x) by colchicine treatment. Seeds of each parental species and those obtained by the interspecific crossing were treated with colchicine at 0, 500, 1000 and 2000 mg/l for 7 days, and the ploidy level of the seedlings was determined with flow cytometry. Either amphiploid (10x) or ploidy chimera (5x + 10x) was obtained from interspecific hybrid seeds treated with all colchicine concentrations, while no chromosome-doubled plants were obtained from both parents. Cross direction in interspecific hybridization affected the results of colchicine treatment and amphiploids were obtained only when V. corymbosum was used as the seed parent. In this cross, 17–25% of the seedlings turned to be amphidiploids or ploidy chimeras by treating with 500 mg/l colchicines for 7 days. These results indicate that susceptibility to colchicine may increase in the interspecific hybrid compared with the parental species but only when V. corymbosum was used as female parent.     

Adaptation of endemic mediterranean Sternbergia candida Mathew Et T. Baytop in the continental climate of central anatolia

Sternbergia candida is an important endemic bulbous plant with beautiful white flowers. It grows on the rock slopes of Baba mountains under Lebanon Cedars in the Mugla province of Turkey with Mediterranean climate. The plant is vulnerable and endangered due to illegal collection for local and foreign flower industry. Fast developing tourism industry and rapid urbanization has also shown negative implications on its population. The study reports three years successful adaptation results of S. candida in the continental climate of Ankara in the Central Anatolia. Four groups of bulbs based on diameter (>2.45, 2.15–2.44, 1.85–2.14 and <1.84 cm) were collected from its natural habitat during March, April and May 1998 and were evaluated for frequency (%) of emergence, mean number of leaves per plant, mean plant height, frequency (%) of flowering, frequency (%) of fruit setting. mean number of bulbs per plant, and mean diameter (cm) of regenerated bulbs. All categories of bulbs could be multiplied and adapted successfully with variable frequency of fruit setting. However, the highest fruit set was recorded from 2.15–2.44 cm April collected bulbs. This indicated that the bulb diameter and collection time had clear implications on multiplication, propagation and adaptation of the plant.     

Micropropagation of Dendrobium draconis Rchb. f. from thin cross-section culture

An efficient procedure is outlined for rapid and mass in vitro propagation of an orchid, Dendrobium draconis Rchb. f. through in vitro culture of thin cross-sections (TCSs) derived from young stems. The TCS explants were excised along the stem from the base to shoot tip of 6-month-old plantlets and cultured on Murashige and Skoog (MS) medium supplemented with 20 g/l sucrose and different concentrations of N⁶-benzyladenine (BA), kinetin (Kn) and 1-naphthaleneacetic acid (NAA), either individually or in combination. Protocorm-like bodies (PLBs) were directly induced from the TCS explants and completely developed into shoots within 6–7 weeks. The optimal growth regulators combination for maximal PLB development was 2 mg/l BA and 1.0 mg/l NAA, giving rise to 68% of responding explants with an average 11 PLBs per explant. Shoot development was best achieved on MS medium containing sucrose and coconut water. Plantlets, 6–8 cm height were transplanted into coconut husk peat with 92% survival rate in a nursery.     

Combining endophytic fungi and bacteria for the biocontrol of Radopholus similis (Cobb) Thorne and for effects on plant growth

Eight endophytic fungal and bacterial isolates with antagonistic activity against Radopholus similis were evaluated in vivo for their individual and combined effects on biocontrol of R. similis and on the growth of “Grand Naine” cultivar banana plantlets in the greenhouse. Penetration efficiency (PE) of R. similis was between 3 and 21% in 29 biological agents (BAs) treatments, less than the 29% of the nematode-alone control (p ≤ 0.0001); 24 of the BAs treatments did not differ from the PE of 5% for a nematicide control. Twenty nine BAs treatments exhibited antagonistic activity against nematodes which reduced final population levels between 18 and 93%, relative to those on nematode-alone control plants (p ≤ 0.0001), and 14 BAs treatments were statistically similar to the nematicide treatment (88% reduction). Twenty four BAs treatments had increments of plant root biomass ranging from 20 to 58%, greater than the control plants; 37% of the treatments with single and combined BAs inoculations had root length increments ranging from 29 to 54% compared with control and chemical treatment. The nematicide, Terbufos 10GR, did not affect plant growth.     

