Application of molecular tools for detection of plant viruses

Grapevine leafroll disease (GLRD) is one of the most important virus diseases of grapevines worldwide, causing major economical impact. The disease has a complex aetiology and currently eleven phloem-limited viruses, termed in general Grapevine leafroll-associated virus (GLRaVs), have been identified. Two of the GLRaVs, GLRaV-1 and GLRaV-3, are included in the European certification scheme of propagation material. However, the flawed notion that GLRaV-3 is more frequent than GLRaV-1 and that all other GLRaVs are possibly not as relevant for GLRD, has until now precluded the development of specific serological and molecular detection assays and limited the scope of molecular characterization of the viruses known to be associated with the disease. Hence, few studies have addressed the phylodynamics of GLRaVs or even characterized the genetic structure of their natural populations. This generalized lack of molecular information, in turn underlie the deficient capacity to detect the viruses. The phylogenetic analyses were conducted on the basis of the heat shock protein 70 homologue (HSP70h) and the coat protein (CP) genes for GLRaV-1 and the HSP70h, the heat shock protein 90 homologue (HSP90h) and the CP genes for GLRaV-5. The data obtained for GLRaV-1 contributed 83 new CP sequences. This information was combined with previous analysis by other authors and used for the production of new polyclonal IgG, capable of detecting CP variants from all the phylogroups observed. Successful testing of this new tool included tissue print immunoblotting (TPIB) and in situ immunoassay (ISIA). The data obtained for GLRaV-5, contributed 61 new CP and 28 new HSP90h gene sequences. Eight phylogenetic groups were identified on the basis of the CP. Characterization of the genetic structure of the isolates revealed a higher diversity than previously reported and allowed the identification of dominant virus variants. For both GLRaV-1 and GLRaV-5, the effect of vegetative propagation on the virus transmission dynamics was addressed.

Development of mechanical methods for cell-tray propagation and field transplanting of dwarf napiergrass (Pennisetum purpureum Schumach.)

Since dwarf napiergrass (Pennisetum purpureum Schumach.) must be propagated vegetatively due to lack of viable seeds, root splitting and stem cuttings are generally used to obtain true-to-type plant populations. These ordinary methods are laborious and costly, and are the greatest barriers for expanding the cultivation area of this crop. The objectives of this research were to develop nursery production of dwarf napiergrass in cell trays and to compare the efficiency of mechanical versus manual methods for cell-tray propagation and field transplanting. After defoliation of herbage either by a sickle (manually) or hand-mowing machine, every potential aerial tiller bud was cut to a single one for transplanting into cell trays as stem cuttings and placed in a glasshouse over winter. The following June, nursery plants were trimmed to a 25–cm length and transplanted in an experimental field (sandy soil) with 20,000 plants ha^(−1) either by shovel (manually) or Welsh onion planter. Labour time was recorded for each process. The manual defoliation of plants required 44% more labour time for preparing the stem cuttings (0.73 person-min. stemcutting^(−1)) compared to using hand-mowing machinery (0.51 person-min. stem-cutting^(−1)). In contrast, labour time for transplanting required an extra 0.30 person-min. m^(−2) (14%) using the machinery compared to manual transplanting, possibly due to the limited plot size for machinery operation. The transplanting method had no significant effect on plant establishment or plant growth, except for herbage yield 110 days after planting. Defoliation of herbage by machinery, production using a cell-tray nursery and mechanical transplanting reduced the labour intensity of dwarf napiergrass propagation.

The effects of photoperiod and light spectrum on stock plant growth and rooting of cuttings of Cotinus coggygria 'Royal Purple'

Extending the season of production and improving the scheduling of ornamental crops are key commercial objectives for nurserymen. In some woody species, the period in which cuttings can be rooted successfully is transient, thus limiting the opportunities for scheduled production. Optimum rooting often occurs in early- to mid-summer coinciding with periods of active shoot growth. The relationship between this shoot activity and root initiation was investigated in Cotinus coggygria 'Royal Purple'. Shoot growth on stock plants was manipulated by altering the photoperiod or light quality. Results indicated there were seasonal effects on rooting, but the importance of shoot activity varied with harvest time. Cuttings harvested in August had high rooting percentages, irrespective of photoperiod, and despite shoot growth terminating in response to the short-day treatment. In contrast, by September, rooting percentage was highest in cuttings from plants under long-days, which had maintained greatest shoot growth activity. Cotinus shoots grown in vitro under 16 h days showed reduced shoot growth and increased rooting competence compared with shoots grown under 8 h days. Growing stock plants under polythene films, which altered the amount and quality of the incident light, influenced the rooting of cuttings harvested in August, but no consistent relationship with shoot activity was apparent. From a practical viewpoint, maintaining shoot activity late in the season may prolong the period for propagation by cuttings; but, from a scientific viewpoint, processes associated with an active shoot apex do not provide a complete explanation of seasonal variation in rooting.

Numerical modeling of the propagation environment in the atmospheric boundary layer over the Persian Gulf

Strong vertical gradients at the top of the atmospheric boundary layer affect the propagation of electromagnetic waves and can produce radar ducts. A three-dimensional, time-dependent, nonhydrostatic numerical model was used to simulate the propagation environment in the atmosphere over the Persian Gulf when aircraft observations of ducting had been made. A division of the observations into high- and low-wind cases was used as a framework for the simulations. Three sets of simulations were conducted with initial conditions of varying degrees of idealization and were compared with the observations taken in the Ship Antisubmarine Warfare Readiness/Effectiveness Measuring (SHAREM-115) program. The best results occurred with the initialization based on a sounding taken over the coast modified by the inclusion of data on low-level atmospheric conditions over the Gulf waters. The development of moist, cool, stable marine internal boundary layers (MIBL) in air flowing from land over the waters of the Gulf was simulated. The MIBLs were capped by temperature inversions and associated lapses of humidity and refractivity. The low-wind MIBL was shallower and the gradients at its top were sharper than in the high-wind case, in agreement with the observations. Because it is also forced by land–sea contrasts, a sea-breeze circulation frequently occurs in association with the MIBL. The size, location, and internal structure of the sea-breeze circulation were realistically simulated. The gradients of temperature and humidity that bound the MIBL cause perturbations in the refractivity distribution that, in turn, lead to trapping layers and ducts. The existence, location, and surface character of the ducts were well captured. Horizontal variations in duct characteristics due to the sea-breeze circulation were also evident. The simulations successfully distinguished between high- and low-wind occasions, a notable feature of the SHAREM-115 observations. The modeled magnitudes of duct depth and strength, although leaving scope for improvement, were most encouraging.

Propagation, growth, and carbohydrates of Dendrobium Second Love (Orchidaceae) in vitro as affected by sucrose, light, and dark

In general, plant material grown in vitro has low photosynthetic ability to achieve positive carbon balances. Therefore, a continuous supply of carbohydrates from the culture medium is required, and sucrose has been the most commonly used carbon source. In this paper, we investigate the effects of different sucrose concentrations and the presence and absence of light on the endogenous levels of soluble carbohydrates and starch as well as on the proliferation and growth of Dendrobium Second Love (Orchidaceae) in vitro. The possibility of using etiolated stem segments as a means for micropropagating this hybrid was also verified. The results obtained indicated that the presence and absence of light and the sucrose concentrations used influenced the amounts of soluble carbohydrates and starch and the proliferation of D. Second Love shoots and roots. An increase in sucrose concentration caused a progressive increase in the amounts of total carbohydrates and starch. Under both light conditions, sucrose was the main sugar found in the shoots followed by glucose and fructose. The addition of sucrose to the culture medium up to 2% and 4% was advantageous to the number of shoots produced per explant and the root longitudinal growth in the presence and absence of light, respectively. Shoot and root dry matter and the number of roots formed per explant increased as sucrose concentration was raised up to 6% in both light treatments. The use of dark-grown shoot segments proved to be a useful and reliable alternative for the micropropagation of this hybrid.