Use of a cyclic high-pressure sodium lamp to inhibit flowering of chrysanthemum and velvet sage

Photoperiod is commonly controlled in the commercial production of ornamental crops to induce or prevent flowering. Flower induction in short-day (SD) plants can be prevented or delayed when the natural daylength is short by providing low-intensity lighting during the dark period. A stationary high-pressure sodium (HPS) lamp with an oscillating aluminum parabolic reflector (cyclic HPS) has been developed to provide intermittent lighting to greenhouse crops. We determined the efficacy of a cyclic HPS lamp at preventing flowering in SD plants garden chrysanthemum [Chrysanthemum × grandiflorum (Ramat.) Kitam.] ‘Bianca’, pot chrysanthemum ‘Auburn’, and velvet sage (Salvia leucantha L.) relative to traditional night interruption (NI) lighting strategies. Plants were grown in a glass-glazed greenhouse at a mean daily temperature of 19.5–20.7 °C with natural SD photoperiods. NI lighting was delivered during the middle of the night (2230–0230 h) from a 600 W cyclic HPS lamp mounted at one gable end of the greenhouse or from incandescent (INC) lamps that were illuminated for the entire 4 h (CONT INC) or for 6 min every 30 min for 4 h. Plants under cyclic HPS were grown at lateral distances of 1, 4, 7, 10, or 13 m from under the lamp. Control plants were grown under an uninterrupted 15 h skotoperiod. As the distance from the cyclic HPS lamp increased from 1 to 13 m, the maximum irradiance measured during the NI decreased from 25.4 to 0.3 μmol m⁻² s⁻¹ and time to visible inflorescence (VI) and the number of nodes at VI decreased. All species had a VI within 54 d, but ≤10% of plants flowered when grown at a lateral distance of 1 or 4 m from the cyclic HPS lamp or under CONT INC. Plants grown without NI had a VI 2 to 15 d earlier and flowered 7 to 24 d earlier than plants grown at 10 or 13 m from the cyclic HPS. All garden chrysanthemums flowered under cyclic INC, whereas velvet sage and pot chrysanthemum had 15% and 35% flowering, respectively. These results indicate that a cyclic HPS lamp can be used effectively to delay flower induction and prevent flowering in these species when NI is delivered at ≥2.4 μmol m⁻² s⁻¹.     

Production of intraspecific hybrids between wild-type and petaloid-sepal cultivars in Habenaria radiata

Orchids are commercially important plants with flowers that are unique and very specialized in shape and color. The flowers consist mostly of sepals, lateral petals, lip (labellum) and column, and are zygomorphic and resupinate. Whereas most orchid species have petaloid tepals in the first and second whorls, Habenaria radiata has a flower with greenish sepals and white lateral petals and lip. ‘Hishou’, one of the cultivars of H. radiata, is a floral homeotic mutant and has a petaloid median sepal and lip-like lateral sepals in the first whorl. Additionally, this cultivar often has non-resupinate flowers whereas wild-type H. radiata flowers are resupinate. In the present study, we investigated the genetic inheritance of these characters in the ‘Hishou’ cultivar by crossing it with wild-type plants. Some intraspecific hybrids, which were confirmed by PCR-RFLP analysis, had flowers with a petaloid median sepal and lip-like lateral sepals in the first whorl, indicating that these were dominant characters. Since the remainder of the intraspecific hybrids had wild-type flowers, these characters must be heterozygous in ‘Hishou’ plants. Although ‘Hishou’ plants had non-resupinate flowers, intraspecific hybrid flowers were resupinate, even though they had the petaloid median sepal and lip-like lateral sepals. This result indicates that non-resupination must be a recessive character. Since sepal-petalization and triple lip characters of ‘Hishou’ inherited dominantly, these characters can be utilized for the breeding of Habenaria species by intra- and interspecific crosses.     