In Vitro Propagation of Heliconia bihai (L.) L. from Zygotic Embryos

(In vitro Propagation of Heliconia bihai L. from Zygotic Embryos). The internal morphology of embryos from immature and mature fruits of Hcliconia bihai (L.) L. cv. Lobster Claw Two was examined. Embryos were inoculated into MS media (full MS and 1/2 MS) and GA(1) (0.2.5 and 5 mg L(-1)) with either sucrose or glucose. These plantlets were then replicated and transferred to MS medium (full MS or 1/2 MS) with 0 or 2.5 mg L(-1) BAP and their multiplication was evaluated 30 and 45 days after inoculation. The genetic variability of the multiplied plants was estimated using isoenzyme analyses. The internal morphology of the mature embryos revealed their tissues to be in more advanced stages of differentiation than immature embryos. In the conversion phase, 85% of the inoculated embryos developed into plants in the 1/2 MS medium with sucrose, in contrast to only 41% of the embryos that were cultivated with glucose. In the multiplication phase, plants cultivated in 1/2 MS medium with 2.5 mg L(-1) BAP demonstrated more buds. Isoenzyme analyses showed pattern changes in terms of the color intensity and the migration of some of the bands. These results may be associated with differences in the ages of the mother plants and of the plantlets obtained in vitro.

Effect of juvenility on cutting propagation of red pitaya

Introduction. Pitaya (Hylocereus undatus) is an exotic fruit species little known in Brazil and which needs basic studies about plant nutrition, propagation and physiology. Emphasizing the co-existence of juvenile and adult stages in the pitaya canopy, the plant is generally propagated by cuttings. Materials and methods. A completely randomized design with four treatments and five replications was adopted. Each treatment was represented by the part of the canopy from which the cutting was taken ( upper, middle and lower cutting and cuttings from young plants). The following variables were registered: % cuttings with roots, % of live cuttings, root density, root diameter, root area, root length and root dry mass. Results were submitted to variance analyses, Tukey's test at 0.01 probability error and simple correlation analysis. Results and discussion. The results indicated that the position from which the cutting is taken had a quantitative effect on rooting formation of pitaya cuttings. Juvenile cuttings presented 35% more cuttings with roots than adult cuttings. Root density, root area, root length and root dry mass depended on juvenility, the highest results being registered for juvenile cuttings, independently of the variable. Conclusion. Juvenile and adult stages co-exist in the pitaya canopy. Juvenility is an important rooting factor for red pitaya cuttings.

In vitro propagation of Maytenus ilicifolia (Celastraceae) as potential source for antitumoral and antioxidant quinomethide triterpenes production. A rapid quantitative method for their analysis by reverse-phase high-performance liquid chromatography

Cell culture of Maytenus ilicifolia were established in order to produce and to quantify the antitumoral and antioxidant quinonemethide triterpenes. In vitro calli were induced from leaf explants of native plants and cultured in semi-solid medium under controlled conditions of humidity, temperature and photoperiod. The quinonemethide triterpenes showed maximum accumulation in the logarithmic phase growth of the cell culture. A rapid, sensitive and reliable reverse-phase HPLC method was used for quantitative determination of the antitumoral and antioxidant quinonemethide triterpenes, 22β-hydroxymaytenin and maytenin in callus of Maytenus ilicifolia. Well resolved peaks with good detection response and linearity in the range 1.0 - 100 μg/mL were obtained. This quantitative work was performed by an external standard method.