In vitro induced tetraploid of Dendranthema nankingense (Nakai) Tzvel. shows an improved level of abiotic stress tolerance

The tetraploid of Dendranthema nankingense (Nakai) Tzvel. was induced by the colchicine treatment using nodal segments. Ploidy level was determined by an analysis of flow cytometry and chromosome counting. The morphological characteristics such as the stomata, leaves, flowers and pollen grains of the tetraploid were significantly larger than those of the diploid. The tolerance responses of the diploid and tetraploid were compared under the imposition of heat, cold, drought and salinity stress. Semi-lethal temperatures suggest that cold tolerance is improved by polyploidization, but the heat tolerance is reduced. Under drought and salt stress, the activity of peroxidase (POD) and relative water content (RWC) in the tetraploid were higher than those in the diploid. Accordingly its malondialdehyde (MDA) content maintained a lower level. The content of chlorophyll (a + b) in the tetraploid was higher, and decrease of its content was postponed in tetraploid compared with the diploid under salt stress. It suggested that polyploidization could alleviate oxidative stress, maintain good water balance and higher chlorophyll (a + b) content, thereby enhanced the drought and salt tolerance in colchicine induced tetraploid.     

Clone identification and genetic relationship among vine cacti from the genera Hylocereus and Selenicereus based on RAPD analysis

Clones of Hylocereus and of Selenicereus species were distinguished from each other by the banding pattern generated by one to nine 10-mer oligonucleotide primers in the random amplified polymorphic DNA (RAPD) reaction. RAPD analysis was also applied to estimate the genetic relationship among five Hylocereus and nine Selenicereus species. A dendrogram was constructed based on a data matrix of 173 polymorphic bands originated by nine primers. Two groups were identified, one consisting of Hylocereus species and the other consisting of Selenicereus species. These results are consistent with the accepted taxonomic classification of the genera studied. The principal coordinate analysis (PCO), i.e. the plot drawn on the basis of the RAPD data, clearly distinguished between three groups, namely, Hylocereus species, S. megalanthus and the rest of the Selenicereus species studied. PCO thus strongly support the notion that the tetraploid S. megalanthus is an exception among the Selenicereus group. The RAPD results support our hypothesis regarding the allopolyploid (rather than autopolyploid) origin of this species.     

Reproductive organography of Bougainvillea spectabilis Willd

Bougainvillea spectabilis Willd. is of prime importance for horticulture, as well as potentially for pharmaceutical industries, agriculture and environmental industries. However, its floral development is not yet well understood. A detailed study on floral structure and floral organography in the species was first completed using microscopy of paraffin microtome sections of buds. The results were indicated as follows: first, the three trumped flowers in the cymose inflorescence develop asynchronously. Secondly, Varieties with multi-whorl bracts do not develop any sexual organs, i.e., perianth, pistil and stamens. Thirdly, the wall of the two-loculus anther consists of two kinds of cells: the inner wall, consisting of thick-cytoplasmed cells and the outer wall, consisting of fibrous cells. Fourthly, the pollen grains, with three germination colpi, vary substantially in the form and size in summer under the highest day temperature of 40 °C. Fifthly, the pistil is characterized with betalain-acumulating stylar brush. Followed the developmental course, only one basal ovule is developed in the superior ovary. Finally, organs of one flower develop consecutively from the outer to the inner, i.e., from bracts, to calyx, stamen, and carpel while the three flowers bloomed one by one in one cymose inflorescence. It almost takes 1 week from first bud to the third flower blooming. Our research showed a series of special characteristics of reproduction organography of B. spectabilis which can be useful for understanding its reproduction biology and its sterility.     

Anatomical and biochemical studies of anthocyanidins in flowers of Anagallis monelli L. (Primulaceae) hybrids

Violet, lilac and red flower colors segregated in an F₃ population obtained from hybridizing blue and orange breeding lines of Anagallis monelli at UNH. One individual per color was studied, as well as “true-blue” cultivar ‘Skylover Blue’. Anatomical examination revealed typical petal layout with upper epidermis, loose mesophyll and lower epidermis. Cells in upper and lower epidermis were categorized by their vacuole color. Blue and red individuals had mostly blue and red cells, respectively. Lilac and violet individuals had blue and bicolored (red and blue) cells on both surfaces, and red cells on the lower epidermis only. Violet individuals had more blue cells on the upper epidermis than lilac individuals. Anthocyanidins were determined by HPLC for each petal epidermis. Blue flowers had only malvidin in both petal surfaces, red flowers had mostly delphinidin with traces of malvidin. Lilac and violet flowers had more malvidin than delphinidin. For violet and lilac flowers respectively, 2 and 3% delphinidin in upper petal surfaces result in a reddish tone while in the lower surface 33 and 25% delphinidin result in a red color. pH in upper and lower petal surfaces were significantly different for each individual, which may affect final flower color.     

Study of long-distance movement of Plum pox virus (Sharka) as an alternative resistance-evaluation method in Prunus

During the breeding programs for Plum pox virus (PPV, Sharka) resistance in Prunus, the evaluation of the new releases through symptoms observation on leaves has been contradictory and represents one of the main handicaps in these programs. In order to increase the accuracy of this traditional evaluation method, we here analyze an alternative method based on the study of the ability of a genotype to allow the long-distance movement of the virus through its vascular vessels. Two different plant models have been assayed: (a) in Model I, the inoculation was performed in the ‘GF305’ rootstock with a later grafting of the genotype under evaluation and a scion of healthy control ‘GF305’, to evaluate the long-distance movement through the studied genotype from the rootstock to the scion (xylem transport), and (b) in Model II, the inoculation with ‘GF305’ diseased scions was performed by grafting these diseased scions onto the studied genotypes, which were grafted onto healthy ‘GF305’ peach seedlings, to evaluate the long-distance movement through the studied genotype from the scion to the rootstock (phloem transport). The results show that, regardless of the presence of symptoms, susceptible genotypes allowed the movement of the virus through their vascular vessels in both directions studied. However, the resistant apricot ‘Stark Early Orange’ did not allow this movement. We propose the study of the ability of a genotype to allow the long-distance movement of the virus as an alternative and more accurate method for the evaluation of PPV resistance. However, this protocol is much more tedious than the traditional one and could be used mainly in the evaluation of a reduced number of more interesting genotypes.     

Intergeneric hybridizations between Opisthopappus taihangensis and Chrysanthemum lavandulifolium

For the first time, reciprocal intergeneric hybridizations were produced between Opisthopappus taihangensis and Chrysanthemum lavandulifolium without emasculation. About 20% seed set was similarly obtained from reciprocal hybridization. Only 5 and 6 of the viable plants observed from 45 and 78 seedling survived present some conspicuous intermediate characteristics. Phenotypic evaluation among the progenies of the parents and the putative hybrids was performed carefully since an average of 5.3% seed set was produced in the type of self-pollination using pollen from the same flower and >10% seed set was similarly obtained in the types of self-pollination using pollen from different flowers in a plant and flowers in individual plants from different seeds. One individual of each hybrid shared the inflorescence habit with the pollen plant was confirmed further by the sequence of ncpGS. The two hybrids might be used as bridges of breeding of multi-generic hybrids.     

Development of a disease severity rating scale for plane tree anthracnose

Anthracnose caused by the fungus Apiognomonia veneta (Sacc. et Speg.) Höhnel., is the most important and frequent disease affecting mature ornamental plane trees (Platanus×hybrida Brot.=P.×acerifolia (Ait.)Willd.) of central Spain. Symptoms of the disease are: leaf vein and petiole necrosis, bud death, defoliation, proliferation of shoots growing in whorls, short internodes, cankers, necrotic lesions and twigs and branches growing in angles. Based on our previous experience, a disease severity rating scale (DSRS) has been established, consisting of six levels: 0=healthy, 1=initial, 2=low, 3=medium, 4=high and 5=dead. In order to achieve a better and more efficient measure of disease intensity we tried to quantify this visual scale using mathematical criteria. In 2000, 610 mature trees up to 200 years old, divided into 4 age classes, were selected and systematically evaluated using a visual estimation based method. The trees were located in central Spain, mainly in two periurban areas of the town of Aranjuez (south of Madrid) and in three urban areas of Madrid city. Trees were observed in late spring, summer and winter. We recorded visual estimation of disease severity, foliage cover, healthy new shoots, dead branches, shoot growth in whorls and branch growth in angles. The statistical relationship between the parameters, disease severity and all the variables recorded (defoliation, healthy new shoots, etc.) has been evaluated. “Leaf density”, “dead branches” and “healthy shoots” were the variables that help to discriminate better between the different levels of the DSRS. A clearer definition of the different phases of disease severity will facilitate the application of possible control methods and the prediction of the behaviour of other Platanus spp.