Dovyalis hebecarpa propagation by the use of cuttings

The vegetative propagation of Dovyalis hebecarpawas studied using herbaceous cuttings of a hybrid introduced in Brazil by the College of Agriculture, Campus of Jaboticabal-UNESP. The treatments consisted of (1) evaluating the effect of five 3-Indolebutyric acid (IBA) doses (0 (control), 1,000, 3,000, 5,000 and 7,000 mg.L -1); (2) the influence of two kinds of herbaceous cuttings (apical and sub-apical) and (3) the collection position on the plant (upper and lower part of the canopy) at two different times of the year (autumn and spring). The experimental design was completely randomized with four replicates of 10 cuttings each; the analysis was on a 5 × 2 × 2 factorial layout. The growth regulator (IBA) did not influence the rooting of cuttings in either sampling season. The best season for the rooting was spring. Apical cuttings were desirable for rooting in both seasons. In autumn cuttings taken from the lower portion of the plant showed significantly higher rooting values than the ones from the upper portion; and in spring cuttings taken from the upper portion had higher rooting percentages. © 2007 by The Haworth Press. All rights reserved.

Lychee propagation (Litchi chinensis Sonn.): A comparative study of rootstocks

The lychee (Litchi chinensis Sonnerat), of the family Sapindaceae, is a highly valued fruit throughout the world. It is native to southern China, but it is cultivated in many parts of the world. In Brazil, the cultivation of the lychee was initiated in the 1970s in the State of São Paulo and has been increasing in production even though the crop size still averages only 1.4 ha per producer. The low crop size is attributed to the high cost of seedlings and the amount of time required for the plant to bear fruit. These factors discourage planting in large areas. The purpose of this research is to study some aspects of what is vitally important for the production of lychee seedlings on a large scale, with low cost and good genetic quality. Tests were performed using rootstocks from seeds of lychee and longan (Euphoria longan Lam.). Scions for grafting were taken from plants of lychee 'Bengal' in the collection of fruit species from FCAV-UNESP, Campus of Jaboticabal, Brazil. The experiments were started in September 2007 and the grafting process was performed in each month of the year finishing in August 2008. During this period the influence of these criteria were evaluated: 1) time of the year; 2) species of rootstock; 3) percentage of grafting success; 4) stem height at grafting point; 5) stem diameter at grafting point. Therefore, through statistical analysis we obtained significant differences in relation to the rootstock used, the months of the year in which the grafting was performed and the interaction between rootstock and the month in which the grafting was performed.

Propagation of Camptosema grandiflorum native to Brazil by seeds and cuttings

Camptosema grandiflorum Benth., belonging to Fabaceae, is a voluble climber plant native to Brazil. Plants bloom in autumn-winter, producing long and hanging inflorescences with showy red flowers, which are much visited by hummingbirds. The leaves are also attractive, composed by three leaflets. It can be propagated by seeds or cuttings, but both seed germination and cutting rooting percentages are very low. Thus, the objective of this work was to study the effect of different temperatures on seed germination and of different indolebutyric acid (IBA) concentrations on the rooting of cuttings of C. grandiflorum. The experiment was set up at the São Paulo State University, located in Jaboticabal, São Paulo State, Brazil. The germination study was conducted in an entirely randomized design with six different temperatures (constant at 20, 25, 30 and 35°C; and alternated at 20-30 and 25-35°C, with a photoperiod of 12 hours) and four replications of 25 seeds each, placed in plastic boxes with vermiculite. The percentage of germination and the speed germination index (SGI) were evaluated. An entirely randomized block design was adopted for the cutting rooting evaluation, with four IBA concentrations (0; 1,000; 2,000; and 3,000 mg kg-1) and five replications of ten cuttings each, comprising 200 cuttings. After 30 days from the beginning of the rooting experiment, data referring to rooting percentage, number and length of roots and dry weight of roots were collected. For the seed germination experiment, fastest germination and highest germination percentage (87%) were obtained when seeds were maintained under the constant temperature of 30°C. For the cutting experiment, the concentrations of 2,000 and 3,000 mg kg-1 of IBA promoted the highest rooting percentages (98.5 and 94.1%, respectively) and number of roots. There were no statistical differences among the IBA concentrations for length of roots and dry weight of roots.

Growth regulators for in vitro propagation of Tillandsia gardneri (Bromeliaceae)

Tillandsia gardneri Lindi, is a herbaceous perennial with ornamental value. However, in Brazil there is no report about this species' cultivation on a commercial scale. The low multiplication rate of T. gardneri (in average one offshoot/plant/year) leads to illegal over-collection in the wild to meet commercial demands. The development of protocols for in vitro propagation of T. gardneri may be useful for increasing multiplication rate, producing enough plants to supply the ornamental market and also to reduce the pressure over plants collection in the wild. The present study evaluated the effect of growth regulators (6-benzylaminopurine-BA alone or in combinations with naphthaleneacetic acid-NAA) on shoots development from seedlings pre-established in vitro, from seed germination on 1/4 MS medium without growth regulators. Seedlings (with about 1.0 cm long) were re-cultured to solid 1/2 MS media supplemented with growth regulators. After 30 days on the induction medium seedlings were re-cultured to MS basal medium. The experiment was conducted in a complete randomized design with four replications and ten treatments: control (free of growth regulators), BA (0.5, 1.0 and 2.0 mg/L), BA (0.5 mg/L) + ANA (0.1 mg/L), BA (1.0 mg/L) + ANA (0.1 mg/L), BA (1.0 mg/L) + ANA (0.5 mg/L), BA (2.0 mg/L) + ANA (0.1 mg/L), BA (2.0 mg/L) + ANA (0.5 mg/L), and BA (2.0 mg/L) + ANA (1.0 mg/L). The outgrowth of shoots did not occur on medium devoid of growth regulators (control). Regression analysis for some evaluated parameters, such as percentage of seedlings responsive to shoot formation and number of shoots/seedling, and regulators concentrations (BA or ANA) were significant, allowing the establishment of the growth regulators concentration for obtaining the best multiplication rate. Some seedlings maintained in media with ANA (0.5 or 1.0 mg/L) were completely converted into callus masses that turned dark brown leading to seedlings death.

Adventitious shoot induction from leaf segments in Anthurium andreanum is affected by age of explant, leaf orientation and plant growth regulator

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Auxin Regulates the Initiation and Radial Position of Plant Lateral Organs

Leaves originate from the shoot apical meristem, a small mound of undifferentiated tissue at the tip of the stem. Leaf formation begins with the selection of a group of founder cells in the so-called peripheral zone at the flank of the meristem, followed by the initiation of local growth and finally morphogenesis of the resulting bulge into a differentiated leaf. Whereas the mechanisms controlling the switch between meristem propagation and leaf initiation are being identified by genetic and molecular analyses, the radial positioning of leaves, known as phyllotaxis, remains poorly understood. Hormones, especially auxin and gibberellin, are known to influence phyllotaxis, but their specific role in the determination of organ position is not clear. We show that inhibition of polar auxin transport blocks leaf formation at the vegetative tomato meristem, resulting in pinlike naked stems with an intact meristem at the tip. Microapplication of the natural auxin indole-3-acetic acid (IAA) to the apex of such pins restores leaf formation. Similarly, exogenous IAA induces flower formation on Arabidopsis pin-formed1-1 inflorescence apices, which are blocked in flower formation because of a mutation in a putative auxin transport protein. Our results show that auxin is required for and sufficient to induce organogenesis both in the vegetative tomato meristem and in the Arabidopsis inflorescence meristem. In this study, organogenesis always strictly coincided with the site of IAA application in the radial dimension, whereas in the apical–basal dimension, organ formation always occurred at a fixed distance from the summit of the meristem. We propose that auxin determines the radial position and the size of lateral organs but not the apical–basal position or the identity of the induced structures.